Author ORCID Identifier

0000-0002-3899-8924

Date of Graduation

8-2022

Document Type

Thesis (MS)

Program Affiliation

Cancer Biology

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Curtis Pickering, Ph.D.

Committee Member

Faye Johnson, M.D./Ph.D.

Committee Member

Heath Skinner, M.D./Ph.D.

Committee Member

Danielle Garsin, Ph.D.

Committee Member

Kunal Rai, Ph.D.

Abstract

INDUCED CYOTOXICTY IN CREBBP/EP300mut HEAD AND NECK SQUAMOUS CELL CARCINOMA

Thomaia Pamplin

Advisor: Curtis Pickering, Ph.D.

Background: Head and neck squamous cell carcinoma HNSCC is the most common malignancy in the head and neck. Most cases are found in advanced stages and depending on the location can be treated with surgical resection and/or radiation (XRT), chemotherapy, or chemoradiation. Our lab groups have identified that HNSCC with a mutation in its CREBBP/EP300 genes can be sensitized to XRT when the histone acetyltransferase activity of CREBBP/EP300 is inhibited. This radiosensitization manifests in the form of increased cell death for CREBBP/EP300mut HNSCC treated with XRT. This project looks at the effect of two chemotherapeutic agents: cisplatin (CDDP) and doxorubicin (DOX) and the response that CREBBPmut HNSCC has to them when combined with a HAT inhibiting (HATi) drug called A485. We hypothesize that when combined with A485, CREBBPmut cells will undergo an induced apoptotic death when treated with DOX, and not cisplatin, because DOX induces DNA damage similar to that of XRT, in that homologous recombination is needed to repair the damage done by DOX.

Methods: CREBBP-mut HNSCC cell lines UMSCC-22A and UMSCC-17B and CREBBP-wt HNSCC lines HN31 and FADU were used in cell culture. Clonogenic survival assays were used to cell growth and death with the different agents (DOX, CDDP, XRT with or without A485) used in single and combination treatments. Flow cytometry was used to measure cell death and apoptosis. Cell Titer Glo was used to measure cell viability after using the three different agents in alone or in combination with A485. Western Blot was used to determine if apoptosis occurred with these treatments in the four cell lines by probing for cleaved caspase 3 and PARP products.

Results: Using clonogenic survival assays to observe cell growth on inhibition of growth, a consistent and conclusive of an enhancement of cell death with cisplatin+A485 combination for CREBBPmut and wt HNSCC cells was not observed. Using real-time quantitative live-cell imaging and analysis platforms through an Incucyte, cell death was measured. Significant differences in cell death between CREBBPmut and wt cell lines treated with DOX+A485 was not observed. Western Blot analysis also did not lead to consistent apoptosis cleavage products in CREBBPmut HNSCC cell lines when treated with DOX, XRT, or CDDP alone or in combination with A485.

Conclusion: An enhancement of cell death with DOX+A485 was not observed, nor was apoptosis consistently observed in the cell death response for CREBBPmut HNSCC cell lines treated with DOX alone in or in combination with A485; therefore, the hypothesis was not proven to be true. Some of the experiments performed in this project have been reproducible in the hands of others, so perhaps with more time and support in trouble shooting, our group may be able to reproduce the XRT results seen in previous findings and more trustworthily determine if DOX leads to a similar phenotype. This project has the potential to help characterize the mode of cell death and DNA damage that occurs in induced cytotoxic response which could eventually help with patient outcomes and quality of life, as chemotherapy and XRT come with many toxicities.

Keywords

Cell death, HAT, HATi, CREEBP, EP300, HNSCC, Cytotoxicity, Radiation, Doxorubicin, Cisplatin

Available for download on Monday, February 20, 2023

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