Sharad S. Singhal, Ph.D.

Sharad Singhal, Ph.D., is a research professor in the Department of Medical Oncology & Therapeutics Research at Beckman Research Institute of City of Hope, and an affiliate full member of City of Hope's comprehensive cancer center and Developmental Cancer Therapeutics Program in Duarte, California. 

Dr. Singhal received his B.Sc. in biology (1982) and M.Sc. in chemistry (1984) from Agra University in India, and his Ph.D. in biochemistry (1989) from Central Drug Research Institute in Lucknow, India. He received Council of Scientific and Industrial Research fellowship during his Ph.D. He did his postdoctoral training at the University of Texas Medical Branch in Galveston, Texas (1989-1993). 

Dr. Singhal's research interests are in glutathione metabolism and its role in stress-defense, carcinogenesis, drug-resistance, radiation-resistance and cell-signaling mechanisms. At City of Hope, Dr. Singhal has conducted carcinogenesis studies in multiple cancers which add to his collective expertise in cancer prevention and control research. The focus of his current research is the development of natural compounds that broadly target cancer-specific signaling networks into the next generation of cancer preventive and therapeutic drugs that have the advantageous side effect of lowering insulin-resistance. He has successfully employed a highly effective in-silico signaling model based on the mercapturic acid pathway, which has already yielded three novel, natural source, lead compounds with very high efficacy in tumors of prostate, breast, renal and neuroblastoma. 

Laboratory 

In addition to cancer prevention and carcinogenesis studies, Dr. Singhal's lab also focuses on multidrug resistance in cancer and the role of non-ABC drug transporter (RLIP/RalBP1) in tumor progression. Loss of p53, perhaps the most powerful tumor suppressor gene, predisposes rodents to spontaneous cancer and humans to familial, as well as acquired, cancers. In solid cancers, RLIP levels are significantly higher in tumor cells than in surrounding normal cells. Homozygous RLIP knockout (RLIP-/-) mice have no spontaneous malignancies and are highly resistant to carcinogen-induced malignancies, however, homozygous p53 knockout (p53-/-) mice are highly susceptible, developing spontaneous malignancies by ~6 months of age and carcinogen-induced malignancies much earlier than wildtype mice.

Until recently, no oncogenes had been identified that, when knocked down, could abrogate spontaneous carcinogenesis in p53-/- rodent models. However, recently, we reported the astounding observation that p53-/- mice were significantly protected from spontaneous malignancy for up to eight months of age by the partial depletion of RLIP (an oncoprotein overexpressed in cancer cells) via antisense therapy. This protection was associated with nearly complete reversal of the major epigenetic and associated gene expression changes that normally occur in p53-/- mice. RLIP-/- and p53-/- mice are polar opposites in the spectrum of cancer susceptibility (Awasthi S et al., PNAS 2018; Singhal J et al., Cancer Lett. 2018, 2019; Nagaprashantha L et al., J Proteomics 2019; Krishna BM et al., Cancers 2020; Singhal J et al., Mol Carcinogenesis 2021; Carcinogenesis 2021; Singhal SS et al., BBA Reviews on Cancer 2022).

These studies demonstrate that depleting or inhibiting RLIP is a novel and highly strategic targeted therapy for treatment of cancer and indicate that RLIP is a high-value target for cancer therapy and provides sound mechanistic and preclinical rationale for its clinical development. In summary, Dr. Singhal has attracted multiple federal and foundation grants and has authored over 200 publications.

Research Interests

Protein chemistry, enzymology, expression, regulation of glutathione S-transferases and their structural and functional interrelationships, and in the field of chemotherapy drug resistance, particularly in the mechanisms involved in mediating drug resistance to a commonly used important chemotherapy drug, adriamycin (doxorubicin). Our laboratory is interested in glutathione metabolism and its role in stress defense, carcinogenesis, drug resistance, radiation resistance, insulin resistance, metabolic diseases, and cell-signaling mechanisms. Recently, we have demonstrated that RLIP76 (RALBP1, ral-binding protein) is the major glutathione-electrophile conjugate (GS-E) and drug transporter in mammalian cells.  RLIP76 is a crucial stress-protective protein that regulates signaling in the Ral, Ras, EGF, insulin and other pathways. Inhibition or depletion of RLIP76 causes cancer cell apoptosis in cell culture, and regression of melanoma, neuroblastoma, lung, prostate, kidney, breast and colon cancer in xenograft.

RLIP76 is a membrane-associated non-ATP binding cassette (ABC) multispecific transporter of chemotherapeutic drugs as well as of glutathione-electrophile conjugates (GS-E) and is involved in signaling pathways. This transporter is ubiquitously expressed in humans as well as in rodent tissues. It is frequently overexpressed in malignant cells and plays a prominent anti-apoptotic role selectively in cancer cells through its ability to control the cellular concentration of pro-apoptotic oxidized lipid by-products. Inhibition of RLIP76, using antibodies, or depletion of RLIP76 using either siRNA or antisense phosphorothioate oligonucleotides kills cancer cells or causes apoptosis in malignant cells preferentially. Recently, we have discovered that RLIP knockout mice are resistant to cancer and that depletion of RLIP by antisense in p53 knockout homozygous mice completely prevents the normal inevitable formation of lymphoma. On the other hand, all p53 knockout control animals developed lymphoma in the thymus or testis as expected, confirming our prediction that RLIP depletion/inhibition can actually inhibit carcinogenesis.  

In addition, we have investigated the anti-cancer effects of flavonoids like 2´-hydroxyflavanone, vicenin, and didymin in chemoprevention studies on various cancers of kidney, lung, prostate, breast and neuroblastomas. We employ a sound combination of genetic, molecular and proteomic approaches to specifically characterize the impact of candidate chemopreventive drugs on the core signaling networks of importance in oncogenic transformation and metastatic progression of respective cancers. This advanced and multi-disciplinary approach has made possible the integration of key essential investigative parameters which expand the understanding of the molecular basis for anticancer effects of novel chemopreventive agents in the cancers tested.  
 

• American Association for Cancer Research
• American Society for Biochemistry and Molecular Biology

• Singhal SS, Awasthi YC and Awasthi S. (2006) Regression of melanoma in a murine model by RLIP76 depletion. Cancer Res 66: 2354-2360 (PMID 16489041).
• Singhal SS, Singhal J, Yadav S, Dwivedi S, Boor P, Awasthi YC and Awasthi S. (2007) Regression of lung and colon cancer xenografts by depleting or inhibiting RLIP76 (RALBP1). Cancer Res 67: 4382-9 (PMID 17483352).
• Awasthi S, Singhal SS, Awasthi YC, Martin B, Woo J-H., Cunningham CC, and Frankel AE. (2008) RLIP76 and Cancer.  Clin Cancer Res 14: 4372-4377 (PMID 18628450).
• Singhal SS, Singhal J, Yadav S, Sahu M, Awasthi YC and Awasthi S. (2009) RLIP76: A target for kidney cancer therapy. Cancer Res 69: 4244-4251 (PMID 19417134) .
• Singhal SS, Sehrawat A, Sahu M., Singhal, P., Vatsyayan, R., Lelsani, P., Yadav, S., and Awasthi, S. (2010) RLIP76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer.  Int J Cancer 126: 1327-1338 (PMID 19626587).
• Awasthi S, Singhal SS, Yadav S, Singhal J, Vatsyayan R, Zajac E, Luchowski R, Borvak J, Gryczynski K and Awasthi YC. (2010) A central role of RLIP76 in regulation of glycemic control. Diabetes 59: 714-725 (PMID: 20007934).
• Singhal SS, Wickramarachchi D, Yadav S, Singhal J, Leake K, Vatsyayan R, Lelsani P, Chaudhary P, Suzuki S, Yang S, Awasthi YC and Awasthi S. (2011) Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis.  Mol Cancer Therapeutics 10: 16-28 (PMID: 21220488).
• Lee S, Wurtzel J, Singhal SS, Awasthi S and Goldfinger LE. (2012) RALBP1/RLIP76 depletion in mice suppresses tumor growth by inhibiting tumor neo-vascularization. Cancer Res 72: 5165-5173 (PMID:22902412).
• Awasthi S, Tompkins J, Singhal J, Riggs AD, Yadav S, Wu X, Singh S, Warden C, Liu Z, Wang J, Slavin TP, Weitzel JN, Yuan Y-C, Awasthi M, Srivastava SK, Awasthi YC and Singhal SS.  (2018) Rlip depletion prevents spontaneous neoplasia in TP53 null mice.  Proc Natl Acad Sci 115: 3918-3923 (PMID: 29572430).
• Singhal SS, Horne D, Singhal J, Vonderfecht S, Salgia R and Awasthi S (2019) Synergistic efficacy of RLIP inhibition and 2'-hydroxyflavanone against DMBA-induced mammary carcinogenesis in SENCAR mice.  Molecular Carcinogenesis 58: 1438-1449 (PMID: 31006917).
• Jana S, Krishna BM, Singhal J, Horne D, Awasthi S, Salgia R and Singhal SS. (2020) Therapeutic targeting of miRNA-216b in cancer. Cancer Letters 484: 16-28 (PMID: 32387443)
• Singhal J, Chikara S, Horne D, Awasthi S, Salgia R and Singhal SS. (2021) Targeting RLIP with CRISPR/Cas9 controls tumor growth. Carcinogenesis 42: 48-57 (PMID: 32426802).
• Singhal J, Kulkarni P, Horne D, Awasthi S, Salgia R and Singhal SS. (2021) Prevention of mammary carcinogenesis in MMTV-neu mice by targeting RLIP.  Molecular Carcinogenesis 60: 213-223 (PMID: 33544936). 
• Singhal SS, Mohanty A, Kulkarni P, Horne D, Awasthi S and Salgia R. (2021) RLIP depletion induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells. Carcinogenesis 42: 742-752 (PMID: 33623991).
• Singhal SS, Horne D, Singhal S, Awasthi S and Salgia R. (2022) RLIP: A necessary transporter protein for translating oxidative stress into pro-obesity and pro-carcinogenic signaling. Biochim Biophys Acta (PMID: 36150564).