AMP-activated protein kinase promotes human prostate cancer cell growth and survival

Abstract

The molecular mechanisms underlying the development and progression of prostate cancer are poorly understood. AMP-activated protein kinase (AMPK) is a serine-threonine kinase that is activated in response to the hypoxic conditions found in human prostate cancers. In response to energy depletion, AMPK activation promotes metabolic changes to maintain cell proliferation and survival. Here, we report prevalent activation of AMPK in human prostate cancers and provide evidence that inhibition or depletion of AMPK leads to decreased cell proliferation and increased cell death. AMPK was highly activated in 40% of human prostate cancer specimens examined. Endogenous AMPK was active in both the androgen-sensitive LNCaP cells and the androgen-independent CWR22Rv1 human prostate cancer cells. Depletion of AMPK catalytic subunits by small interfering RNA or inhibition of AMPK activity with a small-molecule AMPK inhibitor (compound C) suppresses human prostate cancer cell proliferation. Apoptotic cell death was induced in LNCaP and CWR22Rv1 cells at compound C concentrations that inhibited AMPK activity. The evidence provided here is the first report that the activated AMPK pathway is involved in the growth and survival of human prostate cancer and offers novel potential targets for chemoprevention of human prostate cancer.

Figure 1. AMPK is activated in human prostate cancer

Phosphorylated-ACC expression in normal human prostate (A) and primary human prostate cancers (B) was analyzed by immunohistochemical staining using a polyclonal anti-phospho-ACC antibody in paraffin-embedded tissue sections. C, Staining intensity of phosphorylated ACC in normal and malignant prostate tissues. D, Western blot analyses of extracts from various human prostate cancer cell lines. Cell lysates were analyzed by immunoblotting with anti-phospho-AMPK, anti-AMPK, anti-phospho-ACC or anti-ACC antibody. Blots were probed with anti-β-actin antibody to normalize for protein loading.

Figure 2. AMPK is highly activated in human prostate cancer cells

A, Time course of AMPK activation by AICAR in RWPE-1 and CWR22Rv1 cells. Cells were treated for the indicated hours with AICAR (0.5 mM). Cell lysates were resolved by 4−12% SDS-PAGE and probed with anti-phospho-AMPK, anti-AMPK, anti-phospho-ACC or anti-ACC antibody. Blots were probed with anti-β-actin antibody to normalize for protein loading. B, Inhibition of AMPK activity promotes nuclear localization of TORC2 in human prostate cancer cells. LNCap or CWR22Rv1 cells transiently expressing EGFP-TORC2 were exposed to either vehicle control, CC (10 μM) or AICAR (100 μM) for 2 hours. EGFP-TORC2 was visualized by fluorescence microscopy.

Figure 3. Downregulation of AMPK's catalytic subunits by siRNA decreases cellular proliferation in human prostate cancer cells

LNCap or CWR22Rv1 cells were transfected with a mixture of siRNA targeting the two AMPK catalytic subunits (AMPK) or luciferase (Luc). A, Western blot analysis showing 72 hours after transfection of LNCap or CWR22Rv1 with siRNA targeting the AMPK catalytic subunits or Luc. Cell lysates, prepared from cancer cell lines, were resolved by 4−12% SDS-PAGE and probed with anti-AMPK, anti-phospho-ACC or anti-ACC antibody. B, At the indicated number of days post-transfection, cells were collected and counted. Results are representative of three independent experiments. *, Significantly different (P < 0.05) from control cells.

Figure 4. Dose-dependent and time-dependent inhibition of AMPK activity by Compound C

CWR22Rv1 cells were treated with various concentrations of CC for 24 hours (A) or with CC (10 μM) for various times as indicated (B). Samples were collected and analyzed by Western blots using antibodies for phospho-ACC, ACC, phospho-AMPK and AMPK. Blots were probed with anti-beta-actin antibody to normalize for protein loading.

Figure 5. Inhibition of AMPK activity by CC decreases cellular proliferation in human prostate cancer cells

CWR22Rv1 (A) or LNCap (B) cells were treated with various concentration of CC as indicated. At the indicated number of days post-initiation of treatment, cells were collected and counted. C, CWR22Rv1 cells treated with various concentrations of CC for 24 hours were assayed for BrdU incorporation. The number in each panel indicates percentage of BrdU positive cells.

Figure 6. Inhibition of AMPK activity by CC induces apoptotic cell death in human prostate cancer cells

Western blot analyses of PARP-1 cleavage in CWR22Rv1 cells after exposure to various concentrations of CC for 24 hours (A) or 10 μM of CC for the indicated times (B). Samples were collected and analyzed using an antibody to PARP-1. Blots were probed with anti-beta-actin antibody to normalize for protein loading. C, CC induced apoptosis in CWR22Rv1 cells as determined by flow cytometry. Cellular apoptosis were detected as an increased hypodiploid fraction (sub G1) after treatment with DMSO or CC (10 μM) for 48 hours.