Prolactin, signaling and the mammary gland
The hormone prolactin has long been understood to play a vital role in breast growth and development and the production of milk during pregnancy. Prolactin (PRL) is a polypeptide hormone produced primarily by cells known as lactotrophs, which are located in the anterior pituitary gland of all vertebrates. In humans, PRL is also produced at multiple extra-pituitary sites and functions as a circulating cytokine hormone with both autocrine and paracrine actions. The biological activities of PRL are mediated by membrane PRL receptors (PRLR), members of the cytokine receptor superfamily with conserved features of cytokine receptors within the extracellular domain. Ligand binding and activation of PRLR leads to downstream induction of the canonical Jak2/STAT5 or Jak1/STAT3 pathways, feeding into multiple signaling cascades including PI3K/Akt and Raf/Mek/Erk. Jak/Stat independent PRLR signaling can also be mediated through the Src kinases and focal adhesion kinases (FAK) controlling cellular shape.
In the mammary gland, PRL plays a decisive role in epithelial cell proliferation and milk production. The generation of PRL and PRLR gene knockout (KO) mice demonstrated that PRL and PRLR pathways are key regulators in mammary gland development. In addition to its essential function in mammary gland biology, PRL has reproductive, metabolic, osmoregulatory, and immunoregulatory actions in diverse tissues. The importance of PRL in breast cancer development and risk is less well-defined as compared to ovarian steroids. Similar to estrogen and progesterone, high PRL levels can augment mammary tumor development in mice and in women, elevated PRL levels are correlated with increased breast cancer risk and metastasis. Interestingly, PRL and nuclear PRLR can enhance the expression of the estrogen (ERα) and progesterone (PR) receptors. In contrast, studies have also shown that activation of PRLR can suppress the mesenchymal phenotype and reduce invasive behavior.
Loss of PRLR expression in breast cancer can be associated with poor differentiation and larger tumors whereas the gene expression signatures of an activated PRL/PRLR pathway are associated with welldifferentiated tumors, reduced metastasis and higher overall survival. Such results indicate that PRL may have both a pro-oncogenic as well as a metastasis suppressor role in breast cancer. They also support the idea of cross-talk between the actions of PRL and the steroid hormones. As mentioned above, major downstream effectors of PR and PRLR signaling include the STAT family of latent transcription factors. The STAT proteins STAT1, STAT3 and STAT5 are involved in all stages of mammary gland development. Genetic deletion experiments suggest that these proteins are most important in postnatal development. The STAT5 protein isoforms, encoded by the genes STAT5A and STAT5B, are necessary and sufficient for alveologenesis and expression of milk protein genes during late pregnancy and lactation.
Biodialogue between prolactin and progesterone
Both progesterone and PRL are important regulators of STAT proteins as well. The functions of specific STAT proteins have important implications for their potential role in breast cancer progression. For example, STAT1 is believed to be a tumor suppressor that is often lost in ER+ tumors. High levels of pSTAT1 in ER+ breast cancers from post-menopausal women are associated with greater disease-free survival. This suppressive role may be dependent on menopausal status since some clinical studies have observed poorer overall and disease-free survival in premenopausal women with elevated STAT1. The role of STAT5 in breast cancer is complex. Phosphorylation and nuclear localization of
STAT5 in breast cancer is a positive predictor of response to endocrine therapy and patients with more activated STAT5 have decreased risk of disease recurrence and death. However, STAT5 also appears to have a role in tumor formation,
Similar to STAT3, STAT5 and PR interact and STAT5a interact with progesterone receptor at PRE sites. Only isoform PR-B enables STAT5a signaling. Phosphorylation of PR-B at serine 81 (a site absent from PR-A) induces expression of STAT5a and in a feed forward signaling loop, STAT5a activation cooperates with phosphorylated PR-B to drive expression of a specific gene set co-regulated by STATs, including WNT1, thus linking this PR-B signaling program to PRL/PRLR signaling. In breast cancer cells, it has been discovered that PRL-induced expression of MMP9 is dependent on co-expression of PR-B. Metalloproetase 9 (MMP-9) is a protease that breaks down the extracellular matricx (ECM) allowing mobility and malignant cells to escape as metastasis. These data illustrate a cooperative role for progesterone and prolactin in pro-tumorigenic signaling relevant to altered hormone action in a model of mammographically dense breast tissue.
Understanding the details of cross-talk between these two signaling pathways may provide further insight into the role of hormonal regulation in the context of increased mammary density, thereby opening new avenues of research into a potential means of blocking the relevant interactions and signaling pathways in order to prevent breast cancer development in high risk women.
The role of prolactin-induced protein (PIP) in breast cancer
At the end of the 20th century, scientists described Gross Cystic Disease Fluid Protein 15 (GCDFP-15) as one of the proteins present in the cystic ﬂuid from mastopathy. Later this acidic protein was found in the culture medium from breast cancer T47D cells as two glycoforms. Its level was signifcantly increased following prolactin (hPRL) induction, which ultimately lead to naming it prolactin-induced protein (PIP). The expression of PIP is generally restricted to cells with apocrine properties, like in apocrine glands of the axilla, vulva, eyelid, ear canal, and seminal vesicle. However, PIP was also detected in serous cells of salivary glands, submucosal glands of the bronchi, and accessory lacrimal glands. PIP is generally expressed by normal breast tissues, but is highly present in metaplastic/ hyperplastic apocrine epithelium of breast cyst and breast cyst ﬂuid.
In early studies, it was shown that PIP is absent in normal breast epithelium or its expression is very low and/or diffcult to detect, whereas in breast cancers PIP is frequently expressed and present in large amounts. Several early studies revealed that PIP is especially present in breast carcinomas with apocrine features. Also later studies showed that expression of PIP decreases in advanced apocrine carcinomas and was signifcantly lower in infltrating carcinomas, especially node-positive tumors, than in situ carcinomas. Similarly, signifcantly lower levels of PIP were found in invasive breast tissue then in adjacent normal tissue.
PIP has protease activity. As fbronectin is one of the substrates of PIP, it is believed that PIP participates in extracellular matrix degradation, and therefore is engaged in breast cancer progression. In addition to fbronectin, PIP binds numerous proteins including cellular (actin, β-tubulin) and plasmatic (fibrinogen, serum albumin and zinc α2- glycoprotein). In ER-negative breast cancer, the expression of PIP is auto-regulated by the positive feedback loop between PIP and ERK, Akt/PkB signaling. In human molecular apocrine breast cells the secreted PIP, by its proteolytic activity degrades fbronectin to peptides which activates β1-integrins. These in turn ignite intracellular signals (tyrosine kinases and mitogenic kinases) that promote cell survival and metastasis.
With the introduction of new molecular classifcation based on gene expression profling,the following molecular subtypes of breastcancer were discriminated: basal-like, HER2-enriched, luminal A, luminal B and normal-like. According to this nomenclature, thehighest amounts of PIP mRNA were found inthe luminal A subtype, then in HER2-enrichedand normal-like subtypes, and the lowest expression was observed in basal-like subtype. Since in many studies, PIP expression correlated with low grade breastcancers, it was proposed that high expressionof this marker is a predictor of good prognosis. From clinical point of view, isalso important that cases with high PIP expression were characterized by longer disease-free survival and overall survival.
More recently, it was showed that a high level of PIP expression is positively correlated with the response of breast cancer patients to standard adjuvant chemotherapy (doxorubicin + cyclophosphamide). Additionally, we showed that the levels of PIP protein and mRNA decreases along with tumor malignancy grade, and that PIP expression is the lowest in triple negative (ER-, PR-, HER2-) cases with poor prognosis. Finally, as PIP was not found in gastrointestinal cancers, bronchopulmonary structures, and genitourinary cancers, this specifc marker of breast cancer and its metastases can be used to distinguish distant breast metastases from other primary and secondary tumors, being also a potential marker for breast micrometastases to auxilliary lymph nodes.
Latest basic science developments
Lately, recent studies conducted at VCU Massey Cancer Center finds strong evidence that prolactin also acts as a major contributor to breast cancer development and that the hormone could inform the creation of targeted drugs to treat multiple forms of the disease. Hormones have proteins on their cell surface called receptors that receive and send biological messages and regulate cell function. Through research published in npj Breast Cancer, Dr. Charles Clevenger, MD, PhD, and his lab discovered a new altered form of the prolactin receptor called the human prolactin receptor intermediate isoform (hPRLrI) that directly drives breast cancer. The researchers observed that this modified version of the prolactin receptor interacted with other forms of the receptor to turn benign breast cells into malignant ones, and the presence of hPRLrI in breast cancer cells was associated with triple negative breast cancer, a rapid rate of cell reproduction and poor outcomes.
Through a separate study, Clevenger’s lab substantially prevented tumor growth in preclinical models of ER-positive breast cancer using an inhibitor oh histone deacetylase 6 (HDAC6), an enzyme in cytoplasm associated with prolactin. The team has previously found success in the lab stunting breast cancer growth by deactivating STAT-5, the transcription factor responsible for the production of prolactin. In this new research, the scientists found that the prolactin-regulating function of STAT-5 is dually dependent on the enzyme HDAC6 and the gene HMGN2 that codes another DNA-binding protein. They also discovered that the estrogen receptor (ER-alpha) was highly and almost exclusively interactive with STAT-5 at sites where HDAC6 and HMGN2 were also present. This suggests that both the estrogen receptor and prolactin receptor can cooperate through the activation of Stat5 to initiate the development of breast cancer.
Indeed, treating ER-positive breast cancer cells with an HDAC6 inhibitor drastically hindered tumor progression. Dr. Clevenger said these findings support the argument that future approaches in drug design may need to specifically target hPRLrI, and could ultimately inform advanced diagnostic applications for breast cancer as well.
- edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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