The role of PTHrP and Apo2L/TRAIL in breast cancer

© 2012 Dr. Vanessa CheungParathyroid hormone-related protein (PTHrP) is a key component in breast tumour biology. PTHrP is one of the main factors implicated in breast cancer osteolysis. Clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis. Furthermore, PTHrP has been described to cause tumour cell death both in vitro and in vivo. A promising new anti-cancer agent is Apo2L/TRAIL due to its ability to selectively induce apoptosis in cancer cells whilst sparing most normal cells. However, some cancer cells are resistant to Apo2L/TRAIL-induced apoptosis thus limiting its therapeutic efficacy. Since both PTHrP and Apo2L/TRAIL has profound effects on breast cancer, the actions of these two factors were assessed. The effect of PTHrP on gene expression in breast cancer cells was studied. PTHrP was overexpressed in two breast cancer cell lines; MCF-7 and MDA-MB-231, and knocked down in MDA-MB-231, and analysed gene expression by microarray analysis. Cytochrome p450 1B1 (CYP1B1) was identified as a potential factor differentially altered by PTHrP in breast cancer cells. To assess the relationship of PTHrP with factors in the cell death pathways, PTHrP overexpressing breast cancer cells were treated with Apo2L/TRAIL. PTHrP overexpression increased MDA-MB-231 cells sensitivity to Apo2L/TRAIL-induced apoptosis. Interestingly, PTHrP sensitised MCF-7 cells to Apo2L/TRAIL-induced apoptosis. Apo2L/TRAIL-induced apoptosis in PTHrP overexpressing cells through the activation of caspase-10 resulting in caspase-9 activation and induction of apoptosis through the effector caspases, caspase-6 and -7. Studies with PTHrP peptides 1-34, 38-94, 67-86, 107-111, 106-139, 120-139 and 107-139 on MDA-MB-231 cells showed no effect on Apo2L/TRAIL-induced apoptosis, indicating that PTHrP’s action were unlikely to result from extracellular receptor signalling. PTHrP did not alter mRNA expression of Apo2L/TRAIL receptors; TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and OPG, however increased cell surface expression the death receptors TRAIL-R1 and TRAILR2 was observed. Antagonistic antibodies against the death receptors demonstrated that Apo2L/TRAIL preferentially bound to TRAIL-R2 to signal apoptosis. To identify potential genes involved in Apo2L/TRAIL resistance and/or sensitivity gene microarray experiments were performed comparing two breast cancer cell lines with differential sensitivities to Apo2L/TRAIL. CYP1B1 and CARD10 were identified to be differentially expressed. CARD10 was regulated by Apo2L/TRAIL in MDA-MB-231 cells, however there was no associated with PTHrP. CYP1B1 mRNA expression was decreased in PTHrP overexpressing cells. CYP1B1 mRNA expression was further down regulated by Apo2L/TRAIL in PTHrP overexpressing breast cancer cells. Overexpression studies of CYP1B1 in PTHrP overexpressing cells did not affect Apo2L/TRAIL-induced apoptosis, indicating that CYP1B1 is not a direct mediator of PTHrP action. However, overexpression of CYP1B1 sensitised MCF-7 cells to Apo2L/TRAIL-induced cell death. These studies have revealed a novel role for PTHrP with Apo2L/TRAIL that maybe important for future diagnosis and treatment of breast cancer