Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Xianjue Fang

Abstract

Lysophosphatidic acid (LPA) and transforming growth factor beta (TGFβ) are platelet-derived intercellular mediators of cell proliferation and motility. LPA is a general growth, survival and motility-stimulating factor in mammalian cells. TGFβ prevents proliferation of normal epithelial cells. However, the growth-inhibitory effect of TGFβ is lost or reduced in most malignant cells. Instead, TGFβ promotes migration and invasion of advanced cancer cells. Since LPA and TGFβ are both present in the blood and tumor microenvironments, we were interested in signal integration and functional outcomes in malignant epithelial cells in an LPA and TGFβ co-stimulatory context. In a subset of breast and ovarian cancer cell lines which remain sensitive to the cytostatic effect of TGFβ, we found that LPA up-regulated expression of the cyclin-dependent kinase inhibitor p21Waf1. But this up-regulation was not observed in TGFβ-resistant ones. We examined the possibility that LPA-induced p21 might contribute to the cytostatic response to TGFβ. Indeed, TGFβ alone induced p21 expression weakly in TGFβ-sensitive cells. Serum or serum-borne LPA cooperated with TGFβ to elicit the maximal p21 induction. LPA stimulated p21 via LPA1 and LPA2 receptors and Erk-dependent activation of the CCAAT/enhancer-binding protein beta (C/EBPβ) transcription factor independent of p53. Loss or gain of p21 expression led to a shift between TGFβ sensitive and resistant phenotypes in breast and ovarian cancer cells, indicating that LPA-induced p21 is a key determinant of the growth inhibitory activity of TGFβ. The p21-stimulatory action of LPA is absent from most breast and ovarian cancer cells, leading to their resistance to TGFβ. Therefore we reveal a novel crosstalk between LPA and TGFβ that underlies TGFβ sensitive and resistant phenotypes of breast and ovarian cancer cells. In the next part of our study, we examined the role of interactions between LPA and TGFβ in regulation of tumor cell motility. LPA and, to a much less extent, TGFβ stimulate chemotactic migration and invasion of breast and ovarian cancer cells. However, when combined together with LPA, TGFβ strongly attenuated LPA-driven migration and invasion of breast and ovarian cancer cells. This inhibitory effect was most likely mediated through TGFβ downregulation of expression of LPA1, the major receptor subtype responsible for LPA-regulated cell migration. Knockdown of Smad3 or Smad4 with small hairpin RNA (shRNA) eliminated the inhibitory effects of TGFβ on the LPA1 expression and LPA-dependent cell migration. There are two potential TGFβ inhibitory elements (TIE) (-40 bp and -401 bp) present in the human LPA1 gene promoter. Deletion or point mutation of the distal TIE at around -401 bp abolished the inhibitory effect of TGFβ on the LPA1 promoter activity as revealed by luciferase assays. A DNA pull-down assay showed that the -401-TIE-E2F4/5 sequence was capable of binding Samd3, Smad4, and E2F4/5 in TGFβ-treated cells. The binding of the Smad complex to the native TIE-E2F4/5 sequences of the LPA1 gene promoter was further verified by chromatin immunoprecipitation assay. Our results identify a novel role of TGFβ in the control of LPA1 expression and LPA1-coupled biological activities, adding LPA1 to the list of TGFβ-repressed target genes.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

4-30-2012

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