The Current Therapeutic Landscape for Metastatic Prostate Cancer.

In 2024, there will be an estimated 1,466,718 cases of prostate cancer (PC) diagnosed globally, of which 299,010 cases are estimated to be from the US. The typical clinical approach for PC involves routine screening, diagnosis, and standard lines of treatment. However, not all patients respond to therapy and are subsequently diagnosed with treatment emergent neuroendocrine prostate cancer (NEPC). There are currently no approved treatments for this form of aggressive PC. In this review, a compilation of the clinical trials regimen to treat late-stage NEPC using novel targets and/or a combination approach is presented. The novel targets assessed include DLL3, EZH2, B7-H3, Aurora-kinase-A (AURKA), receptor tyrosine kinases, PD-L1, and PD-1. Among these, the trials administering drugs Alisertib or Cabozantinib, which target AURKA or receptor tyrosine kinases, respectively, appear to have promising results. The least effective trials appear to be ones that target the immune checkpoint pathways PD-1/PD-L1. Many promising clinical trials are currently in progress. Consequently, the landscape of successful treatment regimens for NEPC is extremely limited. These trial results and the literature on the topic emphasize the need for new preventative measures, diagnostics, disease specific biomarkers, and a thorough clinical understanding of NEPC.

STAT3 Inhibition Attenuates MYC Expression by Modulating Co-Activator Recruitment and Suppresses Medulloblastoma Tumor Growth by Augmenting Cisplatin Efficacy In Vivo.

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.

Elevating SOX2 Downregulates MYC through a SOX2:MYC Signaling Axis and Induces a Slowly Cycling Proliferative State in Human Tumor Cells.

Slowly cycling/infrequently proliferating tumor cells present a clinical challenge due to their ability to evade treatment. Previous studies established that high levels of SOX2 in both fetal and tumor cells restrict cell proliferation and induce a slowly cycling state. However, the mechanisms through which elevated SOX2 levels inhibit tumor cell proliferation have not been identified. To identify common mechanisms through which SOX2 elevation restricts tumor cell proliferation, we initially performed RNA-seq using two diverse tumor cell types. SOX2 elevation in both cell types downregulated MYC target genes. Consistent with these findings, elevating SOX2 in five cell lines representing three different human cancer types decreased MYC expression. Importantly, the expression of a dominant-negative MYC variant, omomyc, recapitulated many of the effects of SOX2 on proliferation, cell cycle, gene expression, and biosynthetic activity. We also demonstrated that rescuing MYC activity in the context of elevated SOX2 induces cell death, indicating that the downregulation of MYC is a critical mechanistic step necessary to maintain survival in the slowly cycling state induced by elevated SOX2. Altogether, our findings uncover a novel SOX2:MYC signaling axis and provide important insights into the molecular mechanisms through which SOX2 elevation induces a slowly cycling proliferative state.

Subgroup-Specific Diagnostic, Prognostic, and Predictive Markers Influencing Pediatric Medulloblastoma Treatment.

Medulloblastoma (MB) is the most common malignant central nervous system tumor in pediatric patients. Mainstay of therapy remains surgical resection followed by craniospinal radiation and chemotherapy, although limitations to this therapy are applied in the youngest patients. Clinically, tumors are divided into average and high-risk status on the basis of age, metastasis at diagnosis, and extent of surgical resection. However, technological advances in high-throughput screening have facilitated the analysis of large transcriptomic datasets that have been used to generate the current classification system, dividing patients into four primary subgroups, i.e., WNT (wingless), SHH (sonic hedgehog), and the non-SHH/WNT subgroups 3 and 4. Each subgroup can further be subdivided on the basis of a combination of cytogenetic and epigenetic events, some in distinct signaling pathways, that activate specific phenotypes impacting patient prognosis. Here, we delve deeper into the genetic basis for each subgroup by reviewing the extent of cytogenetic events in key genes that trigger neoplastic transformation or that exhibit oncogenic properties. Each of these discussions is further centered on how these genetic aberrations can be exploited to generate novel targeted therapeutics for each subgroup along with a discussion on challenges that are currently faced in generating said therapies. Our future hope is that through better understanding of subgroup-specific cytogenetic events, the field may improve diagnosis, prognosis, and treatment to improve overall quality of life for these patients.

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.

Elevating SOX2 in prostate tumor cells upregulates expression of neuroendocrine genes, but does not reduce the inhibitory effects of enzalutamide.

Prostate cancer (PCa) is one of the leading causes of cancer deaths in men. In this cancer, the stem cell transcription factor SOX2 increases during tumor progression, especially as the cancer progresses to the highly aggressive neuroendocrine-like phenotype. Other studies have shown that knockdown of RB1 and TP53 increases the expression of neuroendocrine markers, decreases the sensitivity to enzalutamide, and increases the expression of SOX2. Importantly, knockdown of SOX2 in the context of RB1 and TP53 depletion restored sensitivity to enzalutamide and reduced the expression of neuroendocrine markers. In this study, we examined whether elevating SOX2 is not only necessary, but also sufficient on its own to promote the expression of neuroendocrine markers and confer enzalutamide resistance. For this purpose, we engineered LNCaP cells for inducible overexpression of SOX2 (i-SOX2-LNCaP). As shown previously for other tumor cell types, inducible elevation of SOX2 in i-SOX2-LNCaP inhibited cell proliferation. SOX2 elevation also increased the expression of several neuroendocrine markers, including several neuropeptides and synaptophysin. However, SOX2 elevation did not decrease the sensitivity of i-SOX2-LNCaP cells to enzalutamide, which indicates that elevating SOX2 on its own is not sufficient to confer enzalutamide resistance. Furthermore, knocking down SOX2 in C4-2B cells, a derivative of LNCaP cells which is far less sensitive to enzalutamide and which expresses much higher levels of SOX2 than LNCaP cells, did not alter the growth response to this antiandrogen. Thus, our studies indicate that NE marker expression can increase independently of the sensitivity to enzalutamide.

Sox2 dosage: A critical determinant in the functions of Sox2 in both normal and tumor cells.

The stem cell transcription factor Sox2 is widely recognized for its many roles during normal development and cancer. Over the last several years, it has become increasingly evident that Sox2 dosage plays critical roles in both normal and malignant cells. The work described in this review indicates that the dosage of Sox2 influences cell fate decisions made during normal mammalian development, as well as cell fate decisions in cancer, including those that influence the tumor cell of origin and progression of the cancer. Equally important, Sox2 dosage is a key determinant in the proliferation of both normal cells and tumor cells, where proliferation is restricted in Sox2high cells. Collectively, the studies reviewed here indicate that tumor cells utilize the fundamental effects of Sox2 dosage to suit their own needs. Finally, we speculate that elevated expression of Sox2 helps establish and maintain tumor dormancy in Sox2-positive cancers.

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.

Generation of Functional Human Retinal Ganglion Cells with Target Specificity from Pluripotent Stem Cells by Chemically Defined Recapitulation of Developmental Mechanism.

Glaucoma is a complex group of diseases wherein a selective degeneration of retinal ganglion cells (RGCs) lead to irreversible loss of vision. A comprehensive approach to glaucomatous RGC degeneration may include stem cells to functionally replace dead neurons through transplantation and understand RGCs vulnerability using a disease in a dish stem cell model. Both approaches require the directed generation of stable, functional, and target-specific RGCs from renewable sources of cells, that is, the embryonic stem cells and induced pluripotent stem cells. Here, we demonstrate a rapid and safe, stage-specific, chemically defined protocol that selectively generates RGCs across species, including human, by recapitulating the developmental mechanism. The de novo generated RGCs from pluripotent cells are similar to native RGCs at the molecular, biochemical, functional levels. They also express axon guidance molecules, and discriminate between specific and nonspecific targets, and are nontumorigenic. Stem Cells 2017;35:572-585.

Promoter Hypomethylation and Expression Is Conserved in Mouse Chronic Lymphocytic Leukemia Induced by Decreased or Inactivated Dnmt3a.

DNA methyltransferase 3a (DNMT3A) catalyzes the formation of 5-methyl-cytosine in mammalian genomic DNA, and it is frequently mutated in human hematologic malignancies. Bi-allelic loss of Dnmt3a in mice results in leukemia and lymphoma, including chronic lymphocytic leukemia (CLL). Here, we investigate whether mono-allelic loss of Dnmt3a is sufficient to induce disease. We show that, by 16 months of age, 65% of Dnmt3a(+/-) mice develop a CLL-like disease, and 15% of mice develop non-malignant myeloproliferation. Genome-wide methylation analysis reveals that reduced Dnmt3a levels induce promoter hypomethylation at similar loci in Dnmt3a(+/-) and Dnmt3a(Δ/Δ) CLL, suggesting that promoters are particularly sensitive to Dnmt3a levels. Gene expression analysis identified 26 hypomethylated and overexpressed genes common to both Dnmt3a(+/-) and Dnmt3a(Δ/Δ) CLL as putative oncogenic drivers. Our data provide evidence that Dnmt3a is a haplo-insufficient tumor suppressor in CLL and highlights the importance of deregulated molecular events in disease pathogenesis.

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.

Concise review: The Sox2-Oct4 connection: critical players in a much larger interdependent network integrated at multiple levels.

The transcription factors Sox2 and Oct4 have been a major focus of stem cell biology since the discovery, more than 10 years ago, that they play critical roles during embryogenesis. Early work established that these two transcription factors work together to regulate genes required for the self-renewal and pluripotency of embryonic stem cells (ESC). Surprisingly, small changes (∼twofold) in the levels of either Oct4 or Sox2 induce the differentiation of ESC. Consequently, ESC must maintain the levels of these two transcription factors within narrow limits. Genome-wide binding studies and unbiased proteomic screens have been conducted to decipher the complex roles played by Oct4 and Sox2 in the transcriptional circuitry of ESC. Together, these and other studies provide a comprehensive understanding of the molecular machinery that sustains the self-renewal of ESC and restrains their differentiation. Importantly, these studies paint a landscape in which Oct4 and Sox2 are part of a much larger interdependent network composed of many transcription factors that are interconnected at multiple levels of function.

Elevating SOX2 levels deleteriously affects the growth of medulloblastoma and glioblastoma cells.

Medulloblastomas and glioblastomas are devastating tumors that respond poorly to treatment. These tumors have been shown to express SOX2 and overexpression of SOX2 has been correlated with poor prognosis. Although knockdown of SOX2 impairs the growth and tumorigenicity of brain tumor cells, it was unclear how elevating SOX2 levels would affect their fate. Interestingly, studies conducted with neural stem cells have shown that small increases or decreases in the level of this transcription factor significantly alter their fate. Here, we report that elevating SOX2 3-fold above endogenous levels in U87 and U118 glioblastoma, and DAOY medulloblastoma cells significantly impairs their ability to proliferate. We extended these findings and determined that elevating SOX2 in DAOY cells remodels their cell-cycle profile by increasing the proportion of cells in the G1-compartment, and induces the expression of genes associated with differentiation. Furthermore, we show that elevating SOX2 leads to a dramatic induction of CD133 expression in DAOY cells, yet inhibits the ability of both CD133(+) and CD133(-) cells to form neurospheres. Together, these findings argue that SOX2 levels must be carefully controlled in glioblastomas and medulloblastomas to maintain their fate. Equally important, our data suggests that increases in the expression of SOX2 during brain tumor progression are likely to be linked closely with changes in other critical genes that work in concert with SOX2 to enhance the tumorigenicity of brain tumors. Importantly, we demonstrate that this is also likely to be true for other cancers that express SOX2. Moreover, these studies demonstrate the advantage of using inducible promoters to study the effects of SOX2 elevation, as compared to gene expression systems that rely on constitutive expression.

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.

The transcription factor encyclopedia.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

Determination of protein interactome of transcription factor Sox2 in embryonic stem cells engineered for inducible expression of four reprogramming factors.

Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated ∼2-3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC.