Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Pathology and Laboratory Medicine

Supervisor

Nichols, Anthony C

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Infection by human papillomavirus (HPV) has caused a rise in HNSCC cases. Unfortunately, a cohort of HPV+ HNSCC patients exhibit treatment resistance. At present, no known treatment resistant biomarkers have been identified. We completed genomic characterization of a local HPV+ HNSCC cohort and observed MACROD2 deletions in the treatment failure dataset. Functional validation revealed siRNA and shRNA MACROD2 knockdown in HNSCC cells led to elevated cell viability and colony formation. Interestingly, shMACROD2 caused no change in cisplatin sensitivity, but a significant increase in radiation resistance in HPV+ HNSCC. Transcriptomic, proteomic, and immunofluorescence studies demonstrated that MACROD2 depleted HPV+ HNSCC cells displayed elevated hypoxia and altered DNA damage response signatures suggestive of possible mechanisms of radioresistance. Further work is needed to understand how to overcome these molecular mechanisms of radiation resistance in MACROD2 deficient HPV+ HNSCC.

Summary for Lay Audience

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Recently, infection by human papillomavirus (HPV) has caused a rapid rise in HNSCC cases. Although patients with HPV-associated HNSCC (HPV+ HNSCC) generally respond well to chemotherapy and radiation treatment, a cohort of patients exhibit treatment resistance resulting in cancer recurrence. At present, no known molecular drivers for treatment resistance in HPV+ HNSCC have been identified. We have completed a genomic characterization of HPV+ HNSCC tumours from patients from our treatment area. We observed that genetic changes in a gene called MACROD2 were more frequent in patients who failed treatment. Thus, we hypothesized that mimicking the genetic change in MACROD2 in cell line models would prove that this gene drives resistance to chemotherapy and radiation in HPV+ HNSCC.

To accomplish this, we used HPV+ HNSCC cells and inhibited the expression of the MACROD2 gene. Inhibiting MACROD2 led to elevated cell aggressiveness in the HPV+ HNSCC cell lines. Interestingly, MACROD2 inhibited cells were not resistant to chemotherapy, but had a significant increase in radiation resistance across several HPV+ HNSCC cell lines.

To understand how loss of MACROD2 gene expression leads to radiation resistance, we examined our cells at the transcript and protein level. Our results show MACROD2 deficient HPV+ HNSCC cell lines exhibit a distinct hypoxia signature, describing a low oxygen environment. Tumour hypoxia has been previously associated with radiation resistance. Also, MACROD2 inhibition was associated with an altered DNA damage response (DDR), also implicated in cancer aggressiveness and treatment resistance. Consequently, a suggested mechanism of radiation resistance in MACROD2 inhibited cells is an elevated hypoxia and altered DDR.

These findings provide a better understanding of the molecular basis of treatment resistance in HPV+ HNSCC. Our findings have the potential to stratify patients based on MACROD2 expression, so those harbouring MACROD2 mutations may seek alternatives to radiation therapy in the treatment of their HPV+ HNSCC.

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