PTHrP induces STAT5 activation, secretory differentiation and accelerates mammary tumor development

Parathyroid hormone-related protein (PTHrP) was originally discovered as a cause of elevated calcium levels in patients with cancer [1‐3]. It is evolutionarily related to parathyroid hormone (PTH) and the amino-terminal portions of both proteins are highly homologous, allowing them to bind and activate the same Type 1 PTH/PTHrP receptor (PTH1R) [2, 3]. As a result, when PTHrP is secreted by tumors, it mimics PTH, leading to excessive bone resorption and hypercalcemia. PTHrP also contributes to the development and physiologic functions of a variety of tissues, and it has been shown to affect cell proliferation and cell death in a number of settings [2‐5]. While many of its functions are mediated by the PTH1R, PTHrP can also remain within the cell to regulate proliferation, differentiation and survival through an intracrine mode of action requiring the translocation of PTHrP into the nucleus [3, 6‐10]. Although nuclear translocation appears to be important for PTHrP biology, details of this signaling pathway remain obscure.

PTHrP and the PTH1R are expressed throughout the life cycle of the mammary gland as well as in breast tumors. Both molecules are required for fetal breast development in mice and humans [11‐14]. PTHrP also has important functions during lactation. Its production is greatly upregulated in alveolar epithelial cells, and it is secreted into both milk and the maternal circulation [15‐17]. In the maternal circulation, PTHrP acts on bone cells to mobilize calcium from the skeleton that is subsequently used by the mammary gland for milk production. In addition, PTHrP in milk regulates total body calcium accrual in suckling neonates, acting to coordinate maternal and neonatal calcium economy [18].

PTHrP is also produced by breast cancers, contributing both to their growth and to tumor-induced changes in systemic metabolism [5, 15, 19]. When produced by breast cancer cells within the bone microenvironment, PTHrP contributes to osteolytic bone destruction and the expansion of bone metastases [5, 20, 21]. In addition, genome-wide association (GWAS) studies have implicated the PTHLH (PTHrP) gene as a breast cancer susceptibility locus [15, 22‐24], suggesting that it may contribute to early steps in transformation and/or cancer progression. However, the exact functions of PTHrP in breast cancer biology remain unsettled. Different studies have reported that its expression either correlates with increased or decreased metastases and survival [10, 25‐29]. Moreover, studies have variably reported that PTHrP either stimulates or inhibits the proliferation, differentiation and survival of breast cancer cells [3, 5, 10, 21, 30‐33]. These contradictory results concerning the role and prognostic value of PTHrP expression in breast cancer underscore the need to better understand how it modulates breast tumor growth and/or breast cancer susceptibility.

In order to examine the effects of PTHrP on mammary tumor development in mice, we developed a tetracycline-regulated, MMTV-driven model of PTHrP overexpression in mammary epithelial cells (MMTV-rtTA;TetO-PTHrP). We found that overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium enabling virgin mice to produce milk. This phenotype was associated with activation of STAT5, and increased expression of Elf5 (E74-like factor-5), both important regulators of alveolar secretory differentiation [34‐37]. Furthermore, overexpression of PTHrP in epithelial cells in MMTV-PyMT mice dramatically promoted the formation of mammary tumors by shortening tumor latency and accelerating tumor growth, ultimately reducing overall survival. Interestingly, tumors overproducing PTHrP expressed markers of secretory differentiation and expressed milk constituents. These data suggest that PTHrP promotes pathways leading to secretory differentiation in both normal mammary epithelial cells and in breast tumor cells.

The data presented in this study demonstrate that overexpression of PTHrP in mammary epithelial cells activates a program of secretory differentiation. When treated with Dox to induce human PTHrP(1–141) expression, the mammary glands of virgin, Tet-PTHrP mice develop alveolar hyperplasia, express histological markers of secretory differentiation, activate milk protein gene expression, and accumulate intracellular lipids. These secretory changes are accompanied by the phosphorylation of STAT5 and an increase in the expression of Elf5, two pioneering transcription factors well described to cooperate in driving gene expression necessary for milk production [34‐37, 60]. Consistent with the activation of these transcription factors, we found that PTHrP upregulates patterns of gene expression previously associated with lactation. It is also possible that PTHrP increases the pool of alveolar progenitors available for secretory differentiation rather than directly causing differentiation of the cells. However, alveolar hyperplasia and the expression of secretory differentiation markers are significantly reversed in response to the withdrawal of Dox, suggesting that they depend on the continuing presence of PTHrP. Previous results from our lab demonstrated that, although PTHrP expression is normally activated during lactation, mammary gland specific ablation of PTHrP affects systemic calcium metabolism during lactation but does not interfere with alveolar development or with overall milk secretion [16, 45]. Given the importance of lactation to mammalian reproduction, it is not surprising that the pathways leading to secretory differentiation would be redundant. Nevertheless, these new data clearly demonstrate that PTHrP is sufficient to induce secretory differentiation in luminal epithelial cells in the absence of pregnancy.

PTHrP overexpression also drives secretory differentiation in tumor cells in the MMTV-PyMT model of breast cancer. Tet-PTHrP;PyMT mice continuously exposed to Dox develop tumors in all mammary glands by 3–4 weeks of age, a dramatic decrease in tumor latency in comparison to Tet-PTHrP;PyMT mice not treated with Dox. While PTHrP promoted premature growth of PyMT-associated mammary tumors, overexpression of PTHrP alone did not efficiently induce tumors. Therefore, in this setting, PTHrP appeared primarily to promote tumor growth rather than initiate transformation. The decrease in tumor latency was accompanied by increased rates of proliferation in the tumors. As noted previously in vascular smooth muscle cells and in human and murine breast tumor cells, increased proliferation was associated with decreased expression of the cell cycle inhibitor, p27Kip1 [30, 76]. This acceleration of tumor development is consistent with the reciprocal results of Li et al., who showed that ablation of PTHrP expression in MMTV-PyMT tumors slowed tumor growth and was associated with reduced proliferation and increased apoptosis [31]. They are also consistent with prior results from our group demonstrating that ablation of the CaSR in MMTV-PyMT tumors or in human BT474 and MDA.MB-231.1833 cells reduced PTHrP expression, which was associated with a reduction in proliferation and increased expression of p27kip1 [30]. Importantly, these effects of PTHrP do not appear to result from an increase in PyMT expression although we cannot exclude the possibility that nuclear PTHrP affected the direct biological effects of PyMT itself. Thus, although there have been variable reports on the effects of PTHrP on human breast cancer cell lines, in mice, PTHrP clearly promotes proliferation in mammary tumor cells expressing PyMT.

While PTHrP accelerates the growth of PyMT tumors, it also promotes secretory differentiation. This is associated with an increase in the expression of differentiation markers, milk protein genes, Elf5, and pSTAT5. Despite an apparent shift to a more differentiated state, tumors continued to metastasize, and cells derived from the tumors overexpressing PTHrP were able to form new tumors when transplanted into non-transgenic mice. The histological appearance of the tumors in Tet-PTHrP;PyMT mice, their expression of pSTAT5 and the accumulation of milk-like secretions is reminiscent of a rare type of human breast cancer known as “secretory carcinoma of the breast” [77‐81]. The majority of these tumors have been shown to contain t(12;15)(p13;q25) chromosomal translocations that results in a fusion oncogene (ETV6-NTRK3) consisting of the oligomerization domain of ETV6 fused to the protein tyrosine kinase domain of the neurotropin 3 receptor (NTRK3). Although most secretory carcinomas behave in an indolent manner, some patients develop metastatic lesions. When an ETV6-NTRK3 construct was knocked into mice, they developed mammary alveolar hyperplasia, followed by the development of multifocal tumors with short latency, again reminiscent of the effects of overexpressing PTHrP on PyMT-mediated tumorigenesis [80]. Although there is no known link between PTHrP expression and the expression or activity of NTRK3 or other neurotropin receptors, it has been suggested that the transforming ability of the ETV6-NTRK3 fusion oncogene relies on activation of the AP1 transcription complex [80]. Given the similarities between PTHrP overexpression in PyMT tumors and this model of secretory carcinomas, as well as the fact that PTHrP has been shown to activate AP1 signaling by increasing c-fos and/or JunB expression in several cell types, further study of potential interactions between PTHrP and AP1 signaling in breast cancer may be revealing [82, 83].

Multiple lines of evidence suggest that the effects of PTHrP on activating secretory differentiation pathways as well as on promoting tumor cell proliferation are mediated by an intracrine pathway rather than through its cell surface receptor. First, previous experiments overexpressing PTHrP in mammary gland myoepithelial cells did not lead to alveolar hyperplasia and secretory differentiation although, similar to the results reported here, it did inhibit ductal elongation during puberty [39, 73]. These differences are not compatible with a typical paracrine mode of action given that the 2 cell types overexpressing PTHrP in these different models are adjacent to each other. Instead, the different phenotypes in these models suggest that a cell-autonomous and cell-restricted mechanism of action drives the secretory differentiation. Second, in cells derived from Tet-PTHrP;PyMT mammary tumors, inducing PTHrP expression by treating them with Dox stimulates cell proliferation, activates STAT5 and increases milk protein gene expression, but treating the cells with exogenous PTHrP does not. Thus, PTHrP is sufficient to induce secretory differentiation, but only if produced within the tumor cells, suggesting a cell autonomous and intracrine mechanism. Third, reducing PTH1R expression in tumor cells does not alter tumor growth or secretory differentiation of the tumor cells, demonstrating that tumor expression of the PTH1R is not required for the observed phenotype. Lastly, treating tumor-bearing Tet-PTHrP;PyMT mice with anti-PTH1R antibodies corrects hypercalcemia but does not reverse STAT5 activation or reduce the expression of secretory markers, demonstrating that secreted PTHrP does not act systemically or on non-tumor cells in the microenvironment to induce paracrine cascades supporting secretory differentiation. These results are consistent with the observations of Tran et al., who previously reported that nuclear PTHrP staining correlates with nuclear pSTAT5 staining in human breast cancers [10]. In addition, Johnson et al. showed that PTHrP overexpression in MCF7 cells results in the downregulation of several pro-dormancy genes and suggested that these actions may occur through PTH1R-independent actions [74]. Finally, prior results from our laboratory have demonstrated that intracrine/nuclear actions of PTHrP downstream of the calcium-sensing receptor are important in modulating cell proliferation and survival in human breast cancer cell lines and in PyMT-induced mouse mammary tumors [30].

PTHrP is widely recognized to be important for the progression of osteolytic bone metastases from breast cancer [20, 84], although its role in the initiation, growth or progression of primary breast tumors is less clear. The results we report here agree with those of Li et al., demonstrating that PTHrP stimulates mammary tumor progression and results in shorter survival in MMTV-PyMT mice [31]. As compared to studies in mice, PTHrP has been variably suggested to either promote or to inhibit breast cancer cell proliferation, differentiation and death in human breast cancer cell lines [3, 5, 10, 30, 33]. Likewise, studies examining PTHrP staining in human breast cancers have reported differing correlations between PTHrP and tumor behavior. Some studies have reported that PTHrP expression correlates with estrogen receptor and progesterone receptor expression, a more differentiated histology, fewer metastases and a better prognosis [10, 26]. In contrast, other studies have suggested that increased PTHrP expression predicts worse survival and increases brain or bone metastases when measured in all breast tumors, in triple-negative breast cancers or in circulating tumor cells [25, 28, 85, 86]. One possible explanation for these conflicting results may be related to differing effects of PTHrP in luminal vs. triple negative sub-types of breast cancer. Another may relate to our observation that PTHrP overexpression results in the upregulation of STAT5 activation. STAT5 is critical to the proliferation and secretory differentiation of normal breast epithelial cells during pregnancy and lactation, but it seems to mirror PTHrP in having different effects on tumor progression in mice and humans. Loss of STAT5 impedes the development of tumors in T-antigen-dependent mouse models, while overexpression of wild-type or constitutively active STAT5 accelerates tumor formation in these models [36, 87‐89]. By contrast, the activation of STAT5 in human breast cancers has generally been observed to be an indicator of more differentiated tumors and a better prognosis [10, 87, 90]. Our findings and those of Tran et al. mirror the previous literature in that PTHrP expression increases STAT5 and tumor progression in MMTV-PyMT mice, but PTHrP expression correlates with nuclear STAT5 expression and better outcome in human breast cancer. This may not be the entire bottom line given the recent report from Assaker and colleagues suggesting that tumor PTHrP expression at the time of diagnosis correlated with subsequent brain metastases and poor survival in patients with triple negative breast cancer (TNBC) [85]. Interestingly, we found the highest numbers of cells with elevated PTHrP gene expression in TNBC’s using single cell sequencing data. Furthermore, genes potentially involved in Stat5 signaling were enriched in TNBC cells expressing higher levels of PTHrP. Therefore, it is possible that interactions between PTHrP and STAT5 may have different consequences depending on the sub-type of breast cancer. Sorting out the details of when and how PTHrP affects different breast cancers in different fashions will be critical to understanding the reported association between the PTHLH gene and breast cancer in GWAS studies [22‐24].

In summary, we report that PTHrP overexpression activates STAT5, increases Elf5 expression, and leads to increased proliferation and secretory differentiation of both normal mammary epithelial cells and mammary tumor cells in mice. This is the result of an intracrine pathway rather than a function of secreted PTHrP. In addition, the greatest proportion of tumor cells with elevated PTHrP expression was found in triple negative breast cancers, where higher PTHrP mRNA levels correlated with an enrichment of STAT5-related gene expression. Our data confirm previous findings that nuclear PTHrP localization correlates with STAT5 activation and increased differentiation in human breast cancers [10]. Therefore, further work on PTHrP and breast cancer should focus on understanding how intracrine PTHrP signaling interacts with STAT5 signaling to modulate programs of secretory differentiation. In addition, it will be helpful to examine whether PTHrP plays an important role downstream of the ETV6-NTRK3 oncogene in secretory carcinomas of the breast. We believe that these lines of investigation may help to resolve conflicting published data regarding the overall effects of PTHrP on tumor behavior and patient survival. Given that PTHLH has been defined as a breast cancer susceptibility gene in GWAS studies, it is important to clarify the molecular mechanisms by which PTHrP affects breast cancer cell behavior.