Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth.

BACKGROUND: Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. METHODS: To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. RESULTS: Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. CONCLUSIONS: Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

The dark side of SOX2: cancer - a comprehensive overview.

The pluripotency-associated transcription factor SOX2 is essential during mammalian embryogenesis and later in life, but SOX2 expression can also be highly detrimental. Over the past 10 years, SOX2 has been shown to be expressed in at least 25 different cancers. This review provides a comprehensive overview of the roles of SOX2 in cancer and focuses on two broad topics. The first delves into the expression and function of SOX2 in cancer focusing on the connection between SOX2 levels and tumor grade as well as patient survival. As part of this discussion, we address the developing connection between SOX2 expression and tumor drug resistance. We also call attention to an under-appreciated property of SOX2, its levels in actively proliferating tumor cells appear to be optimized to maximize tumor growth - too little or too much SOX2 dramatically alters tumor growth. The second topic of this review focuses on the exquisite array of molecular mechanisms that control the expression and transcriptional activity of SOX2. In addition to its complex regulation at the transcriptional level, SOX2 expression and activity are controlled carefully by microRNAs, long non-coding RNAs, and post-translational modifications. In the Conclusion and Future Perspectives section, we point out that there are still important unanswered questions. Addressing these questions is expected to lead to new insights into the functions of SOX2 in cancer, which will help design novels strategies for more effectively treating some of the most deadly cancers.

SOX2 functions as a molecular rheostat to control the growth, tumorigenicity and drug responses of pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly malignancy. Expression of the stem cell transcription factor SOX2 increases during progression of PDAC. Knockdown of SOX2 in PDAC cell lines decreases growth in vitro; whereas, stable overexpression of SOX2 in one PDAC cell line reportedly increases growth in vitro. Here, we reexamined the role of SOX2 in PDAC cells, because inducible SOX2 overexpression in other tumor cell types inhibits growth. In this study, four PDAC cell lines were engineered for inducible overexpression of SOX2 or inducible knockdown of SOX2. Remarkably, inducible overexpression of SOX2 in PDAC cells inhibits growth in vitro and reduces tumorigenicity. Additionally, inducible knockdown of SOX2 in PDAC cells reduces growth in vitro and in vivo. Thus, growth and tumorigenicity of PDAC cells is highly dependent on the expression of optimal levels of SOX2 - a hallmark of molecular rheostats. We also determined that SOX2 alters the responses of PDAC cells to drugs used in PDAC clinical trials. Increasing SOX2 reduces growth inhibition mediated by MEK and AKT inhibitors; whereas knockdown of SOX2 further reduces growth when PDAC cells are treated with these inhibitors. Thus, targeting SOX2, or its mode of action, could improve the treatment of PDAC.

Sox2/Oct4: A delicately balanced partnership in pluripotent stem cells and embryogenesis.

Considerable progress has been made in understanding the roles of Sox2 and Oct4 in embryonic stem cells and mammalian embryogenesis. Specifically, significant progress has been made in answering three questions about the functions of Sox2 and Oct4, which are the focus of this review. 1) Are the first or second cell lineage decisions during embryogenesis controlled by Oct4 and/or Sox2? 2) Do the levels of Oct4 and Sox2 need to be maintained within narrow limits to promote normal development and to sustain the self-renewal of pluripotent stem cells? 3) Do Oct4 and Sox2 work closely together or is the primary role of Sox2 in pluripotent cells to ensure the expression of Oct4? Although significant progress has been made in answering these questions, additional studies are needed to resolve several important remaining issues. Nonetheless, the preponderance of the evidence suggests there is considerable crosstalk between Sox2 and Oct4, and further suggests Sox2 and Oct4 function as molecular rheostats and utilize negative feedback loops to carefully balance their expression and other critical genes during embryogenesis. This article is part of a Special Issue entitled: The Oct transcription factor family, edited by Dr. Dean Tantin.

Context-dependent function of the deubiquitinating enzyme USP9X in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadly malignancies. Recently, the deubiquitinating protease USP9X has been shown to behave as an oncogene in a number of neoplasms, including those of breast, brain, colon, esophagus and lung, as well as KRAS wild-type PDAC. However, other studies suggest that USP9X may function as a tumor-suppressor in a murine PDAC model when USP9X expression is depleted during early pancreatic development. To address the conflicting findings surrounding the role of USP9X in PDAC, we examined the effects of knocking down USP9X in five human PDAC cell lines (BxPC3, Capan1, CD18, Hs766T, and S2-013). We demonstrate that knocking down USP9X in each of the PDAC cell lines reduces their anchorage-dependent growth. Using an inducible shRNA system to knock down USP9X in both BxPC3 and Capan1 cells, we also determined that USP9X is necessary for the anchorage-independent growth. In addition, knockdown of USP9X alters the cell cycle profile of BxPC3 cells and increases their invasive capacity. Finally, we show that an inhibitor of deubiquitinating proteases, WP1130, induces significant cytotoxicity in each of the five PDAC cell lines tested. Overall, our work and the work of others indicate that the function and role of USP9X is highly context-dependent. Although USP9X may function as a tumor-suppressor during the establishment of PDAC, data presented here argue that USP9X promotes cell growth in advanced PDAC cells when PDAC is typically diagnosed. Hence, USP9X may be a promising therapeutic target for the treatment of advanced PDAC.

Sox2 expression is regulated by a negative feedback loop in embryonic stem cells that involves AKT signaling and FoxO1.

The self-renewal and pluripotency of embryonic stem cells (ESC) is regulated by a highly integrated network of essential transcription factors, which includes Sox2. Previous studies have shown that elevating Sox2 on its own in mouse ESC induces differentiation and inhibits the expression of endogenous Sox2 at the protein and mRNA level. These findings led us to hypothesize that increases in Sox2 activate a negative feedback loop that inhibits the transcription of the endogenous Sox2 gene. To test this hypothesis, we used i-OSKM-ESC, which elevate Sox2 in conjunction with Oct4, Klf4, and c-Myc when treated with doxycycline (Dox). Elevating the expression of these four transcription factors in i-OSKM-ESC does not induce differentiation, but it represses expression of endogenous Sox2. We determined that increases of Sox2 in i-OSKM-ESC lead to increases in activated AKT and inactivation of FoxO1 (an activator of Sox2), as well as decreases in binding of FoxO1 to the 5'flanking region of Sox2. Importantly, we determined that inhibition of AKT in Dox-treated i-OSKM-ESC leads to re-expression of endogenous Sox2 at the mRNA and protein level and reactivation of FoxO1. These findings argue that AKT signaling is part of the negative feedback loop that helps carefully control the transcription of Sox2 in ESC by modulating the binding of FoxO1 to the Sox2 gene. Collectively, our findings provide new insights into the mechanisms that enable ESC to carefully regulate the levels of Sox2 and retain their stem cell properties.

The SOX2-interactome in brain cancer cells identifies the requirement of MSI2 and USP9X for the growth of brain tumor cells.

Medulloblastomas and glioblastomas, the most common primary brain tumors in children and adults, respectively, are extremely difficult to treat. Efforts to identify novel proteins essential for the growth of these tumors may help to further our understanding of the biology of these tumors, as well as, identify targets for future therapies. The recent identification of multiple transcription factor-centric protein interaction landscapes in embryonic stem cells has identified numerous understudied proteins that are essential for the self-renewal of these stem cells. To identify novel proteins essential for the fate of brain tumor cells, we examined the protein interaction network of the transcription factor, SOX2, in medulloblastoma cells. For this purpose, Multidimensional Protein Identification Technology (MudPIT) identified >280 SOX2-associated proteins in the medulloblastoma cell line DAOY. To begin to understand the roles of SOX2-associated proteins in brain cancer, we focused on two SOX2-associated proteins, Musashi 2 (MSI2) and Ubiquitin Specific Protease 9x (USP9X). Recent studies have implicated MSI2, a putative RNA binding protein, and USP9X, a deubiquitinating enzyme, in several cancers, but not brain tumors. We demonstrate that knockdown of MSI2 significantly reduces the growth of DAOY cells as well as U87 and U118 glioblastoma cells. We also demonstrate that the knockdown of USP9X in DAOY, U87 and U118 brain tumor cells strongly reduces their growth. Together, our studies identify a large set of SOX2-associated proteins in DAOY medulloblastoma cells and identify two proteins, MSI2 and USP9X, that warrant further investigation to determine whether they are potential therapeutic targets for brain cancer.

Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells.

Recent studies have shown that the RNA binding protein Musashi 2 (Msi2) plays important roles during development. Msi2 has also been shown to be elevated in several leukemias and its elevated expression has been linked with poorer prognosis in these cancers. Additionally, in embryonic stem cells (ESC) undergoing the early stages of differentiation, Msi2 has been shown to associate with the transcription factor Sox2, which is required for the self-renewal of ESC. These findings led us to examine the effects of Msi2 on the behavior of ESC. We determined that ESC express two isoforms of Msi2, the larger canonical isoform (isoform 1) and a shorter, splice-variant isoform (isoform 2). Using multiple shRNA lentiviral vectors, we determined that knockdown of Msi2 disrupts the self-renewal of ESC and promotes their differentiation into cells that express markers associated with mesoderm, ectoderm, and trophectoderm. Moreover, our studies indicate that the extent of differentiation and the loss of self-renewal capacity correlate with the levels to which Msi2 levels were decreased. We extended these findings by engineering ESC to inducibly express either Msi2 isoform1 or isoform 2. We determined that ectopic expression of Msi2 isoform 1, but not isoform 2, enhances the cloning efficiency of ESC. In addition, we examined how Msi2 isoform 1 and isoform 2 affect the differentiation of ESC. Interestingly, ectopic expression of either Msi2 isoform 1 or isoform 2 does not affect the pattern of differentiation induced by retinoic acid. Finally, we show that ectopic expression of either isoform 1 or isoform 2 is not sufficient to block the differentiation that results from the knockdown of both isoforms of Msi2. Thus, it appears that both isoforms of Msi2 are required for the self-renewal of ESC.