Rlip Depletion Suppresses Growth of Breast Cancer

Abstract

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Haploinsufficiency Interactions between RALBP1 and p53 in ERBB2 and PyVT Models of Mouse Mammary Carcinogenesis

“…Rlip-LNA and LNA control antisense (CAS) were purchased from Exiqon (Woburn, MA, USA). Sources of reagents for qRT-PCR, western blot, and genotyping were the same as previously described [62][63][64]. All reagents were of analytical grade.…”

“…Lungs were removed, air-evacuated, and fixed in 10% buffered formalin phosphate before automated paraffin embedding for metastasis quantification. Tumor tissues were cut into ∼5 mm thick slabs followed by H&E staining of embedded tissue by the Pathology Core Facility at TTUHSC [62]. The number of pulmonary metastases was counted under a camera-equipped stereomicroscope.…”

Haploinsufficiency Interactions of RALBP1 and TP53 in Carcinogenesis

“…Concomitant deficiencies of GS-E efflux, GSH-linked antioxidant defenses and CDE-linked vesicular trafficking in RALBP1-null (RALBP1 −/− ) mice show that RALBP1 functions provide an essential link between stress-induced apoptosis defenses and RAL/RAS/RHO/RAC-regulated pathways that promote cancer cell growth [ 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ]. Our recent studies in lung and breast cancers as well as melanoma have confirmed that the inhibition or depletion of RALBP1 blocks CDE and inhibits signaling by cancer-promoting peptides, including EGF, WNT and FGF [ 10 , 11 , 13 , 14 ]. The role of RALBP1 in regulating intracellular vesicular traffic, and the signaling proteins, such as ARF6, ARNO, PI3K and RAC, that regulate vesicular traffic, is reviewed in one paper presented in this symposium issue.…”

“…In stark contrast, mice lacking the RALBP1 ((RalA Binding Protein 1) gene, which encodes RLIP76 (also known as Rlip (RALBP1), used here for both gene and protein), a stress-responsive, anti-apoptotic protein, are highly resistant to carcinogenesis by even the most potent chemical carcinogens [ 10 ]. Targeted inhibition or depletion of RALBP1 causes regression of many types of cancer [ 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. In studies to examine the effect of combined RALBP1 and TP53 deficiency, we discovered that hemizygous RALBP1 deficiency was sufficient to exert a strong dominant negative effect on the spontaneous carcinogenesis phenotype of homozygous TP53-null mice [ 10 ].…”

“…The highly reactive alkylating agent 4HNE, with inherently greater pro-apoptotic effects against malignant as compared with non-malignant cells, is metabolized primarily to a glutathione-conjugate (GS-HNE) that is removed from cells by RALBP1 [ 18 , 26 , 27 , 36 , 37 , 52 , 53 , 54 ]. Elevated 4HNE production from the high levels of oxidative stress imposed on cancer cells by their high metabolic rates renders them very dependent on this mechanism of metabolizing and disposing of 4HNE, as is evident from the dramatic and sustained regression of multiple histological types of cancer upon depletion or inhibition of RALBP1 in mouse models without any toxicity [ 10 , 11 , 55 ]. Our findings of inhibition of cancer growth in vitro and in vivo have been confirmed by several independent groups [ 44 , 48 , 49 , 56 , 57 , 58 ].…”