Professor, Eppley Institute
Ph.D. - SUNY Stony Brook, 1974
- Distinguished Teaching Award
- Outstanding Faculty Mentor of Graduate Students Award
- UNMC Distinguished Scientist Award
Editorial Review Boards:
- International Journal of Developmental Biology - Associate Editor for the Americas
- Molecular Reproduction and Development - Associate Editor
- In Vitro Cellular and Developmental Biology - Associate Editor
- Cytotechnology - Editorial Board
Research in this laboratory focuses on the molecular mechanisms that control fundamental fate decisions surrounding the self-renewal of pluripotent stem cells and tumor-initiating cells. For this purpose a wide range of experimental approaches are used including genetic engineering of cells, proteomic analysis of key transcription factors, and kinome and gene expression analysis. Studies in this laboratory concentrate heavily on the transcription factor Sox2, because it regulates both the self-renewal of pluripotent stem cells and the growth of 20 different cancers and their tumor-initiating cells. Moreover, Sox2 has been directly implicated in resistance to drugs used clinically to treat many of these cancers. Currently, our research is investigating the molecular mechanisms by which Sox2: 1) promotes tumor cell drug-resistance, 2) regulates the proliferation of tumor-initiating cells, and 3) contributes to reprogramming somatic cells into induced pluripotent stem cells (iPS cells).
Overview of Major Findings: Over the past seven years, we have significantly expanded our understanding of the roles of played by Sox2 in both pluripotent stem cells and cancer. More specifically, our studies have led to six important conclusions: 1) small increases in the levels of Sox2 in embryonic stem cells repress transcription of the endogenous Sox2 gene; 2) small increases in the levels of Sox2 (2-fold) promote the differentiation embryonic stem cells; 3) Sox2 is part of a highly interconnected protein-protein interaction network, which is integrated at multiple levels; 4) mining of Sox2-interactomes provides a new strategy of identifying novel targets for cancer therapy as well as identifying additional proteins required for the self-renewal of embryonic stem cells and tumor cells; 5) small increases in the levels of Sox2 (from an inducible promoter) in multiple human tumor cell types (medulloblastoma, glioblastoma, breast, prostate and pancreatic ductal adenocarcinoma) lead to growth inhibition, rather than growth stimulation; and 6) Sox2 can promote drug resistance in tumor cells without directly increasing tumor cell proliferation.
Recent and Ongoing Work: Early work established that Sox2 and Oct4 are required for mammalian embryogenesis as well as the self-renewal and pluripotency of embryonic stem cells. Shortly thereafter, interest in Sox2 and Oct4 rose dramatically with discovery that ectopic expression of Sox2 and Oct4 along with Klf4 and cMyc could reprogram somatic cells into induced pluripotent stem cells. As a consequence, significant effort was made to understand the mechanisms by which Sox2 and Oct4 mediate their effects. These efforts led to the conclusion that Sox2 and Oct4 work together to activate their own transcription and the transcription of a large set of Sox2/Oct4 target genes (Figure 1).
More recently, two studies conducted in our laboratory established that the roles of Sox2 in embryonic stem cells, like the roles of Oct4 in embryonic stem cells, are far more complex. In one study, we demonstrated that small increases in the levels of Sox2 in embryonic stem cells lead to transcriptional repression of the endogenous Sox2 gene as well as the transcription of other essential pluripotency-associated transcription factors, including Oct4 (Figure 2) (Boer et al., Nucleic Acids Research, 2007). Thus, depending on the levels of Sox2, it can activate or repress gene transcription. In another study, we determined that small increases in the levels of Sox2 (2-fold), similar to small decreases in Sox2, trigger rapid differentiation of embryonic stem cells to specific cell types (Figure 3) (Kopp et al., Stem Cells, 2008). Importantly, these two studies established that Sox2 behaves as a molecular rheostat – the self-renewal and pluripotency of embryonic stem cells require the maintenance of Sox2 levels within narrow limits (Rizzino, Stem Cells, 2013).
Subsequently, we determined that small increases in Sox2 lead to the induction of silent genes, such as the bivalent gene Sox21 (Chakravarthy et al., FASEB Journal, 2011), which in turn promote the rapid differentiation of embryonic stem cells (Mallana, Ormsbee et al., Stem Cells, 2010). Moreover, small increases in Sox2 activate a negative feedback loop that involves AKT and FoxO1, and which represses transcription of the endogenous Sox2 gene in embryonic stem cells (Ormsbee, Wuebben et al., PLoS One, 2014).
Although it is evident that Sox2 plays key roles in regulating the growth and survival of both normal stem cells and tumor-initiating cells, the molecular mechanisms by which Sox2 controls cell fate are far from clear. To better understand the molecular action of Sox2, we examined the Sox2-interactome in several different cellular contexts using a highly sensitive proteomic method known as Multidimensional Protein Identification Technology (MudPIT). These studies were prompted by the finding that Sox2 is present in large protein complexes, some larger than 800 kDa (Cox et al., PLoS One, 2010). Interestingly, the Sox2-interactome in undifferentiated embryonic stem cells and embryonic stem cells undergoing differentiation consist of nearly the same number of Sox2-associated proteins (>70 and >60 proteins, respectively). However, the Sox2-interactome changes significantly when embryonic stem cells initiate differentiation (Mallanna, Ormsbee et al., Stem Cells, 2010; Gao, Cox et al., Journal of Biological Chemistry, 2012). Importantly, in embryonic stem cells, Sox2 associates with many other transcription factors required for the self-renewal and pluripotency of embryonic stem cells. Integration of the Sox-interactome in embryonic stem cells with the interactomes of eight other transcription factors in embryonic stem cells demonstrated that Sox2 is part of a highly interconnected protein-protein interaction network (Figure 4). Inspection of this network indicated that the nine pluripotency-associated transcription factors associate with many of the same proteins, including those that participate in transcription, signal transduction, and genome stability. Thus, Sox2 is part of a highly interdependent transcriptional network that helps regulates most, if not all, major cellular processes (Rizzino Stem Cells, 2013).
We extended our work on the network of Sox2-associateed proteins by determining the Sox2-interactome in brain tumor cells (Cox et al., PLoS one, 2013). Interestingly, integration of the Sox2-interactome in brain tumor cells with the Sox2-interactomes in embryonic stem cells and embryonic stem cells undergoing differentiation identified a small set of proteins that associate with Sox2 in all three cellular contexts. Study of two of these associated Sox2 proteins [Musashi 2 and ubiquitin specific protease 9X (USP9X)] demonstrated that each is required for the growth and survival of both glioblastoma and medulloblastoma tumor cells (Cox et al., PLoS One, 2013). These studies strongly suggest that Musashi2 and USP9X should be evaluated as possible therapeutic targets for the treatment of the most lethal adult brain cancer (glioblastoma) and the most common pediatric brain cancer (medulloblastoma). Equally important, our strategy of comparing Sox2-interactomes in different cellular contexts represents a powerful method for identifying novel proteins required for the growth of specific tumor cells.
More recently, the role of USP9X was examined in pancreatic ductal adenocarcinoma cells, which also express Sox2. Five pancreatic tumor cell lines were shown to require USP9X for cell proliferation (Cox et al., Cancer Biology and Therapy, 2014). In addition, a pan-specific inhibitor of several ubiquitin specific proteases (WP1130), including USP9X, was shown to dramatically reduce the growth of each pancreatic tumor cell line. This work together with the work of others strongly suggests that the roles of USP9X in pancreatic ductal adenocarcinoma change during tumor progression – during the early stages of pancreatic cancer, USP9X behaves as a tumor suppressor, but, during the later stages of this cancer, USP9X switches its function and promotes tumor cell growth. Moreover, these studies suggest that drugs like WP1130, when used in combination with other therapeutics, may improve the survival of patients afflicted with this highly lethal cancer.
Improvement in the treatment of cancer depends on obtaining a much deeper understanding of tumor cell growth. Toward that goal, we engineered the tumor cells derived from five different tumor types for inducible overexpression of Sox2. In each case (medulloblastoma, glioblastoma, breast, prostate and pancreatic ductal adenocarcinoma), small increases in Sox2 led to decreases in cell growth (Cox et al., PLoS One, 2012). Moreover, in two of these tumor cell types (medulloblastoma and pancreatic ductal adenocarcinoma), we also engineered the same tumor cells for knockdown of Sox2. These studies determined that small decreases in Sox2 also lead to reduction in cell growth. Thus, as in the case of embryonic stem cells, small increases as well as small decreases in the levels of Sox2 significantly influence cell fate.
Improvements in the treatment of cancer will also require a far deeper understanding of drug resistance, which represents one of the largest challenges in cancer biology. Recently, we have begun to explore the effects of Sox2 on the drug responses of tumor cells. These studies have determined that Sox2 reduces the efficacy of at least three drugs used clinically to treat several different cancers. Remarkably, this work indicates that small increases in the levels of Sox2 reduce the efficacy of drugs under conditions where increases in Sox2 reduce tumor cell growth. Currently, we are exploring the molecular mechanisms by which Sox2 reduces the efficacy of drugs used in clinical cancer trials. The goal of these studies is to help develop new therapies that reduce cancer drug resistance and prolong the life of patients with highly malignant tumors.