Recent fate-mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that these studies failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumour cells in a paracrine manner, in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI 926, a Smoothened inhibitor, blocks this effect and inhibits growth in PDX models. In human breast tumours, the EMT-transcription factors strongly correlate with activated Hedgehog/GLI signalling but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis via non-cell autonomous effects that activate the Hh pathway. Although all Hh inhibitors may act against tumours with canonical Hh/GLI signalling, only GLI inhibitors would act against non-canonical EMT-induced GLI activation.

Eya genes encode a unique family of multifunctional proteins that serve as transcriptional co-activators and as haloacid dehalogenase-family Tyr phosphatases. Intriguingly, the N-terminal domain of Eyas, which does not share sequence similarity to any known phosphatases, contains a separable Ser/Thr phosphatase activity. Here, we demonstrate that the Ser/Thr phosphatase activity of Eya is not intrinsic, but arises from its direct interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme. Importantly, Eya3 alters the regulation of c-Myc by PP2A, increasing c-Myc stability by enabling PP2A-B55α to dephosphorylate pT58, in direct contrast to the previously described PP2A-B56α-mediated dephosphorylation of pS62 and c-Myc destabilization. Furthermore, Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer, opposing the canonical tumor suppressive function of PP2A-B56α. Our study identifies Eya3 as a regulator of PP2A, a major cellular Ser/Thr phosphatase, and uncovers a mechanism of controlling the stability of a critical oncogene, c-Myc.

Lymph nodes are the most common sites of metastasis in cancer patients. Nodal disease status provides great prognostic power, but how lymph node metastases should be treated is under debate. Thus, it is important to understand the mechanisms by which lymph node metastases progress and how they can be targeted to provide therapeutic benefits. In this review, we focus on delineating the process of cancer cell migration to and through lymphatic vessels, survival in draining lymph nodes and further spread to other distant organs. In addition, emerging molecular targets and potential strategies to inhibit lymph node metastasis are discussed.

Structure-based drug repositioning in addition to random chemical screening is now a viable route to rapid drug development. Proteochemometric computational methods coupled with kinase assays showed that mebendazole (MBZ) binds and inhibits kinases important in cancer, especially both BRAFWT and BRAFV600E. We find that MBZ synergizes with the MEK inhibitor trametinib to inhibit growth of BRAFWT-NRASQ61K melanoma cells in culture and in xenografts, and markedly decreased MEK and ERK phosphorylation. Reverse Phase Protein Array (RPPA) and immunoblot analyses show that both trametinib and MBZ inhibit the MAPK pathway, and cluster analysis revealed a protein cluster showing strong MBZ+trametinib - inhibited phosphorylation of MEK and ERK within 10 minutes, and its direct and indirect downstream targets related to stress response and translation, including ElK1 and RSKs within 30 minutes. Downstream ERK targets for cell cycle, including cMYC, were down-regulated, consistent with S- phase suppression by MBZ+trametinib, while apoptosis markers, including cleaved caspase-3, cleaved PARP and a sub-G1 population, were all increased with time. These data suggest that MBZ, a well-tolerated off-patent approved drug, should be considered as a therapeutic option in combination with trametinib, for patients with NRASQ61mut or other non-V600E BRAF mutant melanomas.

Tumor heterogeneity is a major obstacle to the development of effective therapies and is thus an important focus of cancer research. Genetic and epigenetic alterations, as well as altered tumor microenvironments, result in tumors made up of diverse subclones with different genetic and phenotypic characteristics. Intratumor heterogeneity enables competition, but also supports clonal cooperation via cell-cell contact or secretion of factors, resulting in enhanced tumor progression. Here, we summarize recent findings related to interclonal interactions within a tumor and the therapeutic implications of such interactions, with an emphasis on how different subclones collaborate with each other to promote proliferation, metastasis and therapy-resistance. Furthermore, we propose that disruption of clonal cooperation by targeting key factors (such as Wnt and Hedgehog, amongst others) can be an alternative approach to improving clinical outcomes.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg 2þ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic muta-tions, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.

It is well established that a subset of cells within primary breast cancers can undergo an epithelial to mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via non-canonical paracrine-mediated activation of GLI activity that is dependent on SIX1 expression in the EMT cancer cells. However, the mechanism by which these SIX1-expressing EMT cells activate GLI signaling remained unclear. In this study, we demonstrate a novel mechanism for activation of GLI-mediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C. We show that VEGF-C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine-mediated increases in proliferation, migration and invasion of epithelial breast cancer cells, via non-canonical activation of GLI-signaling. We further show that the aggressive phenotypes, including metastasis, imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2, a receptor which interacts with VEGF-C, in neighboring epithelial cancer cells. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, demonstrating that VEGF-C strongly correlates with activation of Hedgehog signaling and EMT in the human disease. Our study suggests that the VEGF-C/NRP2/GLI axis is a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease.

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