STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Abstract

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Keywords: AMPK; AR; CREB; LKB1; Metformin; Prostate Cancer; STAT3; mTORC1.

Fig. 1

Loss of STAT3 accelerates metastatic progression and exhibits decreased LKB1/AMPK signaling. A High occurrence of PTEN and STAT3 deletions in plasma samples of PCa patients with aggressive PCa (n = 95). B Kaplan–Meier cumulative survival analysis revealed a significant (p = 0.0026; log-rank test) increase in lifespan of Pten^pc−/−Stat3^C/+ compared to Pten^pc−/−Stat3^pc−/− mice; WT and Pten^pc−/− mice served as controls (n = 68). C Prostate weights of 19-week-old WT, Pten^pc−/−, Pten^pc−/−Stat3^pc−/− and Pten^pc−/−Stat3^C/+ mice (n = 99). Mean values are shown; Data were analyzed by one-way analysis of variance with Tukey’s multiple comparison test; error bars: s.d. D IHC of muscle and mesentery, 52 weeks of age WT, Pten^pc−/−, Pten^pc−/−Stat3^C/+ and Pten^pc−/−Stat3^pc−/− mice. MET- metastasis, Scale bars, 100 μm; insets: × 600 magnification. Percentage of mice with distant PCa metastases (n = 76). E Haematoxilin/eosin (H&E) stains show only high-grade PIN in Pten^pc−/−Stat3^C/+ mice compared with Pten^pc−/− and Pten^pc−/−Stat3^pc−/− mice. Scale bars, 100 μm. IHC analysis of Stat3, Lkb1 and p-Ampk in prostates from 19-week-old WT, Pten^pc−/−, Pten^pc−/− Stat3^pc−/− and Pten^pc−/−Stat3^C/+ mice. Scale bars, 100 μm. Bar graphs indicate percentage of cells positive for Stat3, Lkb1 and p-Ampk in prostates of 19-week-old WT, Pten^pc−/−, Pten^pc−/−Stat3^pc−/− and Pten^pc−/−Stat3.^C/+. Protein levels quantification was done with HistoQuest software (n ≥ 3)

Fig. 2

STAT3 and LKB1 cooperate to suppress mTORC1. A Western blot analysis of STAT3 and LKB1 in 22Rv1 cells transfected with non-targeting (NT) or shRNAs specific for STAT3 and/or LKB1. B qRT–PCR analysis of STAT3 and STK11 in 22Rv1 cells transfected with NT or shRNA specific for STAT3 (n = 3 each). C ChIP analysis of STAT3 binding to LKB1 (STK11) promoter. 22Rv1 cells harboring NT or two different shSTAT3 constructs were stimulated with IL-6 and immunoprecipated with STAT3 antibody or IgG as a negative control. Bars represent mean ± s.d. from 2 technical replicates. Precipitated DNA is presented as % of input. D IHC analysis of p-4E-BP1, 4E-BP1, p-S6 in prostates from 19-week-old WT, Pten^pc−/−, Pten^pc−/− Stat3^pc−/− and Pten^pc−/−Stat3^C/+ mice. Scale bars, 100 μm. E Western blots analysis of p-4E-BP1, 4E-BP1, p-S6 and S6 expression in prostates from 19-week-old WT, Pten^pc−/−, Pten^pc−/− Stat3^pc−/− and Pten^pc−/−Stat3^C/+ mice. β-actin serves as a loading control. F H&E and IHC analyses of Stat3, p-S6 and p-4E-BP1 in prostates and lung metastases from Pten^pc+/− and Pten^pc+/− Stk11^pc−/− mice, scale bars, 100 μm. Quantification of cells positive for STAT3 and p-4E-BP1 in 19-week-old Pten^pc+/− and Pten^pc+/− Stk11^pc−/− prostate tissue or lung metastases using HistoQuest software (n = 3). Data were analyzed by Student’s t-test and are shown as mean ± s.d

Fig. 3

STAT3 is a central regulator of mTORC1 and CREB signaling. A, B Signature and heatmap of Stat3 and Stat3-regulated proteins in FFPE laser-microdissected prostates of 19-week-old WT, Pten^pc−/−, Pten^pc−/−Stat3^pc−/− and Pten^pc−/−Stat3^C/+ (n = 3 each) using unbiased LC–MS/MS proteomics. C The heatmap shows reprogramming of mTOR and CREB metabolic pathways in PCa with significant enrichment (hypergeometric test, q-value < 0.05). D Western blot analysis of p-CREB, CREB, p-4E-BP1, 4E-BP1, STAT3, p-S6, S6, AR and PSA of LNCaP xenograft tumors treated with vehicle or ruxolitinib (50 mg kg⁻¹) for 22 days. β-actin serves as a loading control. E Western blot analysis of p-CREB, CREB, AR, AR-V7 in 22Rv1 cells transfected with non-targeting (NT) or shRNAs specific for STAT3. Western blot analysis of STA3, p-CREB and CREB in PC3 cells transfected with an empty vector (EV) or STAT3 add-back. PC3 cells lacks STAT3 expression

Fig. 4

Clinically relevant dose of metformin inhibits CREB/mTORC1 in a manner that requires AR and STAT3 signaling. A IHC of radical prostatectomy specimens (benign and cancer core) stained with STAT3 and p-4E-BP1. TMA including benign and cancerous tissue with either metformin (n = 41) or patients without antidiabetic medication (n = 39) was employed as described previously [46]. B Gross anatomy assessment of representative 22Rv1 and PC3 xenograft tumors treated with vehicle or metformin (50 mg/kg; i.p.). Scale bars, 10 mm. Mean values are shown; error bars: s.d. (n = 5). C 22Rv1 and PC3 cells were implanted subcutaneously in mice and grown until tumors reached the size of approximately 100mm³. Xenografted mice were randomized and then received (n = 5 per group) vehicle or 50 mg/kg metformin i.p. daily. Mean tumor volume ± s.d. is shown. D Tumor weights assessment of representative 22Rv1 and PC3 xenograft tumors treated with vehicle or metformin (50 mg/kg; i.p.). Scale bars, 10 mm. Mean values are shown; error bars: s.d. (n = 5). E Comparison of IC₅₀ values of metformin for human PCa cell lines (22Rv1, DU-145 and PC3) and untransformed human prostate cell line RWPE-1. F Western blot analysis of STAT3, LKB1, p-CREB, CREB, p-4E-BP1, 4E-BP1, p-S6 and S6 in 22Rv1 and PC3 xenograft tumors. β-actin serves as a loading control. Sal…physiological saline solution; Met…metformin. G Western blot analysis of AR and PSA in 22Rv1 and PC3 xenograft tumors treated with vehicle or metformin (50 mg/kg; i.p.). β-actin serves as a loading control

Fig. 5

STAT3 and PTEN are negatively correlated with mTORC1 in PCa. A Heatmaps depicting significant downregulation of STAT3 and PTEN mRNA levels and concomitant upregulation of EIF4EBP1 mRNA expression in prostate carcinoma patient samples (n = 13) compared with healthy prostate gland tissues (n = 8). Colors were normalized to depict relative mRNA expression values (log2 median-centered intensity) within each row; dark blue represents the lowest relative expression level and dark red represents the highest relative expression level. Data were extracted from the Oncomine™ Platform [59] and from the Arredouani Prostate study [55]. B Gene expression levels depicting significant downregulation of STAT3 (-1.57-fold) and PTEN (-1.26-fold) mRNA and concomitant upregulation of EIF4EBP1 mRNA (1.77-fold) in prostate carcinoma patients (n = 13) compared with normal prostate gland samples (n = 8). Data (log2 median-centered intensity) were extracted from the Oncomine™ Platform from the Arredouani Prostate dataset. Representation: boxes as interquartile range, horizontal line as the mean, whiskers as lower and upper limits. C Heatmap depicting significant downregulation of STAT3 and PTEN mRNA levels and concomitant upregulation of EIF4EBP1 mRNA expression in prostate carcinoma patients compared with normal prostate gland samples (log2 median-centered intensity). Data were extracted from the Oncomine™ Platform from the Lapointe Prostate dataset. D Gene expression levels depicting significant downregulation of STAT3 (-1.44-fold) and PTEN (-1.72-fold) mRNA and concomitant upregulation of EIF4EBP1 mRNA (1.37-fold) in prostate carcinoma patients (n = 59–62) compared with normal prostate gland samples (n = 37–41). Data (log2 median-centered intensity) were extracted from the Oncomine™ Platform from the Lapointe Prostate dataset. Representation: boxes as interquartile range, horizontal line as the mean, whiskers as lower and upper limits. E Boxplots representing protein expression of STAT3 and 4E-BP1 in cytoplasmatic or nuclear stainings detected by IHC in normal-like glands or tumors in PCa patient TMAs (n = 83)

Fig. 6

CREB signaling predicts ADT-resistance and metastatic progression in PCa patients. A Representative IHC images and boxplots representing protein expression of CREB in nuclear stainings detected by IHC in normal-like glands or tumor cells in PCa patient TMAs (n = 83). B Kaplan–Meier analysis of BCR-free survival ratio based on CREB protein expression in a panel of 59 PCa patients (PSA $\ge$ 0.2 ng/L). C Association of CREB expression at predicting time to biochemical recurrence of high/low-risk disease in the resection cohort. Reduced progression-free survival in months of the “high-risk” subgroup (blue) of 161 patients when compared with the “low-risk” subgroup (red) of 161 patients (HR = 1.56 [1.13–2.16]; p < 0.0073). D Association of CREB expression at predicting time to metastatic disease recurrence of high/low-risk disease in the resection cohort. Reduced progression-free survival in months of the “high-risk” subgroup (red) of 161 patients compared with the “low-risk” subgroup (blue) of 161 patients (HR = 2.09 [1.15–3.83]; p < 0.0164). HR = hazard ratio. (E) LNCaP xenograft model was serially passaged in castrated NSG males. WB analysis of AR, p-CREB, CREB, STAT3 protein expression in LNCAP AD/AI tumors. β-actin serves as a loading control

Fig. 7

Schematic representation of the role of STAT3 in prostate cancer. In a mouse model of PCa with PTEN loss, deletion of STAT3 results in decreased levels of LKB1/pAMPK but increased activation of mTOR signaling, leading to the development of mPCa. In contrast, constitutively activation of STAT3 results in high levels of LKB1/pAMPK and decreased activity of the mTORC1/CREB pathway, thus, preventing the development of cancer in mice with PTEN loss