Reconstitution of amphiregulin-EGFR signaling in lung squamous cell carcinomas activates PTHrP gene expression and contributes to cancer-mediated diseases of the bone

Parathyroid hormone-related protein (PTHrP) is the causative factor of the paraneoplastic syndrome humoral hypercalcemia of malignancy (HHM) and it also contributes to osteolytic metastases, both of which are common complications of squamous carcinomas of the lung. Inhibition of autocrine EGFR signaling has been shown to reduce plasma calcium and PTHrP concentrations in two lung squamous cell carcinoma xenograft models of HHM. The purpose of this study was to investigate the mechanism by which EGFR is activated and stimulates PTHrP gene expression in lung squamous carcinoma cell lines (SCC). Amphiregulin (AREG) was the only EGFR-ligand that could be consistently detected in conditioned media from the SCC lines and reduction of its expression either by siRNA or by precipitating antibodies reduced PTHrP mRNA expression as effectively as EGFR targeted inhibition. Using siRNA knockdown or inhibitors to upstream regulators of AREG shedding including TACE, Src/Lck and G i/o , also reduced PTHrP mRNA expression. We determined that blockade of autocrine AREG-EGFR signaling does not affect PTHrP mRNA stability. Of the three PTHrP promoters (P1, P2, P3), P1 mRNA could be reduced by nearly 100% with an EGFR inhibitor, and both EGF and AREG stimulated P1 mRNA by ∼5-fold. Finally, ectopic expression of EGFR in a receptor-low but AREG expressing cell line increased PTHrP mRNA levels in vitro , and induced the capability to cause HHM and rapid osteolytic growth in vivo . Taken together, we provide evidence that AREG stimulation of EGFR results in high levels of PTHrP gene expression, contributing to cancer-associated bone pathology.