Functional prostacyclin synthase promoter polymorphisms. Impact in pulmonary arterial hypertension.

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 PAH-relevant 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.

Estrogens and Progestins Cooperatively Shift Breast Cancer Cell Metabolism.

Metabolic reprogramming remains largely understudied in relation to hormones in estrogen receptor (ER) and progesterone receptor (PR) positive breast cancer. In this study, we investigated how estrogens, progestins, or the combination, impact metabolism in three ER and PR positive breast cancer cell lines. We measured metabolites in the treated cells using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Top metabolic processes upregulated with each treatment involved glucose metabolism, including Warburg effect/glycolysis, gluconeogenesis, and the pentose phosphate pathway. RNA-sequencing and pathway analysis on two of the cell lines treated with the same hormones, found estrogens target oncogenes, such as MYC and PI3K/AKT/mTOR that control tumor metabolism, while progestins increased genes associated with fatty acid metabolism, and the estrogen/progestin combination additionally increased glycolysis. Phenotypic analysis of cell energy metabolism found that glycolysis was the primary hormonal target, particularly for the progestin and estrogen-progestin combination. Transmission electron microscopy found that, compared to vehicle, estrogens elongated mitochondria, which was reversed by co-treatment with progestins. Progestins promoted lipid storage both alone and in combination with estrogen. These findings highlight the shift in breast cancer cell metabolism to a more glycolytic and lipogenic phenotype in response to combination hormone treatment, which may contribute to a more metabolically adaptive state for cell survival.

Advances in insulin-like growth factor biology and -directed cancer therapeutics.

The insulin and insulin-like growth factor (IGF) family of proteins are part of a complex network that regulates cell proliferation and survival. While this system is undoubtedly important in prenatal development and postnatal cell growth, members of this family have been implicated in several different cancer types. Increased circulating insulin and IGF ligands have been linked to increased risk of cancer incidence. This observation has led to targeting the IGF system as a therapeutic strategy in a number of cancers. This chapter aims to describe the well-characterized biology of the IGF1R system, outline the rationale for targeting this system in cancer, summarize the clinical data as it stands, and discuss where we can go from here.

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.

Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the 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 messenger RNA 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 (TamR) MCF-7L 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, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the 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.