Humoral hypercalcemia of malignancy (HHM) is a debilitating syndrome seen in patients with neoplasia of squamous epithelial cell origin. The highest prevalence of HHM is associated with squamous-cell carcinoma of the lung (SCCs) and ranges from 27-66%. HHM results from increased synthesis and secretion of parathyroid hormone-related hormone (PTHrP). The mechanisms that activate PTHrP gene expression in tumors associated with HHM have yet to be identified.
The contribution of the epidermal growth factor receptor (EGFR) to HHM in the human lung SCC cell lines, RWGT2 and HARA was investigated. To test the relationship between EGFR activity and PTHrP gene expression, PTHrP mRNA levels were measured by Q-RT-PCR following treatment of lung SCC lines with the EGFR tyrosine kinase inhibitor (TKI) PD153035, anti-amphiregulin antibodies as well as with EGF-ligands. Overall, PTHrP expression was significantly increased with EGF-ligand treatment. The in vivo relationship between EGFR and PTHrP gene expression was investigated using xenograft HARA and RWGT2 HHM models. Hypercalcemic mice were treated with the TKI, gefitinib. RWGT2 plasma calcium levels were significantly reduced at all time points when compared to pretreatment and control values. In conclusion, autocrine activation of PTHrP gene expression is mediated through the EGFR in the RWGT2 line, however, our results indicated that the major mechanism of HHM induction in the HARA model was not through EGFR but rather the high concentrations of PTHrP secreted by the HARA line were significantly influenced by exogenous factors. The role of a known regulator of calcium homeostasis in humans, the calcium-sensing receptor (CaR) was investigated. Our experiments evaluate evidence for the expression of the CaR in human lung SCC. We examined if PTHrP secretion and HHM occurs in response to CaR stimulation in the RWGT2, HARA and BEN Australia SCCs. We find that CaR is expressed in lung SCCs and stimulation with extracellular calcium increases PTHrP mRNA expression and secretion in all the lines. Mouse xenograft models revealed that CaR is necessary for the rapid development of HHM and overexpression of CaR in a cell line that does not induce hypercalcemia in a xenograft model will cause HHM.