1. Introduction: PTHrP is a Multidomain and Multifunctional Protein

Parathyroid hormone-related protein (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 promoters. The initial translation products are three isoforms of 139, 141, and 173 amino acids with distinct C-terminals, displaying sequence homology with PTH at the extreme N-terminus, which allows the binding to the same G protein-linked receptor PTH1R. Indeed, PTHrP is a prohormone, the post-translational processing of which generates a family of mature secretory forms of the peptide. Apart from PTHrP (1–36), which contains homology with PTH and is able to activate the PTH/PTHrP receptor, a midregion fragment, i.e., PTHrP (38–94), and a C-terminal fragment of PTHrP surmised to be comprised of PTHrP (107–139), have also been identified as mature secretory forms of the protein. It is now known that the latter acts on skin, heart and bone cells. PTHrP (38–94), whose cell receptor is still unknown, is able to activate intracellular Ca⁺⁺ pathways at low concentrations, being critical for driving Ca⁺⁺ transport from the maternal to the fetal circulation through the placenta, which is the sole normal tissue in which this PTHrP domain has been shown to exert an effect ^[1]. Noteworthy, midregion PTHrP has also been shown to exert effects in malignant tissues ^[2], which will be discussed in greater detail later. It is widely-acknowledged that midregion PTHrP possesses a lysine/arginine-rich bipartite sequence, encompassing amino acids 87–107, which is homologous to the nuclear/nucleolar targeting signal (NTS) present in SV40 large tumor antigen, able to direct importin β/Ran GTPase-mediated import thereby allowing the peptide to play the so-called “intracrine” role that supplements the “autocrine/paracrine” one. In the case of intracrine pathway, the endogenous PTHrP is synthesized starting from alternative translation start sites bypassing the PTHrP N-terminal signal sequence. The translated protein cannot enter the endoplasmic reticulum, but translocates to the nucleoplasm due to its NTS ^[3]. Several published papers have reported the activity of PTHrP or its discrete domains as inducers of cell proliferation or, conversely, cell death and apoptosis in non pathological model systems [4-7]^[4][5][6][7].

Interestingly, one of the original tumors from which PTHrP was purified and sequenced, because of the elevated concentration of its circulating form, was breast cancer [8,9]^[8][9] and it is now well-known that PTHrP exerts multiple effects on different neoplastic cytotypes via cytosolic and nuclear targets, thereby participating to initiate and promote tumor growth and dissemination ^[10]. It is known that PTHrP participates in various complex signaling pathways via its membrane and nuclear effects: evidence exists that the protein and/or its discrete fragments can activate protein kinase C and A routes as well as protein G βγ dimer-mediated mechanisms and that p21, AKT and NFκB are among the final targets of such signals ^[11].

2. PTHrP as an Anti-Proliferative and Death-Promoting Factor

2.1. PTHrP and Osteosarcoma Cells

In 1997, Valin and co-workers ^[12] demonstrated the growth inhibitory effect of the PTHrP (107–111) and (107–139) domains on the rat osteosarcoma cell line UMR 106 through a protein kinase C- (PKC) dependent mechanism. This effect, ascribed to the sole C-terminal fragments, was apparently mediated by a then-unknown receptor, different from the “classical” PTH/PTHrP receptor, binding the N-terminal sequence shared by the two hormones. It is now strongly suggested that C-terminal PTHrP intracellular signaling occurs via the putative TRSAW receptor, recognizing to the Thr¹⁰⁷ Arg¹⁰⁸ Ser¹⁰⁹Ala¹¹⁰ Tyr¹¹¹ portion of the peptide, which is able to activate membrane–bound PKC activity but not the adenylate cyclase ^[13]; interestingly, a similar effect was also recorded in rodent and human skin keratynocytes. In addition, PTHrP (107–111) was also found able to induce the intracellular Ca⁺⁺ signal via transient opening of L-type Ca⁺⁺ channels, thereby affecting the expression of genes whose promoters contain Ca⁺⁺-responsive elements and/or other intracellular pathways such as those dependent upon Ca⁺⁺-calmodulin [1,10,14]^[1][10][14].

2.2. PTHrP and MDA-MB231 Breast Cancer Cells

In 1995, Luparello and collaborators ^[15] tested the effect of different PTHrP domains on the proliferative behavior of 8701-BC, a cell line derived from a primary ductal infiltrating carcinoma (d.i.c.) of the human breast and thereby being more representative of the heterogeneity present in the tumor cell population in vivo ^[16]. The results obtained indicated that PTHrP (67–86) and, more prominently (107–138) and (1–34), exerted anti-proliferative but pro-invasive effects to different degrees, which were abolished by incubation with anti-PTHrP antibody. In addition, experiments with clonal cell lines isolated from the parental 8701-BC line and endowed with different proliferative and invasive properties in vitro demonstrated the heterogeneity of the growth and invasive response of the different subpopulations to administration of PTHrP fragments, thereby suggesting the existence of complex PTHrP-breast cancer cell interplays in the affected tissue ^[17]. Further data obtained after the exposure of 8701-BC cells to PTHrP (67–86) indicated the effect on the modulation of gene expression, in particular identifying heat shock factor binding protein-1 and 90 kDa-heat shock protein as the up-regulated genes. In turn, such over-expression was found to be involved in the modulation of the expression of urokinase-plasminogen activator and matrix metalloprotease-1 and, consequently, in the acquisition of an invasive behavior in vitro by this cell line ^[18].

Another set of data was obtained using the estrogen receptor (ER)-negative and highly malignant MDA-MB231 breast cancer cell line as a model system. In Luparello and coworkers' paper in 2001 ^[19], cell viability, proliferation, invasiveness and growth in nude mice was examined following administration of the midregion (38–94) fragment of PTHrP, which was proven to reduce markedly breast cancer growth and invasion in vitro and in vivo, thereby representing an attractive target for molecular modeling of smaller, orally active anti-neoplastic agents. Subsequent studies revealed that this midregion peptide is imported in the cell nucleoplasm and is endowed with DNA-binding properties in vitro, thus potentially acting as a transcription factor-like molecule. An effect at the gene expression level was reported, especially addressed to the regulation of some apoptosis-related genes (Bad, Bcl-xS, Receptor-interacting protein 1, caspase-2, -5, -6, -7 and -8) that may be responsible, at least in part, for the lethal effect imparted by PTHrP (38–94) on MDA-MB231 cells [20-22]^[20][21][22].

Interestingly, some opposite results, i.e., growth-promoting, were found using the ER-positive MCF-7 cell line. In particular, in 2009, Alokail ^[23] investigated the effect of different N-terminal PTHrP domains on epidermal growth factor receptor (EGFR)-transfected cells and demonstrated that the receptor induced ERK activation via heregulin β1 and PTHrP and both ERK and PKC induced the mitogenic effects via MAPKs, thereby potentially explaining the PTHrP-dependent proliferative effects.

2.3 PTHrP and Lung Cancer Cells

Although listed among the anti-proliferative and death-promoting examples, conclusions on the effect of PTHrP on lung cancer cells cannot be drawn as in the literature this topic is still under discussion. In 1990, Burton and collaborators ^[24] reported the growth stimulatory effect of PTHrP (1–34) on BEN squamous cell lung carcinoma cells in vitro, whereas in 2001, Hastings and co-workers ^[25] demonstrated that the peptide inhibits the growth of the same cells and that exposure to anti-PTHrP antibody stimulated the growth of BEN cell carcinomas in athymic mice, thereby highlighting the paracrine growth inhibitory effects of N-terminal PTHrP fragment on lung tumor. This discrepancy is probably a consequence of clonal heterogeneity of the cell line. Moreover, anti-apoptotic effects of PTHrP (1–34) and (140–173) on BEN cells have been reported ^[26] and could contribute to lung cancer progression. Interestingly, Hastings and coworkers ^[27] have also documented that cyclic thiourea compounds inhibit PTHrP expression mediated by the P3 promoter in BEN cells and that they may inhibit growth of lung cancer cells through the same mechanism. A patient population study ^[28] reported the longer survival of women affected with PTHrP-secreting lung carcinomas; in addition, more recently Monego and collaborators ^[29] showed that the expression of both PTHrP (1–34) and PTH1R are independent prognostic markers of a worse clinical outcome in lung adenocarcinoma patients.

When PTHrP production-lacking lung adenocarcinoma cells, transfected with with a pciNeo-PTHrP 1–87 expression plasmid, were examined for their growth and intracellular signalization aspects, the reduced mitogenesis observed was found linked to a block in G₁ and, from a molecular point of view, to the decreased expression of cyclin D2 and cyclin A2, increased expression of p27, decreased association of cyclin A2 and CDK2, and increased activation of ERK ^[30]. The authors, therefore, stressed the necessity of further investigation to explore this promising association between N-terminal PTHrP, slowing of tumor progression and increased patient survival. Noteworthy, the cited works did not consider the acknowledged intracrine effect mediated by PTHrP NTS, whose impact on lung cancer cell viability and proliferation awaits further investigations.

3. PTHrP as a Stimulator of Cell Survival and Proliferation of Tumor Cells: A Promising Target for Therapeutic Intervention

A number of experimental data obtained on different neoplastic model systems have brought evidence that PTHrP is a pro-survival, anti-apoptotic and proliferation-promoting factor, thereby highlighting the potential therapeutic benefit of the modulation of PTHrP production.

Already in 1995, in fact, Rabbani and coworkers ^[31] had observed that the application of PTHrP antisense strategy to an animal model of Leydig cell tumor produced a significant decrease of doubling time in vitro and the lowering of tumor volume when antisense-transfected cells were inoculated into recipient rats. Few years later, PTHrP was shown to exert a positive influence also on the size of primary prostate carcinoma in rats and its protection against apoptotic stimuli on neoplastic cells was first suggested ^[32]. Dealing with MCF-7 ER-positive breast cancer cells, in 2000, Falzon and Du ^[33] proposed that “intracrine” and “autocrine/paracrine” pathways could transduce opposite instructions to cells, the former being proliferation-restraining whereas the latter inducing cell growth, thus adding a further level of complexity to the biological responses of cells to PTHrP. Additional experiments on the effect of intracrine signalization demonstrated that over-expression of wild-type vs. NTS-mutated PTHrP determined the increase of the percentage of cells in G₂/M phases of the cell cycle, and Bcl-2/Bax and Bcl-xL/Bax ratios, indicative of protection from apoptosis ^[34].

More recently, investigation on the mechanism of action of PTHrP has been focused mainly on prostate, colon and renal cancer cells, although scattered reports of interest on chondrosarcoma, medulloblastoma, anaplastic thyroid and adrenocortical tumor cells have also appeared.