Regression of ovarian cancer xenografts by depleting or inhibiting RLIP.

Ovarian cancer (OC) is the most prevalent and deadly cancer of the female reproductive system. Women will continue to be impacted by OC-related morbidity and mortality. Despite the fact that chemotherapy with cisplatin is the main component as the first-line anticancer treatment for OC, chemoresistance and unfavorable side effects are important obstacles to effective treatment. Targets for effective cancer therapy are required for cancer cells but not for non-malignant cells because they are expressed differently in cancer cells compared to normal cells. Targets for cancer therapy should preferably be components that already exist in biochemical and signalling frameworks and that significantly contribute to the development of cancer or regulate the response to therapy. RLIP is an important mercapturic acid pathway transporter that is crucial for survival and therapy resistance in cancers, therefore, we examined the role of RLIP in regulating essential signalling proteins involved in relaying the inputs from upstream survival pathways and mechanisms contributing to chemo-radiotherapy resistance in OC. The findings of our research offer insight into a novel anticancer effect of RLIP depletion/inhibition on OC and might open up new therapeutic avenues for OC therapy.

The innate effects of plant secondary metabolites in preclusion of gynecologic cancers: Inflammatory response and therapeutic action.

Gynecologic cancers can make up the bulk of cancers in both humans and animals. The stage of diagnosis and the type of tumor, its origin, and its spread are a few of the factors that influence how effectively a treatment modality works. Currently, radiotherapy, chemotherapy, and surgery are the major treatment options recommended for the eradication of malignancies. The use of several anti-carcinogenic drugs increases the chance of harmful side effects, and patients might not react to the treatments as expected. The significance of the relationship between inflammation and cancer has been underscored by recent research. As a result, it has been shown that a variety of phytochemicals with beneficial bioactive effects on inflammatory pathways have the potential to act as anti-carcinogenic medications for the treatment of gynecologic cancer. The current paper reviews the significance of inflammatory pathways in gynecologic malignancies and discusses the role of plants-derived secondary metabolites that are useful in the treatment of cancer.

Phase I trial of intravenous fenretinide (4-HPR) plus safingol in advanced malignancies.

PURPOSE: Fenretinide (4-HPR) is a synthetic retinoid that induces cytotoxicity through dihydroceramide production. Safingol, a stereochemical-variant dihydroceramide precursor, exhibits synergistic effects when administered with fenretinide in preclinical studies. We conducted a phase 1 dose-escalation clinical trial of this combination. METHODS: Fenretinide was administered as a 600 mg/m2 24-h infusion on Day 1 of a 21-day cycle followed by 900 mg/m2/day on Days 2 and 3. Safingol was concurrently administered as a 48-h infusion on Day 1 and 2 using 3 + 3 dose escalation. Primary endpoints were safety and maximum tolerated dose (MTD). Secondary endpoints included pharmacokinetics and efficacy. RESULTS: A total of 16 patients were enrolled (mean age 63 years, 50% female, median three prior lines of therapy), including 15 patients with refractory solid tumors and one with non-Hodgkin lymphoma. The median number of treatment cycles received was 2 (range 2-6). The most common adverse event (AE) was hypertriglyceridemia (88%; 38% ≥ Grade 3), attributed to the fenretinide intralipid infusion vehicle. Other treatment-related AEs occurring in ≥ 20% of patients included anemia, hypocalcemia, hypoalbuminemia, and hyponatremia. At safingol dose 420 mg/m2, one patient had a dose-limiting toxicity of grade 3 troponinemia and grade 4 myocarditis. Due to limited safingol supply, enrollment was halted at this dose level. Fenretinide and safingol pharmacokinetic profiles resembled those observed in monotherapy trials. Best radiographic response was stable disease (n = 2). CONCLUSION: Combination fenretinide plus safingol commonly causes hypertriglyceridemia and may be associated with cardiac events at higher safingol levels. Minimal activity in refractory solid tumors was observed. TRIAL REGISTRATION NUMBER: NCT01553071 (3.13.2012).

Prognostic significance of natural products against multidrug tumor resistance.

Cancer is a pervasive, constantly evolving, and significant public health concern. The number of new cancer cases has risen dramatically in the last decades, making it one of the top causes of poor health and mortality worldwide. Although various treatment strategies, including surgery, radiation, and pharmaceutical therapies, have evolved into more sophisticated, precise methods, there is not much improvement in the cancer-related death toll. Consequently, natural product-based therapeutic discoveries have recently been considered an alternative approach. According to an estimate, one-third of the top twenty medications in today's market have a natural plant-product-based origin. Accordingly, primary prevention is an essential component of worldwide cancer control. This review provides an overview of the mechanisms of action of bioactive ingredients in natural dietary products that may contribute to the prevention and management of multiple malignancies.

RLIP: A necessary transporter protein for translating oxidative stress into pro-obesity and pro-carcinogenic signaling.

Previously, we showed that knockout mice homozygous for deficiency of the mercapturic acid pathway (MAP) transporter protein, RLIP (RLIP-/-), are resistant to chemical carcinogenesis, inflammation, and metabolic syndrome (MetS). We also found that RLIP-/- mice are highly resistant to obesity caused by a high-fat diet (HFD). Interestingly, these studies showed that kinase, cytokine, and adipokine signaling that are characteristics of obesity were blocked despite the presence of increased oxidative stress in RLIP-/- mice. The deficiencies in obesity-inducing kinase, cytokine, and adipokine signaling were attributable to a lack of clathrin-dependent endocytosis (CDE), a process that is severely deficient in RLIP-/- mice. Because CDE is also necessary for carcinogenic signaling through EGF, WNT, TGFß and other cancer-specific peptide hormones, and because RLIP-/- mice are cancer-resistant, we reasoned that depletion of RLIP by an antisense approach should cause cancer regression in human cancer xenografts. This prediction has been confirmed in studies of xenografts from lung, kidney, prostate, breast, and pancreatic cancers and melanoma. Because these results suggested an essential role for RLIP in carcinogenesis, and because our studies have also revealed a direct interaction between p53 and RLIP, we reasoned that if RLIP played a central role in carcinogenesis, that development of lymphoma in p53-/- mice, which normally occurs by the time these mice are 6 months old, could be delayed or prevented by depleting RLIP. Recent studies described herein have confirmed this hypothesis, showing complete suppression of lymphomagenesis in p53-/- mice treated with anti-RLIP antisense until the age of 8 months. All control mice developed lymphoma in the thymus or testis as expected. These findings lead to a novel paradigm predicting that under conditions of increased oxidative stress, the consequent increased flux of metabolites in the MAP causes a proportional increase in the rate of CDE. Because CDE inhibits insulin and TNF signaling but promotes EGF, TGFß, and Wnt signaling, our model predicts that chronic stress-induced increases in RLIP (and consequently CDE) will induce insulin-resistance and enhance predisposition to cancer. Alternatively, generalized depletion of RLIP would antagonize the growth of malignant cells, and concomitantly exert therapeutic insulin-sensitizing effects. Therefore, this review focuses on how targeted depletion or inhibition of RLIP could provide a novel target for treating both obesity and cancer.

Correction: 2'-Hydroxyflavanone effectively targets RLIP76-mediated drug transport and regulates critical signaling networks in breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.24720.].

Case report of recurrent fibromatosis with laryngeal involvement: Treatment based on network analyses of NGS data.

Aggressive fibromatosis (AF) is a rare, benign neoplasm originating from musculoaponeurotic stromal structures characterized by aggressive growth and infiltration of local tissues. To date, only six previous cases of AF involving the larynx have been reported. The present case was that of a 70-year-old female patient with a 5-year history of hoarseness and an enlarging neck mass consistent with aggressive recurrent fibromatosis. MRI displayed a large, solid mass arising from the left anterior cervical space displacing the trachea and upper airway to the right. At one year after the initial radical resection, the patient presented with recurrence. A second radical excision was performed not including a laryngectomy. Radiation therapy was considered for possible local treatment to prevent subsequent tumor recurrence. According to the literature, AF has recurrence rates of up to 40-70% within 18 months. There is only sparse literature to guide treatment. Using the mutations detected in the patient's AF tissue, an Ingenuity Pathway Analysis (IPA) was used to guide treatment of the recurrence. In the present case, the IPA analysis indicated the use of pazopanib to treat the patient's cancer. In general, surgery appears to be the treatment of choice for head and neck AF, but the management of recurrence is controversial.

Rlip Depletion Alters Oncogene Transcription at Multiple Distinct Regulatory Levels.

Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.

Anticancer Activity of Ω-6 Fatty Acids through Increased 4-HNE in Breast Cancer Cells.

Her2-amplified breast cancers resistant to available Her2-targeted therapeutics continue to be a challenge in breast cancer therapy. Dox is the mainstay of chemotherapy of all types of breast cancer, but its usefulness is limited by cumulative cardiotoxicity. Because oxidative stress caused by dox generates the pro-apoptotic Ω-6 PUFA metabolite 4-hydroxynonenal (4-HNE), we surmised that Ω-6 PUFAs would increase the effectiveness of dox chemotherapy. Since the mercapturic acid pathway enzyme RALBP1 (also known as RLIP76 or Rlip) that limits cellular accumulation of 4-HNE also mediates dox resistance, the combination of Ω-6 PUFAs and Rlip depletion could synergistically improve the efficacy of dox. Thus, we studied the effects of the Ω-6 PUFA arachidonic acid (AA) and Rlip knockdown on the antineoplastic activity of dox towards Her2-amplified breast cancer cell lines SK-BR-3, which is sensitive to Her2 inhibitors, and AU565, which is resistant. AA increased lipid peroxidation, 4-HNE generation, apoptosis, cellular dox concentration and dox cytotoxicity in both cell lines while sparing cultured immortalized cardiomyocyte cells. The known functions of Rlip including clathrin-dependent endocytosis and dox efflux were inhibited by AA. Our results support a model in which 4-HNE generated by AA overwhelms the capacity of Rlip to defend against apoptosis caused by dox or 4-HNE. We propose that Ω-6 PUFA supplementation could improve the efficacy of dox or Rlip inhibitors for treating Her2-amplified breast cancer.

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

We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

Targeting the mercapturic acid pathway for the treatment of melanoma.

The treatment of metastatic melanoma is greatly hampered by the simultaneous dysregulation of several major signaling pathways that suppress apoptosis and promote its growth and invasion. The global resistance of melanomas to therapeutics is also supported by a highly active mercapturic acid pathway (MAP), which is responsible for the metabolism and excretion of numerous chemotherapy agents. The relative importance of the MAP in melanoma survival was not recognized until demonstrated that B16 melanoma undergoes dramatic apoptosis and regression upon the depletion or inhibition of the MAP transporter protein RLIP. RLIP is a multi-functional protein that couples ATP hydrolysis with the movement of substances. As the rate-limiting step of the MAP, the primary function of RLIP in the plasma membrane is to catalyze the ATP-dependent efflux of unmetabolized drugs and toxins, including glutathione (GSH) conjugates of electrophilic toxins (GS-Es), which are the precursors of mercapturic acids. Clathrin-dependent endocytosis (CDE) is an essential mechanism for internalizing ligand-receptor complexes that promote tumor cell proliferation through autocrine stimulation (Wnt5a, PDGF, ßFGF, TNFα) or paracrine stimulation by hormones produced by fibroblasts (IGF1, HGF) or inflammatory cells (IL8). Aberrant functioning of these pathways appears critical for melanoma cell invasion, metastasis, and evasion of apoptosis. This review focuses on the selective depletion or inhibition of RLIP as a highly effective targeted therapy for melanoma that could cause the simultaneous disruption of the MAP and critical peptide hormone signaling that relies on CDE.

Prevention of mammary carcinogenesis in MMTV-neu mice by targeting RLIP.

The overexpression and amplification of the protooncogene neu (ERBB2) play an important role in the development of aggressive breast cancer (BC) in humans. Ral-interacting protein (RLIP), a modular stress-response protein with pleiotropic functions, is overexpressed in several types of cancer, including BC. Here, we show that blocking RLIP attenuates the deleterious effects caused by the loss of the tumor suppressor p53 and inhibits the growth of human BC both in vitro and in vivo in MMTV-neu mice. In addition, we show that treatment with the diet-derived, RLIP-targeting chemotherapeutic 2'-hydroxyflavanone (2HF), alone or in combination with RLIP-specific antisense RNA or antibodies, significantly reduced the cumulative incidence and/or burden of mammary hyperplasia and carcinoma in MMTV-neu mice. 2HF treatment correlated with reduced tumor cell proliferation and increased apoptosis, and the average number of Ki67-positive (proliferating) cells was significantly lower in the tumors of 2HF-treated mice than in the tumors of control mice. Furthermore, targeting RLIP also resulted in the overexpression of E-cadherin and the infiltration of CD3+ T cells into mammary tumors. Taken together, these results underscore the translational potential of RLIP-targeting agents and provide a strong rationale to validate them in the clinic.

RLIP depletion induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells.

The incidence of malignant melanoma, a neoplasm of melanocytic cells, is increasing rapidly. The lymph nodes are often the first site of metastasis and can herald systemic dissemination, which is almost uniformly fatal. RLIP, a multi-specific ATP-dependent transporter that is over-expressed in several types of cancers, plays a central role in cancer cell resistance to radiation and chemotherapy. RLIP appears to be necessary for cancer cell survival because both in vitro cell culture and in vivo animal tumor studies show that the depletion or inhibition of RLIP causes selective toxicity to malignant cells. RLIP depletion/inhibition triggers apoptosis in cancer cells by inducing the accumulation of endogenously formed glutathione-conjugates. In our in vivo studies, we administered RLIP antibodies or antisense oligonucleotides to mice bearing subcutaneous xenografts of SKMEL2 and SKMEL5 melanoma cells and demonstrated that both treatments caused significant xenograft regression with no apparent toxic effects. Anti-RLIP antibodies and antisense, which respectively inhibit RLIP-mediated transport and deplete RLIP expression, showed similar tumor regressing activities, indicating that the inhibition of RLIP transport activity at the cell surface is sufficient to achieve anti-tumor activity. Furthermore, RLIP antisense treatment reduced levels of RLIP, pSTAT3, pJAK2, pSrc, Mcl-1 and Bcl2, as well as CDK4 and cyclin B1, and increased levels of Bax and phospho 5' AMP-activated protein kinase (pAMPK). These studies indicate that RLIP serves as a key effector in the survival of melanoma cells and is a valid target for cancer therapy. Overall, compounds that inhibit, deplete or downregulate RLIP will function as wide-spectrum agents to treat melanoma, independent of common signaling pathway mutations.

Activating p53 function by targeting RLIP.

Aberrations in RLIP, p53, and PKCα represent essentially the entire spectrum of all human neoplasms. Elevated PKCα expression, failure of the cell cycle checkpoint (p53 dysfunction), and abnormal glutathione (GSH) metabolism are fundamental hallmarks of carcinogenesis and drug/radiation resistance. However, a lack of investigations into the interactions between these important regulatory nodes has fundamentally limited our understanding of carcinogenesis and the development of effective interventions for cancer prevention and therapy. Loss of p53, perhaps the most powerful tumor suppressor gene, predisposes rodents to spontaneous cancer and humans to familial, as well as acquired, cancers. Until recently, no genetic manipulation of any oncogene had been reported to abrogate spontaneous carcinogenesis in p53-/- rodent models. However, the overexpression of RLIP, a GSH-electrophile conjugate (GS-E) transporter, has been found to enhance cancer cell proliferation and confer drug/radiation resistance, whereas its depletion causes tumor regression, suggesting its importance in cancer and drug/radiation resistance. Indeed, RLIP is an essential effector of p53 that is necessary for broad cancer-promoting epigenetic remodeling. Interestingly, through a haploinsufficiency mechanism, the partial depletion of RLIP in p53-/- mice provides complete protection from neoplasia. Furthermore, RLIP-/- mice exhibit altered p53 and PKCα function, marked deficiency in clathrin-dependent endocytosis (CDE), and almost total resistance to chemical carcinogenesis. Based on these findings, in this review, we present a novel and radical hypothesis that expands our understanding of the highly significant cross-talk between p53, PKCα, and GSH signaling by RLIP in multiple tumor models.

Haploinsufficiency Interactions of RALBP1 and TP53 in Carcinogenesis.

Mutagenic environmental chemical or radiant stresses directly damage DNA and amplify the formation of potent endogenous mutagens from lipid peroxidation, leading to cancers that cause millions of deaths and impose enormous financial and social burdens [...].

Targeting RLIP with CRISPR/Cas9 controls tumor growth.

Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the 'guardian of the genome' because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo.

Association Between Tumor Mutation Profile and Clinical Outcomes Among Hispanic-Latino Patients With Metastatic Colorectal Cancer.

In the United States, CRC is the third most common type of cancer and the second leading cause of cancer-related death. Although the incidence of CRC among the Hispanic population has been declining, recently, a dramatic increase in CRC incidents among HL younger than 50 years of age has been reported. The incidence of early-onset CRC is more significant in HL population (45%) than in non-Hispanic Whites (27%) and African-Americans (15%). The reason for these racial disparities and the biology of CRC in the HL are not well understood. We performed this study to understand the biology of the disease in HL patients. We analyzed formalin-fixed paraffin-embedded tumor tissue samples from 52 HL patients with mCRC. We compared the results with individual patient clinical histories and outcomes. We identified commonly altered genes in HL patients (APC, TP53, KRAS, GNAS, and NOTCH). Importantly, mutation frequencies in the APC gene were significantly higher among HL patients. The combination of mutations in the APC, NOTCH, and KRAS genes in the same tumors was associated with a higher risk of progression after first-line of chemotherapy and overall survival. Our data support the notion that the molecular drivers of CRC might be different in HL patients.

Multi-Omic Analysis Reveals Different Effects of Sulforaphane on the Microbiome and Metabolome in Old Compared to Young Mice.

Dietary factors modulate interactions between the microbiome, metabolome, and immune system. Sulforaphane (SFN) exerts effects on aging, cancer prevention and reducing insulin resistance. This study investigated effects of SFN on the gut microbiome and metabolome in old mouse model compared with young mice. Young (6-8 weeks) and old (21-22 months) male C57BL/6J mice were provided regular rodent chow ± SFN for 2 months. We collected fecal samples before and after SFN administration and profiled the microbiome and metabolome. Multi-omics datasets were analyzed individually and integrated to investigate the relationship between SFN diet, the gut microbiome, and metabolome. The SFN diet restored the gut microbiome in old mice to mimic that in young mice, enriching bacteria known to be associated with an improved intestinal barrier function and the production of anti-inflammatory compounds. The tricarboxylic acid cycle decreased and amino acid metabolism-related pathways increased. Integration of multi-omic datasets revealed SFN diet-induced metabolite biomarkers in old mice associated principally with the genera, Oscillospira, Ruminococcus, and Allobaculum. Collectively, our results support a hypothesis that SFN diet exerts anti-aging effects in part by influencing the gut microbiome and metabolome. Modulating the gut microbiome by SFN may have the potential to promote healthier aging.

Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling.

Age-associated mitochondrial dysfunction and oxidative damage are primary causes for multiple health problems including sarcopenia and cardiovascular disease (CVD). Though the role of Nrf2, a transcription factor that regulates cytoprotective gene expression, in myopathy remains poorly defined, it has shown beneficial properties in both sarcopenia and CVD. Sulforaphane (SFN), a natural compound Nrf2-related activator of cytoprotective genes, provides protection in several disease states including CVD and is in various stages of clinical trials, from cancer prevention to reducing insulin resistance. This study aimed to determine whether SFN may prevent age-related loss of function in the heart and skeletal muscle. Cohorts of 2-month-old and 21- to 22-month-old mice were administered regular rodent diet or diet supplemented with SFN for 12 weeks. At the completion of the study, skeletal muscle and heart function, mitochondrial function, and Nrf2 activity were measured. Our studies revealed a significant drop in Nrf2 activity and mitochondrial functions, together with a loss of skeletal muscle and cardiac function in the old control mice compared to the younger age group. In the old mice, SFN restored Nrf2 activity, mitochondrial function, cardiac function, exercise capacity, glucose tolerance, and activation/differentiation of skeletal muscle satellite cells. Our results suggest that the age-associated decline in Nrf2 signaling activity and the associated mitochondrial dysfunction might be implicated in the development of age-related disease processes. Therefore, the restoration of Nrf2 activity and endogenous cytoprotective mechanisms by SFN may be a safe and effective strategy to protect against muscle and heart dysfunction due to aging.

Rlip Depletion Suppresses Growth of Breast Cancer.

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.