Professor, Eppley Institute
Courtesy Appointment - Department of Biochemistry and Molecular Biology
PhD - SUNY Stony Brook, 1974
- Distinguished Teaching Award
- Outstanding Faculty Mentor of Graduate Students Award
- UNMC Distinguished Scientist Award
Research InterestsOverall Focus and Long-term Goals: The major focus of this laboratory over the past 20 years has been to understand the molecular mechanisms by which the stem cell transcription factor, SOX2, regulates the self-renewal of stem cells and the growth of human tumor cells.1-5 We are focusing on SOX2, because it is expressed in at least 20 types of human cancer, including their cancer stem cell populations.6 Starting in the mid 1990s, we began to focus on the transcriptional activity of SOX2 in embryonic tumor cells. More recently, we have directed our attention to the surprising finding that the effect of SOX2 on the proliferation of tumor cells, as well as that of normal cells, is highly dependent on the dosage of SOX2.6-9 Importantly, we discovered that SOX2 functions as a biphasic molecular rheostat in tumor cells. More specifically, we determined that both elevating SOX2 and knocking down SOX2 in tumor cells lead to dramatic reductions in tumor growth. Consistent with our findings, there is now a significant body of evidence directly linking SOX2 to tumor cell quiescence and dormancy. This is significant, because dormant tumor cells play a critical role in tumor recurrence and drug resistance, which are major clinical challenges. To meet these challenges, we are focusing on the molecular mechanisms by which elevated levels of SOX2 induce growth arrest. With this in mind, our long-term goal is to identify strategies to eradicate dormant tumor cells, by identifying their vulnerabilities.
Increasing SOX2 Levels in SOX2-Expressing Tumor Cells Reduces Their Proliferation: In 2008, we demonstrated that elevating SOX2 by as little as 2-fold in embryonic stem cells disrupts their self-renewal and rapidly triggers differentiation.3 This is due, at least in part, to transcriptional repression of multiple stem cell transcription factors (e.g. Oct4 and Nanog).4 Our work in embryonic stem cells led us to focus heavily on the roles of SOX2 in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC), prostate cancer (PCa), glioblastoma, and medulloblastoma. Remarkably, we determined that SOX2 levels in each of these cancers are optimized to maximize tumor growth – SOX2 levels that are too high or too low reduce tumor growth.1-5 Importantly, our work with these cancers indicate that this is a defining feature of SOX2.6,7 This surprising property of SOX2 is fully consistent with the wide array of molecular mechanisms used to tightly control the expression, subcellular localization, and activity of SOX2.6 This includes regulation at the transcriptional level by the action of multiple distal enhancers, at the post-transcriptional level by more than 16 microRNAs, and by more than a dozen post-translational modifications.6
Over the past ten years, there have been many studies reporting that increasing the expression of SOX2 in a wide range of SOX2-positive tumor cells [e.g. glioblastoma, prostate cancer, and pancreatic ductal adenocarcinoma (PDAC)] enhances their proliferation both in vitro and in vivo.6 Our recent studies argue strongly that these findings need to be reevaluated. In each of the earlier studies, tumor cells were engineered to stably overexpress SOX2. Remarkably, we have observed completely different results when tumor cells are first engineered to express SOX2 from a doxycycline inducible promoter and later doxycycline is added to the culture medium to elevate SOX2. Importantly, our studies demonstrate that elevating SOX2 with the aid of an inducible promoter in medulloblastoma and glioblastoma, as well as in breast tumor cells, reduces proliferation of each tumor cell type.1 More recently, we demonstrated that this is also true for PDAC tumor cells2 and for PCa tumor cells5. For our PDAC and PCa studies, we engineered three different PDAC tumor cell lines and five PCa cell lines for inducible overexpression of SOX2. For each PDAC cell line, increasing SOX2 (~3- to 10-fold) from a doxycycline inducible promoter leads to a reduction in proliferation (in monolayer and in suspension) and the degree of growth inhibition increases as the level of SOX2 is increased. Moreover, we determined that the tumorigenicity of PDAC and PCa cells is blocked once SOX2 expression is elevated in vivo (Fig 1).2, unpublished results Importantly, knocking down SOX2 also reduces the tumorigenicity of PDAC cells (Fig 2).2 Others have shown that this is also true in PCa cells.
The contrasting results obtained when SOX2 is stably overexpressed versus our inducible overexpression studies are due to a fundamental difference in experimental design. Our tumor cell lines engineered for inducible overexpression of SOX2 are generated via drug selection of lentiviral-transduced cells, which occurred at frequencies greater than 70%. In our studies, SOX2 levels are only elevated after the transduced cells have been established and the non-transduced cells have been eliminated by drug selection (puromycin). In direct contrast, cell lines engineered to stably overexpress SOX2 are subjected to drug selection when SOX2 levels are ectopically elevated from the beginning. As a result, any cells that are unable to proliferate or grow more slowly due to elevated levels of SOX2 will be lost during the drug selection period as the faster proliferating cells expand. Consequently, the cells present in these drug-selected populations represent only a subpopulation of the parental cells. Our studies indicate that this is an exceedingly small subpopulation.
SOX2 is part of a highly integrated transcriptional Network9: As part of our efforts to understand why small changes in the levels of SOX2 influence self-renewal of tumor cells, we conducted unbiased proteomic screens to identify the SOX2-interactome in multiple cell types, including embryonic cells, glioblastoma and medulloblastoma cells.10-12 These studies demonstrate that SOX2 interacts with a wide range of proteins involved in transcription, DNA repair/replication, and signal transduction. They also established that SOX2 is part of a highly interdependent protein:protein interaction network that integrates cellular function at multiple levels (Fig 3).11 As a result, when the levels of SOX2 increase by relatively small amounts, multiple regulatory processes are influenced and cell fate is altered. Not surprisingly, when Sox2 is elevated in embryonic stem cells and differentiation is induced, the SOX2-interactome changes dramatically.10
We also developed conceptually novel methods to mine the SOX2-interactome. Specifically, we predicted that proteins associated with SOX2 in different tumor cell types would also be essential for each of these cell types. Our analysis of the SOX2-interactome in ES cells, glioblastoma cells and medulloblastoma cells identified critical proteins not previously known to affect the growth of tumor cells.10-12 For example, our studies demonstrated that knockdown of USP9X in glioblastoma cells and medulloblastoma cells drastically reduces their proliferation.12 We have determined that this is also true for PDAC cells.13
1) Cox JL, Wilder PJ, Desler M, Rizzino, A. Elevating Sox2 levels deleteriously affects the growth of medulloblastoma and glioblastoma cells. PLOS One, 7:e44087, 2012.
2) Wuebben EL, Wilder PJ, Cox JL, Grunkemeyer JA, Caffrey T, Hollingsworth MA, Rizzino A. SOX2 functions as a molecular rheostat to control the growth, tumorigenicity and drug responses of pancreatic ductal adenocarcinoma cells. Oncotarget, 7:34890-34905, 2016.
3) Kopp J L, Ormsbee BD, Desler M, Rizzino A. Small Increases in the Level of Sox2 Trigger the Differentiation of Mouse Embryonic Stem Cells. Stem Cells, 26:903-11, 2008. Accepted 23 January 2008.
4) Boer B, Kopp J, Mallanna S, Desler M, Chakravarthy H, Wilder PJ, Bernadt C and Rizzino A. Elevating the Levels of Sox2 in Embryonal Carcinoma Cells and Embryonic Stem Cells Inhibits the Expression of Sox2:Oct-3/4 Target Genes. Nucleic Acids Research, 35:1773-1786, 2007.
5) Ethan P. Metz, Phillip J. Wilder, Jixin Dong, Kauatubh Datta, Angie Rizzino. Elevating SOX2 in prostate tumor cells upregulates expression of neuroendocrine genes, but does not reduce the inhibitory effects of enzalutamide. Journal of Cellular Physiology, 235:3731-3740, 2020.
6) Wuebben EL, and Rizzino A. The dark side of SOX2: Cancer – a comprehensive overview. Oncotarget 8:44917-44943, 2017.
7) Ethan P. Metz Angie Rizzino. Sox2 Dosage: A Critical Determinant in the Functions of Sox2 in Both Normal and Tumor Cells. Journal of Cellular Physiology, 234:19298-19306, 2019.
8) Rizzino A Sox2/Oct4: A delicately balanced partnership in pluripotent stem cells and embryogenesis. Biochemica et Biophysica Acta-Gene Regulatory Mechanisms, 1859:780-91, 2016.
9) Rizzino A. Concise review: The Sox2-Oct4 connection: critical players in a much larger interdependent network integrated at multiple levels. Stem Cells 31:1033-1039, 2013.
10) Mallanna SK, Ormsbee B, Iacovino M, Gilmore JM, Cox JL, Kyba M, Washburn MP, Rizzino A. Proteomic analysis of Sox2-associated Proteins During the Early Stages of Embryonic Stem Cell Differentiation Identifies Sox21 as a Novel Regulatory of Stem Cell Fate. Stem Cells, 28:1715-27, 2010.
11) Gao Z, Cox JL, Gilmore JM, Ormsbee BD, Mallanna SK, Washburn MP, Rizzino A. Determination of protein interactome of transcription factor Sox2 in embryonic stem cells engineered for inducible expression of four reprogramming factors. J. Biol. Chem., 287:11384-97, 2012.
12) Cox JL, Wilder PJ, Gilmore JM, Wuebben EL, Washburn MP, Rizzino A. The SOX2-interactome in brain cancer cells identifies the requirement of MSI2 and USP9X in brain tumor cells. PLoS One, 8(5):e62857, 2013.13) Cox JL, Wilder PJ, Wuebben EL, Ouellette MM, Hollingsworth MA, Rizzino A. Context-dependent function of the deubiquitinating enzyme USP9X in pancreatic ductal adenocarcinoma. Cancer Biology and Therapy, 15:1042-52, 2014.