Lai Sum Leoh

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.

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.

Alternative Splicing and Caspase-Mediated Cleavage Generate Antagonistic Variants of the Stress Oncoprotein LEDGF/p75

There is increasing evidence that an augmented state of cellular oxidative stress modulates the expression of stress genes implicated in diseases associated with health disparities such as certain cancers and diabetes. Lens epithelium–derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, is emerging as a survival oncoprotein that promotes resistance to oxidative stress–induced cell death and chemotherapy. We previously showed that LEDGF/p75 is targeted by autoantibodies in prostate cancer patients and is overexpressed in prostate tumors, and that its stress survival activity is abrogated during apoptosis. LEDGF/p75 has a COOH-terminally truncated splice variant, p52, whose role in stress survival and apoptosis has not been thoroughly investigated. We observed unbalanced expression of these proteins in a panel of tumor cell lines, with LEDGF/p75 generally expressed at higher levels. During apoptosis, caspase-3 cleaved p52 to generate a p38 fragment that lacked the NH2-terminal PWWP domain and failed to transactivate the Hsp27 promoter in reporter assays. However, p38 retained chromatin association properties and repressed the transactivation potential of LEDGF/p75. Overexpression of p52 or its variants with truncated PWWP domains in several tumor cell lines induced apoptosis, an activity that was linked to the presence of an intron-derived COOH-terminal sequence. These results implicate the PWWP domain of p52 in transcription function but not in chromatin association and proapoptotic activities. Consistent with their unbalanced expression in tumor cells, LEDGF/p75 and p52 seem to play antagonistic roles in the cellular stress response and could serve as targets for novel antitumor therapies. (Mol Cancer Res 2008;6(8):1293–307)

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.

Efficacy of an Anti-transferrin Receptor 1 Antibody Against AIDS-related Non-Hodgkin Lymphoma: A Brief Communication.

The transferrin receptor 1 (TfR1), also known as CD71, is a target for antibody-based cancer immunotherapy due to its high expression on the surface of cancer cells and its ability to internalize. We have previously developed a mouse/human chimeric IgG3 specific for human TfR1 genetically fused to avidin, as a vector to deliver biotinylated anticancer agents into malignant cells. However, we found that this fusion protein (ch128.1Av), and to a lesser extent the same antibody without avidin (ch128.1), exhibits direct cytotoxic activity in vitro against certain malignant hematopoietic cells through the induction of TfR1 degradation and lethal iron starvation. Importantly, both ch128.1 and ch128.1Av have also shown significant anticancer activity in 2 xenograft models of the B-cell malignancy multiple myeloma. It is interesting to note that ch128.1 exhibited superior anticancer activity in both models compared with ch128.1Av, even against malignant cells that show no sensitivity to ch128.1 in vitro. In the present study, we evaluated the efficacy of ch128.1 against an AIDS-related human Burkitt lymphoma cell line (2F7) to determine if ch128.1 can eliminate these cells in vitro and in an in vivo model of AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Even though 2F7 cells expressed high TfR1 levels, these cells lacked sensitivity to the cytotoxicity induced by ch128.1 in vitro. However, ch128.1 showed significant anticancer activity against these AIDS-NHL cells in vivo by significantly prolonging the survival of immunodeficient mice bearing 2F7 tumors. Therefore, ch128.1 warrants further study as a candidate for the treatment of AIDS-NHL and other B-cell malignancies.

Abstract 73: Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma

The transferrin receptor 1 (TfR1), also known as CD71, is a membrane glycoprotein involved in cellular iron uptake and regulation of cell growth. The high level of TfR1 expression on malignant cells and its key role in cancer cell pathology make this receptor an attractive target for antibody cancer therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1). This antibody exhibits direct cytotoxicity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. Importantly, ch128.1 shows remarkable anti-tumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige). Interestingly, this anti-tumor protection was observed even against MM cells (KMS-11 cells) that show no sensitivity to this antibody in vitro, suggesting the in vivo contributions of antibody effector functions. This possibility was supported by the lack of anti-tumor protection observed using a ch128.1 Fc mutant with impaired binding to FcγRs and to the complement component C1q. To examine host effector functions involved in ch128.1-mediated protection in our mouse model bearing KMS-11 tumors, depletion studies of complement and macrophages were performed. Complement depletion using cobra venom factor (CVF) did not affect protection, suggesting that complement-mediated cytotoxicity (CDC) is not a relevant mechanism of action. Notably, we now report that macrophage depletion using clodronate liposomes (clodrolip) significantly reduced protection, suggesting that these effector cells play a relevant role in the anti-tumor activity. Consistent with this result, we also report that ch128.1 is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against KMS-11 cells in the presence of the murine macrophage cell line J774.2 or murine bone marrow-derived macrophages. To examine the role of iron deprivation in ch128.1-mediated tumor death in vivo, animals treated with ch128.1 were systemically supplemented with iron in a preliminary study. However, no difference in survival was observed, suggesting that iron deprivation is not a contributor to the effects of ch128.1 in our in vivo model or the iron dose tested was not optimal. Our results suggest that macrophages play a key role in ch128.1-mediated anti-tumor protection in our model and that ch128.1 can be an effective therapy of incurable human B-cell malignancies such as MM. Citation Format: Lai Sum Leoh, Yoon Kyung Kim, Pierre V. Candelaria, Otoniel Martinez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2017-73

Abstract 1335: Dissecting the mechanisms of anti-tumor protection mediated by an antibody targeting the transferrin receptor 1

The transferrin receptor 1 (TfR1/CD71) is a type II transmembrane homodimeric protein involved in iron uptake and regulation of cell growth. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, make TfR1 an attractive target for antibody therapy. We developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits direct cytotoxic activity against certain human malignant B cells in vitro. This anti-tumor activity is due to the induction of TfR1 degradation and lethal iron deprivation. Importantly, ch128.1 also shows extraordinary anti-tumor activity in two xenograft models of the incurable B-cell malignancy multiple myeloma in immunosuppressed mice (SCID-Beige) resulting in up to 100% survival using a single dose. Interesting, this anti-tumor activity occurs even against malignant cell lines that show limited or no sensitivity to ch128.1 in vitro. The mechanism of in vivo protection of ch128.1 is currently unknown. In order to explore the possible role of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) in the anti-tumor mechanism of ch128.1 and to delineate the residues involved in these effector functions, we developed three constructs with mutations in the ch128.1 heavy chain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. We now show that ch128.1 is capable of inducing ADCC and CDC against malignant B cells in vitro suggesting the potential contribution of these antibody effector functions to its effects in vivo. Additionally, we show that the triple mutant consistently displayed significant ADCC reduction in vitro, while the other mutants showed a weaker impairment, suggesting that all three residues are relevant contributors to ADCC. Lack of CDC in the P331S mutant confirmed the critical role of this residue, while the other residues show a lesser role in CDC in vitro. These results provide, for the first time, insights into the human IgG3 effector functions in the context of TfR1 targeting of malignant cells. Intriguingly, we have recently observed that the strong in vivo protection of ch128.1 against disseminated malignant B cells is abolished when the triple ch128.1 mutant (L234A/L235A/P331S) is used as a therapeutic at the same dose as that of ch128.1. These results point to a functional role for the antibody Fc fragment in anti-tumor protection mediated by ch128.1. Further studies aimed to provide a better understanding of the in vivo activity exhibited by ch128.1 are in progress. This antibody may be potentially used in the therapy of incurable human B-cell malignancies, such as multiple myeloma and certain non-Hodgkin lymphomas. Citation Format: Lai Sum Leoh, Tracy R. Daniels-Wells, Otoniel Martinez-Maza, Manuel L. Penichet. Dissecting the mechanisms of anti-tumor protection mediated by an antibody targeting the transferrin receptor 1. [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 1335. doi:10.1158/1538-7445.AM2015-1335

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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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/p75s 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/p75s 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/p75s 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.