Shree Ram Singh, Ph.D.
Stem cells have an inherent ability to self-renew and to differentiate into several specialized cell types as well as control the maintenance of our tissues and organs. Failure of such maintenance results in several degenerative diseases and cancer. Stem cells have recently attracted significant attention, mainly because of their potential medical benefits in the fields of therapeutic cloning and regenerative medicine. Our current research is directed toward understanding the molecular genetic mechanism by which stem cells regulate tissue homeostasis, regeneration, and tumorigenesis. We are utilizing Drosophila and mouse models to understand the above mechanisms. Specifically, we are using adult testis, kidney and gastrointestinal tissues to characterize the genes/signaling pathways that regulate stem cell behavior and tumor formation. Further, we are focusing on lipid metabolism (lipolysis) on stem cell fate. The knowledge gained from investigating stem cell regulation in Drosophila and mouse models will provide a basis for understanding how human adult stem cells respond during normal and pathological conditions.
Stem cells are undifferentiated cells and play a critical role in tissue development, homeostasis, and regeneration. Stem cells self-renewal divisions are controlled by intrinsic and extrinsic factors. Failure of stem cells function in tissue maintenance results in degenerative diseases, on the other hand, the overproliferation of stem cells results in tumor development (Singh et al., Cell Research, 2005; Singh et al., Journal of Cellular and Molecular Medicine, 2011; Singh, Current Medicinal Chemistry, 2012; Singh, Cancer Letters, 2013). Our current research is directed toward understanding the molecular genetic mechanisms by which stem cells regulate tissue homeostasis, regeneration, and tumorigenesis. We are utilizing Drosophila and mouse models to understand the above mechanisms.
Germline stem cell
In 2006, we identified a novel gene, namely GEF (a small GTPase guanine nucleotide exchange factor), and demonstrated that a Rap-GEF/Rap signaling pathway regulates stem cell anchoring to the niche and organ formation by regulating DE-cadherin-mediated cell adhesion (Wang, Singh et al., Developmental Cell, 2006; Singh. et al., Development, Growth & Differentiation, 2006). Further, we demonstrated that the Drosophila homologue of the human tumor suppressor gene BHD regulates male germline stem cells maintenance and functions downstream of the JAK/STAT and Dpp signal transduction pathways. These findings suggest that BHD regulates tumorigenesis by modulating stem cells in human (Singh et al., Oncogene, 2006). Furthermore, we also demonstrated that germline and somatic stem cells coordinate their self-renewal and differentiation through the JAK/STAT signaling pathway during Drosophila spermatogenesis (Singh et al., Journal of Cellular Physiology, 2010). Later, we found that (Mtor)/Tpr, a nuclear matrix protein that regulates germline stem cell asymmetric division and maintenance through the spindle assembly checkpoint (SAC) complex in Drosophila testis. (Liu, Singh et al., PLoS Genet, 2015). Recently, we demonstrated that Mlf1-adaptor molecule (Madm), a novel tumor suppressor, regulates the competition between germline stem cells and somatic cyst stem cells for niche occupancy in Drosophila testis (Singh et al., Nature Communications, 2016).
Kidney and gastrointestinal stem cell system
Our lab identified kidney stem cells in Drosophila and characterized the signaling pathways that are responsible for maintenance of these stem cells in renal tubules (Singh et al., Cell Stem Cell, 2007; Singh and Hou, Journal of the American Society of Nephrology, 2008; Singh and Hou, Journal of Experimental Biology, 2009; Zeng, Singh et al., Journal of Cellular Physiology, 2010). Recently, we also identified gastric stem cells in the adult Drosophila (Singh et al., Cell Cycle, 2011). We further found that JAK-STAT signaling regulates gastric stem cell proliferation, Wingless signaling regulates gastric stem cell self-renewal, and Hedgehog signaling regulates gastric stem cell differentiation. Our studies also demonstrate that JAK-STAT signaling controls intestinal stem cell (ISC) proliferation and this ability is negatively regulated by Notch, at least through transcriptional control of the JAK-STAT signaling ligand, unpaired (Liu, Singh and Hou, Journal of Cellular Biochemistry, 2010).
Genome-wide screening in germline and gastrointestinal stem cells
In the last few years, we have been focusing on identifying the genes responsible for gastrointestinal stem cell self-renewal and differentiation. Recently, we found that JAK-STAT signaling controls ISC proliferation and this ability is negatively regulated by Notch, at least through transcriptional control of the JAK-STAT signaling ligand, unpaired (Liu, Singh and Hou, Journal of Cellular Biochemistry, 2010). More recently, we finished a genome-wide RNAi screen in Drosophila gastrointestinal tissues (Zeng, Han, Singh et al., Cell Reports, 2015) as well as in Drosophila testis (Liu et al. Nature Communications, 2016) and identified novel regulators in these two tissue systems. We are currently focusing on characterizing these genes.
Lipid metabolism, stem cell and cancer stem cell: Flies to human
Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor propagation, relapse, and the eventual death of most cancer patients (Singh, Cancer Letters, 2013; Hou and Singh, Topics in Developmental Biology, 2017). However, very little is known about the biology behind this resistance to therapeutics. Recently, we reported a novel mechanism of stem-cell and transformed stem cells (TSCs) death using adult Drosophila digestive system. We found that knockdowns of the COPI/Arf1 pathways selectively killed normal and TSCs through necrosis, by attenuating the lipolysis pathway. The dying stem cells were engulfed by neighboring differentiated cells through a Draper-Mbc/Rac1-JNK-dependent autophagy pathway. We further found that Arf1 inhibitors also killed CSCs in human cancer cell lines. These findings together suggest that normal or CSCs, like hibernating animals, primarily rely on lipid reserves for energy and blocking lipolysis starves them to death (Singh et al., Nature, 2016).
Protocols in stem cell and modeling diseases
We developed immunofluorescence labeling, lineage tracing, and in situ hybridization techniques for the identification and characterization of stem cells and differentiated cells in Drosophila germline (Singh and Hou, Methods Mol Biol, 2008; Singh et al., Methods Mol Biol, 2013) and gastrointestinal tissues (Singh et al., Methods Mol Biol, 2012; Pinto et al. Methods Mol Biol, 2018). Further, we published the method of ESC culture and differentiation, and the expression of MMP9 and its inhibitor, TIMP4 in differentiating ESC (Mishra et al. Methods Mol Biol, 2013). In additon, provided the method to generate double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans (Chavali et al., Methods Mol Biol, 2014).
Collaborative network in stem cell, cancer, microRNA and precision medicine
In collaborations, we have investigated the differentiation potential of osteoarthritic chondrocytes (OC) into iPSCs using defined transcription factors and explored the possibility of using these OC-derived iPSCs for chondrogenesis. We found that iPSCs could be generated from OCs using defined factors and that in vitro co-culture of TGF-β1-transfected OC-derived iPSCs with articular cartilages (ACs) in alginate matrix results in significantly improved chondrogenesis of iPSCs. In addition, in vivo study also revealed the obvious cartilage tissue formed in the co-culture of TGF-β1- transfected OC-derived iPSCs with ACs in alginate matrix. This combinational strategy will promote the use of iPSC-derived tissue in tissue engineering (Wei et al., European Cells & Materials, 2012; Yin et al., Stem Cell Reviews and Reports, 2015). Recently, we showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells could be a good source for spinal cord repair (Obara et al. Stem Cell Reviews and Reports, 2019).
Further, we are also focusing on the patient-derived xenograft (PDX) model in colon cancer (Seol et al., Cancer Letters, 2014), interaction of PhIP with curcumin in breast epithelial cells (Jain et al. Cancer Letters, 2015), role of microRNAs in metabolism and tumor development (Chan et al., Cancer Letters, 2015; Singh et al., Cancer Letters 2015) such as miR-373 in non-small cell lung cancer (Seol et al., Cancer Letters, 2014), miR-155 in breast cancer (Kim et al., Cancer Letters, 2015), microRNAs in stem cell aging (Dietrich et al. Adv Exp Med Biol, 2018) hypoxia and hypoxia inducible factors in tumor metabolism (Zeng et al., Cancer Letters, 2015) and bone tumor (Zeng et al. Cancer Letters, 2011), markers for gastric cancer stem cells (Fagoonee et al., Minerva Biotecnologica, 2015), role of histone demethylase KDM1A in oral cancer (Narayanan et al., Cancer Letters, 2015), role of Riluzole on hepatocellular carcinoma (HCC) therapy (Seol et al., Cancer Letters, 2016), generation and characterization of PDX models of pancreatic ductal adenocarcinoma (Jung et al., Oncotarget, 2016), role of complement proteins C7 and CFH on stemness of liver cancer cells (Seol et al., Cancer Letters, 2016), targeting tumor microenvironment in cancer therapy (Singh. et al. Cancer Letters, 2016), endothelial progenitor cells in Crohn's disease (Dietrich and Singh, Digestive Diseases and Sciences, 2017), and demonstrated the power of patient-derived orthotopic xenograft (PDOX) model to identify effective therapy for undifferentiated spindle cell sarcoma (USCS) and the potential of recombinant methioninase (rMETase) to overcome doxorubicin (DOX) resistance (Igarashi et al., Cancer Letters, 2018). Our recent data suggest that tumor-targeting Salmonella typhimurium A1-R can be used as a highly effective general therapeutic for undifferentiated soft tissue sarcoma (Igarashi et al. BBRC, 2018), a combination of tumor-Selective bacterial therapy and cancer regressed an Ewing's sarcoma (Miyake et al, Chemotherapy, 2019). . Recently, we generated PDX of triple-negative breast cancer (TNBC) and our RNA-seq and western blot analysis showed that these PDXs are heterologous nature. We found frequent Notch1 variant and AZGP-GJC3 gene fusion. The TNBC PDX could be a valuable preclinical model in individual therapy (Jung et al., Cancer Letters, 2018). More recently, we examined the effect of several chemotherapeutic drugs such as carboplatinum (CAR), paclitaxel (PAC), gemcitabine (GEM), and 5-fluorouracil (5-FU) and together with Salmonella A1-R on cancers of unknown primary (CUP) (Miyake et al. Signal Transduction and Targeted Therapy, 2018). We also also tested several chemotherapeutic drugs such as MEK inhibitor trametinib (TRA) in combination with gemcitabine (GEM) for pancreatic cancer PDOX (Kawaguchi et al. Tissue and Cell, 2018), Regorafenib on a doxorubicin-resistant Ewing's sarcoma (Miyake et al. Cancer Chemother Pharmacol, 2019), rMETase together with palbociclib (PAL) against a doxorubicin (DOX)-resistant dedifferentiated liposarcoma (Igarashi et al. BBRC, 2019), combination of gemcitabine and nab-paclitaxel for undifferentiated soft-tissue sarcoma (Higuchi et al. Biomedicine & Pharmacotherapy, 2019), and tested the efficacy of osimertinib in an EGFR-mutant cisplatinum-resistant lung adenocarcinoma (Higuchi et al. Translational Oncology, 2019).
Current Student: Cristopher Aguilar Ortiz [The Werner H. Kirsten Student Intern Program (WHK SIP)-from June 2018 to May 2019.
Selected Recent Publications
- Nature. 538: 198-113, 2016. [ Journal Article ]
- Nature Communications. 7: 10473, 2016. [ Journal Article ]
The Nuclear Matrix Protein Megator Regulates Stem Cell Asymmetric Division through the Mitotic Checkpoint Complex.PLoS Genetics. 11: e1005750, 2015. [ Journal Article ]
Novel cancer gene variants and gene fusions of triple-negative breast cancers (TNBCs) reveal their molecular diversity conserved in the patient-derived xenograft (PDX) model.Cancer Letters. 428: 127-138, 2018. [ Journal Article ]
- Cell Stem Cell. 1: 191-203, 2007. [ Journal Article ]
Dr. Shree Ram Singh obtained his Ph.D. in genetics from the Department of Zoology, Banaras Hindu University, India in 2001. Dr. Singh was a guest scientist at the Department of Molecular Cell Biology Biocenter, Johann Wolfgang Goethe University, Frankfurt, Germany in 1998 and in 2001 he received a UNESCO Biotechnology Action Council (BAC) fellowship at the Department of Plant Molecular Biology Biocenter, Johann Wolfgang Goethe University. Dr. Singh pursued his postdoctoral research studies at the University of Haifa, Israel, and at the National Cancer Institute at Frederick. Since 2011, he has served as a staff scientist at National Cancer Institute at Frederick. Dr. Singh was a Lead Guest Editor in 2012 for a special issue of Current Medicinal Chemistry on Stem cells in regenerative medicine and cancer, in 2013 for a special issue of Cancer Letters on Cancer Stem Cells, in 2015 for a special issue of Cancer Letters on Cancer Metabolism, and in 2016 for a special issue of Cancer Letters on Tumor Microenvironment. Dr. SIngh is author and co-authors of scientific paper published in several reputed journals including Nature, Cell Stem Cell, Developmental Cell, Nature Communications, Cell Reports, PLoS Genetics, and Oncogene. He also serves as an editorial board member of Cancer Letters, Scientific Reports, and Signal Transduction and Targeted Therapy, and an Academic Editor of PLoS One and PeerJ, Associate Editor of BMC Genetics, and Review Editor of Frontiers in Cell and Developmental Biology.
Lipolysis Pathway: The Achilles’ Heel of Cancer Stem Cells (CCR Fellows & Young Investigators Newsletter).
Gastric stem cells: Of flies and men
Self-Renewal in the Fly Kidney (Previewed in Developmental Cell)
Multipotent stem cells in the adult fly kidney (Highlighted in Kidney International)
Platinum publication (NCI Poster) Nature 538(7623):109-113, 2016.
Platinum publication (NCI Poster) Methods Mol Biol 1194:385–400, 2014
Platinum publication (NCI Poster) PLoS Genet 11(12):e1005750, 2015.
Platinum publication (NCI Poster) Oncogene 25(44):5933– 5941, 2006.