Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Cao, Deliang

Abstract

RelA/p65 is a main subunit of nuclear factor-kappaB (NF-kappaB) that regulates expression of genes involved in cell growth and survival, stress response, and inflammation, but its oncogenic or tumor-suppressive function in tumorigenesis has been highly controversial. Hundreds of NF-kappaB inhibitors have been developed for targeted cancer therapy, but fail to achieve anticipated anticancer efficacy. Complexity of posttranslational modifications may contribute to tumorigenic diversity of RelA/p65, but mechanisms of action remain unclear. Here we show that phosphorylation of RelA/p65 at Ser536 functions as a tumor suppressor. In normal human colon mucosa, RelA/p65 phosphorylation at Ser536 is gradually increased with the maturation and apoptotic shedding of cryptic cells, but the phosphorylation is significantly decreased in colon tumors. In RelA/p65-silenced breast and colon cancer cells, reconstitution of a constitutively active RelA/p65, whose Ser536 was replaced by aspartic acid (named p65/S536D), triggered dramatic apoptosis and autophagy or senescence upon the cell context, by affecting the expression of a range of cell death/survival genes. Reconstitution with a phosphorylation-deficient RelA/p65 whose Ser536 was replaced by alanine (called p65/S536A) had no effect on cell growth and survival. Intratumoral delivery of p65/S536D effectively suppressed tumor growth in nude mice and was marked with apoptotic cell death. Together our data suggest that it is the phosphorylation at Ser536 that confers NF-kappaB RelA/p65 a tumor-suppressive role. In addition to driving cell death, the phosphorylation of RelA/p65 on Ser536 is also beneficial for chemosensitivity in cancer therapy. Literature reports that NF-kappaB activation contributes to chemoresistance by regulating oncogenic pathways and multi-drug resistance genes, but recent studies on senescence and chemotherapy have suggested that NF-kappaB activation is essential for chemotherapy induced senescence and tumor regression. The mechanisms underlying the contradictory observations are elusive. Here we show that site-differential phosphorylation of NF-kappaB RelA/p65 modulates chemosensitivity and results in distinct outcomes in cancer cells. Phospohrylation of RelA/p65 on Ser276, Ser536 and Ser468 were detected in RITA (reactivation of p53 and induction of tumor cell apoptosis) treated cells and RITA-resistant sublines, and they showed distinct dynamics. Ectopic expression of p65/S536D dramatically enhanced RITA sensitivity while the constitutively phosphorylated form of S276 (S276D) compromised RITA effects in treated cells. The constitutive phosphorylation of Ser468 (S468D) showed very mild effects on RITA sensitivity. In addition, p65/S536D resensitized RITA-resistant sublines to RITA treatment. P65/S536D also enhanced the doxorubicin sensitivity, but not paclitaxel in these multi-drug resistant sublines. ATP-binding cassette (ABC) transporters are critical multidrug resistant factors. We found that in the RITA-resistant sublines, multiple ABC transporter genes were upregulated, particularly the ABCC6. Further studies dissected that ABCC6 was involved in site-specific phosphorylation- related chemoresistance. P65/S536D decreased ABCC6 expression and sensitized cells to doxorubicin and RITA, but not paclitaxel that is not the substrate of ABCC6 transporter. Conversely, p65/S276D enhanced chemoresistance by upregulating ABCC6. The p65/S536D enhanced RITA chemosensitivity is also confirmed in in vivo study using tumor xenografts in nude mice. Taken together, phosphorylation of p65 on Ser536 contributes to chemosensitivity by targeting ABCC6 while Ser276 leads to chemoresistance. These findings solve the controversial issues of NF-kappaB RelA/p65 in tumorigenesis and in chemosensitivity, and are important in developing NF-kappaB targeted cancer therapy, such as inhibitors. This study also highlights that better understanding of distinct active sites on NF-kappaB RelA/p65 is necessary for discussing the tumorigenic roles of NF-kappaB and developing efficient NF-kappaB targeted therapies.

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