Transcription factor OCT1 regulates adult and malignant stem cells

For many years the Oct1 transcription factor was though to behave as a static regulator of housekeeping genes and was constitutively bound to the octamer binding sites of its targets. However, recently there has been a significant amount of evidence from our lab and others that has shifted the paradigm and caused the scientific community to change the way they think about this transcription factor. Oct4, a close family member to Oct1, is well known to be a regulator of pluripotency in embryonic stem cells. Oct4 and Oct1 have similar binding site specificity, similar gene targets and they are both observed to display stem cell properties. However, the role of Oct1 in stem cell identity was unknown. I have established that Oct1 is dynamically regulated and critical for somatic stem cell identity and stem cells in cancer. I have determined that high Oct1 protein levels correlate with stem cell markers in multiple epithelial tissues, both normal and malignant. Using multiple stem cell matrices, I determined that loss of Oct1 results in a decrease in the stem cell population. Reciprocally, a gain in Oct1 results in an increase in the stem cell population. Also, I found that Oct1 transcriptionally regulates multiple functional stem cell markers, including Aldh1a and Abcg2. Previous studies identified multiple posttranslational modifications to Oct1 protein in HeLa cells, including phosphorylation, glycosylation, and ubiquitination. Here I established that K9 and K403 ubiquitin sites of Oct1 are important for degradation. I determined that Oct1 protein levels are significantly reduced by ~80% in the presence of wildtype BRCA1. However, Oct1 mRNA levels are only reduced less than 2 fold with BRCA1. By mutating the ubiquitin sites from Lysine to Arginine (K9/403R) and transducing stable expression of these mutants, I found that Oct1 protein levels greatly increase in the presence of BRCA1. This indicates that mutating the ubiquitin sites protects Oct1 from BRCA1 dependent degradation, thus supporting my hypothesis that Oct1 is a BRCA1 E3 ubiquitin ligase target.

TRANSCRIPTION FACTOR OCT1 REGULATES ADULT AND MALIGNANT STEM CELLS by Jessica Maddox A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Department of Pathology The University of Utah December 2013 Copyright © Jessica Maddox 2013 All Rights Reserved The Univers i ty of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Jessica Maddox has been approved by the following supervisory committee members: Dean Tantin , Chair 06/03/13 Date Approved Janis Weis , Member 06/03/13 Date Approved Jerry Spangrude , Member 06/03/13 Date Approved Jerry Kaplan , Member 06/03/13 Date Approved Alana Welm , Member 06/03/13 Date Approved and by Peter Jensen , Chair/Dean of the Department/College/School of Pathology and by David B. Kieda, Dean of The Graduate School. ABSTRACT For many years the Oct1 transcription factor was though to behave as a static regulator of housekeeping genes and was constitutively bound to the octamer binding sites of its targets. However, recently there has been a significant amount of evidence from our lab and others that has shifted the paradigm and caused the scientific community to change the way they think about this transcription factor. Oct4, a close family member to Oct1, is well known to be a regulator of pluripotency in embryonic stem cells. Oct4 and Oct1 have similar binding site specificity, similar gene targets and they are both observed to display stem cell properties. However, the role of Oct1 in stem cell identity was unknown. I have established that Oct1 is dynamically regulated and critical for somatic stem cell identity and stem cells in cancer. I have determined that high Oct1 protein levels correlate with stem cell markers in multiple epithelial tissues, both normal and malignant. Using multiple stem cell matrices, I determined that loss of Oct1 results in a decrease in the stem cell population. Reciprocally, a gain in Oct1 results in an increase in the stem cell population. Also, I found that Oct1 transcriptionally regulates multiple functional stem cell markers, including Aldh1a and Abcg2. Previous studies identified multiple posttranslational modifications to Oct1 protein in HeLa cells, including phosphorylation, glycosylation, and ubiquitination. Here I established that K9 and K403 ubiquitin sites of Oct1 are important for degradation. I determined that Oct1 protein levels are significantly reduced by ~80% in the presence of wildtype BRCA1. However, Oct1 mRNA levels are only reduced less than 2 fold with BRCA1. By mutating the ubiquitin sites from Lysine to Arginine (K9/403R) and transducing stable expression of these mutants, I found that Oct1 protein levels greatly increase in the presence of BRCA1. This indicates that mutating the ubiquitin sites protects Oct1 from BRCA1 dependent degradation, thus supporting my hypothesis that Oct1 is a BRCA1 E3 ubiquitin ligase target. iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF FIGURES .......................................................................................................... vii CHAPTERS 1. INTRODUCTION ...........................................................................................................1 Somatic stem cells ........................................................................................2 Stem cells properties ....................................................................................4 Stem cells in cancer .....................................................................................8 CSC model: a shifting paradigm ..................................................................8 Octamer transcription factors .......................................................................9 Oct1 stem cell characteristics .....................................................................13 Oct1 posttranslational modifications .........................................................16 BRCA1 and Oct1 .......................................................................................18 Dissertation overview ................................................................................19 References ..................................................................................................20 2. OCT1 IS A SOMATIC AND CANCER STEM CELL DETERMINANT ...................26 Abstract .....................................................................................................27 Introduction ................................................................................................27 Author summary ........................................................................................28 Results ........................................................................................................28 Discussion ..................................................................................................35 Materials and methods ...............................................................................37 Supporting information ..............................................................................38 Acknowledgements ....................................................................................39 Author contributions ..................................................................................39 References .................................................................................................39 3. OCT1, OCT4, AND CANCER STEM CELLS .............................................................41 Abstract .....................................................................................................42 Malignant stem cell self-renewal and gene expression ..............................43 Oct4 in pluripotency, malignancy, and CSCs ............................................44 Oct1 as an Oct4 surrogate ..........................................................................49 Oct1 and Oct4 working in concert .............................................................58 vi Conclusions ................................................................................................58 Acknowledgements ...................................................................................59 References ..................................................................................................60 4. OCT1 PROTEIN DEGRADATION IS REGULATED BY BRCA1 DEPENDENT UBIQUITINATION ......................................................................................................65 Abstract ......................................................................................................66 Introduction ................................................................................................66 Results ........................................................................................................68 Discussion ..................................................................................................72 Materials and methods ...............................................................................77 Acknowledgements ....................................................................................79 Reference ...................................................................................................80 5. DISCUSSION ................................................................................................................82 Oct1 is a stem cell transcription factor ......................................................83 Oct1 regulates somatic and cancer stem cell identity ................................85 Regulation of Oct1 protein in stem cells ....................................................86 Future Directions: Hypothesis and model ..................................................87 References ..................................................................................................92 APPENDICES A: IN VIVO MOUSE MODELS OF OCT1 FUNCTION IN NORMAL AND MALIGNANT STEM CELLS ..........................................................................................95 B: NOTCH SIGNALING UPSTREAM OF OCT1 .........................................................107 LIST OF FIGURES Figures 1.1. Schematic view of stem cell definition .........................................................................5 1.2. Schematic view of stem cell properties ....................................................................... 6 1.3. Schematic view of Oct1 DNA binding domains bound to canonical octamer motif . 12 1.4. Oct1 regulates multiple stem cell mechanisms .......................................................... 15 1.5. Schematic view of Oct1 posttranslational modifications ........................................... 17 2.1. Association of Oct1 with normal somatic stem cells ................................................. 29 2.2. Oct1 protein levels correlate with a stem cell phenotype in primary human malignancy. ................................................................................................ 31 2.3. Oct1 controls the AldefluorHI population in human tumor cell lines ......................... 32 2.4. Oct1 RNAi diminishes the side population of A549 cells ......................................... 34 2.5. Oct1 controls tumor initiation frequency in A549 and MDA-MB-231 cells ............. 35 2.6. Loss of Oct1 interferes with hematopoietic engraftment ........................................... 36 3.1. ESC, SSC, and CSC characteristics ........................................................................... 46 3.2. Emerging roles for Oct1 ............................................................................................. 51 3.3. Defined Oct1 target genes with relevance to CSCs ................................................... 53 3.4. Oct1 modes of transcription regulation ...................................................................... 56 4.1. Schematic view of BRCA1 protein domains ............................................................. 67 4.2. Oct1 ubiquitin sites K9 and K403 are functional ....................................................... 70 4.3. Oct1 protein is reduced in the presence of BRCA1 ................................................... 71 4.4. Oct1 ubiquitin double mutants are protected from BRCA1 dependent degradation . 73 4.5 Schematic view of the functional classification of transcription factors from the Lgr5+ intestinal stem cell signature ........................................................... 76 5.1. Schematic view of hypothetical Oct1 protein regulation model ................................ 88 A.1 Loss of Oct1 results in collapse of epithelium ........................................................... 99 B.1 Inhibition of Notch reduces Oct1 protein expression independent of BRCA1 ........ 110 viii 1 CHAPTER 1 INTRODUCTION 2 Somatic stem cells As mammals, we have the remarkable, but limited potential to regenerate most tissues. Each organ of the body has different regeneration capacity. This capacity is thought to depend on somatic (or adult) stem cell (SSC) activity and maintenance. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are programmed to differentiate into all three germ layers of the developing embryo. The pluripotent nature of ESCs is unique. Unlike ESCs, SSCs are a subgroup of stem cells that are retained postdevelopment and are multipotent. Their potency is limited to the tissue they reside in, but is responsible for all tissue-specific cell types. Historically, SSCs have been difficult to study because of a lack of definitive markers. However, the past decade has yielded substantial progress in identification and function of SSCs, albeit in some tissues more than others. For instance, the most well characterized SSC population that has been described is the hematopoietic system. Hematopoietic stem cells were first purified and characterized in 1988 (Spangrude et al., 1988) and since then most, if not all, of the lineages of the blood have been identified (Orkin, 2000) . SSCs have also been described in most epithelial tissues, including small intestine, skin, breast, and lung (Barker et al., 2007; Clayton et al., 2007; Ema et al., 2000; Kim et al., 2005; Santos et al., 2013). SSC maintenance and proliferation are fundamentally governed by a tightly regulated gene expression network. Identifying transcription factors that are critical to this network is the key to unraveling this complex web. Several recent studies indicate that forkhead O (FoxO) transcription factors are necessary for the regeneration potential of long-term hematopoietic stem cells through regulating the response of HSCs to 3 oxidative stress, quiescence, and survival (Miyamoto et al., 2007; Tothova et al., 2007). FoxO family members participate in various cellular processes including stress resistance, metabolism, cell-cycle arrest, and differentiation (Bluher, 2003; Holzenberger et al., 2003; Medema et al., 2000). Because of their great functional diversity, FoxO proteins are tightly controlled by posttranslational modifications, such as phosphorylation, glycosylation, acetylation, and ubiquitination (Brenkman et al., 2008; Ho et al., 2010; van der Heide et al., 2005). Another critical transcriptional regulator of HSC long-term maintenance is BMI-1, a polycomb complex protein (Rizo et al., 2008). Similar to FoxO proteins, BMI-1 participates in DNA damage response and metabolism (Liu et al., 2009). BMI-1 is also responsible in part for creating a poised gene expression state (Oguro et al., 2010). Both FoxO proteins and BMI-1 transcriptional control of stem cell function are not restricted to HSCs. FoxO proteins have been implicated in pluripotency of ESCs and maintenance of neural stem cells (NSCs) (Schmidt-Strassburger et al., 2012; Zhang et al., 2011). BMI-1 is functionally relevant in the maintenance of several stem cell compartments including small intestine, lung, and skin (Lacroix et al., 2010; Sangiorgi and Capecchi, 2008; Zacharek et al., 2011). Our lab has found that Oct1, a POU domain transcription factor, also plays pivotal roles in DNA damage, stress resistance, and poised gene states, and not surprisingly stem cell identity (Maddox et al., 2012; Shakya et al., 2011a; 2009a; Tantin, 2005). Developing a more comprehensive conceptual framework of the regulation of SSC's will be crucial to understanding age-related degeneration and diseases. 4 Stem cell properties Stem cells are defined by their ability to self-renew and give rise to progeny that are destined to differentiate and eventually die (Figure 1.1). Additionally, they tend to exhibit properties that discriminate them from more differentiated cell types. Although there are discerning characteristics between stem cell types, all stem cells (ESCs, SSCs, CSCs) display some common traits (Figure 1.2). For instance, stem cells frequently use glycolysis as their primary energy source (Schieke et al., 2008; Suda et al., 2011). Recently, work by Takubo et al. provided evidence for a metabolic switch during hematopoietic stem cell proliferation. Specifically, they found that quiescence stem cells use glucose as their primary source of energy, but shift to aerobic respiration when signaled to proliferate (Takubo et al., 2012). Stem cells are typically relatively quiescent while also maintaining the capacity to rapidly proliferate with appropriate upstream signaling. To accomplish this, epigenetic modifiers and transcription factors generate a poised gene expression state. For example, in ESCs, transcriptionally active genes associated with pluripotency are located within euchromatin and are marked by H3K4me3 and H3K9ac. Transcriptionally silent genes associated with differentiation are located in heterochromatin and are marked by H3K9me3. Genes associated with lineage-commitment are in a transcriptionally poised state and have bivalent epigenetic modifications such as the active H3K4me3 and the repressive H3K27me3 marks. Upon signaling of lineage-commitment, genes that direct a distinct lineage preserve the active H3K4me3 marks and remove the repressive H3K27me3 marks to activate transcription. Genes that control other lineages retain the H3K27me3 mark and acquire an additional repressive H3K9me3 mark, preventing transcription (Mohn and Schübeler, 2009). Stem 5 Stem Cell Progenitor Cell Self-renewal Differentiation specialized cell specialized cell specialized cell specialized cell Figure 1.1. Schematic view of stem cell definition. Stem cells are defined by their ability to self-renew and give rise to progeny that differentiate. Most somatic stem cells differentiate into an intermediate, highly proliferative progenitor cell population. Progenitor cell progeny then differentiate into specialized cells. 6 Figure 1.2. Schematic view of stem cell properties. All stem cells tend to exhibit common properties such as poised gene expression, glycolytic metabolism, and stress resistance. Embryonic stem cells (ESCs) are highly proliferative and divide symmetrically. Somatic stem cells (SSCs) and Cancer stem cells (CSCs) are maintained through a quiescent state and divide asymmetrically. Highly proliferative Quiescent Asymmetric cell division Poised gene expression Glycolytic metabolism SSC Stress resistance CSC ESC Symmetric cell division 7 cells create a poised gene expression state for an appropriate and robust response to proliferation and differentiation signals Most stem cell populations maintain a high degree of resistance to oxidative and genotoxic stress. Multiple mechanisms work in concert to develop stress resistance. First, most stem cells are relatively quiescent, or are slowly cycling. Quiescence may play a protective role for stem cells by preventing exhaustion of their proliferative capacity and through limited rounds of DNA synthesis prevent mutations in DNA (Diehn et al., 2009; Inaba and Yamashita, 2012; Moore and Lyle, 2011). Additionally, by adopting a glycolytic metabolic profile, stem cells inherently maintain low levels of damaging reactive oxygen species. As mentioned earlier, recent work by Takubo et al. has found a link between quiescence and glycolytic metabolism in stem cells. They found that HSCs generate ATP by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Pdk overexpression in HSCs defective of glycolysis restored glycolysis, cell cycle quiescence, and stem cell capacity, while Pdk2 and Pdk4 loss attenuated HSC quiescence, glycolysis, and transplantation capacity (Takubo et al., 2012). Finally, stem cells upregulate efflux pumps, such as ABC (ATP-Binding Cassette) transporters, which accumulate at the cell surface (Zhou et al., 2001). Efflux pumps evict toxins and drugs, such as chemotherapeutic cytotoxic compounds, from the cytoplasm preventing cell damage (Davidson and Maloney, 2007). Cumulatively, SSCs and CSCs tend to exhibit stress and drug resistance, are relatively quiescence until signaled to proliferate, require a glycolytic metabolic profile for stem cell maintenance and maybe quiescence, and contain a poised gene expression state. 8 Stem cells in cancer The recent discovery of cancer stem cell (CSC) markers has brought forth new life into an old theory of cancer initiation and drug resistance. CSCs are thought to represent a stem-like population of cells present in many developmentally heterogeneous neoplasms. They are characterized by two essential properties: the ability to self-renew and the ability to produce more differentiated progeny. These properties are shared with two other cell types: pluripotent embryonic stem cells (ESCs) and somatic stem cells (SSCs). CSCs have undergone intense scrutiny, providing insights into pathways governing CSC function. For example, there is evidence for activation of pluripotent gene expression programs associated with CSCs (Ben-Porath et al., 2008; Kim et al., 2010; Somervaille et al., 2009). Other properties of CSCs include relative quiescence, chemo-and radioresistance, and a glycolytic metabolic profile with simultaneous reduced reactive oxygen species levels. CSC model: a shifting paradigm The CSC hypothesis postulates that heterogeneous tumors are organized in a hierarchical manner with a subpopulation of cells that possess the capacity to self-renew and the potential for multilineage differentiation. Therefore, the CSC model implies that tumors are organized similarly, although obviously distorted and dysregulated, to the tissue of origin. The CSC hypothesis is a very attractive idea due to its potential to explain key remaining questions of cancer initiation and progression. For example, a subpopulation of cells in cancer that hold core stem cell qualities, such as stress and drug resistance, could explain recurrence of cancer after chemotherapeutic treatment. 9 Although this model is alluring, controversy remains due to caveats of known CSC functional assays. For instance, one of the current best available functional readouts of stem cell self-renewal and differentiation capacity requires transplantation into a recipient animal. Some argue that xenotransplantations select for survival in hostile and foreign environments and/or for a greater capacity for cellular attachment. Furthermore, in melanoma, tumor formation in xenografts is dependent on the degree of immune deficiency of recipient animals. These results suggested that the ability to transplant is not dependent on "stemness of the tumor," but fitness of the recipient. Recently, work using lineage tracing analysis in spontaneous tumor models in mice provides more robust evidence for the CSC model. Three different groups modeling glioma, intestinal and skin cancer found that heterogeneous tumors form specifically from adult stem cells (Chen et al., 2012; Driessens et al., 2012; Schepers et al., 2012). Chen et al. found that Nestin+ stem cells gave rise to glioma and were resistant to chemotherapeutic drug and responsible for tumor recurrence. Schepers et al. found that Lgr5+ stem cells gave rise to intestinal adenomas and Driessens et al. found that Keratin 14+ stem cells gave rise to skin squamous cell carcinoma. These findings give potent evidence to support the CSC hypothesis, although more work is needed to establish a more precise definition of CSC identity and function. Octamer transcription factors Oct proteins represent a substantial portion of the POU (Pit-1, Oct1/2, UNC-86) domain transcription factor family. POU transcription factors are defined by their classic, bipartite POU DNA binding domain (Herr et al., 1988). POU domain transcription 10 factors are classified into subclass I-VI based on sequence similarity in the DNA binding domain (Table 1.1). This DNA binding domain is divided into two subdomains, known as the POU-specific domain (POUS) and POU-homeodomain (POUH). The subdomains are connected by a nonconserved flexible linker sequence allowing Oct proteins to bind to DNA with great conformational variability and subdomain reorganization (Figure 1.3). In fact, each subdomain recognizes distinct DNA sequences or half sites (Ryan and Rosenfeld, 1997). Oct transcription factors attain their names from the eight base pair DNA sequence (5'ATGCAAAT3') that they recognize. The linker sequence allows Oct proteins to bind cooperatively to the combined half sites. Oct proteins also recognize alternative (noncanonical) DNA sequences (Reményi et al., 2001), which enhances the versatility of Oct1 protein binding and effector functions. The ability of the DNA binding subdomains to spatially reorganize themselves relative to each other can be regulated by upstream signals (Kang et al., 2009). The most widely recognized Oct transcription factor is Oct4. This is partially due to the role of Oct4 in embryonic pluripotency. Oct4 expression is restricted, in early development being limited to the germline, and the blastocyst inner cell mass and epiblast. Oct4 is highly expressed in ESCs, which are derived from the inner cell mass. Loss of Oct4 in knockout mouse models results in loss of pluripotency of the inner cell mass and inappropriate specification as trophectoderm (Nichols et al., 1998). Interestingly, when Oct4 protein is overexpressed, pluripotency is also lost, suggesting tight regulation of Oct4 activity in stem cells (Niwa et al., 2000). Furthermore, Oct4 plays a powerful role in induction of pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). The underpinnings 11 Table 1.1 The mammalian non-octamer-binding POU domains Factor (synonym) Gene symbol Known expression sites (s) Class I Pit-1 (GHF-1) Pou1f1 Pituitary Class IV Brn-3a (Brn-3.0) Pou4f1 Sensory ganglia, retina Brn-3b (Brn-3.2) Pou4f2 Retinal ganglion cells Brn-3c (Brn-3.1) Pou4f3 Inner ear hair cells, retina Class VI Brn-5 Pou6f1 CNS Atypical HNF-1alpha Hnf1a Liver The mammalian octamer-binding POU proteins (Oct proteins) Factor (synonym) Gene symbol Major known expression site (s) Class II Oct1 (NF-A1/OTF-1) Pou2f1 Ubiquitous Oct2 (NF-A2) Pou2f2 Blood/brain Oct11 (Skn-1a) Pou2f3 Skin Class III Oct6 (SCIP/Tst-1) Pou3f1 Early embryo/brain (Schwann cells)/PNS/testes/skin Oct7 (Brn-2/N-Oct3) Pou3f2 Brain (endocrine hypothalamus, pituitary) Oct8 (Brn-1) Pou3f3 Brain Oct9 (Brn-4) Pou3f4 Brain, pancreas Class V Oct4 (Oct3) Pou5f1 Early embryo/ESCs/PGC/germline Sprm-1 Pou5f2 Developing spermatids Table 1.1 POU domain transcription factors with gene symbol and expression patterns. Table 1.1 POU domain transcription factors with gene symbol and expression patterns. The mammalian non-octamer-binding POU domains. ________________________________________________________________ 12 13 involved in the upstream signal regulators, posttranslational modifications, and crosstalk of Oct4 with other Oct proteins remain unknown. Delineating this conceptual framework is vital to understanding pluripotency at a molecular level. Oct1 is a prototypic member of this family and, unlike Oct4, is expressed in most, if not all, developing and adult mammalian tissues. Investigations into the regulatory regions of housekeeping genes such as H2B and U1/U2/U6 snRNA yielded the discovery of conserved octamer sites and Oct1 regulatory activity (Zheng et al., 2003). Oct1 was also found to act similarly in immunological targets, IL-2 and immunoglobulin (Garrity et al., 1994). Because of these original findings, the central functional dogma of Oct1 was that it was a general, static transcription factor for housekeeping genes. Intriguingly, recent discoveries by our lab and others have brought fascinating new light into the dynamic and complex nature of Oct1. Oct1 stem cell characteristics Oct1 and Oct4 are close family members of the POU domain transcription factor family. Indeed, both factors have similar DNA binding specificity and share a large number of gene targets (Kang et al., 2009). As stated above, Oct4 expression is restricted to the inner cell mass of the blastocyst, however, Oct1 is widely and abundantly expressed in adult tissues. For years, Oct1 activity was thought to be fixed, consistent with its role in the regulation of housekeeping genes such as TBP-associated factor 12 (Taf12) and RNA polymerase II polypeptide A (Polr2a) (Murphy et al., 1989). Surprisingly, loss of Oct1 in mice does not result in early embryonic lethality. Rather, Oct1-/- embryos complete implantation and gastrulation, but fail later during 14 development (Sebastiano et al., 2010; Wang et al., 2004b). In addition, Oct1 knockdown has no affect on proliferation of A549 cells (Shakya et al., 2009a), and Oct1 knockout MEFs proliferate at normal rates (Wang et al., 2004b). Further challenging the static activity paradigm, studies have shown that Oct1 responds to different stimuli. For example, Oct1 promotes resistance to genotoxic and oxidative stress (Tantin, 2005) and regulates potent stress response effectors and cell-cycle regulators such as Gadd45a and Cdkn1b (Dalvai et al., 2010; Hirose et al., 2003) (Figure 1.4). Oct1 also responds to genotoxic and oxidative stress by associating with alternative regulatory targets (Kang et al., 2009). Changes in cyclic AMP (cAMP) levels result in translocating of Oct1 between the cytoplasm and nucleus (Wang and Jin, 2010). Recently, additional Oct1 functions have been discovered which reflect a possible new paradigm where Oct1 adopts a subset of Oct4 functions, not in ESCs but in SSCs and CSCs. For example, Oct1 enforces poised transcriptional states (Shakya et al., 2011b) and promotes a glycolytic metabolic profile associated with dampened mitochondrial function and reactive oxygen species (ROS) levels. From those studies our lab established that Oct1 transcriptionally regulates Pdk (Shakya et al., 2009b). Interestingly, as mentioned earlier, Pdk4 has been shown to be responsible for a metabolic switch in hematopoietic stem cells (Takubo et al., 2012). Although Oct1 depletion has little impact on cell growth and viability in culture, or on immortalization by serial passage, loss of Oct1 opposes oncogenic transformation in vitro and tumorigenicity in vivo (Shakya et al., 2009). These studies contribute to the mounting evidence supporting a role for Oct1 in controlling stem cell phenotype. 15 Poised gene expression state Metabolism Stress Resistance ABC transporters ALDH1 nutrient sensing & cell cycle control Oct1 nucleus Figure 1.4. Oct1 regulates multiple stem cell mechanisms. Oct1 upregulates genes that drive glycolysis, stress resistance, and cell cycle control. Additionally, it regulates Abcg2 and ALDH1, two functional stem cell markers. 16 Oct1 posttranslational modifications Posttranslational modifications of proteins give insight into the mechanisms involved in several cellular processes including upstream signaling, partner binding, mass spectrometry methodologies our lab has definitively mapped several phosphorylation, glycosylation, and ubiquitination sites (Figure 1.5) (Kang et al., 2009). Jinsuk Kang, a former graduate student, investigated the functional roles of both phosphorylation and glycosylation of Oct1. Interestingly, he established that following genotoxic and oxidative stress, Oct1 binding specificity is mediated by phosphorylation of Serine 385 and switches from a monomeric into a dimeric confirmation on particular binding sequences (Kang et al., 2009). Additionally, phosphorylation of a different amino acid residue, Serine 335, is induced in both mitotic and stressed cells and becomes enriched in mitotic spindle poles and midbodies. In this form, Oct1 is displaced from mitotic chromatin and directly regulates mitosis (Kang et al., 2011). Dr. Kang also identified two O-GlcNAc sites that regulate response of cells to overgrowth conditions. Specifically, Oct1 reduces Gadd45a induction in response to chronic overgrowth but augments Gadd45a activation in response to acute nutrient starvation. Oct1 dissociates from the nuclear periphery and dissociates with Lamin B during overgrowth conditions, consistent with a model in which Oct1 is reversibly sequestered into a compartment inaccessible for Gadd45a binding in subconfluent cells. O-GlcNAc modifications of Oct1 prevent sequestration, but surprisingly do not sensitize its transcriptional activity in acute nutrient depletion (Kang et al., 2013). Lastly, Oct1 has at least two ubiquitination sites, at Lysine 9 and Lysine 403. Overall, further investigation into Oct1 17 K9 T255 K403 phosphorylation ubiquitination glycosylation N POUS POUH C T270 T276 S278 S283 S335 S385 S448 S728 Figure 1.5. Schematic view of Oct1 posttranslational modifications. Blue lines identify ubiquitination sites, orange lines identify glycosylation sites, and green green lines are phosphorylation sites. 18 posttranslational modification sites may yield a greater depth of understanding into the mechanisms from which Oct1 is dynamically regulated. BRCA1 and Oct1 BRCA1, breast cancer susceptibility gene, is mutated in more than 50% of hereditary breast cancers. Most mutations result in a loss of one allele of BRCA1 and tumorigenesis is dependent on loss of the remaining full length BRCA1 allele, or loss of heterozygosity (LOH). Because of this, BRCA1 functions as a tumor suppressor. BRCA1 acts as a hub for multiple complex processes, but most work has focused on its role in maintenance of genomic stability (Elia and Elledge, 2012). BRCA1, among other known functions, is thought to be a transcriptional regulator with several downstream gene targets. Interestingly, one of those targets is Gadd45, which acts as a downstream effector in DNA damage response and maintenance of genomic stability. BRCA1 both activates and represses Gadd45 through interactions with positive and negative transcriptional cofactors. BRCA1 activates Gadd45 by physically associating with Oct1 and NF-YA at Oct and CAAT binding sequences in the promoter region of Gadd45 (Fan, 2002). Additionally, BRCA1 and Oct1 binding is necessary for transcriptional activation of the estrogen receptor (ESR1) (Hosey et al., 2007), Mad2 (key component of the spindle checkpoint during mitosis) (Wang et al., 2004a), and base excision repair (BER) enzymes (OGG1, NTH1 and REF1/APE1) in response to oxidative stress (Saha et al., 2010). Along with its transcription regulator function, BRCA1 also acts as an E3 ubiquitin ligase. Indeed, the E3 ubiquitin ligase function of BRCA1 is its only source of enzymatic activity and is thought to play a powerful role in the majority of BRCA1- 19 dependent pathways, including DNA damage, stress response, cell cycle regulation, and housekeeping gene regulation (Ohta et al., 2104; Shabbeer et al., 2012; Wu et al., 2007; Yu, 2006). Because Oct1 and BRCA1 physically interact and because BRCA1 E3 ligase functions in similar pathways as Oct1, I hypothesized that BRCA1 may be acting as an E3 ubiquitin ligase to ubiquitinate Oct1 and target it for proteasome degradation. Dissertation overview My work, and the work of others has shown that Oct1 plays a critical role in many stem cell related properties and therefore operates in a vital and highly regulated manner in somatic and cancer stem cells. I have established that Oct1 protein levels are higher in somatic and cancer stem cells and that Oct1 is functionally important for stem cell identity. Additionally, I have found that Oct1 regulates key functional stem cell markers. I hypothesize that Oct1 is posttranslationally modified in stem cells leading to protein stabilization. To elucidate the regulatory mechanism of Oct1, I investigated how Oct1 protein is degraded. 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Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., et al. (2001). The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7, 1028-1034. CHAPTER 2 TRANSCRIPTION FACTOR OCT1 IS A SOMATIC AND CANCER STEM CELL DETERMINANT Jessica Maddox, Arvind Shakya, Samuel South, Dawne Shelton, Jared N. Anderson, Stephanie Chidester, Jinsuk Kang, Keith M. Gligorich, David A. Jones, Gerald J. Spangrude, Bryan E. Welm, Dean Tantin Reprinted from PLoS Genet 8(11): e1003048. doi:10.1371/journal.pgen.1003048 Copyright: © 2012 Maddox et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 27 28 29 30 31 32 33 34 35 36 37 38 39 40 CHAPTER 3 OCT4, OCT1 AND CANCER STEM CELLS Jessica Maddox and Dean Tantin 42 Abstract The identification of master transcription regulators governing cancer stem cell function could yield tremendous therapeutic potential for the treatment of cancer. In this chapter, we will discuss the role of two transcription factors, Oct4 (POU5F1) and Oct1 (POU2F1), in cancer stem cell identity and function. Oct4 is a key regulator of pluripotency in embryonic stem cells and is an obvious candidate regulator of cancer stem cells. Nevertheless, evidence of Oct4 expression and function in many forms of cancer is controversial. In contrast, the Oct4 paralog Oct1 is now known to function as a somatic and cancer stem cell determinant. Oct1 has also been implicated in resistance to cytotoxic stresses, maintenance of glycolytic metabolism, and regulation of poised transcriptional states, all of which are recognized characteristics of stem cells. A number of identified Oct1 target genes are potent controllers of cancer stem cell activity. Other, perhaps noncanonical, Oct1 functions underlying the cancer stem cell phenotype are also possible. The current data are therefore consistent with a conceptual framework in which Oct1, possibly in response to oncogenic signals, adopts a subset of Oct4 functions in cancer stem cells. More investigations, including causal and genome-wide experiments, are necessary to fully substantiate this model. Abbreviations are as follows: cAMP Cyclic AMP CSC Cancer stem cell ESC Embryonic stem cell iPSC Induced pluripotent stem cells SSC Somatic stem cell 43 Malignant stem cell self-renewal and gene expression The history and complexities of the cancer stem cell (CSC) field are covered in more detail elsewhere in this work. In brief, CSCs represent a stem-like population of cells present in many developmentally heterogeneous neoplasms. They are characterized by two essential properties: the ability to self-renew and the ability to produce more differentiated progeny. These properties are shared with two other cell types: pluripotent embryonic stem cells (ESCs) and somatic stem cells (SSCs). CSCs have undergone intense scrutiny, providing insights into pathways governing CSC function. For example, there is evidence for activation of pluripotency gene expression programs associated with CSCs (Ben-Porath et al., 2008; Somervaille et al., 2009; Kim et al., 2010). Other properties of CSCs include relative quiescence, chemo- and radioresistance, and a glycolytic metabolic profile with simultaneous reduced reactive oxygen species levels. Multiple questions must be addressed in order to understand and target critical pathways underlying the CSC phenotype. For example: which master regulatory factors control gene expression to promote CSC maintenance and self-renewal? How are these factors proximally and ultimately controlled by cancer-causing mutations and activated oncogenic pathways? To what extent do the gene expression programs activated by these factors overlap with programs that maintain embryonic pluripotency? The goal of this chapter is to provide an introduction to the transcription factors Oct1 and Oct4, and to provide a snapshot of some current ideas regarding the roles of these factors in the maintenance and self-renewal of CSCs. 44 Oct4 in pluripotency, malignancy, and CSCs Oct proteins Oct proteins comprise a large part of the POU (Pit-1, Oct1/2, UNC-86) domain transcription factor family (Kang et al., 2009b). This metazoan-specific transcription factor family is defined by the presence of a POU DNA binding domain: a bipartite structure divided into two subdomains, known as the POU-specific domain (POUS) and POU-homeodomain (POUH). A flexible linker connects the two subdomains, enabling Oct proteins to bind to DNA with great conformational variability. Oct proteins recognize the classical octamer DNA sequence (5'ATGCAAAT3'), but can also recognize alternative (noncanonical) DNA sequences (Remenyi et al., 2001). The ability of the DNA binding subdomains to spatially reorganize themselves relative to each other can be regulated by upstream signals (Kang et al., 2009a). Oct1 is a prototypic member of this family and is expressed in most, if not all, developing and adult mammalian tissues. Oct4 expression is much more restricted, traditionally being limited to the germline, and the blastocyst inner cell mass and epiblast. Oct4 is highly expressed in ESCs, which are derived from the inner cell mass (Nichols et al., 1998). Oct4, pluripotency and stem cells The most notable function of Oct transcription factors is the powerful role of Oct4 in the maintenance of ESC identity (Nichols et al., 1998). Oct4 is also a nearly ubiquitous component of transcription factor cocktails now routinely used to generate induced pluripotent stem cells (iPSCs), which possess morphologic, growth and differentiation properties of ESCs (Takahashi and Yamanaka, 2006). Hundreds of Oct4 target genes 45 have been identified in ESCs (Chen et al., 2008). Many of the gene targets fall into just a few functional categories, three of which are itemized below. First, in combination with other critical regulators of ESC identity, Oct4 maintains expression of the "core" pluripotency factors (including Oct4 itself, Nanog and Klf4) in a self-sustaining network (Boyer et al., 2005). The extent to which SSCs and CSCs also require the expression of core pluripotency circuitry components is not clear. Second, Oct4 associates with an array of silent but developmentally inducible targets. Examples include Gata2, Pax6, Hoxa5 and Hoxc6. These targets are frequently characterized by a "bivalent" chromatin structure simultaneously marked by repressive and activating nucleosome modifications and a concurrent lack of DNA methylation at CpG dinucleotides (Bernstein et al., 2006; Lister et al., 2009). This chromatin state helps maintain genes in poised configurations such that in response to the correct developmental cues, they can be rapidly activated. Activation of the gene occurs simultaneously with loss of the repressive chromatin marks. In contrast, repressive marks in genes specific for other developmental trajectories are reinforced by de novo DNA methylation. Poised gene expression states are also a characteristic of stem cells, which must maintain key genes in silent but poised configurations (Figure 3.1). Third, Oct4 regulates genes controlling metabolism (e.g., Pcx, Hk1, Ndufa3 and Atp5d). It is now appreciated that ESCs harbor a glycolytic metabolic profile (Kondoh et al., 2007; Folmes et al., 2011). As with poised gene expression states, this feature also appears to be present in SSCs and CSCs (Diehn et al., 2009). However, not all ESC properties are readily transferable to CSCs. ESCs, for example, are highly proliferative, 46 47 whereas CSCs are thought to be relatively quiescent. ESCs are pluripotent, and in the absence of differentiation signals undergo symmetric cell divisions. SSCs and CSCs are multi- or unipotent and may undergo asymmetric cell divisions in a manner regulated signals from the tissue-specific microenvironment (Morrison and Kimble, 2006). Therefore, only a subset of embryonic pluripotency characteristics can be consistent with "pluripotency" programs associated with CSCs (Figure 3.1) Oct4 expression in SSCs As stated above, Oct4 is strongly expressed in germline and pluripotent tissues. Numerous studies have also detected Oct4 in SSCs. For example, Oct4 has been detected in mesenchymal stem cells where it works in concert with Nanog to upregulate Dnmt1, a DNA methyltransferase. Upregulated Dnmt1 represses expression of p16, p21 and genes associated with development and differentiation (Tsai et al., 2012). These data support a functional role for Oct4 in one type of SSC. On balance, however, current evidence supporting a role for Oct4 in SSCs is weak. Transcriptional analysis of ESCs and neural and hematopoietic SSCs demonstrates that the gene expression profiles of these three stem cell types are considerably divergent. Also, Oct4 and other transcription components of the core ESC transcription factor network are not detected in SSCs (Ivanova et al., 2002; Ramalho-Santos et al., 2002). Based on these findings and evidence from SSC regulatory networks (Miranda-Saavedra and Gottgens, 2008), it is likely that transcription regulation of SSC multipotency is largely tissue-specific. In cases where a functional role for Oct4 in SSC maintenance has been rigorously tested, the results have been negative (Lengner et al., 2007). 48 Oct4 expression in malignant cells A cursory literature search of Oct4 expression and cancer will return hundreds of articles supporting or refuting the claim of Oct4 expression in almost all types of cancer, including lung, bladder and colon (Atlasi et al., 2007; Yasuda et al., 2011; Li et al., 2012). The inconsistencies in the literature are due in part to the nature of the Oct4 detection methods. Analysis of Oct4 expression in cancerous lesions typically applies immunohistochemistry, Western blotting, or qRT-PCR to formalin-fixed, paraffin-embedded tissue. Few studies relate their findings to Oct4 expression in ESCs, where it is endogenously found at meaningfully high levels (Mathieu et al., 2011). An obvious exception is germ cell tumors, which are derived from tissues that naturally express Oct4 at high levels. Oct4 functional variants and pseudogenes may also contribute to false-positives in studies that lack appropriate controls (Liedtke et al., 2008). Also, because of the similarities between Oct transcription factors, there is a possibility of antibody cross-reactivity with Oct1, Oct2, Oct6 or Oct11. Beyond these technical caveats, the functional relevance of Oct4 expression in a given malignant tissue must also be addressed. Various studies have suggested a functional role for Oct4 in cancer based on ectopic expression (Hochedlinger et al., 2005; Chang et al., 2011). Proof of a requirement for Oct4 requires loss-of-function studies. Interestingly, some poorly differentiated neoplasms demonstrate expression of downstream stem cell-related Oct4 target genes, but not Oct4 itself (Ben- Porath et al., 2008). Based on this line of thought, an attractive possibility is that limited Oct4 expression may be a consequence rather than a cause of an activated pluripotency program, e.g., a component rather than controller of a malignant "pluripotency" profile. 49 Oct4 expression in CSCs Ascertaining the role of Oct4 in CSCs confronts the same hurdles as in cancer itself. Again, many reports use ectopic Oct4 expression to support its functional role (Chiou et al., 2010; Linn et al., 2010; Beltran et al., 2011; Kumar et al., 2012). Additionally, indirect support for a role for Oct4 in CSCs comes from studies suggesting that the pathways governing the reprogramming of somatic cells to iPSCs overlap with those governing the transformation of somatic cells to CSCs (Utikal et al., 2009; Mosca et al., 2010). Recently, Bourguignon et al. published evidence supporting a functional role for Oct4 and other pluripotency factors (Nanog and Sox2) in CSCs of head and neck squamous cell carcinoma. These factors form a complex in the cytosol and translocate to the nucleus upon stimulation by Hyaluronan via CD44v3, a Hyaluronan receptor and known CSC marker (Bourguignon et al., 2012). Cumulatively, the evidence in support of a functional Oct4 role in CSCs is mixed. Oct1 as an Oct4 surrogate Oct1: a natural candidate Oct1 and Oct4 have similar DNA binding specificity and share a large number of gene targets (Kang et al., 2009b). Unlike Oct4, Oct1 is widely and abundantly expressed in adult tissues. For many years, Oct1 activity was thought to be static, consistent with its role in the regulation of housekeeping genes such as TBP-associated factor 12 (Taf12) and RNA polymerase II polypeptide A (Polr2a). Surprisingly, loss of Oct1 does not result in early embryonic lethality. Instead Oct1 deficient embryos undergo implantation and gastrulation, with the embryos failing later during development (Wang et al., 2004; 50 Sebastiano et al., 2010). Further challenging the static activity paradigm, studies have shown that Oct1 responds to different stimuli. For example, Oct1 promotes resistance to genotoxic and oxidative stress (Tantin et al., 2005), and regulates potent stress response effectors and cell-cycle regulators such as Gadd45a and Cdkn1b (Hirose et al., 2003; Dalvai et al., 2010). Oct1 responds to genotoxic and oxidative stress by associating with alternative regulatory targets (Kang et al., 2009a), and also responds to cyclic AMP (cAMP) levels by translocating between the cytoplasm and nucleus (Wang and Jin, 2010). The last few years have witnessed the identification of additional Oct1 functions (Figure 3.2) consistent with a model in which Oct1 adopts a subset of Oct4 functions in CSCs. For example, Oct1 enforces poised transcriptional states (Shakya et al., 2011) and promotes a glycolytic metabolic profile associated with dampened mitochondrial function and reactive oxygen species (ROS) levels (Shakya et al., 2009). Loss of Oct1 has little impact on cell growth and viability in culture, or on immortalization by serial passage, but antagonizes oncogenic transformation in vitro and tumorigenicity in vivo (Shakya et al., 2009). These studies contribute to the mounting evidence supporting a role for Oct1 in controlling stem cell phenotypes. Oct1 is a somatic and CSC determinant We recently found that Oct1 controls multiple stem cell phenotypes in normal and tumor cells (Maddox et al., 2012). Oct1 deficient fetal liver hematopoietic progenitors manifest engraftment defects in competitive and serial transplantation experiments. Additionally, strong Oct1 protein expression spatially correlates with normal stem cell niches in multiple epithelial tissue types, and correlates with high levels of the known 51 Figure 3.2. Emerging roles for Oct1. Based on this model, we tested whether Oct1 is an upstream mediator of stem cell functions, including critical parameters of CSCs. We also tested whether Oct1 may further serve as an authentic CSC maker. 52 stem cell markers Lgr5, Lrig1 and Aldh1. Elevated Oct1 protein expression also correlates with elevated ALDHHI and CD24LOCD44HI stem cell-like populations in tumor cell lines and primary breast cancer samples. Interestingly, the correlation with Oct1 mRNA expression is poor, suggesting that these differences reflect regulation of Oct1 at the protein level. Loss of Oct1 selectively depletes stem-like populations in multiple human tumor cell lines based on ALDH and dye efflux activity as readouts. Overexpression of Oct1 promotes the stem-like populations. These different assays measure markers associated with CSCs, but do not provide a readout of CSC function. One commonly used functional measure of CSC activity is the ability to initiate tumors in immunodeficient mice. Stable Oct1 knockdown in two different tumor cell lines reduces tumor initiating cell frequency using limiting dilution assays in immunodeficient mice, while Oct1 ectopic expression promotes tumor initiation. Cumulatively these results indicate that Oct1 protein levels can be used as a stem cell marker, and that Oct1 is an upstream SSC and CSC determinant. Aldh1a1, Abcg2, Hmgb3, Abcb1, Abcb4 and Foxo4 are direct Oct1 gene targets (Figure 3.3). These and other targets have been associated with stem cell identity and self-renewal. Abcg2, Abcb1 and Abcb4 encode drug efflux pumps, which mediate an important facet of CSC chemoresistance. Another target is Aldh1a1, which encodes an enzyme that oxidizes toxic aldehydes and synthesizes a key intermediate in retinoic acid biosynthesis. Hmgb3 and Foxo4 are implicated in hematopoietic and germline stem cell function, respectively. Additional target genes remain to be identified. Delineating such targets will aid in deciphering CSC function and may provide additional opportunities for therapeutic intervention. In addition to the 53 Figure 3.3 Defined Oct1 target genes with relevance for CSCs. Transcription start sites are shown with black arrows. Position of the Oct1 binding site relative to the transcription start site is shown above. Data source is referenced under each example. 54 target genes, the underlying mechanisms that drive divergent Oct1 function in stem cell compartments are not understood. For example, Oct1 protein but not mRNA levels are elevated in stem cell compartments. The mechanism(s) underlying this observation are unknown. Posttranslational modifications of Oct1 in CSCs Oct1 protein levels and posttranslational modification states in stem cell compartments and malignancy have not been studied, but are the logical next target of investigation. Precedent exists for posttranslational regulation of proteins important for self-renewal, differentiation and proliferation in CSCs. Beta-catenin, a member of the Wnt signaling pathway, becomes stabilized and translocates to the nucleus to induce expression of genes associated with proliferation and differentiation in somatic epithelial stem cells, and is dysregulated in CSCs (Takahashi-Yanaga and Kahn, 2010). As noted above, CSC-like populations display increased Oct1 protein, but not mRNA transcript levels. Oct1 is known to be ubiquitinated and phosphorylated (Kang et al., 2009a; Kang et al., 2011). The presence of ubiquitination sites suggests that regulation of protein stablility underlies the increased steady-state levels in CSCs. Potential phosphorylation sites within Oct1 that may be differentially modified in CSCs include canonical targets for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA). In primary T cells, ERK is required for Oct1 coactivator switching to regulate gene expression and the establishment of poised transcriptional states (Shakya et al., 2011). In endocrine cells, PKA phosphorylates Oct1 in response to cAMP signaling (Wang et al., 2009). 55 Oct1 mechanisms of action in CSCs Oct1 appears to act in multiple ways to regulate specific gene targets in CSCs. In many cases the mode of regulation differs from canonical transcription factor functions (Figure 3.4). For example, a number of highly expressed ("housekeeping") Oct1 targets are unexpectedly unaffected by Oct1 loss-of-function. These targets are inducibly bound by Oct1 following exposure to genotoxic and oxidative stresses, where the function of Oct1 appears to be to insulate the gene from inappropriate repression. In the absence of Oct1, these targets become inappropriately repressed. The failure to maintain adequate expression in the absence of Oct1 may contribute to the increased stress sensitivity of Oct1 deficient cells (Tantin et al., 2005). Other active targets such as Aldh1a display an interesting property: Oct1 ablation or overexpression does not shift the degree of expression, but rather shifts the proportion of cells within a population that harbor highly elevated activity. This "digital" rather than "analog" mode of gene regulation also suggests that Oct1 need not behave as a canonical transcription factor. Finally, in the absence of Oct1 some transcriptionally silent targets remain silent but manifest poor induction following exposure to the normal induction cues. These findings indicate that Oct1 can help establish poised gene expression states. All three modes of regulation can be explained, in part, by what is known about the mechanisms that Oct1 uses to regulate gene expression. Oct1 can recruit a cofactor activity known as Jmjd1a (KDM3A), a histone lysine demethylase capable of removing an inhibitory nucleosome modification (dimethylated K9 of histone H3). This finding indicates that Oct1 need not behave as a traditional activator or repressor, but can also function as an "antirepressor" preventing 56 Figure 3.4 Oct1 modes of transcriptional regulation. See text for details. Data source is referenced under each example. Figure 3.4. Oct1 modes of transcriptional regulation. 57 the accumulation of negative epigenetic marks and stable gene repression (Shakya et al., 2011). Oct1 could be utilizing these mechanisms to regulate CSC identity. Possible noncanonical functions of Oct1 In addition to the mechanisms postulated above, Oct1 may use additional mechanisms to control CSC activity. For example, during M-phase Oct1 is phosphorylated at Ser335 in the DNA binding domain by the kinase Nek6 (Kang et al., 2011). Phosphorylation excludes Oct1 from mitotic chromatin. Instead, Oct1pS335 concentrates at mitotic spindle pole bodies, kinetochores and the midbody (Kang et al., 2011). Both Oct1 ablation and overexpression can result in mildly abnormal mitoses. Of interest, asymmetric cell division is a hallmark of some SSC and CSC populations. The asymmetric segregation of cell-fate determinants to generate mother and daughter cells relies on the correct orientation of the mitotic spindle. Although there is no direct evidence as yet, it is possible that Oct1 affects asymmetric cell division through regulation of the cell division apparatus. Oct1 has also been associated with the nuclear envelope, particularly in close association with Lamin B1. Nuclear lamins are potent organizers of gene expression and it is thought that Lamin B1 sequesters Oct1 and regulates target genes in response to oxidative stress (Malhas et al., 2009). Intriguingly, some nuclear lamins are associated with genetic syndromes known as laminopathies. Specific mutations in the human gene encoding Lamin A or in the Lamin A-processing enzyme, Zmpste24, cause premature aging syndromes and cancer predisposition. Interestingly Oct1 and Lamin B1 also co-localize at different mitotic structures, with both proteins being required for the proper localization of the other (Kang et al., 2011). 58 It is unknown whether Oct1 utilizes any of these relationships in CSCs. Elevated Oct1 protein has been detected in a number of stem cell compartments, and in purified CSC-like populations from clonal cell lines (Maddox et al., 2012). Surprisingly, much of this additional Oct1 protein appears to be localized to the cytoplasm, rather than the nucleoplasm or nuclear periphery. The function of this cytoplasmic Oct1, if any, is currently unknown, but regulated Oct1 nuclear/cytoplasmic shuttling in response to cAMP and oxidative stress signals has been reported (Wang et al., 2009; Wang and Jin, 2010). Oct1 and Oct4 working in concert As discussed earlier, Oct1 and Oct4 have similar in vitro binding specificity and interact with and regulate common targets. Examples include housekeeping genes, developmental regulators, inflammatory mediators and metabolic genes (Kang et al., 2009b). Because some Oct target sites are complex and can simultaneously interact with multiple Oct proteins, Oct1 and Oct4 can simultaneously bind the same site. Evidence for just such simultaneous interactions exists in ESCs (Ferraris et al., 2011). In addition, Oct1 and Oct4 may regulate other targets in an alternating fashion. More work is required to clarify these interactions and their regulation, and their role if any in CSCs. Conclusions The identification of transcriptional regulators controlling CSC self-renewal, differentiation and proliferation is key to unraveling their aberrant biology, as well as to developing potential therapeutics. Oct1 and Oct4 are natural candidates based on the 59 commanding function of Oct4 in pluripotency, and the similar binding specificity, targets and mechanisms shared by Oct4 and Oct1. The expression and functional role of Oct4 in CSCs is controversial. More studies are needed to fully demarcate the contribution of Oct4 to different types of CSCs. In addition, accumulating evidence indicates that Oct1 acts as a somatic and CSC determinant. In this case the next step is to unlock the upstream signals to which Oct1 responds, and the downstream targets modulated by Oct1, to specifically control stem cell function and malignancy. Acknowledgements We thank J. Spangrude and C. Murtaugh for critical reading of the manuscript. This work was supported by a grant from the National Institutes of Health/National Cancer Institute (1R21CA141009) and a grant from the Concern Foundation. 60 References Atlasi, Y., Mowla, S. J., Ziaee, S. A. and Bahrami, A. R. 2007. 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A. and Tantin, D. 2004. Embryonic lethality, decreased erythropoiesis, and defective octamer-dependent promoter activation in Oct-1-deficient mice, Mol Cell Biol 24(3): 1022-32. Yasuda, H., Tanaka, K., Okita, Y., Araki, T., Saigusa, S., Toiyama, Y., Yokoe, T., Yoshiyama, S., Kawamoto, A., Inoue, Y. et al. 2011. CD133, OCT4, and NANOG in ulcerative colitis-associated colorectal cancer, Oncol Lett 2(6): 1065-1071. CHAPTER 4 OCT1 PROTEIN DEGRADATION IS REGULATED BY BRCA1 DEPENDENT UBIQUITINATION Jessica Maddox, Zuolian Shen, Karina Vasquez-Arreguin, Jinsuk Kang, Dean Tantin 66 Abstract BRCA1, along with its binding partner BARD1, demonstrates E3 ubiquitin ligase activity and participates in multiple cellular functions including cell cycle control, DNA repair and transcriptional regulation (Horwitz et al., 2007; Shabbeer et al., 2012; Starita and Parvin, 2003; Wu et al., 2007). Oct1 and BRCA1 physically interact and evidence suggests that they work in concert to directly regulate transcription of downstream gene targets including Gadd45 (DNA damage), base excision repair (BER) enzymes (OGG1, NTH1 and REF1/APE1), ESR1 (Estrogen receptor), and Mad2 (key component of the spindle checkpoint during mitosis) (Fan, 2002; Saha et al., 2010; Wang et al., 2004). Although Oct1 and BRCA1 act together as transcriptional regulators, whether Oct1 is a target for BRCA1 E3 ubiquitin ligase activity is still unknown. Here we report that Oct1 protein is reduced in the presence of BRCA1, while Oct1 transcript levels remain the same. Mutating Oct1 ubiquitin sites protect Oct1 from BRCA1-dependent degradation. These results indicate that Oct1 degradation is BRCA1 E3 ubiquitin ligase dependent. Introduction BRCA1 germline mutations account for 30% of familial breast cancer cases (King, 2003). BRCA1 is an E3 ubiquitin ligase enzyme and tumor suppressor, which is important for maintenance of genomic stability (Hashizume, 2001). The BRCA1 protein structure consists of an N-terminal ring domain containing E3 ubiquitin ligase activity and C-terminal BRCT repeat domains (Figure 4.1). BRCA1's partner, BARD1, binds to the ring domain and is necessary for BRCA1 enzymatic activity (Hashizume, 2001). BRCA1 E3 ubiquitin ligase participates in multiple cellular pathways and its' substrates 67 BRCA1 1,863 aa RING finger NLS BRCT repeats Figure 4.1. Schematic view of BRCA1 protein domains. N-terminal RING finger domain contains E3 ubiquitin ligase activity. The NLS (nuclear localization signal) is used for nuclear import. The C-terminal BRCT repeat domains bind to DNA damage associated proteins. N C 68 include CtBP interacting protein, cell cycle proteins (cyclin B and Cdc25c), and the estrogen receptor (ER) and progesterone receptor (PR) proteins (Hosey et al., 2007; Ohta et al., 2104; Shabbeer et al., 2012). Interestingly, ubiquitination of ER and PR may act to block the ability of estrogen and progesterone receptors to bind to promoters and therefore repress transcription (Hosey et al., 2007). Using mass spectrometry, our lab has identified multiple sites of posttranslational modification on the Oct1 protein from HeLa cells including phosphorylation, glycosylation and ubiquitination. Phosphorylation and glycosylation sites have been characterized and found to modify Oct1 transcriptional activity under certain stimuli and cell types (Kang et al., 2009; 2013; 2011). However, the function of the Oct1 ubiquitin sites is still unknown. Among the diverse binding partners of BRCA1, Oct1 has been shown to physically cooperate with BRCA1 at specific promoters associated with the DNA damage response (Fan, 2002; Saha et al., 2010; Wang et al., 2004). Whether Oct1 is a target of BRCA1 E3 ligase activity has yet to be determined. Here, we demonstrate that the ubiquitin sites on Oct1 mediate the stability of the protein, and BRCA1 is important for Oct1 degradation. Results Oct1 ubiquitin sites are important for degradation Using mass spectrometry, our lab previously reported posttranslational modifications of Oct1 in HeLa cells including phosphorylation, glycosylation and 69 ubiquitination. While we have previously characterized the function of other posttranslational modifications of Oct1 {Kang:2009ho, Kang:2011im}, the importance of the two ubiquitin sites, Lysine 9 and Lysine 403, remained unknown. (Figure 4.2A). To determine whether these sites played a role in ubiquitin-mediated degradation of Oct1, we constructed a retroviral transduction plasmid containing a mutant version of Oct1 with Lysine 9 and 403 changed to Arginine (K9/403R) (Figure 4.2B). Virus containing either empty vector, wildtype Oct1 or Oct1(K9/403R) was used to transduce both HeLa cells and A549 lung adenocarcinoma cells. Cells transduced with the wildtype Oct1- containing virus showed a modest increase in Oct1 protein levels, while the Oct1(K9/403R) showed a substantial increase in Oct1 protein levels by Western blot analysis. These results indicated that the ubiquitin sites K9 and K403 play an important role in normal Oct1 protein degradation. Oct1 protein levels are reduced in the presence of BRCA1 To determine whether Oct1 is a target of BRCA1 E3 ligase ubiquitination, we first determined the levels of Oct1 protein in cells that express wildtype or mutant BRCA1. Specifically, HCC1937 breast cancer cells that endogenously express the mutant BRCA1 5832insC (BRCA-) and HCC1937 cells that express retrovirally mediated wildtype full-length BRCA1 (BRCA+) were used. Immunoblotting for Oct1 revealed ~80% reduction of Oct1 in the cells that express wildtype BRCA1 compared to the BRCA1- cells (Figure 4.3A,B). Interestingly, Oct1 mRNA transcripts were reduced less than 2 fold in the presence of BRCA1 (Figure 4.3C). 70 A "K9/403R" B Oct1 K9/403R wtOct1 EV GAPDH Oct1 HeLa 1 2 3 GAPDH Oct1 Oct1 K9/403R wtOct1 EV A549 1 2 3 C K9 K403 phosphorylation ubiquitination glycosylation POUS POUH N C Figure 4.2. Oct1 ubiquitin sites K9 and K403 are functional. (A) Schematic view of Oct1 phosphorylation, glycosylation, and ubiquitination sites with K9/403R double mutants. (B, C) HeLa and A549 cells were retrovirally transduced with wildtype (wt) Oct1, Oct1 K9/403R double mutants. Oct1 and GAPDH were detected using Western blotting. EV: Empty vector. 71 α-GAPDH HCC1937 -BRCA 1 HCC1937 +BRCA1 α-Oct1 α-BRCA1 Oct1 (% of control) *P = 0.046 0 20 40 60 80 HCC1937 -BRCA1 HCC1937 +BRCA1 A B C Figure 4.3. Oct1 protein is reduced in the presence of BRCA1. (A) Oct1 was detected using an immunoblot of Oct1 and GAPDH (control) in HCC1937 BRCA- and HCC1937 BRCA+ cell lines. (B) Oct1 and GAPDH protein levels were quantified using Odyssey Software by LiCor. Experiments were performed in triplicate and error bars depict standard deviation. (C) Pou2f1 (Oct1) mRNA levels were measured relative to B-actin in HCC1937 BRCA- and HCC1937 BRCA+ cells using qRT-PCR with intron-spanning primers. Experiments were performed in triplicate and error bars depict standard deviation. Pou2f1 mRNA (per 1000 β-actin) 0 0.1 0.2 HCC1937 -BRCA1 HCC1937 +BRCA1 72 Oct1 ubiquitin double mutants are protected from BRCA1 dependent degradation To investigate whether Oct1 ubiquitin sites K9 and K403 were targeted by BRCA1 ubiquitin E3 ligase, we transduced our virus containing the Oct1(K9/403R) into HCC1937 BRCA- and HCC1937 BRCA+ cells. In cells that express wildtype BRCA1 we see a dramatic increase in Oct1 protein levels indicating stabilization and protection from BRCA1 dependent degradation. In cells lacking BRCA1 we saw a modest overexpression upon transduction with either wildtype or K9/403R Oct1 virus (Figure 4.4). These results indicate that Oct1 ubiquitin mutant stabilization is BRCA1 dependent. Discussion Posttranslational modifications of proteins give great insight into the mechanisms regulating protein function and turnover. We know from previous work in our lab that Oct1 can be phosphorylated, glycosylated, and ubiquitinated (Kang et al., 2013). While the functions of phosphorylation and glycosylation sites of Oct1have been investigated, the role of the ubiquitin sites remained unknown. Here, we show that the ubiquitin sites on Oct1 are important for regulating stability of the Oct1 protein, and mutating these sites is sufficient to increase Oct1 protein levels in both HeLa and A549 cells. Although literature suggests Oct1 and BRCA1 cooperate to transcriptionally regulate gene targets during DNA damage, whether BRCA1 E3 ligase activity targets Oct1 for degradation was not known. Our data shows that Oct1 protein levels were significantly reduced in the presence of wildtype BRCA1 while Oct1 mRNA levels were only reduced less than 2 73 Oct1 GAPDH -BRCA1 +BRCA1 Oct1 K9/403R 1 2 3 4 5 6 EV wtOct1 Oct1 K9/403R EV wtOct1 Oct1 (% of control) 0 10 20 30 40 EV wtOct Oct1 K9/403R Oct1 K9/403R wtOct EV -BRCA1 +BRCA1 A B Figure 4.4. Oct1 ubiquitin double mutants are protected from BRCA1 dependent degradation. (A) Oct1 was detected using an immunoblot of Oct1 and GAPDH (control) in HCC1937 BRCA- and HCC1937 BRCA+ cell lines transduced with retroviral plasmids expressing wildtype Oct and K9/403R Oct1 double mutant. (B) Oct1 and GAPDH protein levels were quantified using Odyssey Software by LiCor. Experiments were performed in triplicate and error bars depict standard deviation. 74 fold. Furthermore, transduction of Oct1 (K9/493R) into cell lines expressing full length BRCA1 resulted in a significant elevation of Oct1 protein levels compared to cells transduced with wildtype Oct1. These results indicate that the ubiquitin sites on Oct1 are targets of BRCA1 E3 ubiquitin ligase, as BRCA1- cells did not show a similar effect. Previous studies have shown that Oct1 may regulate itself by binding to Oct sites in its own promoter. It is possible that a reduction in Oct1 protein levels would result in a slight decrease in Oct1 mRNA. Another possibility is that BRCA1 both ubiquitinates and downregulates Oct1. Future studies to validate the role of BRCA1 E3 ligase activity are needed including Oct1 protein decay assays (by inhibiting protein translation and the proteasome), pulse chase analysis and in vitro ubiquitination assays to validate that Oct1 is ubiquitinated by BRCA1 specifically. It is possible that Oct1 ubiquitination may be important for processes other than protein destruction. The primary function of ubiquitin is to mark proteins for degradation by the 26S proteasome. However, other nonproteolytic functions of ubiquitin have been reported. Ubiquitin has recently been shown to participate in membrane trafficking, protein kinase activation, DNA repair, and chromatin dynamics (reviewed in Chen and Sun, 2009). Some of these processes involve ubiquitin acting as a signal transducer that recruits proteins with ubiquitin-binding domains (Hurley et al., 2006). In addition, ubiquitination of some transcription factors blocks DNA binding and therefore dampens transcriptional activity and function (Horwitz et al., 2007). Determining whether ubiquitin alters Oct1 transcriptional activity will be imperative to fully understanding the nature of ubiquitination of Oct1. Classification of breast cancer subtypes based on gene expression profiling has indicated that basal-like tumors have an expression profile 75 similar to basal/myoepithelial cells of the breast and a transcriptome similar to tumors arising from BRCA1 germline mutation carriers. These tumors tend to be one of the most aggressive subtypes and are associated with poor prognosis. Additionally, they are typically "triple negative" for ER, PR, and HER2, which indicates poor response to currently available hormone targeted therapy (Perou et al., 2000). Interestingly, these tumors also tend to be associated with high levels of CD44hi/CD24lo cancer stem-like cell populations (Hwang-Verslues et al., 2009). While investigating the role of Oct1 in stem cells, I observed that Oct1 protein levels are higher in some stem cell populations while Oct1 transcript levels remain static (Chapter 2). Specifically, I established that Oct1 protein, but not RNA, levels were higher in primary human breast carcinoma samples with elevated CD44hi/CD24lo stem cell populations. Additionally, Oct1 protein, but not RNA, was elevated in ALDH1hi cells sorted from A549 lung adenocarcinoma cells. This discordant phenomenon was not surprising and gave a possible explanation as to why Oct1 target genes were upregulated in stem cell and cancer microarrays, while Oct1 levels remained static. Furthermore, the Clevers lab conducted proteomic analysis of Lgr5+ cells, an intestinal stem cell population, and found that Oct1 protein levels, but not RNA, were enhanced and therefore part of the Lgr5+ stem cell signature (Figure 4.5) (Mu Ntilde Oz et al., 2012). Because of these findings, it is possible that Oct1 protein is stabilized in specific stem cell populations. To determine the functional role of Oct1 in the cancer stem-like cell population in relation to BRCA1, future studies will be conducted to determine whether Oct1 protein levels are higher in CD44hi/CD24lo cancer stem cells. Additionally, I would like to 76 Figure 4.5. Schematic view of the functional classification of transcription factors from the Lgr5+ intestinal stem cell signature. Adapted from Munoz et al., 2012. The intestinal stem cell signature was constructed using PANTHER (www.pantherdb.org) by molecular function. Gene functions were validated manually and re-assigned based on literature. Of the 279 total genes identified, the transcription factors are shown here. The red circle and red arrow indicates Pou2f1 (Oct1) as a part of the intestinal stem cell signature. nucleus cytoplasm TRANSCRIPTION FACTORS HOMEOBOX Hmbox1 Pou2f1 Pbx1 Zfhx3 Tgif1 Tgif2 ZINC FINGERS Bcl11a Uhrf2 Zfp90 Fhl2 Znf280d Znf22 Phf20 Znf346 Znf24 Rnf2 Znf451 Znf48 Sp5 Znf512 Znf618 Bcl11b Znf740 Znf629 HMG BOX Hmbg3 Sox4 Sox9 bHLH Ascl2 Hes1 Mlxip Myc Mycl1 OTHER Cbx2 Phc1 Scml4 Cbx6 Smad5 Mga Dach1 Nfib Tead2 Ehf Nfic Mkl2 Gtf2i Nfia Rbm38 Foxp4 L3mbtl3 plasma membrane 77 determine whether Oct1 protein stabilization, through mutated ubiquitin sites, elevates the CD44hi/CD24lo stem cell population with and without BRCA1. This data would establish a clear link between Oct1 posttranslational modifications, BRCA1, and cancer stem-like cell identity. Cumulatively this supporting data leads us to an attractive hypothesis for the molecular foundations of Oct1 regulation in stem cells in that BRCA1 E3 ubiquitin ligase activity degrades Oct1 as part of normal protein turnover. However, in stem cells, upstream signaling may lead to posttranslational modification of Oct1, protecting it from ubiquitination. Blocking of ubiquitination, therefore, results in Oct1 protein stabilization and accumulation. Consequently, regulated Oct1 protein elevation contributes to stem cell identity through multiple mechanisms, including increased glycolytic metabolism, poised gene expression, and stress resistance. In cancers lacking BRCA1, Oct1 protein levels elevate and contribute to a dysregulated stem cell-like fate. Along with validating BRCA1 E3 ligase activity on Oct1, future studies will focus on testing different modules of this hypothesis. Materials and methods Cell culture A549 and HeLa cells were maintained in DMEM (Invitrogen) and HCC1937 in RPMI (Invitrogen) supplemented with 10% serum (1:1 calf:fetal calf, Atlanta Biologicals), 6mM L-glutamine, 50 U/ml penicillin, 50mg/ml streptomycin (Invtirogen) and 50mM ß-mercaptoethanol (Sigma). Retroviral plasmids pBabe-empty vector, pBabe- 78 wtOct1, and pBabe-Oct1 K9/403R were used to express wildtype and ubiquitin mutant Oct1. Mixed populations were selected for with puromycin (2ug/ml). Retrovirus production and transduction Phoenix-Ampho 293T cells were plated the day prior to transfection onto poly-lysine coated tissue culture dishes at a density of 50% in antibiotic-free media. The following day, the media was replaced with fresh antibiotic-free media and cells were transfected at approximately 70-80% confluency. Phoenix-Ampho cells are a packaging cell line that contain genomic integrations of the packaging genes gag, pol and env. Cells were transfected with pBabe empty vector, pBabe with wildtype Oct1, and pBabe with K9/402R Oct1 mutant plasmids using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions and grown at 37°C. Posttransfection (24hrs), the media was replaced with fresh media containing antibiotics and the cells were incubated overnight. Supernatant containing virus was removed at 48h and 72h posttransfection, filtered through a 0.45 μm filter and administered directly to HCC1937 -BRCA and +BRCA cells. The day before infection HCC1937 cells were resuspended at 0.5x106/mL to ensure actively dividing cells for transduction. Polybrene (Sigma-Aldrich) was used at a final concentration of 8μg/mL. Three days after infection, cells were analyzed for puromycin resistance. Where indicated, Phoenix-Ampho cells at 60% confluency were transduced with virus as a positive control, as these cells are readily infected by retrovirus. 79 qRT-PCR RNA was isolated using Trizol (Invitrogen), followed by RNAeasy clean up (Qiagen). cDNA was synthesized using RevertAid First Strand cDNA synthesis kit (Fermentas). For Pou2f1 RT-PCR, 100ng of cDNA was used to for quantitative RT-PCR using a LightCycler 480 (Roche). ΔCt values were determined by subtracting input DNA, and ΔΔCt values were determined by subtracting the ΔCt value for control primers. The ΔΔCt were converted to fold change using the formula change = 2-ΔΔCt and were averaged. Primers for Pou2f1 qRT-PCR were: Pou2f1 forward, 5 AAAAGAATCAACCCACCAAGC-3; Pou2f1 reverse, 5 GCTAGTCACAAGGCTTGGTGT-3. Primers for Gapdh qRT-PCR were: Gapdh forward, 5 GGCCAAGGTCATCCATGACAA-3; Gapdh reverse 5 AGGGGCCATCCACAGTCTTCT-3. Acknowledgements I would like to thank Jinsuk Kang for the original discovery of Oct1 protein modification sites and constructing the ubiquitin mutant plasmids. I would also like to acknowledge Vicente Planelles for gifting us the HCC1937 cell line with and without wildtype BRCA1. 80 References Chen, Z.J., and Sun, L.J. (2009). Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 33, 275-286. Fan, W. (2002). BRCA1 regulates GADD45 through its interactions with the OCT-1 and CAAT motifs. Journal of Biological Chemistry 277, 8061-8067. Hashizume, R. (2001). The RING Heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. Journal of Biological Chemistry 276, 14537-14540. Horwitz, A.A., Affar, E.B., Heine, G.F., Shi, Y., and Parvin, J.D. (2007). A mechanism for transcriptional repression dependent on the BRCA1 E3 ubiquitin ligase. Proc Natl Acad Sci USA 104, 6614-6619. 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Cancer Research 67, 951-958. 79 CHAPTER 5 DISCUSSION 83 For many years, the dogma of Oct1 function was as a transcriptional regulator of housekeeping genes. Work from our lab and others have shown that Oct1 is quite a bit more dynamic than expected. Here I established that Oct1 regulates somatic and cancer stem cell identity (Chapter 2). To investigate the regulation of Oct1 protein, I found that the known ubiquitin sites of Oct1 are functional and Oct1 protein degradation may be facilitated by the E3 ubiquitin ligase BRCA1 (Chapter 4). Additionally, I wrote a review discussing the most recent evidence for the participation of Oct4 and Oct1 in stem cells and malignancy (Chapter 3). Throughout my dissertation, I have identified novel and important functions of Oct1 and established a conceptual framework from which the Oct1 stem cell pathway can be built upon. Oct1 is a stem cell transcription factor Others in the lab have demonstrated a role for Oct1 in diverse but related pathways such as glycolytic metabolism, stress sensing, and gene poising (Shakya et al., 2009; Shakya, Kang, Chumley, Williams, and Tantin, 2011; Tantin, 2005). This intricate functional profile of Oct1 is not unlike other SSC related transcription factors. As mentioned earlier, FoxO transcription factors and BMI-1 also play a heavy hand in the molecular mechanisms of metabolism, stress resistance, and gene poising, all resulting in a functional readout of stem cell maintenance and proliferation (Liu et al., 2009; Oguro et al., 2010; Rizo, Dontje, Vellenga, de Haan, and Schuringa, 2008; Tothova et al., 2007; Tran, 2002). Interestingly, HSCs depleted of BMI-1 severely lose the ability to transplant into recipient mice indicating a catastrophic loss of HSC maintenance and self-renewal (Park et al., 2003). However, both Dnmt1 deficiency and combined FoxO1/3/4 deficiency 84 result in milder hematopoietic defects similar to the effects of Oct1 deficiency described here (Tothova et al., 2007; Trowbridge, Snow, Kim, & Orkin, 2009). Given that FoxO transcription factors function in similar contexts, it is possible that Oct1 and FoxO family members compensate when either one or the other is lost. The ability of Oct1 to act in a variety of pathways is dependent on two primary mechanisms: 1. Oct transcription factor DNA binding is inherently flexible and variable due to a nonconserved linker between the two subdomains of the POU DNA binding domain, 2. Oct1 posttranslational modifications drive preferential binding to specific Oct binding sequences and in some cases block Oct1 transcriptional activity (Kang et al., 2009; 2013; Reményi et al., 2001). For instance, a former graduate student in our lab showed that upon stress treatment, Oct1-pS385 is increased resulting in preferred binding to the nonclassical Oct MORE sequence (Kang et al., 2009). In this way, Oct1 protein levels are modified and/or stabilized for differential functions according to stimuli or cell type. FoxO proteins are also tightly regulated by posttranslational modifications. For example, FoxO is phosphorylated by JNK and deacetylated by SIRT1, both resulting in nuclear localization and activation of FoxO (Brunet, 2004; Essers et al., 2004). In addition, FoxO proteins are ubiquitinated by Mdm2 and are targeted for degradation by the proteasome (Brenkman, de Keizer, van den Broek, Jochemsen, & Burgering, 2008). Our lab has shown that Oct1 can be modified in many ways including phosphorylation, glycosylation, and ubiquitination. Teasing out the posttranslational Oct1 regulatory network will give greater insight into its role in stem cell identity and how it interacts with other important stem cell related factors. 85 Oct1 regulates somatic and cancer stem cell identity Oct1 functions in multiple stem cell related mechanisms. Oct1 is also closely related to Oct4, a transcription factor specifically expressed in and necessary for pluripotency in embryonic stem cells. Both Oct1 and Oct4 share DNA binding specificity, gene targets, and upstream modules of regulation. Embryonic pluripotency gene expression signatures lacking Oct4 expression have been identified in myeloid leukemia stem cells and aggressive breast carcinomas, suggesting a potential role for Oct4 paralogs (Ben-Porath et al., 2008; Somervaille et al., 2009). Therefore, an attractive hypothesis is that in some instances of cancer, elevated levels/activity of Oct1 can functionally replace Oct4 to direct the common functional properties of ESCs, SSCs, and CSCs. This may be particularly true if, in response to signals, Oct1 assumes additional or augmented functionalities. Based on the rationale that Oct1 behaves similarly to Oct4, I investigated whether Oct1 played a role in both somatic stem cells and stem cells in cancer. By using double immunofluorescence, I initially established that high Oct1 protein levels colocalized with stem cell markers in multiple (normal and malignant) epithelial tissues including gut, mammary, and lung. Additionally, loss of Oct1 reduced engraftment potential in competitive and serial hematopoietic repopulation assays, and compromised the LSK stem/progenitor cell compartment in competitive transplants. These results are consistent with fetal HSC deficiency although, because LSK is an impure population it is formally possible that Oct1 deficient HSCs engraft but function poorly. I used multiple CSC detection parameters, including CD24/44 levels, dye efflux, ALDH activity and tumor initiating frequency in xenograft models, as an indicator of Oct1 function in stem cells. In 86 all assays conducted, (ALDHhi, dye efflux, tumor initiating frequency) loss of Oct1 in cancer cell lines resulted in loss of the stem cell population and Oct1 overexpression resulted in an elevated stem cell population. Additionally, Oct1 protein levels are elevated in sorted ALDH1hi populations. Although each of these assays has their individual limitations, the common finding of an underlying role for Oct1 suggests that it is a controller of the CSC phenotype. Regulation of Oct1 protein in stem cells As stated earlier, Oct1 protein is elevated in normal and malignant stem cells, but mRNA is not. This may be due to increased rates of protein synthesis, decreased rates of degradation, or both. We have found that Oct1 is most likely ubiquitinated by BRCA1 in a breast cancer cell line. These data lead us to believe that regulated Oct1 protein stability may be important in stem cells, but the mechanism is unknown. Interestingly, a large amount of Oct1 protein expression in gut stem cells appears to be cytoplasmic. The function of Oct1 in the cytoplasm of stem cells is unknown, but there is evidence of Oct1 nuclear-cytoplasmic shuttling in response to oxidative stress (Wang & Jin, 2010). Transcription factor and cofactor nuclear-cytoplasmic translocation is a well-characterized mechanism that stem cells employ for poised and rapid signal transduction. For example, both b-catenin and FoxO transcription factors are sequestered and degraded in the cytoplasm. Upon upstream stem cell-related signaling both factors become posttranslational modified allowing for translocation to the nucleus (Korinek et al., 1997; van der Heide, Jacobs, Burbach, Hoekman, & Smidt, 2005). It is possible that Oct1 is regulated in a similar fashion. In addition, transcriptionally active Oct1 protein residing in 87 the nucleus may also be modified, altering its activity. Oct1 activity is regulated by cyclic AMP (Boulon et al., 2002), cellular stress signals (Kang et al., 2009), and MAP kinase activity (Shakya et al., 2011). Oct1 protein modifications in stem cells remain unknown, but are an active topic of investigation. Future directions Hypothesis and model As demonstrated in Figure 5.1, our working hypothesis of regulated Oct1 protein stabilization postulates that during normal protein turnover Oct1 is ubiquitinated by BRCA1 E3 ligase activity. This ubiquitination targets Oct1 for proteasome degradation. However, in stem cells it is possible that upstream signaling results in an Oct1 protein modification that blocks ubiquitination. Prevention of degradation results in accumulation of Oct1, augmenting Oct1 transcriptional activity on stem cell related genes. Although highly speculative, this model is very attractive due to its simplicity and parallel nature of another important stem cell transcription factor family, FoxO. In a breast cancer cell line, I have established that Oct1 protein levels are reduced in the presence of BRCA1. I hypothesize that Oct1 protein is ubiquitinated by BRCA1, however, more assays are needed to validate this hypothesis, including tracking Oct1 expression after inhibition of translation and proteasome inhibition. In vitro ubiquitination would demonstrate that BRCA1 is sufficient to ubiquitinate Oct1 specifically. In addition, reduction of Oct1 expression after knockdown of BRCA1 in nontransformed cells, such as mouse embryonic fibroblasts, would establish a common, not just malignant mechanism of Oct1 degradation. Furthermore, FACS analysis of the CD44hi/CD24lo stem cell 88 stem cell niche Stem cell signaling: Notch? Oct1 Oct1 Oct1 M Oct1 M Oct1 M Oct1 M M BRCA1 BARD1 proteasome Oct1 Figure 5.1. Schematic view of hypothetical Oct1 protein regulation model. My model postulates that Oct1 is degraded by BRCA1 E3 ubiquitin ligase as a part of normal protein turnover. In stem cells, Oct1 is posttranslationally modified preventing Oct1 from being ubiquitinated resulting in accumulation of Oct1. In stem cells, elevated levels of Oct1 contribute to its function as a transcriptional regulator of stem cell identity. Ubiquitin M Post-translational modification 89 population of cell lines expressing mutant Oct1 ubiquitin sites could be used to determine whether inhibition of Oct1 ubiquitination and hence accumulation effects the stem cell population. Oct1 regulation in stem cells is still unclear. I have established convincing preliminary data suggesting that Oct1 is regulated posttranslationally. Determining whether known modulation sites block ubiquitination would give insight into possible stabilization mechanisms and upstream modulators. Also, mapping possible kinases that phosphorylate Oct1 in stem cells would allow for more clarity and further validate our hypothesis. Oct1 is phosphorylated by protein kinase A during mitosis and inhibits Oct1 transcriptional activity (Segil, Roberts, & Heintz, 1991). It is possible that Oct1 is phosphorylated to either dampen or enhance its transcriptional functions. In addition, although mRNA levels are static in tested assays, it is possible that in vivo Oct1 regulation is more complex and uses both transcription and translation in stem cells. Stem cells are physiologically poised to respond to upstream modulators for maintenance and rapid proliferation after injury. Wnt and Notch signaling are well characterized as stem cell signals and are necessary for stem cell self-renewal, differentiation and proliferation (Fre et al., 2009). To demonstrate whether Notch signaling is upstream of Oct1 expression, I administered a Notch inhibitor, γ-secretase inhibitor, to a breast cancer cell line. From preliminary data, Notch inhibition results in reduction of Oct1 expression (see Appendices). Further analysis of the possible role of Notch signaling on Oct1 expression is needed. Although our model is appealing, other hypotheses are plausible. As mentioned earlier, it is possible that Oct1 is both upregulated transcriptionally and stabilized 90 posttranslationally in vivo. It is also possible that Oct1 is regulated posttranscriptionally, through microRNA-mediated downregulation. However, a cursory examination for putative microRNA bindings sites in the 3' UTR of Oct1 was not successful. Lastly, there are 5 alternatively spliced isoforms of Oct1. Recent proteomic and phosphoproteomic analysis of leukemia stem cells have found that Oct1 isoform 3 is phosphorylated in these cell types (Trost et al., 2012). It is possible that these isoforms play a distinct role in stem cells. Future analysis from our lab will focus on investigation of the physiological role and regulatory network of Oct1 in stem cells and cancer. I established through primary breast cancer patient samples, multiple cell line-based stem cell assays, and murine transplant assays that Oct1 is critical for SSC and CSC identity. However, to truly define Oct1 function in SSCs and CSCs, a physiological read out of Oct1 in vivo is necessary. Using an Oct1 conditional knockout mouse model, I am currently evaluating the intestinal architecture of gut stem cells depleted of Oct1. Details on these experiments are described in Appendix A. To evaluate the role of Oct1 during tumor initiation and progression, I am currently crossing our Oct1 conditional knockout mouse model to three mouse models of cancer. In all three models, both Oct1 depletion and cellular transformation are spatiotemporally controlled by Cre recombinase. These models will determine whether Oct1 is necessary for transformation in particular tissue compartments and cell types. Details on these experiments are also described in Appendix A. Oct1 functions as a stem cell determinant, most likely through acting as a stress sensor and driver of glycolytic metabolism. Stress resistance and glycolytic metabolism are hallmarks of stem cells and are necessary for tumor progression and drug resistance 91 (reviewed in Delude, 2011). Mouse embryonic fibroblasts and cancer cell lines that lack Oct1, or have significantly reduced Oct1, have normal growth and viability, but antagonize oncogenic transformation in vitro and tumorigenicity in vivo (Shakya et al., 2009). Based on these data, our lab predicts that Oct1 is a potential chemotherapeutic target for cancer treatment. Ultimately, we hypothesize that Oct1 inhibition, while having minimal effect on normal tissue, could sensitize drug resistant cell populations within a tumor to standard chemotherapeutic drug administration. Future investigations will evaluate these possibilities. 9 2 References Ben-Porath, I., Thomson, M. W., Carey, V. J., Ge, R., Bell, G. W., Regev, A., Weinberg, R. A. 2008. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. 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