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Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine cutaneous cancer whose molecular biology is poorly characterized. Our broad research objective is to identify microRNAs (miRNA) that are biologically and/or clinically important in MCC. While attempting to establish an MCC-specific miRNA signature, we observed that microRNA-375 was the most highly expressed miRNA in primary MCC tumours relative to normal skin – an observation that I propose reflects miR-375’s specific association with neuroendocrine (NE) and secretory subpopulations within normal tissues. Here, I report that miR-375 is strikingly elevated in a range of NE tumour types compared with tissue-matched cancers of non-neuroendocrine origin. Furthermore, I show that miR-375 is expressed abundantly in a subset of MCC cell lines that possess the biochemical and immunohistochemical characteristics of NE cells, but is silenced in cell lines that fail to retain these markers. I demonstrate that the enforced expression of miR-375 induces a NE gene expression signature – a phenomenon that is mechanistically driven by the post-transcriptional repression of multiple Notch pathway components by miR-375. This work identifies the Notch pathway as a novel mechanistic link between the association of miR-375 and a NE cell fate, provides new insights into the cellular ancestry of MCC, and suggests that miR-375 could facilitate clinicopathological diagnosis of MCC and other NE tumours as a novel biomarker. miR-375 is silenced in “variant” MCC cell lines, and inversely correlates with cell doubling time and overall aggressiveness. Therefore, despite its high expression in most MCC tumours, I propose that miR-375 is an endogenous tumour suppressor. I show that the enforced expression of miR-375 inhibits cell viability, impairs cell migration and invasion, can oppose survival under stress, and represses the AKT pro-survival signaling pathway. Only siRNA-mediated inhibition of Notch2 and Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) phenocopied the effects of miR-375 overexpression. Because variant (miR-375low) cell lines originate from more aggressive tumours in both MCC and small cell lung carcinoma, I postulate that miR-375 silencing occurs in a subset of MCC patients and might predispose them to a highly virulent clinical course through the disinhibition of Notch signaling. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-08-15 09:59:09.832

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.

Previous data have unveiled a novel autoregulatory feedback loop between iASPP and p63 in the stratified epithelia; this involves two microRNAs, miR-574-3p and miR-720, and is critical for epidermal homeostasis. The iASPP oncoprotein, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is a key inhibitor of p53 and NF-κB and is highly expressed in many cancers. Non-melanoma skin cancer, comprising of cutaneous squamous carcinoma (cSCC) and basal cell carcinoma, is currently the most common malignancy in the UK. In view of this newly-identified iASPP-p63 axis, I hypothesised a potential role for dysregulation of this feedback loop in the pathogenesis of cSCC and aimed to assess the role of iASPP in human cSCC. Protein and mRNA expression patterns were assessed in a panel of 10 cSCC cell lines generated by our group. In addition, immunostaining of iASPP and p63 was performed in 107 cSCC clinical samples of variable differentiation status. The data reveal an overall increase in expression of iASPP and ΔNp63 in cSCC but also suggest a significant alteration of the cellular localisation of iASPP dependent on the differentiation status of the tumour. To further assess the effects mediated by the iASPP/p63 axis, iASPP and p63 have been silenced by RNAi technology in a subset of cSCC cell lines. Whilst data shows the direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop at the microRNA level may be occurring via a novel p63 regulator, miR-211-5p. Functionality of iASPP in cSCC (proliferation, apoptosis, cell motility/migration and invasiveness) provides evidence for a role of iASPP in preventing epithelial-mesenchymal transition in cSCC via a p63/miR-205-5. These findings provide potential future directions for development of clinical biomarkers and novel therapeutic targets for cSCC and may ultimately provide the tools for tackling the increasing morbidity and mortality associated with this malignancy.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. It is a result of numerous aetiological factors such as genetic predisposition, smoking, excessive alcohol consumption and viruses such as the human papilloma virus. Due to late diagnosis it has a high mortality and morbidity rates which has remained unchanged over the last 5 decades. Currently no screening is available for high risk patients for better monitoring. Diagnosing OSCC relies on histopathology of biopsy tissue, reviewed for dysplasia and advancing lesions. Although the technique has been used for decades for successful diagnosis it fails to identify the molecular signature of OSCC which appears much before the visual signs. It also falls short in predicting the malignant transformation of pre-malignant oral lesions. Identifying the molecular and genetic changes leading to OSCC lesion will aid in more specific (quantitative) and early diagnosis of the disease reducing the financial burden of treating late-stage OSCC patients on the healthcare system. This study focuses on developing new adjuncts which can be used alongside histopathology for early diagnosis. There is a need to monitor high risk patients through non-invasive methods causing less patient discomfort. We therefore explored the potentials of exosomes which are extracellular vesicles secreted by normal and tumour cells. They can be isolated from body fluids such as blood and saliva. In cancer biology exosomes offer both diagnostic and therapeutic advantage. Their involvement in cell-cell communication indicates their influence in tumour development, progression, metastasis and therapeutic efficacy. Exosomes released by cancerous cells carry numerous biomarkers, which are passed on to healthy cells via microenvironment, causing stromal and angiogenic activation along with immune escape. In this study exosomes were successfully isolated from body fluids (blood, saliva and plasma) and cell line supernatant through ultracentrifugation and characterised by visual and particle size quantification techniques including Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), Zetasizer and Nanosight Tracking Analysis (NTA). Exosomal specific membrane proteins were identified through Western blotting. 5 We report the presence of a potential protein biomarker located exclusively on the outer membrane of cancer exosomes. Since body fluids consist of a heterogeneous population of exosomes derived from multiple cell types, such surface biomarker can potentially be used to isolate OSCC exosomes. Characterisation of exosomal mRNA cargo was done using Agilent Bioanalyzer (for RNA quantity and quality assurance) and reverse transcription-quantitative PCR (RT-qPCR; for gene specific quantitation). Functional significance of exosomes was studied by transfecting normal oral keratinocyte cells with self and cancer-derived exosomes. Through gene-expression microarray and subsequent RT-qPCR verification, we report a panel of differentially expressed genes involved in essential cellular functions being modulated by exosome transfection. A previously developed molecular diagnostic system by our research group called quantitative malignancy index diagnostic system (qMIDS) based on FOXM1 oncogene and its downstream targets was validated on archival formalin fixed paraffin embedded OSCC patient biopsy samples. We report that qMIDS index successfully correlates with the disease stages including dysplasia, tumour and lymph node metastasis. Furthermore, through meta-analysis of 8 OSCC microarray studies we identified a panel of six genes including PLAU, FN1, CDCA5, CRNN, CLEC3B and DUOX1 (q6) which are able to identify two clinically distinct sub-groups of OSCC patient population. Through RT-qPCR the expression of q6 biomarkers was established in 100 OSCC biopsy samples. This information can be of immense importance in developing personalized treatment strategies based on the molecular makeup of the presenting tumour.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Renal cell carcinoma (RCC) accounts for 85% of kidney cancer incidence in the US. Since 1950 there has been a 126% increase in kidney cancer incidence in the US. Thirty percent of new patients present with a localized easily treatable carcinoma while 30% of patients present with a high-grade metastatic carcinoma. Five-year survival rates for metastatic RCC is 6-12 months (Lipworth et al, 2006). Current disease treatment options for metastasis include chemotherapy and radiation (8% response rate), immunotherapy (15-30% response rate) and newly developed angiogenesis inhibitors which are in phase III trials (Staehler et el, 2005). In RCC cells, it has been shown that PPARγ agonists inhibit cell proliferation, induce apoptosis, and induce anti-angiogenic effects in vitro. Unlike most tumor types, PPARγ is downregulated in tissue samples from 47 RCC patients. However, in cell culture studies PPARγ expression does not correlate with growth inhibitory or apoptotic effects of PPARγ agonists in renal cell lines indicating that PPARγ independent responses may play a large role in actions associated with the PPARγ agonists (Yuan et al, 2006). 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl, p-t-butyl and p-phenyl substituents activate peroxisome proliferator-activated receptor (PPAR) and inhibit growth of ACHN and 786-0 renal cell carcinoma cell lines. PPAR is overexpressed in ACHN cells and barely detectable in 786-0 cells, and treatment with the t-butyl analog (DIM-C-pPhtBu) induces cell cyle inhibition. DIM-C-pPhtBu also induced several common PPAR-independent proapoptotic responses in ACHN and 786-0 cells, including increased expression of nonsteroidal antiimflammatory drug-activated gene-1 (NAG-1) and endoplasmic reticulum stress which activates death receptor 5 and the extrinsic pathway of apoptosis. In addition, DIM-C-pPhtbu (40 mg/kg/d) also inhibited tumor growth in an orthotopic mouse model for renal carcinogenesis, and this was accompanied by induction of apoptosis in renal tumors treated with DIM-C-pPhtBu but not in tumors treated with the corn oil vehicle (control). Thus, DIM-C-pPhtBu and related compounds represent a novel class of mechanism-based drugs that have potential for treatment of renal adenocarcinoma for which there are currently limited options for successful chemotherapy.

WWOX is a tumor suppressor and the down-regulation of WWOX has been demonstrated in prostate, lung, breast, gastric cancers. However, the role of WWOX in renal cell carcinoma (RCC) remains unknown. It has been demonstrated that WWOX addressed in mitochondria, golgi apparatus, rough ER, lysosome, plasma membrane and nuclear. The Subcellular localization of WWOX has been controversial. There are two parts in this study: (I) The expression of WWOX in RCC and the probability of WWOX to be a diagnostic and a prognostic marker. (II) The regulation of WWOX by phosphorylation. For the study of WWOX expression in RCC, we prepared polyclonal WWOX antibody and characterized the specificity of the antibody. We applied this specific antibody to 33 NT paring RCC tissue specimen for immunoblotting study and 138 cases of paraffin-embedded specimens for IHC, respectively. Our results demonstrated that hWOX1 was specifically down-regulated in clear cell type RCC (p=0.018). The percentage of down-regulation in patient specimen is 60.7 % and 90.7 % in immunoblotting and IHC study, respectively. And in clear cell and clear-granular combined type RCC, down-regulation of WWOX was significantly correlated with the survival rate of patients (p=0.0482). Therefore, WWOX could be used as a diagnostic and a prognostic marker in clear cell type RCC. Besides, we performed bioinformatics to predict the phosphorylation site of WWOX and investigated the effect of phosphorylation on WWOX subcellular localization. Our results demonstrated that hWOX1 was phosphorylated by PKC at Thr49 and Thr102 and the phosphorylation regulated the subcellular localization of WWOX.

Renal cell carcinoma (RCC) accounts for 85% of kidney cancer incidence in the US. Since 1950 there has been a 126% increase in kidney cancer incidence in the US. Thirty percent of new patients present with a localized easily treatable carcinoma while 30% of patients present with a high-grade metastatic carcinoma. Five-year survival rates for metastatic RCC is 6-12 months (Lipworth et al, 2006). Current disease treatment options for metastasis include chemotherapy and radiation (8% response rate), immunotherapy (15-30% response rate) and newly developed angiogenesis inhibitors which are in phase III trials (Staehler et el, 2005). In RCC cells, it has been shown that PPARγ agonists inhibit cell proliferation, induce apoptosis, and induce anti-angiogenic effects in vitro. Unlike most tumor types, PPARγ is downregulated in tissue samples from 47 RCC patients. However, in cell culture studies PPARγ expression does not correlate with growth inhibitory or apoptotic effects of PPARγ agonists in renal cell lines indicating that PPARγ independent responses may play a large role in actions associated with the PPARγ agonists (Yuan et al, 2006). 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl, p-t-butyl and p-phenyl substituents activate peroxisome proliferator-activated receptor (PPAR) and inhibit growth of ACHN and 786-0 renal cell carcinoma cell lines. PPAR is overexpressed in ACHN cells and barely detectable in 786-0 cells, and treatment with the t-butyl analog (DIM-C-pPhtBu) induces cell cyle inhibition. DIM-C-pPhtBu also induced several common PPAR-independent proapoptotic responses in ACHN and 786-0 cells, including increased expression of nonsteroidal antiimflammatory drug-activated gene-1 (NAG-1) and endoplasmic reticulum stress which activates death receptor 5 and the extrinsic pathway of apoptosis. In addition, DIM-C-pPhtbu (40 mg/kg/d) also inhibited tumor growth in an orthotopic mouse model for renal carcinogenesis, and this was accompanied by induction of apoptosis in renal tumors treated with DIM-C-pPhtBu but not in tumors treated with the corn oil vehicle (control). Thus, DIM-C-pPhtBu and related compounds represent a novel class of mechanism-based drugs that have potential for treatment of renal adenocarcinoma for which there are currently limited options for successful chemotherapy.

Head and neck cancer (HNC) makes up 6 ¢H of the cancer patients in the world every year. This disease usually occurs in males and the incidence is increasing year by year. According to statistical analysis, HNC has less than 50 ¢H five-year survival rate. Therefore, the research of HNC seems imminent so that may lead to the development of new approaches of diagnosis and therapy. Recent research had shown that expression of podoplanin caused cellular proliferation, and may be associated with tumor invasion, metastasis and malignant prognosis. Podoplanin, a mucin-type transmembrane sialoglycoprotein, is highly expressed in lymphatic endothelial cells but not expressed in vascular endothelial cells. The purpose of this study was to determine the clinical and pathological significance of podoplanin in oral squamous cell carcinoma (OSCC). Therefore, we collected clinical specimen and associated patient history of OSCC. Further, we used the human cell lines of HNSCC (Fadu, Hep2) to investigate the molecular regulation of podoplanin. Podoplanin expression was analyzed by RT-PCR and Western blot assay firstly. As shown, podoplanin was found to be overexpressed in tumors compared with normal adjacent tissues. Further, immunohistochemical analysis revealed the location of podoplanin expression in OSCC tissues. The results showed that podoplanin had higher expression in T4 stage tumor section than in normal adjacent tissues of OSCC samples, or in T1 stage. Here, podoplanin was highly expressed in the OSCC tumor cell and lymphatics of stage T4 OSCC tissue. Furthermore, we found that overexpression of podoplanin in OSCC patients was associated with decreased five-years survival rate. In the univariate analysis, several factors were statistically significantly associated with disease-specific survival rate, including Tumor stage, Nodal stage, and podoplanin expression. In the subsequent multivariate analysis, only Tumor stage and Nodal stage showed a trend toward worse disease-specific survival. To further investigate the regulatory mechanism of podoplanin and its position of expression within the cell, immunofluorescence and transfection were utilized to assay. The results showed that podoplanin was expressed in the nuclear membrane of the Fadu and Hep2 cell lines, and the PI3K/AKT signaling pathway was involved. We suggest that the role of podoplanin in OSCC should be further investigated for potential future treatment.