Abstract 3182: Decreased expression of PTEN enhances CXCR4-mediated proliferation and tumorigenesis in prostate cancer cells

Abstract The progression of human prostate cancer is a result of metastasis from the primary tumor to vital organs. Metastasis is a complex process that involves invasion, intravasation, extravasation, and metastatic colonization. To date, the biology underlying the various mechanisms of metastasis has not been fully elucidated. Chemokines are pro-inflammatory molecules that bind to chemokine receptors, which are members of the G-protein coupled receptor (GPCR) family. The interaction between chemokines and their receptors results in a diverse array of biological and biochemical functions, such as chemotaxis, hematopoiesis and angiogenesis. Likewise, neoplastic cells employ chemokines and their receptors to promote metastasis, and encourage cell survival. It has been observed that the CXCR4 chemokine receptor is overexpressed on the cell surface of prostate cancer cells, which directed metastatic cells to tissues where its CXCL12 ligand is overexpressed, such as the bones and lungs. The Phosphate and Tensin homolog deleted on chromosome 10 (PTEN) is the second most mutated tumor suppressor in human cancer, and has been shown to be mutated in metastatic prostate cancer cells. Gao et al demonstrated that reconstituted PTEN in lymphocytes down-regulated CXCR4-mediated chemotaxis towards its ligand, CXCL12. Additionally, progenitor PTEN-deficient smooth muscle cells (SMC) increased migration towards CXCL12-producing SMC, suggesting that an alteration in PTEN expression negatively regulated CXCR4-mediated events. We analyzed prostate cancer cells for the expression of CXCR4 by flow cytometry, and observed that CXCR4 was expressed in human metastatic prostate cancer cell lines, PC3 and LnCaP. We analyzed for the expression of PTEN, and observed no expression of PTEN in PC3 and LnCaP cells at the protein level by western blot, nor at the mRNA level in PC3 cells by PCR. Furthermore, when reconstituted with PTEN, PC3 cells demonstrated a mesenchymal to epithelial-like phenotypic change in morphology. Additionally, we observed that reconstituted PTEN inhibited CXCR4-meditated proliferation via an MTT assay. Therefore, we hypothesize that the absence of PTEN expression correlates with an up-regulation of CXCR4-mediated proliferation and tumorigenesis in prostate cancer cells. In summary, our results showed that PTEN induced an epithelial-like morphological change, and inhibited CXCR4-mediated proliferation in metastatic prostate cancer cell lines. In future studies, we will investigate if the reconstituted expression of functional PTEN regulates CXCR4-mediated metastatic events in prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3182.</jats:p

PTEN regulation of ERK1/2 signaling in cancer

Since its discovery, the tumor suppressor phosphatase and tensin homolog (PTEN) has become a molecule with a wide spectrum of functions, which is typically meditated through its lipid phosphatase activity; however, PTEN also functions in a phosphatase-independent manner. It is well established that PTEN regulates several signaling pathways, such as phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), janus kinase (JAK)/signal transducers and activators of transcription (STAT), focal adhesion kinase (FAK), and more recent, extracellular signal-regulated kinase (ERK)1/2, where activation of these pathways typically leads to cancer development and progression. In regard to most of these pathways, the underlining molecular mechanism of PTEN-mediated regulation is well established, but not so much for the ERK1/2 pathway. Indeed, accumulating evidence has shown an inverse correlation between PTEN expression and ERK1/2 in several malignancies. However, the detailed mechanism by which PTEN regulates ERK1/2 is poorly understood. In this review, we discuss the role of PTEN in regulating ERK1/2 by directly targeting shc/Raf/MEK and PI3K/AKT cascades, and a putative cross-talk between the two

ROS enhances CXCR4-mediated functions through inactivation of PTEN in prostate cancer cells

Inactivation of the tumor suppressor phosphatase and tensin homolog 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. Studies have shown that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells. Loss of PTEN function is typically due to genetic and epigenetic modulations, as well as active site oxidation by reactive oxygen species (ROS); likewise ROS upregulates CXCR4 expression. Herein, we show that ROS accumulation permitted CXCR4-mediated functions through PTEN catalytic inactivation. ROS increased p-AKT and CXCR4 expression, which were abrogated by a ROS scavenger in prostate cancer cells. ROS mediated PTEN inactivation but did not affect expression, yet enhanced cell migration and invasion in a CXCR4-dependent manner. Collectively, our studies add to the body of knowledge on the regulatory role of PTEN in CXCR4-mediated cancer progression, and hopefully, will contribute to the development of therapies that target the tumor microenvironment, which have great potential for the better management of a metastatic disease

Abstract 538: ROS differentially regulates prostate cancer cell survival

Abstract In vitro and in vivo studies have shown that ROS accumulation correlates with increasing aggressiveness of prostate cancer cells, and increased expression of oncogenic molecules, such as CXCR4. CXCR4 is a G-Protein Coupled Receptor that is heavily implicated in the metastatic dissemination of prostate tumor cells to bone marrow. In cancer and other cell models, CXCR4 expression and activity increases in response to hypoxic environments and/or oxidative stress. Cells react by inducing expression of a HIF1α transcription factor, which enhances expression of targeted genes involved in adjusting to oxidative stress and cell survival. HIF1α has been shown to directly induce the expression of CXCR4 in prostate cancer cells. Therefore, generation of ROS in tumor tissues may critically influence CXCR4-mediated expression and functions, ultimately encouraging cancer progression. In this study, we elucidate a mechanism by which ROS induces CXCR4 expression and function in prostate cancer cells. We initially investigated the role of ROS on the phosphorylation of AKT (p-AKT) and ERK1/2 (p-ERK1/2) in DU145 and 22Rv1 prostate cancer cell lines. Using H2O2 as our model of ROS, we found that H2O2 induced a gradual expression of p-AKT and p-ERK1/2 by western blot analysis, which was abrogated by the ROS scavenger N-acetyl cysteine (NAC). It has been shown that PTEN is a negative regulator of AKT and ERK1/2 signaling, and that ROS accumulation inactivates PTEN phosphatase function; hence, we determined whether the increase in p-AKT and p-ERK1/2 expression was due to loss of PTEN function. By performing a phosphatase assay, we found that H2O2 inhibited PTEN catalytic activity compared to untreated cells, while NAC abrogated these effects in both cell lines. The increase in ROS and loss of PTEN has been shown to correlate with CXCR4-mediated function; therefore, we determined the role of H2O2 on CXCR4 expression. We found that H2O2 induced CXCR4 expression in DU145 cells, but not in 22Rv1 cells. Furthermore, we investigated whether ROS-mediated induction of CXCR4 was HIF1α dependent. Interestingly, H2O2 induced expression of HIF1α in DU145 cells, which was abrogated by siRNA for HIF1α. HIF1α expression decreased in 22Rv1 cells, which correlated with apoptosis, as confirmed by Annexin-V analysis. Finally, we observed that the H2O2-mediated increase in CXCR4 enhanced transendothelial invasion and migration in DU145 cells in a CXCR4 dependent manner. Our study suggests that ROS differentially regulates CXCR4 expression and function in prostate cancer cells, suggesting that ROS plays a differential role on heterogeneous tumor mass. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 538. doi:1538-7445.AM2012-538</jats:p

Abstract B23: Cysteine (C)-X-C receptor 4 induces oxidative stress through NADPH oxidase-2 in prostate cancer cells

Abstract Reactive oxygen species (ROS), and the coupled oxidative stress, have long been implicated in the onset and progression of many cancers, including prostate cancer (PCa). One of the mechanisms by which ROS contributes to cancer is by acting as a second messenger, relaying signals from the cell surface to important signaling proteins, thereby regulating a variety of cellular processes. We have demonstrated that ROS increased the expression and activity of the chemokine (CXC) receptor, type 4 (CXCR4), which resulted in proliferation, invasion and trans-endothelial migration of PCa cells. Similarly, a study in human hepatoma cells revealed that CXCR4, and its ligand, stromal cell derived factor 1 alpha (SDF-1α), promoted ROS accumulation; however, the source (s) of ROS following growth factor stimulation have not been investigated. Identifying the sources of ROS that leads to oxidative stress in the tumor microenvironment remain areas of intense research. A recent study demonstrated that the increased ROS generation observed in PCa cell lines of varying aggressiveness was largely contributed by the NADPH oxidase family of enzymes. Herein, we observed that SDF-1α promoted ROS accumulation in C4-2 human PCa cells. We also established that NOX, an extramitochondrial ROS-generating system, was the primary source of ROS instead of the mitochondria. Moreover, expression of the isoform, NOX2, was preferentially regulated by CXCR4 at transcriptional and post-transcriptional levels. Finally, NOX2 expression correlated with PCa aggressiveness in microarray datasets. Therefore, we hypothesize that the SDF-1α /CXCR4 signaling axis mediates ROS production in PCa through NADPH-oxidases, resulting in oxidative stress that encourages tumor progression. Results generated from this study will further elucidate the mechanism (s) by which growth factor signaling promotes oxidative stress to enhance tumor development. Understanding the relationship between known cancer promoting elements, such as CXCR4, ROS and NOX, will facilitate the development of more comprehensive therapeutics to enhance current clinical strategies to combat cancer metastasis. Citation Format: Kia J. Jones, Mahandranauth A. Chetram, Danaya A. Bethea, Cimona V. Hinton. Cysteine (C)-X-C receptor 4 induces oxidative stress through NADPH oxidase-2 in prostate cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B23.</jats:p

Abstract 846: ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells .

Abstract Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as cell migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS increased expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, with concomitant decreased expression of HIF1α. Further investigation into pVHL during apoptosis revealed that the tumor suppressor becomes phosphorylated at S111 prior to activity; S111 is within a target motif (RXXS) forphosphorylation by AKT kinase. We then determined that pVHL and p-AKT associated in vitro via co-immunoprecipitation assay. Moreover, knockdown of pVHL by siRNA rescued HIF1α protein expression and rescued cells from apoptosis, which was determined by Annexin-V and cleaved-PARP. Collectively, our study suggests that in the context of oxidative stress, activatedAKT facilitated apoptosis by inducing pVHL function. Moreover, our studies suggest a novel paradigm for AKT-that it may participate in tumor suppressor rather than oncogenesis. Citation Format: Mahandranauth A. Chetram, Kia J. Jones, Christopher Coke, Ayesha Don-Salu-Hewage, Danaya A. Bethea, Cimona V. Hinton. ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 846. doi:10.1158/1538-7445.AM2013-846</jats:p

Abstract 5118: Cysteine (C)-X-C receptor 4 undergoes transportin 1 dependent nuclear localization and is functional at the nucleus of metastatic prostate cancer cells.

Abstract The G-Protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4) plays an important role in prostate cancer (PCa) metastasis. To date, considerable evidence support the presence of GPCRs at the plasma membrane (PM), or within the perinuclear/nuclear compartments of cells and have been suggested to regulate a number of physiological processes including cell proliferation, survival, inflammatory responses and tumorigenesis. In accordance with previous clinical observations in diverse tumor tissues, we observed ubiquitous expression of CXCR4 in metastatic PCa tissues and several PCa cells lines. Additionally, Gαi immunoprecipitation and calcium mobilization studies elucidated that nuclear CXCR4 was functional and participated in G-protein signaling. However, a mechanism by which CXCR4 is translocated to the nucleus is poorly understood.An initial search of the CXCR4 sequence via the PSORT II NLS prediction software program revealed a putative, nontraditional nuclear localization sequence (NLS), “RPRK” in CXCR4.Additionally,the members of karyopherin beta (β) family, Karyopherin-β2 or Transportin 1 (TRN1) were reported to be involved in transportation of various cargos into the nucleus, including the chemokine GPCRs. Collectively, based on our results and previous studies, we suggest that the NLS, “RPRK”and TRN1 play a role in translocation of CXCR4 into the nucleus of advanced PCa. To determine the involvement of “RPRK," manipulations of the NLS revealed that single point mutations of basic resides within the NLS was insufficient to eliminate to abrogate nuclear translocation of CXCR4. However, when the NLS was deleted, nuclear CXCR4 was not detected. Additionally, TRN1 immunoprecipitation and siRNA-TRN1 studies delineated an association between CXCR4 and TRN1,and that nuclear translocation of CXCR4 depended on TRN1. Little information is available about nuclear GPCRs and nuclear CXCR4 in cancer. Thus, the significance of the study is that since CXCR4 is a major signaling receptor involved in PCa metastasis, the dualistic localization pattern and function at the nucleus may encourage recurrence,metastasis and poorer prognosis after tumors have been treated with therapy that targets CXCR4.In conclusion, our studies will provide insight to the development of therapeutics towards CXCR4 and transport mechanisms, as current therapy targets membrane receptors, which may be ineffective if CXCR4 is translocated and functional inside of the cell. Citation Format: Ayesha S. Don-Salu-Hewage, Siu Y. Chan, Kathleen McAndrews, Mahandranauth A. Chetram, Michelle R. Dawson, Cimona V. Hinton. Cysteine (C)-X-C receptor 4 undergoes transportin 1 dependent nuclear localization and is functional at the nucleus of metastatic prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2013-5118</jats:p

Nuclear CXCR4 Expression in Prostate Cancer Cell Lines.

<p><b><i>A</i></b>, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean <u>+</u>S.E. from three independent experiments. *, P<0.05. <b><i>B</i></b>, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.</p

Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.

<p><b><i>A</i></b>, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T₂N₀M₀, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T₄N₁M₁, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. <b><i>B</i></b>, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 null) cell lines. <b><i>C</i></b>, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. <b><i>D</i></b>, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.</p

Nuclear CXCR4 was Functional at the Nucleus.

<p><b><i>A</i></b>, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. <b><i>B</i></b>, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for G_αi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. <b><i>C</i></b>, PC3 nuclei were isolated, incubated with FluoForte dye Ca²⁺ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca²⁺ was measured on a microplate reader at ex = 490 nm/em = 525 nm.</p