EMBO/soda-nel huggingface dataset:
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 7

Figure 7. TRIM46 Promotes Breast Carcinogenesis and Chemoresistance.

A. MCF-7 cells or MCF-10A cells were infected with lentiviruses carrying the indicated constructs. Cells were split into 96-well plates and then harvested at the indicated day. Cell growth curves were measured with MTS assays.

B. MCF-7 cells stably transfected with indicated shRNAs or expression plasmids or MCF-10A cells stably transfected with expression plasmids were tested for colony formation assay.

C. MCF-7 cells infected with lentiviruses carrying indicated plasmids were inoculated into the left abdominal mammary fat pad (5 × 106 cells) of 6-week-old immunocompromised female SCID beige mice. Tumor size was measured after 8 weeks (mammary tumors, n = 6).The efficiency of overexpression or knockdown of the gene was verified by western blotting.

D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined.

E. Control MCF-10A cells or MCF-10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin, VP16, or adriamycin for 72 h, and the proportions of viable cells were examined.

Data information: In (A-B, and D-E), each bar represents the mean ± SD for biological triplicate experiments (*P<0.05, **P<0.01, two-tailed unpaired t-test). In (C), each bar represents the mean ± SD (n=6, *P<0.05, two-tailed unpaired t-test).
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 8

Figure 8. The Clinicopathological Significance of the TRIM46-HDAC1 Axis in Breast Cancer.

A. Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples (grade I: n=29, grade II: n=69, grade III: n=44) and 20 normal mammary tissues for TRIM46 and HDAC1 expression. Representative images are shown on the left panel. The positively stained cells were analyzed, and the mean intensity was scored by Image-Pro Plus software on the right panel. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively. (*P<0.05, **P<0.01, ****P < 0.0001, one-way ANOVA).

B. Total proteins from 20 paired samples of breast cancer "T" versus adjacent normal breast tissues "A" were extracted for western blotting analysis with antibodies against TRIM46 or HDAC1.

C. Quantification of the results in B by normalizing the protein level of TRIM46 or HDAC1 to that of GAPDH in "T" and "A" groups (n = 20). *P < 0.05, paired two-tailed Student's t-test.

D. Bioinformatics analysis of the public dataset from TCGA in breast carcinoma samples (n=1093) and normal mammary tissues (n=112) for the expression of TRIM46, BRCA1, or BLM based on the indicated stratifications (****P < 0.0001, two-tailed unpaired t-test). In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively.

E. Kaplan-Meier survival analysis of TCGA BRCA dataset for the correlation between overall survival of breast cancer patients and TRIM46, BRCA1, or BLM expression.
Androgen Receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling (2021)
Figure 1

Figure 1. AR regulates SDH enzyme through AREs.

A. Schematic representation of SDH composition (catalytic subunits: SDHA, SDHB and membrane anchor subunits: SDHC, SDHD) and SDH activity.

B. ARPI (CSS, ENZA, ODM-201, 14449) decreased, while R1881 increased, SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells. PSA is used as a positive control.

C, D. ARPI treatment (CSS or ENZA) for 12 h in LNCaP cells decreased SDH activity (C) and increased intracellular succinate levels (D) compared to vehicle treated control.

E. ENZA decreased (left panel), whereas R1881 increased (right panel), transcript levels of SDHA and SDHB in LNCaP cells.

F. Quantitative PCR on DNA immuno-precipitated with AR antibody in ChIP assay showed increased signal for SDHA, SDHB along with PSA after treatment with R1881 compared to androgen deprivation in LNCaP cells. PSA is used as positive control.

G. EMSA shows dose-dependent interaction between SDHA and SDHB AREs and AR-DNA binding protein, which was disrupted with mutated ARE probes (SDHA-M, SDHB-M). Quantification of binding of three independent experiments is shown at the bottom panel.

Data information: ENZA: enzalutamide, CSS: charcoal stripped serum, ODM-201: darolutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test (G) or one-way ANOVA followed by Tukey's test (C, D, E, F). *, p<0.05, **, p<0.01, ****, p<0.0001 compared between groups. Exact p values are reported in the Appendix Table S7.
Androgen Receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling (2021)
Figure 2

Figure 2. Inhibition of SDH activity increases AR protein levels and activity.

A. SDHA and SDHB silencing significantly increased AR full length protein levels in LNCaP, LAPC4, C4-2 and MR49F cells as well as both AR full length and V7 protein levels in 22Rv1 cells.

B. ENZA acutely decreases AR levels (up to 18 h) while SDHB silencing rescued the early ENZA-mediated AR downregulation in LNCaP cells.

C. SDHA/SDHB silencing maintains higher levels of AR transcriptional activity during acute phase of ENZA stress (18 h) in LNCaP cells.

D. SDH repression during acute phase of ENZA stress increased transcription of different AR-regulated genes without altering AR mRNAs levels in LNCaP cells.

E-F. Overexpression of SDHA and SDHB subunits decreased AR protein levels (E) and AR transactivation (F) in LNCaP cells.

G-H. LNCaP cell viability is increased by SDHA/SDHB silencing (G) and reduced by SDHA/SDHB overexpression (H) compared to siScr and empty vector, respectively.

Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test (D) or one-way ANOVA followed by Tukey's test (C, F, G, H).*, p<0.05 and **, p<0.01 compared between groups. Exact p values are reported in the Appendix Table S7.
Androgen Receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling (2021)
Figure 3

Figure 3. Hsp27 regulates the expression and activity of AR post SDH repression.

A. LNCaP cells silenced for SDHA or SDHB showed approximately 20-30% longer AR half life compared to ENZA alone in the presence of 50 µg/ml cycloheximide(left panel). Right panel shows quantification of AR protein normalized by loading control.

B. Co-silencing of SDHB and Hsp27 reversed siSDHB-mediated AR protein upregulation in LNCaP cells.

C. SDH-repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay.

D. Hsp27 silencing in LNCaP cells showed dramatically high levels of cleaved-PARP compared to ENZA, which were rescued to some extent by SDH-repression.

E. Treatment with p-p38 inhibitors: SB203580 (10 µM) or Genistein (20 µM) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells (left panel). Bar graph in the right panel shows band intensity normalized to loading control.

Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels C (end points) and E . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, Exact p values are reported in the Appendix Table S7.
Androgen Receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling (2021)
Figure 4

Figure 4. Succinate increases intracellular calcium flux to activate p-CaMKK2/p-AMPK in LNCaP cells.

A Extracellular flux analysis by measuring oxygen consumption rate (OCR) (left panel) and extracellular acidification rate (ECAR) (right panel) in LNCaP cells indicates that SDHA/SDHB silencing increased both activities compared to ENZA alone.

B. Comparison of OCR vs ECAR clearly shows significant upregulation of glycolytic capacity (2 fold) in SDH-repressed cells compared to ENZA alone..

C. Silencing of SDHA/SDHB subunits in LNCaP cells increased p-AMPK, as well as p-p38/p-Hsp27 levels. Co-silencing of AMPK inhibited the upregulation of AR mediated by SDH repression.

D. SDHA and SDHB overexpression decreased p-CaMKK2 and p-AMPK levels in LNCaP cells.

E. SDHA and SDHB silencing increased AR, p-CaMKK2 and p-AMPK axis in LNCaP cells, which was inhibited by CaMKK2 silencing.

F.. Treatment with 5 µM STO-609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA/SDHB subunit silencing.

G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.

Data information: ENZA: enzalutamide, 2-DG: 2-deoxyglucose, DMM: Dimethyl malonate, DMS: Dimethyl succinate. Data shown as mean ± SEM of three independent experiments. Scale bar: 10µm.
Androgen Receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling (2021)
Figure 5

Figure 5. IHC of human prostate TMA identifies increases in p-CAMKK2/p-AMPK/p-p38/p-Hsp27 axis after ADT.

A-C. Human patient samples demonstrate relative differences in the immunoexpression of AR, SDHA, p-CaMKK2/p-AMPK, p-p38, and p-Hsp27 in untreated (n=70), neoadjuvant-treated (NHT) (n=130) and CRPC (n=24) samples. Representative images (A), dot plots (B), compiled bar graph (C) support the AR-SDH loop where inhibition of AR axis in NHT samples reduced SDHA levels along with upregulation of p-CaMKK2/p-AMPK/p-p38/p-Hsp27 axis. AR-positive CRPC samples restored SDHA as well as the whole axis.

Data information: Data shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. **, p<0.01; ***, p<0.001 and ****, p<0.0001. Scale bar: 100µm. All the TMAs were constructed from minimum 2 cores per patient.
Androgen Receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling (2021)
Figure 6

Figure 6: IVM decreases AR levels and activity in both in vitro and in vivo studies.

A. In LNCaP xenografts, AR and SDHB protein levels (left panel) and SDH activity (right panel) were reduced acutely after castration (3 days) but returned to baseline after castration resistance (3 months). SDH assay was performed on tumour tissues from 5 mice per group.

B. IVM reduced AR activity in combination with ENZA in LNCaP cells.

C. ENZA reduced cell proliferation more potently when combined with IVM in LNCaP cells.

D. IVM reversed the cell proliferation benefit mediated by SDH repression in LNCaP cells. E. LNCaP xenografts treated with IVM (n=10) (10mg/kg) demonstrated lower serum PSA values (upper panel) and growth of tumours after castration (lower panel) compared to vehicle (n=10). PSA was measured every week. The data has been taken from our published study in Nappi. L, et al, JCI, 2020.

F. IVM treatment reduced p-Hsp27 levels and therefore AR protein level, with subsequent p-CAMKK2 axis activation in castrated LNCaP xenografts. Bar graph in the right panel shows quantification of data from western blot normalized to loading control.

Data information: ENZA: enzalutamide, IVM: ivermectin. Data shown as means ± SEM (C,D, E) or means ± SD (A, B, F). In vitro data represent three independent experiments. Statistical analysis was performed for the last data point (C,D). *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, one-way ANOVA followed by Tukey's test. Exact p values are reported in the Appendix Table S7.
TSC2 regulates tumor susceptibility to TRAIL-mediated T cell killing by orchestrating mTOR signaling (2022)
Figure 1

Figure 1: Whole-genome CRISPR-Cas9 knockout screen identifies TSC2 as a negative regulator of tumor sensitivity to CTL killing.

A) Schematic outline of in vitro genome-wide CRISPR-Cas9 knockout screen in IFNγ receptor-deficient (IFNGR1-KO) D10 melanoma cells under CD8 T cell challenge (Vredevoogd et al, 2019).

B) Volcano plot of depleted and enriched sgRNAs in tumor cells treated with MART-1 T cells versus Ctrl T cells.

C) Schematic illustration of in vitro competition assay.

D) Representative flow cytometry plot of in vitro competition assay (results are quantified in (E)).

E) In vitro tumor T cell co-culture competition assay in IFNGR1-KO D10 cells containing sgRNAs targeting TSC1, TSC2 or both. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of four biological replicates with different T cell donors (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

F) In vitro tumor T cell co-culture competition assay in multiple melanoma cell lines containing indicated sgRNAs. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of six biological replicates with different cell lines (n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

G) Western blot analysis of TSC2 expression in cells used in (H).

H) T cell co-culture competition assay performed in sgCtrl- or sgTSC2-expressing D10 cells, and reconstituted with TSC2 expression. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of five biological replicates with different T cell donors (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

I) In vitro T cell co-culture competition assay of sgCtrl and sgTsc2-expressing lung cancer cell lines. Statistics was done by Mann-Whitney test. Error bars indicate SD of five biological replicates with different T cell donors (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
TSC2 regulates tumor susceptibility to TRAIL-mediated T cell killing by orchestrating mTOR signaling (2022)
Figure 2

Figure 2: TSC2 ablation sensitizes melanoma cells to CTL killing in an in vivo tumor ACT model.

A) Schematic outline of in vivo competition assay.

B) Tumor growth upon adoptive cell transfer with Ctrl (n = 9) or MART-1 (n = 8) T cells. Data points represent average tumor volume, and error bars represent SEM.

C) Flow cytometry plot of both tumor mix input and representative output of the in vivo competition assay. Note that in bulk tumor digest non-tumor tissue, such as stroma cells, express neither GFP nor mCherry, showing up in the flow cytometry plots as double-negative cells.

D) Quantification of the in vivo competition assay (output) at end point by flow cytometry analysis. Error bars indicate SD. Statistical analysis was performed by Mann-Whitney test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. For this specific experiment, mice that deceased before the analysis (n=1, Ctrl T cell group), with failed ACT injection (n=1, MART-1 T cell group) or were completely tumor-free after ACT (n=1, MART-1 T cell group) were excluded from the analysis.
TSC2 regulates tumor susceptibility to TRAIL-mediated T cell killing by orchestrating mTOR signaling (2022)
Figure 3

Figure 3: TSC2 depletion-induced deregulation of mTOR signaling increases tumor cell death by CTL attack.

A) Western blot analysis of baseline mTOR signaling in sgCtrl- or sgTsc2-expressing melanoma and lung cancer cell lines. Representative plot of 2-6 independent experiments. All blots shown in this panel were run in parallel.

B) Pooled data of Western blot quantification on mTORC1/mTORC2 signaling ratio from all 8 cell lines show in Fig. EV3B. Statistical analysis was performed by Mann-Whitney test. Error bars indicate SD of mTOR ratio from eight different cell lines, with more than three independent experiments per cell line (n = 8). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

C) Western blot analysis of mTOR signaling in sgCtrl- or sgTSC2-expressing D10 melanoma cells upon MART-1 T cell challenge for indicated time. Representative of three biological replicates with different T cell donors (n = 3).

D) In vitro competition assay of sgCtrl- and sgTSC2-expressing D10 cells co-cultured with MART-1 T cells in the presence of LY2584702 (5uM), normalized to DMSO-treated groups. Statistical analysis was performed by Mann-Whitney test. Error bars indicate SD of four biological replicates with different T cell donors (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

E) Western blot analysis of sgCtrl- or sgTSC2-expressing D10 melanoma cells upon MART-1 T cell challenge for indicated time. Representative of three biological replicates with different T cell donors (n = 3).

F) In vitro competition assay of sgCtrl- and sgTsc2-expressing D10 cells co-cultured with MART-1 T cells in the presence of necroptosis (Nec1-s, 20uM) or apoptosis inhibitors (Q-VD-Oph, 50uM), normalized to inhibitor plus Ctrl T cell-treated groups, showing the net MART-1 T cell effect. Statistical analysis was performed by one-way ANOVA with Holm-Sidak's multiple comparisons test. Error bars indicate SD of three biological replicates with different T cell donors (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

G) Cell viability analysis by CellTiter-Blue assay of T cell sensitivity in multiple melanoma and lung cancer cell lines in the presence of Tautomycin (TC, 150nM), Akt Inhibitor V (AktV, 1.5uM), or the combination (TC, 150nM+ AktV, 1.5uM) compared to DMSO-treated group. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of independent experiments with seven different cell lines. Each data point represents the average mean from three biological replicates with different T cell donors (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
TSC2 regulates tumor susceptibility to TRAIL-mediated T cell killing by orchestrating mTOR signaling (2022)
Figure 4

Figure 4: TSC2 depletion enhances TRAIL receptor expression and sensitizes melanoma cells to TRAIL-induced death.

A) Cell viability analysis by CellTiter-Blue assay of sgCtrl- or sgTSC2-expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days. Statistical analysis was performed by two-tailed unpaired Student t test. For D10, error bars represent SD from three independent experiments (n = 3). For A549, error bars represent SD of pooled data from two independent experiments with three technical replicates each (n = 2). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

B) Cell viability analysis by CellTiter-Blue assay of sgCtrl- or sgTSC2-expressing D10 melanoma cells treated with TRAIL agonistic antibody Conatumumab, at indicated concentrations for two days. Statistical analysis was performed by two-tailed paired Student t test. Error bars indicate SD from three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

C) Flow cytometry analysis of TRAIL-R2 surface expression level in multiple sgTSC2- expressing tumor cell lines compared to their sgCtrl-expressing controls. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test. Error bars represent SD of three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

D) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2, TRAIL receptors (TNFRSF10A and TNFRSF10B), or both in a co-culture with MART-1 T cells. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of four biological replicates with different T cell donors. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

E) Flow cytometry analysis of TRAIL-R2 surface expression level in D10 melanoma cells after a three-day treatment with Tautomycin (TC, 150nM), Akt Inhibitor V (AktV, 1.5uM), or the combination (TC, 150nM + AktV, 1.5uM) compared to DMSO-treated cells. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of four biological replicates (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

F) Cell viability analysis by CellTiter-Blue assay of TRAIL (100ng/ml) sensitivity in multiple parental melanoma and lung cancer cell lines in the presence of Tautomycin (TC, 150nM) and Akt Inhibitor V (AktV, 1.5uM) treatment, compared to DMSO-treated group. Statistical analysis was performed by Wilcoxon matched-pairs signed rank test of seven different cell lines. Representative graph of three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
TSC2 regulates tumor susceptibility to TRAIL-mediated T cell killing by orchestrating mTOR signaling (2022)
Figure 5

Figure 5: Low TSC2 expression: TRAIL signaling ratio is associated with immune checkpoint blockade response in melanoma patients.

A) Heat-map of Spearman correlation between TSC2, TNFRSF10A/ TNFRSF10B expression, mTORC1, and TRAIL signaling in TCGA SKCM (Skin Cutaneous Melanoma) baseline patient cohort. HALLMARK_MTORC1_SIGNALING (Liberzon et al, 2015), PID_TRAIL_PATHWAY (Schaefer et al, 2009) gene sets were taken from the Molecular Signatures Database (MSigDB).

B) TSC2 expression: TRAIL signaling expression ratio for predicting ICB treatment responses in pre-treatment patient samples (Riaz et al, 2017; Gide et al, 2019). Combo, anti-PD-1 plus anti-CTLA-4 combination treatment. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

C) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment. Early during treatment (EDT), anti-PD-1 + anti-CTLA-4, and anti-PD-1 alone treatment cohorts. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

D) ROC curve analysis showing the probability of the indicated signatures as classifiers of ICB treatment response. Statistical significance between signatures and no predictive value (AUC=0.5) was calculated with bootstrapping.

Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).