Sharad S. Singhal, Ph.D.

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.