Esophageal squamous cell carcinoma (ESCC), the major histological subtype of esophageal cancer, is one of the most common malignancies with poor prognosis in the world. Despite continued development of diagnosis and treatment, ESCC remains the sixth leading cause of cancer death worldwide. Current treatment regimens in ESCC are often characterized by ineffectiveness and poor selectivity. Therapeutic methods directed at cancer-associated genes or cancer stem cells (CSCs) may be effective approaches to cure this deadly cancer. Therefore, this study aims to identify specific ESCC-related genes and cancer stemness genes which help us to develop new targeted agents to achieving objective, long-lasting therapeutic responses in ESCC. To obtain an accurate overview of genetic changes occurring in ESCC patients, our group performed microarray-based mRNA expression profiling and high-throughout transcriptome sequencing (RNA-Seq) to compare differentially expressed genes between ESCC tumors and their corresponding non-tumorous tissues. Prostate stem cell antigen (PSCA) was considered to be a candidate of primary interest due to significantly reduced expression in both microarray and RNA-Seq data. In this study, we examined the role of PSCA on the pathogenesis of esophageal cancer. Our results showed that PSCA was frequently down-regulated in ESCC. Its expression was negatively regulated by transcription factor SOX5. Also, we provided evidence that down-regulation of PSCA was associated with poor clinical outcomes of patients with ESCC. Both in vitro and in vivo assays revealed that PSCA could arrest cell cycle progression and promote differentiation. To further elucidate the mechanism involved in biological function of PSCA, we performed co-immunoprecipitation and mass spectroscopy to identify proteins that associate with PSCA. This study found that RB1CC1, a key signaling node to regulate cellular proliferation and differentiation, interacted specifically with PSCA both in vitro and in vivo. Binding of PSCA and RB1CC1 in cytoplasm resulted in stabilization and translocation of RB1CC1 into nucleus and then further regulates the crucial cell cycle and differentiation genes. Furthermore, in order to identify the cancer stemness genes specifically expressed in CSCs of ESCC, we utilized gene expression analysis to profile 34 stemness-associated genes in ESCC specimens. Developmental pluripotency associated 4 (DPPA4), a well known pluripotent marker of stem cell, was considered as the best candidate. Our following histopathological study demonstrated that DPPA4 rigorously marked the rare CSCs, in contrast to core stemness factors (OCT4 and SOX2) and previous reported CSC markers (CD90 and CD44), which expressed in a large population of cancer cells. Moreover, the expression of DPPA4 was also found to have prognostic value in ESCC, as the appearance of DPPA4+ cells was significantly associated with poor differentiation, advanced stage and higher incidences of lymph node metastasis. Finally, our functional studies showed that ESCC cells expressing exogenous DPPA4 conferred an enhanced ability to initiate tumor, self-renew, resist chemotherapy and metastasize through lymphatic system. In summary, this study provide evidence indicating that novel tumor suppressor gene PSCA and cancer stemness gene DPPA4 may contribute to the development and progression of ESCC. Additionally, they may serve as potential targets for development of effective therapeutic strategies. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Hong Kong, situated in the Southern part of China, is an endemic area where the incidence of nasopharyngeal carcinoma (NPC) is among the highest in the world. The cancer, which is located at the deepest part of the human skull, represents one of the most difficult tumours to treat in the head and neck region. The management of tumour recurrence after radiotherapy is even more challenging. Epstein-Barr virus (EBV) is a human herpes virus and it is the first virus that is discovered to be associated with human malignancy. Over the past few decades, the role of EBV serology in the management of NPC has been extensively investigated. More recently, a series of EBV encoded microRNAs, which are short, non-coding RNAs, are found to be commonly expressed in NPC. In our studies, we have investigated the role of EBV DNA and EBV miRNA BART7 in the management of recurrent NPC. Plasma EBV DNA is accepted as a tumour marker for NPC. We found that in patients with recurrent NPC, the pre-operative level of plasma EBV DNA reflects the tumour load and correlates with the T-stage of the tumour. It also predicts the chance of resection margins that are histologically positive for malignancy. When measured serially after surgery, it is useful to detect tumour recurrence. However, we found that up to 15.5% of our patients, who had tumour recurrence in the nasopharynx, were seronegative for EBV DNA. In such circumstances, plasma EBV miRNA BART7, which is expressed independently of EBV DNA, may be used for such purpose. Moreover, using the in-vitro model, we demonstrated that the expression of EBV miRNA BART7 in the HONE1 cell line increases the rate of proliferation, migration and invasion of tumour cells. By using the same in-vitro model, we found that the EBV miRNA BART7 increases the sensitivity of the HONE1 cells to ionizing radiation in a dose-dependent manner. This is confirmed with the in-vivo experiments using the zebra fish model. In our previous study on the multivariate analysis of prognostic factors in salvage nasopharyngectomy for recurrent NPC, we found that the resection margin status is one of the most important independent factors influencing the local tumour control and overall survival. In order to improve the chance of obtaining clear margins after surgery, we have to depend on the intra-operative frozen section analysis and the post-operative histological examination of the resection margin specimen. In our current study, we showed that contrast MRI is accurate in assessing the local extent of recurrent nasopharyngeal carcinoma. During nasopharyngectomy, a radial resection margin of 15mm should be taken with the underlying medial pterygoid muscle. For tumours with parapharyngeal extension, the pharyngobasilar fascia should be resected enbloc with the specimen. The chance of local recurrence after salvage nasopharyngectomy in patients with histologically uninvolved margins was 20.0%. Tissue EBV miRNA BART7 is useful to identify a subgroup of patients with histologically close margins who are at increased risk of subsequent local tumour recurrence. Post-operative adjuvant treatment is warranted for these patients. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Non-small cell lung cancer (NSCLC) causes most of the cancer deaths worldwide. Tyrosine kinase inhibitors (TKIs), like erlotinib and crizotinib, are commonly used as specific treatments targeting epidermal growth factor receptor (EGFR)-mutated and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Autophagy is a highly conserved cellular process in response to stress. Tumor microenvironment (TME) is composed of both tumor cells and stromal cells. This study aimed to investigate whether autophagy could confer intrinsic and acquired resistance to TKIs in NSCLC, and its role in the presence of TME or in animal models. In the first part of this study, the effect of EGFR TKI or ALK TKI on sensitive NSCLC cells to generate autophagy was investigated, and manipulation of autophagy in these cell lines was performed. Autophagy inhibition was shown to enhance apoptotic effect of TKIs in sensitive NSCLC cells. This part provided strong evidence that TKIs and autophagy inhibitor chloroquine (CQ) work synergistically in sensitive NSCLC cells. Autophagy induction by erlotinib treatment was observed in a HCC827 (lung adenocarcinoma, EGFR exon 19 del) xenograft model, which was in line with the in vitro observation. Correspondingly, the combination of erlotinib (12.5 mg/kg) with CQ (50 mg/kg) in the HCC827 xenograft model achieved greater tumor growth suppression, compared with single drug treatments. In the second part of this study, a model of TME was established to allow study of autophagy under such circumstances. An activated TME with cytokine production, autophagy induction and epithelial-to-mesenchymal transition (EMT) was generated by co-culturing NSCLC cells and human fibroblasts. Sensitivity to TKI under TME was not affected, and combination of chloroquine with TKI under TME remained synergistic compared with single treatments. In the third part of this study, erlotinib-resistant (ER) HCC827 cells were acquired by stepwise exposure to increasing concentrations of erlotinib in cell culture. Common acquired resistance mechanisms to EGFR TKI (EGFR T790M or c-MET amplification) were excluded in this ER HCC827 model, except EMT. Autophagy status in ER HCC827 cells was studied and autophagy manipulation was performed. It was found that CQ and erlotinib worked synergistically to induce cell death even in ER HCC827 cells. In an ER HCC827 xenograft model, significant degree of autophagy and EMT was evident. Interestingly, combining erlotinib (25 mg/kg) with CQ (50 mg/kg) showed better inhibitory effect on tumor growth compared with single treatments. In summary, TKIs induced both apoptosis and autophagy in EGFR-mutated and ALK-rearranged NSCLC cells. Autophagy inhibition by CQ enhanced TKI-induced cell death in sensitive cells. The presence of TME did not confer TKI resistance. Autophagy was highly activated in EGFR-mutated NSCLC cells with acquired resistance to erlotinib. Combination of CQ with erlotinib remained synergistic in the presence of TME and acquired resistance, both in vitro and in vivo. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

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Every year more than 10 million people develop cancers globally. Ovarian cancer, specifically, results in more than 22,000 new cases and 16,000 deaths from this disease yearly, more than any other cancer of the female reproductive system. In addition, because of non-specific symptoms and poor screening techniques, most ovarian cancer cases are discovered after the disease is in an advanced state. Consequently, aggressive and effective treatment options that incur minimal toxic effects to healthy tissue are in great need. In the present research, stealth biodegradable nanoparticles were developed as vehicles for the controlled and targeted delivery of chemotherapeutic agents for the treatment of ovarian cancer. The design of this delivery system consisted of nanoparticles of biodegradable polymers of the poly(lactic-co-glycolic acid) family loaded with the chemotherapeutic agent doxorubicin or the imaging agents rhodamine 6G, indocyanine green or gadopentetic acid. Nanoparticles were modified by incorporation of functional poly(ethylene glycol) on their surface to improve the stability of the colloidal suspension, increase their circulation lifetime in vivo, and provide a site for conjugation of targeting agents specific to ovarian tissue. Various methods were evaluated for this surface modification, including the use of polymer blends, the chemical conjugation of the polymers, and the polymerization of lactide and glycolide monomers initiated by heterofunctional poly(ethylene glycol). Nanoparticles incorporating poly(ethylene glycol) presented improved characteristics compared to unmodified particles including smaller size, higher stability and slower release of the chemotherapeutic agent doxorubicin The actual drug or agent content was decreased in the case of doxorubicin and rhodamine, but increased for indocyanine green as a result of improved agent-polymer interactions. Poly(ethylene glycol)-containing nanoparticles were conjugated to monoclonal antibody mAb106-105, which is specific to the extracellular domain of human folliclestimulating hormone (FSH) receptors. These receptors are only expressed in ovarian cells in women, thus providing a system that is highly specific to ovarian tissue. The interaction and therapeutic potential of nanoparticles with or without targeting antibodies were tested on OVCAR-3, Caov-3, and MDA-MB-231 cancer cells.

Colorectal cancer is the third most common cancer and cause of death due to cancer in the United States. Death due to colorectal cancer is generally caused by hepatic metastasis rather than the primary tumor itself. The five-year survival rate is only 10% for patients whose colorectal cancer metastasized, which indicates the need for more effective therapies to treat colon cancer. The diet contains (1) preformed vitamin A as retinyl esters in animal-derived food sources and (2) provitamin A carotenoids in plant-derived food sources. Once absorbed, retinol is re-esterified and transported to the liver, the major site of vitamin A storage. Therefore, dietary vitamin A supplementation can increase retinol levels in the colon and liver, potentially affecting both primary colon tumors and liver metastases of the primary tumors. All-trans--retinoic acid (ATRA) is thought to regulate most of the effects of retinoids, via the ATRA/RAR/RARE pathway exerting an inhibitory effect on cancer growth and progression. As cancer progresses, colon cancer acquires the resistance to ATRA. The purpose of this study is to understand the mechanism by which retinol decreased the growth and progression of ATRA-resistant human colon cancer in vivo and in vitro. We first demonstrated that retinol decreased the growth of ATRA-resistant colon cancer cells by arresting cell cycle progression independent of the ATRA/RAR/RARE pathway. Next, we showed retinol inhibited ATRA-resistant human colon cancer cell invasion by decreasing MMP-2, -9 and PI3K activity in vitro. Finally, dietary vitamin A supplementation decreased the incidence and multiplicity of liver metastases in nude mice intrasplenically injected with ATRA-resistant human colon cancer cells. Taken together, these data suggest the possibility of dietary vitamin A supplementation for colon cancer therapy and prevention.

Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background. Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically. The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations.

No description available.

Background: Many well-known pharmacologic agents have been shown to have toxic effects to the cochleo-vestibular system. Examples of such ototoxic agents include cisplatin and aminoglycoside antibiotics. Ototoxicity monitoring consists of a comprehensive pattern of audiological assessments designed to detect the onset of any hearing loss. Three main methods have emerged over the past decade, and include the basic audiological assessment, extended high frequency (EHF) audiometry, and otoacoustic emission (OAE) measurement. These measures can be used separately or in combination, depending on clinical purpose and patient considerations. It is suggested by the American Academy of Audiology Position Statement and Clinical Practice Guidelines: Ototoxicity Monitoring, that baseline testing be done in a fairly comprehensive manner, including pure-tone thresholds in both the conventional- and extended high frequency ranges, tympanometry, speech audiometry, and the testing of OAEs (AAA, 2009). Anecdotal evidence suggests that New Zealand Audiologists do not currently follow a national ototoxicity monitoring protocol. Therefore the main aim of this study was to explore the current status of ototoxicity monitoring within New Zealand. Hypothesis: It was hypothesized that hospital based Audiology departments across New Zealand each followed their own internal ototoxicity monitoring protocol based, to a large extent, on the guidelines proposed by the American Academy of Audiology and by the American Speech-Language-Hearing Association. Method: Through the use of a Telephone Interview Questionnaire, 16 charge Audiologists were interviewed to establish their current state of knowledge regarding ototoxicity monitoring at 16 out of 20 district health boards in New Zealand. Enquiries about the current systems and procedures in place at their departments together with any suggestions and recommendations to improve on these systems were made. Results: This study found that only 9 of the 16 DHBs interviewed currently follow an ototoxicity monitoring protocol. Furthermore, other than initially hypothesized the origin of the protocols followed by the remaining 7 departments were reported to have ranged from independently developed protocols to historically adopted protocols. One department implemented an adapted version of a protocol by Fausti et al. (Ear and Hearing 1999; 20(6):497-505). This diversity in origin however, does confirm our initial suspicion that no universal and standardized monitoring protocol is currently being followed by Audiologists working in the public health sector of New Zealand.

No description available.