Cancer cells can disseminate during very early and sometimes asymptomatic stages of tumor progression. Though biological barriers to tumorigenesis have been identified and characterized, the mechanisms that limit early dissemination remain largely unknown. We report here that the orphan nuclear receptor nuclear receptor subfamily 2, group F, member 1 (NR2F1)/COUP-TF1 serves as a barrier to early dissemination. NR2F1 expression was decreased in patient ductal carcinoma in situ (DCIS) samples. High-resolution intravital imaging of HER2+ early-stage cancer cells revealed that loss of function of NR2F1 increased in vivo dissemination and was accompanied by decreased E-cadherin expression, activation of wingless-type MMTV integration site family, member 1 (WNT)-dependent β-catenin signaling, disorganized laminin 5 deposition, and increased expression of epithelial–mesenchymal transition (EMT) genes such as twist basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box binding homeobox 1 (ZEB1), and paired related homeobox 1 (PRRX1). Furthermore, downregulation of NR2F1 promoted a hybrid luminal/basal phenotype. NR2F1 expression was positively regulated by p38α signaling and repressed by HER2 and WNT4 pathways. Finally, early cancer cells with NR2F1LOW/PRRX1HIGH staining were observed in DCIS samples. Together, these findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 in early-stage breast cancer cells. Significance: During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.

BMP7 evokes acute chemotropic, PI3K-dependent responses, such as growth cone collapse and monocyte chemotaxis, as well as classical Smad-dependent gene transcription. That these divergent responses can be activated in the same cell raises the question of how the BMP-dependent signaling apparatus is manipulated to produce chemotropic and transcriptional signals. RNA interference and site-directed mutagenesis were used to explore functional and structural BMP receptor requirements for BMP7-evoked chemotropic activity. We show that specific type II BMP receptor subunits, ActRIIA and BMPR2, are required for BMP7-induced growth cone collapse in developing spinal neurons and for chemotaxis of monocytes. Reintroduction of wild-type ActRIIA into monocytic cells lacking endogenous ActRIIA restores BMP7-evoked chemotaxis, whereas expression of an ActRIIA K76A receptor variant, fails to rescue. BMP7-evoked Smad-dependent signaling is unaffected by either ActRIIA knockdown or expression of the ActRIIA K76A variant. In contrast, BMP7-evoked PI3K-dependent signaling is significantly disturbed in the presence of ActRIIA K76A. These results support a model for selective engagement of chemotropic BMPs with type II BMP receptors, through specific residues, that results in strict regulation of PI3K-dependent signal transduction.

A major challenge in melanoma treatment is the emergence of aggressive metastases from previously dormant disseminated cancer cells (DCCs). Here we identify Midkine (MDK) as a key factor that disrupts melanoma DCC dormancy by triggering an autocrine signal that suppresses the dormancy inducer NR2F1 and alters the p-p38/p-ERK ratio, via ALK signaling. Dormant melanoma DCCs in lymph nodes and visceral tissues exhibit an MDK ^LOW /NR2F1 ^HIGH phenotype. Transient NR2F1 activation for one month with a potent agonist counteracts MDK-driven mTORC1 activity and suppresses lung metastasis in mice bearing human melanoma DCCs. Dual targeting of MDK (genetic blockade) and NR2F1 (activation) markedly limits metastatic outgrowth and extends survival for 6 months in mouse models independent of BRAF status. These findings support the MDK-NR2F1 axis as a promising therapeutic target to sustain dormancy and prevent melanoma recurrence. Statement of Significance: This work identifies Midkine as a key disruptor of tumor dormancy in melanoma, driving metastatic awakening via NR2F1 suppression. Dual targeting of Midkine (inhibition) and NR2F1 (activation) prolongs survival in preclinical models.

Supplementary Data from NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells

Supplementary Data from NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells