Poorna Chandra Rao Lelsani

The Sensors and Regulators of Cell-Matrix Surveillance in Anoikis Resistance of Tumors

Normal cells continuously monitor the nature of their respective cellular microenvironment. They are equipped with an inherent molecular defense to detect changes that can precipitate and trigger an oncogenic cascade in the internal and external environment of cells. The process called anoikis unleashes many a characteristic molecular change in the cells which eventually program to cell death in response to cell detachment and inappropriate cellular attachment, both of which can otherwise potentiate the ability of cells to preferentially pursue a malignant course due to the release of molecular discipline which conforms them to a benign structural and functional spectrum. The initiation and propagation of signaling that serves as a switch to cell survival or cell death mediated by surveillance of cell microenvironment is comprised of many heterogeneous sets of molecules interacting mainly at the interface of cell–extracellular matrix. Transforming cells continuously reprogram their signaling characteristics in sensing and modulating the stimuli from cell surface molecules like integrins, cadherins and immunoglobulin family of cell adhesion molecules at adhesion complexes, which enables them to resist anoikis and metastasize to different organs. Actin cytoskeleton binds BIM and Bcl2 modifying factor (BMF), which are regulated by the adhesion status and consequent conformation of cytoskeleton in the cells. This review aims at an integrated synopsis of fundamental mechanisms of the critical interactions of cell surface molecules to facilitate a focused analysis of the differential regulation of signaling processes at cell‐ECM junctions that collectively rein the anoikis resistance, which in turn impacts metastatic aggressiveness and drug resistance of tumors originating from respective organs.

RLIP76 Transports Sunitinib and Sorafenib and Mediates Drug Resistance in Kidney Cancer

RLIP76 is a stress‐responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It represents the predominant glutathione‐conjugate (GS‐E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin‐mediated apoptosis, and that its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. We demonstrated for the first time that the striking anti‐neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects are also demonstrable for the antibody, antisense and siRNA in a renal cell xenografts model of Caki‐2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS‐E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti‐apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma.

RLIP76: A versatile transporter and an emerging target for cancer therapy

In the last few years, extensive research has been made to elucidate the functional significance of RLIP76. The resulting novel breakthroughs have helped us understand its transport and signaling functions. RLIP76 is a ubiquitously expressed, key stress-defensive, anti-apoptotic, multi-functional protein that transports glutathione-conjugates of electrophilic compounds, thus controlling the intracellular concentration of pro-apoptotic oxidized lipid byproducts and other xenobiotics such as chemotherapeutic agents. These properties place RLIP76 at a very important position in the hierarchy of the stress defense mechanism adopted by the cell. Selective over-expression of RLIP76 in malignant cells of diverse origin is one of the possible mechanisms by which these cells overcome chemotherapy and radiation induced oxidative damage. RLIP76 has also been shown to be an effective transporter of many conventional chemotherapeutic drugs. Such transport, if inhibited, can lead to increased cellular accumulation of drugs which in turn translates to enhanced drug sensitivity. Recent studies have shown that inhibition and/or depletion of RLIP76 by antibodies, siRNA, or antisense can lead to drastic and sustained regression of lung, kidney, melanoma, colon, and prostate cancer xenografts with no observed recurrence of tumors. All these findings converge on the fact that such inhibition/depletion of RLIP76 can be used clinically to terminate cancer growth and progression. In the present review, we will discuss the role of RLIP76 as a multi-drug transporter, its involvement in cancer, and the prospects of using RLIP76 inhibition as an emerging treatment for cancer.

Glutathione-Conjugate Transport by RLIP76 Is Required for Clathrin-Dependent Endocytosis and Chemical Carcinogenesis

Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76−/− MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis—RLIP76−/− mice will be deficient in chemical neoplasia—was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76−/−. PMA-induced skin carcinogenesis in RLIP76+/+ mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76−/− mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76−/− mice. In RLIP76−/− mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis. Mol Cancer Ther; 10(1); 16–28. ©2011 AACR.

The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4.

It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 μM HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 μM HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 μM HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce angiogenic signaling mechanism in the endothelial cells. These findings may suggest a role of HNE and GSTA4-4 in oxidative stress induced proliferative retinopathies.

Abstract 3702: 2-Hydroxyflavanone inhibits renal cancer growth by inhibiting tumor cell proliferation and angiogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Renal cell carcinoma (RCC) is one of the top ten cancers prevalent in USA. Loss-of-function mutation in the VHL is an established risk factor for RCC contributing to 75% of total reported cases of RCC. Loss of VHL leads to highly vascularized phenotype of renal tumors. Intake of oranges has been proven to reduce the risk of RCC in multi-center international clinical trials. Hence, we studied effects of 2HF, an active anti-cancer compound of oranges, in VHL- mutant RCC. Our in vitro investigations revealed that 2HF selectively inhibits VHL-mutant RCC by inhibiting EGFR signaling which is increased due to VHL mutations in RCC. Our results also revealed for the first time, that 2HF inhibits GSTπ activity. 2HF inhibited cyclin B1 and CDK4, and induced G2/M phase arrest in VHL-mutant RCC. Importantly, 2HF inhibited the angiogenesis in VHL-mutant RCC by decreasing VEGF expression. Our in vivo studies in mice xenografts confirmed our in vitro results as evident by decreased levels of proliferation marker, Ki67 and angiogenic marker CD31, in 2HF treated (0.01%, w/w) mice xenografts of VHL-mutant RCC. 2HF also increased the expression of E-cadherin in VHL-mutant RCC which would be of significance in restoring the normal epithelial phenotype. Collectively, our in vitro and in vivo results revealed the potent anti-proliferative and anti-angiogenic effects of 2HF in VHL-mutant RCC, sparing normal cells, which could have significant implications not only in the specific management of VHL-mutant RCC but also in treating other VHL syndromes with highly vascularized phenotype. (Supported in part by NIH grant CA 77495 (SA), CA 155350, Cancer Research Foundation of North Texas, and Institute for Cancer Research & the Joe & Jessie Crump Fund for Medical Education (SSS)) 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 3702. doi:10.1158/1538-7445.AM2011-3702

Abstract 1525: RLIP76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer

RLIP76 is a stress-responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It functions as the predominant glutathione-electrophile conjugate (GS-E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin-mediated apoptosis, and its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. Recently, we have demonstrated for the first time the striking anti-neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects for the antibody, antisense and siRNA in a renal cell xenografts model of Caki-2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS-E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies support the validity of RLIP76 as a target in kidney cancer therapy, and the functional model in which RLIP76 provides protection from chemical and radiant stress through its transport activity. Results of these studies revealed for the first time that sorafenib as well as sunitinib, receptor tyrosine kinase inhibitors (RTKIs), are substrates for transport by RLIP76. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti-apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma. (Supported in part by NIH Grants CA 77495 and CA 104661 (to SA), Cancer Research Foundation of North Texas (to SSS & SY), Institute for Cancer Research and the Joe & Jessie Crump Fund for Medical Education (to SSS)) 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 1525.