Prolactin (PRL) acting through the prolactin receptor (PRLR) has an important role in breast carcinogenesis through its effects on mitogenesis and proliferation. PRL acts in an endocrine and an autocrine/paracrine manner and both PRL and PRLR are found in about 80% of human breast cancer biopsies. We are examining the role of the autocrine PRL system in metastasis. For these studies we used the highly metastatic MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. The non-metastatic cell line MCF7 was used as a control. PRL and its receptors have been identified in normal and cancerous human breast tissues and cell lines; however, much of the evidence implicating a role for PRL in mammary tumorigenesis is from rodent models. The extent of PRLs involvement in human breast cancer is less well documented. To evaluate the role autocrine PRL plays in tumor growth, progression, and metastasis, we stably transfected the three breast cancer cell lines with a human prolactin cDNA. We found that PRL confers a more aggressive phenotype in vitro and in vivo. In vitro, it inhibited apoptosis and increased mitogenesis on plastic and on extracellular matrices, enhanced growth on soft agar and increased cell migration. In vivo, over-expression of PRL increased angiogenesis and tumorigenesis in an orthotopic xenograft model. Microarray analyses, confirmed by immunohistochemistry, revealed that PRL differentially regulated various members of the Wnt pathway resulting in increased Wnt signaling. The Wnt inhibitor Dkk-1 reversed the growth promoting effects of PRL. This is the first evidence demonstrating that PRL acts through activation of the canonical Wnt pathway. We also examined the role that the P-regulated Hox-related homeobox-containing gene, MSX2, plays during tumorigenesis where mining of published databases found a strong correlation between elevated MSX2 expression and estrogen receptor (ER) positive breast cancer. NMuMG cells, a normal mouse mammary epithelial cell line, stably-transfected with a MSX2 cDNA showed increased growth in serum-starved conditions, enhanced growth on soft agar and increased cell migration in vitro. These functions were accompanied by constitutive over-expression of cyclin E and cyclin D1. Additionally, the NMuMG-MSX2 cells demonstrated enhanced tumor formation when injected orthotopically into the mammary fat pad of immune compromised mice. 25% of transgenic mice over-expressing MSX2 in the mammary glands developed tumors by 15 months of age;control mice failed to develop tumors. Additionally, immunohistochemistry of human infiltrating breast carcinomas showed positive staining for MSX2 only in the infiltrating tumor cells while the non-infiltrating tumor cells were negative. These results suggest that MSX2 may play a significant role in mouse and human mammary tumor development and invasion. Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. In addition to PRL, signaling by laminin-111 is necessary to restore functional differentiation of mammary epithelia. We showed that in 2D cultures, PRLRs are basolaterally localized and physically segregated from the apically placed ligand. Detachment of the cells exposes the receptor to ligation by PRL leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We showed that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the PRLR and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and beta-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression. Growth of normal breast cells or breast cancer cells as spheres in non-adherent (3D) culture conditions has been used as a surrogate for the presence of stem or progenitor cells. Self-renewal pathways may be activated to increase the number of stem cells contributing to the formation of the spheres. For both primary normal breast cells, cell lines and breast cancer cells, we found that only PRL treatment results in increased sphere formation. P acts in cooperation with PRL to give more, larger and highly disorganized spheres in culture. The signaling pathways and receptor isoforms involved in this hormonally activated self-renewal is currently under investigation. High levels of PRL in plasma correlate with increased risk of breast cancer, especially among postmenopausal women. Several isoforms of PRL exist in human circulation, including a 16 kDa isoform that is an N-terminal cleavage product of the full-length 23 kDa PRL. 16 kDa PRL has been shown to be anti-angiogenic in vitro and in vivo, and to reduce formation of tumors from prostate and colon cancer cell lines. We explored the effect of 16 kDa PRL expression in vitro and in vivo using two breast cancer cell line models (MCF-7 and MDA-MB-231) and also the HCT-116 colon cancer cell line. In vitro, in all three cell lines, 16 kDa PRL expression inhibited cell proliferation compared to empty vector controls. In vivo results were markedly different between the two types of cell lines. HCT-116 cells expressing 16 kDa PRL exhibited reduced vascularization and tumor formation, consistent with published results. The breast cancer cell lines expressing 16 kDa PRL also exhibited inhibition of angiogenesis in vivo but no reduction in tumor size or formation. These results suggest that the effects of 16 kDa PRL on tumor formation may be organ specific and that the unique sensitivity of breast cells to the mitogenic effects of PRL may play a dominant role in tumor formation, thus outweighing the anti-angiogenic effects of 16 kDa PRL. PRLRs exist in multiple isoforms resulting from alternative splicing of PRLR exon 11. We have been studying the long form (LF) and two short isoforms, 1a (SF1a) and 1b (SF1b) that lack components of the intracellular domain of PRLR but maintain the same extracellular domain. Current data suggests that the short form PRLRs act as dominant negatives for differentiation signaling through the LF receptor;their roles in proliferation remain to be determined. Previous work on these isoforms has only examined mRNA levels and established potential roles through transfection studies. We have developed the first antibodies that can distinguish between these three isoforms using differences in the intracellular domains. The specificity of these polyclonal and monoclonal antibodies was demonstrated by western blots and immunocytochemistry on CHO cells stably transfected with the cDNA independently for each of the three isoforms. Subsequently we examined potential clinical uses for these antibodies by immunohistochemistry on ductal and lobular carcinoma samples obtained from the Cooperative Human Tissue Network (CHTN) and a tissue array containing 144 ductal carcinoma and 24 lobular carcinomas. We were able to correlate these results with quantitative polymerase chain reaction (qPCR) on whole tissue samples. We have found differences in expression that demonstrate these antibodies could be used as a new clinical tool to distinguish between different subclasses of breast cancers and aid in diagnosis and possibly prognosis.
