Growth pathways in feline oral squamous cell carcinoma Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/rj430873v

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  • Background Bcl11b, previously CTIP2, is a zinc finger nuclear transcription factor involved in the development of keratinocytes, teeth, T lymphocytes, and nervous tissue. Expression of Bcl11b has been reported in human head and neck squamous cell carcinoma (HNSCC) and increased expression correlates with poorly differentiated tumors. This study aimed to evaluate Bcl11b expression patterns in feline oral squamous cell carcinoma (FOSCC) with the hypothesis that histologic grading of FOSCC would correlate with Bcl11b expression as reported in HNSCC. Previously reported prognostic markers, including EGFR expression, Ki67, and VEGF production were analyzed concurrently. Our hypothesis was that high Bcl11b expression would correlate with other markers of malignancy. Our second objective was to evaluate associated pathways upstream of Bcl11b, to determine their role in FOSCC. Since EGFR requires interaction with Src to become fully activated and stimulate cell growth, subsequent experiments focused exclusively on Src pathways and the effects of Src inhibition on the growth of FOSCC cells. Our aim was to focus on activation status of the entire Bcl11b pathway, from the proximal EGFR to the distal nuclear transcription factor Bcl11b, to determine the role of this pathway in growth of FOSCC. We hypothesized that a Src inhibitor, dasatinib, would inhibit activation of downstream Bcl11b. Methods FOSCC biopsies were assigned a histologic grade based on a modified grading scheme that considered degree of differentiation, nuclear pleomorphism, scirrhous response, and mitotic index. FOSCC biopsies were stained by immunohistochemistry (IHC) for Bcl11b, EGFR, Ki67, and VEGF-D. Bcl11b, total EGFR, pEGFR, pSrc, pERK, and VEGF-D were analyzed by western blot in FOSCC cell lines, under various conditions. Cells were grown in supplemented medium, then serum-starved or stimulated with EGF, and then exposed to dasatinib. Immunoprecipitation and western blotting was used to confirm phosphorylation of Bcl11b in all three FOSCC cell lines with EGF supplementation, and following treatment with dasatinib. Cell lines were exposed to a range of dasatinib doses (0.01-10nM) with and without EGF to determine effects on cell viability by an MTS assay. An ELISA kit was performed to measure production of VEGF by SCC cells and the effects of dasatinib on its production. Principal findings The majority (70%) of FOSCC biopsy samples were assigned an intermediate histologic grade (24/34) based on our modified grading scheme. Bcl11b expression was moderate to strong in 79.4% (27/34) of biopsies, and samples with intense Bcl11b positive staining had a higher number of positively staining cells (p<0.001). All samples analyzed (32/32) expressed VEGF-D, and 18/21 samples had varying expression of EGFR. No correlation was found between Bcl11b expression and tumor grade, Ki67, or VEGF-D. A correlation between Bcl11b score and EGFR expression approached statistical significance (P=0.05598). All three FOSCC cell lines expressed Bcl11b, pEGFR, pSrc, pERK, and VEGF-D. Addition of EGF to the medium increased expression of many of these targets and partially rescued cells from the inhibitory effects of dasatinib. Dasatinib lowered cell viability, total levels of pBcl11b and VEGF production in FOSCC cells. Conclusions/ Significance This is the first description of Bcl11b expression in the cat. Its strong expression in FOSCC strengthens the impact of FOSCC as a model of HNSCC. We have demonstrated activation, from proximal to distal, of an important growth factor signaling pathway that triggers cell division in FOSCC cell lines and biopsies. Phosphorylated Src serves as a major signaling hub in the middle of this pathway, downstream from EGFR and upstream of Bcl11b, making it a strategic target. Dasatinib decreased phosphorylation of Bcl11b in two cell lines and consistently decreased production of VEGF, supporting its use as a direct cytotoxic agent as well as a role in reducing tumor angiogenesis. Future studies should determine the effect of combined inhibition using dasatinib and an EGFR or VEGFR inhibitor to minimize the protective effect EGF provides to FOSCC cells.
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