Additional file 1 of STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway
Creators: name: Pencik, Jan
name: Philippe, Cecile
name: Schlederer, Michaela
name: Atas, Emine
name: Pecoraro, Matteo
name: Grund-Gröschke, Sandra
name: Li, Wen (Jess)
name: Tracz, Amanda
name: Heidegger, Isabel
name: Lagger, Sabine
name: Trachtová, Karolína
name: Oberhuber, Monika
name: Heitzer, Ellen
name: Aksoy, Osman
name: Neubauer, Heidi A.
name: Wingelhofer, Bettina
name: Orlova, Anna
name: Witzeneder, Nadine
name: Dillinger, Thomas
name: Redl, Elisa
name: Greiner, Georg
name: D’Andrea, David
name: Östman, Johnny R.
name: Tangermann, Simone
name: Hermanova, Ivana
name: Schäfer, Georg
name: Sternberg, Felix
name: Pohl, Elena E.
name: Sternberg, Christina
name: Varady, Adam
name: Horvath, Jaqueline
name: Stoiber, Dagmar
name: Malcolm, Tim I.
name: Turner, Suzanne D.
name: Parkes, Eileen E.
name: Hantusch, Brigitte
name: Egger, Gerda
name: Rose-John, Stefan
name: Poli, Valeria
name: Jain, Suneil
name: Armstrong, Chris W. D.
name: Hoermann, Gregor
name: Goffin, Vincent
name: Aberger, Fritz
name: Moriggl, Richard
name: Carracedo, Arkaitz
name: McKinney, Cathal
name: Kennedy, Richard D.
name: Klocker, Helmut
name: Speicher, Michael R.
name: Tang, Dean G.
name: Moazzami, Ali A.
name: Heery, David M.
name: Hacker, Marcus
name: Kenner, Lukas
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplementary Fig. 1. (A) Circos plot of STAT3 and PTEN deletions: inner circle TCGA (n=492) and outer circle plasma-DNA samples from advanced PCa patients (n=43). (B) Gross anatomy of representative prostates isolated at 19 weeks of age from WT, Ptenpc−/−, Ptenpc−/−Stat3pc−/− andPtenpc−/−Stat3C/+ mice. Scale bars, 10 mm. (C) Transmission electron microscopy (TEM) pictures of mitochondria from WT, Ptenpc−/−, Ptenpc−/−Stat3pc−/− and Ptenpc−/−Stat3C/+ prostates, 19 weeks of age mice. Red arrowheads show defected mitochondrial shape. Stat3 activation in Ptenpc−/−Stat3C/+ cells reversed defected mitochondrial phenotype. Supplementary Fig. 2. (A) Western blot analysis of STAT3, LKB1, p-4E-BP1, 4E-BP1, p-S6 and S6 expression in WT and Stat3KO MEFs. β-actin servesas a loading control. (B) qRT–PCR analysis of STAT3 and STK11 transcript levels in WT and Stat3KO MEFs (n=3 each). (C) qRT–PCR analysis of Stk11, Mo25, Strada and Ampkα mRNA expression in prostates of 19-week-old WT, Ptenpc−/−, Ptenpc−/−Stat3pc−/− and Ptenpc−/−Stat3C/+ (n=3 each). Data were analyzed by Student’s t-test and are shown as mean ± s.d. Supplementary Fig. 3. (A) The heatmap of murine PCa proteomics with significant enrichment of genes involved in regulation of oxidative phosphorylation. 19-week-old WT, Ptenpc−/−, Ptenpc−/−Stat3pc−/−and Ptenpc−/−Stat3C/+ mice (n=3). (B, C) Heatmap of murine PCa proteomics with significant enrichment of genes involved in regulation of glutamine metabolism and glycolysis. 19-week-old WT, Ptenpc−/−, Ptenpc−/−Stat3pc−/− and Ptenpc−/−Stat3C/+ mice (n=3). (D) IHC analysis of p-ACC and FASN inprostates from 19-week-old WT, Ptenpc−/−, Ptenpc−/− Stat3pc−/− and Ptenpc−/−Stat3C/+ mice. Scale bars, 100 μm. Bar graphs indicate percentage of cellspositive for p-ACC and FASN in prostates of 19-week-old WT, Ptenpc−/−, Ptenpc−/−Stat3pc−/− and Ptenpc−/−Stat3C/+. Protein levels quantification was done with HistoQuest software (n≥3). (E) Western blot analysis of AR and STAT3 in LNCaP cells transfected with non-targeting (NT) or shRNAs specific for STAT3. Supplementary Fig. 4. (A) IHC and representative images analysis of radical prostatectomy specimens (benign and cancer core) of 80 PCa patients stained with 4E-BP1 and p-S6. 41 used metformin and 39 PCa patients with nopharmacological treatment were included as a control group. (B) HRSM measurement of NAD+, NADH and ATP in22Rv1 and PC3 tumors (n=5). (C) H&amp;E and IHC stainings of Ki-67, Cleaved Caspase 3 (CC3) and STAT3 expression invehicle versus metformin-treated xenografted tumors (n=5), scale bar 50 µm. (D) Colony forming units of 22Rv1 cells (NT (non-targeting), shSTAT3 #446, shSTAT3 #843) using different concentrations of metformin (0 mM (1% PBS solventcontrol), 0.005 mM, 0.05 mM, 0.5 mM) and quantification of formed colonies (n=3 each). (E) Assessment of apoptosis in 22Rv1 cells (NT (non-targeting), shSTAT3 #446, shSTAT3 #843) after metformin (0 mM (1% PBS solventcontrol), 0.005 mM, 0.05 mM, 0.5 mM) or rotenone (0 µM (0.1% DMSO solvent control), 0.5 µM) treatment (n=3 each). (F) Oxygenconsumption rate (OCR) curves of 22Rv1 cells (NT (non-targeting), shSTAT3 #446, shSTAT3 #843) after metformin treatment (50 µM, 5 mM) are shown. Injections of oligomycin (Injection 1), FCCP (Injection 2), and rotenone and antimycin A (R/A; Injection 3) are indicated by arrows. The resulting maximal respiration is shown on the right. Supplementary Fig. 5. (A) TCGA gene expression data for STAT3, PTEN and EIF4EBP1 were obtained from cBioPortal. TPM expression data were processed by adding a constant of 1.1 and subsequently log2 transformation. Boxplots were generated to visualize the difference inexpression between normal and cancer samples in TCGA. P-values are provided which were output using the unpaired Student's t-test to test the significance ofthe difference in mean expression between groups [57]. (B) Scatterplots were generated to visualize the relationship inexpression between each pair of genes (PTEN-STAT3, PTEN-EIF4EBP1 and STAT3-EIF4EBP1) in PCa samples in TCGA. The Pearson correlation ­­(R) and the p-value of this correlation are indicated on each plot. For these correlation analyses, the median summarizedexpression was used. (C) GSE3325 was processed by downloading the raw data as .CEL files from GeneExpression Omnibus (GEO) and performing RMA normalization resulting in log2-RMA normalized expression values. Boxplots are provided using the median summarized expression and at each individual probe set level (in case that is of interest). (D) Scatterplots weregenerated to visualized the relationship in expression between each pair ofgenes (PTEN-STAT3, PTEN-EIF4EBP1 and STAT3-EIF4EBP1) in prostate cancer samplesin GSE3325. The Pearson correlation ­­(R) and the p-value of this correlation are indicated on each plot. For these correlation analyses, the median summarized expression was used. (E) PTEN, EIF4EBP1, EIF4E mRNA levels of 247 patients with localized/locally advanced PCa commencing radical radiotherapy (with/without ADT) analyzed by RNA-Seq (Jain et al., 2018) [58]. Groups were generated by a median split. Significance was estimated by log-rank test (95% confidence interval) and p-value was adjusted with Benjamini-Hochberg method. + = censored. (F) Boxplot representing protein expression of p-4E-BP in cytoplasmatic or nuclear stainings detected by IHC in normal-like glands or tumors in PCa patient TMAs (n=83). (G) Evaluation and Gleason pattern annotations of nuclear and cytoplasmic IHC stainings of p4E-BP1 in PCa patient TMAs (n=83). Boxplots show median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Kruskal-Wallisand Dunn’s all pair tests were performed to assess significance (95% confidence interval). Supplementary Fig. 6. (A) Boxplot representing CREB nuclear protein expression stainings and representative images detected by IHC in PCa patient TMAs (n=83), (low GSS≤7 and high GSC=8-10). (B) Kaplan–Meier analysis of BCR-free survival ratio based on CREB protein expression in a panel of 59 PCa patients (PSA≥0.4ng/mL). Supplementary Table 1.