Breast cancer (BC) is one of the most common cancers among women worldwide that ~25% of new cancer cases diagnosed every year would be BC; moreover, ~15% of cancer deaths per year caused by BC makes it the leading cause of cancer death among women worldwide. To date, though the cause of a large proportion of BC are still unclear, recent studies have revealed that a supportive breast tissue microenvironment is critical for the development and progression of BC, especially the communication with immune cells within breast tissue. Therefore, breast inflammatory microenvironment is currently received a substantial attention in the prevention and treatment of BC. Research on breast cancer immunology suggests that inflammatory mediators, estrogen and several inflammation-related tumorigenic pathways are potentially contributors for inflammatory breast tumorigenesis. It is evidenced that elevated levels of inflammatory mediators, such as cytokines, chemokines, prostaglandins, and enhanced estrogen production while suffering from chronic inflammation is responsible for not only activating oncogenic pathways, for example NF-κB, STAT3 and Wnt signaling pathways, but also reducing the efficacy of cancer-specific immunity against tumor cells. Accordingly, targeting the chronic inflammatory status in breast tissue has become a promising strategy for breast cancer therapy. Recently, due to the annoying side effects accompanying by traditionally anticancer drugs, there is an increased interest in finding out natural sources to treat BC. Herein, we report that antroquinonol (AQ) and/or 4-acetylantroquinonol B (4-AAQB) isolated from Antrodia Camphorata were able to modulate the expression of several inflammatory mediators, IL-6 and IFN-γ in particular, and downregulate the aromatase expression and Wnt signaling responses induced by inflammatory status. Taken together, the present findings provide new insights into the role of AQ and 4-AAQB in inflammatory breast tumors and also suggest them as promising candidates for breast cancer immunotherapy.

Cancers often resurrect embryonic molecular programs to promote disease progression. In melanomas, which are tumors of the neural crest (NC) lineage, a molecular signature of the embryonic NC is often reactivated. These NC factors have been implicated in promoting pro-tumorigenic features like proliferation, migration and therapy resistance. However, the molecular mechanisms that establish and maintain NC identities in melanomas are largely unknown. Additionally, whether the presence of a NC identity has any clinical relevance for patient melanomas is also unclear. Here, using comparative genomic approaches, I have a) identified a novel role for GDF6-activated BMP signaling in reawakening a NC identity in melanomas, and b) identified a NC signature as a clinical predictor of melanoma progression. Like the genomes of many solid cancers, melanoma genomes have widespread copy number variations (CNV) harboring thousands of genes. To identify disease-promoting drivers amongst such huge numbers of genes, I used a comparative oncogenomics approach with zebrafish and human melanomas. This approach led to the identification of a recurrently amplified oncogene, GDF6, that acts via BMP signaling to invoke NC identities in melanomas. In maintaining this identity, GDF6 represses the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, allowing melanoma cells to remain undifferentiated and survive. Functional analysis in zebrafish embryos indicated a role of GDF6 in blocking melanocyte differentiation, suggesting that the developmental function of GDF6 is reiterated in melanomas. In clinical assessments, a major fraction of patient melanomas expressed high GDF6, and its expression correlated with poor patient survival. These studies provide novel insights into regulation of NC identities in melanomas and offer GDF6 and components of BMP pathway as targets for therapeutic intervention. In additional studies, I wanted to test whether a broader NC identity in melanomas had any clinical relevance. In these studies, I performed transcriptome analysis of zebrafish melanomas and derived a 15-gene NC signature. This NC gene signature positively correlated with the expression of SOX10, a known NC marker in human melanomas. Patients whose melanomas expressed this signature showed poor overall survival. These findings identify an important predictive signature in human melanomas and also illuminate the clinical importance of NC identity in this disease.

RUNX1, a transcription factor required for hematopoiesis and lymphocyte differentiation, is one of the most commonly targeted genes in hematopoietic malignancies. Mutations in the RUNX1 gene are associated with a poor prognosis in a subset of T cell acute lymphoblastic leukemia (T-ALL) and RUNX1 has been proposed as a tumor suppressor in TLX1/3-transformed human T-ALL. Recent ChIP-seq studies in human T-ALL cell lines demonstrated that a large portion of TAL1- and NOTCH1- bound regions contain RUNX binding sites in promoter or enhancer regions, suggesting oncogenic roles for RUNX1 in T-ALL. To interrogate RUNX1 functions in leukemogenesis, we depleted RUNX1 in a T-ALL mouse model and in human T-ALL cell lines. We found that RUNX1 is required for the maintenance of mouse T-ALL growth in vivo and the survival of human T-ALL cell lines in vitro. In addition, inhibition of the RUNX1 activity with a small molecule inhibitor impairs the growth of human T-ALL cell lines and primary patient samples. RUNX1 depletion reduces the expression of a subset of TAL1- and NOTCH1- regulated genes including the MYB and MYC oncogenes, respectively. We demonstrate that RUNX1 regulates transcription factor binding and acetylation of H3K27 at the Myb and Myc enhancer loci. These studies provide genetic and pharmacological evidences that RUNX1 supports T-ALL cell survival and suggest RUNX1 inhibitor as a therapeutic strategy in T-ALL treatment.

Tumor heterogeneity observed between patients has made it challenging to develop universal or broadly effective cancer therapies. Therefore, an ever-growing movement within cancer research aims to tailor cancer therapies to individual patients or specific tumor subtypes. Tumor stratification is generally dictated by the genomic mutation status of the tumor cells themselves. Importantly, non-genetic influences – such as interactions between tumor cells and other components of the tumor microenvironment – have largely been ignored. Therefore, in an effort to increase treatment predictability and efficacy, we investigated how tumor-stroma interactions contribute to drug sensitivity and drug resistance. I designed a high throughput co-culture screening platform to measure how tumor-stroma interactions alter drug mediated cell death. I identified tumor-stroma interactions that strongly desensitize or sensitize cancer cells to various drug treatments. The directionality of these observed phenotypes was dependent on the stromal cell tissue of origin. Further study revealed that interactions between tumor cells and fibroblasts modulate apoptotic priming in tumor cells to mediate sensitivity to chemotherapeutics. The principles uncovered in this study have important implications on the use of drugs that are designed to enhance apoptosis. For example, based on our screening data, I hypothesized and experimentally validated that the effectiveness of BH3 mimetic compounds would be strongly dependent on the fibroblast growth environment. Taken together, our study highlights the importance of understanding how environmental interactions alter the drug responses of cancer cells and reveals a mechanism by which stromal cells drive broad spectrum changes in tumor cell sensitivities to common chemotherapeutics.

Melanoma is one of the most aggressive and deadly forms of skin cancer. Arising from melanocytes, a pigment cell population derived from the neural crest, melanomas often adopt characteristics associated with the neural crest – the ability to rapidly proliferate, migrate and invade throughout the body. Historically, these characteristics along with a baseline resistance to chemotherapy have made melanoma extremely difficult to treat. Improvements in targeted and immunotherapeutic options have improved patient outcomes, but many patients still experience limited durable responses to therapy. In order to improve patient outcomes, new potential avenues of therapy must be identified based on the underlying pathogenesis of the disease. We previously identified and characterized the function of a novel melanoma oncogene, GDF6, uncovering a role in promoting melanoma cell survival and dedifferentiation by activating a neural crest identity. Here, we have a) identified a role for GDF6-activated BMP signaling during melanocyte development that forms a basis for its oncogenic role in melanoma, b) determined BMP signaling may play a role in promoting a neural crest-like state during melanoma initiation, and c) assayed novel monoclonal antibodies targeting GDF6 for use as blocking antibodies to treat advanced melanoma. Previous work identified GDF6 as a melanoma oncogene that promotes melanoma progression through suppression of apoptosis and differentiation in melanoma cells, by regulating neural crest factor expression and neural crest identity, suggesting a potential role for GDF6 in the embryonic neural crest. Additional studies had previously identified roles for GDF6 and its orthologous genes in specific biological contexts, including embryonic neuronal cell survival, bone and cartilage development, embryonic eye development, and bone and ligament repair in adult tissue. Furthermore, a study had indicated a role for a GDF6 ortholog, gdf6a, during zebrafish neural crest induction, but had not uncovered any specific role for gdf6a in further development of the neural crest or in any neural crest derivatives. We determined blocking gdf6a-activated BMP signaling acts to increase melanocyte development during embryogenesis by increasing the proportion of neural crest cells activating the pigment cell marker, mitfa. Furthermore, we showed the increase in melanocytes is at the expense of the iridophore population. These results indicate GDF6 function in melanoma is a reiteration of the normal physiological function of GDF6 during embryonic melanocyte development from the neural crest. Given these results and our previous findings of the role of GDF6-activated BMP signaling established melanomas, we hypothesized a potential role for GDF6-activated BMP signaling during melanoma initiation. Previous studies have determined neural crest identity and neural crest-like characteristics to be crucial during multiple phases of melanoma, including initiation, progression, and metastasis. We evaluated melanoma initiating lesions to determine the potential impact of BMP signaling on development and progression of these lesions. We found early lesions in our model to have active BMP signaling and that modulation of BMP signaling could alter the rate of development of these lesions in our animals. Furthermore, BMP modulation ultimately impacted the development of these lesions into melanomas. Together, these results indicate BMP signaling is a potential driving pathway during melanoma initiation and progression. Finally, we wanted to determine the therapeutic potential of targeting GDF6 in order to treat patients with advanced melanoma. Given our previous findings and mechanism of ligand-activated BMP signaling, we hypothesized a monoclonal antibody targeting GDF6 could block GDF6 activity at its receptor on melanoma cells, thus inhibiting GDF6-activated BMP signaling. Monoclonal antibodies have been widely used as therapy in cancer as well as many other rheumatologic and immunologic conditions. We established a panel of GDF6-targeting antibodies via a hybridoma approach. We then assessed the antibodies ability to identify mammalian GDF6 in vitro and performed functional assays to determine if anti-GDF6 antibody treatment yielded the expected results of inhibiting GDF6-activated BMP signaling. We observed decreased pathway activity, decreased cell viability, and increased cell death in melanoma cells treated with anti-GDF6 antibodies in vitro. We further investigated whether these antibodies could exert anti-melanoma effects in vivo. Together, these results indicate potential therapeutic value for our antibodies in treating GDF6-positive melanomas.

The role of vascular endothelial growth factor (VEGF) signaling in cancer is well-known in the context of angiogenesis but is also important in the functional regulation of tumor cells themselves. Notably, autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression and drug resistance. Therefore, the first half of this thesis focuses on the mechanism of VEGF-NRP-mediated support of CSCs. Aberrant activity of the Hippo pathway effector YAP and TAZ are associated with breast CSCs and have been shown to confer stem cell-like properties. I found that VEGF-NRP2 signaling contributes to the activation of YAP/TAZ in various breast cancer cells, which mediates a positive feedback loop that promotes mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, which enabled the activity of YAP/TAZ. In complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) β2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, YAP/TAZ-mediated repression of β2-chimaerin sustained Rac1 activity in CSCs. Depletion of β2-chimaerin in non-CSCs increased Rac1 activity, YAP/TAZ activation and mammosphere formation. Analysis of breast cancer patients revealed an inverse correlation between β2-chimaerin and TAZ expression in tumors. These findings highlight an unexpected role for β2-chimaerin in a feedforward loop of YAP/TAZ activation and the acquisition of CSC properties. Given that CSCs have been implicated in therapy resistance and are enriched in triple negative breast cancer (TNBC), which exhibits VEGF-NRP2 signaling, the second half of this thesis focuses on understanding the mechanism by which VEGF-NRP2 contributes to the chemoresistance of TNBC. I discovered that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double strand break repair. Mechanistically, I found that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. I also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to chemotherapy and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ in response to cisplatin. These findings reveal novel roles for VEGF-NRP2 and YAP/TAZ in DNA repair and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ and Rad51 that contributes to resistance to platinum chemotherapy. In summary, this thesis provides novel insight into the roles of autocrine VEGF-NRP2 signaling in breast CSC function and therapy resistance and provides rationale in inhibiting NRP2 for platinum-resistant tumors that are dependent on YAP/TAZ activation.

<p><a>Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is an FDA-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment.</a> After a short positive response period to enzalutamide, tumors will develop drug resistance. In these studies, we uncovered that NOTCH signaling and DNA methylation are a deregulated in enzalutamide-resistant cells. <i>NOTCH2</i> and<i> c-MYC </i><a>gene expression<i> </i>positively correlated with <i>AR </i>expression in samples from patients with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide-resistance</a>. The expression of Notch signaling components was upregulated in enzalutamide-resistant cells suggesting the activation of the pathway. Inhibition of this pathway <i>in vitro</i> and <i>in vivo</i> promoted an increase in the sensitivity to enzalutamide with an impact on AR expression. On the other hand, DNMT activity and DNMT3B expression were upregulated in resistant lines. Enzalutamide induced the expression of DNMT3A and DNMT3B in prostate cancer cells with a potential role for p53 and pRB in this process. The overexpression of DNMT3B3, a DNMT3B variant, promoted an enzalutamide-resistant phenotype in C4-2 cells. DNA methylation inhibition, using low-concentration decitabine, and <i>DNMT3B</i> knockdown induced a re-sensitization of resistant prostate cancer cells and tumors to enzalutamide. Decitabine treatment in enzalutamide-resistant induced a decrease in the expression of AR-V7 and changes in genes from the apoptosis, DNA repair and mRNA splicing pathways. Decitabine plus enzalutamide treatment of 22RV1 xenografts induced a decrease in tumor weight, KI-67 and AR-V7 expression and an increase in Cleaved-Caspase3 levels. All the above suggest that Notch signaling and DNA methylation pathways are deregulated after enzalutamide resistance onset, and targeting these pathways restores the sensitivity to enzalutamide.<b><u></u></b></p>

RBMS3 belongs to the family of c-myc gene single-strand binding proteins (MSSPs) that play important roles in transcriptional regulation. Here, we show that RBMS3 functions as a tumor promoter in triple-negative breast cancer (TNBC), a highly aggressive BC subtype. Analysis of RBMS3 expression shows that RBMS3 is upregulated at both mRNA and protein levels in TNBC cells. Functionally, overexpression of RBMS3 increases cell migration, invasion and cancer stem cell (CSC) behaviors. Moreover, RBMS3 induces expression of epithelial-mesenchymal transition (EMT) and CSC markers. Conversely, loss of RBMS3 in TNBC BT549 cells inhibits cell proliferation, migration and mesenchymal phenotype. Correlation analysis shows RBMS3 is associated with TGF-β signaling. Mechanistically, RBMS3 interacts with Smad2, Smad3 and Smad4 mRNA and regulates the stability of these transcripts. Importantly, RBMS3 prevents TGF-β-induced cytostasis and apoptosis in premalignant cancer cells. Moreover, RBMS3 inversely correlates with expression of ESRPs, epithelial-specific splicing regulatory proteins that regulate morphogenesis-associated alternative splicing events. ESRPs appear to suppress EMT through distinct mechanisms: ESRP1 restricted cell migration, whereas ESRP2 prevented cell growth. RBMS3 significantly facilitates the EMT process when ESRPs are lost. Collectively, the studies within this dissertation identify RBMS3 as a positive regulator of EMT and breast cancer progression by regulating the TGF-β signaling pathway.

Sphingolipids are a class of lipid molecules that function both as structural membrane components and as bioactive signaling molecules. Sphingolipids can be produced de novo or salvaged and recycled. Despite the established roles of sphingolipids such as sphingosine 1-phosphate and ceramides in regulating signaling involved in pro- and anti-tumorigenic cellular processes, the role of the de novo sphingolipid biosynthesis pathway in cancer is unclear. The main objective of this thesis study was to determine whether there is an essential role for this pathway in cancer and whether its disruption can be a cancer-specific metabolic vulnerability. Here, we find that de novo sphingolipid synthesis through the rate-limiting enzyme serine palmitoyltransferase (SPT) is not required in cancer cells due to their salvage capacity. However, upregulation of SPT in cancer cells creates a requirement to detoxify its product, 3-ketodihydrosphingosine (3KDS), via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). We demonstrate that KDSR is essential in cancer cells both in vitro and in vivo to restrain the levels of its substrate 3KDS, the accumulation of which can disrupt ER structure and function, resulting in proteotoxic stress and cell death. Our findings also reveal that KDSR is essential specifically in cancer cells and not normal cells and that upregulation of SPT in cancer may act as a biomarker for sensitivity to targeting KDSR. Altogether, this thesis study provides new insights into the role of KDSR in the de novo sphingolipid biosynthesis pathway in both cancer and ER homeostasis and demonstrates the potential to exploit this for therapeutic purposes in a cancer-specific manner.

The cJUN NH2-terminal kinase (JNK) pathway responds to environmental stresses and participates in many cellular processes, including cell death, survival, proliferation, migration, and genome maintenance. Importantly, genes that encode components of the JNK signaling pathway are frequently mutated in human breast cancer, but the functional consequence of these mutations in mammary carcinogenesis is unclear. Anoikis – suspension-induced apoptosis – has been implicated in oncogenic transformation and tumor cell metastasis. Anoikis also contributes to lumen formation during mammary gland development and epithelial cell clearance during post-lactational involution. JNK is known to contribute to certain forms of cell death, but the role of JNK during anoikis was unclear. I examined the requirement of JNK in anoikis and discovered that JNK promotes cell death by transcriptional and post-translational regulation of pro-apoptotic BH3-only proteins. This conclusion suggested that JNK signaling may contribute to mammary gland remodeling during involution. Indeed, JNK deficiency in mammary epithelial cells disrupted the remodeling program of gene expression and delayed involution. Finally, I sought to understand the importance of JNK in mammary carcinogenesis. I found that JNK loss in the mammary epithelium was sufficient for genomic instability and tumor formation. Moreover, JNK loss in a model of breast cancer resulted in significantly accelerated tumor development. Collectively, these studies advance our understanding of the JNK pathway and breast biology, and provide insight that informs the design of therapeutic approaches that target the JNK signal transduction pathway.