Modeling luminal breast cancer heterogeneity: combination therapy to suppress a hormone receptor-negative, cytokeratin 5-positive subpopulation in luminal disease.

INTRODUCTION: Many Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+ PR+ ones. One such subpopulation we call "Luminobasal" is ER-, PR- and cytokeratin 5 (CK5)-positive. It is not targeted for treatment. METHODS: To address the relationships between ER+PR+CK5- and ER-PR-CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We used high-throughput screening to identify pLB-specific drugs and examined their efficacy alone and in combination with hormone therapy in mixed-cell tumor models. RESULTS: We show that pLUM and MCF7 cells suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and that pLUM cells suppress growth of pLB cells in mixed-cell xenografts in vivo. High-throughput screening of 89 FDA-approved oncology drugs shows that pLB cells are sensitive to monotherapy with the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib. By exploiting mixed-cell 3D colonies and mixed-cell solid mouse tumors models we demonstrate that combination therapy with gefitinib plus the anti-estrogen fulvestrant constitutes a robust treatment strategy. CONCLUSIONS: We propose that response to combination endocrine/EGFR inhibitor therapies in heterogeneous Luminal cancers may improve long-term survival in patients whose primary tumors have been preselected for appropriate biomarkers, including ER, PR, CK5 and EGFR.

GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast.

17ß-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology.

Expression and differential cell distribution of low-threshold Ca(2+) channels in mammalian male germ cells and sperm.

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca(2+) influx through voltage-gated Ca(2+) (Ca(V)) channels. Although the electrophysiological characterization of Ca(2+) currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (Ca(V)3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of Ca(V)3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca(V)3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca(V)3.1 and Ca(V)3.2 were detected in the head, suggesting its participation in the AR. Ca(V)3.1 and Ca(V)3.3 were found in the principal and the midpiece of the flagella. All Ca(V)3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca(V)3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca(V)3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca(V) channels caused small but significant motility alterations. Antibodies to HVA channels detected Ca(V)1.3 and Ca(V)2.3 in human sperm flagella.