Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
Head and neck squamous cell carcinoma (HNSC) represents the 6th most common cancer type worldwide. A better understanding of the molecular alterations that occur in cells of the tumor and surrounding environment will allow for the identification of new therapeutic targets and biomarkers. This thesis focuses on two distinct but related issues: (1) identification and functional elucidation of epigenetically deregulated genes in oral squamous cell carcinoma (OSCC), and (2) use of tumor-induced changes in non-cancerous epithelial cells adjacent to human papillomavirus-positive oropharyngeal cancers (HPV+ OPCs) as screening biomarkers.OSCC displays a dismal 5-year survival of ~50%. We previously performed whole-genome DNAmethylation and gene expression profiling of tissues from the oral cavity and found the gene SMPD3 to be frequently hypermethylated and downregulated. Overexpression of SMPD3 in oral dysplasia and cancer cell lines did not alter proliferation but decreased migration and invasion and increased resistance to erlotinib. Further, SMPD3 has been linked to the biogenesis of extracellular vesicles. Although SMPD3 overexpression did not alter vesicle size or concentration, it did significantly alter vesicle microRNA content.Once a tumor is established, it extrudes signals in the form of EVs, cytokines, and othermolecules that affect nearby non-malignant cells. Such changes, called malignancy-associated changes (MACs), can be used as biomarkers to screen for the presence of a tumor. We applied this idea to HPV+ OPCs, which are difficult to detect due to their formation at the base of large invaginations. To detect MACs, we compared cells from tumor-adjacent and contralateral normal epithelia, tumors, and patients without cancer in terms of microRNA expression, gene expression, and nuclear morphology. Using RNA from tissue biopsies, we identified 55 genes and 10 microRNAs that fit our criteria for MACs. We then built a machine learning model that could classify nuclei based on measurement of morphological features. Nuclei from tissue and brush biopsies could be classified according to their site of origin with high accuracy. This observation could form the basis of a brush biopsy-based screening method for HPV+ OPC. Together, these data contribute to our understanding of cancer progression at the molecular level
Lung adenocarcinoma (LAC) is the most common subtype of non-small cell lung cancer and is the leading cause of cancer death worldwide. Five-year survival rates of LAC remain dismally low at ~17%, due to the late stage of diagnosis. MicroRNAs (miRNAs) are small RNAs 17-22 nucleotides long and are stable within the serum and when present within extracellular vesicles (EVs), additionally EV miRNAs contribute to tumor-stromal communication. Determining non-invasive novel biomarkers for early disease detection and understanding EV miRNA tumor-stromal communication may aid in improving overall survival. In this thesis, I identify miRNAs that are differentially detected within the serum of patients with LAC using miRNA profiling of patient serum samples and demographically matched controls. I additionally characterize the function of LAC secreted miRNA within EVs when entering normal fibroblast and endothelial cells. My hypotheses are that miRNAs within the serum of LAC patients will show a unique signature that will be distinguishable from non-cancer high risk individuals, and that miRNAs selectively released from LAC cells within EVs will promote tumorigenesis in endothelial cells through stimulating angiogenesis and in fibroblasts through promoting the cancer associated fibroblast phenotype. A unique signature of miRNAs within the serum of LAC patients is found and miRNAs within patient serum is dependent on sex. Several miRNAs within LAC EVs are then functionally characterized in the role they play when signaling to normal stromal cells. Together, this thesis provides a comprehensive analysis of LAC miRNAs and the roles they play as biomarkers within the serum and as tumor-stromal signals when within EVs.
Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer. Although there have been improvements in detection and treatment with the development of targeted therapies, OSCC has a low five-year survival rate which has shown little improvement in recent history. OSCCs are known to secrete extracellular vesicles (EVs) into the extracellular space including into the blood stream. These EVs contain a wide variety of different molecules capable of effecting cancer processes including mRNAs, miRNAs, and proteins. By performing an in depth analysis we will gain a better understanding on how OSCC secreted miRNAs are capable of acting as messages between cancerous and stromal cells. Additionally, I have presented data on how these miRNAs can be exploited for their ability to act as biomarkers.In this thesis I first described which miRNAs are altered in patients with oral cancer or carcinoma in situ and determined that this altered expression is capable of predicting cancer status. After validating the suitability of cell lines as an OSCC model, I examined if altered serum miRNAs overlap with miRNAs which are selectively secreted from oral cancer cell lines. Follow-up functional analysis was performed for miR-142-3p and it was determined that this miRNA was being secreted in order to remove it’s tumor suppressive effect within the cancer cell and additionally to transfer a tumor promoting signal to endothelial cells of the tumor stroma. To confirm that this was not an isolated phenomenon I examined the function of miR-142-3p when secreted from lung cancer cells and noticed a similar effect in endothelial cells and an additional effect on fibroblasts. Effected fibroblasts underwent changes associated with wound healing and tumor promotion.These data taken together provide a comprehensive analysis of the alterations of secreted miRNAs in OSCC and provide insight in the ability of some miRNAs to serve a dual role both within the tumor cells and cells of the tumor stroma. It is possible these results could lead to the creation of a diagnostic test with possible future applications to the diagnosis of oral cancer.
Oral squamous cell carcinoma (OSCC) is the most common subtype of head and neck cancer and has a relatively low five year survival rate of ~50%. One of the reasons for this high mortality rate is that patients are generally diagnosed at late stages. OSCC develops through a typical histological progression and although lesions in the oral cavity are visible at the premalignant stage, it is not possible to predict which lesions will progress based on histology alone. In-depth analysis of genome-wide molecular alterations may identify novel genes or pathways that can be used as biomarkers or therapeutic targets in order to improve survival rates of this disease. In this thesis, I perform DNA methylation, gene expression and miRNA profiling on a panel of patient tissue samples, each with a paired adjacent normal, dysplasia and either a carcinoma in situ or squamous cell carcinoma, taken from a single contiguous disease field within a patient’s oral cavity. My hypotheses are that the epigenetic landscape of OSCC becomes progressively more deregulated throughout the different histological stages and that the most frequently altered molecular events identified at the dysplasia stage may be crucial for premalignant disease development and progression. A high level of deregulation in both methylation and miRNA patterns as the disease progresses is observed, and a number of highly frequent molecular events are identified. Several of these molecular events are then functionally validated to assess the ability to contribute to tumorigenesis in oral premalignant lesions.Taken together, this thesis provides one of the most comprehensive epigenetic analyses of paired normal, dysplasia and CIS/SCC biopsies with regards to DNA methylation and miRNA profiling. In addition to providing a deeper insight into the molecular mechanisms at play within the premalignant lesions, we also validate the ability of these mechanisms to directly contribute to tumorigenesis.
Master's Student Supervision (2010 - 2018)
BACKGROUND: Oral cancer is a devastating disease with a five-year survival rate of 50%. While tobacco remains a key etiological factor for oral cancer, cases in non-smoker patients are also reported. An improved understanding of the molecular basis of oral cancer, including the alterations contributing to disease in non-smokers, is essential. To date, the role of microRNAs (miRNAs) in oral tumorigenesis – and oral premalignant lesions specifically, is largely unknown. The objectives of this study were (1) to identify miRNAs that are deregulated at the premalignant and malignant stages of oral cancer in non-smoker patients, and (2) to elucidate their expression patterns throughout disease progression. METHODS: To remove variation due to timing differences in sampling, we analyzed global miRNA expression in varied stages of precancerous, cancerous and adjacent normal tissue biopsies obtained simultaneously from a single, contiguous field in a patient’s mouth. Total RNA was isolated from each microdissected specimen and profiled for the expression of 742 human miRNAs using Real-Time PCR. The expression of selected candidate miRNAs was further validated in an independent cohort of premalignant and malignant tissues via in situ hybridization (ISH).RESULTS: Overall, the amount of miRNA alterations was associated with lesion severity, suggesting that miRNA changes are accumulated during premalignant progression. In addition, we have identified distinct lists of candidate miRNAs that were consistently deregulated at specific histopathological disease states. Examination of the individual expression profiles of these candidates across sequential premalignant/malignant stages demonstrated that they follow distinct patterns of deregulation over time and may therefore function differently throughout oral tumorigenesis. ISH staining for one of the selected up-regulated candidates, miR-155, corresponded with its previous Real-Time PCR expression data and was further validated in independent dysplastic and malignant tissues.CONCLUSIONS: Our unique sample set allowed us to investigate intralesional progression within a single surgical field and delineate miRNA aberrations that may be driving this process. Collectively, our results suggest that miR-155 may represent a key driver of oral tumorigenesis and that molecular heterogeneity across fields of diseased tissue has significant implications when selecting candidates for development of novel targeted therapies or prognostic screening protocols.
Cancer is a major cause of death worldwide. Patients diagnosed at an early stage have an improved prognosis and therefore efforts have been made into the development of methods to detect tumors at their earliest stages. MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression by interfering with the translation of target mRNAs. Studies have found that miRNAs are present at stable levels in the circulation and that they are differentially expressed in patients with various diseases. In this thesis we used qRT-PCR to assess the utility of 742 serum miRNAs as biomarkers for early cancer detection. In aim 1 we examined the levels of serum miRNAs in patients with high-risk oral lesions. We identified five miRNAs that are significantly deregulated in the serum of these patients compared to demographically matched, non-cancer controls. Additionally, these miRNAs correspondingly decreased or increased after surgical resection of the lesion. In aim 2 we examined the effect of hemolysis, fasting, and smoking on the serum miRNA levels of healthy individuals. We also compared serum miRNA profiles of samples taken from healthy individuals over different time periods. We found that mechanical hemolysis of blood samples simulating blood drawing can significantly alter serum miRNA quantification and should be taken into consideration when identifying endogenous controls and candidate biomarkers for circulating miRNA studies. Fasting, smoking, and a time period up to 17 months between samples were demonstrated to not have a significant effect on the overall serum miRNA profiles of healthy individuals. In aim 3 we compared the miRNA profiles of paired samples collected during surgery from the same patient from a) pulmonary venous effluent draining the tumor vascular bed (tumor associated samples) and b) systemic arterial blood to identify lung adenocarcinoma biomarkers. We found 35 miRNAs that were significantly up-regulated in tumor-associated serum samples. However, when we tested the candidate miRNAs in cancer versus non-cancer peripheral venous blood samples they were not significantly differentially expressed. The results presented in this thesis demonstrate the need for standardized protocols for circulating miRNA studies and provide evidence for the utility of serum miRNAs as biomarkers of disease.