The activation of the Janus-activated kinase 2 (Jak2) tyrosine kinase following ligand binding has remained incompletely characterized at the mechanistic level. We report that the peptidyl-prolyl isomerase (PPI) cyclophilin A (CypA), which is implicated in the regulation of protein conformation, is necessary for the prolactin (PRL)-induced activation of Jak2 and the progression of human breast cancer. A direct correlation was observed between the levels or activity of CypA and the extent of PRL-induced signaling and gene expression. Loss of PRLr-CypA binding, following treatment with the PPI inhibitor cyclosporine A (CsA), or overexpression of a dominant-negative PRLr mutant (P334A) resulted in a loss of PRLr/Jak2-mediated signaling. In vitro, CsA treatment of breast cancer cells inhibited their growth, motility, invasion, and soft agar colony formation. In vivo, CsA treatment of nude mice xenografted with breast cancer cells induced tumor necrosis and completely inhibited metastasis. These studies reveal that a CypA-mediated conformational change within the PRLr/Jak2 complex is required for PRLinduced transduction and function and indicate that the inhibition of prolyl isomerases may be a novel therapeutic strategy in the treatment of human breast cancer. [Cancer Res 2008;68(19):7769-78]

The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.

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Stat5a is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that directly target Stat5a, despite ample evidence that it contributes to breast cancer pathogenesis. Here, deacetylation of the Stat5a coactivator and chromatin remodeling protein HMGN2 on lysine residue K2 by HDAC6 promotes Stat5a-mediated transcription and breast cancer growth. HDAC6 inhibition both in vitro and in vivo enhances HMGN2 acetylation with a concomitant reduction in Stat5a-mediated signaling, resulting in an inhibition of breast cancer growth. Furthermore, HMGN2 is highly acetylated at K2 in normal human breast tissue, but is deacetylated in primary breast tumors and lymph node metastases, suggesting that targeting HMGN2 deacetylation is a viable treatment for breast cancer. Together, these results reveal a novel mechanism by which HDAC6 activity promotes the transcription of Stat5a target genes and demonstrate utility of HDAC6 inhibition for breast cancer therapy. Implications HMGN2 deacetylation enhances Stat5a transcriptional activity, thereby regulating prolactin-induced gene transcription and breast cancer growth.

Background Prolactin is a lactogenic hormone associated with breast cancer risk in prospective studies, which used immunoassays. The immunoassay captures multiple isoforms and may not fully reflect the biological activity of prolactin relevant to breast carcinogenesis. Methods We considered plasma bioactive prolactin levels measured by the Nb2 lymphoma cell bioassay, which is sensitive to the somatolactogenic activity of prolactin and growth hormone, within a nested case-control study of invasive breast cancer in the Nurses’ Health Studies (NHS/NHSII). We also considered associations with breast cancer risk factors. Results We had bioassay measures on 1329 cases and 1329 controls. Bioassay levels were inversely associated with parity (4+ vs. 0 children=−18%, p=0.01), body mass index (30+ vs. <22kg/m2=−16%, p<0.01), and age at menopause (53+ vs. 48yr=−18%, p=0.03) and positively with family history of breast cancer (yes vs. no=14%, p<0.01). The relative risk (RR) comparing the top versus bottom quartile of bioassay levels was 1.19 (95% confidence interval [CI]=0.94–1.51; p-trend=0.18). The association was suggestively stronger for postmenopausal (RR=1.36, 95%CI=0.93–1.98; p-trend=0.12) versus premenopausal women (RR=0.99, 95%CI=0.71–1.37, p-trend=0.71). There was an association for cases diagnosed <4 years after blood draw (RR=2.66, 95%CI=1.45–4.89, p-trend<0.01), but not for cases diagnosed later. We did not observe differential associations by estrogen receptor status or other tumor characteristics. Conclusions Our results show similar associations for prolactin levels measured by bioassay and by immunoassay with both breast cancer risk factors and risk. Impact Future work examining risk prediction model of breast cancer can use the immunoassay to accurately characterize risk.