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., Oncogene, 2006; Singh et al., Cell Stem Cell, 2007; Singh et al., Journal of Cellular and Molecular Medicine, 2011; Singh, Current Medicinal Chemistry, 2012; Singh, Cancer Letters, 2013; Singh et al., Nature, 2016; Singh et al., Advances in Experimental Medicine and Biology, 2019). Our current research is directed toward understanding the molecular genetic mechanisms by which stem cells regulate tissue homeostasis, regeneration, and tumorigenesis. We are utilizing model organisms and using various tissue and cell systems to understand the above mechanisms. We are also involved in cancer drug screening to identify novel regulators of regeneration, and cancer therapy, and exploring the role of tumor-targeting bacteria for cancer therapy.
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, Current Topics in Developmental Biology, 2017; Singh et al., Advances in Experimental Medicine and Biology, 2019). 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 (Singh et al., Nature, 2016). 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). More recently, we identified a pathway that connects dying intestinal stem cells with phagocytic enterocytes (ECs) after ablation of the COPI/Arf1-lipolysis pathway in ISCs (under review).
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 addition, 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). 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). We also reported that COL17A1 is important in differentiation of HAP stem cells (Shirai et al. 2019, Tissue and Cell, 2019). Recently, we demonstrated that HAP pluripotent stem cells derived from cryopreserved intact human hair follicles sustain multilineage differentiation potential (Obara et al., Scientific 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). 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). Recently, we identified BX-795 as a potential anticancer drug for primary pancreatic ductal adenocarcinoma cells (Choi et al., Cancer Letters, 2019).
We also 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). We also tested several chemotherapeutic drugs such as MEK inhibitor trametinib (TRA) in combination with gemcitabine 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). We also identified that olaratumab combined with doxorubicin and ifosfamide overcomes individual doxorubicin and olaratumab resistance undifferentiated soft-tissue sarcoma PDOX (Higuchi et al., Cancer Letters, 2019) and pioglitazone in combination with cisplatinum arrests a chemotherapy-resistant osteosarcoma PDOX Model (Higuchi et al. Cancer Genomics & Proteomics, 2020). Presented the ability of the PDOX models to identify effective approved agents-as well as experimental-therapeutics for sarcoma (Igarashi et al., Cancer Letters, 2020). We also identified novel targets by integrated cancer-stromal interactome analysis of pancreatic adenocarcinoma (Hiroshima et al., Cancer Letters, 2020).
Bacteria in cancer therapy: PDOX model
We are also exploring the role of tumor-targeting bacteria for cancer therapy using PDOX models. Our recent work suggest that tumor-targeting bacteria such as Salmonella typhimurium A1-R together with chemotherapies can be used as a highly effective general therapeutic for cancers of unknown primary (CUP) (Miyake et al. Signal Transduction and Targeted Therapy, 2018; Miyake et al. Tissue and Cell, 2018), gastrointestinal stromal tumors (Miyake et al. Heliyon, 2018), and cervical cancer (Miyake et al. Archives of Gynecology and Obstetrics, 2019), melanoma (Kawaguchi et al. BBRC, 2018), and sarcomas (Kiyuna et al., BBRC, 2018; Igarashi et al. BBRC, 2018; Miyake et al, Chemotherapy, 2019; Igarashi et al. Translational Oncology, 2020).