Role of Tyrosine Kinase Jak2 in Prolactin-induced Differentiation and Growth of Mammary Epithelial Cells

Abstract

Genetic studies in mice have established a critical role for prolactin receptors and transcription factor Stat5 in mammary gland differentiation. However, the enzymatic coupling between prolactin receptors and Stat5 in this process has not been established. In addition to Jak2, several other tyrosine kinases reportedly also are associated with prolactin receptors and may phosphorylate Stat5. Because Jak2 null mice die in utero, we targeted Jak2 in an ex vivo model of prolactin-induced mammary epithelial cell differentiation to determine the role of Jak2 in regulation of cell differentiation and growth. Two independent targeting strategies were used to suppress Jak2 in immortalized HC11 mouse mammary epithelial cells: 1) stable expression of a specific Jak2 antisense construct and 2) adenoviral delivery of a dominant-negative Jak2 gene. We now demonstrate that Jak2 is essential for prolactin-induced differentiation and activation of Stat5 in normal mouse mammary epithelial cells. Furthermore, suppression of Jak2 in HC11 cells was associated with constitutive activation of oncoprotein Stat3 and a hyperproliferative phenotype characterized by increased mitotic rate, reduced apoptosis, and reduced contact inhibition. Collectively, our data suggest that Jak2 is differentiation-inducing and growth-inhibitory in normal mammary epithelial cells, observations that may shed new light on the role of the Jak2-Stat5 pathway in breast cancer.

Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma

“…2,4,5,8,11,12 The binding of prolactin to prolactin receptor initiates multimeric signaling pathways (Figure 4), including but not limited to the Jak/Stat pathway 8,[13][14][15] to produce several endocrine effects on the human breast tissues, such as the regulation of growth and differentiation of ductal epithelium, proliferation and differentiation of lobular units, and initiation and maintenance of lactation. 11 Prolactin has been shown to contribute to the progression and motility of breast cancer by increasing the proliferation of mammary carcinoma cells and actively inhibiting the apoptosis of mammary tumor cells.…”

Human prolactin receptors are insensitive to mouse prolactin: implications for xenotransplant modeling of human breast cancer in mice

“…We also tested whether human prolactin can mimic the more complex biologic activity of mouse prolactininduced differentiation of mammary epithelial cells. Using the HC11 cell differentiation assay (Xie et al 2002), we determined that human prolactin is as effective as mouse prolactin in inducing three-dimensional mammospheres (Fig. 2B).…”

“…For prolactin-induced Stat5 activation studies, 80% confluent HC11 cells were serum-starved for 16 h and then treated with human prolactin (10 nM), or mouse prolactin (10 nM) or vehicle control for 15 min. For mammary epithelial cell differentiation study (Xie et al 2002), confluent HC11 cells were serum-starved in medium containing 2% fetal bovine serum and lacking EGF for 48 h. Cells were then incubated in RPMI 1640 medium containing 10% fetal bovine serum, 0·1 µM dexamethasone, insulin (5 µg/ml) and either no prolactin or 10 nM human or mouse prolactin for 7 days. COS-7 cells (ATCC) were grown in Dulbecco's modified essential medium (DMEM) containing 10% fetal bovine serum, 2 mM -glutamine and penicillin-streptomycin (50 IU/ml and 50 µg/ml respectively).…”

“…For proliferation assay, cells were lactogen-starved for 16 h in lactogen-free medium containing 10% gelded horse serum (Sigma) with no fetal bovine serum or WEHI supernatant prior to treatment with prolactin (DaSilva et al 1994). The mouse mammary epithelial cell line HC11 (Ball et al 1988) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM -glutamine, insulin (5 µg/ml), EGF (10 ng/ml) and penicillin-streptomycin (50 IU/ml and 50 µg/ml respectively), as previously described (Xie et al 2002). For prolactin-induced Stat5 activation studies, 80% confluent HC11 cells were serum-starved for 16 h and then treated with human prolactin (10 nM), or mouse prolactin (10 nM) or vehicle control for 15 min.…”

Targeting Cellular Signaling Pathways in Breast Cancer Stem Cells and its Implication for Cancer Treatment

“…Briefly, the receptor tyrosine kinase Janus kinases (JAK) recruit STAT3, which dimerizes and is translocated to the nucleus where it recognizes specific DNA sequences and activates the transcription of target genes. This transcription factor regulates many cellular processes, including proliferation, apoptosis, invasion, angiogenesis and metastasis (71).…”