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Inadequate nutrient intake leads to oxidative stress disrupting homeostasis, activating signaling, and altering metabolism. Oxidative stress serves as a hallmark in developing prostate lesions, and an aggressive cancer phenotype activating mechanisms allowing cancer cells to adapt and survive. It is unclear how adaptation and survival are facilitated; however, literature across several organisms demonstrates that a reversible cellular growth arrest and the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), contribute to cancer cell survival and therapeutic resistance under oxidative stress. We examined adaptability and survival to oxidative stress following nutrient deprivation in three prostate cancer models displaying varying degrees of tumorigenicity. We observed that reducing serum (starved) induced reactive oxygen species (ROS) which provided an early stress environment and allowed cells to confer adaptability to increased oxidative stress (H2O2). Measurement of cell viability demonstrated a low death profile in stressed cells (starved+H2O2), while cell proliferation was stagnant. Quantitative measurement of apoptosis showed no significant cell death in stressed cells suggesting an adaptive mechanism to tolerate oxidative stress. Stressed cells also presented a quiescent phenotype, correlating with NF-κB expression, suggesting a mechanism of tolerance. Our data suggests that nutrient deprivation primes prostate cancer cells for adaptability to oxidative stress and/or a general survival mechanism to anti-tumorigenic agents.

Inadequate nutrient intake leads to oxidative stress disrupting homeostasis, activating signaling, and altering metabolism. Oxidative stress serves as a hallmark in developing prostate lesions, and an aggressive cancer phenotype activating mechanisms allowing cancer cells to adapt and survive. It is unclear how adaptation and survival are facilitated; however, literature across several organisms demonstrates that a reversible cellular growth arrest and the transcription factor, nuclear factorkappaB (NF-?B), contribute to cancer cell survival and therapeutic resistance under oxidative stress. We examined adaptability and survival to oxidative stress following nutrient deprivation in three prostate cancer models displaying varying degrees of tumorigenicity. We observed that reducing serum (starved) induced reactive oxygen species which provided an early oxidative stress environment and allowed cells to confer adaptability to increased oxidative stress (H2O2). Measurement of cellviability demonstrated a low death profle in stressed cells (starved+H2O2), while cell proliferation was stagnant. Quantitative measurement of apoptosis showed no signifcant cell death in stressed cells suggesting an adaptive mechanism to tolerate oxidative stress. Stressed cells also presented a quiescent phenotype, correlating with NF-?B nuclear translocation, suggesting a mechanism of tolerance. Our data suggests that nutrient deprivation primes prostate cancer cells for adaptability to oxidative stress and/or a general survival mechanism to anti-tumorigenic agents.

The G-protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4) plays an important role in prostate cancer metastasis. CXCR4 is regarded as a plasma membrane receptor, that it transmits signals that support transformation, progression and metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutic approaches such as antagonists and monoclonal antibodies have focused on receptors the located at the plasma membrane. An emerging concept for GPCRs is that they can localize to the nucleus where they may retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 is highly expressed in high grade metastatic prostate cancer tissues. Increased expression of CXCR4 is also detected in several prostate cancer cell lines as compared to normal prostate epithelial cells. Our studies identify a nuclear pool of CXCR4 and also define a mechanism for nuclear targeting of CXCR4. A classical nuclear localization sequence (cNLS), "RPRK", in CXCR4 can contribute to nuclear localization. In addition, CXCR4 interacts with the nuclear transport receptor, Transportin ?i, to promote nuclear accumulation of CXCR4. Importantly, G?i immunoprecipitation and calcium mobilization studies indicate that nuclear CXCR4 is functional and can participate in G-protein signaling revealing that the nuclear pool of CXCR4 can retain function. Localization of functional CXCR4 to the nucleus may be a mechanism by which prostate cancer cells evade treatment, thus contributing to increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4. This study addresses the mechanism of nuclear targeting for CXCR4 and demonstrates that CXCR4 can retain function within the nucleus and provides important new information to illuminate what have previously been primarily clinical observations of nuclear CXCR4.

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), RPRK, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportin?1 and Transportin?1-binding to CXCR4 promoted its nuclear translocation. Importantly, G?i immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4. KEYWORDS: Prostate Cancer, G-Protein Coupled

Initial studies demonstrated that Inhibitor of DNA binding/differentiation protein 4 (Id4) acts as a tumor suppressor in prostate cancer (PCa). To further confirm and investigate the mechanism by which ID4 acts as a tumor suppressor, herein we concentrated on two different approaches. In the first approach we investigated ID4 role as a tumor suppressor by regulating AKT/PI3K pathway. In the second approach, we examined the role ofld4 in blocking the tumorogenic properties of metastatic PC3 cells. Phosphoinositide 3-kinase/Protein kinase B (PB/AKT) pathway regulates multiple biological processes leading to cell survival, proliferation and growth in cancerous cells. We performed immunohistochemistry (IHC) to determine AKT, pAKT and PTEN protein expression in Id4 knockout mouse prostates and PCa cell lines. IHC on Id4 knockout mouse prostates demonstrated a significant decrease in PTEN expression as compared to normal mouse prostates. Consistent with decrease PTEN, the expression of pAKT expression increased. Similar pattern was observed in PCa cell lines: DU145 cells lacking Id4 had low PTEN as compared to ld4 over-expressing DU 145 cells. In addition, Chromatin immunoprecipitation (Ch!P) analysis also demonstrated an increase in the binding of acetylated p53 on PTEN promoter in the presence of Id4. The second approach demonstrated the tumor suppressor function of Id4 in highly tumorogenic and metastatic PC3 cell line. Interestingly, this study demonstrated that overexpression of Id4 in PC3 cells results in decreased tumorogenecity in part through increased expression of Androgen receptor (AR) and its target genes Cyclin dependent inhibitor I (p21) and FK506-binding protein 51 (FKBPS l ). Apoptosis, migration and cell proliferation decreased in the Id4 overexpressed PC3 cells. Mice injected with PC3 + Id4 cells showed decreased tumor size and volume. IHC studies on tumor xenografts demonstrated increased levels of AR, Ki67, and p21 in Id4 overexpressed xenograft. Coll

Inactivation of the tumor suppressor phosphatase and tensin liomolog deleted on chromosome 10 (PTEN) is heavily implicated in the tumorigenesis of prostate cancer. Conversely, the upregulation of the chemokine (CXC) receptor 4 (CXCR4) is associated with prostate cancer progression and metastasis. The inactivation of functional PTEN is typically due to genetic and epigenetic modulations, as well as active site oxidation by reactive oxygen species (ROS); likewise ROS upregulates CXCR4 expression. Furthermore, the pathways of CXCR4 and PTEN converge, leading to the promotion and regulation of turnorigenesis, respectively. Based on these observation we hypothesized that, PTEN inhibits CXCR4-mediated functions in prostate cancer cells. This hypothesis was investigated through the following aims: (1) To determine whether PTEN inhibits CXCR4-mediated functions in prostate cancer cells and, (2) To investigate a mechanism by which PTEN function is lost, and CXCR4 function is gained in prostate cancer cells. Upon screening advanced metastatic prostate cancer cell lines for PTEN, identified a loss of expression in PC3 and LNCaP cells, whereas DU145 expressed wildtype PTEN. All three cell lines were positive for surface expression of CXCR4. Reconstitution of PTEN induced a mesenchymal to epithelial-like morphological change and inhibited CXCR4-mediated migration and proliferation in PC3 cells. Downregulation of PTEN by siRNA enhanced the CXCR4-mediated migratory behavior of DU145 cells. By western blot analysis, we observed that PTEN inhibited basal Protein Kinase B (AKT) phosphorylation, but not extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in PTEN expressing cells. Upon CXCR4 stimulation, PTEN inhibited ERK1/2 phosphorylation, but not phosphorylation of AKT. The CXCR4- mediated migration of PC3 cells was through the ERK1/2 pathway, as confirmed by chemical inhibitors. Based on these studies, we suggest that loss of PTEN permits CXCR4-mediated functions in

Iroquois homeobox gene 3 (Irx3) is a transcription factor belonging to the Iroquois family of homeobox genes that is expressed in the embryonic organs of multiple organisms. Although much is known about Irx3 during embryogenesis, the cross talk of expression and function of Irx3 in peripheral vascular disease (PVD) remains to be investigated. Herein, we demonstrate that Endothelial PAS Domain 1 (EPAS1), which is involved in vasculogenesis and diseases associated with hypoxia, is an upstream regulator of Irx3, and this interaction contributes to the cellular movement of human microvascular endothelial cells (HMVECs). Genetic EPAS1 loss-of-function (LOF) studies in HMVECs resulted in a reduction of Irx3 mRNA, and Irx3-mediated endothelial cell migration in wound healing. In contrast, the effects of EPAS1Gain-of-function (GOF) in HMVECs showed increased Irx3-mediated endothelial cell migration in wound healing. Taken together, these results reveal that Irx3 is an important regulator of cellular movement as a downstream target of EPAS1 signaling in HMVECs, which regulates migration. KEY TERMS: Irx3, EPAS1, Angiogenesis, Cell Migration, HMVEC, Tie2, Biochemistry, Biophysics, and Structural Biology, Biology, Cancer Biology, Cell and Developmental Biology, Diseases, Medicine and Health Sciences, Molecular Biology, Other Cell and Developmental Biology

The expression of C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with increased metastatic potential of cancer cells. CXCR4 increases tumor malignancy by encouraging tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis provides a good strategy to inhibit the metastatic spread of tumor cells and slow cancer progression. Various studies suggest that cannabis may have anti-proliferative as well as anti-metastatic properties, though a biochemical mechanism describing how this occurs has yet to be discovered. Our lab has confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) can form heterodimers that play a role in decreasing cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231 and the prostate cancer cell line PC-3, with CXCL12 and AM1241, a synthetic ligand for CB2, desensitizes the intrinsic cellular response to migrate toward areas of high CXCL12 concentration. Furthermore, through co-immunoprecipitation and proximity ligation assays (PLA), we have determined that there is increased interaction between the two receptors with co-stimulation of respective agonists, providing evidence for the therapeutic notion that treating tumors that endogenously secrete CXCL12 with exogenous ligands for the cannabinoid can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Finally, to determine whether simultaneouslytreated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays to determine G-protein coupled receptor activation were employed. Results showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their indiv

The inhibitor of DNA binding protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Benezra, 1990 #1 Recently, Patel et al., demonstrated that inhibitor of differentiation 4 (ID4) acts as a tumor suppressor and its loss, frequently observed in prostate cancer, promotes castration-resistant prostate cancer (CRPC) through constitutive androgen receptor (AR) activation. Patel, 2014 #4 However, the mechanism by which loss of ID4 promotes constitutively active AR signaling in the CRPC conditions is unknown. The rationale of the present study was to unravel the underlying molecular mechanisms through which loss of ID4 potentiates AR signaling in this setting. Initially, chromatin immunoprecipitation (ChIP) assay results demonstrated a significant increase in binding of AR to its respective response elements on PSA, FKBP51, TMPRSS2, and ETV1 promoters in L(-)ID4 cells, further implicating constitutive AR activity. Among the notable findings, proteomic profiling between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated protein levels of Heat shock protein 27 (Hsp27) and the 52-kDa FK506-binding protein (FKBP52), suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies further confirmed a direct interaction between ID4 and FKBP52 in vitro but not with AR. Recent evidences suggest that FKBP52 is a positive regulator of AR signaling in cellular and whole animal models.Guy, 2015 #5 Thus, we hypothesized that ID4 acts as a tumor suppressor by selectively regulating AR activity through interaction with FKBP52. To address the underlying mechanism, we blocked the FKBP52-AR signaling using a specific inhibitory compound known as MJC13. Storer Samaniego, 2015 #6, De Leon, 2011 #8 The results demonstrated that MJC13 effectively inhibited AR-dependent expression and activity in a do

In recent years, the causal relationship between inflammation and cancer has gained wider acknowledgement and acceptance. While various types of immune cells are involved in the process of inflammation, macrophages represent the major inflammatory component of many tumors. Derived from circulating monocytes, these cells migrate to tumor sites in response to molecular cues present within the tumor microenvironment. Once there, interactions with neoplastic cells shape the differentiation and functional orientation of macrophages into two phenotypically distinct subsets: the classically activated M1 macrophages and the alternatively activated M2 macrophages. The preeminent paradigm in macrophage-related cancer research is that within the tumor stoma, macrophages acquire an M2 phenotype characterized by production of pro-angiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory responses, thereby promoting tumor progression. M1 macrophages, on the other hand are thought to exert anti-tumorigenic effects due to their production of pro-inflammatory cytokines, and reactive oxygen species (ROS). While the generation of ROS during immune responses is an important aspect of immune regulation and host defense, excessive ROS production has been implicated in the pathogenesis of various degenerative diseases, including cancer. Yet, despite the well-established role of M1 macrophages in generating high levels of ROS via NADPH oxidase (NOX), M1 macrophages are still largely viewed as anti-tumorigenic. Hence, this study reevaluates the complex interaction between prostate cancer (PCa) cells and tumor-associated macrophages (TAMs), and operates on the premise that PCa cells promote a pro-tumor microenvironment, denoted by increased inflammation and oxidative stress, in part, through M1 macrophage-mediated, NOX-derived ROS production. Accordingly, immunofluorescent analysis of prostate tissue microarrays demonstrated an influx of M1 macrophages in prostate carc