Regulates Prostate Cancer Cell Growth Research Articles

TRIM65 regulates prostate cancer cell growth and stemness through the WNT/β-catenin axis

TRIM65 regulates prostate cancer cell growth and stemness through the WNT/β-catenin axis

MIRO2 Regulates Prostate Cancer Cell Growth via GCN1-Dependent Stress Signaling.

MIRO2/GCN1/GCN2 constitute a novel mitochondrial signaling pathway that controls androgen-independent and androgen-sensitive prostate cancer cell growth.

LncRNA HAND2-AS1 Regulates Prostate Cancer Cell Growth Through Targeting the miR-106a-5p/RBM24 Axis [Retraction

[This retracts the article DOI: 10.2147/OTT.S246274.].

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

LINC00641 regulates prostate cancer cell growth and apoptosis via the miR-365a-3p/VGLL4 axis.

Long non-coding RNA (lncRNA) was frequently abnormally expressed in cancers. LINC00641 was reported to play crucial roles in regulating tumor progression. However, its role in prostate cancer (PCa) has not been fully explored. In this work, proliferation, invasion and apoptosis assays were performed to detect the biological roles of LINC00641 in PCa. Bioinformatic analyses, Luciferase activity reporter assay, and rescue experiments were performed to investigate the potential mechanisms of LINC00641 in PCa. Expression levels of LINC00641, microRNA-365a-3p (miR-365a-3p), and vestigial like family member 4 (VGLL4) in PCa tissues and normal tissues were analyzed at ENCORI. We found LINC00641 and VGLL4 was reduced, while miR-365a-3p was elevated expression in PCa tissues compared with normal tissues. LINC00641 overexpression inhibited growth and invasion abilities of PCa cells in vitro. Functional assays revealed that miR-365a-3p/VGLL4 pair was the downstream targets of LINC00641. The findings of our work provided evidence that LINC00641 serves as a tumor suppressive lncRNA in PCa by regulating miR-365a-3p/VGLL4 axis.

SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models.

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.

<p>lncRNA HAND2-AS1 Regulates Prostate Cancer Cell Growth Through Targeting the miR-106a-5p/RBM24 Axis</p>

IntroductionIncreasing evidence has shown that abnormally expressed long non-coding RNA (lncRNA) plays crucial roles in prostate cancer (PCa) progression.Materials and MethodsHere, we analyzed the expression level of lncRNA HAND2 antisense RNA 1 (HAND2-AS1) in PCa cells and tissues. Function assays were performed to investigate the biological roles of HAND2-AS1 in PCa cell behaviors. Bioinformatics methods, luciferase activity reporter assay, and RNA pull-down assay were performed to validate the connection of microRNA-106a-5p (miR-106a-5p) with HAND2-AS1. Also, the target of miR-106a-5p was explored using the same methods.ResultsOur results revealed HAND2-AS1 expression was decreased in both PCa cells and tissues. In vitro functional assays showed HAND2-AS1 could inhibit PCa cell proliferation and colony formation through promoting cell apoptosis. Dual-luciferase activity assays showed miR-106a-5p could directly bind with HAND2-AS1 and RNA binding motif protein 24 (RBM24). Mechanistically, we showed that HAND2-AS1 regulates PCa cell behaviors via targeting miR-106a-5p/RBM24 axis.ConclusionIn summary, our results illustrated that HAND2-AS1 functions as miR-106a-5p sponge to regulate RBM24 expression, and to influence PCa progression.

Circ_KATNAL1 regulates prostate cancer cell growth and invasiveness through the miR-145-3p/WISP1 pathway.

Prostate cancer (PCa) is the second leading cause of death in men, and current studies have shown that circular RNAs (circRNAs) play important roles in its occurrence and development. Detection of circRNAs in PCa cells showed that circ_KATNAL1 is down-regulated, mainly located in the cytoplasm, and contains multiple binding sites of miR-145-3p, which is an anticancer miRNA. RNA immunoprecipitation with anti-AGO2 antibody, RNA pull-down assays with biotin-labeled circ_KATNAL1 probe or an miR-145-3p mimic, and dual luciferase reporter gene assays confirmed that circ_KATNAL1 binds directly to miR-145-3p in cells, and that WISP1, which is highly expressed in many types of tumors, is an important target gene of miR-145-3p. Circ_KATNAL1 and miR-145-3p promote each other's expression, and down-regulate the expression of the target gene WISP1. Both circ_KATNAL1 and miR-145-3p inhibit cell proliferation, invasiveness, and migration, down-regulate the expression of MMP-2 and MMP-9, promote cell apoptosis and the activation of caspase-3, caspase-8, caspase-9, and PARP, whereas WISP1 has the opposite effect, and the above-mentioned functions of circ_KATNAL1 were achieved through the miR-145-3p/WISP1 pathway. Therefore, circ_KATNAL1 plays an anticancer role in PCa cells through the miR-145-3p/WISP1 pathway, which could be an important target for the diagnosis and treatment of PCa.

Discovery of a novel long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth

BackgroundVirtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA.MethodsRapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3’UTR of the lymphocyte-specific protein tyrosine kinase (LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth.ResultsIn this study, a previously unannotated lncRNA lying within exon six and 3’UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA “HULLK” for Hormone-Upregulated lncRNA within LCK. Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation.ConclusionsHULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa.

Iron-responsive element-binding protein 2 plays an essential role in regulating prostate cancer cell growth.

Iron-responsive element-binding proteins (IRPs) are master regulators of cellular iron homeostasis. Our previous work demonstrated that iron homeostasis is altered in prostate cancer and contributes to prostate cancer progression. Here we report that prostate cancer cells overexpress IRP2 and that overexpression of IRP2 drives the altered iron phenotype of prostate cancer cells. IRP2 knockdown in prostate cancer cell lines reduces intracellular iron and causes cell cycle inhibition and apoptosis. Cell cycle analysis demonstrates that IRP2-depleted prostate cancer cells accumulate in G0/G1 due to induction of p15, p21, and p27. Activation of these pathways is sufficient to significantly reduce the growth of PC3 prostate tumors in vivo. In contrast, IRP1 knockdown does not affect iron homeostasis and only modestly affects cell growth, likely through an iron-independent mechanism. These results demonstrate that upregulation of IRP2 in prostate cancer cells co-opts normal iron regulatory mechanisms to facilitate iron retention and drive enhanced tumor growth.

LncRNA PVT1 regulates prostate cancer cell growth by inducing the methylation of miR‐146a

Prostate cancer is the third most common causes of death from cancer in men. Our previous study demonstrated that lncRNA PVT1 was overexpressed and played an oncogenic role in the progression of prostate cancer. However, the molecular mechanism of modulating the prostate cancer tumorigenesis was still unknown. In this study, we aim to investigate the interaction between PVT1 and miR‐146a in prostate cancer and reveal the potential mechanism in prostate cancer carcinogenesis. The expression level of miR‐146a was assessed by quantitative RT‐PCR. The correlation analysis and methylation status analysis was made to confirm the interaction between PVT1 and miR‐146a. Biological function analysis was performed through gain‐of‐function and loss‐of‐function strategies. Our results showed that miR‐146a was downregulated and negatively correlated with PVT1 level in prostate cancer. PVT1 mediated miR‐146a expression by inducing the methylation of CpG Island in its promoter. miR‐146a overexpression eliminated the effects of PVT1 knockdown on prostate cancer cells. PVT1 regulated prostate cancer cell viability and apoptosis depending on miR‐146a. Our study suggested a regulatory relationship between lncRNA PVT1 and miR‐146a during the process of the prostate cancer tumorigenesis. PVT1 regulated prostate cancer cell viability and apoptosis depending on miR‐146a. It would contribute to the diagnosis, treatment and prognosis of prostate cancer.

Abstract 979: Identification of a novel long noncoding RNA within the LCK gene locus that regulates prostate cancer cell growth

Abstract Prostate cancer remains the second most common type of cancer and frequent cause of cancer-related mortality in American men. Even though many patients with metastatic prostate cancer will initially respond to androgen deprivation therapy, virtually all patients will relapse and develop lethal castration-resistant prostate cancer. Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and there is increasing evidence demonstrating that dysregulation of lncRNAs is associated with many human cancers, including cancers of the prostate, breast, and lung. We have discovered in a high-throughput RNAi screen identifying regulators of prostate cancer cell growth that knockdown of lymphocyte-specific protein tyrosine kinase (LCK) significantly decreases growth of prostate cancer cells in the presence and absence of androgen. Surprisingly, immunoprecipitation and western blot analyses show that LCK is not expressed at the protein level in prostate cancer cells. Rapid amplification of cDNA ends (RACE) and sequencing have revealed that a previously unannotated lncRNA lies within exon six and the 3’UTR of the LCK gene. While short hairpin RNAs (shRNAs) targeting the carboxy-terminus of the LCK gene decreases cell growth, expression of shRNAs specific for the amino-terminus has no effect on growth. Furthermore, only quantitative polymerase chain reaction (qPCR) primers directed toward the 3’ section of the LCK gene yield detectable levels of transcript. These data provide further validation for the existence of a lncRNA within the LCK gene locus. Remarkably, the lncRNA situated within the LCK gene is dramatically upregulated in response to androgen. Therefore, we have labeled this lncRNA “HULLK” for Hormone-upregulated lncRNA within LCK. Cellular fractionation and qPCR show that HULLK predominantly localizes to the cytoplasm. In addition to the effects on prostate cancer cell growth, we have data that alludes to the increase in transcript levels of several Src family members following depletion of HULLK. Thus, these studies indicate that the LCK gene contains a lncRNA involved in the regulation of prostate cancer cell growth and perhaps transcription. While additional analyses will be required to fully characterize HULLK, our data suggest that it may serve as a novel regulator of prostate cancer proliferation. Citation Format: Huy Q. Ta, Samuel R. Jackson, Hilary Whitworth, Shriti Bhadel, Daniel Gioeli. Identification of a novel long noncoding RNA within the LCK gene locus that regulates prostate cancer cell growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 979.

Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth.

Prostate cancer is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated prostate cancer initially respond to androgen deprivation therapy (ADT), but virtually all patients will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating prostate cancer cell growth led to our discovery that checkpoint kinase 2 (CHK2) knockdown dramatically increased prostate cancer growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects prostate cancer proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. In addition, we provide evidence that CHK2 physically associates with the AR and that cell-cycle inhibition increased this association. Finally, IHC analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and prostate cancer growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation.

Abstract 5049: Checkpoint kinase 2 is a novel regulator of prostate cancer cell growth

Abstract Prostate cancer (PCa) is the second leading cause of cancer death in American men, and the cure for metastatic disease remains a significant challenge. While nearly all patients with disseminated PCa initially respond to androgen deprivation therapy (ADT), virtually every patient will relapse and develop incurable castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling pathways continue to play a crucial role in CRPC progression. Previous studies have shown that signal transduction pathways can stimulate AR activation, suggesting that the ability of signaling cascades to influence AR function may have a significant role in CRPC progression, and that CRPC may not be effectively treated by ligand-directed therapy alone. A high-throughput RNAi screen identifying signaling pathways that regulate PCa cell growth led to our discovery that knockdown of Checkpoint Kinase 2 (CHK2) dramatically increased PCa proliferation in the presence and absence of androgen. Furthermore, CHK2 depletion hypersensitized cells to castrate androgen levels. These CHK2-mediated effects on growth were dependent on the downstream signaling proteins CDC25C and CDK1 and could be blocked by the AR antagonist MDV3100. Immunohistochemical analysis of CHK2 in patient samples demonstrated that reduced CHK2 expression significantly correlated with increased Gleason score indicating the clinical relevance of CHK2 in PCa. Moreover, CHK2 expression is lower in castration-resistant C4-2 and CWR22Rv1 cells compared to androgen-sensitive LNCaP cells consistent with loss of CHK2 expression during PCa progression. CHK2 depletion increased AR transcriptional activity on both androgen-activated and androgen-repressed genes, substantiating that CHK2 affects PCa growth through the AR. Remarkably, quantitative PCR, chromatin immunoprecipitation, and western blot analyses revealed that CHK2 is a novel AR-repressed gene, suggesting a negative feedback loop between CHK2 and the AR. Furthermore, we show that CHK2 physically associates with the AR, and that cellular stress, such as DNA damage and serum starvation, increases this association. Based on these data, we propose that CHK2 is a negative regulator of androgen sensitivity and PCa growth. Thus, alterations to CHK2 signaling may sensitize CRPC to ADT and radiation. Citation Format: Huy Q. Ta, Melissa L. Ivey, Henry F. Frierson, Mark R. Conaway, Jaroslaw Dziegielewski, James M. Larner, Daniel Gioeli. Checkpoint kinase 2 is a novel regulator of prostate cancer cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5049. doi:10.1158/1538-7445.AM2015-5049

TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation.

CYP3A5 regulates prostate cancer cell growth by facilitating nuclear translocation of AR.

The central role of androgen receptor (AR) signaling is established in prostate cancer growth and progression. We propose CYP3A5 is part of a feedback loop that modulates the sensitivity of AR to androgen exposure. The purpose of this study is to elucidate the mechanism of regulation of AR expression by CYP3A5. To identify the role of CYP3A5 in regulating AR signaling, CYP3A5 protein expression was inhibited using CYP3A5 siRNA and azamulin. Both cell fractionation and immunocytochemical approaches in combination with dihydrotestosterone (DHT) and R1881 treatment were used to evaluate changes in AR nuclear translocation. CYP3A5 siRNA blocked growth of LNCaP and C4-2 cells by 30-60% (P ≤ 0.005). Azamulin, a CYP3A pharmacologic inhibitor, reduced the growth of LNCaP, C4-2 and 22RV1 lines by ∼ 40% (P ≤ 0.005). CYP3A5 siRNA inhibited growth in response to DHT and R1881 treatment in LNCaP and C4-2 by decreasing nuclear AR localization and resulting in diminished PSA and TMPRSS2 expression. Decreased AR nuclear localization resulting from CYP3A5 inhibition resulted in growth inhibition comparable to IC60 and IC40 of bicalutamide in LNCaP and C4-2 cell lines. Conversely, the CYP3A inducer rifampicin enhanced AR nuclear localization. As CYP3A5 regulates the nuclear translocation of AR; co-targeting CYP3A5 may provide a novel strategy for enhancing the efficacy of androgen deprivation therapy. Consequentially, these data suggest that concomitant medications may impact androgen deprivation therapy's efficacy.

SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin.

Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth.

Abstract 2450: CaMKK2-AMPK signaling facilitates androgen-mediated prostate cancer cell metabolism

Abstract Prostate cancer is the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. While we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of the disease, it is poorly understood what AR-regulated processes drive this pathology. Previous work from several independent laboratories has suggested AR signaling promotes prostate cancer through a Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKK2)-dependent signaling mechanism. Further, it was demonstrated this enzymatic cascade promotes the use of sugars for cellular energy, a common trait for many cancers. Given CaMKK2's known role as a regulator of various aspects of metabolism, we hypothesized that CaMKK2 could promote prostate cancer cell growth through additional mechanisms beyond glycolysis. To test our hypothesis, we used clinical samples to track the activation of downstream signaling components of CaMKK2 in prostate cancer. The mechanistic role of these components was then identified using a variety of cell-based assays and xenograft mouse models of prostate cancer. Here, we demonstrate that AR-mediated CaMKK2 signaling regulates prostate cancer cell growth via the metabolic sensor 5′-AMP-activated protein kinase (AMPK), a kinase that classically regulates cellular energy homeostasis. In patients, activation of AMPK correlated with prostate cancer progression. Using a combination of radiolabeled assays and emerging metabolomic approaches, we also show that prostate cancer cells respond to androgen treatment by increasing not only rates of glycolysis, as is commonly seen in many cancers, but also glucose and fatty acid oxidation. Importantly, this effect was dependent on androgen-mediated AMPK activity. Our results further indicate that the AMPK-mediated metabolic changes increased intracellular ATP levels and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis, affording distinct growth advantages to the prostate cancer cells. Correspondingly, we used outlier analysis to determine that PGC-1α is overexpressed in a subpopulation of clinical cancer samples. This was in contrast to what was observed in immortalized benign human prostate cells and a testosterone-induced rat model of benign prostatic hyperplasia. Taken together, our findings converge to demonstrate that androgens can co-opt the CaMKK2-AMPK-PGC-1α signaling cascade, a known homeostatic mechanism, to increase prostate cancer cell growth. The current study points to the potential utility of developing metabolic-targeted therapies directed towards the CaMKK2-AMPK-PGC-1α signaling axis for the treatment of prostate cancer. Citation Format: Daniel E. Frigo, Yan Shi, Jenny J. Han, Efrosini Tsouko, Michael M. Ittmann. CaMKK2-AMPK signaling facilitates androgen-mediated prostate cancer cell metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2014-2450

Androgens regulate prostate cancer cell growth via an AMPK-PGC-1α-mediated metabolic switch

Prostate cancer is the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. While we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of the disease, it is poorly understood what AR-regulated processes drive this pathology. Here, we demonstrate that AR regulates prostate cancer cell growth via the metabolic sensor 5′-AMP-activated protein kinase (AMPK), a kinase that classically regulates cellular energy homeostasis. In patients, activation of AMPK correlated with prostate cancer progression. Using a combination of radiolabeled assays and emerging metabolomic approaches, we also show that prostate cancer cells respond to androgen treatment by increasing not only rates of glycolysis, as is commonly seen in many cancers, but also glucose and fatty acid oxidation. Importantly, this effect was dependent on androgen-mediated AMPK activity. Our results further indicate that the AMPK-mediated metabolic changes increased intracellular ATP levels and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis, affording distinct growth advantages to the prostate cancer cells. Correspondingly, we used outlier analysis to determine that PGC-1α is overexpressed in a subpopulation of clinical cancer samples. This was in contrast to what was observed in immortalized benign human prostate cells and a testosterone-induced rat model of benign prostatic hyperplasia. Taken together, our findings converge to demonstrate that androgens can co-opt the AMPK-PGC-1α signaling cascade, a known homeostatic mechanism, to increase prostate cancer cell growth. The current study points to the potential utility of developing metabolic-targeted therapies directed towards the AMPK-PGC-1α signaling axis for the treatment of prostate cancer.

Androgens Upregulate Cdc25C Protein by Inhibiting Its Proteasomal and Lysosomal Degradation Pathways

Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein.