Lens Epithelium-derived Growth Factor P75 Research Articles

DFS70/LEDGFp75: An Enigmatic Autoantigen at the Interface between Autoimmunity, AIDS, and Cancer

Clinical and diagnostic laboratories often encounter patient sera containing antinuclear antibodies (ANAs) that produce a nuclear dense fine speckled immunofluorescence pattern on HEp-2 cells. These autoantibodies usually target the dense fine speckled protein of 70 kDa (DFS70), commonly known as lens epithelium-derived growth factor p75 (LEDGFp75). Anti-DFS70/LEDGFp75 autoantibodies have recently attracted much interest because of their relatively common occurrence in sera from patients with positive ANA tests with no clinical evidence of systemic autoimmune rheumatic disease (SARD). Their presence has been documented primarily in patients with diverse non-SARD inflammatory conditions and “apparently healthy” individuals. While there is circumstantial evidence that depending on the context these autoantibodies could play protective, pathogenic, or sensor roles, their significance remains elusive. DFS70/LEDGFp75 has emerged during the past decade as a stress transcription co-activator relevant to HIV integration, cancer, and inflammation. It is not clear, however, what makes this protein the target of such a common autoantibody response. We suggest that a better understanding of DFS70/LEDGFp75 biology is key to elucidating the significance of its associated autoantibodies. Here, we discuss briefly our current understanding of this enigmatic autoantigen and potential scenarios leading to its targeting by the immune system.

Abstract 775: Targeting the stress survival oncoprotein LEDGF/p75 to overcome prostate cancer resistance to taxane therapy

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the U.S. It presents a significant health burden among African American men, who tend to develop more aggressive, chemoresistant tumors and experience higher mortality rates compared to men from other ethnic groups. Eliminating these mortality disparities will require innovative therapies for the chemoresistant form of the disease. Our group has shown previously that the stress survival protein lens epithelium-derived growth factor p75 (LEDGF/p75) is overexpressed in PCa cells and clinical tumors, and promotes resistance to the taxane drug docetaxel (DTX). This study was designed to evaluate the hypothesis that LEDGF/p75 overexpression promotes taxane chemoresistance in PCa by antagonizing drug-induced caspase-independent cell death, and that targeting this protein with small inhibitory RNAs (siRNAs) or small molecule inhibitors will restore tumor cell sensitivity. We observed that acquisition of DTX-resistance in metastatic PCa cell lines (PC3 and DU145) is associated with increased expression of LEDGF/p75. Exposure of these cell lines to increasing concentrations of the taxanes DTX, paclitaxel (PTX), and cabazitaxel (CTX), and to the non-taxane drug TRAIL for up to 72 hours revealed that DTX-resistant cells also displayed increased resistance to PTX and CTX, compared to sensitive cells, but not to TRAIL. This was assessed by viability and clonogenic assays, and microscopic examination. To determine if this resistance was associated with overexpression of LEDGF/p75, we knocked down the protein with siRNAs. This knockdown attenuated resistance to the taxanes, but had no effects on TRAIL-induced cell death. We then explored if LEDGF/p75-induced taxane resistance was associated with preservation of the structural integrity of this protein. Immunoblotting analysis revealed that LEDGF/p75 was cleaved into distinct fragments by caspases during TRAIL-induced apoptosis, leading to its inactivation. However, LEDGF/p75 remained intact during treatment with taxanes, suggesting that these drugs do not induce caspase-mediated cleavage and inactivation of this protein. To determine if LEDGF/p75 is a druggable target for overcoming taxane chemoresistance in PCa cells, we repositioned a panel of small molecule inhibitors originally designed to inhibit the interaction of the C-terminal region of this protein with HIV-1 integrase. Screening of 130 inhibitors for their cytotoxic properties in DTX-resistant and sensitive prostate cancer cells, we identified a number of inhibitors that induced cell death directly, or when combined with DTX, re-sensitized the resistant cells to the drug. These promising results provide novel insights into mechanisms by which LEDGF/p75 promotes chemoresistance in PCa cells, and have implications for the development of innovative strategies to overcome this resistance. Citation Format: Leslimar Rios Colon, Catherine Elix, Anamika Basu, Tino W. Sanchez, Nouri Neamati, Carlos A. Casiano. Targeting the stress survival oncoprotein LEDGF/p75 to overcome prostate cancer resistance to taxane therapy. [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 775. doi:10.1158/1538-7445.AM2014-775

Association of single nucleotide polymorphisms in the lens epithelium-derived growth factor (LEDGF/p75) with HIV-1 infection outcomes in Brazilian HIV-1+ individuals.

The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.

Abstract 3117: Overexpression of the stress transcription co-activator LEDGF/p75 contributes to the upregulation of the stress protective genes HSP27, ERp57 and CYGB in prostate cancer.

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer related death in men in the US. PCa tumor aggressiveness, response to treatment, and mortality are worst in African-American men, compared to other ethnic groups, thus presenting a major health disparity. Our group reported previously that lens epithelium derived growth factor p75 (LEDGF/p75), a stress survival transcription coactivator, has elevated expression in PCa cells and tissues and promotes resistance to cell death induced by oxidative stress and chemotherapy. This is likely due to its ability to transactivate promoter regions of stress and antioxidant genes such as peroxiredoxin 6 (Prdx6) and heat shock protein 27 (HSP27). We hypothesized that high expression of LEDGF/p75 in prostate tumor cells and tissues contributes to the elevated expression of its putative target genes, some of which were identified in previous studies by our group and others. These include genes associated with stress protection and chemotherapy resistance such as HSP27, endoplasmic reticulum protein 57 (ERp57), and cytoglobin (CYGB). To test this hypothesis we performed an expression analysis of LEDGF/p75, HSP27, ERp57 and CYGB proteins in human PCa cell lines using immunoblotting, and in prostate tumor tissues using immunohistochemistry on tumor tissue microarrays (TMAs). PCa cell lines included stable PC3 clones overexpressing LEDGF/p75, as well as docetaxel (DTX) resistant PC3-DR and DU145-DR cells overexpressing this protein, with their corresponding parental DTX sensitive cell lines. The TMAs contained tissue specimens corresponding to prostate tumor, hyperplasia, normal-adjacent, and disease-free normal prostate tissues. Correlations between the expression of LEDGF/p75 and HSP27 or ERp57 or CYGB in cell lines as well as in tumors and normal tissues was determined. Our results indicated that overexpression of LEDGF/p75 in PC3 stable clones and in DTX resistant PCa cells was associated with increased protein expression of HSP27, ERp57 and CYGB. Statistically significant correlation was also observed between the protein levels of LEDGF/p75 and HSPp27 or ERp57 or CYGB in the TMAs. Luciferase-based transcription reporter assays confirmed that the promoter regions of HSP27 and ERp57 are activated by LEDGF/p75 in PCa cells. Chromatin immunoprecipitation assays also demonstrated binding of LEDGF/p75 to HSP27 promoter in PCa cells. Additional promoter activation studies are in progress to demonstrate that LEDGF/p75 transcriptionally regulates ERp57 and CYGB genes. These results enhance our understanding of the contribution of LEDGF/p75 overexpression to the activation of stress protective genes and its implications for chemotherapy resistance in metastatic PCa. Citation Format: Anamika Basu, Christina Cajigas, Benanette Medina, Heather Rojas, Hiya Banerjee, Melanie Mediavilla-Varela, Laisum Leoh, Carlos Casiano. Overexpression of the stress transcription co-activator LEDGF/p75 contributes to the upregulation of the stress protective genes HSP27, ERp57 and CYGB in prostate cancer. [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 3117. doi:10.1158/1538-7445.AM2013-3117

Abstract 892: The stress oncoprotein LEDGF/P75 promotes selective resistance to multiple chemotherapeutic drugs in prostate cencer cells.

Abstract Prostate cancer (PCa) is the second leading cause of cancer related deaths among men in the United States, with African American men presenting more aggressive tumors and increased mortality compared to other ethnic groups. Eliminating these disparities will require an interdiplinary effort that combines improved access to healthcare and health education, combined with innovative approaches to circumvent therapy resistance. The lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator that transactivates stress protective genes to promote resistance to microenvironmental stressors such as oxidative stress and chemotherapy. Previously, our group established that LEDGF/p75 is overexpressed in PCa cells and clinical tumors and that it promotes resistance to docetaxel (DTX) treatment, the standard of chemotheraoy for metastatic PCa. The present study was designed to determine the effects of LEDGF/p75 overexpression and knockdown on cell viability in different PCa cellular models exposed to various chemotherapeutic drugs used in the treatment of advanced PCa. We also evaluated if exposure to DTX and the other drugs increased expression of LEDGF/p75 in PCa cells. For these studies, we used different cell lines with LEDGF/p75 overexpression and siRNA-mediated knockdown, as well as DTX resistant cell lines with elevated levels of this protein. PCa cells were treated with increasing concentrations of the chemotherapeutic drugs DTX, Doxorubicin (DOXO), Paclitaxel (PTX), and TRAIL for up to 96 hours. Loss of cell viability was assessed by viability assays and morphological microscopic examination. Our preliminary results suggested that elevated expression of LEDGF/p75 promotes selective resistance to chemotherapeutic drugs, particularly to DTX, DOXO, and PTX, but not to TRAIL. This selectivity seems to be related to the ability of LEDGF/p75 to protect against stress-induced lysosomal cell death but not against TRAIL-induced classical death receptor mediated apoptosis. Knockdown of LEDGF/p75 sensitized PCa cells to the chemotherapeutic drugs. In addition, increased cellular stress induced by exposure to DTX treatment resulted in increased expression of LEDGF/p75 in a cell-type dependent manner. These results have implications for the development of novel therapeutic strategies for the treatment of PCa based on targeting LEDGF/p75 and its target genes for the sensitization of tumor cells to chemotherapeutic agents. Citation Format: Leslimar Rios-Colon, Anamika Basu, Catherine Elix, Carlos A. Casiano, Marino De León. The stress oncoprotein LEDGF/P75 promotes selective resistance to multiple chemotherapeutic drugs in prostate cencer 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 892. doi:10.1158/1538-7445.AM2013-892

Abstract B76: Circumventing chemoresistance as a strategy to reduce the mortality disparities associated with advanced prostate cancer.

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death among men in the U.S., with African American men presenting a disproportionately high incidence and mortality from this disease. Although these disparities result from the combination of multiple factors (socioeconomic, health care, lifestyle, biological, etc.), there is growing consensus that PCa in African-American men is in general more aggressive than in other ethnic groups. Reducing PCa mortality disparities will therefore require novel strategies for the effective treatment of advanced, aggressive tumors characterized by chemotherapy resistance. Currently, chemotherapy with docetaxel (DTX), the standard of care for advanced PCa, is limited because most patients treated with the drug ultimately develop chemoresistance and succumb to the disease. Circumventing this resistance is therefore essential for increasing the effectiveness of DTX and other emerging anti-PCa drugs, and reducing the high mortality associated with advanced PCa. We established previously that lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival oncoprotein, is overexpressed in clinical prostate tumors, and that its overexpression in PCa cell models promotes resistance to DTX treatment via inhibition of lysosomal cell death. In the present study we evaluated if overexpression of LEDGF/p75 in PCa cells also promotes resistance to other chemotherapeutic drugs, and if its depletion sensitizes chemoresistant PCa cells to drug-induced cell death. We treated PC-3 cells (with and without stable LEDGF/p75 overexpression) with increasing concentrations of the anti-cancer drugs DTX, paclitaxel, doxorubicin, and TRAIL for up to 48 hrs. We then assessed cell viability by MTT assays and morphological examination. LEDGF/p75 overexpressing PC-3 cells showed higher viability than control cells in response to DTX and doxorubicin. However, LEDGF/p75 did not protect against paclitaxel and TRAIL, indicating selectivity in its ability to promote chemoresistance. We also observed by immunoblotting and quantitative PCR that metastatic PCa cell lines selected for DTX resistance, designated PC3-DR and DU-145DR, naturally express high levels of endogenous LEDGF/p75 relative to their sensitive parental cell lines. To determine if the elevated expression of LEDGF/p75 in these two cell lines contribute to DTX resistance, we knocked down this protein with a specific small inhibitory RNA (si-p75). Cell viability significantly decreased in both cell lines in response to combined treatment with DTX and si-p75, relative to siRNA controls. We also observed that these chemoresistant PCa cell lines express high levels of proteins associated with inflammation and cellular response to oxidative stress, such as IL-6, IL-6R, Hsp27, PRDX6, and cytoglobin, and that the levels of these proteins decreased upon LEDGF/p75 knockdown. Our preliminary results suggest that LEDGF/p75 overexpression promotes selective chemoresistance in cellular models of advanced PCa, and that its inhibition increases sensitization to DTX associated with downregulation of inflammation/oxidative stress-related genes. These promising results have implications for the development of novel therapeutic strategies for chemoresistant PCa. Studies are in progress to assess the expression levels of LEDGF/p75 in sera and prostate tissues from African-American and Caucasian PCa patients, and correlate these levels with advanced disease and chemotherapy resistance in these patients. Citation Format: Leslimar Ríos-Colón, Anamika Basu, Christina Cajigas, Catherine Elix, Carlos A. Casiano. Circumventing chemoresistance as a strategy to reduce the mortality disparities associated with advanced prostate cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B76.

LEDGF (p75) promotes DNA-end resection and homologous recombination

Lens epithelium-derived growth factor p75 splice variant (LEDGF) is a chromatin-binding protein known for its antiapoptotic activity and ability to direct human immunodeficiency virus into active transcription units. Here we show that LEDGF promotes the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. Depletion of LEDGF impairs the recruitment of C-terminal binding protein interacting protein (CtIP) to DNA DSBs and the subsequent CtIP-dependent DNA-end resection. LEDGF is constitutively associated with chromatin through its Pro-Trp-Trp-Pro (PWWP) domain that binds preferentially to epigenetic methyl-lysine histone markers characteristic of active transcription units. LEDGF binds CtIP in a DNA damage-dependent manner, thereby enhancing its tethering to the active chromatin and facilitating its access to DNA DSBs. These data highlight the role of PWWP-domain proteins in DNA repair and provide a molecular explanation for the antiapoptotic and cancer cell survival-activities of LEDGF.

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.

Expression of the Stress Response Oncoprotein LEDGF/p75 in Human Cancer: A Study of 21 Tumor Types

Oxidative stress-modulated signaling pathways have been implicated in carcinogenesis and therapy resistance. The lens epithelium derived growth factor p75 (LEDGF/p75) is a transcription co-activator that promotes resistance to stress-induced cell death. This protein has been implicated in inflammatory and autoimmune conditions, HIV-AIDS, and cancer. Although LEDGF/p75 is emerging as a stress survival oncoprotein, there is scarce information on its expression in human tumors. The present study was performed to evaluate its expression in a comprehensive panel of human cancers. Transcript expression was examined in the Oncomine cancer gene microarray database and in a TissueScan Cancer Survey Panel quantitative polymerase chain reaction (Q-PCR) array. Protein expression was assessed by immunohistochemistry (IHC) in cancer tissue microarrays (TMAs) containing 1735 tissues representing single or replicate cores from 1220 individual cases (985 tumor and 235 normal tissues). A total of 21 major cancer types were analyzed. Analysis of LEDGF/p75 transcript expression in Oncomine datasets revealed significant upregulation (tumor vs. normal) in 15 out of 17 tumor types. The TissueScan Cancer Q-PCR array revealed significantly elevated LEDGF/p75 transcript expression in prostate, colon, thyroid, and breast cancers. IHC analysis of TMAs revealed significant increased levels of LEDGF/p75 protein in prostate, colon, thyroid, liver and uterine tumors, relative to corresponding normal tissues. Elevated transcript or protein expression of LEDGF/p75 was observed in several tumor types. These results further establish LEDGF/p75 as a cancer-related protein, and provide a rationale for ongoing studies aimed at understanding the clinical significance of its expression in specific human cancers.

Pathway specific gene expression profiling reveals oxidative stress genes potentially regulated by transcription co‐activator LEDGF/p75 in prostate cancer cells

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells. A "Human oxidative stress and antioxidant defense" qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting. Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs. Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death.

Abstract 2092: Pathway specific gene profiling analysis reveals potential target genes of the stress oncoprotein LEDGF/p75 in prostate cancer cells

Abstract Chronic inflammation in the prostate microenvironment, with its associated increase in oxidative stress, is critical for prostate carcinogenesis since it leads to DNA mutations and epigenetic changes, as well as deregulation of stress and antioxidant proteins that protect prostate cells against oxidative damage and cell death. The lens epithelium-derived growth factor p75 (LEDGF/p75), an oxidative stress-induced transcription coactivator, is overexpressed in prostate tumors and other tumor types. LEDGF/p75 is emerging as a stress oncoprotein that protects cancer cells against lysosomal cell death and promotes resistance to chemotherapeutic drugs. We hypothesize that the pro-survival function of LEDGF/p75 is associated with its ability to promote the transcriptional activation of protective stress genes. To date, only very few candidate target genes of LEDGF/p75 have been identified in cancer cells. Towards this goal we initiated a stress focused gene profiling analysis of PC3 prostate cancer cells overexpressing or lacking LEDGF/p75, using the Real Time PCR Array System (SABiosciences). We selected an array that contained 80 genes involved in the cellular stress response and antioxidant defense. siRNA oligos targeting the C-terminus of the LEDGF/p75 mRNA sequence were used to induce transient LEDGF/p75 knockdown in PC3 cell. LEDGF/p75 was also stably overexpressed in PC3 cells by transduction with a lentiliviral vector. Both transient siRNA-mediated knockdown and overexpression of LEDGF/p75 were associated with significant changes in the expression of several stress/antioxidant genes, using a cutoff fold change value of 1.5 (P&amp;lt;0.001). We selected for further analysis common genes that were significantly downregulated when LEDGF/p75 was knocked down and upregulated when this protein was overexpressed. These genes are phosphoinositide-binding protein E, cytoglobulin, superoxide dismutase 3, thyroid peroxidase, titin, glutathione transferase zeta1 and albumin. The expression of these genes in response to LEDGF/p75 expression was further validated using real-time RT-PCR. This study identifies candidate stress genes potentially regulated by LEDGF/p75 in PC3 cells, and provides a rationale for investigating the mechanisms by which this emerging stress oncoprotein promotes resistance to lysosomal cell death and chemotherapy in prostate tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2011-2092

Abstract 2083: The stress oncoprotein LEDGF/p75 attenuates oxidative stress-induced necrosis but not apoptosis in prostate cancer cells

Abstract The lens epithelium-derived growth factor p75 (LEDGF/p75) is emerging as a stress response oncoprotein in human cancer. Our group and others showed previously that this transcription coactivator is overexpressed in major human cancers, including prostate cancer, and protects against lysosomal cell death and chemotherapeutic drugs. Although LEDGF/p75 is considered a novel anti-apoptotic protein due to its ability to attenuate lysosomal cell death, previous studies from our group demonstrated its inactivation by caspases during apoptosis. We hypothesized that LEDGF/p75 promotes resistance to stress-induced caspase-independent cell death, but not to apoptosis, in cancer cells. To test this hypothesis, we established PC3 and RWPE-2 prostate cancer cell lines stably overexpressing LEDGF/p75, and evaluated its ability to protect these cells against non-apoptotic and apoptotic insults. LEDGF/p75 significantly attenuated cytotoxicity induced by tert-butyl hydroperoxide (TBHP), a strong inducer of oxidative stress. Characterization of TBHP-induced cell death showed that it was caspase-independent since it could not be inhibited with the broad caspase inhibitor z-VAD-fmk and did not involve caspase-3 activation. Furthermore, TBHP-treated cells displayed a necrotic morphology characterized by extensive cytoplasmic fragmentation and shrunken, non-fragmented nuclei. Necrosis was also confirmed by the appearance in immunoblots of a 45 kD cleavage fragment of DNA topoisomerase that was previously established by our group as a marker of necrosis. Overexpression of LEDGF/p75 attenuated the generation of reactive oxygen species (ROS) in TBHP-treated cells. Interestingly, LEDGF/p75 also attenuated ROS in cells treated with the classical apoptosis inducers staurosporine and TRAIL, but failed to protect against these apoptotic insults. These results indicated that the ability of LEDGF/p75 to attenuate ROS is not sufficient for its pro-survival function. Analysis of LEDGF/p75 integrity by immunoblotting analysis showed that the protein remained intact in cells treated with TBHP, but was cleaved into its signature 65 kd apoptotic fragment in cells treated with staurosporine and TRAIL. Our group has shown previously that this caspase-generated fragment lacks pro-survival activity. Taken together, these results strongly suggest that overexpression of LEDGF/p75 can protect tumor cells from caspase-independent cell death processes in which this protein remains structurally intact. This study provides the basis for investigating the contribution of LEDGF/p75 to prostate cancer resistance to therapeutic modalities that induce caspase-independent cell death, as well as its potential as a novel therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2011-2083

Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

BackgroundThe presence of the cellular Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) protein is essential for integration of the Human immunodeficiency virus type 1 (HIV-1) cDNA and for efficient virus production. In the absence of LEDGF/p75 very little integration and virus production can be detected, as was demonstrated using LEDGF/p75-knokdown cells.ResultsHere we show that the failure to infect LEDGF/p75-knockdown cells has another reason aside from the lack of LEDGF/p75. It is also due to inhibition of the viral integrase (IN) enzymatic activity by an early expressed viral Rev protein. The formation of an inhibitory Rev-IN complex in virus-infected cells can be disrupted by the addition of three IN-derived, cell-permeable peptides, designated INr (IN derived-Rev interacting peptides) and INS (IN derived-integrase stimulatory peptide). The results of the present work confirm previous results showing that HIV-1 fails to infect LEDGF/p75-knockdown cells. However, in the presence of INrs and INS peptides, relatively high levels of viral cDNA integration as well as productive virus infection were obtained following infection by a wild type (WT) HIV-1 of LEDGF/p75-knockdown cells.ConclusionsIt appears that the lack of integration observed in HIV-1 infected LEDGF/p75-knockdown cells is due mainly to the inhibitory effect of Rev following the formation of a Rev-IN complex. Disruption of this inhibitory complex leads to productive infection in those cells.

High-resolution profiling of the LEDGF/p75 chromatin interaction in the ENCODE region

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a transcriptional coactivator involved in stress response, autoimmune disease, cancer and HIV replication. A fusion between the nuclear pore protein NUP98 and LEDGF/p75 has been found in human acute and chronic myeloid leukemia and association of LEDGF/p75 with mixed-lineage leukemia (MLL)/menin is critical for leukemic transformation. During lentiviral replication, LEDGF/p75 tethers the pre-integration complex to the host chromatin resulting in a bias of integration into active transcription units (TUs). The consensus function of LEDGF/p75 is tethering of cargos to chromatin. In this regard, we determined the LEDGF/p75 chromatin binding profile. To this purpose, we used DamID technology and focused on the highly annotated ENCODE (Encyclopedia of DNA Elements) regions. LEDGF/p75 primarily binds downstream of the transcription start site of active TUs in agreement with the enrichment of HIV-1 integration sites at these locations. We show that LEDGF/p75 binding is not restricted to stress response elements in the genome, and correlation analysis with more than 200 genomic features revealed an association with active chromatin markers, such as H3 and H4 acetylation, H3K4 monomethylation and RNA polymerase II binding. Interestingly, some associations did not correlate with HIV-1 integration indicating that not all LEDGF/p75 complexes on the chromosome are amenable to HIV-1 integration.

Abstract 2945: Methyl CpG binding protein MeCP2 interacts with the stress-response transcription coactivator LEDGF/p75 and modulates its survival and transcription functions

Abstract The lens epithelium derived growth factor p75 (LEDGF/p75) is an emerging oncoprotein identified as a key player in the cellular response to oxidative stress. LEDGF/p75 is induced by oxidative stress and promotes resistance to stress-induced cell death presumably by transcriptionally activating specific stress and survival genes. LEDGF/p75 has a splice variant, p52, which induces cell death when overexpressed. We have shown elevated expression of LEDGF/p75 in advanced stage prostatic adenocarcinomas, most likely as a result of increased oxidative stress in the prostate tumor microenvironment. To understand the mechanisms by which LEDGF/p75 transcriptionally activate stress genes and promotes protection against oxidative stress-induced cell death, it was necessary to identify endogenous cellular interacting partners of this protein. Screening of a transcription factor protein array identified the Methyl CpG binding Protein 2 (MeCP2) as a possible interacting partner of LEDGF/p75. MeCP2 is mutated in patients with Rett syndrome, both activate and repress transcription and has been implicated in prostate tumor growth. This study was designed to validate MeCP2 as an interacting partner of LEDFG/p75 and to explore its influence on LEDGF/p75's function. To examine the interaction of MeCP2 and LEDGF, pull down assays were performed with purified GST-MeCP2 incubated with His-LEDGF/p75 or His-p52, and with GST-MeCP2 or GST-p75 incubated with U2OS cell lysates. The results suggested binding of MeCP2 to both LEDGF/p75 and p52. Co-immunoprecipitation assays using U2OS cell lysates revealed binding of proteins, detected by immunoblotting using specific anti-LEDGF/p75 or MeCP2 antibodies, suggesting interaction of both proteins in the cellular microenvironment. We also examined the intracellular localization of both proteins in U2OS cells by immunofluorescence microscopy. Both MeCP2 and LEDGF/p75 co-localized in the chromatin, showing a similar dense fine speckled staining pattern. To examine if MeCP2 influences LEDGF/p75's transcriptional activity, luciferase reporter assays were performed. Even though LEDGF/p75, p52 and MeCP2 transactivated the Hsp27 promoter (pr) individually, co-expression of LEDGF/p75 with MeCP2 increased Hsp27pr activity, while co-expression of LEDGF/p52 with MeCP2 decreased activity. In addition, LEDGF/p75 overexpression protected PC3 cells from tert-butyl hydrogen peroxide (TBHP) treatment; but siRNA knockdown of MeCP2 in these cells sensitized cells to TBHP treatment compared to control siRNA. In conclusion, our data suggest that LEDGF/p75 and MeCP2 are interacting partners and MeCP2 influences LEDGF/p75's transcription and survival function. Future studies will explore modulating the interaction between LEDGF/p75 and MeCP2 to sensitize prostate cancer cells to therapy-induced cell death. 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 2945.

Abstract 4667: Elevated expression of the stress oncoprotein LEDGF/p75 in major human cancers

Abstract Long-term cumulative exposure to environmental factors (i.e., dietary habits, unhealthy lifestyle, infectious agents, etc) lead to chronic inflammation and an augmented state of cellular oxidative stress, which contribute to the etiology and progression of various human cancers. We reported previously that lens epithelium derived growth factor p75 (LEDGF/p75), a transcription coactivator, has an elevated expression in human prostate tumors and cell lines. We hypothesize that LEDGF/p75 might be highly expressed in tissues with elevated oxidative stress such as tumors, because of its ability to transcriptionally activate stress genes that promote resistance to stress- and chemotherapy-induced cell death. To date, there have not been any comprehensive studies aimed at investigating the expression of LEDGF/p75 in human cancers. We performed an expression analysis of LEDGF/p75 protein in a panel of human cancers using immunohistochemistry on commercially available tissue microarrays (TMAs). Paraffin embedded tissue specimens were deparaffinized, rehydrated and stained with either a rabbit anti-LEDGF/p75 serum or the rabbit pre-immune serum as an antibody control, using a Biogenic i6000 auto stainer (Biogenex Corp.). Images were acquired using an Olympus BX50 microscope equipped with a Spot RT3TM camera. The TMA slides were assessed for LEDGF/p75 immunoreactivity by a pathologist who was blinded for the tumor/normal status of the tissues. A 4-tier grading system (0-negative, 1-weak, 2-moderate and 3-strong staining intensity) was used to evaluate the staining intensity. Associations between LEDGF/p75 expression in tumors and normal tissues were analyzed using Kruskall Wallis's rank sum test. We used TMAs (Imgenex and NDRI) containing specimens corresponding to over 20 major tumor types and normal adjacent tissues. Our data revealed high expression of this protein in tumors of prostate, pancreas, lungs, liver, thyroid, colon, breast and kidney, as compared to corresponding normal adjacent tissues. To further validate our findings, we analyzed LEDGF/p75 expression in the above-mentioned cancers using TMAs (US BIOMAX), each containing multiple tissues specimens of a specific major tumor type, as well as non-cancerous inflammatory disease and normal post-mortem control tissues. Immunohistochemical analysis revealed high expression of LEDGF/p75 in most of the tumor sections as compared to normal control tissues. The normal tissues showed minimal or no staining. The anti-LEDGF/p75 staining was mostly confined to the nucleus, although cytoplasmic staining was also evident in many cells. These results further validate the high expression of LEDGF/p75 in major human cancers and constitute the first step towards defining the biological significance of LEDGF/p75 expression in human cancers. 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 4667.

LEDGF Hybrids Efficiently Retarget Lentiviral Integration Into Heterochromatin

Correction of genetic diseases requires integration of the therapeutic gene copy into the genome of patient cells. Retroviruses are commonly used as delivery vehicles because of their precise integration mechanism, but their use has led to adverse events in which vector integration activated proto-oncogenes and contributed to leukemogenesis. Here, we show that integration by lentiviral vectors can be targeted away from genes using an artificial tethering factor. During normal lentivirus infection, the host cell-encoded transcriptional coactivator lens epithelium-derived growth factor/p75 (LEDGF/p75) binds lentiviral integrase (IN), thereby targeting integration to active transcription units and increasing the efficiency of infection. We replaced the LEDGF/p75 chromatin interaction-binding domain with CBX1. CBX1 binds histone H3 di- or trimethylated on K9, which is associated with pericentric heterochromatin and intergenic regions. The chimeric protein supported efficient transduction of lentiviral vectors and directed the integration outside of genes, near bound CBX1. Despite integration in regions rich in epigenetic marks associated with gene silencing, lentiviral vector expression remained efficient. Thus, engineered LEDGF/p75 chimeras provide technology for controlling integration site selection by lentiviral vectors.

Integration of HIV-1 DNA Is Regulated by Interplay between Viral Rev and Cellular LEDGF/p75 Proteins

The present work describes a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Rev protein and the cellular lens epithelium-derived growth factor p75 (LEDGF/p75) protein in vitro and in virus-infected cells. Here we show, for the first time, that formation of an Rev-LEDGF/p75 complex is a crucial step in regulating viral cDNA integration. Coimmunoprecipitation experiments at various times after virus infection revealed that, first, an integrase enzyme (IN)-LEDGF/p75 complex is formed, which is then replaced by a Rev-LEDGF/p75 and Rev-IN complexes. This was supported by in vitro experiments showing that Rev promotes dissociation of the IN-LEDGF/p75 complex. Combination of the viral IN and the cellular LEDGF/p75 is required for proper integration of the viral cDNA into the host chromosomal DNA. Our findings demonstrate that integration of HIV-1 cDNA is regulated by an interplay between viral Rev and the host-cell LEDGF/p75 proteins.

Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

BackgroundHormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality.ResultsWe investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity.ConclusionThese results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC.

Lens Epithelium-derived Growth Factor/p75 Interacts with the Transposase-derived DDE Domain of PogZ

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function.