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Investigating Biological Mechanisms of Radiation Resistance in Advanced Stage Cervical Cancer
Date of Award
Doctor of Philosophy (PhD)
The current standard of care treatment for locally advanced cervical cancer is curative intent pelvic radiation with concurrently administered platinum chemotherapy (CRT). This treatment strategy is effective for many patients, but 33-50% of patients treated with CRT develop disease recurrence. Metastatic and recurrent cervical cancer is an incurable condition, and many of the currently available treatments are associated with significant morbidity and mortality. Identifying these patients upfront is a challenge that clinicians face when developing treatment strategies. Previous studies used to catalog the genomic and transcriptomic landscape of cervical cancer lacked high quality corresponding clinical follow up data for patients, and patients were not treated uniformly, making the clinical interpretation of these alterations difficult. The goal of this thesis was to pair next-generation sequencing technology with well annotated prospectively collected clinical follow up data from patients uniformly treated with CRT to identify biomarkers present in pretreatment cervical tumor biopsies that correlate with disease recurrence and investigate the mechanism by which these biomarkers result in radiation treatment failure.In this thesis I identified both human host and Human Papilloma Virus (HPV) related biomarkers that correlate with disease recurrence after standard of care CRT in a cohort of 88 advanced stage cervical cancer patients. Mutations in the human host genes FBXW7 and TP53 were associated with metastatic disease recurrence. All patients with NSD1 mutant tumors recurred exclusively within the pelvis, including two patients with co-occurring mutations of either FBXW7 or TP53; therefore, this gene was selected for further in vitro validation studies of radiation resistance. NSD1 is a histone methyltransferase (HMT) that has been previously identified as a poor prognostic indicator for both neuroblastoma and breast cancer patients. Engineered NSD1 knockdown cervical cancer cell lines exhibited decreased H3K36 methylation and transcriptional reprogramming of genes involved in cell extrinsic inflammatory pathways and cell intrinsic p53, apoptosis and estrogen response pathways. Loss of NSD1 was sufficient to induce resistance to single agent carboplatin and combination CRT treatment, validating it as a reliable biomarker for CRT treatment failure. In HPV positive cervical cancer cases, HPV genotype and high viral transcript expression were predictive of treatment failure after CRT. Downstream validation to determine whether high viral transcript expression is sufficient to induce radiation treatment resistance was achieved by overexpressing HPV 16 E6 and E6*I viral transcripts in the Siha cervical cancer cell line. Using this system, I demonstrated that increased E6*I expression led to the stabilization of p53 and p21 inducing cellular senescence, whereas E6 overexpression led to increased p53 degradation. Modulating either transcript resulted in reduced cell sensitivity to radiation treatment providing a biological basis for the observed clinical effect of these alterations.Lastly, I collaborated with Dr. Akin Ojesina from the University of Alabama Birmingham to genomically characterize cervical cancer cases where HPV was undetectable using currently available technology. HPV negative cervical cancer accounts for 7-11% of all cervical cancer cases and these patients have worse survival outcomes compared to patients with HPV positive cervical cancer after standard of care CRT. We found that HPV negative tumors had an enrichment of mutations in genes that are involved in regulating entry into the cell cycle. One of these significantly mutated genes was CCND1, which was also overexpressed in HPV negative tumors. Palbociclib is an FDA approved drug that targets the G1 cell cycle checkpoint through inhibition of CDK 4/6, which are the activating binding partners to cyclin D1. Using a panel of HPV negative and positive head and neck squamous cancer cell lines I confirmed that HPV negative cancer cell lines were uniquely sensitive to single agent Palbociclib treatment. These results suggest that the addition of Palbociclib may be a viable treatment strategy for this poor prognostic group.Altogether the results of this dissertation demonstrate the power of employing next-generation sequencing to a cohort of patients who were uniformly treated and had well annotated, prospectively collected clinical follow up data. Using this resource, I was able to identify novel biomarkers of disease recurrence after standard of care CRT and determine the proposed biological mechanism of CRT resistance in each case. I identified and validated that mutations in NSD1 and high HPV transcript expression can be used to identify patients prior to treatment who are likely to fail the standard of care. These patients may benefit from the addition of other chemotherapies or targeted agents into their initial treatment plan or alternatively radiation dose escalation could be employed. Additionally, in the case of cervical tumors in which HPV is undetected, these patients would likely benefit from Palbociclib treatment.
Chair and Committee
Julie K. Schwarz
Kareem Azab, Katherine Fuh, Robert Mercer, Nima Mosammaparast,
Ruiz, Fiona, "Investigating Biological Mechanisms of Radiation Resistance in Advanced Stage Cervical Cancer" (2019). Arts & Sciences Electronic Theses and Dissertations. 2016.