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 À/À 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 À/À mice will be deficient in chemical neoplasia-was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76 À/À . PMA-induced skin carcinogenesis in RLIP76 þ/þ mouse was suppressed completely by depletion of either PKCa or RLIP76by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76 À/À mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76À/À mice. In RLIP76 À/À 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.

Normal cells continuously monitor the nature of their respective cellular microenvironment. They are equipped with an inherent molecular defense to detect changes which can precipitate and trigger an oncogenic cascade in the internal and external environment of cells. The process called Anoikis unleashes many a characteristic molecular changes in 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 release of molecular discipline which conforms them to a benign structural and functional spectrum. The initiation and propagation of signaling which 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 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 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 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.Renal cell Carcinoma (RCC) affects~55,000 Americans each year, resulting in~15,000 deaths in the United States and 120,000 deaths worldwide annually, making RCC one of the most lethal urologic cancers. RCC accounts for 2% of all cancers. The incidence of RCC has been steadily rising over the past 30 years. RCC has been classified histologically as clear cell, papillary, chromophobe, collecting duct and medullary.The majority (75%) of cases are clear-cell RCC. These are characteristically associated with loss of function of the von Hippel-Lindau (VHL) gene (e.g., chromosome 3p depletion, suppressed expression, or loss-of-function base substitutions). VHL mutations occur in~60% of RCCs.1 RCC is a highly treatment-resistant tumor type; however, advances in elucidating the molecular pathophysiology underlying RCC have led to the identification of promising targets for therapeutic intervention. In clear-cell RCC, mutations to the VHL gene involved with cellular responses to hypoxia, results in the upregulation of many proteins necessary for tumor growth and survival, such as vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and plateletderived growth factor (PDGF), which are involved in tumorinitiated angiogenesis. The mTOR pathway seems to be important primary or alternativepathway in RCC, disrupting phosphoinositide 3-kinase (PI3K)/AKT signaling. Temsirolimus fo...

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.