Mechanisms of receptor-mediated hypercalcemia in human lung squamous cell carcinoma

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 precise mechanisms that activate high concentrations of PTHrP gene expression in tumors associated with HHM have yet to be identified and were investigated in these studies.;The first study evaluates the contribution of the epidermal growth factor receptor (EGFR) to HHM in the human lung SCC cell lines, RWGT2 and HARA. 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. Significant reductions of PTHrP/GAPDH mRNA ratios were noted with either anti-amphiregulin antibodies or TKI 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. HARA plasma calcium levels were reduced after the 78 h treatment whereas calcium concentrations were significantly reduced at all time points when compared to pretreatment and control values in the RWGT2 mice. 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 the tumor microenvironment. Our findings from the EGFR studies lead us to the second series of studies which investigates the role of a known regulator of calcium homeostasis in humans, the calcium-sensing receptor (CaR). Currently, it is unknown if CaR is expressed in normal human lung epithelia or in lung SCC. Our experiments evaluate evidence for the expression of the CaR in human lung SCC. As CaR can use the Ras MAPK pathway for downstream signaling and as increased PTHrP expression and secretion in squamous epithelia results from upstream Ras MAPK signaling, 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 (Ca2+o) increases PTHrP mRNA expression and secretion in all the lines. Furthermore, stimulation of the lung SCCs with Ca2+o results in different patterns of cytosolic calcium oscillations dependant on the variant of CaR. Using mouse xenograft models, we determined that CaR is necessary for the rapid development of HHM and furthermore, that overexpression of CaR in a cell line that does not induce hypercalcemia in a xenograft model will cause HHM. Finally, we present evidence that the known gain of function mutation, R990G, when expressed in an otherwise normocalcemic xenograft model predisposes to an earlier onset of hypercalcemia. Overall, our findings contribute to the understanding of the mechanisms that lead to increased PTHrP secretion and subsequent HHM in patients diagnosed with lung SCC.