Ctr9, a key subunit of PAFc, affects global estrogen signaling and drives ERalpha-positive breast tumorigenesis

The human RNA polymerase II-associated factor complex (hPAFc) and its individual subunits have been implicated in human diseases including cancer. However, its involvement in breast cancer cells awaits investigation. Using data mining and human breast cancer tissue microarrays, we found that Ctr9, the key scaffold subunit in hPAFc, is highly expressed in ERα+ luminal breast cancer and the high expression of Ctr9 correlates with poor prognosis. Knockdown of Ctr9 in ERα+ breast cancer cells almost completely erased estrogen regulated transcriptional response. At the molecular level, Ctr9 enhances ERα protein stability, promotes recruitment of ERα and RNAPII and stimulates transcription elongation and transcription-coupled histone modifications. Knockdown of Ctr9, but not other hPAFc subunits, alters the morphology, proliferative capacity and tamoxifen-sensitivity of ERα+ breast cancer cells. Together, our study reveals that Ctr9, a key subunit of hPAFc, is a central regulator of estrogen signaling that drives ERα+ breast tumorigenesis, rendering it a potential target for the treatment of ERα+ breast cancer. To globally identify Ctr9 regulated genes, MCF7-tet-on-shCtr9 cells were cultured in DMEM supplemented with 10% FBS in the absence or presence of 500 ng/ml Dox for 4 days, followed by continuing cultured in stripped medium in the absence or presence of 500 ng/ml Dox for another 3 days. Cells were then treated with DMSO or 10 nM E2 for 4 hours prior to cell collection. Total RNA was extracted using a Qiagen RNeasy Plus Kit according to manufacturer protocol, and three independent experiments were performed. Purified total RNA was submitted to the University of Wisconsin Madison Biotechnology Center for RNA quality analysis, reverse transcription, labeling, and hybridization to the Affymetrix human transcriptome array 2.0 containing >6.0 million distinct probes covering 44,699 protein coding genes and 22,829 non-protein coding genes. Data analysis and visualization were performed using the Affymetrix Transcriptome Analysis Console (TAC) Software.