Cytokeratin 5 alters ß-catenin dynamics in breast cancer cells.

Estrogen receptor (ER) positive breast cancers often contain subpopulations of cells that express the intermediate filament protein cytokeratin 5 (CK5). CK5+ cells are enriched in cancer stem cell (CSC) properties, can be induced by progestins, and predict poor prognosis in ER+ breast cancer. We established through CK5 knockout and overexpression in ER+ breast cancer cell lines that CK5 is important for tumorsphere formation, prompting us to speculate that CK5 has regulatory activity in CSCs. To interrogate CK5 interacting proteins that may be functionally cooperative, we performed immunoprecipitation-mass spectrometry for CK5 in ER+ breast cancer cells. Focusing on proteins with signaling activity, we identified ß-catenin, a key transcription factor of the Wnt signaling pathway and cell adhesion molecule, as a CK5 interactor, which we confirmed by co-immunoprecipitation in several breast cancer models. We interrogated the dual functions of ß-catenin in relation to CK5. Knockout or knockdown of CK5 ablated ß-catenin transcriptional activity in response to progestins and Wnt stimuli. Conversely, CK5 induced by progestins or overexpression was sufficient to promote the loss of ß-catenin at the cell membrane and total E-cadherin loss. A breast cancer patient-derived xenograft showed similar loss of membrane ß-catenin and E-cadherin in CK5+ but not intratumoral CK5- cells and single-cell RNA sequencing found the top enriched pathways in the CK5+ cell cluster were cell junction remodeling and signaling. This report highlights that CK5 actively remodels cell morphology and that blockade of CK5-ß-catenin interaction may reverse the detrimental properties of CK5+ breast cancer cells.

Breast Cancer Suppression by Progesterone Receptors Is Mediated by Their Modulation of Estrogen Receptors and RNA Polymerase III.

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. Cancer Res; 77(18); 4934-46. ©2017 AACR.