Background-Formation of long-term memories is critically dependent on extracellular-regulated kinase (ERK) signaling. Activation of the ERK pathway by the sequential recruitment of mitogenactivated protein kinases is well understood. In contrast, the proteins that inactivate this pathway are not as well characterized.

Summary Prolactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2). Jak2 associates with PRLr, phosphorylates c-terminus of the PRLr, which leads to Stat5 recruitment and activation. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase (PPI), which is constitutively bound to the PRLr that catalyze the cis-trans interconversion of proline imide bonds. Treatment with Cyclosporine (CsA) inhibited CypA binding to the PRLr and blocked PRLr-driven activation of Jak2/Stat5. Recently, Waters et al., utilizing Fluorescence Resonance Energy Transfer (FRET) with transfectants expressing CFP and YFP-tagged forms of the growth hormone receptor (GHR) showed that GHR activation induced a rotational movement in C-terminus of the GHR, resulting in a loss of baseline FRET signal. Like the GHR, PRL stimulation of transfectants expressing CFP/YFP-tagged PRLr constructs resulted in a loss of FRET efficiency. In contrast, treatment with NIM811 (a non-immunosuppressive form of CsA) or siRNA knockdown of CypA resulted in a return of FRET signal in the presence of PRL. These studies reveal that ligand stimulation of the PRLr results in a conformational change as measured by FRET signal of the receptor that is reversed by CypA inhibition or knockdown, implicating CypA as the mediator of this conformational change and ligand-induced signaling. To further assess the consequences of CypA inhibition or knockdown on the PRLr/Jak2 mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated in breast cancer cells. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates in ER+/PR+ T47D cells in a time dependent manner. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL induced genes. NIM811 inhibited ER-/+, and HER2+ breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent pre-clinical testing of NIM811 in relevant mouse mammary cancer models has found that NIM811 treatment of a TNBC xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA (by constitutive genetic deletion) in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates conformational change in the C-terminus of the PRLr through its PPI activity, and alters PRLr/Jak2 complex signaling/functions in breast cancer and mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention as a chemo-preventive and as an inhibitor of metastasis. Citation Format: Shawn Hakim, Shannon E. Hedrick, Justin M. Craig, Charles V. Clevenger. Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4508.

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