Efrosini Tsouko

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. Although 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 toward the AMPK-PGC-1α signaling axis for the treatment of prostate cancer.

Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth.

Cancer cells display an increased demand for glucose. Therefore, identifying the specific aspects of glucose metabolism that are involved in the pathogenesis of cancer may uncover novel therapeutic nodes. Recently, there has been a renewed interest in the role of the pentose phosphate pathway in cancer. This metabolic pathway is advantageous for rapidly growing cells because it provides nucleotide precursors and helps regenerate the reducing agent NADPH, which can contribute to reactive oxygen species (ROS) scavenging. Correspondingly, clinical data suggest glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, is upregulated in prostate cancer. We hypothesized that androgen receptor (AR) signaling, which plays an essential role in the disease, mediated prostate cancer cell growth in part by increasing flux through the pentose phosphate pathway. Here, we determined that G6PD, NADPH and ribose synthesis were all increased by AR signaling. Further, this process was necessary to modulate ROS levels. Pharmacological or molecular inhibition of G6PD abolished these effects and blocked androgen-mediated cell growth. Mechanistically, regulation of G6PD via AR in both hormone-sensitive and castration-resistant models of prostate cancer was abolished following rapamycin treatment, indicating that AR increased flux through the pentose phosphate pathway by the mammalian target of rapamycin (mTOR)-mediated upregulation of G6PD. Accordingly, in two separate mouse models of Pten deletion/elevated mTOR signaling, Pb-Cre;Ptenf/f and K8-CreERT2;Ptenf/f, G6PD levels correlated with prostate cancer progression in vivo. Importantly, G6PD levels remained high during progression to castration-resistant prostate cancer. Taken together, our data suggest that AR signaling can promote prostate cancer through the upregulation of G6PD and therefore, the flux of sugars through the pentose phosphate pathway. Hence, these findings support a vital role for other metabolic pathways (that is, not glycolysis) in prostate cancer cell growth and maintenance.

MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer

MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.

Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer

The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC.

Glutamine Transporters are Targets of Multiple Oncogenic Signaling Pathways in Prostate Cancer

Despite the known importance of androgen receptor (AR) signaling in prostate cancer, the processes downstream of AR that drive disease development and progression remain poorly understood. This knowledge gap has thus limited the ability to treat cancer. Here, it is demonstrated that androgens increase the metabolism of glutamine in prostate cancer cells. This metabolism was required for maximal cell growth under conditions of serum starvation. Mechanistically, AR signaling promoted glutamine metabolism by increasing the expression of the glutamine transporters SLC1A4 and SLC1A5, genes commonly overexpressed in prostate cancer. Correspondingly, gene expression signatures of AR activity correlated with SLC1A4 and SLC1A5 mRNA levels in clinical cohorts. Interestingly, MYC, a canonical oncogene in prostate cancer and previously described master regulator of glutamine metabolism, was only a context-dependent regulator of SLC1A4 and SLC1A5 levels, being unable to regulate either transporter in PTEN wild-type cells. In contrast, rapamycin was able to decrease the androgen-mediated expression of SLC1A4 and SLC1A5 independent of PTEN status, indicating that mTOR complex 1 (mTORC1) was needed for maximal AR-mediated glutamine uptake and prostate cancer cell growth. Taken together, these data indicate that three well-established oncogenic drivers (AR, MYC, and mTOR) function by converging to collectively increase the expression of glutamine transporters, thereby promoting glutamine uptake and subsequent prostate cancer cell growth. Implications: AR, MYC, and mTOR converge to increase glutamine uptake and metabolism in prostate cancer through increasing the levels of glutamine transporters. Mol Cancer Res; 15(8); 1017–28. ©2017 AACR.

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 CaMKK2s 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

Abstract 217: Mitochondrial reprogramming regulated cancer pathway in triple negative breast cancer

Compared to other subtypes of tumors, triple negative breast cancers (TN BCa) currently suffer from limited knowledge on its etiology and treatment options. Transmitochondrial cybrids (cybrid) and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in TN BCa. Analysis of cybrids and established BCa cell lines showed that metastatic TN BCa maintain high levels of ATP through fatty acid β-oxidation and activate Src oncoprotein by its autophosphorylation. Inhibition and induction of β-oxidation including the shRNA mediated knockdown strategies, and analysis of patient derived xenograft (PDX) models confirmed the role of mitochondrial β-oxidation in Src activation and metastasis. Analysis of BCa clinical data further reaffirmed the role of mitochondrial β-oxidation in Src regulation and their significance in BCa metastasis. This study is innovative in showing the mitochondrial reprogramming mediated regulation of a major cancer pathway by its post-translation modification. Citation Format: Jun H. Park, Sajna Vithayathil, Danli Wu, Vasanta Putluri, Pi-Lin Sung, Efrosini Tsouko, Vadiraja B. Bhat, Cristian Coarfa, Daniel E. Frigo, Michael T. Lewis, Arun Sreekumar, Patricia Yotnda, Chad J. Creighton, Nagireddy Putluri, Lee-Jun C. Wong, Benny A. Kaipparettu. Mitochondrial reprogramming regulated cancer pathway in triple negative breast cancer. [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 217.

Abstract 1136: Regulation of AMPK by androgen receptor signaling and its role in promoting prostate cancer through the use of autophagy

While we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of prostate cancer, it is poorly understood what AR-regulated processes drive this pathology. Autophagy, an enigmatic cellular recycling process, has received increased attention as of late due to its potential oncogenic role in late-stage cancers. One of the master regulators of autophagy is the 5′-AMP-activated protein kinase (AMPK). Previous work from several independent laboratories has suggested AR signaling promotes cancer progression through an AMPK-dependent signaling mechanism. Further, it was demonstrated this enzymatic cascade was specifically regulated by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Given AMPK9s known role as a regulator of autophagy, we hypothesized that AR-mediated CaMKK2-AMPK signaling could promote prostate cancer through increasing cellular rates of autophagy. To test this hypothesis, we initially assessed whether AR signaling could regulate autophagy and if so, what effect(s) this had on prostate cancer cell growth and tumor formation. We then used a variety of pharmacological and molecular approaches to determine whether CaMKK2-AMPK signaling was required and sufficient to regulate autophagy and subsequent prostate cancer cell growth in vitro and in vivo. Here, we have determined that 1) AR regulates cell metabolism and cell growth in part by increasing autophagy in prostate cancer cells, 2) functional autophagy was clinically detected in metastatic, castration-resistant cancers but not treatment-naive, localized tumors and 3) autophagy is required for prostate cancer progression in preclinical animal models. Additionally, AR-mediated autophagy was mediated through the direct expression of CaMKK2 and subsequent phosphorylation/activation of AMPK. Correspondingly, levels of both CaMKK2 and phosphorylated/activated AMPK correlated with prostate cancer progression in genetic mouse models and patients. Mechanistically, AR-mediated autophagy appears to promote cell growth by augmenting intracellular lipid accumulation, a hallmark of prostate cancers. Taken together, our findings converge to demonstrate that AR signaling can co-opt the AMPK signaling cascade, a known homeostatic mechanism, to promote prostate cancer by increasing autophagy. The current study points to the potential utility of developing metabolic-targeted therapies directed towards the CaMKK2-AMPK signaling axis for the treatment of prostate cancer. Further, an inhibitor of this signaling cascade could serve as an alternative, more specific therapeutic compared to existing inhibitors of autophagy that, to date, have demonstrated limited efficacy in clinical trials due to their toxicity and poor pharmacokinetics. Citation Format: Yan Shi, Efrosini Tsouko, Alicia M. Blessing, Jayantha Tennakoon, Jenny J. Han, Michael M. Ittmann, Daniel E. Frigo. Regulation of AMPK by androgen receptor signaling and its role in promoting prostate cancer through the use of autophagy. [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 1136. doi:10.1158/1538-7445.AM2015-1136