The UFL1-AKT positive feedback loop promotes breast cancer progression by enhancing lipid synthesis

Domain: biology
Published: 2026-01-20
Editorial outcome: minor_revision

Abstract
UFMylation, a ubiquitin-like modification, is crucial for cellular processes and is linked to human diseases, including cancer. However, its role in cancer remains unclear. Here, we report that UFL1 promotes breast tumor growth by remodeling lipid metabolism. Mechanistically, UFL1 interacts with and UFMylates AKT, enhancing its localization at the endoplasmic reticulum and phosphorylation by PDK1 and mTORC2, thereby increasing AKT-mediated lipid synthesis. Moreover, AKT phosphorylates UFL1, boosting its activity. Thus, UFL1 and AKT form a positive feedback loop, accelerating lipid synthesis and breast tumor growth. Clinically, UFL1 levels are increased in human breast tumors and are associated with poor clinical outcomes in breast cancer patients. Importantly, UFMylation inhibitors sensitize breast cancer cells to AKT inhibitors and anticancer drugs. Our findings reveal a critical role for UFMylation in lipid metabolism and identify the UFL1-AKT axis as a potential therapeutic target in breast cancer.

Introduction
UFMylation, a ubiquitin-like modification, is sequentially catalyzed by E1 (UBA5, ubiquitin-like modifier activating enzyme 5), E2 (UFC1, ubiquitin-fold modifier-conjugating enzyme 1), and E3 (UFL1, UFM1-specific ligase 1), leading to the covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to the lysine residues of substrates1–3. Currently, UFL1 is the sole E3 ligase for UFMylation; however, by itself, UFL1 is unstable and inactive4,5. UFBP1 (UFM1 binding protein 1) is essential for UFL1 stability and activity1,6,7. UFMylation is a highly dynamic process, and UFM1 can be removed from UFMylated substrates by UFSP1/2 (UFM1-specific proteases 1/2) to prevent excessive UFMylation8–10. A growing body of evidence highlights the functional importance of UFMylation in various biological processes, such as DNA damage repair11–14 and endoplasmic reticulum (ER) stress response15,16. Although UFMylation dysregulation has been implicated in cancers, only a few UFMylation substrates have been identified in cancer cells, and the functions and molecular mechanisms of UFMylation in cancer remain largely uncharted. Moreover, UFMylation exhibits different, or even opposing, functions depending on cancer types or subtypes17. Therefore, identifying novel UFMylation substrates and deciphering their functions, as well as the underlying mechanisms, will advance our understanding of the fundamental roles of UFMylation in cancer and may potentially benefit cancer treatment. Recent research shows that UFL1 depletion in bovine mammary epithelial cells dramatically decreases the expression of lipid synthesis-related genes, while UFBP1 ablation increases lipid accumulation in hepatocytes18,19, suggesting potential roles for UFMylation in lipid metabolism under physiological conditions, although the underlying mechanisms remain elusive. However, whether and how UFMylation modulates lipid metabolism in cancer remains to be explored.

Increased de novo lipid synthesis contributes to the unlimited proliferation and survival of cancer cells and is therefore considered a hallmark of cancer20,21. The serine/threonine protein kinase AKT (also known as protein kinase B, PKB) plays a central role in de novo lipogenesis by directly regulating the activity of ACLY (ATP citrate lyase) or indirectly modulating the expression of ACC1 (acetyl-CoA carboxylase 1), FASN (fatty acid synthase), ACSS2 (acyl-CoA synthetase short-chain family member 2), SCD1 (stearoyl-CoA desaturase), and HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), which are rate-limiting enzymes in de novo lipid synthesis22,23. In mammalian cells, there are three AKT isoforms (AKT1, AKT2, and AKT3), which possess redundant as well as unique functions23. AKT1 is an oncogenic kinase that is hyperphosphorylated and overactive in most human cancers24, with elevated AKT1 activity observed in approximately 40% of breast cancers25. The recruitment of AKT1 to the plasma membrane or intracellular membrane through binding of its PH (pleckstrin homology) domain to PIP3 (phosphatidylinositol 3,4,5-trisphosphate) is a prerequisite for AKT1 phosphorylation at threonine (T) 308 by PDK1 and at serine (S) 473 by Rictor-mTOR complex (mTORC2)26,27. Phosphorylation at T308 and S473 leads to full AKT1 activation26. Despite reports that ubiquitination28, methylation29,30, and SUMOylation31 control AKT1 activity, the mechanisms underlying AKT1 membrane localization and activity remain poorly understood.

In the present study, we report that a positive feedback loop between UFL1 and AKT1 promotes breast tumor growth by reprogramming lipid metabolism. Mechanistically, UFL1 UFMylates AKT1 at lysines (K) 189, K276, and K297, disrupting the interaction between the PH domain and KD (kinase domain) of AKT1, thereby facilitating AKT1 localization at the ER and subsequent phosphorylation at T308 and S473 by PDK1 and mTORC2, respectively. Interestingly, AKT1 phosphorylation stimulates UFSP2-mediated AKT1 deUFMylation. On the other hand, AKT1 phosphorylates UFL1 at T426, enhancing UFL1 activity and AKT1 UFMylation by UFL1. Activated AKT1 sustains breast tumor growth by phosphorylating ACLY and inducing the expression of several lipogenic genes to reinforce lipid synthesis. Clinically, the mRNA and protein levels of UFL1 are both elevated in breast cancer tissues and negatively correlate with the overall survival of breast cancer patients. Intriguingly, the levels of phosphorylated UFL1 and phosphorylated AKT1 display a positive correlation in human breast tumors. Importantly, targeting UFMylation and AKT1 exhibits a synergistic anticancer effect in vitro and in vivo. Our findings delineate a mechanism in lipid metabolism via the UFL1-AKT1 positive feedback loop and uncover this regulatory loop as a potential therapeutic target in breast cancer.

Results
UFL1 possesses oncogenic potential in breast cancer
UFL1 is the only E3 ligase in the UFMylation system, but its expression and function in cancers are highly context-dependent. Previous studies on UFL1 in breast cancer, even within the same breast cancer subtype, have led to controversial conclusions32,33. To comprehensively evaluate the clinical relevance of UFL1, we first assessed its expression levels in human breast tumors. Analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) breast cancer dataset revealed that UFL1 mRNA levels were significantly increased in human breast tumors compared with normal breast tissues (Fig. 1A). This elevation was observed in all breast cancer subtypes, including Luminal A, Luminal B, human epidermal growth factor receptor 2-positive (HER2+), and triple-negative breast cancer (TNBC) (Supplementary Fig. 1A). The Gene Expression Omnibus (GEO) dataset also indicated the highest levels of UFL1 mRNA in stage III breast cancers (Supplementary Fig. 1B). Importantly, GEPIA (Fig. 1B) and GEO (Supplementary Fig. 1C) datasets both showed that high levels of UFL1 mRNA were associated with decreased overall survival in breast cancer patients. Furthermore, we performed immunohistochemistry (IHC) assays and found that UFL1 protein levels were markedly upregulated in breast tumors (Fig. 1C, D), and all breast cancer subtypes showed elevated UFL1 protein levels (Supplementary Fig. 1D). Consistently, moderate and high levels of UFL1 protein were associated with poor clinical outcomes in breast cancer patients (Fig. 1E). A similar negative correlation was found in the Kaplan–Meier Plotter dataset (Fig. 1F). Collectively, UFL1 expression is increased in breast tumors and negatively correlates with clinical outcomes in breast cancer patients.

The aforementioned findings prompted us to hypothesize that UFL1 is an oncogenic protein in breast cancer. To confirm this hypothesis, UFL1 was depleted in MCF-7 (Luminal A), BT474 (Luminal B), SK-BR-3 (HER2+), MDA-MB-231 (TNBC), and MDA-MB-468 (TNBC) breast cancer cells using two independent shRNAs targeting UFL1 (shUFL1#1 and shUFL1#2) for cell growth assays. Compared with scrambled shRNA (shSC), shUFL1#1 and shUFL1#2 dramatically suppressed breast cancer cell growth (Fig. 1G, H; Supplementary Fig. 1E–G). Due to the lack of effective therapeutic targets, TNBC is much more aggressive than other breast cancer subtypes. Therefore, we mainly focused on TNBC cells. Cell growth is coordinately controlled by cell proliferation and cell death. Immunostaining assays showed that UFL1 depletion dramatically downregulated Ki-67 (a cell proliferation marker) protein levels (Supplementary Fig. 1H, I), while increasing the protein levels of cleaved caspase-3 (CC3, a cell death marker) (Supplementary Fig. 1J, K). Consistently, the intensity of Annexin-V (a marker of early apoptotic cells) and propidium iodide (PI, a marker of necrotic or late apoptotic cells) staining was significantly increased by UFL1 ablation, as evidenced by flow cytometry assays (Supplementary Fig. 1L, M). All these results suggested a critical role for UFL1 in promoting proliferation and suppressing cell death. Next, we conducted xenograft mouse assays. shSC, shUFL1#1, and shUFL1#2 MDA-MB-468 cells were implanted into the mammary fat pads of female NOD-SCID mice. As expected, UFL1 depletion significantly inhibited MDA-MB-468 tumor growth (Fig. 1I, J; Supplementary Fig. 1N). Consistently, immunohistochemistry (IHC) staining revealed that UFL1 knockdown markedly enhanced cell death and impeded cell proliferation, as evidenced by increased CC3-positive cell numbers and decreased Ki-67-positive cell numbers, respectively (Fig. 1K–N). Taken together, these findings indicate that UFL1 promotes breast tumor growth by increasing cell proliferation and repressing cell death.

UFL1 is indispensable for lipid accumulation in breast cancer cells
To delineate the mechanism of UFL1-mediated breast tumor growth, we conducted Gene Set Enrichment Analysis (GSEA) using the Biomarker Exploration for Solid Tumors (BEST, https://rookieutopia.com/) database to identify biological processes potentially regulated by UFL1 in breast cancer34. Fatty acid metabolism emerged as a strong candidate (Supplementary Fig. 2A), as lipid synthesis and lipid accumulation are usually elevated in cancer cells and positively correlate with poor clinical outcomes35–37. Additionally, previous studies have shown that dysfunction of the UFMylation system alters lipid accumulation, although the underlying mechanisms remain poorly understood18,19. Based on these findings, we hypothesized that UFL1 may modulate lipid metabolism in breast cancer cells. To test this hypothesis, we analyzed lipid content by performing Oil Red O (ORO) staining and ORO absorbance assays in UFL1 knockdown TNBC cells38. As expected, the absorbance of eluted ORO at 510 nm was significantly downregulated by UFL1 ablation in MDA-MB-231 and MDA-MB-468 cells (Fig. 2A, B). Furthermore, we quantified lipid content using the fluorescent lipid dye BODIPY 493/503. Consistent with the ORO staining results, UFL1 depletion significantly decreased lipid accumulation, as evidenced by decreased BODIPY 493/503 fluorescence intensity in MDA-MB-231 and MDA-MB-468 cells (Fig. 2C–F). In accordance with these findings, the levels of total free fatty acids and cholesterol were both decreased by UFL1 ablation in MDA-MB-231 and MDA-MB-468 cells (Fig. 2G–J). Importantly, lipid abundance in MDA-MB-468 breast tumors was markedly decreased by UFL1 knockdown (Fig. 2K, L). Taken together, these results demonstrate that UFL1 regulates lipid accumulation in breast cancer cells.

To confirm whether UFL1 controls breast cancer cell growth by modulating lipid accumulation, control and UFL1 knockdown breast cancer cells were cultured in low-serum medium (1% FBS) for 24 h and subsequently incubated with or without oleic acid (OA), one of the most abundant fatty acids that promote breast cancer cell proliferation at low concentration39. Our results showed that the decreased growth of breast cancer cells due to UFL1 depletion was almost completely restored by OA supplementation (Supplementary Fig. 2B, C), indicating that UFL1 promotes breast cancer cell growth by elevating lipid abundance. SREBP1 controls the expression of several rate-limiting enzymes involved in lipid synthesis, including ACLY, ACC1, SCD1, and FASN40. Therefore, MDA-MB-231 and MDA-MB-468 cells stably expressing UFL1 were established (Supplementary Fig. 2D) and cultured in the presence or absence of the SREBP1 inhibitor Fatostatin. As expected, UFL1 overexpression significantly increased the levels of total free fatty acids and cholesterol, and this increase was almost completely abolished by Fatostatin (Supplementary Fig. 2E–H), suggesting that UFL1 may modulate lipid synthesis in breast cancer cells. This conclusion was well supported by the results of BODIPY 493/503 staining assays (Supplementary Fig. 2I–L). Importantly, UFL1 overexpression-induced cell proliferation was diminished by Fatostatin treatment (Supplementary Fig. 2M, N). Collectively, these data indicate that UFL1 promotes breast cancer cell growth by increasing lipid synthesis.

To further validate the prediction that UFL1 exerts its oncogenic functions through modulating lipid accumulation in vivo, xenograft assays were performed by injecting MDA-MB-231 cells harboring an empty vector (EV) or a vector encoding UFL1 into the mammary fat pads of female NOD-SCID mice, followed by intraperitoneal (IP) injection of vehicle or Fatostatin. In keeping with the in vitro findings, ectopically expressed UFL1 promoted breast tumor growth (Fig. 2M, N; Supplementary Fig. 2O), increased lipid abundance (Fig. 2O, P), suppressed cell death (Fig. 2Q, R), and enhanced cell proliferation (Fig. 2Q, S), without affecting the body weight of tumor-bearing mice (Supplementary Fig. 2P). These effects of UFL1 in breast tumors were effectively reversed by Fatostatin (Fig. 2M–S; Supplementary Fig. 2O–R). Taken together, these results demonstrate that UFL1 promotes breast tumor growth by regulating lipid synthesis.

UFL1 interacts with and UFMylates AKT
To elucidate the mechanism of UFL1-mediated lipid accumulation, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays using MDA-MB-231 cells stably expressing Flag-UFL1 to identify potential substrates of UFL1. LC-MS/MS analysis revealed that AKT1 is a high-confidence potential UFL1-binding protein (Supplementary Fig. 3A). Notably, several key components of the UFMylation system, such as DDRGK1 (UFBP1), CDK5RAP3, UFM1 and UFC1, as well as more than 10 well-known UFL1 substrates, including PARP114, RPL2616, MRE1112, 13 and p5341, were also identified in the list of putative UFL1-interacting proteins (Supplementary Fig. 3A), confirming the feasibility of our approach. Moreover, GSEA analysis via the BEST database indicated that UFL1 may regulate the PI3K-AKT-mTOR signaling pathway, which is critical for lipid metabolism (Supplementary Fig. 3B). Consistent with the LC-MS/MS analysis, exogenous UFL1 showed a robust interaction with ectopically expressed AKT1 but weakly associated with AKT1’s paralogs, AKT2 or AKT3, in HEK293T cells (Supplementary Fig. 3C). Hence, we focused on AKT1. The endogenous interaction between UFL1 and AKT1 was also observed in MDA-MB-231 (Fig. 3A, B) and MDA-MB-468 (Fig. 3C, D) cells. To further determine whether UFL1 directly binds to AKT1, we performed in vitro GST pull-down assays using purified GST-AKT1 and immunoprecipitated UFL1 from breast cancer cells. UFL1 was pulled down by GST-AKT1 but not by GST alone (Fig. 3E), suggesting a direct interaction between UFL1 and AKT1. Given that UFL1 predominantly localizes to the perinuclear ER6,7, we performed immunofluorescence staining and found that UFL1 and AKT1 mainly colocalized at the perinuclear region of MDA-MB-231 cells (Fig. 3F; Supplementary Fig. 3D), suggesting potential ER colocalization of UFL1 and AKT1 in breast cancer cells. Next, we sought to identify the domains that mediate the UFL1-AKT1 interaction. Domain-mapping assays indicated that UFL1 associates with AKT1 via its N-terminus (Fig. 3G, H; Supplementary Fig. 3E, F). Additionally, AKT1 associated with UFBP1 and UFC1 (Supplementary Fig. 3G, H), suggesting that UFL1 may modulate AKT1 in a UFMylation-dependent manner.

Indeed, AKT1 was UFMylated in breast cancer cells, and this modification was significantly decreased by UFL1 ablation in MDA-MB-231 and MDA-MB-468 cells (Fig. 3I, J). In contrast, ectopic expression of UFL1 in MDA-MB-231 and MDA-MB-468 cells led to upregulation of AKT1 UFMylation (Supplementary Fig. 3I, J). UFL1 is inactive on its own and is activated by forming a complex with UFBP15,42,43. Mutation of leucine (L) 45 to arginine (R) in UFL1 (L45R) disrupts the UFL1-UFBP1 interaction5,42,43. To further assess the impact of UFL1 activation on AKT1 UFMylation, we compared the effect of wild-type (WT) and L45R mutant UFL1 and found that only WT UFL1, not L45R UFL1, was capable of UFMylating AKT1 (Supplementary Fig. 3K). These data confirm that UFL1 UFMylates AKT1 in breast cancer cells. To identify potential UFMylation sites in AKT1, we assessed UFMylation within AKT1’s conserved domains, including the PH domain, KD, and CD (C-terminal domain). Our results showed that UFMylation levels were significantly higher in the KD compared to the PH domain and CD (Fig. 3K), suggesting that the primary UFMylation sites are located within the KD. The KD contains six conserved lysine (K) residues (Supplementary Fig. 3L), and we constructed Flag-AKT1 variants with single mutations at each of these K residues to assess UFMylation in HEK293T cells. Notably, single mutations of K to R at positions K189, K276, or K297 significantly reduced AKT1 UFMylation (Supplementary Fig. 3M), indicating that these sites are key for AKT1 UFMylation. Indeed, a triple mutation of these residues (referred to as 3K/R) nearly abolished AKT1 UFMylation (Supplementary Fig. 3M). To further confirm that AKT1 is UFMylated at K189, K276, and K297 in TNBC cells, we expressed both WT and 3K/R mutant Flag-AKT1 at comparable levels in MDA-MB-231 and MDA-MB-468 cells. Compared with Flag-AKT1-WT, the UFMylation levels of Flag-AKT1-3K/R were significantly lower in these TNBC cells (Fig. 3L, M). Similar results were obtained from MCF-7, BT474, and SK-BR-3 breast cancer cells (Supplementary Fig. 3N–P). These findings demonstrate that K189, K276, and K297 are key UFMylation sites in AKT1 in breast cancer cells. Although K189, K276, and K297 are conserved in AKT2 and AKT3, their UFMylation levels were lower than those in AKT1 (Supplementary Fig. 3Q) potentially due to their weak association with UFL1 (Supplementary Fig. 3C), suggesting that AKT1 is the primary substrate of UFL1. Intriguingly, other lipid synthetic enzymes, such as FASN and Rictor (Supplementary Fig. 3A), were also identified as potential UFL1-interacting proteins in LC-MS/MS analysis, prompting us to test their UFMylation. Indeed, UFL1 associated with and UFMylated FASN and Rictor in breast cancer cells (Supplementary Fig. 3R–W). Compared with FASN and Rictor, AKT1 possesses a much more comprehensive and profound impact on lipid synthesis, Hence, we focused on AKT1. Collectively, our data demonstrate that UFL1 directly associates with AKT1 and UFMylates AKT1 at K189, K276, and K297 in breast cancer cells.

AKT UFMylation is a prerequisite for AKT activation and AKT-mediated lipid synthesis
UFMylation regulates protein stability or activity6,7, prompting us to assess its impact on AKT1 protein levels and AKT1 phosphorylation at T308 (p-AKT1(T308)) and S473 (p-AKT1(S473)), both of which are critical for AKT1 activation and function. Remarkably, we observed a reduction in p-AKT1(T308) and p-AKT1(S473) in MDA-MB-231 and MDA-MB-468 cells upon UFL1 depletion, while the AKT1 protein levels remained unchanged (Fig. 4A; Supplementary Fig. 4A). Similar results were obtained in MCF-7, SK-BR-3, and BT474 cells (Supplementary Fig. 4B). In line with these findings, depletion of UBA5, the unique E1 enzyme of the UFMylation system, led to decreased AKT1 phosphorylation (Supplementary Fig. 4C). Thus, AKT1 UFMylation regulates its phosphorylation without affecting its stability. Moreover, when MDA-MB-231 and MDA-MB-468 cells expressing comparable levels of Flag-AKT1-WT and Flag-AKT1-3K/R were analyzed for phosphorylation, Flag-AKT1-WT showed robust phosphorylation at T308 and S473, whereas p-AKT1(T308) and p-AKT1(S473) were nearly undetectable in Flag-AKT1-3K/R (Fig. 4B). These results indicate that UFMylation at K189, K276, and K297 is essential for AKT1 phosphorylation and full activation.

It has been suggested that AKT1 membrane localization and the disruption of AKT1 autoinhibition are critical for its full activation, but the underlying mechanisms remain poorly understood44–51. As mentioned above, UFL1 is a perinuclear ER-resident protein that colocalizes with AKT1 in the perinuclear region of breast cancer cells (Fig. 3F), suggesting that UFL1 may influence AKT1’s ER localization. Consistent with this hypothesis, our immunostaining experiments showed that UFL1 knockdown significantly impaired the colocalization of AKT1 with the well-known ER marker Calreticulin (Fig. 4C; Supplementary Fig. 4D). Furthermore, subcellular fractionation assays revealed that the abundance of AKT1, p-AKT1(T308), and p-AKT1(S473) on the ER was decreased by UFL1 depletion in MDA-MB-231 and MDA-MB-468 cells, regardless of insulin treatment, which induces PIP3 generation (Fig. 4D; Supplementary Fig. 4E–H). By contrast, the protein levels of p-AKT1(T308) and p-AKT1(S473), but not AKT1, were reduced in the cytosol following UFL1 ablation (Fig. 4D; Supplementary Fig. 4E–H). In contrast, UFL1 overexpression enhanced AKT1 ER localization and activation in MDA-MB-231 cells (Supplementary Fig. 4I). Moreover, PIP3-conjugated beads were used for co-IP assays with lysates of ER fractions isolated from control and UFL1 knockdown breast cancer cells, with or without insulin treatment. UFL1 depletion markedly reduced the association between AKT1 and PIP3 on the ER (Fig. 4E). These findings suggest that UFL1 promotes AKT1 ER localization and activation. Intramolecular interaction between the PH domain and KD maintains AKT1 in an inactive state46,52–54. Notably, AKT1-3K/R mutation remarkably strengthened the interaction between the PH domain and KD in breast cancer cells (Fig. 4F), indicating that AKT1 UFMylation triggers a conformation change in AKT1, thereby abolishing AKT1 autoinhibition. Taken together, UFL1 enhances AKT1 ER localization, relieves AKT1 autoinhibition, and consequently triggers AKT1 activation in breast cancer cells.

PDK1 and mTORC2 are responsible for the phosphorylation of AKT1 at T308 and S473, respectively55,56. We predicted that UFL1 might influence the interaction between AKT1 and mTORC2 or PDK1. As shown in Fig. 4G, the endogenous interactions of AKT1 with Rictor and PDK1 were both dramatically mitigated by UFL1 knockdown in TNBC cells. Conversely, UFL1 overexpression enhanced the interaction of AKT1 with Rictor and PDK1 in TNBC cells (Supplementary Fig. 4J). Notably, neither depletion nor overexpression of UFL1 influenced the protein levels of PDK1 and Rictor (Fig. 4G; Supplementary Fig. 4J). These findings suggest that UFL1 strengthens the interaction between AKT1 and Rictor, as well as between AKT1 and PDK1, in breast cancer cells. It is worth noting that UFMylation seemed to have no impact on the molecular weight of phosphorylated AKT1, suggesting that AKT1 phosphorylation may promote its deUFMylation. To verify this prediction, endogenous AKT1 and phosphorylated AKT1(S473) were examined by western blotting analysis using an anti-AKT1 antibody or an anti-p-AKT1(S473) antibody. After a long exposure, weak p-S473 and AKT1 bands, whose molecular weights were approximately 27 kDa greater than that of their corresponding lower bands, were observed in shSC cells, and these upper bands nearly disappeared in UFL1 knockdown cells (Supplementary Fig. 4K). On the contrary, the intensity of the upper bands was robustly increased by depletion of UFSP2 (Fig. 4H; Supplementary Fig. 4L), which catalyzes deUFMylation. These data indicated that phosphorylated AKT1 may undergo deUFMylation. Indeed, the UFMylation levels of AKT1-WT were much lower than those of mutant AKT1 (AKT1-DM), which harbors T308A and S473A mutations (Fig. 4I). Moreover, the interaction between UFSP2 and AKT1-WT was stronger than the UFSP2-AKT1-DM association, and the former, but not the latter, interaction was increased by insulin treatment (Fig. 4J). Notably, UFL1 depletion also downregulated the protein levels of phosphorylated but not total PLK1 (Supplementary Fig. 4M), which is a substrate of mTORC2, probably due to UFL1-dependent Rictor UFMylation (Supplementary Fig. 3T, U, W). In contrast, UFL1 ablation did not influence total and phosphorylated PKCα (Supplementary Fig. 4M), which is a substrate of PDK1. These results indicated that UFL1 may modulate mTORC2 activity by UFMylating Rictor. Taken together, UFMylation promotes PDK1- and mTORC2-mediated AKT1 phosphorylation, which in turn accelerates UFSP2-mediated AKT1 deUFMylation.

Active AKT1 is essential for de novo lipid synthesis through the phosphorylation of ACLY and activation of SREBP1, a transcription factor that drives the transcription of several genes involved in lipid synthesis, including ACLY, ACC1, SCD1, and FASN40. UFL1 depletion significantly decreased the protein levels of phosphorylated ACLY (p-ACLY (S455)), although the total ACLY protein levels were slightly reduced (Fig. 4K; Supplementary Fig. 4N, O). Additionally, the protein levels of ACC1, ACSL3, and FASN were also downregulated by UFL1 knockdown, likely due to impaired SREBP1 maturation (Fig. 4K; Supplementary Fig. 4N, O). In contrast, UFL1 overexpression elevated the levels of p-ACLY (S455), ACLY, ACSL3, ACC1, and FASN (Supplementary Fig. 4P). Consistently, UFL1 ablation suppressed the mRNA levels of these lipid synthesis-related genes (Supplementary Fig. 4Q), without perturbing the transcription of genes involved in lipid export (Supplementary Fig. 4R), uptake (Supplementary Fig. 4S), or oxidation (Supplementary Fig. 4T). Collectively, these results indicate that UFL1 promotes AKT1-mediated ACLY phosphorylation and the transcription of lipid synthesis-related genes, thereby enhancing lipid synthesis and accumulation.

Deficiency in AKT UFMylation impairs its oncogenic potential
Since AKT1 activation is impaired in the absence of AKT1 UFMylation, we hypothesized that AKT1-mediated lipid accumulation and breast tumor growth would be attenuated by the 3K/R mutation. Indeed, ORO and BODIPY 493/503 staining assays showed that the decreased lipid accumulation due to AKT1 ablation was completely restored by AKT1-WT, but not AKT1-3K/R, in TNBC cells (Fig. 5A–D; Supplementary Fig. 5A–E). In line with these findings, the reduction in free fatty acids and cholesterol abundance due to AKT1 depletion was rescued by AKT1-WT, but not by the AKT1-3K/R, in TNBC cells (Fig. 5E, F; Supplementary Fig. 5F, G). Additionally, overexpressed AKT1-WT, but not AKT1-3K/R, elevated lipid abundance in MCF-7, BT474, and SK-BR-3 cells (Supplementary Fig. 5H–M). Hence, UFL1 promotes lipid accumulation through UFMylated AKT1. To explore the role of AKT1 UFMylation in tumorigenesis in vivo, we conducted xenograft mouse assays. As expected, the retarded growth of MDA-MB-231 breast tumors caused by AKT1 ablation was restored by AKT1-WT, but the 3K/R mutant AKT1 failed to do so (Fig. 5G, H; Supplementary Fig. 5N). Consistent with the results in Fig. 5A–F, the disturbed lipid accumulation caused by AKT1 knockdown in breast tumors was rescued by AKT1-WT, but not by AKT1-3K/R, as evidenced by ORO staining (Fig. 5I, J). IHC staining indicated that AKT1 depletion in MDA-MB-231 tumors led to increased CC3 and decreased Ki-67 signals, which were reversed by reconstituted AKT1-WT, but not by AKT1-3K/R (Fig. 5K–M). These findings suggest that AKT1 UFMylation plays a critical role in regulating cell proliferation and cell death in breast tumors. Importantly, AKT1-WT, but not AKT1-3K/R, in shAKT1 breast tumors displayed comparable levels of p-AKT1(T308) and p-AKT1(S473) to those observed in shSC control tumors (Fig. 5K, N–P). This indicates that AKT1 UFMylation is essential for AKT1 phosphorylation and activation in breast tumors. Given that UFL1 also UFMylates FASN and Rictor, we further evaluated lipid abundance in UFL1-overexpressed breast cancer cells treated with or without the FASN inhibitor C75 or the Rictor inhibitor JR-AB2-011. Unsurprisingly, increased lipid accumulation by ectopic UFL1 was dramatically ameliorated by C75 and JR-AB2-011 (Supplementary Fig. 5O–T), suggesting that UFL1 also controls lipid accumulation via FASN and Rictor. Taken together, our results highlight that AKT1 UFMylation is crucial for AKT1’s oncogenic functions, both in vitro and in vivo.

UFL1 phosphorylation by AKT is required for UFL1-mediated breast tumor growth
Given that AKT1 interacts with UFL1, which possesses a canonical AKT consensus phosphorylation motif (Fig. 6A), we speculated that AKT1 might phosphorylate UFL1. Indeed, immunoblotting using a commercial antibody that specifically recognizes phosphorylated AKT substrates (referred to as p-AKT-sub) indicated that UFL1 was phosphorylated in MDA-MB-231 cells, and this phosphorylation was enhanced by insulin, which activates AKT1 (Fig. 6B). More importantly, AKT1 depletion completely abolished this p-AKT-sub signal (Fig. 6B). The in vitro kinase assay demonstrated that Flag-UFL1 immunoprecipitated from MDA-MB-231 cells was phosphorylated by purified GST-AKT1, but not by GST alone (Fig. 6C). These results establish UFL1 as an AKT1 substrate in breast cancer cells. To determine whether T426 in UFL1 was the phosphorylation site targeted by AKT1, WT and T426A mutant Flag-UFL1 were stably expressed alone or in combination with Myc-AKT1 in MDA-MB-231 and MDA-MB-468 cells, followed by immunoblotting with a specific antibody for T426 phosphorylation (referred to as p-UFL1(T426)). The p-UFL1(T426) signal was observed in UFL1-WT, but not in UFL1-T426A (Fig. 6D, E). Similar results were observed in MCF-7, SK-BR-3, and BT474 breast cancer cells (Supplementary Fig. 6A). Thus, AKT1 phosphorylates UFL1 at T426 in breast cancer cells. Since UFL1 modulates AKT1 activation, we next examined whether p-UFL1(T426) affects AKT1 UFMylation and phosphorylation. As expected, UFL1-WT, but not UFL1-T426A, increased AKT1 UFMylation (Supplementary Fig. 6B), as well as p-AKT1(T308) and p-AKT1(S473) (Fig. 6F, G), in MDA-MB-231 and MDA-MB-468 cells, suggesting that phosphorylation is critical for UFL1 activity. Notably, T426A mutation had no impact on total and UFMylated RPL26 (Supplementary Fig. 6B), indicating that p-UFL1(T426) may specifically modulate AKT signaling. Together, these results indicate that AKT1 and UFL1 form a positive feedback loop to mutually enhance their activities.

The above finding prompted us to investigate the impact of p-UFL1(T426) on UFL1-mediated lipid accumulation and tumorigenesis. To this end, we first assessed lipid abundance in breast cancer cells using ORO and BODIPY 493/503 staining. Our results showed that the reduction in lipid abundance due to UFL1 depletion was restored by UFL1-WT, but not by UFL1-T426A, in both MDA-MB-231 and MDA-MB-468 cells (Fig. 6H–K; Supplementary Fig. 6C–F). Moreover, UFL1-WT, but not UFL1-T426A, elevated lipid accumulation in MCF-7 and BT474 cells (Supplementary Fig. 6G–J). Next, we performed xenograft mouse assays with mammary fat pad injections. The inhibitory effect of shUFL1#1 on MDA-MB-231 breast tumor growth and lipid abundance in the tumors was completely reversed by WT, but not T426A, UFL1 (Fig. 6L–O; Supplementary Fig. 6K). IHC analysis further revealed that UFL1’s ability to promote cell proliferation and suppress cell death was significantly impaired by the T426A mutation (Fig. 6P, Q; Supplementary Fig. 6L). Notably, the T426A mutation also abolished p-UFL1(T426) signal in breast tumors, compared to UFL1-WT (Fig. 6R, S; Supplementary Fig. 6L). Intriguingly, the levels of p-UFL1(T426) and p-AKT1(S473) exhibited a positive correlation in TNBC tissues (Fig. 6T; Supplementary Fig. 6M), suggesting a rationale for the combination of UFMylation and AKT1 inhibition in breast cancer treatment. Collectively, these findings demonstrate that p-UFL1 (T426), mediated by AKT1, is essential for UFL1 to control lipid accumulation and drive breast tumor growth.

UFMylation inhibitors synergize with AKT inhibitors in breast cancer treatment
Although several ongoing clinical trials are investigating AKT inhibitors in advanced solid tumors, the clinical benefits of these AKT inhibitors have been limited due to their low efficacy, non-selectivity, and the development of resistance57,58, necessitating a combination of AKT inhibitors with other chemotherapies. UFMylation inhibitor DKM 2-93 and AKT inhibitor MK-2206 both significantly decreased AKT1 UFMylation and phosphorylation (Fig. 7A–D). Co-treatment with DKM 2-93 and MK-2206 displayed a synergistic inhibitory effect on AKT1 UFMylation (Fig. 7A, B), AKT1 phosphorylation (Fig. 7C, D; Supplementary Fig. 7A), and lipid accumulation (Fig. 7E–H; Supplementary Fig. 7B–E) in MDA-MB-231 and MDA-MB-468 cells. Consistently, DKM 2-93 and MK-2206 synergistically inhibited the viability of these breast cancer cells (Supplementary Fig. 7F, G). Activated AKT1 has been reported to alleviate the sensitivity of breast cancer cells to clinical drugs, including cisplatin and etoposide59. Since DKM 2-93 markedly suppresses AKT1 activation, we examined whether DKM 2-93 could sensitize breast cancer cells to these chemotherapeutic agents. As expected, DKM 2-93 sensitized MDA-MB-231 and MDA-MB-468 breast cancer cells to both cisplatin and etoposide (Supplementary Fig. 7H–K). Furthermore, in breast tumor-bearing mice treated with DKM 2-93 and MK-2206, we observed a significant reduction in tumor volume and lipid abundance compared to tumors treated with DKM 2-93 or MK-2206 alone (Fig. 7I–K, M; Supplementary Fig. 7L). Of note, the body weights of the mice remained identical (Supplementary Fig. 7M). Similarly, the combination of DKM 2-93 and MK-2206 synergistically suppressed p-AKT1(S473) (Fig. 7L, N), p-UFL1(T426) (Fig. 7L, O), and cell proliferation (Fig. 7L, P), while promoting cell death (Fig. 7L, Q) in the tumors. Notably, DKM 2-93 and MK-2206 did not alter the protein levels of AKT or UFL1 (Supplementary Fig. 7N, Q). In summary, targeting AKT1 UFMylation represents a promising strategy for sensitizing breast cancer cells to AKT inhibitors or chemotherapeutic agents.

Methods
Cell culture
MDA-MB-231, MDA-MB-468, MCF-7, SK-BR-3, BT474, and HEK293T cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (CM15019, Macgene), RPMI 1640 (Macgene, CM10041), or McCoy’s 5A (Macgene, CM10051) medium, supplemented with 10% FBS (ExCell, FSP500), 1% penicillin, and 1% streptomycin. All cells were confirmed to be mycoplasma-free and cultured at 37 °C with 5% CO2.

Plasmids
Full-length and truncated cDNAs of human UFL1, UFBP1, UFM1, UFC1, AKT1, AKT2, and AKT3 were PCR-amplified and subsequently cloned into lentiviral vectors, including pLVX-IRES-Flag, pLVX-IRES-HA, and pLVX-IRES-Myc. For protein purification, full-length and truncated AKT1 were subcloned into the pGEX-6P-1 vector. Site-directed mutagenesis was employed to generate mutant UFL1 and AKT1. The shRNA sequences targeting human UFL1 (shRNA#1 targets 3’UTR: GGAACTTGTTAATAGCGGA; shRNA#2 targets CDS: GAGGAGTAATTTTTACGGA), AKT1 (shAKT1 targets 3’UTR: CGTGCCATGATCTGTATTTAA), UBA5 (shUBA5 targets CDS: CCTCAGTGTGATGACAGAAAT), and UFSP2 (shUFSP2#1 targets CDS: GACTGATGCTTCTGCTTGTAA; shUFSP2#2 targets CDS: GCTGAAGACCTGCAAGTTATT) were obtained from Sigma-Aldrich Advanced Genomics (https://www.sigmaaldrich.cn/CN/en/products/molecular-biology-and-functional-genomics/gene-editing-and-functional-genomics#products) and subsequently incorporated into the pLKO.1 lentiviral vector. Additional plasmids used in this study have been described in previous publications63. All constructs were verified through DNA sequencing. Plasmid transfection was conducted using PolyJet (SignaGen, SL100688) according to the manufacturer’s instructions.

Lentivirus production and establishment of stable cell lines
Lentivirus was generated as described previously64. Briefly, HEK293T cells were co-transfected with individual expression plasmids and the packaging vectors pMD2.G and psPAX2 using PolyJet (SignaGen, SL100688) according to the manufacturer’s protocol. Forty-eight hours post-transfection, virus particles were harvested and filtered using a 0.45 μm syringe filter. Breast cancer cells were co-cultured with the lentivirus for 48 h, followed by selection with puromycin (1.0 μg/mL, Abcam, ab141453) or G418 (3 mg/mL, MedChemExpress, HY-17561) for an additional 4–8 days. All knockdown and overexpression breast cancer cell lines are stable.

Antibody
Anti-Flag antibody (20543-1-AP; RRID: AB_11232216; 1:8000 for WB (western blotting)), anti-Flag antibody (66008-4-Ig; RRID: AB_2918475; 1:8000 for WB), anti-HA antibody (51064-2-AP; RRID: AB_11042321; 1:5000 for WB), anti-Myc antibody (16286-1-AP; RRID: AB_11182162; 1:5000 for WB), anti-Myc antibody (60003-2-Ig; RRID: AB_2734122; 1:5000 for WB), anti-β-actin antibody (66009-1-Ig; RRID: AB_2687938; 1:10000 for WB), anti-GAPDH antibody (10494-1-AP; RRID: AB_2263076; 1:10000 for WB), anti-Rictor antibody (27248-1-AP; RRID: AB_2880817; 1:1000 for WB), anti-ACC1 antibody (21923-1-AP; RRID: AB_11042445; 1:1000 for WB), anti-ACLY antibody (15421-1-AP; RRID: AB_2223741; 1:1000 for WB), anti-ACSL3 antibody (20710-1-AP; RRID: AB_10697670; 1:1000 for WB), anti-FASN antibody (10624-2-AP; RRID: AB_2100801; 1:1000 for WB), anti-RPL26 antibody (17619-1-AP; RRID: AB_2878420; 1:3000 for WB), anti-UBA5 antibody (12093-1-AP; RRID: AB_2211743; 1:1000 for WB), anti-UFSP2 antibody (16999-1-AP; RRID; AB_2214070; 1:1000 for WB), and anti-Ki-67 antibody (27309-1-AP, RRID: AB_2756525; 1:6000 for IHC (immunohistochemistry); 1:500 for IF (immunofluorescence)) were obtained from Proteintech. Anti-PKC alpha antibody (R014277), anti-Phospho-PKC alpha (Thr638) antibody (R014974; 1:1000 for WB), anti-PLK1 antibody (R011618; 1:1000 for WB), anti-Phospho-PLK1 (Thr210) antibody (R015061; 1:1000 for WB), and anti-PDK1 antibody (R013172; 1:1000 for WB) were purchased from Epizyme. Anti-AKT1 antibody (sc-5298; RRID; AB_626658; 1:1000 for WB), anti-SREBP1 antibody (sc-17755; RRID: AB_628283; 1:1000 for WB), anti-PDI antibody (sc-376370; RRID: AB_10988269; 1:200 for WB), and normal mouse IgG (sc-2025; RRID: AB_737182) were obtained from Santa Cruz Biotechnology. Anti-AKT1 antibody (2938; RRID; AB_915788; 1:1000 for WB; 1:100 for IF), anti-Phospho-Akt (Ser473) (D9E) antibody (4060; RRID: AB_2315049; 1:1000 for WB; 1:200 for IHC), anti-Phospho-Akt (Thr308) (D25E6) antibody (13038; RRID: AB_2629447; 1:1000 for WB; 1:100 for IHC), anti-Phospho-ATP-Citrate Lyase (Ser455) antibody (4331; RRID: AB_2257987; 1:1000 for WB), anti-Phospho-(Ser/Thr) Akt Substrate antibody (9611; RRID: AB_330302; 1:800 for WB), normal rabbit IgG antibody (2729; RRID: AB_1031062), and anti-cleaved caspase 3 (Asp175) antibody (9661; RRID: AB_2341188; 1:100 for IF; 1:300 for IHC) were obtained from Cell Signaling Technology. HRP-conjugated goat anti-rabbit secondary antibody (AB0101; RRID: AB_2941855; 1:10000 for WB) and HRP-conjugated goat anti-mouse antibody (AB0102; RRID: AB_2904147; 1:10,000 for WB) were obtained from Abways. Anti-UFL1 antibody (HPA030559, RRID: AB_10600537; 1:1000 for WB; 1:100 for IHC; 1:150 for IF) and anti-Flag-M2 antibody (F1804; RRID: AB_262044; 1:5000 for WB) were obtained from Sigma-Aldrich. Anti-UFM1 antibody (ab109305; RRID: AB_10864675; 1:1000 for WB), anti-Calreticulin antibody (ab92516; RRID: AB_10562796; 1:300 for WB; 1:500 for IF) were obtained from Abcam. Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (A21206; RRID: AB_2535792; 1:1000 for IF) and Alexa Fluor 594-conjugated donkey anti-mouse secondary antibody (A21203; RRID: AB_141633; 1:1000 for IF) were obtained from Thermo Fisher Scientific. For all immunoprecipitation (IP) assays, 1–4 μg primary antibody was incubated with 1–3 mg total protein.

Cell growth assays
Breast cancer cells (1 × 105) were suspended in medium supplemented with or without 20 μM oleic acid (OA) and seeded in 6-well plates (Jet Bio-Filtration, TCP011006). The cells at each time point were detached using 0.05% Trypsin-EDTA, resuspended in medium, stained with trypan blue, and counted using a TC20 automated cell counter (Bio-Rad).

Cell viability assays
Breast cancer cells (2 × 103) were seeded in 96-well plates (Jet Bio-Filtration, TCP011096). The next day, the cells were treated with 2 μM DMK 2-93 (MedChemExpress, HY-101836), 0.5 μM MK-2206 (MedChemExpress, HY-10358), 0.2 μM etoposide (Sigma, E1383), 2 μM or 1 μM cisplatin (Sigma, 232120), or a combination of these inhibitors for four days. Viable cells were measured using a Cell Counting Kit-8 (CCK-8; MedChemExpress, HY-K0301) according to the manufacturer’s instructions.

Immunofluorescence staining
For antibody staining, breast cancer cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA, Sangon Biotech, E672002) at room temperature for 20 min. These cells were washed twice with PBS, permeabilized with 0.2% Triton X-100 solution for 20 min, and then blocked with 3% BSA at room temperature for 30 min. Subsequently, the cells were incubated overnight with primary antibodies at 4 °C. The next day, cells were washed with PBS and incubated with secondary antibodies for 1 hour at room temperature. To analyze protein colocalization, Pearson’s correlation coefficients (Rr) were calculated using the Fiji Colocalization Finder plugin in ImageJ software. The Rr values represent colocalization (0.5 < r < 1.0) or no colocalization (−1 < r < 0.5). Additionally, the intensities of green and red were plotted in a scatter plot to visualize fluorescence colocalization. Liner and diagonal distributions indicate good colocalization. For BODIPY staining, after treatment with 1 mM sodium citrate for 12 h in the absence or presence of 1 μM Fatostatin (MedChemExpress, HY-14452A), 45 μM C75 (MedChemExpress, HY-12364), 50 μM JR-AB2-011 (MedChemExpress, HY-14452A), 6 μM MK-2206 (MedChemExpress, HY-12464), or 20 μM DKM 2-93 (MedChemExpress, HY-122022), breast cancer cells were washed with PBS after fixation and stained with 0.25 μg/mL BODIPY 493/503 (Thermo Fisher, D3922) for 15 min. Finally, cells were stained with DAPI (Sigma-Aldrich, 10236276001). Immunofluorescence images were captured using a Leica TCS SPE confocal microscope (Leica). Quantification of BODIPY average intensity was performed using Image-Pro Plus (Media Cybermetics).

Oil Red O (ORO) staining
For cell ORO staining, after treatment with 1 mM sodium citrate for 12 h, breast cancer cells were wash by PBS and fixed with 4% PFA (Sangon Biotech, E672002). The cells were then stained with ORO staining solution (Sangon Biotech, E607319) according to the standard protocol. To quantify lipid accumulation, ORO was eluted with 100% isopropanol, and the optical densities of the eluates were measured with a spectrophotometer at a wavelength of 510 nm. To visualize lipid accumulation in breast tumors, tumor was fixed in 4% PFA (Sangon Biotech, E672002) overnight at 4 °C and then sequentially dehydrated with 15% and 30% sucrose at 4 °C. Tumor tissues were embedded in Tissue-Tek OCT compound and frozen in 2-methylbutane precooled with liquid nitrogen. Afterward, tissues were sectioned into 10 μm thick slides using a cryostat microtome. Tumor sections were placed on SuperFrost glass slides, air-dried, and soaked in 60% isopropanol for 5 min, followed by incubation in ORO solution (Sangon Biotech, E607319) according to the standard protocol. Sections were then decolorized in 60% isopropanol solution for 20 s, washed three times with ddH2O, and incubated in hematoxylin for 10 s. The slides were rinsed three times with ddH2O, air-dried, and sealed with glycerol-gelatin mounting solution. Images were captured using a Leica microscope. Quantification of ORO intensity in tumors was performed using Image-Pro Plus (Media Cybermetics).

Flow cytometry
The experimental procedure for detecting apoptosis was performed using an Annexin-V-YS Fluor 647/PI Apoptosis Detection Kit (YEASEN, 40304ES). Briefly, cells were harvested using trypsin (EDTA-free), washed twice with cold PBS, and resuspended at a density of 1 × 106 cells/mL in 100 μL 1X Annexin-V binding buffer. Subsequently, the cell suspension was incubated with 5 μL YS Fluor 647-conjugated Annexin-V and 10 μL propidium iodide (PI) for 15 min at room temperature in the dark. Following the incubation, 400 μL binding buffer was added to each sample. The stained cells were then immediately analyzed using a flow cytometer (BD Biosciences, LSR Fortessa), collecting a minimum of 10,000 events per sample. Median fluorescence intensity was used for statistical analyses to mitigate the influence of outliers and skewed distributions. The resulting data was interpreted using FlowJo software (BD Biosciences, version 11) to distinguish viable (Annexin-V⁻/PI⁻), early apoptotic (Annexin-V⁺/PI⁻), late apoptotic (Annexin-V⁺/PI⁺), and necrotic or damaged cells (Annexin-V⁻/PI⁺) based on their differential fluorescence signals in the FL1 and FL2 channels.

Immunoprecipitation and immunoblotting
Cells were resuspended in NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 0.5% NP-40, protease inhibitors, and phosphatase inhibitors), followed by sonication and centrifugation at 12,000 g for 10 min. Whole-cell lysates were subjected to immunoblotting (IB) analysis. For immunoprecipitation assays, cell lysates were incubated with the indicated antibodies and protein A/G magnetic beads (Bio-Rad) or PIP3 coated magnetic beads (Echelon Biosciences, P-B345A) at 4 °C overnight. Subsequently, the beads were gently washed three times with NETN lysis buffer. The immunoprecipitates were boiled in 1 x Laemmli buffer and separated by SDS-PAGE. IB was subsequently performed using the indicated antibodies.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay
MDA-MB-231 cells stably expressing Flag-UFL1, treated with MG132 for 6 h before harvest, were lysed using NETN lysis buffer. The supernatant was collected and incubated with anti-Flag magnetic beads (BioLinkedIn, L1103) at 4 °C for 2 h. The putative UFL1-interacting proteins in the immunoprecipitates were fractionated by SDS-PAGE and subjected to LC-MS/MS analysis conducted by PTM Biolabs Inc. Briefly, the tryptic peptides were analyzed using an NSI source, followed by MS/MS in a Q ExactiveTM Plus (Thermo) system coupled online to the EASY-Nlc 1000 UPLC system. The resulting MS/MS data were processed using Proteome Discoverer 1.3. Tandem mass spectra were searched against Homo Sapiens database (SwissProt, 20395 sequences). Trypsin/P was specified as the cleavage enzyme, allowing up to 2 missing cleavages. Mass error tolerance was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethyation on cysteine residues was specified as a fixed modification, and oxidation on methionine residues was specified as variable modification. Peptide confidence was set to high, and the peptide ion score threshold was set to > 20.

Protein purification and pull-down assay
Full-length and truncated GST-AKT1 proteins were expressed in BL21 competent E. coli cells and purified with glutathione-Sepharose 4B (GE Healthcare, 17-0756-01) following standard protocols. Purified GST or GST-fusion proteins were incubated with immunoprecipitated UFL1 from breast cancer cells. Subsequently, the beads were washed extensively five times, boiled in 1 x Laemmli buffer, and analyzed by IB to assess the protein interaction.

In vivo UFMylation assay
In vivo UFMylation assays were performed as described previously41,63. Briefly, HEK293T or breast cancer cells were collected and resuspended in a buffer containing 150 mM Tris-HCl (pH 8.0), 5% sodium dodecyl sulfate (SDS), and 30% glycerol, followed by boiling for 10 min. Cell lysates were diluted with buffer A (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 2 mM N-ethylmaleimide, and proteinase inhibitors) at a 1:20 ratio and incubated with the indicated antibodies and protein A/G magnetic beads (Bio-Rad) at 4 °C overnight. The immunoprecipitates were boiled in 1 x Laemmli buffer, separated by SDS-PAGE, and analyzed by IB.

In vitro UFMylation assay
In vitro UFMylation assays were performed according to the previous description41. Briefly, HEK293T cells were co-transfected with plasmids encoding HA-UBA5, HA-UFC1, HA-UFBP1, and HA-UFM1, and lysed with NETN lysis buffer for immunoprecipitation using anti-HA magnetic beads, followed by elution with HA peptide. Meanwhile, Flag-UFL1 expressed in HEK293T cells was immunoprecipitated with anti-Flag magnetic beads and subsequently eluted with Flag peptide. GST or GST-AKT1 bound to glutathione-Sepharose 4B (GE Healthcare, 17-0756-01) was incubated with immunoprecipitated HA-UBA5, HA-UFC1, HA-UFBP1, HA-UFM1, and Flag-UFL1 in a buffer containing 0.05% bovine serum albumin, 50 mM HEPES (pH 7.5), 5 mM ATP, and 10 mM MgCl2 at 30 °C for 2 h. The immunoprecipitates were boiled in 1x Laemmli buffer, separated by SDS-PAGE, and analyzed by IB.

In vivo phosphorylation assay
Breast cancer cells were lysed with NETN buffer supplemented with protease and phosphatase inhibitors. For AKT1 and ACLY, cell lysates were subjected to SDS-PAGE, followed by IB analysis using anti-p-AKT1 (S473), anti-p-AKT1 (T308), or anti-p-ACLY (S455) antibodies. For UFL1, endogenous UFL1 or exogenous Flag-UFL1 was immunoprecipitated using anti-UFL1 or anti-Flag antibody, respectively. Immunoprecipitated UFL1 or Flag-UFL1 was subjected to SDS-PAGE, followed by IB analysis using anti-p-AKT-sub or anti-p-UFL1 (T426) antibodies.

In vitro phosphorylation assay
Immunoprecipitated UFL1 from breast cancer cells was mixed with purified GST or GST-AKT1 in 1x kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, and 100 mM ATP), and incubated at 37 °C for 2 h. The mixture was separated by SDS-PAGE, followed by IB analysis using specific antibodies.

Reverse transcription (RT)-qPCR
Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen, 15596018) and treated with DNase I (Ambion, AM2224). cDNA was synthesized using iScript Reverse Transcription Supermix (BioRad, 1708841). RT-qPCR assays were performed in triplicate using Hieff qPCR SYBR Green Master Mix (YEASEN, 11201ES08) according to the standard protocol. Primers for RT-qPCR were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/): ACLY (Fw: ATCGGTTCAAGTATGCTCGGG; Rv: GACCAAGTTTTCCACGACGTT), FASN (Fw: ACAGCGGGGAATGGGTACT; Rv: GACTGGTACAACGAGCGGAT), ACC1 (Fw: CATGCGGTCTATCCGTAGGTG; Rv: GTGTGACCATGACAACGAATCT), SCD1 (Fw: TCTAGCTCCTATACCACCACCA; Rv: TCGTCTCCAACTTATCTCCTCC), ACSS2 (Fw: CAAGTGTGTCAGTTCAGCAATG; Rv: CCACAAGCTCTGGGATCATAGG), CD36 (Fw: CCAAGCTCTGGGATCATAGG, Rv, GCATTTGCTGATGTCTAGCACA), FATP2 (Fw: TTTCCGCCATCTACACAGTCC; Rv: CGTAGGTGAGAGTCTCGTCG), FATP5 (Fw: CATGGCGTGACAGTGATCCT; Rv: CAGCCCGTAGTCCATTGCC), CPT1 (Fw: TCCAGTTGGCTTATCGTGGTG; Rv: TCCAGAGTCCGATTGATTTTTGC), CPT2 (Fw: CTGGAGCCAGAAGTGTTCCAC; Rv: AGGCACAAAGCGTATGAGTCT), ACOX1 (Fw: ACTCGCAGCCAGCGTTATG; Rv: AGGGTCAGCGATGCCAAAC), LDLR (Fw: ACGGCGTCTCTTCCTATGACA; Rv: CCCTTGGTATCCGCAACAGA), MTTP (Fw: ATTGTAAAGTGACCTACCAGGCT; Rv: ACCTCGCTATTTTGCATGAATCC), 18S (Fw: CGCGGTTCTATTTTGTTGGT; Rv: AGTCGGCATCGTTTATGGTC), RPL13A (Fw: GAGGTCGGGTGGAAGTACCA; Rv: TGCATCTTGGCCTTTTCCTT).

Cytosol and endoplasmic reticulum (ER) isolation
ER was isolated according to an optimized approach65. Briefly, breast cancer cells were washed with PBS, resuspended in ice-cold MIB-S buffer (0.25 M D-Sucrose, 25 mM KCl, 10 mM HEPES, 1 mM EGTA (pH 8.0)), centrifuged at 600 g for 5 min, and homogenized using a 15 cm3 homogenizer with a tight pestle for 20 strokes. Cell lysates were centrifuged at 600 g for 5 min, and supernatant I was collected. Supernatant I was centrifuged at 1200 g for 5 min, and supernatant II was retained. Supernatant II was centrifuged at 20,000 g for 35 min, and supernatant III was retained. Supernatant III was centrifuged at 100,000 g for 65 min, and supernatant IV (cytosol) and the ER pellet were kept for protein detection.

Quantification of free fatty acid (FFA) and total cholesterol
The content of FFA and total cholesterol were quantified using the FFA content assay kit (Solarbio, BC0595) and the total cholesterol content assay kit (Solarbio, BC1985), respectively. Briefly, breast cancer cells were resuspended in extraction solutions and sonicated, and the supernatant was collected. Absorbance of the supernatant was measured using a microplate reader at 545 nm for FFA and 500 nm for total cholesterol. All experiments were conducted in triplicate, and the data were normalized to cell numbers.

Animal studies
Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Shandong Normal University (SDNU). Female nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice (6–8 weeks old, Strain NO. T001492) were purchased from GemPharmatech and housed in a specific pathogen-free (SPF) facility, where they were maintained on a 12-h light/12-h dark cycle at a controlled ambient temperature of 20–24 °C and relative humidity of 40–60%. According to the policy of the IACUC at SDNU, the volume of xenograft tumors in mice should not exceed 2000 mm3. 2 × 106 MDA-MB-231 or MDA-MB-468 cells, resuspended in 50 μL PBS, were mixed with an equal volume of Matrigel (ABW Bio, 82704) and injected into the second left mammary fat pad of female NOD-SCID mice. To assess the effect of Fatostatin, DKM 2-93, and MK-2206, MDA-MB-231 tumor (approximately 100 mm3)-bearing mice were randomly assigned to four treatment groups (five mice per group) and received either a vehicle solution (2% DMSO, 40% PEG 400, 5% Tween-80, and 53% saline), Fatostatin (30 mg/kg, intraperitoneal, once every three days), MK-2206 (100 mg/kg, oral, once every three days), DKM 2-93 (40 mg/kg, intraperitoneal, once every three days), or a combination of MK-2206 and DKM 2-93. Tumor size was measured every three days with a caliper when tumor size reached approximately 100 mm3 and analyzed according to the formula: V=0.52 × L × H × W (V: volume, L: length, H: height, W: width). Mice were euthanized with carbon dioxide, and primary tumors were surgically removed, weighted, and photographed when their volume reached approximately 1500 mm3 in any cohort.

Immunohistochemistry (IHC)
IHC assays were performed as described previously66. Briefly, tumors were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned. Tissue ribbons were placed onto slides, which were then baked, deparaffinized using xylene, and hydrated in graded ethanol, followed by antigen retrieval in Citrate Antigen Retrieval solution. Endogenous peroxidase activity was blocked with 3% peroxide-methanol, and non-specific staining was blocked with 10% goat serum. The tissues were incubated with the indicated primary antibodies overnight at 4 °C and followed by HRP-conjugated secondary antibody for 1 h at room temperature. The following primary antibodies were used: anti-Ki-67 (1:5000), anti-cleaved caspase-3 (CC3, 1:500), anti-AKT1 (1:100), anti-p-AKT1 (S473) (1:100), anti-p-AKT1 (T308) (1:100), anti-UFL1 (1:150), and anti-p-UFL1 (T426) (1:150). The staining was visualized using the Pannoramic MiDi II slide scanner (3D Histech, Budapest, Hungary). The number of CC3-positive cells or Ki-67-positive cells per field was counted manually, and the average intensity (integrated intensity/area) of AKT1, p-AKT1 (S473), anti-p-AKT1 (T308), UFL1, and p-UFL1 (T624) in xenograft tumors was determined using Image-Pro Plus (Media Cybermetics). The human breast tumor tissue microarrays (TMAs) consisting of 120 human breast cancer tissues and 46 adjacent normal breast tissues were purchased from the Tissue Resource at The First Affiliated Hospital of Jinan University, which provided only the status of ER (estrogen receptor), HER2 (human epidermal growth factor receptor 2), and PR (progesterone receptor) of breast cancer tissues. Ethical approval (JNUKY-2023-0062, 2023/01/01-2026/12/31) and informed consent have been obtained regarding the use of patient tissues. The IHC staining of TMAs was quantitatively scored by Drs. Yan Chen and Junjie Liang on a four-grades scale (0–3) based on the percentage of positive cells and the overall staining intensity, as previously described67. The sections were assigned by the following proportion scores: 0 if 0% of the tumor cells displayed positive staining, 1 for 0–1%, 2 for 2–10%, 3 for 11–30%, 4 for 31–70%, 5 for 71–100%. Additionally, the intensity of staining was assigned a four-graded scale (0–3). Grades 0–3 represent: no expression (0), weak expression (1), moderate expression (2), and strong expression (3). A total score (range, 0–8) was obtained by combining the proportion and intensity scores. GraphPad Prism 8.0 was employed to perform the Pearson correlation analysis (two-tailed). Note that the scores for some samples overlap.

Statistical analysis
At least 3 samples prepared from independent experiments were used to ensure adequate power for all studies. Statistical analysis was carried out by one-way or two-way ANOVA with multiple testing correlation within multiple groups, two-tailed t-test within two groups, two-tailed Pearson correlation test, or log-rank test. Data were presented as mean ± SD. Significance was established at p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary:\nThis manuscript from Fei Meng et al presents a mechanistic study uncovering a positive feedback loop between UFL1 and\nAKT1 in triple-negative breast cancer. The authors show that UFL1 promotes AKT1 activation through UFMylation at lysines\nK189, K276, and K297, facilitating AKT1’s ER localization and interaction with upstream kinases PDK1 and mTORC2. In\nturn, AKT1 phosphorylates UFL1 at T426, enhancing UFL1 activity and thereby amplifying UFMylation. This regulatory axis\nis shown to control lipid metabolism and tumor growth, and the study further demonstrates that pharmacological inhibition of\nUFMylation sensitizes cells to AKT inhibition and chemotherapy. These mechanistic findings came from a comprehensive\nand rigorous experimental design, in which the authors combined in vitro biochemistry, genetic models and in vivo xenograft\nmouse model. Moreover, the identification and functional dissection of UFMylation (3K/R) and phosphorylation (T426) sites\nare well executed and supported by multiple lines of evidence. The use of AKT and UFMylation inhibitors, and the\ndemonstration of synergy, provide a translational angle that strengthens the manuscript. Finally, the emphasis on AKT1-\nmediated lipid accumulation connects this signaling axis to a well-established hallmark of cancer. However, several key\npoints need to be addressed to fully support the proposed model.\n________________________________________\nMajor Points for Revision:\n+ Biochemical Evidence for AKT1 UFMylation in AKT activation: A key mechanistic claim of the manuscript is that\nUFMylation of AKT1 at K189, K276, and K297 is required for its phosphorylation at T308 and S473. However, in standard\ncellular models (including our own lab’s experience and widely published studies), insulin or IGF-1 stimulation induces\nrobust AKT1 phosphorylation without any observable upward shift in AKT1 molecular weight on Western blot with the\nphospho-AKT1 specific antibodies. This raises an important question: if UFMylation were required for AKT activation, why is\nthe fully activated AKT with both p-T308 and p-S473 readily observed in the absence of a UFM1-dependent band shift in\nthese common settings? Please clarify whether the proposed UFL1-AKT1 UFMylation axis is prior to AKT activation and\nwhether the de-UFMylation of fully activated AKT1 happened.\n+ Breast cancer subtype specificity: While experiments include multiple breast cancer subtypes, the core mechanistic and\ntherapeutic data focus largely on TNBC cell lines. Some conclusions imply pan-breast cancer relevance, but the\nexperimental data supporting this are relatively limited. Please clarify whether the UFL1-AKT1 feedback loop and its impact\non lipid metabolism are broadly active across subtypes, or primarily relevant in TNBC.\n+ Clinical relevance and correlation analysis: The manuscript refers to a positive correlation between pUFL1 and pAKT1 in\nbreast tumor tissues. However, the correlation coefficient (r) and significance (p-value) are not clearly presented in the main\ntext or figure legends. Please include the quantitative data (r, p-value) and consider moving the correlation plot to a main\nfigure to better support the clinical relevance of the proposed axis.\n+ Functional role of UFMylation beyond AKT1: The focus is tightly centered on AKT1, but the authors note that other\ncomponents of the lipid metabolism and PI3K-AKT-mTOR pathway may interact with UFL1. Please comment more fully on\nwhether UFMylation of other components (e.g., ACLY, SREBP1 regulators) may contribute to the lipid phenotype, or\nwhether this is uniquely AKT1-driven.\n________________________________________\nMinor points:\n+ Improve clarity in the Discussion to avoid repetition and to sharpen the distinction between known and novel findings.\n+ Specify whether AKT2 and AKT3 were experimentally evaluated for UFMylation and ruled out since it is surprisingly that\nUFL1 only targets AKT1 but negligible with AKT2 and 3.\n+ Provide consistent cross-referencing between figures and legends, especially for supplemental data.", "reviewer_2": "The UFL1-AKT positive feedback loop promotes breast cancer progression by enhancing lipid synthesis and drug\nresistance” by Meng, Du and Liang et al., nicely describes a new regulatory mechanism of AKT based on UFMylation. The\nimportance of UFMylation of AKT is supported by convincing IP and in vivo rescue experiments. While the data supporting\nthe posttranslational modifications is convincing the more phenotypic data are unclear.\nMain concerns:\nThe title: The title claims that UFL1-AKT feedback loop promotes breast cancer progression. There is very little evidence\nthat the pathway can actually drive the disease. There is a lot of evidence to suggest that the pathway is required for\nprogression. The only ectopic overexpression I could find was lipid related in supp figure 2B-I. However, these do not\ninclude any in vivo or in vitro tumor outcome data.\nER localization: The manuscript claims that UFL1 mediates an ER localization of AKT supported by Immunofluorescence\nimages (3F and 4C) and western blotting on cellular fractionation (4D). The IF images should be shown at higher\nmagnification, and the co-localization should be quantified. In the same manner, the western blots should be repeated and\nquantified.\nWestern blot quantification: In general, western blots should be quantified where possible. I understand this might be hard\nfor some of the IP’s, but it should at the very least be done for regular depletion studies as 4H. The figure legends\nconsistently state at these blots have been repeated independently three times. If so, please provide the quantification.\nRequirement of lipid synthesis: The requirement of lipid synthesis to be the driver of the UFL1 pathway needs more\nvalidation. Currently, the claim is backed by in vitro proliferation rescue with OA (Supp 2J,K). Given the multitude of AKT\neffects on cancer cells, more conclusive evidence is needed to back this claim.\nMinor concerns:\nAll bar plots should display individual values.\nLine 110: In Figure 1E, I am guessing that the separation is based on the quantification in 1D. If so, please relabel to low\nUFL1 and moderate/strong UFL1 (rather than simply high/low).\nLine 111: Figure 1F. I believe this data is from the Tang data set, not Liu?\nLine 212: (data not shown). Please provide the data.\nLine 498: Why are the cells treated with 1mM sodium citrate for 12 hours before staining for Bodipy and oil red O? And how\ndoes that impact the measurements? I am guessing citrate will boost the acetyl-CoA levels and thus possibly the lipid\nsynthesis. However, the citrate incubation is not typically required to identify lipid droplets. Does the data hold up without the\npreincubation?", "reviewer_3": "The manuscript by Meng et al reported the critical role of the UFL1-AKT axis in breast cancer progression and drug\nresistance. Specifically, the authors found that AKT1 was UFMylated by UFL1, an UFMylation-specific E3 ligase, in breast\ncancer cells. AKT1 UFMylation promoted its ER recruitment and activation by both PDK1 and mTORC2, which in turn\nenhanced lipid biogenesis and cancer progression. Moreover, the authors showed that AKT1 phosphorylated UFL1 and\nboosted UFL1’s activity. Finally, UFL1 expression was elevated in breast cancer samples and associated with poor\nprognosis in human patients. Targeting both UFMylation and AKT1 had a synergetic effect on TNBC in xenograft models.\nOverall, the finding of the positive feed-back regulation between AKT1 and UFL1 appears to be novel and may represent a\npotential target for cancer therapy. However, there are some significant flaws in data analysis and interpretation that should\nbe addressed. As described below, this reviewer failed to duplicate some of the results as claimed by the authors regarding\nthe association between UFL1 mRNA level and overall survival of human BC patients. In addition, a few pieces of\nexperimental data appeared to conflict to each other, and some results were difficult to explain. Finally, the evidence to\nsupport the role of lipid metabolism in tumor progression is relatively insufficient.\nSpecific comments:\n1. Concerns on bioinformatic analyses of BC databases and clinical samples.\na. This reviewer failed to duplicate KM survival curves using GEPIA database (http://gepia.cancer-pku.cn). A simple search\nusing UFL1 and BRCA dataset in GEPIA yielded no significant association between Ufl1 mRNA levels and overall survival\nof BC patients. The number of patients also differed from what the authors reported.\nb. In Fig.S1C, GSE9693 dataset was about “Clostridioides difficile”, which was nothing to do with breast cancer.\nc. In Fig.1F, this reviewer could not replicate the KM survival plot as reported using KM plotter database (kmplot.com). There\nwas no reference or GSE number for Liu-2014 dataset (as indicated in Fig.1F). Additionally, none of BC datasets in KMplot\ncould generate similar survival plots showing the significant correlation between Ufl1 mRNA level and overall survival.\nPlease provide more information on the dateset and UFL1 (named as KIAA0776 in KM plot) probe set used in the analysis\nreported by the authors.\n2. Specificity of Ufl1 antibody in IHC\nThe authors did not provide the source of UFL1 and other antibodies. To the knowledge of this reviewer, some commercial\nUFL1 antibodies recognized multiple bands in immunoblotting. Even though Fig.1K may have indirectly addressed this\nissue, more stringent test should be done to examine the specificity of UFL1 antibody. Meanwhile, the knockdown efficiency\nof UFL1 shRNAs should be shown.\n3. For cell death assay of tissue culture cells, Annexin V staining or TUNEL should be done in addition\nto CC3 staining.\n4. The claim that UFL1 is an oncogenic driver (Fig.1) appears to be an overstatement. Even though UFL1 overexpression\npromoted cell proliferation, no experimental evidence supported its oncogenic activity.\n5. The claim that UFL1 promotes breast cancer cell growth by increasing lipid accumulation was backed up by the only\nfunctional assay that was shown in Fig.S2J and K, in which addition of OA reversed UFL1 knockdown phenotype. Since\nlipid metabolism was such a big part of this study, more functional experiments should be done to firmly establish the\nrelationship between lipid metabolism, the UFL1-AKT axis and tumor progression.\n6. High-quality of confocal imaging in Fig.3F should be provided. The current one appeared to be fuzzy, especially\nUFL1.\n7. As shown in Fig.3I and J, knockdown of UFL1 led to reduced AKT UFMylation but had no effect on overall UFMylation\n(especially RPL26 UFMylation, around 27kD and higher), a result that seems to contradict to previous studies.\nPlease explain why.\n8. In Fig.3I and J and other similar figures, please provide the results of IgGs as negative controls in those IPs.\n9. In Fig.3, it was showed that UFM1 antibody could detect UFMylated endogenous and exogenous AKT1. How about AKT1\nand Flag antibodies?\n10. In Fig. 4A and B, it would be interesting to examine if phosphorylation of other mTORC2 and PDK1 targets is altered by\nUFL1 knockdown.\n11. In Fig.4C, co-localization of AKT1 and calreticulin should be quantitated.\n12. In Fig. 4D, AKT1 levels in ER fractions were obviously lower in UFL1 knockdown ER fraction. However, in Fig. 4E,\nAKT1 levels remained unchanged in UFL1 KD ER fractions. Please explain this discrepancy. Additionally, no increase of\nAKT1 in cytoplasmic fractions was observed in UFL1 KD cells. Why?\n13. In Fig.4F, more negative controls are needed.\n14. in Fig.5A, please show the protein levels of shRNA-resistant AKT1s.\n15. In Fig.6, it would be interesting to test the effect of UFL1(T426A) mutant on other UFM1 targets such as RPL26.\n16. In Fig.7A and B, UFMylation inhibitor (Uba5 E1 inhibitor) DKM 2-93 dramatically reduced AKT1 UFMylation but had no\neffect on overall UFMylation. Any explanation? Furthermore, if both inhibitors work through the route of AKT1, how was the\nsynergy achieved?\n17. Please provide more experimental details, including antibody sources and RIDDs, web links, designs of shRNA\nresistant genes, cell lines in each experiment, transient or stable lines, and etc."}, "author_response": "Response to Editor and Reviewers\nEditor and Reviewers’ comments are highlighted in blue, and the response is provided below the\nreferee comments.\nReviewer #1 (AKT activation and posttranslational modifications, Remarks to the Author):\nSummary:\nThis manuscript from Fei Meng et al presents a mechanistic study uncovering a positive feedback loop\nbetween UFL1 and AKT1 in triple-negative breast cancer. The authors show that UFL1 promotes AKT1\nactivation through UFMylation at lysines K189, K276, and K297, facilitating AKT1’s ER localization\nand interaction with upstream kinases PDK1 and mTORC2. In turn, AKT1 phosphorylates UFL1 at\nT426, enhancing UFL1 activity and thereby amplifying UFMylation. This regulatory axis is shown to\ncontrol lipid metabolism and tumor growth, and the study further demonstrates that pharmacological\ninhibition of UFMylation sensitizes cells to AKT inhibition and chemotherapy. These mechanistic\nfindings came from a comprehensive and rigorous experimental design, in which the authors combined\nin vitro biochemistry, genetic models and in vivo xenograft mouse model. Moreover, the identification\nand functional dissection of UFMylation (3K/R) and phosphorylation (T426) sites are well executed\nand supported by multiple lines of evidence. The use of AKT and UFMylation inhibitors, and the\ndemonstration of synergy, provide a translational angle that strengthens the manuscript. Finally, the\nemphasis on AKT1-mediated lipid accumulation connects this signaling axis to a well-established\nhallmark of cancer. However, several key points need to be addressed to fully support the proposed\nmodel.\nMajor Points for Revision:\n1. Biochemical Evidence for AKT1 UFMylation in AKT activation: A key mechanistic claim of the\nmanuscript is that UFMylation of AKT1 at K189, K276, and K297 is required for its phosphorylation\nat T308 and S473. However, in standard cellular models (including our own lab’s experience and\n1\nwidely published studies), insulin or IGF-1 stimulation induces robust AKT1 phosphorylation without\nany observable upward shift in AKT1 molecular weight on Western blot with the phospho-AKT1\nspecific antibodies. This raises an important question: if UFMylation were required for AKT\nactivation, why is the fully activated AKT with both p-T308 and p-S473 readily observed in the\nabsence of a UFM1-dependent band shift in these common settings? Please clarify whether the\nproposed UFL1-AKT1 UFMylation axis is prior to AKT activation and whether the de-UFMylation of\nfully activated AKT1 happened.\nResponse: We thank the reviewer for these constructive suggestions.\n(1). Based on our findings that UFL1 depletion, UBA5 ablation, and mutation of AKT’s UFMylation\nsites (3K/R) all significantly decreased AKT phosphorylation (Fig. 4A, B; Supplementary Fig. 4A-C\nin the revised manuscript), we concluded that AKT UFMylation occurs prior to AKT phosphorylation.\n(2). We agree that insulin or IGF-1-activated AKT1 displays no changes to its molecular weight, which\nseems to conflict with the fact that UFMylation is required for AKT activation and UFM1’s molecular\nweight is approximately 9 kDa. We hypothesized that AKT phosphorylation promotes its\ndeUFMylation. To address this, we performed the following experiments, and these results support\nour prediction from different angles.\n(i). Similar to other post-translational modifications, UFMylation is dynamic and reversible, and this\nfeature ensures that UFMylated AKT1 accounts for a tiny portion of total AKT1. Therefore,\nendogenous AKT1 and p-S473 were examined using an anti-AKT1 antibody or an anti-p-S473 antibody\nin MDA-MB-231 and MDA-MB-468 cells with or without UFL1 depletion. After a long exposure,\nweak p-S473 and AKT1 bands, whose molecular weights were approximately 27 kDa greater than that\nof their corresponding lower bands due to UFMylation at K189, K276, and K297, were observed in\nshSC cells, and these upper bands nearly disappeared in UFL1 knockdown cells (Supplementary Fig.\n4K in the revised manuscript). Hence, a tiny amount of AKT1 is maintained in a UFMylation state,\nperhaps due to deUFMylation.\n2\n[FIGURE REDACTED]\n(ii). Covalently bound UFM1 is primarily cleaved from substrates by the cysteine protease UFSP2\n(Christopher P. Walczak et al., 2019 PNAS. PMID: 30626644; Jessie Kulsuptrakul et al., 2021 Cell\nRep. PMID: 33730579)(Fig. 1 in this letter). Thus, UFSP2 was depleted by shRNA in MDA-MB-231\nand MDA-MB-468 cells to assess whether inhibited deUFMylation leads to an increase in AKT1\nUFMylation and an upward shift of the p-S473 band. As expected, the intensity of the upper p-S473\nband and the UFMylated AKT1 band was both significantly increased in UFSP2 knockdown cells (Fig.\n4H in the revised manuscript). These findings indicate that AKT1 undergoes deUFMylation.\n(iii). p-T308 and p-S473 result in full AKT1 activation. A plasmid encoding a mutant AKT1 (referred\nto as AKT1-DM), which harbors T308A and S473A mutations, was generated. MDA-MB-231 cells\nexpressing comparable levels of wild-type (AKT1-WT) and AKT1-DM were established. Compared\nwith AKT1-WT, AKT1-DM possessed higher levels of UFMylation (Fig. 4I in the revised manuscript),\nsuggesting that AKT1 phosphorylation enhances its deUFMylation.\n(iv). Breast cancer cells expressing AKT1-WT and AKT1-DM were treated with or without insulin and\nsubjected to co-immunoprecipitation (co-IP) assays. The interaction between UFSP2 and AKT1-WT\nwas stronger than the UFSP2-AKT1-DM association, and the former, but not the latter, interaction was\n3\nincreased by insulin treatment (Fig. 4J in the revised manuscript). Hence, AKT1 phosphorylation\npromotes the UFSP2-AKT1 interaction.\nCollectively, these findings indicate that AKT1 UFMylation promotes AKT1 phosphorylation, which\nin turn enhances the UFSP2-AKT1 interaction, thereby deUFMylating AKT1.\nNotably, a similar phenomenon has been reported in AKT1 polyubiquitination (Wei-Lei Yang et al.,\nC H Lin, S Y Liu, and E H Y Lee, 2016 Oncogene. PMID: 25867063), which significantly increase\nAKT1’s molecular weight, while p-T308 and p-S473 bands with an upward shift are barely detectable.\nFurther investigations are needed to clarify whether ubiquitination, SUMOylation, and other post-\ntranslational modifications of AKT1 are also removed by de-conjugation enzymes after AKT1 is\nphosphorylated.\n2. Breast cancer subtype specificity: While experiments include multiple breast cancer subtypes, the\ncore mechanistic and therapeutic data focus largely on TNBC cell lines. Some conclusions imply pan-\nbreast cancer relevance, but the experimental data supporting this are relatively limited. Please clarify\nwhether the UFL1-AKT1 feedback loop and its impact on lipid metabolism are broadly active across\nsubtypes, or primarily relevant in TNBC.\nResponse: We thank the reviewer for these valuable comments. The UFL1 mRNA and protein levels\nare both increased in all breast cancer subtypes, including Luminal A, Luminal B, HER2+, and TNBC\n(Supplementary Fig. 1A, D in the revised manuscript). UFL1 ablation suppressed the proliferation of\nall breast cancer cell types (Fig. 1G, H; Supplementary Fig. 1E-G in the revised manuscript). All these\nfindings indicate that the UFL1-AKT1 feedback loop and its impact on lipid metabolism may be\nbroadly active across subtypes. To further test this hypothesis, we carried out the following\nexperiments:\n(1). MCF-7 (Luminal A), BT474 (Luminal B), and SK-BR-3 (HER2+) cells were transfected with\nvectors encoding AKT1-WT or AKT1-3K/R and subjected to UFMylation detection. Our results\n4\nindicated that AKT1-WT, but not AKT1-3K/R, was UFMylated in MCF-7, BT474, and SK-BR-3 cells\n(Supplementary Fig. 3N-P in the revised manuscript), suggesting that K189, K276, and K297 are key\nUFMylation sites in Luminal A, Luminal B, and HER2+ breast cancer cells. Additionally, we found\nthat AKT1-WT, but not AKT1-3K/R, enhanced lipid abundance in these breast cancer cells, as\nevidenced by BODIPY staining assays (Supplementary Fig. 5H-M in the revised manuscript).\n(2) MCF-7, BT474, and SK-BR-3 cells stably expressing UFL1-WT or UFL1-T426A were lysed for\nUFL1 phosphorylation detection using an antibody that specifically recognizes phosphorylated UFL1\nat T426 (referred to as p-UFL1 (T426) antibody). As expected, phosphorylation signals were only\nobserved in UFL1-WT, but not UFL1-T426A (Supplementary Fig. 6A in the revised manuscript). Thus,\np-UFL1 (T426) also occurs in Luminal A, Luminal B, and HER2+ breast cancer cells. Furthermore,\nUFL1-WT increased lipid accumulation in MCF-7 and BT474 cells, whereas UFL1-T426A had no\nimpact on these cells (Supplementary Fig. 6G-J in the revised manuscript).\nTaken together, these findings suggest that the UFL1-AKT1 feedback loop-mediated lipid metabolism\napplies to all breast cancer subtypes. However, as we mentioned in the revised manuscript, TNBC is\nmore aggressive and lacks effective therapeutic targets; therefore, we mainly focused on TNBC in this\nstudy.\n3. Clinical relevance and correlation analysis: The manuscript refers to a positive correlation between\npUFL1 and pAKT1 in breast tumor tissues. However, the correlation coefficient (r) and significance\n(p-value) are not clearly presented in the main text or figure legends. Please include the quantitative\ndata (r, p-value) and consider moving the correlation plot to a main figure to better support the clinical\nrelevance of the proposed axis.\nResponse: We thank the reviewer for these constructive suggestions. We quantitatively rescored the\nspecimens according to the percentage of immunopositive cells and the staining intensity, as\npreviously described (Ke Wu et al., 2023 Nat Cell Biol, PMID: 37156912). The sections were assigned\nthe following proportion scores: 0 if 0% of the tumor cells displayed positive staining, 1 for 0-1%, 2\n5\nfor 2-10%, 3 for 11-30%, 4 for 31-70%, 5 for 71-100%. Additionally, the intensity of staining was\nassigned a four-grade scale (0-3). Grades 0-3 represent: no expression (0), weak expression (1),\nmoderate expression (2), and strong expression (3). A total score (range, 0-8) was obtained by\ncombining the proportion and intensity scores. GraphPad Prism 8.0 was employed to perform the\nPearson correlation analysis (two-tailed). We have included these informations in the “Materials and\nMethods” section of the revised manuscript. Additionally, as suggested, we have moved the correlation\nplot from Supplementary Fig. 6 to Fig. 6T to better support the clinical relevance of the proposed axis,\nand the correlation coefficient (r) and significance (p-value) have been presented in the Fig. 6T in the\nrevised manuscript.\n4.Functional role of UFMylation beyond AKT1: The focus is tightly centered on AKT1, but the authors\nnote that other components of the lipid metabolism and PI3K-AKT-mTOR pathway may interact with\nUFL1. Please comment more fully on whether UFMylation of othercomponents (e.g., ACLY, SREBP1\nregulators) may contribute to the lipid phenotype, or whether this is uniquely AKT1-driven.\nResponse: We thank the reviewer for this recommendation. Rictor is a critical component of the\nmTORC2 complex, and FASN catalyzes the conversion of Acetyl-CoA and Malonyl-CoA into long-\nchain saturated fatty acids. Thus, they are essential for lipid synthesis. Our LC-MS/MS analysis\nshowed that Rictor and FASN are potential UFL1-interacting proteins (Supplementary Fig. 3A in the\nrevised manuscript). Co-IP and western blotting assays showed that UFL1 associates with and\nUFMylates FASN and Rictor (Supplementary Fig. 3R-W in the revised manuscript). Moreover, UFL1\noverexpression elevated lipid abundance, and this elevation was reversed by the FASN inhibitor C75\nand Rictor inhibitor JR-AB2-011 (Supplementary Fig. 5O-T in the revised manuscript), suggesting that\nUFL1 modulates lipid metabolism through multiple layers of mechanisms. Further investigations\nshould be performed to elucidate the mechanisms by which UFL1 controls FASN- and Rictor-mediated\nlipid metabolism. The mass spectrometry raw data have been deposited to the ProteomeXchange\nConsortium via the PRIDE repository with the dataset identifier PXD062297. To access the dataset,\n6\nreviewers may log in to the PRIDE website using the username and password that have been submitted\nto the editor.\nNotably, AKT1 not only phosphorylates ACLY but also modulates the expression of multiple lipid\nsynthesis-related genes via SREBP1, therefore possessing a broad and profound impact on lipid\nmetabolism. Hence, we focused on the UFL1-AKT1 feedback loop, but not on FASN or Rictor in this\nresearch.\nMinor points:\n1. Improve clarity in the Discussion to avoid repetition and to sharpen the distinction between known\nand novel findings.\nResponse: Thank you for this suggestion. As suggested, we have rephrased the Discussion to avoid\nrepetition and to sharpen the distinction between known and novel findings in the revised manuscript.\n2. Specify whether AKT2 and AKT3 were experimentally evaluated for UFMylation and ruled out\nsince it is surprisingly that UFL1 only targets AKT1 but negligible with AKT2 and 3.\nResponse: Thank you for your suggestion. AKT isoforms exhibit high sequence similarity, with\napproximately 80% homology across the family, thereby possessing redundant functions. However,\nAKT isoforms also possess unique or even opposing functions, suggesting that they may have different\nregulators and downstream effectors. In this study, we found that the interaction between UFL1 and\nAKT1 was stronger than the UFL1-AKT2 and UFL1-AKT3 associations (Supplementary Fig. 3C in the\nrevised manuscript). As suggested, we have detected the UFMylation of AKT2 and AKT3. The results\nindicated that UFL1 also UFMylated AKT2 and AKT3, but their UFMylation was weaker than that of\nAKT1 (Supplementary Fig. 3Q in the revised manuscript), probably due to their weak interaction with\nUFL1. Collectively, these data demonstrate that AKT1 is the primary substrate of UFL1 in breast cancer\ncells. The mechanism by which AKT isoforms display different binding affinity toward UFL1 should\nbe investigated in future studies.\n7\n3. Provide consistent cross-referencing between figures and legends, especially for\nSupplementarydata.\nResponse: Thank you for your suggestion.We have carefully checked the cross-referencing between\nfigures and legends to ensure the consistency.\nReviewer #2 (Breast cancer and lipid metabolism, Remarks to the Author):\n\"The UFL1-AKT positive feedback loop promotes breast cancer progression by enhancing lipid\nsynthesis and drug resistance” by Meng, Du and Liang et al., nicely describes a new regulatory\nmechanism of AKT based on UFMylation. The importance of UFMylation of AKT is supported by\nconvincing IP and in vivo rescue experiments. While the data supporting the posttranslational\nmodifications is convincing the more phenotypic data are unclear.\nMain concerns:\n1.The title: The title claims that UFL1-AKT feedback loop promotes breast cancer progression. There\nis very little evidence that the pathway can actually drive the disease. There is a lot of evidence to\nsuggest that the pathway is required for progression. The only ectopic overexpression I could find was\nlipid related in supp figure 2B-I. However, these do not include any in vivo or in vitro tumor outcome\ndata.\nResponse: We thank the reviewer for these constructive suggestions. To better support the conclusion\nthat the UFL1-AKT feedback loop promotes breast cancer progression, we performed experiments as\nfollows:\n(1). Cell proliferation and BODIPY staining assays in vitro and xenograft assays in mice were\nperformed using parental and UFL1-overexpressed MDA-MB-231 or MDA-MB-468 breast cancer\ncells (Fig. 2M-S; Supplementary Fig. 2D-R in the revised manuscript), and we found that UFL1\nincreased breast cancer cell proliferation and lipid accumulation in vitro (Supplementary Fig. 2D-N in\nthe revised manuscript) and breast tumor growth and lipids accumulation in vivo (Fig. 2M-S;\nUFL1 was impaired by the SREBP1 inhibitor Fatostatin (Fig. 2M-S; Supplementary Fig. 2O-R in the\nrevised manuscript), which abolishes lipid synthesis. Together, these findings indicate that UFL1\nfunctions as an oncogenic driver by modulating lipid synthesis in breast cancer.\n(2). BODIPY staining assays indicated that AKT1-WT, but not AKT1-3K/R, enhanced lipid abundance\nin MCF-7, BT474, and SK-BR-3 breast cancer cells, as evidenced by BODIPY staining assays\n(Supplementary Fig. 5H-M in the revised manuscript). Moreover, AKT1-WT overexpression\nsignificantly increased lipid abundance in MDA-MB-231 and MDA-MB-468 cells (Supplementary Fig.\n5O-T in the revised manuscript).\n(3). BODIPY staining assays indicated that UFL1-WT increased lipid accumulation, whereas UFL1-\nT426A had no effect in MCF-7 and BT474 breast cancer cells (Supplementary Fig. 6G-J in the revised\nmanuscript).\nCollectively, these results suggest that the UFL1-AKT feedback loop promotes breast cancer\nprogression.\n2. ER localization: The manuscript claims that UFL1 mediates an ER localization of AKT supported\nby Immunofluorescence images (3F and 4C) and western blotting on cellular fractionation (4D). The\nIF images should be shown at higher magnification, and the co-localization should be quantified. In\nthe same manner, the western blots should be repeated and quantified.\nResponse: We appreciate you raising these points. As suggested, the IF images have been shown at\nhigher magnification, and the co-localization has been quantified using the Fiji Colocalization Finder\nplugin in ImageJ software (Fig. 3F; Supplementary Fig. 3D; Fig. 4C; and Supplementary Fig. 4D in\nthe revised manuscript). Meanwhile, repeated western blots in regular depletion studies, including Fig.\n4A, 4D, 4H, 4K, 7C, and 7D, have been quantified (Supplementary Fig. 4A, E-H, L, N, O and\nSupplementary Fig. 7A in the revised manuscript).\n9\n3. Western blot quantification: In general, western blots should be quantified where possible. I\nunderstand this might be hard for some of the IP’s, but it should at the very least be done for regular\ndepletion studies as 4H. The figure legends consistently state at these blots have been repeated\nindependently three times. If so, please provide the quantification.\nResponse: We thank the reviewer for these suggestions. As suggested, repeated western blots in\nregular depletion studies, including Fig. 4A, 4D, 4H, 4K, 7C, and 7D, have been quantified\n(Supplementary Fig. 4A, E-H, L, N, O and Supplementary Fig. 7A in the revised manuscript).\n4. Requirement of lipid synthesis: The requirement of lipid synthesis to be the driver of the UFL1\npathway needs more validation. Currently, the claim is backed by in vitro proliferation rescue with\nOA (Supp 2J,K). Given the multitude of AKT effects on cancer cells, more conclusive evidence is\nneeded to back this claim.\nResponse: We thank the reviewer for these insightful suggestions. SREBP1 is the master transcription\nfactor that governs the expression of most rate-limiting enzymes involved in lipid synthesis. Thus,\nSREBP1 depletion or inhibition is widely used for downregulating lipid accumulation. To strengthen\nthe conclusion that UFL1 exerts its oncogenic functions through lipid synthesis, we performed the\nfollowing experiments:\n(1). Breast cancer cells stably expressing UFL1 were treated with or without the SREBP1 inhibitor\nFatostatin for subsequent lipid content measurement, BODIPY staining, and proliferation assays, and\nthe results showed that UFL1-induced lipid accumulation and proliferation were decreased by\nFatostatin (Supplementary Fig. 2D-N in the revised manuscript).\n(2). Breast cancer cells harboring an empty vector (EV) or a vector encoding UFL1 were implanted\ninto the mammary fat pad of female NOD-SCID mice. When tumor volume reached approximately\n200 mm3, Fatostatin was administered by intraperitoneal (IP) injection. In line with the in vitro results,\nUFL1 promoted breast tumor growth and lipid accumulation in tumor tissues, and these phenotypes\n10\nwere alleviated by Fatostatin (Fig. 2M-S and Supplementary Fig. 2O-R in the revised manuscript),\nsuggesting that UFL1 enhances tumor growth by modulating lipid synthesis.\n(3). In cancer cells, lipid synthesis is remarkably enhanced to meet the demand for unlimited cell\nproliferation and metastasis. Hence, increased lipid synthesis is a hallmark of breast cancer cells\n(Miguel Martin-Perez et al., 2022 Cell Metab. PMID: 36261043; Felix C E Vogel, Adriano B Chaves-\nFilho, and Almut Schulze, 2024 Nat Cancer. PMID: 38273023; Alexander R Terry and Nissim Hay,\nbut not AKT1-3K/R, enhanced lipid abundance in MCF-7, BT474, and SK-BR-3 breast cancer cells\n(Supplementary Fig. 5H-M in the revised manuscript). Moreover, AKT1-WT overexpression\nsignificantly increased lipid abundance in MDA-MB-231 and MDA-MB-468 cells (Supplementary Fig.\n5O-T in the revised manuscript).\n(4) BODIPY staining assays indicated that UFL1-WT increased lipid accumulation, whereas UFL1-\nT426A had no effect in MCF-7 and BT474 breast cancer cells (Supplementary Fig. 6G-J in the revised\nmanuscript).\nCollectively, these results indicate that UFL1 exerts its oncogenic functions through enhancing lipid\nsynthesis.\nMinor concerns:\n1. All bar plots should display individual values.\nResponse: Thank you for your suggestions. As suggested, all bar plots now display individual data\npoints in the revised manuscript.\nLine 110: In Figure 1E, I am guessing that the separation is based on the quantification in 1D. If so,\nplease relabel to low UFL1 and moderate/strong UFL1 (rather than simply high/low).\nResponse: Thank you for your suggestions. As suggested, “low UFL1” and “moderate/strong UFL1”\nhave been relabeled in Fig. 1E to maintain the consistency with Fig. 1D in the revised manuscript.\n11\n2. Line 111: Figure 1F. I believe this data is from the Tang data set, not Liu?\nResponse: Thank you for your reminder. The data were indeed from the Tang_2018 dataset, and we\nhave corrected this error in the revised manuscript.\n3. Line 212: (data not shown). Please provide the data.\nResponse: Thank you for your suggestion. AKT isoforms exhibit high sequence similarity, with\napproximately 80% homology across the family, thereby possessing redundant functions. However,\nAKT isoforms also possess unique or even opposing functions, suggesting that they may have different\nregulators and downstream effectors. In this study, we found that the interaction between UFL1 and\nAKT1 was stronger than the UFL1-AKT2 and UFL1-AKT3 associations (SupplementaryFig. 3C in the\nrevised manuscript). As suggested, we have detected the UFMylation of AKT2 and AKT3. The results\nindicated that UFL1 also UFMylated AKT2 and AKT3, but their UFMylation was weaker than that of\nAKT1 (SupplementaryFig. 3Q in the revised manuscript), probably due to their weak interaction with\nUFL1. Collectively, these data demonstrate that AKT1 is the primary substrate of UFL1 in breast cancer\ncells. The mechanism by which AKT isoforms display different binding affinity toward UFL1 should\nbe investigated in future studies. As suggested, we have included these data in Supplementary Fig.\n3Q in the revised manuscript.\n4. Line 498: Why are the cells treated with 1mM sodium citrate for 12 hours before staining for Bodipy\nand oil red O? And how does that impact the measurements? I am guessing citrate will boost the acetyl-\nCoA levels and thus possibly the lipid synthesis. However, the citrate incubation is not typically\nrequired to identify lipid droplets. Does the data hold up without the preincubation?\nResponse: Thank you for your questions. Except for liver cells, the intensity of BODIPY and Oil Red\nO (ORO) staining in most other cell types, including cancer cells, is very low. For example, the ORO\nand BODIPY signals are very weak in MDA-MB-231, MDA-MB-468, and MCF-7 cells (Heather J\nWright et al., 2017 PNAS. PMID: 28739932; Yangjing Zhao et al., 2022 Adv Sci. PMID: 34747157;\nJiamin Lin et al., 2023 eLife. PMID: 38078907) (Fig. 2A, B; Fig. 3A; and Fig. 5A in this letter).\nand BODIPY signals (Yanjie Tan et al., 2024 Nat Commun. PMID: 39333556; Jiamin Lin et al., 2023\neLife. PMID: 38078907) (Fig. 3 and Fig. 5A in this letter). However, low dose OA (100 or 200 μM)\ncan dramatically increase the ability of breast cancer cells to proliferate and invade (Jiamin Lin et al.,\n2023 eLife. PMID: 38078907) (Fig. 5A-D in this letter). In contrast, 1 mM sodium citrate (SCT)\nsignificantly increases lipid droplet accumulation (Yangjing Zhao et al., 2022 Adv Sci. PMID:\n34747157) (Fig. 4A in this letter) without affecting breast cancer cell proliferation and apoptosis\n(Yangjing Zhao et al., 2022 Adv Sci. PMID: 34747157) (Fig. 4B, C in this letter). Therefore, we utilized\n1 mM SCT to enhance the ORO and BODIPY signals in this study.\nTo rule out the possibility that 1 mM SCT affects our conclusion, we compared the intensity of\nBODIPY staining in shSC and shUFL1#1 MDA-MB-231 cells in the presence or absence of 1 mM SCT.\nWe found that 1 mM SCT markedly increased BODIPY signals, and UFL1 depletion significantly\ndecreased BODIPY signals with or without SCT treatment (Fig. 6 in this letter). This is rational,\nbecause UFL1 activates AKT, thereby enhancing the activity of ACLY, which catalyzes the conversion\nof citrate into Acetyl-CoA. Thus, 1 mM SCT treatment does not change our conclusion that UFL1 is\n[FIGURE REDACTED]\n13\nessential for lipid accumulation. These results are non-essential for this study; therefore, we have\nprovided them here for your review.\nThe manuscript by Meng et al reported the critical role of the UFL1-AKT axis in breast cancer\nprogression and drug resistance. Specifically, the authors found that AKT1 was UFMylated by UFL1,\nan UFMylation-specific E3 ligase, in breast cancer cells. AKT1 UFMylation promoted its ER\nrecruitment and activation by both PDK1 and mTORC2, which in turn enhanced lipid biogenesis and\ncancer progression. Moreover, the authors showed that AKT1 phosphorylated UFL1 and boosted\nUFL1’s activity. Finally, UFL1 expression was elevated in breast cancer samples and associated with\npoor prognosis in human patients. Targeting both UFMylation and AKT1 had a synergetic effect on\nTNBC in xenograft models.\nOverall, the finding of the positive feed-back regulation between AKT1 and UFL1 appears to be novel\nand may represent a potential target for cancer therapy. However, there are some significant flaws in\ndata analysis and interpretation that should be addressed. As described below, this reviewer failed to\nduplicate some of the results as claimed by the authors regarding the association between UFL1 mRNA\nlevel and overall survival of human BC patients. In addition, a few pieces of experimental data\nappeared to conflict to each other, and some results were difficult to explain. Finally, the evidence to\nsupport the role of lipid metabolism in tumor progression is relatively insufficient.\nSpecific comments:\n1. Concerns on bioinformatic analyses of BC databases and clinical samples.\na. This reviewer failed to duplicate KM survival curves using GEPIA database (http://gepia.cancer-\npku.cn). A simple search using UFL1 and BRCA dataset in GEPIA yielded no significant association\nbetween Ufl1 mRNA levels and overall survival of BC patients. The number of patients also differed\nfrom what the authors reported.\ninto High and Low expression groups. When the High and Low expression groups are denoted as\nCutoff-High (50%) and Cutoff-Low (50%), the numbers of the High and Low groups are 535 and 533,\nrespectively. Thus, the total number of samples is 1068 (Fig. 7A in this letter). In fact, the KM analysis\nwas performed by assigning the high and low expression groups with Cutoff-High (22%) and Cutoff-\nLow (78%), respectively. Thus, the number of the High group and the Low group should be 235\n(1068*22%) and 833 (1068*78%), respectively (Fig. 7B in this letter). However, the numbers of the\nHigh group and Low group were both incorrectly labeled as 835 on the GEPIA website, and we\noverlooked correcting them in the initial submission. This error has been corrected in Fig. 1B in the\nrevised manuscript.\nb. In Fig.S1C, GSE9693 dataset was about “Clostridioides difficile”, which was nothing to do with\nbreast cancer.\nResponse: Thank you for your reminder. GSE9693 is a typo; it should be GSE9893 (Fig. 8A in this\nletter). We have corrected this error in Supplementary Fig. 1C in the revised manuscript.\nc. In Fig. 1F, this reviewer could not replicate the KM survival plot as reported using KM plotter\ndatabase (kmplot.com). There was no reference or GSE number for Liu-2014 dataset (as indicated in\nFig. 1F). Additionally, none of BC datasets in KMplot could generate similar survival plots showing\n16\nthe significant correlation between Ufl1 mRNA level and overall survival. Please provide more\ninformation on the dateset and UFL1 (named as KIAA0776 in KM plot) probe set used in the analysis\nreported by the authors.\nResponse: Thank you for your reminder. The data were derived from the Tang_2018 dataset (Fig. 8B\nin this letter), and we have corrected this copy-and-paste error in Fig. 1F in the revised manuscript.\n2. Specificity of Ufl1 antibody in IHC. The authors did not provide the source of UFL1 and other\nantibodies. To the knowledge of this reviewer, some commercial UFL1 antibodies recognized multiple\nbands in immunoblotting. Even though Fig.1K may have indirectly addressed this issue, more stringent\ntest should be done to examine the specificity of UFL1 antibody. Meanwhile, the knockdown efficiency\nof UFL1 shRNAs should be shown.\nResponse: Thank you for your reminder.\n(1). We have provided the catalog number and RRID of all antibodies in the “Materials and Methods”\nsection of the revised manuscript.\n(2). Anti-UFL1 antibody (HPA030559, RRID: AB_10600537) was purchased from Sigma-Aldrich,\nand the specificity of this antibody has been confirmed in vitro and in vivo in multiple published papers,\nsuch as Nat Cell Biol (Jiang Liu et al., PMID: 32807901), Adv Sci (Jiongjie Jing et al., PMID:\n39985286), and PNAS (Junzhi Zhou et al., PMID: 36893266). To further examine the specificity of\nthis anti-UFL1 antibody, we are providing the whole WB membranes of UFL1 in shSC and shUFL1\nbreast cancer cells. These membranes indicate that this antibody specifically recognizes UFL1 bands\n17\n(Fig. 9 in this letter), and the UFL1 knockdown efficiency is very high (Fig. 9in this letter; Fig. 1G, H\nand Supplementary Fig. 1E-G in the revised manuscript). Moreover, the IHC staining in Fig. 1K in the\nrevised manuscript also validates the specificity of this anti-UFL1 antibody. Since the specificity of\nthe UFL1 antibody has been verified in published papers, we will not include the whole WB\nmembranes of UFL1 in shSC and shUFL1 breast cancer cells in the revised manuscript. Instead, we\nhave provided them here for your review.\n3. For cell death assay of tissue culture cells, Annexin V staining or TUNEL should be done in addition\nto CC3 staining.\nResponse: Thank you for your suggestion. As suggested, Annexin-V and propidium iodide (PI)\nstaining have been performed for flow cytometry assays (Supplementary Fig. 1L, M in the revised\nmanuscript), and the results were consistent with those from CC3 staining.\n4. The claim that UFL1 is an oncogenic driver (Fig.1) appears to be an overstatement. Even though\nUFL1 overexpression promoted cell proliferation, no experimental evidence supported its oncogenic\nactivity.\nResponse: Thank you for these constructive suggestions. To better support the conclusion that UFL1\npromotes breast cancer progression, we performed experiments as follows:\n(1). Cell proliferation and BODIPY staining assays in vitro and xenograft assays in mice were\nperformed using parental and UFL1-overexpressed MDA-MB-231 or MDA-MB-468 breast cancer\ncells (Fig. 2M-S and Supplementary Fig. 2D-R in the revised manuscript), and we found that UFL1\nincreased breast cancer cell proliferation and lipid accumulation in vitro (Supplementary Fig. 2D-N\n18\nand Supplementary Fig. 5O-T in the revised manuscript) and breast tumor growth and lipid\naccumulation in vivo (Fig. 2M-S and Supplementary Fig. 2O-R in the revised manuscript).\nInterestingly, the cancer-promoting effect of UFL1 was impaired by the SREBP1 inhibitor Fatostatin\n(Fig. 2M-S and Supplementary Fig. 2O-R in the revised manuscript), which abolishes lipid synthesis.\nTogether, these findings indicate that UFL1 functions as an oncogenic driver by modulating lipid\nabundance in breast cancer.\n(2). BODIPY staining assays indicated that AKT1-WT, but not AKT1-3K/R, enhanced lipid abundance\nin MCF-7, BT474, and SK-BR-3 breast cancer cells, as evidenced by BODIPY staining assays\n(Supplementary Fig. 5H-M in the revised manuscript). Hence, UFL1 enhances lipid accumulation in a\nUFMylation-dependent manner.\nCollectively, these results suggest that UFL1 promotes breast cancer progression via modulation of\nlipid synthesis.\n5. The claim that UFL1 promotes breast cancer cell growth by increasing lipid accumulation was\nbacked up by the only functional assay that was shown in Fig.S2J and K, in which addition of OA\nreversed UFL1 knockdown phenotype. Since lipid metabolism was such a big part of this study, more\nfunctional experiments should be done to firmly establish the relationship between lipid metabolism,\nthe UFL1-AKT axis and tumor progression.\nResponse: We thank the reviewer for these constructive suggestions. SREBP1 is the master\ntranscription factor that governs the expression of most rate-limiting enzymes involved in lipid\nsynthesis. Thus, SREBP1 depletion or inhibition is widely used for downregulating lipid accumulation.\nTo strengthen the conclusion that UFL1 exerts its oncogenic functions through lipid synthesis, we\nperformed the following experiments:\n(1). Breast cancer cells stably expressing UFL1 were treated with or without the SREBP1 inhibitor\nFatostatin for subsequent lipid content measurement, BODIPY staining, and proliferation assays, and\nthe results showed that UFL1-induced lipid accumulation and proliferation were decreased by\nFatostatin (Supplementary Fig. 2D-N in the revised manuscript).\n(2). Breast cancer cells harboring an empty vector (EV) or a vector encoding UFL1 were implanted\ninto the mammary fat pad of female NOD-SCID mice. When tumor volume reached approximately\n200 mm3, Fatostatin was administered by intraperitoneal (IP) injection. In line with the in vitro results,\nUFL1 promoted breast tumor growth and lipid accumulation in tumor tissues, and these phenotypes\nwere alleviated by Fatostatin (Fig. 2M-S and Supplementary Fig. 2O-R in the revised manuscript),\nsuggesting that UFL1 enhances tumor growth by modulating lipid synthesis.\n(3). In cancer cells, lipid synthesis is remarkably enhanced to meet the demand for unlimited cell\nproliferation and metastasis. Hence, increased lipid synthesis is a hallmark of breast cancer cells\n(Miguel Martin-Perez et al., 2022 Cell Metab. PMID: 36261043; Felix C E Vogel, Adriano B Chaves-\nFilho, and Almut Schulze, 2024 Nat Cancer. PMID: 38273023; Alexander R Terry and Nissim Hay,\nbut not AKT1-3K/R, enhanced lipid abundance in MCF-7, BT474, and SK-BR-3 breast cancer cells,\nas evidenced by BODIPY staining assays (Supplementary Fig. 5H-M in the revised manuscript).\nMoreover, AKT1-WT overexpression significantly increased lipid abundance in MDA-MB-231 and\nMDA-MB-468 cells (Supplementary Fig. 5O-T in the revised manuscript).\n(4). BODIPY staining assays indicated that UFL1-WT increased lipid accumulation, whereas UFL1-\nT426A had no effect in MCF-7 and BT474 breast cancer cells (Supplementary Fig. 6G-J in the revised\nmanuscript).\nCollectively, these results indicate that UFL1 exerts its oncogenic functions through enhancing lipid\nsynthesis.\n6. High-quality of confocal imaging in Fig.3F should be provided. The current one appeared to be\nfuzzy, especially UFL1.\nhigh-quality confocal images, the colocalization of UFL1 and AKT1 was quantified using the Fiji\nColocalization Finder plugin in ImageJ software (Fig. 3F and Supplementary Fig. 3D in the revised\nmanuscript), and selected regions were magnified to improve the visual effect.\n7. As shown in Fig.3I and J, knockdown of UFL1 led to reduced AKT UFMylation but had no effect\non overall UFMylation (especially RPL26 UFMylation, around 27kD and higher), a result that seems\nto contradict to previous studies. Please explain why.\nResponse: Thank you for your reminder. Overall UFMylation was not affected by UFL1 ablation,\nwhich may be due to two reasons: the specific lysis buffer for UFMylation detection was not fresh\nenough, and too much protein was loaded for UFM1 detection. To avoid these problems mentioned\nabove, we repeated all these assays using the following fresh lysis buffer: HEK293T or breast cancer\ncells were collected and resuspended in a buffer containing 150 mM Tris-HCl (pH 8.0), 5% sodium\ndodecyl sulfate (SDS), and 30% glycerol, followed by boiling for 10 minutes. Cell lysates were diluted\nwith buffer A (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 2 mM N-ethylmaleimide, and\nprotease inhibitors) at a 1:20 ratio and incubated with the indicated antibodies and protein A/G\nmagnetic beads (Bio-Rad) at 4 °C overnight. The immunoprecipitates were boiled in 1 x Laemmli\nbuffer, separated by SDS-PAGE, and analyzed by WB. For overall UFMylation (UFM1) detection, 5-\n10 μg of whole cell lysates were loaded. In this case, a reduction in overall UFMylation was observed\nupon UFL1 ablation (Fig. 3I, J in the revised manuscript) and UFMylation inhibitor treatment (Fig. 7A,\nB in the revised manuscript). Notably, UFL1 overexpression only had a minimal impact on overall\nUFMylation (Supplementary Fig. 3I, J in the revised manuscript) probably due to high levels and\nactivity of endogenous UFL1 in breast cancer cells (Fig. 1C, D; Supplementary Fig. 1D; Fig. 6T; and\nSupplementary Fig. 6M in the revised manuscript).\n8. In Fig.3I and J and other similar figures, please provide the results of IgGs as negative controls in\nthose IPs.\nnegative controls in Fig. 3I, J, L, M; Supplementary Fig 3I, J, N-Q; Fig. 4F, G, I; Supplementary Fig.\n4J; Supplementary Fig. 6B; and Fig. 7A, B in the revised manuscript.\n9. In Fig.3, it was showed that UFM1 antibody could detect UFMylated endogenous and exogenous\nAKT1. How about AKT1 and Flag antibodies?\nResponse: Thank you for your valuable suggestions. Similar to other post-translational modifications,\nUFMylation is dynamic and reversible, and this feature ensures that UFMylated AKT1 accounts for a\ntiny portion of total AKT1. Therefore, endogenous AKT1 was examined using an anti-AKT1 antibody\nin MDA-MB-231 and MDA-MB-468 cells with or without UFL1 depletion. After a long exposure,\ntwo AKT1 bands were observed in the parental cells (Supplementary Fig. 4K in the revised manuscript).\nThe molecular weight of the upper AKT1 (UFMylated AKT1) bands was approximately 27 kDa greater\nthan that of their corresponding lower bands, and the upper bands were very weak (Supplementary\nFig. 4K in the revised manuscript). These upper bands nearly disappeared in UFL1 knockdown cells\n(Supplementary Fig. 4K in the revised manuscript). In contrast, UFL1 overexpression significantly\nincreased the intensity of upper Flag-AKT1 (UFMylated Flag-AKT1) in MDA-MB-231 and MDA-\nMB-468 cells (Fig. 10 in this letter). Covalently bound UFM1 is primarily cleaved from substrates by\nthe cysteine protease UFSP2 (Christopher P. Walczak et al., 2019 PNAS. PMID: 30626644; Jessie\nKulsuptrakul et al., 2021 Cell Rep. PMID: 33730579) (Fig. 1 in this letter). Thus, UFSP2 was depleted\nby shRNA in MDA-MB-231 and MDA-MB-468 cells to assess whether inhibited deUFMylation leads\nto an increase in AKT1 UFMylation and an upward shift of the AKT1 band. As expected, the intensity\nof the UFMylated AKT1 bands was significantly increased in UFSP2 knockdown cells (Fig. 4H in the\nrevised manuscript).\n22\n10. In Fig. 4A and B, it would be interesting to examine if phosphorylation of other mTORC2 and\nPDK1 targets is altered by UFL1 knockdown.\nResponse: Thank you for your suggestions. PKCα and PLK1 are well-known targets of mTORC2\n(Timothy R Baffi et al., 2021 Sci Signal. PMID: 33850054; Tsuneo Ikenoue et al., 2008 EMBO J.\nPMID: 18566587) and PDK1 (Jing Tan et al., 2013 Cancer Discov. PMID: 23887393), respectively.\nHence, we determined the phosphorylation of PKCα and PLK1 in shSC and shUFL1 MDA-MB-231\nand MDA-MB-468 cellsand found that UFL1 depletion significantly downregulated the protein levels\nof phosphorylated PKCα but had no impact on PLK1 phosphorylation (Supplementary Fig. 4M in the\nrevised manuscript). It is reasonable that UFL1 affected PKCα phosphorylation because UFL1\ninteracted with and UFMylated Rictor, which is a critical component of the mTORC2 complex\n(Supplementary Fig. 3T, U, W in the revised manuscript).\n11. In Fig.4C, co-localization of AKT1 and calreticulin should be quantitated.\nResponse: Thank you for your suggestions. As suggested, the colocalization of AKT1 and calreticulin\nhas been quantified using the Fiji Colocalization Finder plugin in ImageJ software (Supplementary\nFig. 4D in the revised manuscript).\n12. In Fig. 4D, AKT1 levels in ER fractions were obviously lower in UFL1 knockdown ER fraction.\nHowever, in Fig. 4E, AKT1 levels remained unchanged in UFL1 KD ER fractions. Please explain this\ndiscrepancy. Additionally, no increase of AKT1 in cytoplasmic fractions was observed in UFL1 KD\ncells. Why?\nResponse: Thank you for your reminder. This discrepancy may be caused by excess loading of protein\nlysate. After reducing protein loading, decreased AKT1 levels in ER fractions were observed (Fig. 4D\nin the revised manuscript). In quiescent cells, AKT1 predominantly localizes in the cytosol. Growth\nfactors and other stimuli stimulate PH domain-dependent accumulation of AKT1 at the respective\nmembranes. AKT1 membrane accumulation is transient, typically peaking around 2-5 min after\nstimulation (Véronique Calleja et al., 2007 PloS Biol. PMID: 17407381; Michael Ebner et al., 2017 Mol\n(Hiroyuki Kubota 2012 Mol Cell. PMID: 22633957; Michael Ebner et al., 2017 Mol Cell. PMID:\n28157504). This observation, among others, has led to the prevailing model of Akt signaling, whereby\nit is activated at the membrane but dissociates from the membrane and freely diffuses in the cytosol in\na “locked” active state, phosphorylating its numerous substrates (Corina E Antal and Alexandra C\nNewton, 2013 Mol Cell Proteomics. PMID: 23788531; Véronique Calleja et al., 2007 PloS Biol. PMID:\n17407381; Véronique Calleja, Michel Laguerre, and Banafshé Larijani, 2009 J Chem Biol. PMID:\n19568789; R Meier et al., 1997 J Biol Chem. PMID: 9374542). From this perspective, all inactive AKT1\nand most active AKT1 exist in the cytosol, and the influence of UFL1 depletion on the AKT1 levels in\nthe cytoplasmic fraction is negligible. In contrast, UFL1 depletion dramatically affected p-T308 and p-\nS473 levels in both the cytosol and ER membrane.\n13. In Fig.4F, more negative controls are needed.\nResponse: Thank you for your suggestions. As suggested, we have provided the results of IgG as a\nnegative control in those immunoprecipitation experiments (Fig. 4F in the revised manuscript).\n14. in Fig.5A, please show the protein levels of shRNA-resistant AKT1s.\nResponse: Thank you for your suggestions. AKT1 was knocked down by shAKT1\n(CGTGCCATGATCTGTATTTAA) targeting its 3’UTR. Therefore, shAKT1 only depletes\nendogenous AKT1, without affecting ectopically expressed AKT1. We have included this information\nin the “Materials and Methods” section of the revised manuscript and presented the protein levels of\nAKT1 in shSC, shAKT1, shAKT1+AKT1-WT, and shAKT1+AKT1-3K/R in Supplementary Fig. 5A in\nthe revised manuscript.\n15. In Fig.6, it would be interesting to test the effect of UFL1 (T426A) mutant on other UFM1 targets\nsuch as RPL26.\nResponse: Thank you for your suggestions. The protein levels of total RPL26 (Supplementary Fig.\n6B in the revised manuscript) and UFMylated RPL26 (as indicated by asterisks in Supplementary Fig.\n24\n6B in the revised manuscript) were both comparable in MDA-MB-231 and MDA-MB-468 cells\nexpressing UFL1-WT and UFL1-T426A, suggesting that p-UFL1 (T426) may specifically modulate\nthe AKT signaling pathway. Notably, AMPK phosphorylates UFL1 at T536, disrupting the UFL1-PD1\ninteraction and PD1 UFMylation to trigger its degradation in T cells (Chuan He et al., 2024 Mol Cell.\nPMID: 38377992). Upon DNA damage, ATM phosphorylates UFL1 at S462 to enhance its ligase\nactivity and promote ATM activation in a positive feedback loop, thereby facilitating DNA damage\nrepair (Bo Qin et al., 2019 Nat Commun. PMID: 30886146). Thus, UFL1 phosphorylation at distinct\namino acids may dictate substrate specificity, ultimately determining its biological function.\n16. In Fig.7A and B, UFMylation inhibitor (Uba5 E1 inhibitor) DKM 2-93 dramatically reduced AKT1\nUFMylation but had no effect on overall UFMylation. Any explanation? Furthermore, if both\ninhibitors work through the route of AKT1, how was the synergy achieved?\nResponse: Thank you for your constructive questions.\n(1). Similar to your question #7, overall UFMylation was not affected by UBA5 and AKT inhibitors,\nwhich may be due to two reasons: the specific lysis buffer for UFMylation detection was not fresh\nenough, and too much protein was loaded for UFM1 detection. To avoid these problems, we repeated\nall these assays using the following fresh lysis buffer: HEK293T or breast cancer cells were collected\nand resuspended in a buffer containing 150 mM Tris-HCl (pH 8.0), 5% sodium dodecyl sulfate (SDS),\nand 30% glycerol, followed by boiling for 10 minutes.Cell lysates were diluted with buffer A (50 mM\nTris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 2 mM N-ethylmaleimide, and protease inhibitors) at a\n1:20 ratio and incubated with the indicated antibodies and protein A/G magnetic beads (Bio-Rad) at\n4 °C overnight. The immunoprecipitates were boiled in 1 x Laemmli buffer, separated by SDS-PAGE,\nand analyzed by WB. For overall UFMylation (UFM1) detection, 5-10 μg of whole cell lysates were\nloaded. In this case, a reduction in overall UFMylation was observed upon UFMylation inhibitor\ntreatment (Fig. 7A, B in the revised manuscript). Notably, UFL1 overexpression only had a minimal\nimpact on overall UFMylation (Supplementary Fig. 3I, J in the revised manuscript) probably due to\n25\nhigh levels and activity of endogenous UFL1 in breast cancer cells (Fig. 1C, D, Supplementary Fig. 1D;\nFig. 6T, Supplementary Fig. 6M in the revised manuscript).\n(2). Synergistic combinations of two or more agents can overcome toxicity and other side effects\nassociated with high doses of single drugs by countering biological compensation, allowing reduced\ndosage of each compound or accessing context-specific multitarget mechanisms (Joseph Lehár et al.,\nRev Cancer. PMID: 38627557). Multiple promising combinatorial strategies have been developed to\nachieve synergistic effects, such as vertical inhibition, disruption of positive feedback loops, blocking\nparallel signaling pathways, and preventing compensatory bypass signaling. ① Vertical inhibition\nconsists of inhibiting the main oncogenic driver, which is genetically altered, often mutated or\namplified, together with an upstream or downstream component of the same oncogenic pathway, to\nachieve a deeper suppression of the pathway (Naiara Perurena, Lisa Situ, and Karen Cichowski, 2024\nNat Rev Cancer. PMID: 38627557; David Shahbazian, Joshua Sznol, and Harriet M Kluger, 2012 Adv\nPharmacol. PMID: 22959021). From this perspective, AKT1 is the main oncogenic driver, UFL1 (or\nother components of the UFMylation system) is an upstream component of the AKT1 pathway; the\ncombination of an AKT1 inhibitor and a UBA5 inhibitor achieved a deeper suppression of the AKT1\npathway (Fig. 7 in the revised manuscript). Thus, the combination of an AKT1 inhibitor and a UBA5\ninhibitor constitutes a vertical inhibition. Similarly, PDK1 phosphorylates AKT1 at T308, and a PDK1\ninhibitor (GSK2334470) and an AKT1 inhibitor (Ipatasertib and MK-2206) achieve a synergistic effect\n(Emily K Slotkin et al., 2019 Cancer Discov. PMID: 30877085; Kristyna Kupcova et al., 2024 Exp\nHematol Oncol. PMID: 39487517). PRMT5 methylates and subsequently activates AKT1, and a\nPRMT5 inhibitor (GSK3326595) and an AKT inhibitor (MK-2206) display a synergistic effect on\npromoting cell death (Shasha Yin et al., 2021 Nat Commun. PMID: 34103528). ② When Protein A\nand Protein B directly or indirectly activate each other (forming a positive feedback loop), their\ninhibitors frequently exhibit strong synergistic effects. This occurs because blocking either protein\n26\nalone leaves the other free to reactivate its partner, while dual inhibition almost collapses the entire\nsignaling circuit (Joseph Lehár et al., 2009 Nat Biotechnol. PMID: 19581876;Deborah Plana, Adam C\nPalmer, and Peter K Sorger, 2022 Cancer Discov. PMID: 34983746). In this regard, AKT1 and UFL1\nreciprocally activate each other to form a positive feedback loop; therefore, their inhibitors exert a\nsynergistic anti-cancer effect. In the same manner, an AKT inhibitor (MK-2206) and an mTOR\ninhibitor (Everolimus) (Yvonne Yeung et al., 2017 Mol Oncol. PMID: 28544747), an EGFR inhibitor\n(Gefitinib) and a MEK inhibitor (Trametinib) (João M Fernandes Neto et al., 2020 Nat Commun.\nPMID: 32572029), and a STAT3 inhibitor (Napabucasin) and an IL-6 inhibitor (Tocilizumab) (Anibal\nMéndez-Clemente et al., 2022 Oncol Rep. PMID: 35703345) exhibit synergistic anti-cancer effects. In\nsummary, the UBA5 inhibitor sensitizes breast cancer cells to the AKT1 inhibitor, and their\ncombination achieves a deeper suppression of AKT1 signaling and breast tumor growth, thereby\nexhibiting a synergistic effect.\n17. Please provide more experimental details, including antibody sources and RIDDs, web links,\ndesigns of shRNA resistant genes, cell lines in each experiment, transient or stable lines, and etc.\nResponse: Thank you for your reminder. (1) We have provided the catalog numbers, RRIDs, and\nsources of antibodies in the “Materials and Methods” section of the revised manuscript. (2) We have\nprovided the web links of Sigma-Aldrich Advanced Genomics, PrimerBank, Gene Expression\nProfiling Interactive Analysis (GEPIA), Gene Expression Omnibus (GEO), ProteomeXchange\nConsortium, PRoteomics IDEntifications Database (PRIDE), and Kaplan-Meier plotter in the\n“Materials and Methods” and “Data Availability” sections of the revised manuscript. (3) UFL1 and\nAKT1 were depleted by shUFL1#1 (GGAACTTGTTAATAGCGGA) and shAKT1\n(CGTGCCATGATCTGTATTTAA) targeting their 3’UTR, respectively. Therefore, shUFL1#1 and\nshAKT1 only knockdown endogenous UFL1 and AKT1, without affecting exogenous cDNAs of UFL1\nand AKT1. We have included this information in the “Materials and Methods” section of the revised\n27\nmanuscript. (4) As suggested, the information regarding cell lines and transient or stable lines has been\nincluded in the revised manuscript.\n28"}, {"round": 2, "reviewer_comments": {"reviewer_1": "This manuscript from Fei Meng et al. presents a mechanistic study defining a positive feedback loop\nbetween UFL1 and AKT1 in breast cancer. The authors demonstrate that UFL1 UFMylates AKT1 at\nlysines K189, K276, and K297, promoting its ER localization and phosphorylation by PDK1 and\nmTORC2. In turn, AKT1 phosphorylates UFL1 at T426, enhancing UFL1 ligase activity and\nreinforcing AKT1 UFMylation. This reciprocal regulation drives lipid accumulation and tumor growth,\nwhile pharmacological inhibition of UFMylation synergizes with AKT inhibition and sensitizes cells\nto chemotherapy.\nIn this revised manuscript, the authors have convincingly addressed the previous concerns by\nproviding additional biochemical evidence for the dynamic nature of AKT1 UFMylation and de-\nUFMylation. The authors extended their analyses to multiple breast cancer subtypes, incorporating\nquantitative clinical correlation data, and broadened their discussion of UFMylation substrates beyond\nAKT1. The new experiments, including UFSP2 knockdown, AKT1 phosphorylation-deficient mutants,\nand expanded xenograft and lipid metabolism assays, strengthen the mechanistic model and enhance\nclinical relevance.\nOverall, the study is comprehensive, rigorous, and meaningful. The mechanistic dissection of\nUFMylation and phosphorylation sites is well handled, and the therapeutic angle is compelling. This\nwork significantly improves upon the initial version and now provides strong support for the proposed\nUFL1–AKT1 feedback loop as a driver of lipid metabolism and tumor growth in breast cancer.", "reviewer_2": "The authors have addressed all my concerns. I have no further comments.", "reviewer_3": "All concerns have been satisfactorily addressed in the revision"}, "author_response": "Response: We appreciate the reviewer’s positive comments on our efforts.\nResponse: We appreciate the reviewer for the very positive comments on our research.\nResponse: We appreciate the reviewer’s positive comments.\nResponse: We appreciate the reviewer’s positive comments."}]

Decision letter