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

Abstract

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 posttranslational 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)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98)(99)(100)(101)(102)(103)(104)(105)(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(1NLS) 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. ' 2006 Wiley-Liss, Inc.Key words: PTHrP; breast cancer; NLS; calcium signalling; midregion PTHrP Parathyroid hormone-related protein (PTHrP) was first identified as a tumour-derived protein that was the primary factor in humoral hypercalcemia of malignancy (HHM). 1-3 The first 13 amino acids of PTHrP and parathyroid hormone (PTH) share significant homology 4,5 and it is this sequence which interacts with the parathyroid hormone receptor 1 (PTHR1). 6 As a result, tumour-derived PTHrP enters the circulation and activates PTHR1 in both the bone and kidney to induce PTH-like activity, the outcome of which is hypercalcaemia and osteoclast-mediated bone resorption. 1,2,7 PTHrP is expressed in breast epithelium and is associated with normal mammary gland function, cell growth and differentiation. [8][9][10][11][12] Studies using PTHrP knockout mice resulted in mammary epithelial cell degeneration, 13 emphasising the importance of PTHrP in breast growth. Breast cancers, in most cases, secrete high levels of PTHrP, [14][15][16] and as a result malignancy associated HHM is very common. The expression of PTHrP has also been reported to be higher in breast cancer skeletal metastases than in the primary tumour. 14,17,18 The role of PTHrP may therefore be biolog...

Midregion PTHrP regulates Rip1 and caspase expression in MDA-MB231 breast cancer cells

“…This is consistent with data indicating that midregion PTHrP possesses a nuclear targeting signal (NTS) encompassing aminoacids 87-107 and that the lysine-rich 84-93 domain is per se able to direct importin b/Ran GTPase-mediated import of to the nucleoplasm under the control of phosphorylation/dephosphorylation events in T 85 [24, for review]. Intracrine role for PTHrP in cell growth, adhesion, migration, invasion, and integrin expression regulation has been documented for MCF-7 breast tumor cells over-expressing wild type versus NTS-mutated protein [25,26], and mobilization of intracellular calcium has been observed in different breast cancer cell lines treated with PTHrP (67-101), whose ability to translocate to the nucleus was reported in the same paper by Kumari et al [27]. Noteworthy, intracrine effects of midregion PTHrP domains have been described also in other experimental models [e.g.…”

PTHrP Produced by Myeloma Plasma Cells Regulates Their Survival and Pro-Osteoclast Activity For Bone Disease Progression

“…This approach is in line with the work by other authors to obtain a detectable effect of PTHrP and its fragments in in vitro studies. (19,41,57) In conclusion, we have identified novel pathogenetic functions of PTHrP in MM related to tumor survival, proliferation, and OC hyperactivation. Because serum elevation of PTHrP in patients with solid tumors produces severe skeleton devastation and leads to a poor prognosis, a systematic analysis of PTHrP levels in MM patients would be advisable, to determine whether this peptide is a novel marker of disease progression.…”

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

“…The scrambled sequence (KKVKPKR) was reported as nonfunctional because it is unable to reach the nucleus even when it is localised in the cytoplasm. [44,29,46,47] The bioconjugates of cobaltocenium and ferrocene with NLS scr were totally different in their behaviour towards cellular uptake and nuclear localisation when compared with the wild-type NLS conjugates. These conjugates not only did not gain entry into the nuclei but were also unable to enter the intact cells.…”

“…[45] Various scrambled sequences of NLS in which Lys128 is replaced by other amino acids have been shown to be unable to enter cells and translocate into their nuclei. [29,46,47] Ferrocene (11) and cobaltocenium conjugates (13) of the scrambled NLS sequence, both labelled with FITC, were studied along with a metal-free, FITC-labelled scrambled peptide 9. All three compounds 9, 11 and 13 showed similar behaviour.…”