Effect of βV-Tubulin on Cellular Function in Malignancy

Abstract

This thesis work focuses on the Class V beta tubulin isotype (βV-tubulin) exploring the premise that cellular function or dysfunction may be mediated by the isotype composition of its microtubules. It expands on a previously published study that showed βV-tubulin to be highly expressed in tissue with secretory function. We hypothesize that altered βV-tubulin expression may be associated with the pathogenesis of ovarian cancer that is known to arise from secretory epithelial cells. Specifically, we test the association of βV-tubulin with chemosensitivity, invasion and secretion.

Firstly, to evaluate the association of βV-tubulin expression with ovarian cancer pathogenesis, we performed immunohistochemistry (INC) using a highly specific antibody developed in our laboratory against human βV-tubulin to evaluate expression in the fallopian tube epithelium (FTE) of patients who underwent salpingectomy; dichotomizing analysis for high-risk (BRCA mutant) versus low-risk cohorts (n= 82 in total). These studies were complemented by evaluating βV-tubulin expression in serous ovarian neoplasms (n = 13). Observations were recorded by conventional pathological scoring metrics. We reported that in the FTE, βV-tubulin is expressed solely in the non-ciliated secretory cell type. While βV-tubulin expressing cells were rare in pathologically "normal" FTE, their frequency varied by reason for salpingectomy, and rose dramatically in FTE of patients with BRCA mutations. In tumor tissue, βV- tubulin expression was correlated with differentiation status and was elevated in high-grade serous ovarian carcinoma, relative to borderline serous tumors. Thus, these IHC-based analyses support the hypothesis that deregulated expression of 13V-tubulin is a pathologic feature of FTE from BRCAmutant women at high risk of developing ovarian carcinoma.

A second component of these studies focused on genetically modulating βV-tubulin expression in cancer cell lines with high endogenous expression of the tubulin isotype. Cell models were selected to represent diseases of interest, namely ovarian and breast cancer; and cellular functions of interest, namely secretion and metastasis. The βV-tubulin encoding TUBB6 gene was targeted for knockout using CRISPR gene editing in two metastatic ovarian cancer and one triple negative breast cancer (TNBC) line. Knockout of βV-tubulin was confirmed in select clones by immunoblotting and immunofluorescence. Subsequent characterization evaluated the impact of βV-tubulin modulation on proliferation, chemosensitivity, invasion, mitochondrial density and activity, and secretory output and constituents. Complete knockout of βV-tubulin in cancer cell lines had no significant effect on proliferation rate or gross morphology. Nuanced though reproducible, changes were observed in cancer cell properties, including increased invasion and an increased ratio of polarized mitochondria in the absence of βV- tubulin. Moreover, βV-tubulin expression correlated with differential sensitivity to certain clinically relevant chemotherapeutics including Taxol, specifically conferring increased drug resistance. Finally, semi-quantitative proteomic analysis identified cohorts of differentially secreted proteins following modulation of βV-tubulin, and subsequent gene ontology analysis implicated modulation of functional pathways governing mRNA slicing and reproductive hormone regulation. Ongoing studies will interrogate the potential association of these functional pathways and candidate proteins with βV- tubulin.

Overall, we conclude that expression of βV-Tubulin in secretory cells of the fallopian tube epithelium correlates with cellular atypia and potentially increased risk of BRCA-mutant ovarian cancer; though additional studies are required to corroborate these pilot data. Our findings add to the current understanding of the early pathogenesis of ovarian cancer, and highlight βV-tubulin as worthy of consideration as an IHC-based biomarker for precursor lesions in FTE. Moreover, βV-tubulin has modest but reproducible effects on defined cancer cell functions. The finding that βV-tubulin expression can potentially modulate the secretory output of cancer cells supports a specialized function for this isotype and lays the groundwork for additional studies. Thus, this research furthers understanding of the role of this understudied Class V isotype of β-tubulin in supporting the malignant phenotype of ovarian and breast cancer.