Shannalee R. Martinez

Differential Expression of Peroxiredoxins in Prostate Cancer: Consistent Upregulation of PRDX3 and PRDX4

The peroxiredoxins (PRDXs) are emerging as regulators of antioxidant defense, apoptosis, and therapy resistance in cancer. Because their significance in prostate cancer (PCa) is unclear, we investigated their expression and clinical associations in PCa.

The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

The lens epithelium–derived growth factor p75 (LEDGF/p75) is a transcription coactivator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kDa (DFS70) and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull-down and AlphaScreen assays, coimmunoprecipitation, and nuclear colocalization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS osteosarcoma cells, whereas this effect was more pronounced in PC3 prostate cancer cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75–MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes. Mol Cancer Res; 10(3); 378–91. ©2012 AACR.

LEDGF/p75 Overexpression Attenuates Oxidative Stress-Induced Necrosis and Upregulates the Oxidoreductase ERP57/PDIA3/GRP58 in Prostate Cancer

Prostate cancer (PCa) mortality is driven by highly aggressive tumors characterized by metastasis and resistance to therapy, and this aggressiveness is mediated by numerous factors, including activation of stress survival pathways in the pro-inflammatory tumor microenvironment. LEDGF/p75, also known as the DFS70 autoantigen, is a stress transcription co-activator implicated in cancer, HIV-AIDS, and autoimmunity. This protein is targeted by autoantibodies in certain subsets of patients with PCa and inflammatory conditions, as well as in some apparently healthy individuals. LEDGF/p75 is overexpressed in PCa and other cancers, and promotes resistance to chemotherapy-induced cell death via the transactivation of survival proteins. We report in this study that overexpression of LEDGF/p75 in PCa cells attenuates oxidative stress-induced necrosis but not staurosporine-induced apoptosis. This finding was consistent with the observation that while LEDGF/p75 was robustly cleaved in apoptotic cells into a p65 fragment that lacks stress survival activity, it remained relatively intact in necrotic cells. Overexpression of LEDGF/p75 in PCa cells led to the upregulation of transcript and protein levels of the thiol-oxidoreductase ERp57 (also known as GRP58 and PDIA3), whereas its depletion led to ERp57 transcript downregulation. Chromatin immunoprecipitation and transcription reporter assays showed LEDGF/p75 binding to and transactivating the ERp57 promoter, respectively. Immunohistochemical analysis revealed significantly elevated co-expression of these two proteins in clinical prostate tumor tissues. Our results suggest that LEDGF/p75 is not an inhibitor of apoptosis but rather an antagonist of oxidative stress-induced necrosis, and that its overexpression in PCa leads to ERp57 upregulation. These findings are of significance in clarifying the role of the LEDGF/p75 stress survival pathway in PCa.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) stimulates in vitro proliferation of human fetal B-cell precursors. However, its in vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (−T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in −T mice. Patient-derived xenografts generated from +T as compared to −T mice showed a 3–6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from −T mice. +T/−T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

TSLP or IL‐7 provide an IL‐7Rα signal that is critical for human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) and IL‐7 are cytokines that signal via the IL‐7 receptor alpha (IL‐7Rα) to exert both overlapping and unique functions during early stages of mouse B‐cell development. In human B lymphopoiesis, the requirement for IL‐7Rα signaling is controversial and the roles of IL‐7 and TSLP are less clear. Here, we evaluated human B‐cell production using novel in vitro and xenograft models of human B‐cell development that provide selective IL‐7 and human TSLP (hTSLP) stimulation. We show that in vitro human B‐cell production is almost completely blocked in the absence of IL‐7Rα stimulation, and that either TSLP or IL‐7 can provide a signal critical for the production and proliferation of human CD19+ PAX5+ pro‐B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL‐7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro‐B cells under the influence of mouse IL‐7 in a xenograft scenario is reduced by anti‐IL‐7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL‐7Rα mediated signals for normal human B‐cell production.

MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes

Hypoxia is a common intrauterine stressor, often resulting in intrauterine growth restriction and increased risk for cardiovascular disease later in life. The aim of this work was to test the hypothesis that microRNA-210 (miR-210) mediates the detrimental suppression of glucocorticoid receptor (GR) in response to hypoxia in fetal rat cardiomyocytes. Cardiomyocytes isolated from gestational day 21 Sprague Dawley fetal rats showed increased miR-210 levels and reduced GR abundance after exposure to ex vivo hypoxia (1% O2). In regard to mechanisms, the different contributions of hypoxia response elements (HREs) motifs in the regulation of miR-210 promoter activity and the miR-210-mediated repression of GR expression were determined in rat embryonic heart-derived myogenic cell line H9c2. Moreover, using a cell culture-based model of hypoxia-reoxygenation injury, we assessed the cytotoxic effects of GR suppression under hypoxic conditions. The results showed that hypoxia induced HIF-1α-dependent miR-210 production, as well as miR-210-mediated GR suppression, in cardiomyocytes. Furthermore, inhibition or knockdown of GR exacerbated cell death in response to hypoxia-reoxygenation injury. Altogether, the present study demonstrates that the HIF-1α-dependent miR-210-mediated suppression of GR in fetal rat cardiomyocytes increases cell death in response to hypoxia, providing novel evidence for a possible mechanistic link between fetal hypoxia and programming of ischemic-sensitive phenotype in the developing heart.Hypoxia is a common intrauterine stressor, often resulting in intrauterine growth restriction and increased risk for cardiovascular disease later in life. The aim of this work was to test the hypothesis that microRNA-210 (miR-210) mediates the detrimental suppression of glucocorticoid receptor (GR) in response to hypoxia in fetal rat cardiomyocytes. Cardiomyocytes isolated from gestational day 21 Sprague Dawley fetal rats showed increased miR-210 levels and reduced GR abundance after exposure to ex vivo hypoxia (1% O2). In regard to mechanisms, the different contributions of hypoxia response elements (HREs) motifs in the regulation of miR-210 promoter activity and the miR-210-mediated repression of GR expression were determined in rat embryonic heart-derived myogenic cell line H9c2. Moreover, using a cell culture-based model of hypoxia-reoxygenation injury, we assessed the cytotoxic effects of GR suppression under hypoxic conditions. The results showed that hypoxia induced HIF-1α-dependent miR-210 production, as well as miR-210-mediated GR suppression, in cardiomyocytes. Furthermore, inhibition or knockdown of GR exacerbated cell death in response to hypoxia-reoxygenation injury. Altogether, the present study demonstrates that the HIF-1α-dependent miR-210-mediated suppression of GR in fetal rat cardiomyocytes increases cell death in response to hypoxia, providing novel evidence for a possible mechanistic link between fetal hypoxia and programming of ischemic-sensitive phenotype in the developing heart.

RNA sequencing reveals upregulation of a transcriptomic program associated with stemness in metastatic prostate cancer cells selected for taxane resistance

Patients with metastatic castration-resistant prostate cancer (mCRPC) develop resistance to conventional therapies including docetaxel (DTX). Identifying molecular pathways underlying DTX resistance is critical for developing novel combinatorial therapies to prevent or reverse this resistance. To identify transcriptomic signatures associated with acquisition of chemoresistance we profiled gene expression in DTX-sensitive and -resistant mCRPC cells using RNA sequencing (RNA-seq). PC3 and DU145 cells were selected for DTX resistance and this phenotype was validated by immunoblotting using DTX resistance markers (e.g. clusterin, ABCB1/P-gp, and LEDGF/p75). Overlapping genes differentially regulated in the DTX-sensitive and -resistant cells were ranked by Gene Set Enrichment Analysis (GSEA) and validated to correlate transcript with protein expression. GSEA revealed that genes associated with cancer stem cells (CSC) (e.g., NES, TSPAN8, DPPP, DNAJC12, and MYC) were highly ranked and comprised 70% of the top 25 genes differentially upregulated in the DTX-resistant cells. Established markers of epithelial-to-mesenchymal transition (EMT) and CSCs were used to evaluate the stemness of adherent DTX-resistant cells (2D cultures) and tumorspheres (3D cultures). Increased formation and frequency of cells expressing CSC markers were detected in DTX-resistant cells. DU145-DR cells showed a 2-fold increase in tumorsphere formation and increased DTX resistance compared to DU145-DR 2D cultures. These results demonstrate the induction of a transcriptomic program associated with stemness in mCRPC cells selected for DTX resistance, and strengthen the emerging body of evidence implicating CSCs in this process. In addition, they provide additional candidate genes and molecular pathways for potential therapeutic targeting to overcome DTX resistance.

Abstract 3295: A novel patient-derived xenograft model for evaluating the role of TSLP in CRLF2 B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) with genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL) is associated with poor outcomes. CRLF2 B-ALL is 5 times more common in Hispanic children than others making it a significant biological component of pediatric cancer health disparities. CRLF2 is a component of the receptor complex that is activated by the cytokine, TSLP. Receptor signaling induces Jak/STAT5 and PI3/AKT/mTOR pathway activation and plays a role in the proliferation and differentiation B cell precursors. We found that primary human bone marrow (BM) stroma express TSLP providing an in vivo source of TSLP to stimulate CRLF2 B-ALL cells. Our goal was to develop patient-derived xenograft (PDX) models of CRLF2 B-ALL for studies to understand disease mechanisms and identify therapies to treat CRLF2 B-ALL and reduce the health disparities for Hispanic children with this disease. PDX models are possible because many cytokines produced in the mouse show cross species activity on human cells. However, available data suggests that mouse TSLP does not activate human CRLF2-mediated signals. Using phospho flow cytometry we show that mouse TSLP was unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP. We developed a human TSLP +/- PDX model system by transplanting immune deficient NSG mice with HS-27 stroma transduced to express human hTSLP (hTSLP+ mice) or with control vector (hTSLP- mice). Human TSLP was present at normal human serum levels in hTSLP+ mice but undetectable in hTSLP- mice. To identify genes targeted by TSLP in CRLF2 B-ALL and verify pathway activation, we transplanted primary leukemia cells from a Hispanic patient into hTSLP+ and hTSLP- mice. Whole genome microarray was performed on CRLF2 B-ALL cells isolated from the BM of the hTSLP+ and hTSLP- PDX mice. Microarray identified 280 genes that are upregulated and 281 genes that are downregulated in vivo in leukemia cells from hTSLP+ as compared to hTSLP- PDX mice. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in hTSLP+ as compared to hTSLP- mice, confirming hTSLP activity in the hTSLP+ PDX mice. Our next question was whether cells expanded in hTSLP+ vs. hTSLP- mice would exhibit changes in their ability to respond to TSLP. When we subjected PDX-expanded primary CRLF2 B-ALL cells to ex vivo TSLP stimulation ∼1/3 fewer gene targets were up- and downregulated in the leukemia cells expanded in hTSLP- mice as compared to cells from hTSLP+ mice. This suggests that CRLF2 B-ALL cells expanded in xenograft without TSLP lose some of their ability to respond to TSLP. The hTSLP+ CRLF2 B-ALL PDX mice described here provide a novel preclinical model for studying disease mechanisms and identifying therapies to target signaling pathways activated by TSLP in CRLF2 B-ALL and reduce cancer health disparities for this disease. Citation Format: Olivia L. Francis, Parveen Shiraz, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Karina Mayagoitia, Elizabeth Ginelli, Katherine R. Salcedo-Concepcion, Shannalee Martinez, Xiaobing Zhang, Valeri Filippov, Ruijun J. Su, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model for evaluating the role of TSLP in CRLF2 B-ALL. [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 3295. doi:10.1158/1538-7445.AM2015-3295

Abstract B25: A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL

While the overall survival rate for children with B cell precursor acute lymphoblastic leukemia (B-ALL) is high, a subset of children with this disease are at high risk for relapse and death. Genome-wide analysis has shown that gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome–positive ALL and these are designated Ph-like ALL. Approximately half of Ph-like ALL are characterized by genetic defects resulting in overexpression of CRLF2. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of this receptor complex activation. The activating JAK mutations found in some CRLF2 B-ALL have led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. In preliminary studies to address this question we evaluated the effect of TSLP on a CRLF2 B-ALL cell lines with JAK defects and which have been reported to exhibit constitutive JAK-STAT5 activation. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP is different from most other cytokines produced in the xenograft in that it is species-specific and does not activate the human TSLP receptor complex that includes CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that we believe to be a major factor in CRLF2 B-ALL. To overcome this obstacle we engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show serum hTSLP levels in the hTSLP+ mice that approximate the normal range in human serum. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with a CRLF2 B-ALL. We used this hTSLP+/– xenograft model system to evaluate the in vivo effects of TSLP on survival and proliferation of transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Mice were euthanized at 5 weeks and BM was harvested. Evaluation of BM disease by flow cytometry showed that the percentage of viable human leukemia cells in hTSLP+ mice was twice that observed in hTSLP– mice. Evaluation of cell cycle progression in human CRLF2 B-ALL cells isolated from xenograft BM showed that the percentage of cycling cells in hTSLP+ mice was 2.5 fold higher than in hTSLP– mice. When primary Ph-like ALL cells were transplanted to produce hTSLP+/– xenografts, the viable pre-B ALL cells present in the BM of hTSLP+ mice showed higher expression levels of the TSLPR components (CRLF2 and IL-7Rα) than those in the hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that it impacts primary pre-B ALL cells. Preliminary data obtained from this model suggests that TSLP provides a signal that promotes in vivo survival of CRLF2 B-ALL cells and that it may play a role in selection of leukemia clones during in vivo leukemogenesis. Microarray analysis comparing gene expression in primary CRLF2 B-ALL cells isolated from hTSLP+ and hTSLP– xenograft mice identified 565 that genes are differentially regulated (> 2 fold up or downregulated; p Citation Format: Ruijun Su, Francis L. Olivia, Shannalee R. Martinez, Ineavely Baez, Terry Ann Milford, Terrence Bennett, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B25.

Abstract B21: A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL

While the overall survival rate for children with B cell precursor acute lymphoblastic leukemia (B-ALL) is high, a subset of children with this disease are at high risk for relapse and death. Genome-wide analysis has shown that gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome–positive ALL and these are designated Ph-like ALL. Approximately half of Ph-like ALL are characterized by genetic defects resulting in overexpression of CRLF2. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of this receptor complex activation. The activating JAK mutations found in some CRLF2 B-ALL have led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. In preliminary studies to address this question we evaluated the effect of TSLP on a CRLF2 B-ALL cell lines with JAK defects and which have been reported to exhibit constitutive JAK-STAT5 activation. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP is different from most other cytokines produced in the xenograft in that it is species-specific and does not activate the human TSLP receptor complex that includes CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that we believe to be a major factor in CRLF2 B-ALL. To overcome this obstacle we engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show serum hTSLP levels in the hTSLP+ mice that approximate the normal range in human serum. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with Ph-like B-ALL. First, we used the hTSLP+/– xenograft model system to evaluate the in vivo effects of TSLP on survival and proliferation of transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Mice were euthanized at 5 weeks and BM was harvested. Evaluation of BM disease by flow cytometry showed that the percentage of viable human leukemia cells in hTSLP+ mice was twice that observed in hTSLP– mice. Evaluation of cell cycle progression in human CRLF2 B-ALL cells isolated from xenograft BM showed that the percentage of cycling cells in hTSLP+ mice was 2.5 fold higher than in hTSLP– mice. When primary Ph-like ALL cells were transplanted to produce hTSLP+/– xenografts, the viable pre-B ALL cells present in the BM of hTSLP+ mice showed higher expression levels of the TSLPR components (CRLF2 and IL-7Rα) than those in the hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that it impacts primary pre-B ALL cells. Preliminary data obtained from this model suggests that TSLP provides a signal that promotes in vivo survival of CRLF2 B-ALL cells and that it may play a role in selection of leukemia clones during in vivo leukemogenesis. Microarray analysis comparing gene expression in primary CRLF2 B-ALL cells isolated from hTSLP+ and hTSLP– xenograft mice identified 565 that genes are differentially regulated (> 2 fold up or downregulated; p Citation Format: Olivia Francis, Ruijun Su, Shannalee Martinez, Ineavely Baez, Terry-Ann Milford, Terrence Bennett, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B21.