Yvonne B. Feeney

Prolyl isomerase cyclophilin A regulation of Janus-activated kinase 2 and the progression of human breast cancer.

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 PRL-induced transduction and function and indicate that the inhibition of prolyl isomerases may be a novel therapeutic strategy in the treatment of human breast cancer.

The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

The polypeptide hormone prolactin (PRL) stimulates breast epithelial cell growth, differentiation, and motility through its cognate receptor, PRLr. PRLr is expressed in most breast cancers; however, its exact role remains elusive. Our laboratory previously described a novel mode of PRLr signaling in which Stat5a-mediated transcription is regulated through ligand-induced phosphorylation of the PRLr transactivation domain (TAD). Herein, we used a PRLr transactivation-deficient mutant (PRLrYDmut) to identify novel TAD-specific target genes. Microarray analysis identified 120 PRL-induced genes up-regulated by wild type but not PRLrYDmut. Compared with control, PRLr expression significantly induced expression of approximately 4700 PRL-induced genes, whereas PRLrYDmut ablated induction of all but 19 of these genes. Ingenuity pathway analysis found that the PRLr TAD most profoundly affected networks involving cancer and proliferation. In support of this, PRLrYDmut expression reduced anchorage-dependent and anchorage-independent growth. In addition, pathway analysis identified a link between the PRLr TAD and the estrogen and progesterone receptors (ERα/PR). Although neither ERα nor PR was identified as a PRL target gene, a TAD mutation significantly impaired ERα/PR expression and estrogen responsiveness. TMA analysis revealed a marked increase in nuclear, but not cytoplasmic, PRLr TAD phosphorylation as a function of neoplastic progression. We propose that PRLr TAD phosphorylation contributes to breast cancer pathogenesis, in part through regulation of ERα and PR, and has potential utility as a biomarker in this disease.

HMGN2 Inducibly Binds a Novel Transactivation Domain in Nuclear PRLr to Coordinate Stat5a-Mediated Transcription

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.

Prolactin Receptor-Integrin Cross-Talk Mediated by SIRPα in Breast Cancer Cells

The hormone prolactin (PRL) contributes to the pathogenesis of breast cancer in part through its activation of Janus-activated kinase 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5), a PRL receptor (PRLr)–associated pathway dependent on cross-talk signaling from integrins. It remains unclear, however, how this cross-talk is mediated. Following PRL stimulation, we show that a complex between the transmembrane glycoprotein signal regulatory protein-α (SIRPα) and the PRLr, β1 integrin, and Jak2 in estrogen receptor–positive (ER+) and ER− breast cancer cells is formed. Overexpression of SIRPα in the absence of collagen 1 significantly decreased PRL-induced gene expression, phosphorylation of PRLr-associated signaling proteins, and PRL-stimulated proliferation and soft agar colony formation. In contrast, overexpression of SIRPα in the presence of collagen 1 increased PRL-induced gene expression; phosphorylation of Jak2, Stat5, and Erk; and PRL-stimulated cell growth. Interestingly, overexpression of a tyrosine-deficient SIRPα (SIRPα-4YF) prevented the signaling and phenotypic effects mediated by wild-type SIRPα. Furthermore, overexpression of a phosphatase-defective mutant of Shp-2 or pharmacologic inhibition of Shp-2 produced effects comparable with that of SIRPα-4YF. However, the tyrosine phosphorylation of SIRPα was unaffected in the presence or absence of collagen 1. These data suggest that SIRPα modulates PRLr-associated signaling as a function of integrin occupancy predominantly through the alteration of Shp-2 activity. This PRLr-SIRPα-integrin complex may therefore provide a basis for integrin-PRLr cross-talk and contribute to the biology of breast cancer. Mol Cancer Res; 8(10); 1413–24. ©2010 AACR.

HDAC6 Deacetylates HMGN2 to Regulate Stat5a Activity and Breast Cancer Growth

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. Mol Cancer Res; 14(10); 994–1008. ©2016 AACR.

Hormonal Determinants of Nipple Aspirate Fluid Yield among Breast Cancer Cases and Screening Controls

Background: Nipple aspiration fluid (NAF) use as a biosample is limited by the variable yield across studies. We investigated the endocrine determinants of yield in an ongoing breast cancer case–control study. Methods: One-hundred and eighteen women yielding ≥2 μL NAF and 120 non-yielders were included; serum hormones were measured; differences in median hormones were assessed using the Wilcoxon rank-sum test. ORs and 95% confidence intervals (95% CI) for yielder status relative to hormone levels were estimated using logistic regression, adjusting for parity and lactation, and, in premenopausal women, menstrual cycle phase (MCP). Results: Prolactin concentrations were higher in yielders than non-yielders (premenopausal: 7.6 and 2.5 ng/mL, P < 0.01; postmenopausal 5.3 and 2.2 ng/mL; P < 0.01). Among premenopausal-yielders, estradiol was lower (64.3 vs. 90.5 pg/mL, MCP-adjusted P = 0.02). In separate menopausal status and parity-adjusted models, significant case–control differences persisted in prolactin: case OR 1.93 (95% CI, 1.35–2.77), control OR 1.64 (95% CI, 1.17–2.29). Premenopausal control yielders had higher progesterone (OR, 1.70; 95% CI, 1.18–2.46) and sex-hormone binding-globulin (OR, 2.09; 95% CI, 1.08–4.05) than non-yielders. Among parous women, further adjustment for lactation suggested a stronger positive association of serum prolactin with yield in cases than controls. Conclusion: NAF-yielders show higher prolactin than non-yielders, regardless of menopause and parity; implications of this and other endocrine differences on NAF biomarkers of breast cancer risk deserve further study. Impact: NAF yield is associated with a distinct endocrine environment that must be considered in studies of NAF-based breast cancer risk markers. Cancer Epidemiol Biomarkers Prev; 22(12); 2277–84. ©2013 AACR.

Prolactin Receptor-Integrin Crosstalk Mediated by SIRPα in Breast Cancer Cells

The hormone prolactin (PRL) contributes to the pathogenesis of breast cancer in part through its activation of Janus-activated kinase 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5), a PRL receptor (PRLr)–associated pathway dependent on cross-talk signaling from integrins. It remains unclear, however, how this cross-talk is mediated. Following PRL stimulation, we show that a complex between the transmembrane glycoprotein signal regulatory protein-α (SIRPα) and the PRLr, β1 integrin, and Jak2 in estrogen receptor–positive (ER+) and ER− breast cancer cells is formed. Overexpression of SIRPα in the absence of collagen 1 significantly decreased PRL-induced gene expression, phosphorylation of PRLr-associated signaling proteins, and PRL-stimulated proliferation and soft agar colony formation. In contrast, overexpression of SIRPα in the presence of collagen 1 increased PRL-induced gene expression; phosphorylation of Jak2, Stat5, and Erk; and PRL-stimulated cell growth. Interestingly, overexpression of a tyrosine-deficient SIRPα (SIRPα-4YF) prevented the signaling and phenotypic effects mediated by wild-type SIRPα. Furthermore, overexpression of a phosphatase-defective mutant of Shp-2 or pharmacologic inhibition of Shp-2 produced effects comparable with that of SIRPα-4YF. However, the tyrosine phosphorylation of SIRPα was unaffected in the presence or absence of collagen 1. These data suggest that SIRPα modulates PRLr-associated signaling as a function of integrin occupancy predominantly through the alteration of Shp-2 activity. This PRLr-SIRPα-integrin complex may therefore provide a basis for integrin-PRLr cross-talk and contribute to the biology of breast cancer. Mol Cancer Res; 8(10); 1413–24. ©2010 AACR.

Cyclophilin A Function in Mammary Epithelium Impacts Jak2/Stat5 Signaling, Morphogenesis, Differentiation, and Tumorigenesis in the Mammary Gland

The prolyl isomerase cyclophilin A (CypA) regulates the Jak2/Stat5 pathway, which is necessary for mammary differentiation and the pathogenesis of breast cancer. In this study, we assessed the role of this isomerase during mammary gland development and erbB2-driven tumorigenesis. Genetic deletion of CypA resulted in delayed mammary gland morphogenesis and differentiation with corresponding decrease in Jak2/Stat5 activation; mammary gland cross-transplantation confirmed this defect was epithelial in nature. Analysis of mammary stem and progenitor populations revealed significant disruption of epithelial maturation. Loss of CypA in the erbB2 transgenic mouse model revealed a marked increase in mammary tumor latency that correlated with decreased Stat5 activation, associated gene expression, and reduced epithelial cell proliferation. These results demonstrate an important role for CypA in the regulation of Jak2/Stat5-mediated biology in mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention.Significance: These findings reveal cyclophilin A functions in normal mammary epithelial development and ErbB2-driven mammary tumorigenesis and suggest therapies targeting cyclophilin A may be efficacious for breast cancer treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3877/F1.large.jpg Cancer Res; 78(14); 3877-87. ©2018 AACR.

Abstract P3-06-08: HDAC6 deacetylates HMGN2 to regulate Stat5a activity and breast cancer growth

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. Mol Cancer Res; 14(10); 994-1008. ©2016 AACR.

Abstract 3836: Enhancement of doxorubicin cytotoxicity in breast cancer by NIM811, A non-immunosuppressive cyclosporine A analog

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cyclophilin A (CypA) is a member of the immunophilin family of peptidyl prolyl isomerases (PPI) that catalyze the cis-trans interconversion of proline imide bonds of peptides. In this role, cyclophilins have been found to function as signaling switches, regulating the activity of receptors, kinases and transcription factors. The PPI activity of CypA is inhibited by the immunosuppressive drug cyclosporine A (CsA), and in turn the CypA-CsA complex inhibits calcineurin-mediated NFAT activation. Our laboratory has demonstrated that CypA is necessary for the prolactin (PRL)-induced activation of Jak2/Stat5 signaling and gene expression (Cancer Res 68:7769, 2008). In addition, we have also shown that CsA inhibits the in vitro and in vivo growth and progression of both ER+ and ER- breast cancer cell lines. Here, we show that the non-immunosuppressive CsA analog NIM811 blocked PRL-stimulated activation of the Jak2/Stat5, PI3K/AKT and MAPK pathways in T47D cells. NIM811 also inhibited ER+, ER-, and Her2+ breast cancer cell proliferation, survival, motility and anchorage independent growth in a dose-dependent manner. NIM811 inhibited PRL induced gene expression, such as CISH and Cyclin D1. NIM811 also blocked the PRL-induced association between Stat5 and cMyb, and at higher doses, triggered PARP cleavage and activate Caspase 3. Furthermore, NIM811 increased the effects of doxorubicin in inhibiting monolayer cell growth and soft agar colony growth in vitro. In vivo, NIM811 alone significantly induced the primary tumor central necrosis and inhibited lymph node metastasis; combining NIM811 with a low dose of doxorubicin significantly inhibited MDA-MB-231 breast cancer xenograft tumor growth and distant organ metastasis. In summary, these results indicate that non-immunosuppressive cyclophilin inhibitor NIM811 has significant potential as both a single agent or in combination with cytotoxic agents for breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3836. doi:1538-7445.AM2012-3836