Half of estrogen receptor-positive breast cancers contain a subpopulation of cytokeratin 5 (CK5)-expressing cells that are therapy resistant and exhibit increased cancer stem cell (CSC) properties. We and others have demonstrated that progesterone (P4) increases CK5+ breast cancer cells. We previously discovered that retinoids block P4 induction of CK5+ cells. Here we investigated the mechanisms by which progesterone receptors (PR) and retinoic acid receptors (RAR) regulate CK5 expression and breast CSC activity. After P4 treatment, sorted CK5+ compared to CK5 − cells were more tumorigenic in vivo. In vitro, P4-treated breast cancer cells formed larger mammospheres and silencing of CK5 using small hairpin RNA abolished this P4-dependent increase in mammosphere size. Retinoic acid (RA) treatment blocked the P4 increase in CK5+ cells and prevented the P4 increase in mammosphere size. Dual small interfering RNA (siRNA) silencing of RARα and RARγ reversed RA blockade of P4-induced CK5. Using promoter deletion analysis, we identified a region 1.1 kb upstream of the CK5 transcriptional start site that is necessary for P4 activation and contains a putative progesterone response element (PRE). We confirmed by chromatin immunoprecipitation that P4 recruits PR to the CK5 promoter near the − 1.1 kb essential PRE, and also to a proximal region near − 130 bp that contains PRE half-sites and a RA response element (RARE). RA induced loss of PR binding only at the proximal site. Interestingly, RARα was recruited to the − 1.1 kb PRE and the − 130 bp PRE/RARE regions with P4, but not RA alone or RA plus P4. Treatment of breast cancer xenografts in vivo with the retinoid fenretinide reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR cross talk, may explain the efficacy of retinoids in prevention of some breast cancer recurrences.

Rationale: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, and idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with approximately 20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Because one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers.Objectives: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAHrelevant cell types. To determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in heritable PAH families, and control populations.Methods: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in more than 300 individuals. Prostacyclin synthase promoter haplotypes' transcriptional activities were determined with luciferase reporter assays. Measurements and Main Results:We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH-relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype.Conclusions: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared with affected patients with PAH.

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 β-catenin, a key transcription factor of the Wnt signaling pathway and cell adhesion molecule, as a CK5 interactor, which we confirmed by coimmunoprecipitation in several breast cancer models. We interrogated the dual functions of βcatenin in relation to CK5. Knockout or knockdown of CK5 ablated β-catenin transcriptional activity in response to progestins and Wnt stimuli. Conversely, CK5 induced by progestins or overexpression was sufficient to promote loss of β-catenin at the cell membrane and total Ecadherin loss. A breast cancer patient-derived xenograft showed similar loss of membrane βcatenin 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-β-catenin interaction may reverse the detrimental properties of CK5+ breast cancer cells. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor positive breast cancer, development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic mRNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here we show that the eIF4F translation pathway was hyperactive in tamoxifen resistant MCF-7L (TamR) breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, use of a pharmacologic agent which inhibited eIF4E-eIF4G interaction also decreased the proliferation and anchorage dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and potentially other endocrine therapies.