MET variants with activating N-lobe mutations identified in hereditary papillary renal cell carcinomas still require ligand stimulation.

In hereditary papillary renal cell carcinoma (HPRCC), the MET receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations recently identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, further enhanced by HGF stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon14 skipping, the two N-lobe MET variants His1086Leu, Ile1102Thr further characterized were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation displayed also transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET TKIs opens the way for use of targeted therapies for patients harboring the corresponding mutations.

ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis.

BACKGROUND: The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis. METHODS: Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples. RESULTS: Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer. CONCLUSION: MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.

Ets-1 controls breast cancer cell balance between invasion and growth.

Ets-1 overexpression in human breast cancers is associated with invasiveness and poor prognosis. By overexpressing Ets-1 or a dominant negative mutant in MMT breast cancer cells, we previously highlighted the key role of Ets-1 in coordinating multiple invasive features of these cells. Interestingly, we also noticed that Ets-1 decreased the density of breast cancer cells cultured in three-dimensional extracellular matrix gels. The 3D context was instrumental to this phenomenon, as such downregulation was not observed in cells grown on two-dimensional plastic or matrix-coated dishes. Ets-1 overexpression was deleterious to anchorage-independent growth of MMT cells in soft agar, a standard model for in vitro tumorigenicity. The relevance of this mechanism was confirmed in vivo, during primary tumor growth and in a metastatic assay of lung colonization. In these models, Ets-1 was associated with epithelial-to-mesenchymal transition features and modulated the ratio of Ki67-positive cells, while hardly affecting in vivo apoptotic cell death. Finally, siRNA-mediated knockdown of Ets-1 in human breast cancer cell lines also decreased colony growth, both in anchorage-independent assays and 3D extracellular matrix cultures. These in vitro and in vivo observations shed light on an unsuspected facet of Ets-1 in breast tumorigenesis. They show that while promoting malignancy through the acquisition of invasive features, Ets-1 also attenuates breast tumor cell growth and could therefore repress the growth of primary tumors and metastases. This work also demonstrates that 3D models may reveal mechanisms of tumor biology that are cryptic in standard 2D models.

Loss of a negative feedback loop involving pea3 and cyclin d2 is required for pea3-induced migration in transformed mammary epithelial cells.

UNLABELLED: The Ets family transcription factor Pea3 (ETV4) is involved in tumorigenesis especially during the metastatic process. Pea3 is known to induce migration and invasion in mammary epithelial cell model systems. However, the molecular pathways regulated by Pea3 are still misunderstood. In the current study, using in vivo and in vitro assays, Pea3 increased the morphogenetic and tumorigenic capacity of mammary epithelial cells by modulating their cell morphology, proliferation, and migration potential. In addition, Pea3 overexpression favored an epithelial-mesenchymal transition (EMT) triggered by TGF-ß1. During investigation for molecular events downstream of Pea3, Cyclin D2 (CCND2) was identified as a new Pea3 target gene involved in the control of cellular proliferation and migration, a finding that highlights a new negative regulatory loop between Pea3 and Cyclin D2. Furthermore, Cyclin D2 expression was lost during TGF-ß1-induced EMT and Pea3-induced tumorigenesis. Finally, restored Cyclin D2 expression in Pea3-dependent mammary tumorigenic cells decreased cell migration in an opposite manner to Pea3. As such, these data demonstrate that loss of the negative feedback loop between Cyclin D2 and Pea3 contributes to Pea3-induced tumorigenesis. IMPLICATIONS: This study reveals molecular insight into how the Ets family transcription factor Pea3 favors EMT and contributes to tumorigenesis via a negative regulatory loop with Cyclin D2, a new Pea3 target gene.