Concurrent PIK3CA mutant promotes cachexia through inflammatory signaling in EGFR mutant lung cancer

Domain: biology
Published: 2025-12-16
Editorial outcome: accept

Abstract
PIK3CA mutation is frequently concurrent with known oncogenic drivers such as EGFR mutation in lung cancer, raising an interesting question about its real function. Cachexia is a systemic disease arising from tumor-organ crosstalk, significantly contributing to cancer-related mortality. Through integrative study of genetically engineered mouse models (GEMMs) and clinical data, we find concurrent PIK3CA mutant preferentially drives cachexia in EGFR-mutant lung cancer, promoting malignant progression instead of cancer initiation. PIK3CA mutant-mediated cachexia can be overcome by osimertinib (Osi) treatment in Osi-sensitive GEMM. In contrast, chemotherapy, routinely used in clinic for those relapsed from Osi therapy, fails to ameliorate cachexia in Osi-resistant GEMM despite notable tumor suppression. PIK3CA mutant-driven cachexia is mediated through NF-κB activation and can be dampened by combined aspirin treatment. This work provides insights into PIK3CA mutant biological function and mechanisms behind its clinical impacts, and proposes a potential strategy for clinical management.

Introduction
Lung cancer is one of the most aggressive diseases worldwide, with non-small cell lung cancer (NSCLC) representing the most prevalent histological subtype1. PIK3CA, encoding the p110α catalytic subunit of PI3K, is mutated in approximately 7–16% of NSCLC2,3. PI3K plays a pivotal role in multiple tumorigenic processes in NSCLC, including promoting glucose transport4, facilitating bone metastasis5, mediating cytokine production6, and contributing to an immunosuppressive microenvironment by modulating immune cell infiltration7. PIK3CA mutations are highly enriched in its helical region (E542K, E545K) and kinase domain (H1047R)8. It’s well established that these mutations are oncogenic and frequently cause aberrant activation of downstream AKT signaling, triggering a cascade of responses that drive lung tumorigenesis9–11. Animal model study shows that PIK3CA mutant transgene alone is sufficient to drive lung cancer initiation in mice12. Paradoxically, most PIK3CA mutations are found to occur concurrently with those famous oncogenic drivers13–15, e.g., EGFR mutations, KRAS mutations, and ALK fusions, which are known to be mutually exclusive15–18 and individually suffice to drive lung cancer initiation19–21. Multiple studies have reported that PIK3CA mutations are frequently concurrent with oncogenic EGFR mutations in East Asian NSCLC populations13–15. Osimertinib (Osi), a third-generation EGFR tyrosine kinase inhibitor (TKI), is currently the preferable option for EGFR-mutant NSCLC patients, owing to its high efficacy and well-tolerated safety profile22. However, similar to that of early-generation EGFR-TKIs, Osi resistance inevitably develops. Additional C797S mutation represents one of the most prevalent mechanisms in Osi resistance23. When occurring in conjunction with a sensitizing mutation and in the absence of T790M mutation, C797S mutation confers resistance to Osi while preserving sensitivity to first- and second-generation agents. The presence of triple mutants consisting of the sensitizing mutation, T790M, and C797S, leads to resistance against all three generations of EGFR TKIs24. For these patients, chemotherapy, e.g., pemetrexed (PEM) in combination with cisplatin (CDDP), is the remaining option in clinic25.

High-throughput sequencing analyses of TKI-resistant lung cancer specimens have identified the emergence of additional PIK3CA mutation26,27, indicative of its potential contribution to drug resistance. This is further supported by the observation of worse overall survival (OS) of those EGFR-mutant patients with concurrent PIK3CA mutation in their tumors28. However, detailed analyses of clinical TKI responses show that the concurrence of PIK3CA mutation does not affect the therapeutic efficacy of TKI treatments28,29, e.g., almost no difference of progression-free survival (PFS) in patients with or without PIK3CA mutations. This raises another interesting question regarding the real mechanism behinds the clinical impacts of PIK3CA mutant.

Cancer cachexia is a complex and debilitating syndrome characterized by body weight loss primarily due to skeletal muscle atrophy and adipose tissue wasting30, responsible for more than 20% of cancer-related deaths31. Cachexia occurs in approximately 50% of lung cancer patients32 and is associated with poor life quality33, elevated treatment-related toxicity34,35, reduced therapeutic responses36, and increased risk of mortality37–40. Recent study has begun to focus on the link between specific gene alterations and cachexia development37.

In this work, we uncover an unexpected biological function of PIK3CA mutations in lung tumorigenesis, mainly contributing to cachexia instead of driving lung cancer initiation. Moreover, we provide a reasonable explanation about the clinical observation of paradoxical difference between PFS and OS in link to concurrent PIK3CA mutation. We also propose a potential strategy for clinical management of lung cancer patients with concurrent PIK3CA mutation.

Results
PIK3CA mutant contributes to malignant progression but not lung cancer initiation
To investigate the real function of PIK3CA mutant in lung tumorigenesis, we generated a genetically engineered mouse model (GEMM) by integrating the LoxP-stop-LoxP-PIK3CA E545K transgene into the Rosa26 locus of C57BL/6 mice (Fig. S1a), in which the PIK3CA mutant can be conditionally expressed via intranasal inhalation of Ad-Cre virus41. Unexpectedly, we found no tumor formation even after 40 weeks of Ad-Cre administration (Fig. S1b, c). This indicates that PIK3CA mutant expression alone seems insufficient to drive lung cancer initiation. Given that PIK3CA mutation and TP53 mutation frequently co-occur in human NSCLC (Fig. S1d), we further crossed PIK3CAE545K to Trp53flox/flox mouse to see if there is any lung tumor formation. Again, we found no tumor formation in this model (Fig. S1e, f). These data collectively point to a dispensable role of PIK3CA mutant in driving lung cancer initiation.

It’s well established that either EGFR mutation, KRAS mutation, or ALK fusion alone suffice in driving lung cancer initiation in mice19–21. EGFR mutation is the oncogenic driver most significantly associated with the concurrence of PIK3CA mutation (Fig. S2a). We then crossed PIK3CAE545K mice with EGFRL858R;Trp53flox/flox (EP) mice to generate the EGFRL858R;PIK3CAE545K;Trp53flox/flox (EPP) cohort for further study (Fig. S2b). Both in vitro and in vivo assays demonstrate that PIK3CA mutant can activate the downstream AKT signaling and promotes lung cancer progression (Fig. S2c–k). Further analyses of in situ tumors showed that PIK3CA mutant significantly promoted lung cancer malignant progression, characterized by a greater degree of cellular pleomorphism and nuclear atypia42 (Fig. S2l, m). The tumor-bearing EPP mice exhibited significantly reduced survival compared to the EP mice (Fig. S2n). These data collectively demonstrate that PIK3CA mutant mainly contributes to lung cancer malignant progression instead of driving lung cancer initiation.

PIK3CA mutant drives cachexia in EGFR mutant lung cancer
Interestingly, the tumor-bearing EPP mice displayed a rapid decrease in body weight starting from 6 weeks post Ad-Cre treatment, which was only observed in EP control mice at late stage (Figs. 1a, b and S3a). Further analyses of the body composition revealed that the EPP mice experienced a continuous loss of lean mass along with a decrease in fat mass, aligning with the clinical diagnostic criteria for cachexia as previously established43 (Fig. S3b, c). Moreover, fat imaging visually illustrated a significant decrease of fat tissue in these tumor-bearing EPP mice (Fig. 1c). Compared to WT and EP mice, the EPP mice exhibited significant decrease in muscle mass in gastrocnemius (GAST), tibialis anterior (TA), and quadriceps (QUAD), as well as a decrease in epididymal white adipose tissue (eWAT) in males and the gonadal white adipose tissue (gWAT) in females (Fig. 1d, e). H&E staining analyses revealed the reduction of muscle fiber cross-sectional area (CSA) and adipocyte size in the EPP mice (Fig. S3d–h), histologically confirming these atrophic changes. Moreover, real-time PCR analysis confirmed increased expression of the muscle atrophy-associated marker Atrogin-1 in TA from EPP mice (Fig. 1f).

Metabolic monitoring indicated that EPP mice exhibited a significant decrease in locomotor activity, reduced energy expenditure (EE), and diminished food intake (Figs. 1g, h and S3i), all of which are hallmarks of cachexia in clinical settings32. Moreover, the lower respiratory exchange ratio (RER) in EPP mice indicated a metabolic shift toward the utilization of non-glucose substrates, potentially related to their decreased food intake (Fig. 1i). These characteristics were particularly pronounced at night, coinciding with the activity rhythms of mice (Fig. S3j–l).

To explore the potential impact of tumor burdens upon cachexia, we further analyzed the EP mice and EPP mice with comparable tumor burdens at different time points post Ad-Cre treatment, e.g., the EP mice post 17 weeks of Ad-Cre treatment and the EPP mice post 10 weeks of Ad-Cre treatment (Fig. 1j). Interestingly, a significant difference of cachexia features was still detectable in these two groups, e.g., the EPP mice showed lower levels of mass of skeletal muscles and adipose tissues, and increased Atrogin-1 expression in TA tissues (Figs. 1k, l and S3m). These data suggest that PIK3CA mutant contributes to cachexia through mechanisms other than by promoting tumor burden.

Analyses of cachexia signature proposed by TRACERx study37 highlighted the PI3K-AKT signaling as one of the most significantly enriched pathways (Fig. 1m, n). Compared to patients without cachexia, the PI3K-AKT signature was similarly marginally enriched in those primary tumors from patients with cachexia (Figs. 1o and S4a). Consistently, we observed that PI3K-AKT signaling was similarly enriched in cachectic individuals across two additional datasets of lung tumor RNA sequencing (RNA-seq)44 and serum proteomics45 (Fig. S4b, c). To validate our findings across other cancer types, we further performed Gene Set Enrichment Analysis (GSEA) on a pre-clinical melanoma model46, and observed enrichment of PI3K-AKT signaling in xenografts capable of inducing cachexia (Fig. S4d). These data collectively support the significant role of PI3K-AKT signaling in driving cachexia.

Muscle wasting characteristics in EPP mice recapitulate clinical phenomena
Muscle wasting is the most critical characteristic of cancer cachexia43. We next comparatively analyzed the gene expression profiling of TA derived from WT, EP, and EPP mice (Fig. 2a). Principal component analysis (PCA) revealed separation between TA from EPP mice and those from WT and EP mice (Fig. 2b). Consistently, gene expression pattern of EPP mice differed dramatically from the others (Fig. 2c). We found an enrichment of proteasome and autophagy signaling in TA of EPP mice (Fig. 2d), which has been extensively documented in muscle atrophy47. An enrichment of features associated with muscle disorder-related diseases was also observed, suggesting that muscle atrophy in cancer cachexia may share signaling pathways with other muscle disorders.

We next analyzed two public databases (GSE13056348; GSE13352349) containing expression data from muscle tissues of cachectic patients and their respective controls. We found 42 genes upregulated in the muscles of cachectic patients, with 37 genes shared homologous counterparts in murine tissues with detectable expression (Fig. 2e). We further found that most of these genes were enriched in the muscle tissues of EPP mice (Fig. 2f), suggesting that the muscle wasting features in these mice closely resemble clinical observations.

Osi effectively inhibits lung cancer progression and alleviates cachexia in TKI-sensitive EGFRL858R GEMM with concurrent PIK3CA mutant
Previous studies indicated that PIK3CA mutant drives resistance to TKIs in EGFR-mutant lung cancer26,27. We then assessed the effect of single-agent Osi on mice carrying TKI-sensitive EGFR mutation (EGFRL858R) plus PIK3CA mutation (Fig. 3a). Osi treatment exhibited potent tumor-inhibitory effects, resulting in nearly complete tumor regression supported by dramatic decrease of tumor burdens and tumor numbers (Fig. 3b–d). In parallel, we observed increased weights in the TA, QUAD, GAST skeletal muscles, as well as in WAT tissues, indicative of the notable alleviation of cachexia (Fig. 3e, f). Moreover, Osi treatment mitigated atrophic changes in these tissues, as evidenced by an increase in muscle fiber CSA and adipocyte size (Fig. 3g–k), along with the downregulation of Atrogin-1 in TA tissues (Fig. 3l). These data suggest that, in the TKI-sensitive GEMM, tumor malignancy and cachexia mediated by the concurrent PIK3CA mutant could be effectively suppressed by Osi treatment.

Chemotherapy inhibits tumor growth but fails to alleviate cachexia in Osi-resistant EGFRTLCS GEMM with concurrent PIK3CA mutant
Once relapses from Osi therapy, chemotherapy, e.g., PEM and CDDP, is routinely used as the conventional therapeutic strategy for patients25. We next developed EGFRTLCS mice carrying L858R, T790M, and C797S mutations (Fig. S5a), which confer resistance to all three generations of TKIs used in clinical practice24. We generated the EGFRTLCS;Trp53flox/flox (TLCS-P) and EGFRTLCS;PIK3CAE545K;Trp53flox/flox (TLCS-PP) cohorts for further study, and found PI3K-AKT signaling was significantly enriched in mouse lung tumors derived from TLCS-PP mice (Fig. S5b). Compared to the TLCS-P mice, TLCS-PP mice showed an early onset of weight loss (Figs. 4a, b and S5c). Cachexia development in these mice was further characterized by continuous reduction of both lean and fat mass (Fig. 4c, d), and histological wasting of muscle and adipose tissues (Fig. S5d–g).

We next treated the TLCS-PP mice with the standard second-line chemotherapy regimen of PEM and CDDP (Fig. 4e). We found chemotherapy resulted in a significant reduction of both tumor burdens and numbers, indicative of its effectiveness in suppressing tumor progression (Fig. 4f, g). Consistent with the adverse effects of chemotherapy on cachexia50, the PEM/CDDP combination failed to exert protective effect against cachexia development, as evidenced by the reduced weights of skeletal muscle and adipose tissue and an increased trend of Atrogin-1 expression in TA tissues (Figs. 4h, i and S5h). These data suggest that the PIK3CA mutant-associated cachexia in Osi-resistant cases can’t be alleviated by chemotherapy despite notable tumor regression, emphasizing the need to explore the mechanisms underlying PIK3CA mutant in driving cachexia.

PIK3CA mutant drives cachectic inflammation through NF-κB activation
We then performed RNA-seq on mouse lung tumors derived from both Osi-sensitive and Osi-resistant GEMMs with or without PIK3CA mutant. Enrichment of epithelial-mesenchymal transition and inflammatory response pathways has been documented as a clinical feature of primary lung tumors from cachexic patients37. Interestingly, we observed the enrichment of both pathways in the EPP and TLCS-PP tumors compared to their respective controls (Figs. 5a and S5i, j). Moreover, the EPP and TLCS-PP tumors exhibited the enrichment of NF-κB signaling, which is known as the upstream regulator of multiple cachexia-associated pro-inflammatory factors51 (Figs. 5a and S5k). Upregulation of key factors in these tumors was further confirmed by RNA-seq and real-time PCR analyses (Figs. 5b–d and S5l). Elevated levels of IL-6 were also detected in the serum of EPP and TLCS-PP mice (Fig. S5m, n).

To further clarify the role of NF-κB signaling in this process, we performed immunostaining analyses on the EP cells with ectopic expression of PIK3CA mutant (Fig. S2d). We found that PIK3CA mutant notably increased the nuclear translocation of p65 (Fig. 5e). The enhancement of NF-κB activity was further confirmed by luciferase detection (Fig. S5o). Moreover, the p-p65 levels were elevated in EPP and TLCS-PP tumors (Figs. 5f and S5p). These data indicate that the PIK3CA mutant activates NF-κB pathway. Additionally, cachexia-associated factors were found upregulated following ectopic expression of PIK3CA mutant and attenuated by the treatment with NF-κB inhibitor BAY11-7082 (Fig. 5g).

We further analyzed NSCLC patient data from The Cancer Genome Atlas (TCGA) database. Increased expression levels of cachexia-associated factors were observed in samples exhibiting elevated PI3K-AKT signaling (Fig. 5h). We next consolidated these factors into a cachectic inflammation signature, and found that a higher cachectic inflammation signature was associated with shorter patient overall survival (Fig. 5i). These data indicate that PIK3CA mutant triggers NF-κB signaling, leading to the upregulation of cachexia-associated pro-inflammatory factors, which are associated with poor prognosis of lung cancer patients.

Combined aspirin treatment attenuates cachexia in EGFR mutant lung cancer with concurrent PIK3CA mutant
Aspirin, a well established non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the activity of NF-κB signaling52. To assess the effect of aspirin on inflammation inhibition, we treated EP tumor cells with aspirin in vitro and found that aspirin effectively suppressed NF-κB signaling and the pro-inflammatory factors upregulated by ectopic expression of PIK3CA mutant (Fig. S6a, b). We then treated the TLCS-PP mice with aspirin alone or combined with PEM/CDDP (Fig. 6a). Interestingly, aspirin treatment alone significantly mitigated weight loss without notable tumor regression (Figs. 6b–d and S6c). When combined aspirin with chemotherapy, we observed pronounced tumor regression, and the weight loss became similar to the vehicle group. Notably, we found that aspirin treatment led to increased weights in both skeletal muscle and adipose tissue (Fig. 6e, f), indicating effective suppression of cachexia. Aspirin also resulted in increased muscle fiber CSA and adipocyte size (Fig. S6d–g), histologically confirming the recovery from atrophic changes. Furthermore, aspirin reduced the expression of Atrogin-1 in TA tissues (Fig. 6g). These data suggest that aspirin, despite no tumor inhibition role, effectively mitigates cachexia progression of TKI-resistant lung cancer with concurrent PIK3CA mutant.

To further investigate the potential link between aspirin and the PIK3CA mutant, we established a cachexia model based on the transplantation of Lewis lung carcinoma (LLC) cell line with wild-type PIK3CA following a previously described protocol53. We found that LLC transplantation resulted in reduced tissue mass whereas aspirin administration had no significant impact upon tumor growth as well as tissue mass (Fig. S6h–j). Moreover, we observed no improvement of grip strength (Fig. S6k), no changes of Atrogin-1 level as well as NF-κB signaling in muscle tissue (Fig. S6l, m).

Besides utilizing adjunct anti-inflammatory therapy alongside the current standard chemotherapy regimen, we also evaluated the therapeutic potential of newly approved PI3K inhibitor inavolisib for cachexia management54–56. We found that inavolisib effectively suppressed AKT phosphorylation and NF-κB activation in vitro (Fig. S7a). We then treated TLCS-PP mice with inavolisib and found that inavolisib administration attenuated tumor growth and led to significant increases in both skeletal muscle and adipose tissue weights (Fig. S7b–f). We also observed a reduction of Atrogin-1 expression in TA tissues (Fig. S7g). These findings underscore the important role of PIK3CA mutant in cachexia development and suggest the PI3Kα inhibitor with improved safety might serve as a future therapeutic option for lung cancer patients with PIK3CA mutant.

Methods
Mouse models
The Trp53flox/flox mice were originally provided by Dr. Tyler Jacks (Cambridge, MA). The transgenic mouse models, including PIK3CAE545K, EGFRL858R, and EGFRTLCS mice were generated using CRISPR/Cas9 technology by commercial companies. All mice used were on a C57BL/6J background. The brief process is as follows: homologous recombination vector (donor vector) is composed of a 5′ homology arm, the indicated coding sequence, and a 3′ homology arm. Cas9 mRNA, gRNA, and the donor vector were microinjected into the fertilized eggs of C57BL/6J mice to generate the F0 generation mice.

Mice were maintained in specific pathogen-free facilities and housed in single-sex cages at 20 ± 2 °C, 40–60% humidity under a 12-h light/12-h dark photoperiod with the light on at 7:00. Mice were fed a standard chow diet ad libitum. At 6–8 weeks of age, mice were treated with Ad-Cre virus (2 × 10⁶ p.f.u.) via nasal inhalation. Approximately equal numbers of male and female mice were used. The time points post-Ad-Cre treatment are indicated for each experiment.

Osimertinib (MCE, HY-15772) was prepared in a solution containing 5% DMSO, 40% PEG300, 5% Tween-80, and 50% saline. PEM (MCE, HY-10820) or aspirin (MCE, HY-14654) was prepared in a solution containing 10% DMSO, 40% PEG300, and 50% saline. Cisplatin (MCE, HY-17394) was formulated in saline. Inavolisib (Selleck, S8668) was prepared in a solution containing 0.5% (w/v) methylcellulose and 0.2% (w/v) Tween-80. For osimertinib treatment, EGFRL858R;PIK3CAE545K;Trp53flox/flox mice were gavage daily with osimertinib (5 mg/kg/day) for 28 days, starting 6 weeks after Ad-Cre infection. Control mice were administered the vehicle solution (5% DMSO: 40% PEG300: 5% Tween-80: 50% saline). For chemotherapy and combination therapy, EGFRTLCS;PIK3CAE545K;Trp53flox/flox mice were administered PEM (50 mg/kg/day) combined with cisplatin (4 mg/kg/day) or aspirin (50 mg/kg/day) or both via intraperitoneal injection for 14 days, starting 10 weeks after Ad-Cre infection. PEM and cisplatin were administered weekly, and aspirin was given daily. Control mice were administered the vehicle solution (10% DMSO: 40% PEG300: 50% saline). For inavolisib therapy, EGFRTLCS;PIK3CAE545K;Trp53flox/flox mice were gavage daily with inavolisib (25 mg/kg/day) for 28 days, starting 8 weeks after Ad-Cre infection. Control mice were administered the vehicle solution consisting of 0.5% (w/v) methylcellulose and 0.2% (w/v) Tween-80. All mice were sacrificed for gross inspection and histopathological examination.

Tumor number, tumor burden, and tumor size were analyzed using ImageJ software. To calculate tumor burden, paraffin-embedded mouse lung tissue sections were processed with H&E staining and scanned using ZEISS Axioscan 7 to obtain lung lobe images. Tumor and lung areas for each lobe were delineated and quantified using ImageJ software. The tumor burden was calculated as the ratio of tumor areas to lung area.

Cachexia model based on LLC transplantation
Male C57BL/6 mice (8–10 weeks old) were received 5 × 10⁶ LLC cells in 100 μL PBS. Aspirin (50 mg/kg/day) was administered via intraperitoneal injection daily starting 7 days post-tumor transplantation. Control mice received the vehicle solution (10% DMSO, 40% PEG300, 50% saline).

Assessment of disease progression and humane endpoints
For the subcutaneous tumor model, disease progression in mice was monitored regularly by measuring tumor size with calipers and recording body weight. For in situ tumor model, disease progression was monitored by recording body weight and observing clinical signs, including activity level, posture, grooming, and general behavior. Experimental endpoints were generally defined based on pre-determined study duration or attainment of humane endpoints.

Humane endpoints were defined as a 20% reduction in body weight from tumor induction, cessation of eating and drinking, severe lethargy or moribund state, a maximal tumor diameter of 2 cm, tumor ulceration exceeding 1 cm, severe trauma leading to secondary infection, or significant organ dysfunction. Body weight changes in Figs. 1b and 4b were shown relative to tumor induction, and body weight changes in Fig. 6b were relative to treatment initiation.

Plasmid
The pCDH-PIK3CAE545K plasmid was generated by standard PCR and homologous recombination-based cloning. The psPAX2 plasmid (#12260) and pMD2.G plasmid (#12259) were purchased from Addgene. The NF-κB-Luc plasmid and Renilla plasmid were laboratory-preserved plasmids.

Cell culture and lentivirus infection
293T and EP cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S). Cells were used for experiments within 10 to 20 passages from thawing. All cell lines were routinely tested for mycoplasma. For the establishment of stable overexpression cell line, 293T cells were transfected with a 4:3:2 ratio of pCDH-PIK3CA E545K plasmid, psPAX2 plasmid, and pMD2.G plasmid. Lentiviral particles generated were then transduced into EP cells, followed by puromycin selection (3 μg/mL; Sigma-Aldrich) initiated 48 h post-transfection and continued for an additional 2 days. 293T cell line was purchased from ATCC (CRL-3216), EP cell line was manually established from lung tumors of female EGFRL858R;PIK3CAE545K;Trp53flox/flox mice following Ad-Cre treatment.

Immunoblotting
Whole-cell lysates of cell lines or murine tumors were generated using lysis buffer (10% SDS, 1 mM DTT, and glycerin) and incubated at 100 °C for 10 min. Equal amounts of total protein were separated by SDS-PAGE and transferred onto PVDF membranes. Proteins samples were probed with specific antibodies against p110α (CST, 4249, 1:1000), AKT (CST, 9272, 1:1000), p-AKT (CST, 4070, 1:1000), S6 (CST, 2217, 1:1000), p-S6 (CST, 2215, 1:1000), p65 (CST, 8242, 1:1000), p-p65 (CST, 3033, 1:1000), and β-actin (Abclonal, AC026, 1:200000). Protein expression was assessed by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and detected on SAGECREATION (Sage Creation Science Co.).

For quantification, band intensities were quantified using ImageJ. Background was first subtracted, and rectangular regions were then drawn around each band to measure integrated densities. The signal of each target protein was normalized to its corresponding loading control (actin). Ratios were further normalized to the value of the first lane. For Figs. 5f and S5p–S7a actin was measured on the same blot as the target proteins. For Figs. S2d and S2h, actin was measured on the same samples but on different blots. All samples within each subpanel originated from the same experiment, and the corresponding gels/blots were processed in parallel.

RNA Isolation, reverse transcription, and real-time PCR
Total RNA from cultured cells or tissue samples was extracted using TRIzol (Invitrogen), and complementary DNA was synthesized from 1 μg of RNA using the PrimeScript RT Reagent Kit (TaKaRa). Real-time PCR was performed on a LightCycler® 96 System (Roche) using SYBR Green I Master (Roche). β-actin was utilized as the internal control. The following primers were used: Il1b F: 5′-GCAACTGTTCCTGAACTCAAC-3′, R: 5′-ATCTTTTGGGGTCCGTCAACT-3′. Il6 F: 5′-TCTGCAAGAGACTTCCATCCAGTTGC-3′, R: 5′-AGCCTCCGACTTGTGAAGTGGT-3′. Il11 F: 5′-CTGACGGAGATCACAGTCTGGA-3′, R: 5′-GGACATCAAGTCTACTCGAAGCC-3′. Lif F: 5′-AAAAGCTATGTGCGCCTAACA-3′, R: 5′-GTATGCGACCATCCGATACAG-3′. Osm F: 5′-ACGGTCCACTACAACACCAG-3′, R: 5′-CCATCGTCCCATTCCCTGAAG-3′. Ptgs2 F: 5′-TTCAACACACTCTATCACTGGC-3′, R: 5′-AGAAGCGTTTGCGGTACTCAT-3′. Tnf F: 5′-CTGAACTTCGGGGTGATCGG-3′, R: 5′-GGCTTGTCACTCGAATTTTGAGA-3′. Atrogin-1 F: 5′-CTTCTCGACTGCCATCCTGGAT-3′, R: 5′-TCTTTTGGGCGATGCCACTCAG-3′. Nfkb1 F: 5′-CTGAACAATGCCTTCCGGCT-3′, R: 5′-TGGTACCCCCAGAGACCTCAT-3′. Rela F: 5′-GATCGCCACCGGATTGAAGA-3′, R: 5′-GGGGTTCAGTTGGTCCATTG-3′. Actb F: 5′-GGCTGTATTCCCCTCCATCG-3′, R: 5′-CCAGTTGGTAACAATGCCATGT-3′. Oligonucleotides were obtained by chemical synthesis.

Soft agar colony formation assay
For soft agar assay, a bottom layer of 1% agar with complete medium was first solidified. An upper layer was then added, containing 500 cells suspended in a 0.4% agar medium mixture in 6-well plates. After 2–3 weeks of incubation, cells were stained with 0.005% crystal violet, and the number of colonies was counted.

Immunofluorescence analyses
Cells grown on glass coverslips were washed with cold PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min. After blocking with 4% bovine serum albumin in PBS for 1 h, cells were probed with p65 (CST, 8242, 1:500) overnight at 4 °C. After washing three times with PBST, secondary antibodies were added and incubated for 1 h at room temperature. After washed with PBST, the coverslips were mounted onto glass slides using fluorescent mounting medium. Confocal images were captured using a Leica TCS SP8 system with a HC PL APO CS2 63×/1.40 oil objective.

Luciferase reporter gene assay
293T cells were seeded in 12-well plate at 3 × 10⁵ cells/well. 24 h after seeding, cells were transiently transfected with 1 μg of either PIK3CA E545K plasmid or the control vector, along with 1 μg NF-κB-Luc plasmid and 500 ng Renilla plasmid. 48 h post-transfection, cells were harvested, and the Dual-Luciferase Reporter Assay System (Promega) was used for detection.

Tissue collection
Mice were euthanized, and their body weight was recorded. The lungs with tumors were excised and fixed in 4% formalin; a portion of the tumors was snap-frozen for further analysis. GAST, TA, QUAD, eWAT, and gWAT were dissected and weighed. The white adipose tissues were fixed in 4% formalin for histological examination. One part of the skeletal muscle was snap-frozen for RNA-seq, while the remaining portion was embedded in optimal cutting temperature compound for rapid freezing and subsequent histological examination.

Body composition analysis and fat imaging
Mice were weighed and body composition was measured using Bruker’s minispec LF50 Body Composition Analyzer. The instrument provides measurements of three related components: fat, free body fluids, and lean tissue mass. The imaging of mouse adipose tissue was performed using the NM42-060H-I (Niumag) small animal magnetic resonance imaging scanner.

Metabolic cage
Mice were individually housed in CLAMS metabolic cages (Columbus Instruments) for a duration of 3 days. Oxygen consumption (VO2) and carbon dioxide expiration (VCO2) were measured for 1 min with 14 min intervals at a flow rate set at 0.72 liter per minute. Respiratory exchange ratio (RER) was calculated as the ratio of VCO2 to VO2. Simultaneously, locomotor activity and energy expenditure were recorded using the built-in detection system, while food intake was manually measured at the same designated time each day.

Grip strength measurement
Each mouse was allowed to grasp a bar attached to a force transducer and was steadily pulled horizontally by the tail away from the bar using a KW-ZL-2 grip strength meter. The maximum force exerted before releasing the bar was recorded. Each mouse was tested five times, and the average of the remaining three measurements after exclusion of the highest and lowest values was calculated as the grip strength.

RNA-seq analyses
Raw fastq data from RNA-seq were processed with Trimmomatic79 (v0.39) for adapter trimming and low-quality read filtering. The processed data were then aligned to the mm10 reference genome using STAR80 (v2.5.2b). Genes with zero expression in more than 70% of the samples were filtered out. FPKM normalization and log2 transformation were applied to the raw count data, followed by differential expression analysis using limma81 to identify differentially expressed genes (DEGs) between conditions. Pathway enrichment analysis was conducted using the Enrichr82 method based on the resulting DEGs. In addition, the GSEA analysis was performed using the R package clusterProfiler83 with 1000 permutations and gene_set permutation type (default setting). Genes were ranked based on their fold-change values.

Public datasets analyses
Public datasets were obtained from multiple sources. Gene expression profiles were downloaded from the Gene Expression Omnibus database, including GSE13056348 and GSE13352349, which contain expression data from muscle tissues of cachectic patients and their respective controls. Differential expression analysis was performed by R package limma81. Additional transcriptomic data and gene lists—including the cachexia signature proposed by the TRACEx study37, tumor RNA-seq37,44 and serum proteomics45 data from lung cancer patients, as well as tumor RNA-seq data from melanoma xenografts46—were extracted from supplementary tables of published studies. Pathway enrichment analysis was performed using the Enrichr82 web tool based on downloaded gene lists. GSEA was conducted using the R package clusterProfiler83 with 1000 permutations and gene_set permutation type (default setting). Genes were ranked based on their fold-change values. Normalized enrichment scores (NES) and false discovery rate (FDR) q-values were calculated.

Statistics and reproducibility
GraphPad Prism 9 was used for statistical analyses. Data were presented as mean ± standard deviation (SD) unless otherwise stated. Statistical significance for two experimental samples was determined using two-tailed unpaired Student’s t test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was applied. Survival analysis was performed using the log-rank (Mantel–Cox) test. P value < 0.05 was considered statistically significant. The number of biological replicates (n) is specified in each figure legend and p-values are indicated in the individual figures.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Ethical regulations
The research performed in the present study complies with all ethical regulations. All mice were kept in the specific pathogen-free environment at Shanghai Institute of Biochemistry and Cell Biology and treated in strict accordance with protocols (SIBCB-2101008) approved by the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Yue et al investigated how concomitant PIK3CA mutation in EGFR-mutant lung cancer influences tumor progression and associated cachexia. Using genetically engineered mouse models and based on clinical evidence, the authors found that PIK3CA mutation does not result in the initiation of lung cancer but enables disease progression and cachexia through NF-κB-dependent pro-inflammatory signaling. Although treatment of osimertinib (Osi) suppressed tumor progression and cachexia in TKI-sensitive models, chemotherapy failed to mitigate cachexia in Osi-resistant models even though tumor burden was reduced. Findings also indicated that aspirin suppresses PIK3CA-driven cachexia and argued for the potential use of aspirin in cancer patients with PIK3CA mutant lung tumors in managing inflammation and cachexia. Conclusions of this manuscript are generally interesting and supported by the data, however, the last part on the use of aspirin to block inflammation and cachexia requires additional work. The manuscript can be improved if the following comments are considered.\n\nMajor comments\n1. It is not clear why the authors presented body weight and weight of fat and muscle tissues in relative values. It will be more appropriate if actual value are provided. This applies to Figures 1, 3, 4 ,6 and S3.\n2. The authors should further validate the cachectic state in their mutant mice by measuring the expression of muscle atrophy-related genes in muscle tissues. This applies to Figures 1, 3, 4 and 6.\n3. Activation of the NF-κB pathway should be documented in tumor samples. Protein or mRNA expression levels of the elements of this pathway should be tested in Figures 5 and 6.\n4. The cachexia-inducing effect of PIK3CA mutation in TLCS mice appears to be less pronounced (particularly the lean mass). To confirm that this is not the case, the authors should document the extent of tissue wasting in Figure 4A-D.\n5. The findings presented in Figure 6 and S6 are very interesting. However, important controls are missing. The aspirin group has larger body weight and muscle/adipose tissue weight than the vehicle group. To understand the degree of this rescue effect, a WT or a TLCS-P control group should be included. Does the aspirin’s effect depend on PIK3CA mutation? Aspirin can potentially be tested in other cachexia models such as LLC, B16 or C26. It is also important to demonstrate aspirin’s effects on muscle tissue gene expression (atrophy markers and NF-κB pathway elements) and muscle strength (grip strength or physical activity).\n\nMinor Comments\n6. Please explain the method used to calculate tumor burden.\n7. The GSEA dot plots have inconsistent representations: in Figure 1N, dot size represents count; in Figure 2D, it represents the odds ratio; and in Figure 5A, it represents NES. For the GSEA plots, the FDR value should be reported instead of the p-value (e.g., Figure 1O).\n8. The legend of Figure 2D (line 629) states that the analysis is \"based on significantly differentially expressed genes.\" However, gene set enrichment analysis is traditionally performed using all detected genes.\n9. In Figure 2F, clarify whether the color scale represents only expression values or if it is a z-score.\n10. Please re-visit Figure 3I. The y-axis is labeled as \"% frequency,\" but this is incorrect.\n11. Color scales are missing in Figures 5A, 5B, 5C, 5G and S6A.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "NCOMMS2501615T\nThe study of Yue et al. describes new GEMMs of NSCLC, where a PIK3CAE545K transgene with concurrent mutation(s) in EGFR and loss of Trp53 are combined. The presence of this PIK3CA mutation leads to accelerated cancer progression and onset of a cachexic phenotype observed 6-9 weeks after an intranasal Ad-Cre delivery. Cachexia could be effectively prevented by tumor suppression achieved with osimertinib, a clinical EGFR inhibitor, and was ameliorated by aspirin, a very pleiotropic nonsteroidal anti-inflammatory drug. Chemotherapy with pemetrexed/cisplatin only marginally reduced tumor burden and appeared to aggravate cachexia. The results include in-depth metabolic, transcriptomic, and histological characterization of the generated models. The results convincingly show a cachexic phenotype develops in these mouse models.\n\nGeneral major points:\n1)A causative role of PIK3CA mutations in cachexia has, however, not been completely demonstrated. In vivo experiments using PI3Kα-selective inhibitors are not included in the study. The authors should investigate whether PI3Kα inhibitors after Ad-Cre (for example using GDC-0077 or STX-478 or covalent PI3Kα inhibitors) prevent or delay cachexia in their mouse models with or without altering tumor burden. An effect of a pharmacological inhibition of the PI3Kα pathway would strengthen the conclusions of the study, and further support an important role of mutated PI3Kα in the promotion of cachexia.\nAlternatively, the exploration of cachexic phenotypes could be investigated in the context of PIK3CAH1047R mutations or loss of Pten. This would, however, delay dissemination of the current data, and provide less hard evidence for a connection of cachexia and PI3K overactivation.\n2)The role of inflammation in cachexia is well known. There is also a considerable body of clinical evidence suggestive a benefit of systemic anti-inflammatory therapy in patients with cancer.\nThe role of PIK3CA mutations in mediating cancer progression is also well described. The link between PIK3CA mutations and cachexia is a novel and interesting concept. In the absence of data showing that targeting of PI3Kα modulates cachexia, this link remains somewhat ambiguous. The manuscript is inspiring and points to a novel link of PI3Kα in modulating cachexia, although the molecular and cellular processes are somewhat obscured. The authors should thus put the results in a wider context and tune down some of their statements.\n\nSpecific major points:\nGenerally, the quality of the data is high and the figures are presented in a clear and logical manner. In addition to the in vivo experiments mentioned above, addressing the following comments would improve reproducibility and clarity.\n3)Introduction: The introduction would benefit from more in-depth information on PI3Kα’s biological role in tumorigenesis (NSCLC), beyond genetic status and prevalence. Here effects on tumor metabolism, microenvironment, immune cell infiltration, inflammation should be introduced and put into context.\n4)Introduction, lines 36-37: “PIK3CA mutant transgene alone is sufficient to drive lung cancer initiation in mice (ref. 5)” There are additional studies using lung cancer GEMMs that show the opposite (10.1158/0008-5472.CAN-15-1249, 10.1158/0008-5472.CAN-13-0681). The authors should adapt this statement and provide additional references in the introduction instead of discussion.\n5)Introduction, lines 41-42: “…80% of lung cancer harboring PIK3CA mutations tends to have EGFR mutations in East Asian cohort (ref. 7).”\nThis statement does not precisely reflect the findings in ref. 7. This study found that PIK3CA mutations were found in ~3% (34/1117) of NSCLC patients, of which 61.8% (21/34) harbored concurrent oncogenic mutations, and for 81% (17/21) of those, the driver mutation was in EGFR. Overall, 50% (17/34) PIK3CA-mutated tumors had EGFR driver mutations. The statement should also refer to NSCLC instead of “lung cancer” in East Asian cohorts. The introduction currently implies that PI3Kα mutations are very frequent in NSCLC; and later (line 89) that they are highly associated with EGFR mutations. This needs some adaptation (see also next point).\n6)Introduction, lines 55-56: “This is further supported by the observation of worse overall survival (OS) of those EGFR-mutant patients with concurrent PIK3CA mutation in their tumors (ref 21, 22).”\nThis should be put in the context of low prevalence of concurrent EGFR/PIK3CA mutations and therefore a low statistical power. The studies in refs. 21 and 22 include only a total of 23 and 37 patients with PIK3CA mutations, respectively. In addition, comparisons of PIK3CA-mutant versus PIK3CA-wildtype (without further stratification based on co-occurring oncogenic driver mutations) suggest that the presence of PIK3CA mutations leads to poorer outcomes, independently of other oncogenic drivers.\n7)Results / Fig 1: do the authors have data on a follow-up of EP mice beyond the 10 weeks as shown in Fig 1B? Do EP mice develop cachexia at a later point in time? The same question would be of interest for TLCS-P mice.\n8)Results / Fig 1: The authors use a cachexia gene list proposed in the TRACERx study. This list is highly enriched for genes in the PI3K-AKT signaling pathway (Fig 1M-N). The enrichment of the PI3K-AKT signature in primary tumors from cachectic individuals is, however, not substantial (Fig 1O, NES=1.303) and is not highlighted in the global GSEA analysis of the original study. The authors should provide a detailed methodology for their GSEA analysis, including other gene sets, the number of permutations and the type and report of FDR values. The same applies for the analyses in Figs S4A, S4B.\nWithin the same paragraph, the authors state that “these data collectively support the significant role of PIK3CA mutant in driving cachexia.”. These data, however, do not directly address PIK3CA mutation status, but PI3K-AKT pathway activity. The text referring to this data should be adapted and refined.\n9)Results / Fig 2: The authors should state the time post Ad-Cre delivery for the mice used in the RNA-seq analysis of TA muscles (Fig. 2A-C). Were these mice matched by age or tumor burden?\n10)Results / Fig 5/S6: Color scales in heatmaps from Figs. 5B, 5C, 5G, S6A are missing.\n11)Results /Fig S5: The authors should provide a more detailed methodology for their GSEA analysis in Figs. S5C S5D, S5E, including number of permutations and type, and report the FDR values. Were any other gene sets tested at the same time?\n12)Results / Fig 5: The authors should state the time post Ad-Cre administration for the mice used in the RNA-seq analysis of tumors (Fig. 5A-C). Were these mice matched by age or tumor burden? Could the authors comment on the reason why the hallmark PI3K-Akt-mTOR signaling gene set is not significantly enriched in EPP vs EP tumors? Why was a similar “global” hallmarks GSEA not performed on TLCS-P and TLCS-PP tumor tissue?\n13)Results: The data on NF-kB activity and p65 nuclear localization in response to ectopic expression of PIK3CAE545K are interesting. Is the same true for tumors in vivo? The connection of PI3K activation and NF-kB activity is controversial in the literature. Could the authors refer to some of the discrepancies?\n14)Comment 12 (results): For the analysis of TCGA data in Fig. 5: how were the PI3K-AKT high and low groups defined?\n15)Discussion, lines 266-268: “However, once _patients_ relapse from TKI therapy and the tumors develop TKI resistance, the impact of PIK3CA mutant begins to unleash, with cachexia driven by PIK3CA mutant gradually worsening and ultimately leading to poor patient prognosis.” This statement is highly hypothetical as there is no clinical data to be referred to. This should be rephrased to reflect this. There is also no in vivo data to support that PIK3CA mutations become important only after TKI resistance. The data show that all mice harboring PIK3CAE545K mutations develop cachexia faster, independently of Osimertinib sensitivity.\n16)Discussion, lines 296-299: “To validate our findings across other cancer types, we further perform Gene Set Enrichment Analysis (GSEA) on a recently published pre-clinical melanoma model, and observed enrichment of PI3K-AKT signaling in xenografts capable of inducing cachexia (data not shown).” This belongs to the results section. The data should be shown.\n17)Materials and methods: A section describing in detail the (re-)analyses performed on publicly available datasets should be included.\n18)Results / minor point: Could the authors check whether the error bars in Figs S2F and S2G represent SEM instead of SD (as stated in the figure legend)?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response letter\nReviewer #1 (Remarks to the Author); expert in lung cancer and cancer-associated cachexia:\nIn this manuscript, Yue et al investigated how concomitant PIK3CA mutation in EGFR-mutant lung\ncancer influences tumor progression and associated cachexia. Using genetically engineered mouse\nmodels and based on clinical evidence, the authors found that PIK3CA mutation does not result in the\ninitiation of lung cancer but enables disease progression and cachexia through NF-κB-dependent pro-\ninflammatory signaling. Although treatment of osimertinib (Osi) suppressed tumor progression and\ncachexia in TKI-sensitive models, chemotherapy failed to mitigate cachexia in Osi-resistant models\neven though tumor burden was reduced. Findings also indicated that aspirin suppresses PIK3CA-\ndriven cachexia and argued for the potential use of aspirin in cancer patients with PIK3CA mutant lung\ntumors in managing inflammation and cachexia. Conclusions of this manuscript are generally\ninteresting and supported by the data, however, the last part on the use of aspirin to block inflammation\nand cachexia requires additional work. The manuscript can be improved if the following comments are\nconsidered.\nWe sincerely appreciate your positive feedback and insightful comments, which have substantially\nimproved our manuscript. We have now included the WT control, evaluated atrophy marker expression,\nand employed the LLC-induced cachexia model to further characterize the role of aspirin in cachexia\nmanagement. Additionally, we evaluated NF-κB activation in vivo and characterized the degree of\ncachexia at the histological level. We also conducted further bioinformatic analyses and revised the\nfigure presentation along with the corresponding text. Below are our detailed point-by-point responses.\nMajor comments\n1) It is not clear why the authors presented body weight and weight of fat and muscle tissues in relative\nvalues. It will be more appropriate if actual value are provided. This applies to Figures 1, 3, 4 ,6 and\nS3.\nResponse: Thank you for this valuable suggestion. To ensure balanced representation and minimize\nany potential sex-based bias, we included approximately equal numbers of male and female mice in our\nstudy. Given the substantial influence of sex on body composition, direct cross-sex comparisons would\nnot be appropriate. Thus, we followed the method from a previous cachexia study (Talbert EE et al.,\nCell Rep, 2019, PMID: 31390573) and presented the data in relative values. We recognize that this\napproach may not suit all readers and apologize for any inconvenience. If necessary, we would love to\nprovide a supplementary table containing all the absolute values corresponding to each figure.\n2) The authors should further validate the cachectic state in their mutant mice by measuring the\nexpression of muscle atrophy-related genes in muscle tissues. This applies to Figures 1, 3, 4 and 6.\nResponse: Following the suggestion, we have examined Atrogin-1, one of the most commonly used\nmarkers of muscle atrophy (Gomes MD et al., Proc Natl Acad Sci U S A, 2001, PMID: 11717410;\nSandri M et al., Cell, 2004, PMID: 15109499., Bilgic SN et al., Nature, 2023, PMID: 37165186) in TA\ntissues throughout our study. We have examined its expression and added these new data into the\nrevised manuscript (New Fig. 1F, 3L, S3L, S5G, 6G, S6L).\n3) Activation of the NF-κB pathway should be documented in tumor samples. Protein or mRNA\n or mRNA\nexpression levels of the elements of this pathway should be tested in Figures 5 and 6.\nResponse: Following your suggestion, we have examined NF-κB pathway activation by\nimmunoblotting of in situ tumors from GEMMs used in our study. Compared to EP and TLCS-P tumors,\np-p65 levels were elevated in EPP (New Fig. 5F) and TLCS-PP tumors (New Fig. S5M) respectively,\nindicating that PIK3CA mutant leads to NF-κB pathway activation in vivo.\nFigure 5. F, Immunoblotting analyses of indicated proteins in in situ tumors from EGFRL858R; Trp53flox/flox (EP) and\nEGFRL858R; PIK3CAE545K; Trp53flox/flox (EPP) mice.\nFigure S5. M, Immunoblotting analyses of indicated proteins in in situ tumors from EGFRTLCS; Trp53flox/flox (TLCS-P) and\nEGFRTLCS; PIK3CAE545K; Trp53flox/flox (TLCS-PP) mice.\nWe sincerely thank you for your thoughtful suggestion regarding Figure 6. However, following\ntreatment, the residual lesions were relatively small, which limited our ability to obtain sufficient tissue\nfor the further analysis. We truly appreciate your understanding and hope this limitation is acceptable\ngiven the current context.\n4) The cachexia-inducing effect of PIK3CA mutation in TLCS mice appears to be less pronounced\n(particularly the lean mass). To confirm that this is not the case, the authors should document the extent\nof tissue wasting in Figure 4A-D.\nResponse: By week 12 post intranasal administration of Ad-Cre, we observed significant differences\nin body weight, lean mass, and fat mass between TLCS-PP mice and both WT and TLCS-P groups.\nTherefore, we did not extend the study beyond 12 weeks. Considering that cachexia typically worsens\nas tumor progression advances, we believe the differences would be more pronounced at later time\npoints. To further assess the extent of tissue wasting in Fig. 4A-D, we performed histological analyses\nof muscle (New Fig. S5C-D) and adipose tissues (New Fig. S5E-F), which revealed significant wasting\nin TLCS-PP mice compared to controls.\nFigure S5. C-D, Representative micrographs (C), and average fiber cross-sectional area (CSA) (D) of gastrocnemius tissue in\nWT mice, TLCS-P mice, and TLCS-PP mice following 12 weeks post Ad-Cre intranasal delivery. E-F, Representative\nmicrographs (E), and average size of adipocytes (F) of white adipose tissue in WT mice, TLCS-P mice, and TLCS-PP mice\nfollowing 12 weeks post Ad-Cre intranasal delivery.\n5) The findings presented in Figure 6 and S6 are very interesting. However, important controls are\nmissing. The aspirin group has larger body weight and muscle/adipose tissue weight than the vehicle\ngroup. To understand the degree of this rescue effect, a WT or a TLCS-P control group should be\nincluded. Does the aspirin’s effect depend on PIK3CA mutation? Aspirin can potentially be tested in\nother cachexia models such as LLC, B16 or C26. It is also important to demonstrate aspirin’s effects\non muscle tissue gene expression (atrophy markers and NF-κB pathway elements) and muscle strength\n(grip strength or physical activity).\nResponse: Following your valuable suggestion, we have now included WT mice matched for age and\ngenetic background to better evaluate the rescue effect of aspirin. Our results show that aspirin partially\nalleviates cachexia symptoms in tumor-bearing mice. However, it does not fully restore tissue weights\n(New Fig. 6E-F) or histological features (New Fig. S6C-F) to the levels seen in WT mice. These\nfindings indicate that while aspirin has a beneficial effect, it does not completely reverse cachexia\nprogression.\nFigure 6. E-F, Assessed weights of the gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD) (E), and white\nadipose tissue (WAT) (F) in WT, age-matched mice from the same strain and TLCS-PP mice following intraperitoneal\nadministration of vehicle, aspirin, PEM/CDDP (Chemo), or combined treatment (Combo). Data were normalized based on the\naverage values of corresponding tissue in mice receiving vehicle with the same sex.\nFigure S6. C-D, Representative micrographs (C), and average fiber cross-sectional area (CSA) (D) of gastrocnemius tissue in\nWT, age-matched mice from the same strain and TLCS-PP mice following intraperitoneal administration of vehicle, aspirin,\nPEM/CDDP (Chemo), or combined treatment (Combo). E-F, Representative micrographs (G), and average size of adipocytes\n(H) of white adipose tissue in WT, age-matched mice from the same strain and TLCS-PP mice following intraperitoneal\nadministration of vehicle, aspirin, PEM/CDDP (Chemo), or combined treatment (Combo).\nTo further investigate the relationship between aspirin’s effect and the PIK3CA mutant, we employed\nthe Lewis lung carcinoma (LLC)-induced cachexia model following a previously described protocol\n(Bilgic SN et al., Nature, 2023, PMID: 37165186). Mice with comparable body weights were randomly\nassigned to three groups (Reviewer only Fig. 1A). We found that LLC transplantation resulted in\nreduced tissue mass. Aspirin administration led to a modest but not significant increase in tissue mass\n(Reviewer only Fig. 1B-C), and no affect upon tumor growth (Reviewer only Fig. 1D). Furthermore,\nno improvement was observed in grip strength (Reviewer only Fig. 1E), and expression levels of\nAtrogin‑1 remained unchanged (Reviewer only Fig. 1F), suggesting that aspirin’s efficacy depends on\nPIK3CA mutant. Additionally, aspirin had no effect on NF-κB signaling in muscle tissue (Reviewer\nonly Fig. 1G), supporting that its inhibitory effect relies on NF-κB activation within the tumor.\nReviewer only Figure 1. A, Assessed body weight of mice in the indicated groups before tumor transplantation. B-C, Assessed\nweights of the gastrocnemius (GAST), tibialis anterior (TA), and quadriceps (QUAD) muscles (B), and white adipose tissue\n(WAT) (C) in non–tumor-bearing mice, and LLC-transplanted mice treated with vehicle or 50 mg/kg aspirin. D, Tumor weight\nof LLC-transplanted mice receiving vehicle or 50 mg/kg aspirin. E, Grip strength of non–tumor-bearing mice, and LLC-\ntransplanted mice treated with vehicle or 50 mg/kg aspirin. F-G, Real-time PCR detection of mRNA levels for indicated gene\nin tibialis anterior tissues from non–tumor-bearing mice, and LLC-transplanted mice treated with vehicle or 50 mg/kg aspirin.\nMinor Comments\n6) Please explain the method used to calculate tumor burden.\nResponse: To calculate tumor burden, paraffin-embedded mouse lung tissue sections were processed\nwith H&E staining and scanned using ZEISS Axioscan 7 to obtain lung lobe images. Tumor and lung\nareas for each lobe were delineated and quantified using ImageJ software. The tumor burden was\ncalculated as the ratio of tumor areas to lung area. We have now included this information in the revised\nmanuscript (Lines 369–373).\n7) The GSEA dot plots have inconsistent representations: in Figure 1N, dot size represents count; in\nFigure 2D, it represents the odds ratio; and in Figure 5A, it represents NES. For the GSEA plots,\nthe FDR value should be reported instead of the p-value (e.g., Figure 1O).\nResponse: Sorry for the confusion. We employed two approaches: EnrichR analysis, which uses the\nhypergeometric distribution and predefined gene sets (Fig. 1N, 2D, S4B); and GSEA, which evaluates\nthe entire ranked gene list (Fig. 1O, S4A, 5A, S5B-E). For the enrichR-based analyses (New Fig. 1N,\n2D, S4B), we have now standardized all the representation where dot size consistently represents gene\ncount and color indicates FDR values. For the GSEA-based analysis (New Fig. 5A), we retain the NES\nfor dot size and have revised the plot to display FDR values. Additionally, we have added FDR values\nto the GSEA plots in New Fig. 1O, S4A, S4C, S5B, and S5H-J.\n8) The legend of Figure 2D (line 629) states that the analysis is \"based on significantly differentially\nexpressed genes.\" However, gene set enrichment analysis is traditionally performed using all detected\ngenes.\nResponse: Thank you for pointing out this issue. We performed the pathway enrichment analysis in\nFig. 2D using EnrichR, which is based on the hypergeometric distribution and requires a predefined set\nof significantly differentially expressed genes. We have revised the figure legend accordingly (Lines\n735–739). In parallel, we performed GSEA using the full ranked list of all detected genes and found\nconsistent enrichment of key pathways such as Proteasome and Autophagy (Reviewer only Fig. 2).\nReviewer only Figure 2, Dot plot shows KEGG pathways enriched in tibialis anterior tissues of EPP mice compared to EP\nmice (Top 20 pathways shown). The dot size represents the normalized enrichment score (NES). The dot color indicates\nenrichment significance.\n9) In Figure 2F, clarify whether the color scale represents only expression values or if it is a z-score.\nResponse: We have now included the color scales which represents Z-score values (New Fig. 2F).\n10) Please re-visit Figure 3I. The y-axis is labeled as \"% frequency,\" but this is incorrect.\nResponse: Corrected (New Fig. 3I).\n11) Color scales are missing in Figures 5A, 5B, 5C, 5G and S6A.\nResponse: We have added the missing color scales to New Fig. 5A, 5B, 5C, 5G, S6A.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author); expert in PI3K:\nThe study of Yue et al. describes new GEMMs of NSCLC, where a PIK3CAE545K transgene with\nconcurrent mutation(s) in EGFR and loss of Trp53 are combined. The presence of this PIK3CA\nmutation leads to accelerated cancer progression and onset of a cachexic phenotype observed 6-9\nweeks after an intranasal Ad-Cre delivery. Cachexia could be effectively prevented by tumor\nsuppression achieved with osimertinib, a clinical EGFR inhibitor, and was ameliorated by aspirin, a\nvery pleiotropic nonsteroidal anti-inflammatory drug. Chemotherapy with pemetrexed/cisplatin only\nmarginally reduced tumor burden and appeared to aggravate cachexia. The results include in-depth\nmetabolic, transcriptomic, and histological characterization of the generated models. The results\nconvincingly show a cachexic phenotype develops in these mouse models.\nWe sincerely appreciate your positive feedback and thoughtful comments on our work.\nGeneral major points:\n1) A causative role of PIK3CA mutations in cachexia has, however, not been completely demonstrated.\nIn vivo experiments using PI3Kα-selective inhibitors are not included in the study. The authors should\ninvestigate whether PI3Kα inhibitors after Ad-Cre (for example using GDC-0077 or STX-478 or\ncovalent PI3Kα inhibitors) prevent or delay cachexia in their mouse models with or without altering\ntumor burden. An effect of a pharmacological inhibition of the PI3Kα pathway would strengthen the\nconclusions of the study, and further support an important role of mutated PI3Kα in the promotion of\ncachexia.\nAlternatively, the exploration of cachexic phenotypes could be investigated in the context of\nPIK3CAH1047R mutations or loss of Pten. This would, however, delay dissemination of the current\ndata, and provide less hard evidence for a connection of cachexia and PI3K overactivation.\nResponse: As suggested, we have now treated TLCS-PP mice with Inavolisib (GDC-0077) following\na previously established regimen (Song KW et al., Cancer Discov, 2022, PMID: 34544753; Gough SM\net al., Clin Cancer Res, 2024, PMID: 38819400) (New Fig. S6G). We found that inavolisib treatment\nfor 28 days significantly attenuated tumor growth and resulted in significant increase of skeletal muscle\nand adipose tissue weights (New Fig. S6H-K) whereas reduced the expression of the muscle-atrophy\nmarker, Atrogin-1 (New Fig. S6L). These findings provide further evidence for the critical contribution\nof the PI3Kα pathway to cachexia development.\nFigure S6. G, Schematic diagram of inavolisib treatment strategy. H-I, Statistical analyses of tumor burden (H) and tumor\nnumber (I) in TLCS-PP mice following gavage of vehicle or inavolisib. J-K, Assessed weights of the gastrocnemius (GAST),\ntibialis anterior (TA), and quadriceps (QUAD) muscles (J), and white adipose tissue (WAT) (K) in TLCS-PP mice following\ngavage of vehicle or inavolisib. L, Real-time PCR detection of mRNA levels for indicated gene in tibialis anterior tissues from\nTLCS-PP mice following gavage of vehicle or inavolisib.\nWe appreciate the your suggestion to further explore cachectic phenotypes in models harboring\nPIK3CAH1047R mutation or Pten alteration. We fully agree that such models could offer valuable\ninsights into the role of the PI3Kα pathway in cachexia. However, due to time limitation, generating\nand characterizing these models is not currently feasible. To address this point, we incorporated\npharmacological inhibition experiments as described above.\n2) The role of inflammation in cachexia is well known. There is also a considerable body of clinical\nevidence suggestive a benefit of systemic anti-inflammatory therapy in patients with cancer.\nThe role of PIK3CA mutations in mediating cancer progression is also well described. The link between\nPIK3CA mutations and cachexia is a novel and interesting concept. In the absence of data showing that\ntargeting of PI3Kα modulates cachexia, this link remains somewhat ambiguous. The manuscript is\ninspiring and points to a novel link of PI3Kα in modulating cachexia, although the molecular and\ncellular processes are somewhat obscured. The authors should thus put the results in a wider context\nand tune down some of their statements.\nResponse: We sincerely appreciate your positive assessment of our work. To further elucidate the role\nof PIK3CA mutant in cachexia development, we have now performed pharmacological inhibition\nexperiments with a PI3Kα inhibitor and incorporated these data into revised manuscript (New Fig. S6G-\nL). We fully agree that the role of PIK3 signaling in cachexia progression deserves investigation in a\nbroader context. At this stage, given our focus on the PIK3CA mutant and its notable co-occurrence\nwith EGFR mutant (Fig. S2A), we have centered our study on the function of PIK3CA mutant within\nthe EGFR-mutant genetic background. We also agree that exploring the roles of other genes in this\npathway, such as Pten, and examining the function of PIK3CA mutant in various genetic contexts\nrepresent important directions for future research.\nSpecific major points:\nGenerally, the quality of the data is high and the figures are presented in a clear and logical manner.\nIn addition to the in vivo experiments mentioned above, addressing the following comments would\nimprove reproducibility and clarity.\n3) Introduction: The introduction would benefit from more in-depth information on PIK3α’s biological\nrole in tumorigenesis (NSCLC), beyond genetic status and prevalence. Here effects on tumor\nmetabolism, microenvironment, immune cell infiltration, inflammation should be introduced and put\ninto context.\nResponse: Following your suggestion, we have revised the Introduction accordingly (Lines 39–42).\nGiven the relatively few studies on PIK3α specifically related to inflammation in NSCLC, we expanded\nthis description to the broader functions of PIK3.\n4) Introduction, lines 36-37: “PIK3CA mutant transgene alone is sufficient to drive lung cancer\ninitiation in mice (ref. 5)” There are additional studies using lung cancer GEMMs that show the\nopposite (10.1158/0008-5472.CAN-15-1249, 10.1158/0008-5472.CAN-13-0681). The authors should\nadapt this statement and provide additional references in the introduction instead of discussion.\nResponse: We initially considered integrating the description of these references into the Introduction\nof the manuscript. However, after careful evaluation, we found that doing so might compromise the\ncoherence of the introductory narrative. To maintain the logical progression of ideas and ensure clarity\nfor readers, we have opted to retain this discussion in its current location within the manuscript. We\nhope this arrangement aligns with your expectations while preserving the intended flow of the text.\n5) Introduction, lines 41-42: “…80% of lung cancer harboring PIK3CA mutations tends to have EGFR\nmutations in East Asian cohort (ref. 7).”\nThis statement does not precisely reflect the findings in ref. 7. This study found that PIK3CA mutations\nwere found in ~3% (34/1117) of NSCLC patients, of which 61.8% (21/34) harbored concurrent\noncogenic mutations, and for 81% (17/21) of those, the driver mutation was in EGFR. Overall, 50%\n(17/34) PIK3CA-mutated tumors had EGFR driver mutations. The statement should also refer to\nNSCLC instead of “lung cancer” in East Asian cohorts. The introduction currently implies that PIK3α\nmutations are very frequent in NSCLC; and later (line 89) that they are highly associated with EGFR\nmutations. This needs some adaptation (see also next point).\nResponse: Sorry for the mistake and any confusion. We have now revised the description as follows:\n“Multiple studies have reported that PIK3CA mutations are frequently concurrent with oncogenic EGFR\nmutations in East Asian NSCLC populations (Wang L et al., PLoS One, 2014, PMID: 24533074; Chaft\nJE et al., Mol Cancer Ther, 2012, PMID: 22135231; Skoulidis F et al., Nat Rev Cancer, 2019, PMID:\n31406302).” (Lines 51–52)\n6) Introduction, lines 55-56: “This is further supported by the observation of worse overall survival\n(OS) of those EGFR-mutant patients with concurrent PIK3CA mutation in their tumors (ref 21, 22).”\nThis should be put in the context of low prevalence of concurrent EGFR/PIK3CA mutations and\ntherefore a low statistical power. The studies in refs. 21 and 22 include only a total of 23 and 37 patients\nwith PIK3CA mutations, respectively. In addition, comparisons of PIK3CA-mutant versus PIK3CA-\nwildtype (without further stratification based on co-occurring oncogenic driver mutations) suggest that\nthe presence of PIK3CA mutations leads to poorer outcomes, independently of other oncogenic drivers.\nResponse: Thanks for pointing out this. We fully agree that the relatively small sample size should be\nnoted to avoid potential misleading. However, we found that including the number in Introduction part\nmight disrupt its logical flow and introduce redundancy. After careful consideration, we have instead\nincluded the relevant clarification in the Discussion section (Lines 280–281, Line 292). We hope this\nsolution is acceptable. Additionally, we have removed the inappropriate citation following your\nvaluable suggestion.\n7) Results / Fig 1: do the authors have data on a follow-up of EP mice beyond the 10 weeks as shown\nin Fig 1B? Do EP mice develop cachexia at a later point in time? The same question would be of interest\nfor TLCS-P mice.\nResponse: Yes, EP and TLCS-P mice will develop cachexia at a later point in time. We have now\nanalyzed both EP and TLCS-P mice at late time points. Our H&E staining revealed notable wasting of\nmuscle and adipose tissue in EP mice at 17 weeks (Reviewer only Fig. 3A-B) and TLCS-P mice at 20\nweeks (Reviewer only Fig. 3C-D) after Ad-Cre delivery. In current study, we focused the tumor-\nbearing mice with or without PIK3CA mutant at comparable time points after intranasal induction.\nReviewer only Figure 3. A-B, Representative micrographs of gastrocnemius tissue (A) and white adipose tissue (B) in healthy\nctrl and EP mice after Ad-Cre delivery of 17.0 weeks. C-D, Representative micrographs of gastrocnemius tissue (C) and white\nadipose tissue (D) in healthy ctrl and TLCS-P mice after Ad-Cre delivery of 20.4 weeks.\n8) Results / Fig 1: The authors use a cachexia gene list proposed in the TRACERx study. This list is\nhighly enriched for genes in the PI3K-AKT signaling pathway (Fig 1M-N). The enrichment of the PI3K-\nAKT signature in primary tumors from cachectic individuals is, however, not substantial (Fig 1O,\nNES=1.303) and is not highlighted in the global GSEA analysis of the original study. The authors\nshould provide a detailed methodology for their GSEA analysis, including other gene sets, the number\nof permutations and the type and report of FDR values. The same applies for the analyses in Figs S4A,\nS4B. Within the same paragraph, the authors state that “these data collectively support the significant\nrole of PIK3CA mutant in driving cachexia.”. These data, however, do not directly address PIK3CA\nmutation status, but PI3K-AKT pathway activity. The text referring to this data should be adapted and\nrefined.\nResponse: Thank you for pointing out this issue. In Fig. 1O, we performed GSEA using the KEGG\ngene set instead of the Hallmark gene set applied in the original study. We found that the PI3K-AKT\nsignaling pathway ranked 13th (Reviewer only Fig. 4) with a p-value of 0.023 and an FDR of 0.227\n(New Fig. 1O). We also observed pronounced enrichment of the PI3K-AKT pathway in Fig. 1N, along\nwith consistent enrichment in the cachexia state across multiple gene sets (New Fig. S4A-C),\ncollectively indicating the involvement of this pathway in cancer cachexia.\nReviewer only Figure 4. Dot plot shows KEGG pathways enriched in cancer-associated cachexia (CAC) patients compared\nto non-CAC patients in TRACERx study (Al-Sawaf et al., 2023) cohort.\nSpecifically, For New Fig. 1O, S4A, GSEA was performed using the R package clusterProfiler with\n1,000 permutations and gene_set permutation type (default setting). Genes were ranked by fold-change\nvalues. For New Fig. S4B, the analysis was based on a list of differentially expressed proteins (DEPs)\nreported by Wang et al. (Wang D et al., J Hematol Oncol, 2023, PMID: 36973755), derived from human\nserum proteomics comparing cachectic and non-cachectic lung cancer patients. KEGG pathway\nenrichment analysis was performed using enrichR, which is based on a hypergeometric test. We have\nincluded FDR values and added detailed methodology in the revised manuscript (Lines 460–472).\nMoreover, we have revised the sentence “these data collectively support the significant role of PIK3CA\nmutant in driving cachexia” and focused on the PI3K-AKT pathway (Line 147).\n9) Results / Fig 2: The authors should state the time post Ad-Cre delivery for the mice used in the RNA-\nseq analysis of TA muscles (Fig. 2A-C). Were these mice matched by age or tumor burden?\nResponse: The mice used for RNA-seq analysis were age-matched, and tissue collection was performed\n8 weeks after Ad-Cre delivery (Lines 729–730).\n10) Results / Fig 5/S6: Color scales in heatmaps from Figs. 5B, 5C, 5G, S6A are missing.\nResponse: We have added the color scales in the revised manuscript.\n11) Results /Fig S5: The authors should provide a more detailed methodology for their GSEA analysis\nin Figs. S5C S5D, S5E, including number of permutations and type, and report the FDR values. Were\nany other gene sets tested at the same time?\nResponse: We have included additional details on the GSEA in the revised manuscript (Lines 460–\n472). Specifically, GSEA was performed using the R package clusterProfiler with 1,000 permutations\nand gene_set permutation type (default setting). Genes were ranked by fold-change values. For the\nanalyses shown in New Fig. S5B, H-J, we tested all gene sets from the MSigDB Hallmark Gene Set\nCollection. We have now added FDR values to these figures to more accurately illustrate the statistical\nsignificance of the results.\nFollowing your suggestion, we further analyzed the PI3K-AKT and TNF signaling pathways using the\nKEGG gene set, both of which showed significant enrichment (Reviewer only Fig. 5A-B). Moreover,\nEMT and inflammatory response pathways were highlighted in our study as transcriptomic features of\ntumors from cachectic lung cancer patients in the TRACERx study (Al-Sawaf O et al., Nat Med, 2023,\nPMID: 37045997). Due to the lack of appropriate counterparts in the KEGG gene set, these pathways\nwere not included here for further enrichment analysis.\nReviewer only Figure 5. A-B, GSEA enrichment plot of PI3K-AKT signaling pathway (A) and TNF signaling pathway (B)\nin TCLS-PP tumors compared to TLCS-P tumors.\n12) Results / Fig 5: The authors should state the time post Ad-Cre administration for the mice used in\nthe RNA-seq analysis of tumors (Fig. 5A-C). Were these mice matched by age or tumor burden? Could\nthe authors comment on the reason why the hallmark PI3K-Akt-mTOR signaling gene set is not\nsignificantly enriched in EPP vs EP tumors? Why was a similar “global” hallmarks GSEA not\nperformed on TLCS-P and TLCS-PP tumor tissue?\nResponse: For the RNA-seq analysis shown in New Fig. 5A-C, tumors were collected at time points\nappropriate to each tumor model: 8-10 weeks post Ad-Cre administration for EPP, 10-12 weeks for\nTLCS-PP, and approximately 18 weeks for EP and TLCS-P tumors. While the mice were not strictly\nage-matched, tumors were selected based on comparable size to ensure consistency. Due to technical\nlimitations in isolating early-stage pulmonary tumors, we typically collect individual tumors with a\ndiameter of 3-5 mm, which generally correspond to advanced disease stages.\nIn Figure 5A, the PIK3/AKT/mTOR pathway was significant (Reviewer only Fig. 6), but not on the\ntop 15 as shown in the Figure 5A.\nReviewer only Figure 6. GSEA enrichment plot of PIK3/AKT/mTOR signaling pathway in EPP tumors versus EP tumors.\nRegarding the comparison between TLCS-PP and TLCS-P tumors, we only highlighted four\nrepresentative signaling pathways in New Fig. S5B, H-J. We have now performed a global Hallmark\nGSEA analysis and found that all four highlighted pathways were enriched among the top 15 in the\nTLCS-PP vs. TLCS-P comparison (Reviewer only Fig. 7).\nReviewer only Figure 7. Dot plot shows Hallmark gene sets enriched in TLCS-PP tumors versus TLCS-P tumors based on\nGSEA analysis (Top 15 pathways shown). The dot size represents the normalized enrichment score (NES). The dot color\nindicates enrichment significance.\n13) Results: The data on NF-kB activity and p65 nuclear localization in response to ectopic expression\nof PIK3CAE545K are interesting. Is the same true for tumors in vivo? The connection of PI3K\nactivation and NF-kB activity is controversial in the literature. Could the authors refer to some of the\ndiscrepancies?\nResponse: Following your suggestion, we have examined NF-κB pathway activation by\nimmunoblotting of in situ tumors from GEMMs used in our study. Compared to EP and TLCS-P tumors,\np-p65 levels were elevated in EPP (New Fig. 5F) and TLCS-PP tumors (New Fig. S5M) respectively,\nindicating that PIK3CA mutant leads to NF-κB pathway activation in vivo.\nFigure 5. F, Immunoblotting analyses of indicated proteins in in situ tumors from EGFRL858R; Trp53flox/flox (EP) and\nEGFRL858R; PIK3CAE545K; Trp53flox/flox (EPP) mice.\nFigure S5. M, Immunoblotting analyses of indicated proteins in in situ tumors from EGFRTLCS; Trp53flox/flox (TLCS-P) and\nEGFRTLCS; PIK3CAE545K; Trp53flox/flox (TLCS-PP) mice.\nAdditionally, we have revised the Discussion section to provide further details on the link between PI3K\nactivation and NF-κB signaling (Lines 309–313).\n14) Comment 12 (results): For the analysis of TCGA data in Fig. 5: how were the PI3K-AKT high\nand low groups defined?\nResponse: For the analysis shown in Figure 5, the PI3K-AKT signaling activity was quantified using\nsingle-sample Gene Set Enrichment Analysis (ssGSEA) based on the\nREACTOME_PI3K_AKT_ACTIVATION gene set. The ssGSEA scores were calculated for each\nTCGA sample to represent the pathway activity. To better highlight the differences and more clearly\ndemonstrate the association with relevant outcomes, we defined the high and low PI3K-AKT activity\ngroups as follows: samples with ssGSEA scores greater than the top tertile (2/3) were classified as\n“high” activity, while those with scores below the bottom tertile (1/3) were classified as “low” activity.\n15) Discussion, lines 266-268: “However, once _patients_ relapse from TKI therapy and the tumors\ndevelop TKI resistance, the impact of PIK3CA mutant begins to unleash, with cachexia driven by\nPIK3CA mutant gradually worsening and ultimately leading to poor patient prognosis.” This statement\nis highly hypothetical as there is no clinical data to be referred to. This should be rephrased to reflect\nthis. There is also no in vivo data to support that PIK3CA mutations become important only after TKI\nresistance. The data show that all mice harboring PIK3CAE545K mutations develop cachexia faster,\nindependently of Osimertinib sensitivity.\nResponse: We have adapted the statement accordingly (Lines 298–303).\n16) Discussion, lines 296-299: “To validate our findings across other cancer types, we further perform\nGene Set Enrichment Analysis (GSEA) on a recently published pre-clinical melanoma model, and\nobserved enrichment of PIK3-AKT signaling in xenografts capable of inducing cachexia (data not\nshown).” This belongs to the results section. The data should be shown.\nResponse: We have now added this data into the Results section (New Figure S4C).\nFigure S4. C, GSEA enrichment plot of PIK3-AKT signaling pathway in cachectic melanoma xenografts versus non-cachectic\nmelanoma xenografts in Graca et al. cohort.\n17) Materials and methods: A section describing in detail the (re-)analyses performed on publicly\navailable datasets should be included.\nResponse: We have included the details about the re-analysis of publicly available datasets (Lines 460–\n472).\n18) Results / minor point: Could the authors check whether the error bars in Figs S2F and S2G\nrepresent SEM instead of SD (as stated in the figure legend)?\nResponse: Sorry for the mistake. In the previous version, the error bars represented SEM. We have\ncorrected them to SD in the revised version (New Fig. S2F-G).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nPOINT-BY-POINT RESPONSE\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all of my comments. This is very much appreciated. My only\nremaining recommendation is related to the mutant PIK3CA dependency of aspirin’s effects on\ncancer cachexia. The authors utilized LLC cachexia model and demonstrated little\nimprovements in cachexia phenotypes in response to aspirin treatment arguing that PIK3CA\nmutation status of the tumor matters. I think the authors should include these data in their\nmanuscript and discuss the possible link between aspirin efficacy and PIK3CA activity.\nResearchers from the cachexia field reading this article would like to see these results.\nAlthough the results may be negative, they do not undermine the conclusions of this manuscript.\nPublication of these data could prevent unnecessary repetition of such experiments.\nResponse: We sincerely appreciate your positive assessment of our work. In response to your\nsuggestion, we have now incorporated the LLC cachexia data in revised manuscript (New Fig.\nS6H-M).\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate your great efforts in co-reviewing our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors’ point-by-point responses and the new data provided in the revised manuscript and\nthe reviewer-only figures address the previously raised concerns only partially. Data showing\nthat EP and TLCS-P mice also develop cachexia should be included in the manuscript, as this\nrepresents an important finding. Collectively, the results indicate mutation of PIK3CA\ncontributes to tumor progression in mice and, subsequently, faster cachexia development by 7-\n8 weeks. The evidence that cachexia development is not simply due to faster tumor progression\nin the presence of PIK3CA mutation is not compelling. Cachexia still develops in the absence\nof PIK3CA mutations in these mice, albeit at a slower pace. Furthermore, after revisions, the\nstudy still lacks detailed exploration of intracellular/extracellular signalling and PI3Kα’s exact\nfunction and mechanism leading to NF-κB activation. The overall narrative should be revised\nto balance storytelling with more accurate representation of the data. More specific points are\nbelow.\nMajor points:\n1) The authors should clearly state that PIK3CA mutation accelerates (rather than initiates)\nthe development of cachexia. The cut-off at 10 weeks for body weights, lean mass and fat mass\n(Figures 1B, S3A, S3B), and the relative display of tissue weights in Figures 1D,E and 1K,L\nobscures this fact in the current version of the manuscript. Extended time-course data for EP\nshould be shown in Figures 1B, S3A, S3B. The same applies for TLCS-P mice (Figures 4B-D).\nResponse: We appreciate your insightful comment and fully agree that the PIK3CA mutant\nindeed accelerates rather than initiates cachexia. To make it clear, we have revised the title to:\nConcurrent PIK3CA mutant promotes cachexia through inflammatory signaling in EGFR\nmutant lung cancer. In support of this point, we have now added New Fig. S3A and S5C to\nshow that weight loss is also observed in EP and TLCS-P mice at late stage. Moreover, we have\nalso included WT mice to further support this point (New Fig. 1K-L, S3M). Due to time limit,\nwe are not able to obtain the extended time-course measurements of fat and lean mass at this\nstage and hope you could understand this.\n2) Appropriate WT control mice should also be included in Figures 1K,L; 3E-L; 4H,I; S6J-L.\nResponse: Following your suggestion, we have now included WT control mice data in revised\nmanuscript (New Fig. 1K-L, S3M, 3E-L, 4H-I, S5H, and S6R-T).\n3) What was the reason for using raw values to generate the graphs in Reviewer-only Figure\n1? Since these kinds of data are always normalised (to control groups) in the manuscript, the\nsame normalisation should have been applied here. These data do not show a large effect size\nof aspirin treatment in a different, PIK3CA-independent cachexia model (LLC), but it is\ndifficult to say if this is the case when the data analysis and presentation are not consistent with those\npresented in the manuscript.\nResponse: We followed a previously established protocol for LLC-induced cachexia model,\nwhich was conducted using male mice (Bilgic SN et al., Nature, 2023, PMID: 37165186). We\nthus didn’t perform normalization in the original version. In light of your suggestion, we have\nnow normalized the data and incorporated these in revised manuscript (New Fig. S6I-J).\n4) Reviewer-only Figure 4 should be added to Figure 1, before panel O, as in this case the\nPI3K-AKT signalling pathway is singled out despite marginal significance and not meeting a\nconventional 5% FDR threshold (also in the case of the GSEA analysis shown in Figure S4C).\nResponse: Following your suggestion, we have now incorporated the reviewer-only Figure 4\nin revised manuscript (New Fig. 1O).\n5) Explorations of intersections of PI3K-Akt and NF-κB singling pathways in vitro and/or in\nanimals following different treatments are missing. The experimental models provide some\ntranslational evidence for the use of these inhibitors (osimertinib, inavolisib and aspirin) in\nalleviating cachexia, but the mechanisms for the observed effects are probably unrelated:\nosimertinib and inavolisib more effectively suppress tumor burden, thus delaying cachexia,\nwhile the mechanism of action of aspirin likely involves systemic pleiotropic actions (in both\ntumor and muscle/adipose tissues). Does aspirin also alleviate cachexia when it develops in EP\nand TLCS-P mice (thus independently of mutated tumor PIK3CA)?\nResponse: Thank you for these insightful comments. We fully agree that these inhibitors may\nalleviate cachexia through different mechanisms. As for osimertinib and inavolisib, their\nfunction in reducing tumor burden seems closely linked to their anti-cachectic effects, which\nmakes it difficult to clearly dissect the two. As for aspirin, we agree it’s possible that this drug\nmay exert systemic anti-inflammatory effect in the context of cachexia suppression. To test this,\nwe have performed another transplantation experiment using mouse Lewis lung carcinoma\n(LLC) followed by aspirin treatment and find no inhibition of NF-κB signaling by aspirin in\nmuscle tissues (New Fig. S6M). To further dissect the intersections of PI3K-AKT and NF-κB\nsingling pathways, we have now performed in vitro experiment using EP cells with ectopic\nexpression of FLAG-tagged PIK3CA mutant (New Fig. S6A, S6N). We find that\noverexpression of PIK3CA mutant resulted in increased phosphorylation levels of AKT and\np65 and treatment with aspirin or inavolisib effectively suppressed NF-κB activation, indicating\nthat aspirin exerts certain effects upon tumor cells. Due to time limit, we couldn’t perform deep\nexamination in EP and TLCS-P mice. Although our muscle analyses in LLC allograft model\nshows no positive inhibition of NF-κB by aspirin, we fully understand that these data can’t fully\nexclude the systemic effects of aspirin in the host. Future work is necessary to look deep into\nthe function of aspirin in tumor cells and host environment. We have now added relevant\ndiscussion in revised manuscript (Line 336-338).\nFigure S6. A, Immunoblotting analysis of indicated proteins in EP cell line stably expressing empty vector or\nPIK3CA E545K mutant (E545K) with or without 2mM aspirin treatment for 24h. N, Immunoblotting analysis of\nindicated proteins in EP cell line stably expressing empty vector or PIK3CA E545K mutant (E545K) with or without\n500nM inavolisib treatment for 24h.\n6) Additional mechanistic investigations, for example, of tumor-derived cachexia\nfactors/cytokines secreted in the tumor microenvironment or blood/plasma, or of lung immune\nmicroenvironment are necessary to substantiate the claims that this work uncovers a new\nbiological function of mutant PIK3CA.\nResponse: Thank you for this insightful suggestion. We have now performed ELISA on mouse\nserum and found elevated IL-6 levels in EPP and TLCS-PP mice and included these data into\nrevised manuscript (New Fig. S5M-N).\nFigure S5. M, Serum IL-6 levels measured by ELISA in WT, EP, and EPP mice at 8 weeks post Ad-Cre treatment.\nN, Serum IL-6 levels measured by ELISA in WT, TLCS-P, and TLCS-PP mice at 12 weeks post Ad-Cre treatment.\n7) Since the rescue effect of aspirin is not particularly pronounced after inclusion and\nnormalisation to wildtype controls, and the mechanism mediating the action of aspirin in this\ncase is not defined, the authors should not overstate that it represents a potentially effective\nstrategy for clinical management.\nResponse: We have made corresponding revision in the manuscript (Line 33, 82).\n8) The data in Figure S2K should be averaged for each mouse before statistical analyses are\nperformed to avoid pseudo-replication (in this case the number of measured values vastly\nexceeds the number of genuine replicates).\nResponse: Replaced (New Fig. S2K).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate your thoughtful review and invaluable mentorship.\nPOINT-BY-POINT RESPONSE\nReviewer #3 (Remarks to the Author):\nThe authors’ efforts to improve the manuscript over the course of the revisions is appreciated\nand the overall scientific quality has clearly improved. While the revised title is appropriate,\nseveral sections of the manuscript would still benefit from toning down or rephrasing of certain\nstatements (see detailed comments below). In addition, please provide quantification for the\nwestern blots shown in Figures 5F, S2H, and S5P, and include replicates for the western blot\nquantification presented in Figures S2D, S6A, and S6N.\nResponse: We sincerely appreciate your positive feedback on our work and the detailed\ncomments on our manuscript. Following your insightful suggestions, we have toned down the\nstatements in specific sections of the text and provided quantification for the western blots\nshown in New Fig. 5F, S2D, S2H, S5P, S6A, and S7A. For Fig. S2D, S6A, and S6N (New Fig.\nS7A), we have included additional replicates in the source data file.\nLines 31-32:\n“This work uncovers the biological function of PIK3CA mutant and mechanism behinds its\nclinical impacts”\nThe abstract’s concluding statement is overstating the findings and should be rephrased to\nreflect that the study provides insights rather than fully uncovering mechanisms or establishing\nclinical impact.\nResponse: We have rephrased the sentence as: This work uncovers the biological function of\nPIK3CA mutant, provides insights into mechanism behinds its clinical impacts…\nLine 130:\n“To exclude the potential impact of tumor burdens upon cachexia…”\nIn my view, this experiment does not exclude an impact of tumor burden. Data shown later in\nFigure 3 indicate that cachexia is effectively suppressed by lowering tumor burden (e.g. with\nOsi), which appears inconsistent with this statement. I recommend replacing “exclude” with\n“explore” or similar in line 130 and reformulating the concluding line 136.\nResponse: We have rephrased the sentence as: To explore the potential impact of tumor\nburdens upon cachexia…\nLine 136:\n“These data demonstrate that PIK3CA mutant triggers the cachexia symptom independent of\ntumor burdens”.\nSuggested rephrasing: These data suggest that PIK3CA mutation contributes to cachexia\nthrough mechanisms other than by promoting tumor burden.\nResponse: We have rephrased the sentence as suggested.\nLines 138-147:\nThis paragraph explores several public datasets, however, apart from the enrichment of the\nPI3K-AKT signaling pathway in the TRACERx study-derived gene signature (Figure 1N), most\nother observations have low statistical significance. This limitation is not clearly acknowledged\nin the text.\nResponse: We have rephrased the sentence as: the PI3K-AKT signature showed marginally\nsignificant enrichment in those primary tumors from patients with cachexia\nLines 306-310:\n“We propose that when patients relapse from TKI therapy and the tumors develop TKI\nresistance, the impact of PIK3CA mutant may become evident, with cachexia driven by PIK3CA\nmutant gradually worsening and leading to poor patient prognosis.”\nGiven that the TKI-resistant model is presumably still EGFR-driven but lacks an efficacious\nEGFR inhibitor, the observed effects reflect the consequences of incomplete tumor burden\ncontrol at this stage, rather than a specific role of PIK3CA mutation in the TKI-resistant context.\nI recommend clarifying this distinction in the interpretation.\nSuggested: “We propose that when patients relapse from TKI therapy and the tumors develop\nTKI resistance, the impact of PIK3CA mutant may become evident in the absence of effective\ntumor burden control, with cachexia driven by PIK3CA mutant gradually worsening and\nleading to poor patient prognosis.”\nResponse: We have rephrased the sentence as suggested.\nLines 344-345:\nPlease remove the conclusion that the study “provides new insights into the benefit of aspirin\nfor PIK3CA-mutant patients with colon cancer”. This statement extends beyond the scope of\nthe presented data in lung cancer GEMMs and should not be highlighted as a direct insight.\nResponse: We have removed the sentence.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe sincerely appreciate your efforts and insightful suggestions regarding our manuscript."}]

Decision letter