Title

Regulation of Cellular Communication Network Factor 1 (CCN1) by Signal Transducer and Activator of Transcription 3 (STAT3) and Calcium in Ovarian Adenocarcinoma (OVCAR8) Cells

Date of Award

Fall 2020

Project Type

Thesis

Program or Major

Biochemistry

Degree Name

Master of Science

First Advisor

Paul C Tsang

Second Advisor

Sarah R Walker

Third Advisor

David H Townson

Abstract

Within the ovary, angiogenesis is not only integral to follicular development and the formation of the corpus luteum, but it is also a hallmark of tumorigenesis and tumor progression in ovarian cancers. Formerly known as Cysteine rich 61-Connective tissue growth factor-Nephroblastoma overexpressed 1 or Cysteine-rich 61 (CYR61), CCN1 promotes angiogenesis and is associated with various pathologies, including cancer. However, among the angiogenic factors within the ovary, the regulation of CCN1 remains not well characterized. Therefore, the overall objective of the present study was to identify potential regulators of CCN1 and their mechanisms of action in the ovarian adenocarcinoma cell line, OVCAR-8. The specific objectives were (1) to determine the regulation of CCN1 expression by prostaglandin-F2α (PGF2α), signal transducer and activator of transcription 3 (STAT3) and calcium, and (2) to identify the respective cellular signaling molecules that played a role in regulating CCN1 expression. Generally, cells were grown to 85% confluency and serum-starved for 2 hours prior to treatment. Then, the cells were treated for 2 hours to stimulate the protein kinase C (PKC) pathway via PGF2α and phorbol 12-myristat 13-acetate (PMA), and the calcium signaling pathway via the calcium ionophore (A23187). Total RNA was extracted, cDNA was generated, and CCN1 mRNA was analyzed via quantitative polymerase chain reaction (qPCR). Treatments with PGF2α (0.1, 0.5, 1uM; n=3 independent experiments) and PMA (0.1, 1, 10nM; n=4) had little to no effect on CCN1 expression when compared to the negative control (serum-free medium), whereas treatment with the ionophore (0.1, 0.5, 1uM) resulted in a 2-fold increase in CCN1 expression (p0.05; n=3) increase in CREB1 expression, suggesting a possible inverse relationship between STAT3 and CREB1, relative to CCN1 expression. When STAT3 is lowered, CREB1 is raised and CCN1 expression is increased. Further investigation is needed to determine the precise nature of the molecular interactions between STAT3 and CREB1 to regulate CCN1. Similar to results observed with OVCAR-8 cells, calcium treatment and STAT3 knock-down in two other ovarian cancer cell lines, OVCAR-3 and SK-OV-3 cells, resulted in increased CCN1 expression. In conclusion, the regulation of CCN1 is complex and involves the interplay of several different signaling molecules, including STAT3, calcium, and CREB1

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