Date of Graduation

5-2016

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Kenneth Y. Tsai, M.D., Ph.D

Committee Member

Elsa R. Flores, Ph.D

Committee Member

Russell R. Broaddus, M.D., Ph.D

Committee Member

Jeffrey N. Myers, M.D., Ph.D

Committee Member

Xiaoping Su, Ph.D

Abstract

Skin cancer is the most common malignancy in humans. Annually, in U.S. there are over 3 million cases with an estimated overall economic impact of $2 billion. Cutaneous Squamous Cell Carcinoma (cuSCC) comprises 15-20% of all skin cancers. cuSCC has the best-defined progression from a distinct precancerous lesion, the Actinic Keratosis (AK), to invasive cuSCC. Destructive therapies for AK treatment must be used repetitively, causing significant morbidity. There is a tremendous need for targeted diagnostics and therapy for AKs, representing an important opportunity for secondary skin cancer prevention. Our knowledge of the molecular and cellular events that lead to the transformation of normal skin (NS) to AK and subsequently to cuSCC is very limited, thus representing a fundamental gap in our understanding of this progression.

In order to identify novel targets for molecular chemoprevention, we used isogenic human samples throughout cuSCC development and a UV-driven Hairless mouse model of cuSCC to identify genomic drivers of cuSCC by cross-species analysis. RNA-Seq identified 349 transcripts that were differentially expressed across normal skin (NS), AK, and cuSCC. mRNA profiles primarily distinguished NS from other samples, whereas microRNA profiles could segregate the three groups. Using cross-species functional pair analysis (anti-correlated miRNA-mRNA expression) we identified several miRNAs (miR-21,-31,-221) and their targets as major promoters of cuSCC development. TRANSFAC analysis identified ETS2, MAZ, and TCF3 as core transcriptional drivers of progression. Whole exome sequencing demonstrated that UVB signature mutations dominate in AKs and cuSCCs, with frequent mutations in TP53, NOTCH1/2, and CDKN2A, as previously reported. Surprisingly, NS samples adjacent to the cuSCCs had up to 1200 mutations with no significant overlap with cuSCC. Ingenuity Variant Analysis identified NOTCH and its coactivators as the functional modules most perturbed in cuSCCs. Because cuSCC shares commonly mutated genes with lung SCC and head & neck SCC (HNSCC), we assessed the global similarity of gene expression to other cancers. By this measure, cuSCC is most similar to HNSCC and its mRNA signature predicts survival in non-HPV-related HNSCC. cuSCC is also similar to lung SCC as well as basal subtype of breast cancer. Our cross species analysis has identified key genomic drivers of cuSCC development as potential chemoprevention targets and suggests that our model can serve as the basis for validating chemoprevention targets in cuSCC and molecularly similar cancers such as HNSCC and lung SCC.

Through our initial analysis of 9 sets of matched samples, we identified miR-181a as a potential molecular target; expression of the miR-181a family gradually increases throughout cuSCC progression. Importantly, miR-181a is significantly upregulated in multiple carcinomas. We have focused on the function of TGFΒR3, which is downregulated by miR-181a and which has a tumor suppressor role in many contexts. We hypothesize that upregulation of miR-181a promotes initiation and progression of keratinocyte transformation by targeting TGFΒR3.

Comparison of miR-181a levels in human cuSCCs to normal skin shows that miR-181a has a significantly higher expression (~8.4 folds) in cuSCCs. Our results show that miR-181a overexpression (OE) and TGFΒR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT keratinocytes and in primary normal human epidermal keratinocytes (NHEKs). In addition, OE of miR-181a or direct KD of TGFΒR3 by shRNA is sufficient for enhanced anchorage-independent survival of HaCaTs. Moreover, miR-181a OE or TGFΒR3 KD enhances cellular motility through increase of migration and invasion and upregulation of EMT markers, such as snail, slug, and vimentin. Luciferase assay results demonstrate that miR-181a directly and specifically targets the 3’UTR of TGFBR3. Rescue experiments show that miR-181a phenotype can be partially rescued by TGFBR3 overexpression. In summary, we show that miR-181a regulates susceptibility to apoptosis as well as cellular adhesion and motility at least in part through TGFΒR3.

Keywords

Biological sciences/Genetics/Genomics/Transcriptomics/Molecular biology/miR-181a/TGFBR3