A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2012 / Cell-extracellular matrix (ECM) detachment triggers a cell survival mechanism known as autophagy. A link between attachment and autophagy suggests a form of adhesion-based regulation, involving mechanotransduction of extracellular-originating signals to the cellular machinery controlling autophagy induction. This implies a role for integrin-linked kinase (ILK), which transmits mechanical stimuli to the mammalian target of rapamycin (mTOR) signalling pathway. Cells with a propensity for metastasis may negate these adhesive signals, inducing autophagy inappropriately. Metastasis is a hallmark of transformation frequently associated with human oesophageal squamous cell carcinoma (HOSCC). Additionally, hyperactive mTOR/mTORC1 signalling correlates increasingly with HOSCC. Therefore, the protein expression of significant signal transduction pathway intermediates was investigated in response to both soluble and ECM-originating stimuli. Measurements by SDS-PAGE and western-blotting coupled to semi-quantitative densitometry, during standard tissue culture conditions, revealed that HOSCC’s expressed moderate-to-high levels of mTOR, p-RPS6(Ser 235/236) and mATG-13; indicating elevated levels of autophagy induction despite aberrant signalling through mTOR/mTORC1. Additionally, an 80 kDa mTORβ isoform was identified in HOSCC cells with lower mTOR abundance, presumably to maintain aberrant mTORC1 signalling. A canonical role for the PI3K/PKB pathway was also identified; where autophagy induction accompanied diminished mTORC1 signalling in response to specific PI3K inhibition with LY294002 and serum withdrawal. However, autophagy induction varied in response to a dose-dependent decrease in mTORC1 signalling after exposure of HOSCC cells to rapamycin. Moreover, specific inhibition of p90RSK with BI-D1870, suggests that mTORC1 phosphorylates RPS6(Ser 235/236) in the absence of MAPK signals. Furthermore, ectopic ILK expression indicated an enhanced potential for adhesion-based signalling. Correspondingly, HOSCC cells commonly increased mTOR and p-RPS6(Ser 235/236) expression following growth on fibronectin or collagen. However, co-immunoprecipitation analysis revealed that signals transduction to mTOR precludes a direct interaction with ILK or FAK. Rather, ECM-modulation of mTOR occurs in a integrin-triggered, but PI3K-depedant manner; since specific inhibition of PI3K negated fibronectin-induced increases of mTOR concentration and RPS6(Ser 235/236) phosphorylation. Thus, these data strongly suggest mTOR is a target for adhesion-based signal transduction, where the ECM influences cell survival through mTORC1. Moreover, exploitation of autophagy induction post cell-ECM detachment in HOSCC may promote the survival of metastases during dissemination.

Non-melanoma skin (NMSC) cancer is the most common human malignancy. Cutaneous squamous cell carcinoma (cSCC) and its precursor, actinic keratosis (AK) affect tens of thousands of people each year in the UK. Merkel cell carcinoma is a rare, yet aggressive type of NMSC recently linked with Merkel Cell Polyomavirus (MCPyV). In spite of the clinical burden of NMSC, key molecular regulatory patterns remain largely unknown. The aims of this thesis were to investigate genome-wide genetic, epigenetic and transcriptional changes in AK and cSCC, and assess the prevalence of MCPyV and its effect on methylation in NMSC. Copy-number analysis revealed that AK harbours significantly more genomic aberrations compared to skin, the majority of which occurs on chromosomes 8 and 9. Transcriptional profiling has found 292 and 308 genes as differentially expressed in AK compared to non-sunexposed and sun-exposed skin, respectively, and gene-set enrichment analysis (GSEA) revealed dysregulation of PPAR pathway in this lesion. Expression profiling of cSCC and AK has revealed 346 differentially expressed genes, and GSEA detected dysregulation in several canonical pathways including TGF-β and MAPK pathway. Aberrant methylation in cSCC cell lines occurs in the promoters of many developmental genes. A total of 1085 hyper- and 833 hypomethylated genes were detected in cSCCs, and GSEA revealed dysregulation of critical signalling pathways (WNT, MAPK signalling pathways). Methylation analysis of AK revealed a total of 4194 differentially methylated genes, and implicated FOXF2, PITX2, RUNX1 and SMAD3 transcription factors in this lesions. MiRNA profiling of cSCC and normal skin revealed significant dysregulation of 38 miRNAs including several of viral origin. MCPyV was shown to be common in NMSC, yet MCPyV nor human papillomavirus does not affect cSCC methylation. Taken together, this work provides novel insight into molecular regulation of cSCC oncogenesis, and identifies potential epigenetic targets for functional evaluation in this malignancy.

The Aim of the project was to investigate cell senescence in basal cell carcinoma (BCC). Although the concept of oncogene-induced senescence (OIS) was originally confined to cultured cells, it is now well established that this mechanism has an important role in tumour biology. OIS represents a physiological response that restricts the progression of benign tumours into their malignant counterparts e.g. nevi to melanoma or adenoma to adenocarcinoma. Full malignancy is associated with the loss of important tumour suppressor genes including RETINOBLASTOMA and/or TP53. BCC of the skin is the most common skin tumour and is associated with mutational inactivation of the PTCH1 tumour suppressor gene (and less frequently oncogenic activation of SMOOTHENED). Although BCC does not appear to stem from precursor lesions - though mouse models of BCC display areas of basaloid hyperproliferation - and it is relatively stable at the genomic level, we sought to determine if these unique tumours display any characteristics of OIS. Human BCCs were positive for Senescence-associated β-galactosidase (SA-β-gal) activity (pH 6.0) and expressed known markers of senescence including DCR2, DEC1 (SHARP2) as well as the cell cycle inhibitors p15INK4b and p16INK4a. Interestingly, SA-β-gal activity was observed in stromal cells surrounding the tumour islands and this may account for why BCCs are difficult to culture in vitro as senescent cells are known to express increased levels of growth factors, cytokines and ECM proteins. To determine if OIS is associated with Hedgehog signalling in BCC, I employed a novel in vitro model of BCC created through PTCH1 suppression in human immortalised NEB1 keratinocytes. NEB1-shPTCH1 cells are viable and proliferative (albeit more slowly than control NEB1-shCON cells) and do not display SA-β-gal activity but they express higher levels of several senescent markers including DCR2 and p21WAF1. I also investigated senescence in a Mouse model of BCC in which one allele of Ptch1 is mutated and which on x-ray irradiation results in BCC formation. The expression of known markers of senescence including DCR2, DEC1 (SHARP2) as well as the cell cycle inhibitors p15INK4b, p16INK4a and p53 were all with the exception of p21WAF1 detected in these tumours. Together these data suggest that senescence is a characteristic of BCC and may explain why these tumours rarely metastasise.

Basal cell carcinoma (BCC) of the skin is predominantly associated with mutational inactivation of the PTCH1 tumour suppressor gene resulting in constitutive activation of the Hedgehog (HH) developmental pathway. Tumour formation is linked to induction of the GLI (GLI1 and GLI2) transcription factors via a pathway that is thought to require the transmembrane protein SMOOTHENED (SMO) and accordingly, SMO is attracting much interest as a drug target in cancer therapy. However, although there has been a high degree of success in treating some BCCs with anti-SMO compounds, many tumours are only partially or unresponsive which indicates that SMO-independent mechanisms may contribute to tumour formation and/or viability. To further understand how loss of (or reduced) PTCH1 function contributes to BCC, RNAi (retroviral shRNA) was employed to suppress PTCH1 in NEB1 and N/Tert immortalised human keratinocyte cells. Compared to control (shCON) cells, PTCH1 knockdown (shPTCH1) cells displayed more compact colony formation as well as increased GLI1 (but not GLI2) expression however, whereas the increase of GLI1 was suppressed upon transfection with SMO siRNA in shPTCH1 cells, it was insensitive to the presence of the SMO antagonists Cyclopamine-KAAD and SANT1 in shCON cells. The reason for this is unclear but SMO levels were increased and more nuclear in shPTCH1 cells indicating that SMO may have nuclear signalling capability that is unresponsive to certain SMO antagonists. Indeed, cDNA microarray profiling revealed that 80% of the genes that were differentially expressed in NEB1-shPTCH1 cells (>2-fold, p<0.01) remained differentially expressed in the presence of Cyclopamine-KAAD; this includes the chemokines CXCL10 and CXCL11 which were recently shown to be over-expressed in BCC thus helping validate the efficacy of NEB1-shPTCH1 cells as an in vitro tumour model. In addition, functional gene grouping has predicted novel biological processes downstream of PTCH1 that may be important in BCC biology including NF-κB signalling. In summary, the data presented in this thesis suggests that the mechanism(s) through which the loss of PTCH1 leads to BCC formation may be more complex than has been inferred from previous studies.

Resumo: A incidência do câncer de pele vem aumentando anualmente em homens e em mulheres, tornando-se grave problema se Saúde Pública mundial. Diversos são os fatores que influenciam a formação dos tumores, sendo a radiação ultravioleta um dos principais responsáveis, que vem elevando seu índice graças à redução da camada de ozônio. Ainda assim o câncer de pele poderia ser evitado se as medidas de prevenção fossem tomadas a tempo, como a fotoproteção, cuidados com os horários de exposição ao sol e a detecção precoce dos tumores. Os avanços científicos e tecnológicos têm proporcionado novas abordagens para o desenvolvimento de agentes antineoplásicos e o direcionamento de fármacos a locais de ação específicos, gerando maior eficácia dos tratamentos. Nesta linha, a principal proposta deste trabalho envolve a possibilidade de conjugar as propriedades do fotoprotetor salicilato de octila com a do antineoplásico 5-fluoruracila, por meio do processo de latenciação. Para tanto foi obtido o pró-fármaco succinil octil salicilato de 5- fluoruracila (SOSFlu), a partir de duas etapas sintéticas, cujos compostos foram caracterizados por meio da determinação da faixa de fusão, cromatografia em camada delgada, análises espectroscópicas de infravermelho, de ressonância magnética nuclear de 13C e 1H, determinação dos coeficientes de extinção molar e de partição. Tendo o SOSFlu apresentado melhores valores de coeficientes de extinção molar e de partição em relação ao fotoprotetor salicilato de octila e ao antineoplásico 5-fluoruracila, espera-se que apresente maior efetividade para proteger a pele contra a radiação ultravioleta e maior permeabilidade cutânea. / Abstract: The incidence of the skin cancer comes increasing annually in men and in women, becoming a serious problem if world Public Health. Several they are the factors that influence the formation of the tumors, being the ultraviolet radiation responsible one of the main ones that comes elevating its index thanks to the reduction of the layer of ozone. Nevertheless the skin cancer could be avoided the prevention measures they were taken on time, as the sunscreem, cares with the schedules of exhibition in the sun and the precocious detection of the tumors. The scientific and technological progresses have been providing new bording for the development of agents antineoplasic and the drug delivery to specific action places, generating larger effectiveness of the treatments. In this line, the main proposal of this work involves the possibility to conjugate the property of the sunscreem octil salicilate with an antineoplasic 5-fluoruracil, by means of the latentiation process. For so much it was obtained pro-drug succinoil octil salicilate of 5-fluoruracil derived of the octil salicilate and 5- fluoruracil, starting from two synthetic stages, whose compositions were characterized by means of analyses IR spectra, melting point determination, thin layer chromatography and 13C and 1H MNR, extinction molar coefficient and partition coefficient. Tends SOSFlu presented better values of coefficients of extinction molar and of partition in relation to the sunscreen octil salicilate and to the antineoplasic 5-fluoruracil, it is waited that presents larger effectiveness to protect the skin against the ultraviolet radiation and larger cutaneous permeability. / Orientador: Luis Vitor Silva Sacramento / Coorientador: Márcia da Silva / Banca: Veni Maria Andres Felli / Banca: Maria Luiza Cruz / Mestre

The goal of this thesis is to study the potential of optical spectroscopy as a clinical diagnostic tool for melanoma and nonmelanoma skin cancer. Skin cancer is the most common cancer in the United States. Like most cancers, early diagnosis and treatment improves patient prognosis for both melanoma and nonmelanoma skin cancer. However, current “gold standard” for diagnosis is invasive, costly and time-consuming. A diagnostic procedure consists of a clinical examination of the suspicious lesion, followed by biopsy and histopathology, with an additional turnaround time of approximately one week. There is a need for an accurate, objective, noninvasive, and faster method to aid physician in diagnosing cancerous lesions, increasing diagnosis accuracy while preventing unnecessary biopsies. We propose Spectral Diagnosis, a system capable of noninvasive in vivo spectroscopic examination of human skin. The research objectives are: (1) Probe pressure effects on in vivo spectroscopy measurements of human skin, (2) Clinical trial of Spectral Diagnosis, (3) Design, construction, and characterization of a confocal Raman microspectroscope. Spectral Diagnosis utilizes an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. We identified short term and light probe pressure effects to be minimal on diagnostic parameters, and should not negatively influence diagnostic performance. We conducted a clinical trial at the University of Texas MD Anderson Cancer Center, and our results show that principal components from three spectroscopy modalities (diffuse reflectance spectroscopy, laser induced fluorescence spectroscopy, and Raman spectroscopy) provide excellent melanoma and nonmelanoma skin cancer diagnosis. We also constructed and characterized a Raman microspectroscope, with the goal of developing a physiological-based fitting model to better understand the analysis of in vivo Raman spectroscopy data from human skin tissue. / text

The maintenance and regulation of cellular NAD(P)(H) content and its influence on cell function involves in many metabolic pathways which are poorly understood. Niacin deficiency in humans, which leads to low NAD status, causes sun sensitivity in skin, indicative of DNA repair deficiencies. Animal models of niacin deficiency demonstrate genomic instability and increased cancer development in sensitive tissues including skin. Therefore, we have developed a cell culture model that allows assessment of pathways regulated by NAD(P) content as a way to identify NAD-dependent signaling events that may be critical in early skin carcinogenesis. Using our model, we showed that niacin restriction, and consequent NAD depletion, reversibly alters NAD(P)(H) pools, increases apoptosis, induces G2/M cell cycle arrest, and decreases DNA stability. These alterations are affected by increased expression and activity of NOX leading to an accumulation of ROS, which may provide a survival mechanism as has been shown in cancer cells. Our data also support the hypothesis that glutamine is a likely alternative energy source during niacin deficiency. Here, we also identified the expression of all seven NAD-dependent deacetylase (SIRT) family members in skin cells. We showed that in response to photodamage, the expression of several SIRTs is altered in keratinocytes. Furthermore, we showed that SIRTs responses to photodamage differ between normal and immortalized keratinocytes, which may be indicative of alterations potentially important in skin carcinogenesis. In addition, we have shown that NAD-depleted HaCaT keratinocytes are more sensitive to photodamage. We observed that both poly(ADP-ribose) polymerases (PARPs) and SIRTs are inhibited by the unavailability of their substrate, NAD+, leading to unrepaired DNA damage upon photodamage and subsequent increase in cell death. Our data demonstrate that both SIRTs and PARPs are critical following DNA damage and identify which SIRTs are essential. Finally, we identified for the first time the expression of the nicotinic acid receptor in human skin keratinocytes, mainly in the differentiating keratinocytes of the stratum corneum in the epidermis. This study identifies new roles for niacin as a potential skin cancer prevention agent and demonstrates that niacin status is a critical resistance factor for UV damaged skin cells.

Over one million Americans are afflicted with skin cancer each year. Even though skin cancer has a 95% cure rate, approximately 10,000 people die in the United States each year of this disease. The current ABCDE(F) detection method is not sensitive enough to detect skin cancer in its early stages and requires a biopsy for any suspicious lesions. A lot of unnecessary biopsies, which are painful and costly to the patient, are taken. Therefore, a noninvasive technique is needed that can accurately detect the presence of skin cancer. In this thesis, an optical approach will be presented that has potential to be a noninvasive skin cancer detection technique. Several morphological and biochemical changes occur as tissue becomes cancerous, and therefore the optical properties of the tissue can be used to detect skin cancer. A Mueller matrix imaging system has been developed by our group that measures the 16 or 36-element Mueller matrix, which completely describes the optical properties of the tissue sample. The system is automated and can collect the Mueller matrix in less than one minute. This system will be used to image Sinclair swine, and data analysis techniques will be employed to determine if the system can distinguish between cancerous and noncancerous tissue. System software improvements will also be made, and a new calibration technique will be presented.

Nonmelanoma skin cancer remains the most common diagnosed cancer in the United States. Sunscreen is the most common protective method for UV exposure. For the present study, Sun Exposure and Protection Habits Measurement Study (SEPH) survey data were analyzed to determine patterns of sunscreen application in lifeguards, parents, and children, how the amount of total body coverage differs across the three participant group and the variation in coverage with respect to selected demographic characteristics for each of the 3 participant groups. Results of the study showed that children had the highest percentage of total body coverage (M=86.7, 95% CI: 82.7, 90.6) among the three groups and parents had the least (M = 68.3, 95% CI: 65.2, 71.3). No differences in the total body area covered were noted for intervention or latitude. Further research efforts are needed to determine effective intervention strategies for parents and the need for overall body coverage.

No description available.