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Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanomas striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of cancer. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. My results demonstrate that the combination of ABT-737 and RNA silencing of Mcl-1 induces significant cell death in six different melanoma cell lines, representing a potential new therapeutic strategy. I show that the apoptotic response to the combination treatment involves both the intrinsic pathway and a death receptor-independent role for extrinsic pathway proteins. The combination treatment also induces a number of gene expression changes as assessed by cDNA microarray and follow-up analyses. Based on the results of the array, I investigated the effects of inhibition of MAPK proteins combined with ABT-737 and/or Mcl-1 knockdown. I found that the combination of a p38 MAPK inhibitor and ABT-737 strongly and synergistically induces apoptosis in melanoma cell lines, thus suggesting a second novel treatment combination with potential for melanoma therapy. Finally, I provide novel evidence that Bcl-2 family member PUMA is cleaved in a caspase-dependent fashion during apoptosis and may play a role in treatment response. Currently, there are no effective treatments for metastatic melanoma. My findings describe two potential combination therapies for melanoma as well as provide novel evidence as to the mechanisms involved in treatment response.

Melanoma is among the top ten most common cancers in the US. The health care delivery system patients participate in may affect access to, and utilization of, health care resources that may determine health outcomes. Much of the current literature has addressed lighter-skinned populations since these individuals are at greatest risk of developing melanoma. This Dissertation is focused on melanoma in nonwhite populations and the effect of health care delivery and access to care on outcome. Specifically, our aims were to review the literature in the area of melanoma among nonwhite populations and compare the incidence of melanoma among nonwhite populations in Florida to the US. Additionally, we sought to determine the stage of melanoma diagnosis for a 12 year period in fee-for-service (FFS) and health maintenance organizations (HMO), to determine if differences in mortality exist between these systems of health care delivery and if established involvement in a health care delivery system may, in part, be responsible for any differences seen in stage of diagnosis and survival. We found non-Hispanic black (NHB) females and Hispanic males in Florida had significantly higher incidence rates of invasive melanoma than their counterparts in the US, 60% and 20% respectively. We also found that Hispanic Medicare patients enrolled in HMO health care delivery systems were less likely to receive a diagnosis of regional versus earlier stages of melanoma compared to Hispanic patients enrolled in FFS systems. Additionally, non-Hispanic white (NHW) Medicare patients enrolled in HMO health care delivery systems were less likely to be diagnosed at a later stage of melanoma compared to NHW patients enrolled in FFS systems. Targeted educational interventions and earlier detection of melanoma are fundamental components of cancer prevention and control efforts aimed at decreasing mortality. More comprehensive medical training, expanded public education campaigns, and increased awareness among patients of all skin types to perform self-skin checks are highly recommended. Further studies elucidating the etiology and risk factors for melanoma among minority populations are needed. We recommend investigation of possible differences in the etiology of melanoma among darker-skinned individuals as well as differences between health care delivery systems.

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El Melanoma Maligno Acral (MMA) lentiginoso es una de las 4 variedades clínicas del melanoma maligno. Ocurre en 2-8% de pacientes blancos y es el tipo predominante en afro caribeños y orientales (35-60%). El tumor se encuentra particularmente en los dedos y sitios que soportan peso. La variante subungueal afecta mas comúnmente el primer dedo del pie y la mano, constituyendo el 2% de todos los melanomas cutáneos. Un factor que dificulta el diagnóstico correcto de la variedad clínica del melanoma es el tiempo de enfermedad, ya que los casos localmente avanzados no permiten distinguir claramente su historia natural. El MM es una de las patologías que tiene el mayor retardo en el diagnóstico y en la mayoría de los casos esto sería atribuible al retraso en la búsqueda de atención médica por el paciente, por lo cual es importante reconocer signos y síntomas que advertidos oportunamente nos lleven a realizar un diagnóstico y tratamiento precoz. Planteamos así la presente revisión de los casos presentados en el hospital en los últimos 5 años. Nuestros resultados muestran que el MMA constituye alrededor del 50% del total de MM cutáneos primarios, con 22 de 53 casos registrados. El grupo etáreo afectado con mayor frecuencia está entre 60-70 años, habiéndose encontrado un predominio en varones (60%). El tiempo transcurrido desde el inicio de enfermedad y diagnóstico fue entre 6 y 12 meses en la mayoría de casos. El dolor fue el motivo principal de consulta 99 casos), entre otros, así como sangrado, crecimiento de la lesión, cambio de color y ulceración. La lesión previa mas frecuentemente reportada fue la mancha o lunar en 13 casos. La totalidad de los melanomas se ubicó en los pies: 03 casos se localizaron en la uña. Respecto al tamaño, en el 60% de los casos la lesión medía de 2-3 cms. En lo referente al estadiaje 19 casos (85%) correspondían a estadíos avanzados: Clark IV y V, 10 con Breslow mayor de 1.5 mm y 09 con más de 4 mm. Adicionalmente hubo 03 casos asociados a vitíligo acrofacial. Concluimos que el MMA es el más frecuente de los melanomas cutáneos primarios en nuestros pacientes, la información de las historias clínicas en algunos casos es insuficiente para precisar la variedad, finalmente nuestros resultados concuerdan en su mayoría con lo reportado en la literatura.

Malignant melanoma is cancer arising from melanocytes that have acquired the ability to metastasize and colonize secondary organs such as the lungs, liver, and brain. According to the Melanoma Research Foundation, malignant melanoma is the most rapidly increasing type of cancer with an annual incidence increase of ~ 4% despite the therapeutic and medical breakthroughs in cancer treatment. Melanoma is the most common cancer in young adults ages 20-30, and it is the leading cause of cancer death in females ages 25-30. Non-modifiable risk factors include age, gender, and inherited predisposition to moles. As for modifiable risk factors, exposure to UV rays from the sun is well-established, but obesity has recently emerged as a factor through recent epidemiological and animal studies. Our results showed that obesity modulates the expression of the transcription factor Snai1, which has been shown to be a key gene in the regulation of the Epithelial-to-Mesenchymal Transition (EMT). Serum from obese ob/ob mice, as well as conditioned media from 3T3L1 adipocytes, increased the invasive ability of melanoma cells and the expression of the transcription factor Snai1. Yet, the cytokine IL-6 may not be a critical component of obesity-mediated B16BL6 melanoma cell invasiveness. / text

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Cutaneous melanoma is an increasingly common aggressive malignancy. The molecular mechanisms responsible for melanoma’s initiation and progression are still unclear, but new evidence suggests microRNAs (miRNAs) may be involved. MicroRNAs are small non-coding RNAs that have been shown to act as either oncogenes or tumour suppressors. These short, ~22 nucleotide long, single stranded RNA molecules regulate gene expression post-transcriptionally, through complementary binding to target messenger RNA (mRNA), and mediate mRNA degradation and translational repression. Our laboratory has previously shown that miRNA expression levels are altered through the different stages of melanoma tumourigenesis and has identified numerous significantly dysregulated miRNAs. miR-205 expression is significantly decreased in both primary and metastatic melanoma. Because of this decrease in miR-205 level with increasing cancer aggressiveness, we originally hypothesized that miR-205 may act as a tumour suppressor in melanoma. Unexpectedly, miR-205 re-expression in metastatic melanoma cells has shown oncogenetic potential. Through functional assays, we determined that miR-205 plays a primary role in promoting cellular migration and invasion, and in repressing adhesion. A gene expression analysis was conducted and the target prediction algorithm TargetScan was utilized to determine potential mRNA targets for miR-205. CADM1, PTPRJ and SHIP2 were three of the targets investigated, because of their known functional role in migration and cellular adhesion. CADM1 and PTPRJ were both verified to be directly targeted by miR-205 in an in vitro melanoma system using a luciferase reporter assay. In summary, we have demonstrated a surprising functional role for miR-205 in melanoma. The re-expression of miR-205 promotes malignant phenotypes and therefore is functioning with oncogenic potential within our metastatic melanoma cell culture system. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2012-07-09 12:32:35.235

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