Abstract
It was previously reported that a midregion domain of parathyroid hormone-related protein (PTHrP), that is, [67-86]-amide, is able to restrain growth and promote matrigel penetration by the 8701-BC cell line, derived from a biopsy fragment of a primary ductal infiltrating carcinoma of the human breast, and that cell invasion in vitro is drastically impaired by inactivation of urokinase-plasminogen activator (uPa). In this study we started a more detailed investigation of the possible effects on gene expression arising from the interaction between PTHrP [67-86]-amide and 8701-BC breast cancer cells by a combination of conventional-, differential display-and semi-quantitative multiplex-polymerase chain reaction (PCR) assays. We present here the first evidence that the upregulation of some stress-related genes, most noticeably heat shock factor binding protein-1 (hsbp1) and heat shock protein 90 (hsp-90), is involved in the acquisition of an in vitro more invasive phenotype by cells treated with midregion PTHrP. This is conceivably accomplished by sequestering and inactivating heat shock factor-1 (hsf1) which is able to recognize Ets transcription-factor-binding sites present in some gene promoters, such as those of uPa and matrix metalloprotease-1 (MMP-1). In fact, our data show that incubation of PTHrP [67-86]-amide-treated cells with either antisense hsbp1-oligonucleotide or geldanamycin, an hsp90-inactivating antibiotic, results in downregulation of uPa and upregulation of MMP-1, and in a prominent inhibition of cell invasion in matrigel-containing Transwell chambers. Alternatively, incubation of untreated 8701-BC cells with quercetin, a flavonoid known to decrease the amount of free hsf1, is found to induce upregulation of uPa and downregulation of MMP-1, and an increase of matrigel invasion by cells, thus providing further supporting data of the involvement of hsf unavailability on the modulation of uPa and MMP-1 expression and on cell invasive behaviour. These studies confirm a previous postulate that over-secretion of uPa, rather than of other extracellular proteases, is a primary condition for the increase of invasive activity triggered by PTHrP [67-86]-amide in vitro, and support a role for midregion forms of PTHrP in potentially affecting pathological mammary growth and differentiation. They also identify two new key protagonists in the complex scenario of breast tumor cell invasiveness in vitro, that is, hsbp1 and hsp90, which deserve further and more extensive studies as potential and attractive molecular targets for anti-breast cancer treatments.
Highlights
The parathyroid hormone-related peptide (PTHrP), classically regarded as the mediator of the humoral hypercalcemia of malignancy syndrome, is the product of a gene spanning more than 15 kb of genomic DNA and exhibiting a complex organization in humans, where it generates multiple mRNA variants through alternative splicing events and utilization of different transcriptional start-sites
In this study we demonstrate that treatment of 8701-BC cells with this midregion PTHrP peptide is linked to the upregulation of heat shock factor-binding protein 1, coding for a factor known to interact with heat shock factor1 trimers and negatively regulate hsf1 activity (Satyal et al, 1998; Cotto and Morimoto, 1999), and of some members of heat shock protein family, noticeably hsp90α and -β, and that such over-expression is involved in the modulation of urokinaseplasminogen activator (uPa)- and matrix metalloprotease1 (MMP-1; interstitial collagenase) gene expression and of the invasive behaviour in vitro of 8701-BC breast cancer cells
In consideration of present and literature data, in a third set of assays we investigated whether upregulation of hsbp1 and hsp90 could have some consequence on the levels of expression of uPa and matrix metalloprotease-1 (MMP-1) genes, whose protein products are prominently involved in the acquisition of an invasive phenotype by breast cancer cells
Summary
The parathyroid hormone-related peptide (PTHrP), classically regarded as the mediator of the humoral hypercalcemia of malignancy syndrome, is the product of a gene spanning more than 15 kb of genomic DNA and exhibiting a complex organization in humans, where it generates multiple mRNA variants through alternative splicing events and utilization of different transcriptional start-sites. Translation of PTHrP mRNAs produces three protein isoforms of either 139, 141 or 173 aminoacids with distinct C-terminals, displaying sequence homology with PTH at extreme N-terminus which allows the binding to the same G protein-linked receptor (reviewed by Philbrick et al, 1996). Nuclear targeting sequence which allows nuclear/nucleolar accumulation of expressed PTHrP mediated by the importin β/Ran protein system, by-passing the requirement for binding to cell surface receptors (Lam et al, 1999; Lam et al, 2000); Aarts et al (Aarts et al, 1999; Aarts et al, 2001) have demonstrated the RNA-binding ability of PTHrP and its involvement in ribosome biogenesis, suggesting a role for this protein in intracellular events, such as RNA transcription and/or processing, that influence cell cycle progression (Massfelder et al, 1997). Evidence exist that PTHrP is involved in the control of cell proliferation, differentiation and survival of cartilage and bone cells, its gene being a downstream target for ras and src and an upstream element of the Bcl-2 and c-fos signaling pathways (Li and Drucker, 1994; Amling et al, 1997; McCauley et al, 1997)
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