Nuclear targeting of a midregion PTHrP fragment is necessary for stimulating growth in breast cancer cells

Parathyroid-hormone related protein (PTHrP) is the primary factor in humoral hypercalcemia of malignancy and is highly secreted by breast cancers. The pro-hormone undergoes post-translational processing and cleavage to give rise to mature secretory peptides, one of which is midregion PTHrP (38-94/95/101) containing a nuclear localisation sequence (NLS) in amino acids (87-106). The current study investigates whether the NLS in midregion PTHrP is important in breast cancer growth. PTHrP-(67-101), a midregion PTHrP fragment containing NLS-(87-101) significantly increased growth of MCF-7 and MDA-MB231 cells (126.3 and 121.3% of control respectively in serum conditions), independent of PTHR1 whereas PTHrP-(67-86), which lacks the NLS did not. Fluorescent-labelled PTHrP-(67-101) translocated to the nucleus, whereas PTHrP-(67-86) remained cytosolic and a scrambled(+NLS) peptide was not internalised. In comparison, no growth influence or uptake was seen in non-tumour breast cells (Hs578Bst). Increases in intracellular calcium mobilisation were observed in breast cancer cells stimulated with both PTHrP-(67-101) and PTHrP-(67-86) (EC(50) of 3.2 pM and 2.2 pM respectively for MCF-7 cells), whereas inositide turnover was not detected. Both nuclear uptake and calcium signalling were attenuated in the presence of EGTA, but not with U73122 or N-terminal PTHrP peptides. Our studies indicate that the NLS-containing midregion PTHrP peptide is dependent on both internalisation and nuclear translocation to induce growth in breast cancer cells. These findings highlight the importance of midregion PTHrP and its receptor in breast cancer growth and may provide potential targets for future therapeutic intervention.