The inability to tan properly after sun exposure strongly correlates with increased incidence of skin cancer. The melanocortin 1 receptor (MC1R) is a transmembrane Gs-coupled cell surface receptor found on epidermal melanocytes that transmits pro-survival and pro-differentiation signals mediated by the second messenger cAMP. Humans carrying loss-of-function polymorphisms in MC1R signaling exhibit higher incidences of skin cancers including melanoma. This study focused on the physiologic effects of topical application of forskolin, an adenylate cyclase activator, in extension (Mc1re/e) K14-SCF animals, which model the fair-skinned UV-sensitive human. Twice daily application of the drug promoted accelerated pigmentation, increased skin darkening due to epidermal deposition of melanin pigment, and induced epidermal melanin, which protected the skin against UV injury as judged by “minimal erythematous dose” (MED). Moreover, MC1R signaling regulated the expression of antioxidant enzymes at the transcriptional level. The human melanoma cell line A375, known to harbor a loss-of-function signaling mutation in MC1R, was used to determine effects of cAMP stimulation on the expression of antioxidant enzymes. We observed increases in expression of genes that control the biosynthesis and regulation of glutathione including the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glutathione peroxidase, GPX, and glutathione reductase GSR. In addition, there is an increase in manganese superoxide dismutase (MnSOD) at the protein level. There was accumulation of MnSOD in the mitochondria after pharmacologic induction of cAMP with forskolin. Addition of the oxidative agent H2O2 enhanced the expression of MnSOD at the protein level as early as one hour after MC1R stimulation. Oxygen consumption rate on mitochondria was measured using Seahorse analysis; pharmacologic activation of MC1R/cAMP signaling did not affect mitochondrial metabolism. In addition, topical application of a crude extract of Solidago inhibited UV-induced inflammation in K14-SCF mice. Several UV-induced cytokines, including TNF-α, were down-regulated at the transcriptional level after topical application of Solidago extract. Together, these results indicate that MC1R signaling protects melanocytes from UV damage by regulating antioxidant enzyme expression and suggest that pharmacologic cAMP induction may be a useful preventive mechanism against UV-mediated skin sunburn and oxidative injury.

<p>Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer. Although some TN breast cancers respond initially to neoadjuvant chemotherapy, the majority of patients die within three years of treatment due to recurrent tumor growth. Developing ex vivo models for TN breast cancer recurrence and defining responsible molecules will be crucial to developing effective combination therapies for TN breast cancer patients. We have developed an in vitro model of TN breast cancer dormancy/recurrence. Short-term exposure of tumor cells to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from "recurrent" colonies exhibit increased chemotherapy resistance, resembling therapy resistance of recurrent tumors in patients. Furthermore, we identify a novel signaling axis [nuclear bFGF/DNA-dependent protein kinase (DNA-PK)] supported by chemotherapy-enriched dormant TN breast cancer cells. This signaling axis drives accelerated DNA repair in chemo-residual TN breast cancer cells. Targeting this axis with either with a bFGF shRNA or DNA-PK small molecule inhibitor blocks recurrent colony formation. Using the Oncomine gene expression database, we found that bFGF expression in tumor samples from TN breast cancer patients predicts five year tumor recurrence following neoadjuvant chemotherapy treatment. Finally, we demonstrate that recurrent tumor cells exhibit increased invasiveness, reflecting the aggressive behavior of recurrent tumors in patients. Collectively, these studies identify a novel signaling axis in TN breast cancer that likely contributes to tumor recurrence and provide molecular targets for developing future therapeutics against TN breast cancer.</p> / Dissertation

Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.

Prostate cancer is the most commonly diagnosed malignancy among men, but few genetic factors that drive prostate cancer initiation have been identified. The WD repeat domain 77 (Wdr77) protein is essential for cellular proliferation when it localizes in the cytoplasm of epithelial cells at the early stage of prostate development. In the adult prostate, it is transported into the nucleus and functions as a co-regulator of the androgen receptor to promote cellular differentiation and prostate function. This developmental process is reversed during prostate tumorigenesis i.e., Wdr77 is translocated from the nucleus into the cytoplasm to drive proliferation of prostate cancer cells. In this study, we used in vivo genetic studies to investigate the role of Wdr77 in prostate tumorigenesis. We found that prostate-specific deletion of Wdr77 abolished prostate tumor initiation induced by loss of the tumor suppressor Pten. Mechanistically, Wdr77 ablation inhibited E2F3 activation and enhanced TGFb signaling, leading to attenuated cellular proliferation induced by loss of Pten. These findings establish an essential role of Wdr77 for prostate tumor initiation.

It is estimated that four in five women will give birth while one in eight women will be diagnosed with breast cancer at some point in her lifetime. It is also known that pregnancy at a young age is associated with a marked decrease in the risk of breast cancer and that this protection is different according to breast cancer subtypes. This thesis explores the impact of reproductive history on breast cancer biology and provides the molecular characterization of breast cancer diagnosed during pregnancy. The last part investigates the effect of RANKL inhibition on the biology of breast cancer in young women. In the first study, we investigated the impact of parity and age at first pregnancy on the clinicopathological features, the genomic and transcriptomic landscape, and the immune microenvironment of 313 breast cancers. For the first time, we highlighted a link between reproductive history and the genomic landscape of subsequent breast cancer. We demonstrated that, independently of clinicopathological features, age at first birth is associated with specific genomic alterations that could explain the differences in risk reduction associated with pregnancy according to breast cancer subtypes. This study represents a first step toward the recognition that reproductive factors matter in order to fully understand breast cancer biology and advocates that reproductive history should be routinely collected in future studies addressing the biology of breast cancer but also of other female cancers. The second study is focused on the molecular characterization of breast cancer diagnosed during pregnancy (BCP). We conducted a comparative analysis of a unique cohort of BCP patients and non-pregnant control patients by integrating gene expression, copy number alterations, and whole-genome sequencing data. We showed that BCP has unique molecular characteristics including an enrichment of non-silent mutations, a higher frequency of mutations in mucin gene family and an enrichment of mismatch repair deficiency mutational signature. This provides important insights into the biology of BCP and suggests that these features may be implicated in promoting tumor progression during pregnancy. In addition, it provides an unprecedented resource for further understanding of the biology of breast cancer in young women and how pregnancy could modulate tumor biology. In a previous study, the laboratory had reported up-regulation of RANKL in young and pregnant breast cancer patients. Therefore, in the last chapter, we investigated the biological effect of denosumab, a RANKL inhibitor, in a preoperative study including 27 young primary breast cancer patients. We demonstrated evidence that denosumab induces modulation of the tumor immune microenvironment with an increased level of tumor-infiltrating lymphocytes. This effect was likely due to upregulation of inflammatory cytokines and depletion of immunosuppressive regulatory T cells within the tumor microenvironment. These findings suggest a role for denosumab in reshaping the tumor immune microenvironment of breast cancer and that its use in combination could improve immunotherapy efficacy. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

RNA interference (RNAi) is a natural mechanism of cellular defence against exogenous double stranded RNA (dsRNA). The discovery of small dsRNA molecules which can be processed by the RNAi pathway in mammalian cells was one of the key advances in the study of functional genomics. These molecules can be designed to downregulate the expression of specific genes. Collections or libraries of dsRNA molecules targeting an extensive number of genes are now available. Using these libraries, numerous studies have implemented high-throughput screens for the study of molecular effectors of numerous phenotypes. The process of designing an RNAi screen requires the consideration of several critical factors during both the experimental and analysis phases. The experimental screen should aim to reproduce the biological phenomenon studied as closely as possible by choosing an adequate model and screening conditions. Phenotype evaluation and assessment of knockdown effects need careful consideration. The results obtained from large-scale RNAi screens are often complex. An analysis pipeline should be implemented which integrates the biological basis of the phenomenon and facilitates the interpretation of the data. This project designed and implemented an unbiased shRNA screen in two in vitro models relevant to carcinogenesis and diabetes. The first screen implemented used a model of neighbour suppression to study the molecular effectors of the response in tumorigenic cells to growth suppression cues from the surrounding tissue, a cellular interaction relevant in early tumorigenesis. The second screen studied two phenotypes relevant to diabetes: proliferation and resistance to lipotoxicity of β-cells in a reversibly immortalised  cell line. An integrative analysis pipeline was also developed to apply network biology and functional enrichment analysis methods for the interpretation of the data obtained from both screens.

Cancer cells require survival strategies to respond to microenviromental changes and out-compete their neighbours. They activate stress response mechanisms under extreme microenvironmental conditions, some of which are controlled by the amino acid-sensitive kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1). Exosomes are secreted nanovesicles made inside intracellular endosomal compartments that mediate a specialised and complex form of intercellular signalling that can reprogramme target cells via the action of multiple active cargos. I investigated whether mTORC1 activity might modulate the type of exosome secreted in response to microenvironmental changes. Here I identify a new form of mTORC1-regulated exosome biogenesis and signalling involving recycling multivesicular endosomes (rMVEs), a previously unrecognised site for exosome biogenesis. Reduced activity of a specific form of glutamine-sensitive mTORC1 in HCT116 colorectal cancer cells results in an ‘exosome switch’ in which exosomes are preferentially released from these compartments instead of late endosomes. Importantly, RAB11a is found in association with at least a proportion of rMVEs that generate these alternative exosomes and is loaded on to some of their ILVs, providing a RAB signature of compartmental origin. I provide evidence that this exosome switch is conserved in other cancer cell types. My study also presents a proteomics analysis of extracellular vesicle (EV) preparations from normal and mTORC1-inhibited cells. I demonstrate that EV preparations isolated following exosome switching have enhanced pro-angiogenic properties and novel tumour growth-promoting activities. Activation of the receptor tyrosine kinase c-MET and its downstream mitogen-activated protein kinase (MAPK) ERK via phosphorylation is stimulated by these EVs, providing a potential explanation for their growth-promoting effects. Subsequent studies in the lab have demonstrated that several of these pro-tumorigenic activities are mediated by exosomes. I conclude that stress-induced mTORC1 inhibition allows tumour cells to initiate a novel exosome secretion pathway that potentially mediates a cancer cell survival plan that reverses microenvironmental change and supports tumour adaptation. In the future, blocking this response could improve patient outcome following treatment with mTORC1-inhibitory or anti-angiogenic drugs that have currently met with limited success in the clinic.

Melanoma is the deadliest form of skin cancer due to its high propensity to metastasize and resistance to current therapies. We have created a spontaneous mouse model of metastatic melanoma (Dct-Grm1/K5-Edn3) where metastasis to the lungs is 80% penetrant. The primary tumors of these mice present cellular heterogeneity with cells at varying levels of differentiation. The main goal of this study was to determine the metastatic potential of the primary tumor resident Tyrosinase positive cells and evaluate the dynamic phenotypic changes as those cells move from the primary tumors to the sites of metastasis. To accomplish this aim I crossed the Dct-Grm1/K5-Edn3 mice to CreERT2/mT/mG mice to indelibly label Tyrosinase cell populations within the primary tumor with Green Fluorescent Protein (GFP) by topical application of 4-hydroxytamoxifen (4HT) at the tumor site. In vivo lineage tracing and characterization of GFP+ cells were performed in the metastatic lesions. In the 4HT treated Dct-Grm1/ K5-Edn3/Tyr-CreERT2/mT/mG mice, primary tumor derived Tyrosinase positive cells or their progeny (GFP+) established successful metastases in the distant organs indicating the tumorigenic capacity of the differentiated cell populations. Numerous metastatic melanoma cells were identified in the vasculature of the metastatic organs and established close association with the vascular endothelium. The intravascular cells lost pigmentation and did not express melanocytic markers; however, they mimicked endothelial cell properties and gained the expression of CD31 (also known as platelet endothelial cell adhesion molecule PECAM-1) and vascular endothelial (VE)-Cadherin. In the lung metastatic foci, GFP+ cells resumed pigmentation production and lost the expression of endothelial cell markers. Evidence from other metastatic organs in the mice further supported the phenotypic plasticity of metastatic melanoma cells. The in vivo lineage tracing system established in the melanoma mouse model revealed tumor phenotypic plasticity and will be a powerful model to evaluate and help us understand the etiology and pathogenesis of melanoma metastasis. Further characterization of those more aggressive cells in melanoma will allow for the development of new prognostic tests and novel therapeutic strategies to eliminate metastasis.

Modular domains of proteins are important in cellular signaling processes. Eukaryotic cells are constantly undergoing DNA damage due to exogenous and endogenous sources of damage. The DNA damage response (DDR) involves a complex network of signaling events mediated by modular domains such as the BRCT (BRCA1 C-terminal) domains. Therefore, proteins containing BRCT domains are important for DNA damage detection and signaling. In this dissertation, we focus on two BRCT-containing proteins BRCA1 and PAXIP1. BRCA1 is a gene that is known to be associated with increased risk of hereditary breast and ovarian cancer. Germline variants of BRCA1 are assessed to determine lifetime risk of developing breast and ovarian cancer. This is performed by genetic testing of the BRCA1 sequence and the variants can be classified as pathogenic, non-pathogenic or variants of unknown significance (VUS). Using family history, segregation analysis, co-occurrence and tumor pathology, certain variants have been classified as either pathogenic or non-pathogenic. However, a large majority of the variants are classified as VUS. Functional assays are critical in providing insight in the case of VUS results. We have a developed a visualization resource to aid in functional analysis of BRCA1 missense variants that occur due to single amino acid changes. This tool is known as BRCA1 Circos (http://research.nhgri.nih.gov/bic/circos/) and it aggregates, harmonizes and allows interpretation of data from all published studies on functional analysis of BRCA1 missense variants. Therefore, this is an important tool that will aid in the meta-analysis of functional data needed to better assess VUS. Functional studies of BRCA1 also demonstrate that majority of the variants that have a functional impact on the protein lie in the BRCT region of the protein. This indicates that the BRCT region is important in cancer development. To further analyze the function of BRCT-containing proteins, a study was previously undertaken to evaluate the role of BRCT-containing proteins and their interaction partners in the DNA damage response and consequently, cancer. BRCT domains of seven BRCT-containing proteins were used as baits and their binding partners were demonstrated to be highly enriched in the DDR process. We hypothesized that members of this BRCT-centric protein-protein interaction network could constitute targets for sensitization to DNA damaging chemotherapy agents in lung cancer. Therefore, we probed this established dataset containing the protein-protein interaction network (PPIN) of seven BRCT-containing proteins to identify seventeen kinases. A systematic pharmacological screen was performed to evaluate these kinases as targets to enhance platinum-based chemotherapy in lung cancer and this revealed WEE1, a mitotic kinase, as a potential target. Of the seventeen kinases, inhibition of mitotic kinase, WEE1, was found to have the most effective response in combination with platinum-based compounds in lung cancer cell lines. In the PPIN, WEE1 was shown to interact with PAXIP1 (PTIP), a BRCT-containing protein involved in transcription and in the cellular response to DNA damage. PAXIP1 has been shown to bind DDR proteins, such as 53BP1 and γH2AX, and also shown to be an important part of immune development. In this dissertation, we observe that WEE1 binds to PAXIP1 and PAXIP1 regulates the WEE1-mediated phosphorylation of its main substrate, CDK1. We also demonstrate that ectopic expression of PAXIP1 combined with WEE1 inhibitor, AZD1775, leads to an increase in the mitotic index at the G2/M checkpoint. Overexpression of PAXIP1 combined with AZD1775 treatment in cells with prior DNA damage causes high levels of caspase-3 mediated apoptosis as compared to AZD1775 treatment alone. In summary, we identify the role of PAXIP1 in sensitizing lung cancer cells to the WEE1 inhibitor, AZD1775, in combination with platinum-based therapy and propose the use of WEE1 and PAXIP1 levels as mechanism-based biomarkers. Overall, these studies indicate that BRCT-containing proteins through their role in the DDR and the cell cycle are crucial for both cancer prevention and therapy.

Endothelin receptor b (Ednrb) and its ligand Endothelin 3 (Edn3) have been implicated in melanoma. Several studies have shown an upregulation of EDNRB and EDN3 at both the protein and mRNA levels, as melanoma becomes more aggressive. This study investigated the putative role played by Edn3 over-expression in melanoma progression and angiogenesis in vivo. We crossed Tg(Grm1)Epv transgenic mice that aberrantly express metabotropic glutamate receptor1 under the Dopachrome tautomerase promoter, leading to spontaneous melanocytic lesions in the ears and tails that do not metastasize, with transgenics that overexpress Edn3 under the Keratin 5 promoter (K5-Edn3) or overexpress Ednrb in melanocytes (Tg(Ednrb)1Lk). In both the Tg(Grm1)Epv/K5-Edn3 and Tg(Grm1)Epv/Tg(Ednrb)1Lk mice, tumors appeared earlier and grew significantly larger and faster when compared to Tg(Grm1)Epv mice. Approximately eighty-one percent of Tg(Grm1)Epv/ K5-Edn3 mice and 76% of Tg(Grm1)Epv/Tg(Ednrb)1Lk mice had pigmented lesions in distant organs such as the lung and brain. Real-Time PCR analysis showed higher expression levels of genes involved in cell-cell and cell-matrix interactions and angiogenesis in lesions of Tg(Grm1)Epv/K5-Edn3 when compared to controls. Considering the rapid tumor growth rate of in the Tg(Grm1)Epv/K5-Edn3 mice, differences in the angiogenic response compared to control mice were investigated. Immunofluorescence analysis with the endothelial cell marker CD31 showed that there were more endothelial cells per tumor area in the Tg(Grm1)Epv/K5-Edn3 mice than the controls. Proteome analysis showed that the Dct-Grm1/K5-Edn3 mice had significant increases in other angiogenic related genes such as Angiogenin, CXCL 16 and Endoglin, when compared to controls, while real time PCR analysis of tail tumors also showed higher expression levels of angiogenic related genes such as Hif-1α. The results of this study showed that the EDNRB/EDN3 axis is sufficient to alter the kinetics of melanocytic tumors’ progression, lead them to a fully malignant state, and increase the tumor angiogenic response.