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

Abstract

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.

RLIP76 Protein Knockdown Attenuates Obesity Due to a High-fat Diet

“…Because RLIP76 is the dominant transporter of lipid peroxidation-derived glutathione conjugates (i.e. leukotrienes and alkenal-SG conjugates) and this ATPase-mediated transport activity is directly necessary for clathrin-dependent endocytosis (CDE), RLIP76 functions as a rate-limiting step of both the mercapturic acid pathway and CDE (53). Thus, RLIP76 activity correlates directly with the rate of internalization of peptide hormone-receptor complexes from the plasma membrane through CDE, and indirectly with the accumulation of oxidative stress promoting mercapturic acid precursor glutathione conjugates of lipid peroxidation-derived reactive oxygen species.…”

Novel Anti-cancer Compounds for Developing Combinatorial Therapies to Target Anoikis-Resistant Tumors

“…Extensive studies have characterized the tumor promoting and metastasis favoring nature of Ral-binding protein 1 (RalBP1 or RLIP76), a 76 kDa multi-specific Ral-effector that also predominantly functions as a transporter of glutathioneconjugates (GS-E) of chemotherapy drugs and toxic end products of lipid peroxidation (104,105). In our extensive studies, depletion of RLIP76 by R508 phosphorothioate antisense or inhibition of the transport activity by RLIP76 antibody effectively inhibited the proliferative and migratory capacity of melanomas, neuroblastomas, squamous cell carcinomas and tumors of lung, kidney and prostate.…”

“…In our extensive studies, depletion of RLIP76 by R508 phosphorothioate antisense or inhibition of the transport activity by RLIP76 antibody effectively inhibited the proliferative and migratory capacity of melanomas, neuroblastomas, squamous cell carcinomas and tumors of lung, kidney and prostate. RLIP76 inhibition also substantially sensitized the tumor cells to radiation and chemotherapy drugs like doxorubicin, cisplatin, sorafenib and sunitinib (105,106). Targeting RLIP76 has been very effective in inhibiting anoikis-resistant metastatic progression of bladder and prostate cancers (107).…”

RalA-binding Protein 1 is an Important Regulator of Tumor Angiogenesis

“…RLIP76 knockout mice are highly resistant to chemical carcinogenesis and are even resistant to the growth of subcutaneously implanted cancer cells [20,21,32,33,36,37,41]. Recently, we have been published in RLIP76 regulates tumor angiogenesis in vivo and in vitro, these studies suggest that suppression of RLIP76 can inhibit vascular growth and/or angiogenesis for tumor angiogenesis.…”

“…Introduction RalA-binding protein 1 (RLIP76) plays an important role in cancer, such as melanoma regression, regression of lung and colon cancer, colorectal cancer, breast cancer, and carcinoma cancer [21,24,32,35,36]. RLIP76 knockout mice are highly resistant to chemical carcinogenesis and are even resistant to the growth of subcutaneously implanted cancer cells [20,21,32,33,36,37,41]. Recently, we have been published in RLIP76 regulates tumor angiogenesis in vivo and in vitro, these studies suggest that suppression of RLIP76 can inhibit vascular growth and/or angiogenesis for tumor angiogenesis.…”