T cells are a crucial component of the immune system and play an important role in responses to pathogens, tumours, and transplanted tissues. In many human cancers, elevated numbers of tumour-infiltrating CD8+ killer T cells are associated with favourable outcomes, suggesting that enhancing T-cell responses could provide major therapeutic benefit for cancer patients. Thus, identifying factors that can promote protective T-cell responses is of great clinical importance. The cytokine interleukin-2 (IL-2) is a major inducer of T-cell proliferation and differentiation, and is used clinically to treat melanoma and renal cell carcinoma. The first two chapters of this thesis focus on the biochemical mechanisms by which IL-2 induces T-cell proliferation. By using mutant and chimeric cytokine receptors expressed in lymphocyte cell lines, the interplay between Shc and STAT5, two major mitogenic signaling pathways activated by the IL-2 receptor, are investigated, revealing an essential synergy between the two pathways for optimal lymphocyte proliferation. The third chapter of this thesis describes work done to identify cytokines that promote T-cell responses within the ovarian cancer microenvironment. In human diseases such as HIV/AIDS and cancer, high numbers of “polyfunctional” T cells (i.e., T cells capable of multiple effector functions) are associated with favourable outcomes. Using clinical ovarian cancer samples in a novel ex vivo assay, it was found that the ovarian tumour environment inhibits polyfunctional T-cell responses to varying extents among patients. After surveying a large panel of cytokines, the cytokine combination of IL-2, IL-12, and IL-18 was found to overcome the immunosuppressive environment to potently enhance CD8+ T-cell proliferation and polyfunctionality in all patient samples. The polyfunctional profiles induced by these cytokines are associated with protective immunity in various human conditions. Thus, these findings suggest that given the right signals, T cells can become highly polyfunctional effectors in the ovarian cancer microenvironment, which offers promise for the development of effective T-cell based therapies for this clinically challenging disease. / Graduate / 0982

ABSTRACT Background Cutaneous melanoma is a potentially lethal cancer for which incidence rates have risen dramatically in white-skinned populations worldwide over the past decades. While some risk factors for melanoma have been clearly established, such as pigmentary characteristics, sun exposure, and familial history of the disease, emerging evidence suggests that other factors, such as hormonal and genetic factors, may play a role in the aetiology of this cancer. The present work aimed at 1) examining the relationships between hormonal factors, benign gynaecological conditions, and the risk of melanoma, and 2) to explore shared risk factors for endometriosis and melanoma in a large French prospective cohort; and 3) to study the potential associations between novel naevus-associated gene variants and the risk of melanoma in a large Australian population-based study. Methods The E3N prospective cohort includes 98,995 French women insured by a national scheme mostly covering teachers, and aged 40-65 years at inclusion. Women were followed-up approximately every two years starting in 1990 through self-administered questionnaires. A first investigation focused on the potential association between a personal history of endometriosis or of other benign gynaecological conditions and the risk of melanoma, which was examined in the E3N cohort using Cox proportional hazards regression models. A second study explored the potential relationships between cutaneous phenotypic factors associated with melanoma and the risk of endometriosis in the E3N cohort, using unconditional logistic regression models. A third investigation used data from the Q-MEGA (an Australian study that followed-up four population-based samples of melanoma patients in Queensland, diagnosed between 1987 and 1995), as well as from the BTNS (a study including adolescent twins and their parents), from which the parents of the twins served as healthy controls in the present investigation. The association between novel naevus-associated gene variants and the site- and subtype-specific risk of melanoma was assessed using unconditional multinomial logistic regression models. Results A significantly positive association was observed between a personal history of endometriosis and the risk of melanoma in the E3N cohort, as well as a significantly positive association between a personal history of uterine fibroma and melanoma risk. The association between endometriosis and melanoma was even stronger when restricting to endometriosis reported as treated or diagnosed by laparoscopic surgery. However, a history of ovarian cyst, uterine polyp, breast adenoma/fibro-adenoma or breast fibrocystic disease was not significantly associated with the risk of melanoma. Also, significantly positive dose-effect relationships were found in the E3N cohort between the risk of endometriosis and skin sensitivity to sun exposure, number of naevi, and number of freckles, while no significant associations were found with hair or skin colour. Finally, variants of MTAP, PLA2G6 and IRF4 were significantly associated with the propensity to develop naevi in the Q-MEGA study. There was also a statistically significant association between MTAP rs10757257 and the risk of melanoma. Although there was no evidence that this association varied according to anatomical site of the tumour, the risk alleles of this polymorphism were more common in patients with superficial spreading melanoma or nodular melanoma than in controls, while patients with melanoma of the lentigo maligna type were no more likely than controls to carry these alleles. In contrast, no association was found between PLA2G6 and IRF4 variants and the risk of melanoma, globally or by site or type of melanoma. Conclusion The present findings suggest a positive association between endometriosis and melanoma, for which they constitute the strongest evidence to date. This finding may reflect the existence of shared risk factors between endometriosis and melanoma, which is supported by the finding of significant associations between endometriosis and some cutaneous phenotypic traits that are established risk factors for melanoma. Because these traits are mostly genetically determined, it can be speculated that endometriosis and melanoma share similar genetic characteristics. More research will be needed in order to clarify common pathways between endometriosis and melanoma. The finding of a positive association between uterine fibroma and melanoma risk had not been previously reported and warrants further investigation. The presented results also confirm an association between MTAP, PLA2G6 and IRF4 variants and naevus propensity, as well as an association between MTAP and melanoma. The findings suggest that the relationship is subtype-specific, which confirms and further refines the overarching “divergent pathways” model. Since MTAP is located at the same locus as CDKN2A, which has also been associated with naevus counts, further research will be necessary to determine whether these results can be attributed to MTAP independently of CDKN2A.

Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 66-75).

Diss. Linköping : Univ., 2004.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Thesis (Ph.D.)--Ohio University, August, 2008. / Title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 68-74).

Thesis (M.Ost.)--Unitec New Zealand, 2008. / Coda (electronic version) title-page has 2009 date. Includes bibliographical references (leaves 76-79).