Amy N. Nash

Date of Award

2009

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Laura Richardson

Second Advisor

Beverly Delidow

Third Advisor

Elaine Hardman

Fourth Advisor

Kelley Kiningham

Fifth Advisor

Donald Primerano

Abstract

The proto-oncogene SKI is a transcription factor and a co-repressor of the TGFβ superfamily, including TGF

β and BMP. However, additional data suggests that SKI may function as a tumor suppressor in some cell types. The TGFβ superfamily has been implicated in cancer progression and germ cell migration. Testicular cancer afflicts men during their peak reproductive years and is the most common cancer among men of this age group. Cisplatin-based chemotherapy is the standard treatment for testicular cancer. This treatment can lead to undesirable side effects, including infertility. We have shown that SKI expression is decreased in testicular germ cell tumors compared to normal testis. The hypothesis of this work is that decreased SKI expression promotes testicular cancer progression by allowing unregulated TGFβ superfamily signaling, conferring an increased ability of testicular cancer cells to invade and metastasize. To test the effects of decreased SKI in testicular cancer, we used a human embryonal carcinoma cell line, NCCIT. We found that transient SKI-knockdown confers an increased ability to migrate and invade, but does not affect proliferation, in NCCIT cells. Forced overexpression of SKI in NCCIT cells resulted in decreased migration and invasion compared to control cells. We next investigated whether TGFβ or BMP enhanced the invasive phenotype of stable SKI-knockdown NCCIT cells. NCCIT cells stably transfected with nonspecific shRNA were used as a control. Addition of TGFβ or TGFβ blocking antibody had no effect on the migration of either SKI-knockdown cells or control cells. Addition of BMP4 enhanced migration in SKI-deficient NCCIT cells but had no effect on control cells. Treatment with noggin, a BMP inhibitor, reduced the migration of SKI-deficient NCCIT cells back to the level of control cells. These data imply that in NCCIT cells with decreased SKI expression, the BMP pathway, but not the TGFβ pathway, promotes migration. Microarray analysis was performed to identify downstream targets of SKI that may be involved in cellular invasion. CXCR4 expression was increased in SKI- knockdown cells compared to control cells, but CXCR4 expression was not affected by treatment with BMP. Signaling partners CXCR4 and SDF1 have been implicated in germ cell migration and cancer metastasis. Addition of SDF1 enhanced the invasive potential of SKI-deficient cells but had no effect on control cells. Addition of AMD3100, a potent and specific inhibitor of CXCR4, blocked the effects seen with SDF1. These data, taken together with the BMP data, imply that decreased SKI expression in NCCIT cells leads to enhanced invasion through both BMP-dependent and BMP-independent mechanisms. This research provides insight into the mechanism behind testicular cancer metastasis, and it identifies the BMP signaling pathway and CXCR4/SDF1 as potential targets for the development of new therapies to treat patients with metastatic testicular cancer.

Subject(s)

Metastasis.

Testicular -- Cancer.

Proto-oncogene -- SKI.

Bone morphogenetic proteins.

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