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 androgensensitive 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. [Mol Cancer Ther 2009;8(4):733-41]

BackgroundOur previous work on early PSA kinetics following prostate stereotactic body radiation therapy (SBRT) demonstrated that an initial rapid and then slow PSA decline may result in very low PSA nadirs. This retrospective study sought to evaluate the PSA nadir 5 years following SBRT for low- and intermediate-risk prostate cancer (PCa).Methods65 low- and 80 intermediate-risk PCa patients were treated definitively with SBRT to 35–37.5 Gy in 5 fractions at Georgetown University Hospital between January 2008 and October 2011. Patients who received androgen deprivation therapy were excluded from this study. Biochemical relapse was defined as a PSA rise >2 ng/ml above the nadir and analyzed using the Kaplan–Meier method. The PSA nadir was defined as the lowest PSA value prior to biochemical relapse or as the lowest value recorded during follow-up. Prostate ablation was defined as a PSA nadir <0.2 ng/ml. Univariate logistic regression analysis was used to evaluate relevant variables on the likelihood of achieving a PSA nadir <0.2 ng/ml.ResultsThe median age at the start of SBRT was 72 years. These patients had a median prostate volume of 36 cc with a median 25% of total cores involved. At a median follow-up of 5.6 years, 86 and 37% of patients achieved a PSA nadir ≤0.5 and <0.2 ng/ml, respectively. The median time to PSA nadir was 36 months. Two low and seven intermediate risk patients experienced a biochemical relapse. Regardless of the PSA outcome, the median PSA nadir for all patients was 0.2 ng/ml. The 5-year biochemical relapse free survival (bRFS) rate for low- and intermediate-risk patients was 98.5 and 95%, respectively. Initial PSA (p = 0.024) and a lower testosterone at the time of the PSA nadir (p = 0.049) were found to be significant predictors of achieving a PSA nadir <0.2 ng/ml.ConclusionSBRT for low- and intermediate-risk PCa is a convenient treatment option with low PSA nadirs and a high rate of early bRFS. Fewer than 40% of patients, however, achieved an ablative PSA nadir. Thus, the role of further dose escalation is an area of active investigation.

Voltage-gated sodium (Nav) channels are required for impulse conductance in excitable tissues. Navs have been linked to human cancers, including prostate. The expression and distribution of Nav isoforms (Nav1.1-Nav1.9) in human prostate cancer are not well established. Here, we evaluated the expression of these isoforms and investigated the expression of Nav1.8 in human prostate cancer tissues. Nav1.8 was highly expressed in all examined cells. Expression of Nav1.1, Nav1.2, and Nav1.9 were high in DU-145, PC-3 and PC-3M cells compared to LNCaP (hormone-dependent), C4-2, C4-2B, and CWR22Rv-1 cells. Nav1.5 and Nav1.6 were expressed in all cells examined. Nav1.7 expression was absent in PC-3M and CWR22Rv-1, but expressed in the other cells examined. Immunohistochemistry revealed intensive Nav1.8 staining correlated with more advanced pathologic stage of disease. Increased intensity of nuclear Nav1.8 correlated with increased Gleason grade. Our results revealed that Nav1.8 is universally expressed in human prostate cancer cells. Nav1.8 expression statistically correlated with pathologic stage (P=0.04) and Gleason score (P=0.01) of human prostate tissue specimens. The aberrant nuclear localization of Nav1.8 with advanced prostate cancer tissues warrant further investigation into use of Nav1.8 as a potential biomarker to differentiate between early and advanced disease.

Abstract. Raf-1 protein serine-threonine kinase plays an important role in cell growth, proliferation, and cell survival. Previously, we and others have demonstrated that antisense raf oligonucleotide-mediated inhibition of Raf-1 expression leads to tumor growth arrest, radiosensitization and chemosensitization in vivo. Raf-1 inhibition is also associated with apoptotic cell death. In this study, we inhibited Raf-1 using an antisense raf oligonucleotide (AS-raf-ODN) to identify downstream targets of Raf-1 using microarray gene expression analysis. Treatment of MDA-MB-231 breast cancer cells with 250 nM AS-raf-ODN led to significant inhibition of Raf-1 protein (75.2±9.6%) and c-raf-1 mRNA levels (86.2±3.3%) as compared to untreated control cells. The lipofectin control or mismatch oligonucleotide had no effect on Raf-1 expression. To determine the changes in gene expression profiles that were due to inhibition of Raf-1, we simultaneously compared the gene expression patterns in AS-raf-ODN treated cells with untreated control cells and cells treated with lipofectin alone or MM-ODN. A total of 17 genes (4 upregulated and 13 down-regulated) including c-raf-1 were identified that were altered after AS-raf-ODN treatment. Functional clustering analysis revealed genes involved in apoptosis (Bcl-X L ), cell adhesion (paxillin, plectin, Rho GDI·, CCL5), metabolism (GM2A, SLC16A3, PYGB), signal transduction (protein kinase C nu), and transcriptional regulation (HMGA1), and membrane-associated genes (GNAS, SLC16A3). Real-time PCR, Northern analysis and Western analysis confirmed the microarray findings. Our study provides insight into Raf-1 related signaling pathways and a model system to identify potential target genes.

Abstract. Raf-1 serine/threonine protein kinase plays an important role in cell growth, differentiation and cell survival. Recent reports using c-raf-1 gene-knockouts have observed MEK/ERK independent functions of Raf-1 in cell survival and protection from apoptosis. Raf-1 has also been shown to be involved in counteracting specific apoptotic pathways by restraining caspase activation, although the precise mechanism is unknown. XIAP is a potent inhibitor of apoptosis that blocks both the mitochondria and death receptor mediated pathways of apoptosis by directly binding to and inhibiting the initiator and effector caspases. In our efforts to understand the mechanism by which Raf-1 inhibits caspase activation, we discovered a novel interaction between Raf-1 and XIAP. In this study, we describe the physical interaction between Raf-1 and XIAP in vitro and in vivo in mammalian cells. We also demonstrate that Raf-1 phosphorylates XIAP in vitro and in vivo. Additionally, Raf-1 prevents XIAP degradation in response to different apoptotic triggers. Our studies identify XIAP as a new substrate of Raf-1 and provide potentially important insight into mechanisms underlying Raf-1 effects on cell survival.