Epigenetic silencing of the endothelin-B receptor gene in non-small cell lung cancer.

Endothelin-1 is overexpressed in several tumor types. Activation of the endothelin-A (ETA) receptor may promote cell growth, angiogenesis and invasion, and inhibits the apoptotic process, while activation of the endothelin-B (ETB) receptor may induce cell death by apoptosis and inhibit tumor progression. Hypermethylation and subsequent silencing of the ETB receptor gene promoter has been reported in some cancer types. As the endothelin pathway is subject to research for pharmacological cancer treatment, we investigated the extent of epigenetic deregulation of the ETB receptor gene in non-small cell lung cancer (NSCLC). We scanned 64 NSCLC paired tumor/normal surgical specimens for the ETB receptor promoter for methylation by developing four pyrosequencing assays that covered 24 CpGs. The ETB receptor promoter was significantly hypermethylated in 31 (48%) of tumor samples, presenting considerably higher methylation in 22/24 CpG sites compared with the normal counterpart tissues. ETB receptor mRNA levels were reduced in all lung tumors compared with normal adjacent lung tissue, indicating the potentially important involvement of this gene in lung cancer development. Furthermore, tumor samples with ETB receptor gene methylation tended to have lower receptor mRNA levels compared with unmethylated tumor specimens, suggesting a primary epigenetic role in ETB receptor silencing. Our results point to a significant involvement of ETB receptor epigenetic deregulation in the pathogenesis of lung cancer making the gene a promising candidate biomarker for response to regimens modulating the endothelin axis.

Preclinical anticancer activity of the specific endothelin A receptor antagonist ZD4054.

Endothelins are a family of small peptides (ET-1, ET-2, and ET-3) that mediate various physiological processes of mitogenesis, repair, and tissue differentiation by binding to endothelin A (ETA) and endothelin B (ETB) cell surface receptors. Activation of the ETA receptor by ET-1 has emerged as an important factor promoting tumor cell proliferation, survival, angiogenesis, migration, invasion, and metastasis in several tumor types including prostate, ovary, colon, cervix, breast, and lung. As activation of the ETB receptor has an opposing effect, inducing cell death by apoptosis, a rationale exists for specific antagonism of the ETA receptor as a treatment strategy for cancer. ZD4054 is a specific ETA receptor antagonist currently being evaluated in hormone-resistant prostate cancer in phase III clinical trials. In vitro, ZD4054 reversed ET-1-mediated inhibition of apoptosis in serum-deprived rat A10 and human VLTR-16 cells in a concentration-dependent manner. ZD4054 inhibited ET-1-mediated survival signaling pathways and decreased proliferation in ovarian OVCA 433 and HEY cells and in prostate PPC-1 and LAPC-4 cells. In A673 rhabdomyosarcoma cells, ET-1-induced phosphorylation of FAK, FAK, and paxillin was reversed with ZD4054, inhibiting the invasive phenotype mediated by these adhesion factors. In vivo, ZD4054 led to a significant reduction in tumor growth in animals bearing ovarian tumor xenografts, and significantly inhibited tumor angiogenesis. Pretreatment with ZD4054 also significantly delayed the onset of metastatic events after intracardiac injection of bladder TSU-Pr1-B1 cells in mice. These preclinical data show the potential anticancer effects of the specific blockade of the ETA receptor with ZD4054, supporting a program of clinical investigation.