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A novel molecular relationship between PARN and PLD that, when deregulated, contributes to the aggressive phenotype of breast cancer cell lines.

Miller, Taylor Elaine

Abstract Details

2017, Master of Science (MS), Wright State University, Biochemistry and Molecular Biology.
The removal of mRNA transcript poly(A) tails by 3'-5' exonucleases is the rate-limiting step for controlled mRNA decay in eukaryotes. Poly(A)-specific ribonuclease (PARN) is one such exonuclease that degrades poly(A) tails, and although its in vitro activity is well-characterized, PARN’s patho-physiological roles in the cell are not well understood. Prior studies have found a possible role for PARN in cancer, in that PARN expression levels in human breast cancer tissues are often decreased compared to normal control tissues. Indeed, data mined from the ONCOMINE cancer array database showed that PARN is downregulated in patient invasive breast carcinoma samples compared to adjacent normal control tissue. Interestingly, phospholipase D (PLD), a cell-signaling molecule well known for promoting breast cancer cell growth, proliferation, and metastasis, was upregulated in these same breast carcinoma samples. PARN is known to target mRNA containing AU-rich elements (AREs) for degradation and although not identified in the literature as a target of PARN, PLD1 has large AREs in its 3' UTR. Taken together, as the levels of PLD are elevated in breast cancer and PARN levels are decreased and PLD1 contains AREs that PARN may directly target, we hypothesized that a regulatory connection between PARN and PLD would exist. We investigated whether PARN regulates PLD and if PLD regulates PARN. In non-cancerous cell lines, such as in COS-7 and HMEC, we found that PARN downregulated the expression of both predominant mammalian isoforms of PLD, PLD1 and PLD2. This phenomenon was not observed in the breast cancer cell lines. We also investigated whether the converse was true, if PLD has any regulatory effect on PARN. We found that PLD and its catalytic product, phosphatidic acid (PA) in the exogenous form of dioleoyl-PA (DOPA), increased PARN expression and activity. PARN also co-localized with exogenously-added fluorescent PA in cells, as demonstrated by microscopy. However, DOPA did not bind to recombinant PARN protein in vitro (although PA species with saturated acyl chains did bind to PARN), suggesting that the effect of DOPA on regulating PARN expression and activity is most likely through signaling downstream of PA. We hypothesized that the mechanism by which PARN downregulates PLD in non-cancerous cells would be absent or inhibited in breast cancer cell lines. We determined that PARN directly targets the AU-rich 3' UTR of PLD1 that initiates PLD1 mRNA degradation presumably through PARN deadenylation of the mRNA poly(A) tail. Furthermore, this effect was largely dependent on the presence of a miR-203 targeting site that immediately precedes a specific AU-rich element (ARE) in the 3' UTR of PLD1. Thus, a combination of PARN deadenylase activity and a putative presence of miR-203 synergizes in destabilizing PLD1 transcripts hampering translation. We found that this newly discovered mechanism of PLD regulation was absent in the highly aggressive MDA-MB-231 breast cancer cell line, which may explain the high expression of PLD in this and other breast cancer cell lines. We report herein the novel regulatory relationship between PARN and PLD that, when deregulated, contributes to the phenotype seen in the MDA-MB-231 breast cancer cell line.
Julian Gomez-Cambronero, Ph.D. (Advisor)
Michael Leffak, Ph.D. (Committee Member)
Weiwen Long, Ph.D. (Committee Member)
80 p.

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Citations

  • Miller, T. E. (2017). A novel molecular relationship between PARN and PLD that, when deregulated, contributes to the aggressive phenotype of breast cancer cell lines. [Master's thesis, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1493581959771992

    APA Style (7th edition)

  • Miller, Taylor. A novel molecular relationship between PARN and PLD that, when deregulated, contributes to the aggressive phenotype of breast cancer cell lines. 2017. Wright State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wright1493581959771992.

    MLA Style (8th edition)

  • Miller, Taylor. "A novel molecular relationship between PARN and PLD that, when deregulated, contributes to the aggressive phenotype of breast cancer cell lines." Master's thesis, Wright State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=wright1493581959771992

    Chicago Manual of Style (17th edition)