The endothelins and their receptors are best known for their regulation of the vascular system. Their widespread expression in epithelial cells and their overexpression in some tumors has prompted investigation into their ability to regulate cancer progression. In this study, we assessed the mRNA expression of the major endothelin B receptor gene (EDNRB) isoforms and found differences in both mRNA and protein expression in normal breast cells and breast cancer cell lines. Knocking down the EDNRB gene in breast cancer cells altered invasiveness toward endothelin 3 (ET3), and we observed EDNRB isoform-specific regulation of breast cancer cell invasion and cell signaling, as well as isoform-and subtype-specific differences in breast cancer patient survival. The results reported in this study emphasize the importance of the endothelin B receptor in breast cancer. To our knowledge, this study is the first to clarify the differential expression and roles of specific EDNRB isoforms in breast cancer.

Abstract. Calcium/calmodulin-dependent protein kinase (CaM Kinase) proteins are targets of hormones and growth factors and regulate cancer cell growth, apoptosis and migration. The hormone estrogen (E2) utilizes CaM Kinases to activate the Extracellular-signal regulated kinase (ERK) leading to MCF-7 breast cancer cell growth. The hormone Vitamin D (Vit D) may inhibit breast cancer cell growth however the cellular mechanisms of Vit D action remain to be elucidated. Within the present study we provide data that E2 stimulation of MCF-7 cells activates CaM Kinase Kinase (CaM KK), CaM KI, and ERK and the transcription factors Elk-1 and SRF. E2 treatment of MCF-7 cells potently stimulated Elk-1/SRF directed transcription of SRE-luciferase reporters. E2 activation of ERK, Elk-1, SRF and SRE-dependent luciferase activities were blocked by treating cells with the CaM KK inhibitor, STO-609, and the ERK inhibitor, U0126. Moreover, siRNAs directed against CaM KK and ERK blocked transcription factor phosphorylation and luciferase activity. Treatment of cells with Vit D, the hormone Epinephrine (Epi), or Forskolin, prevented E2 activation of CaM KK and ERK. Interestingly, Vit D promoted a PKA-dependent phosphorylation and inhibition of CaM KK as well as its association with 14-3-3. Epi and Vit D treatment of cells blocked the ability of E2 to active Elk-1 and SRE-luciferase activity. This data suggests an important role for CaM KK and ERK in regulating transcription downstream of E2 in MCF-7 cells. Our results also suggest that Vit D treatment of MCF-7 cells utilizes a unique PKA-dependent mechanism to block E2 activation of CaM KK, ERK and transcription.

No abstract

The endothelin axis, comprised of the active peptides endothelin 1-3 (ET1-3), the endothelin A receptor (ETAR) and endothelin B receptor (ETBR), and endothelin converting enzyme 1 (ECE1) has been studied in numerous cancers. In clinical samples of breast cancer, increased levels of ET1, ECE1, and the endothelin receptors have been reported, and several studies correlate expression of these proteins with poor prognosis. The endothelin pathway is known to induce mitogen activated protein kinase activation, leading to increased survival, proliferation, and migration, but the precise role of ECE1 and the endothelin receptors in breast cancer invasion is not fully understood. We hypothesized that the endothelin axis is instrumental in modulating breast cancer cell invasion. To initially confirm that the endothelin axis regulates breast cancer cell invasion, we measured MDA-MB-231 breast cancer cell invasion using an in vitro assay in the presence of an ECE1 inhibitor, CGS 35066, and found that abrogating ECE1 decreases breast cancer invasion. Next, we measured expression of ECE1 mRNA and protein using RT-PCR and Western blots in normal human mammary epithelial cells (HMECs), low-invading MCF-7 breast cancer cells, and highly invasive MDA-MB-231 breast cancer cells. We found that the major ECE1 isoform, ECE-1c, was most highly expressed on MCF-7 cells and that HMECs expressed more of the intracellular ECE1 isoform, ECE-1b, than either cancer cell line. Western blot analysis confirmed these results, as total ECE1 protein expression was highest in the MCF-7 cells. While multiple studies have been published on the expression of the endothelin receptors ETAR and ETBR in clinical breast cancer samples, less is known about the specific expression and function of these receptors in these breast cancer cell lines. Western blot analysis demonstrated notable up-regulation of ETAR expression in the MCF-7 and MDA-MB-231 cells compared to the HMECs, which is consistent with clinical findings. Interestingly, ETBR expression was expressed in HMECs but its expression was low or negative in both breast cancer cell lines. Previous studies have demonstrated that knockdown of ETAR inhibits breast cancer cell invasion, but whether ETBR functions similarly in breast cancer is unknown. To determine if ETBR might be regulating invasion in mammary cells, we transfected MCF7 cells with ETBR-specific siRNA and found that knocking down the gene encoding for ETBR (EDNRB) resulted in significantly increased cell invasion. This result suggests that while ETAR positively regulates breast cancer cell invasion, as reported in other studies, ETBR may be negatively regulating invasion through the endothelin axis. Together, our findings support an important role for the endothelin axis in breast cancer invasion, and specifically implicate ETAR and ETBR as performing complimentary and opposing roles in the regulation of breast cancer cell invasion. Citation Format: Mary Elizabeth Belles, Meena Halaka, Jessica M. Do, Parth Majmudar, Reem Sidani, Hannah Stephen, Molly Watson, Rebecca E. Conway. Expression and function of endothelin converting enzyme 1 and endothelin receptors in breast cancer invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A58.