The transcriptional coactivator Yes-associated protein (YAP) is a major regulator of organ size and proliferation in vertebrates. As such, YAP can act as an oncogene in several tissue types if its activity is increased aberrantly. Although no activating mutations in the yap1 gene have been identified in human cancer, yap1 is located on the 11q22 amplicon, which is amplified in several human tumors. In addition, mutations or epigenetic silencing of members of the Hippo pathway, which represses YAP function, have been identified in human cancers. Here we demonstrate that, in addition to increasing tumor growth, increased YAP activity is potently prometastatic in breast cancer and melanoma cells. Using a Luminex-based approach to multiplex in vivo assays, we determined that the domain of YAP that interacts with the TEAD/TEF family of transcription factors but not the WW domains or PDZ-binding motif, is essential for YAP-mediated tumor growth and metastasis. We further demonstrate that, through its TEAD-interaction domain, YAP enhances multiple processes known to be important for tumor progression and metastasis, including cellular proliferation, transformation, migration, and invasion. Finally, we found that the metastatic potential of breast cancer and melanoma cells is strongly correlated with increased TEAD transcriptional activity. Together, our results suggest that increased YAP/TEAD activity plays a causal role in cancer progression and metastasis.

The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER⁻/PR⁻breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools.

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here, we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and MENA, an actin regulator, and involves increases in focal complex signaling and tumor cell–mediated extracellular matrix (ECM) remodeling. Compared with MENA, higher levels of the prometastatic MENA^INV isoform associate with α5, which enables 3-D haptotaxis of tumor cells toward the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MENA^INV and FN levels were correlated in two breast cancer cohorts, and high levels of MENA^INV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor cell–intrinsic mechanism that promotes metastasis through ECM remodeling and ECM-guided directional migration. Significance: Here, we provide new insight into how tumor cell:ECM interactions generate signals and structures that promote directed tumor cell migration, a critical component of metastasis. Our results identify a tumor cell–intrinsic mechanism driven by the actin regulatory protein MENA that promotes ECM remodeling and haptotaxis along FN gradients. Cancer Discov; 6(5); 516–31. ©2016 AACR. See related commentary by Santiago-Medina and Yang, p. 474. This article is highlighted in the In This Issue feature, p. 461

Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

When properly employed, targeted therapies are effective cancer treatments. However, the development of such therapies requires the identification of targetable drivers of cancer development and metastasis. The expression and nuclear localization of the transcriptional coactivators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are increased in many human cancers, and experimental evidence indicates that aberrant YAP or TAZ activation drives tumor formation and metastasis. Although these findings make YAP and TAZ appealing therapeutic targets, both have important functions in adult tissues, so directly targeting them could cause adverse effects. The identification of pathways active in cancer cells and required for YAP/TAZ activity could provide a way to inhibit YAP and TAZ. Here, we show that SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) is an important driver of YAP/TAZ activity in human breast cancer and melanoma cells. SRC activation increased YAP/TAZ activity and the expression of YAP/TAZ-regulated genes. In contrast, SRC inhibition or knockdown repressed both YAP/TAZ activity and the expression of YAP/TAZ-regulated genes. We also show that SRC increases the activity of YAP and TAZ by repressing large tumor suppressor homolog (LATS), and we identify the GTPase-activating protein GIT ArfGAP 1 (GIT1) as an SRC effector that regulates both YAP and TAZ. Importantly, we demonstrate that SRC-mediated YAP/TAZ activity promotes tumor growth and enhances metastasis and that SRC-dependent tumor progression depends, at least in part, on YAP and TAZ. Our findings suggest that therapies targeting SRC could help manage some YAP/TAZ-dependent cancers.

Tumor integrin β1 (ITGB1) contributes to primary tumor growth and metastasis, but its specific roles in extravasation have not yet been clearly elucidated. In this study, we engineered a three-dimensional microfluidic model of the human microvasculature to recapitulate the environment wherein extravasation takes place and assess the consequences of β1 depletion in cancer cells. Combined with confocal imaging, these tools allowed us to decipher the detailed morphology of transmigrating tumor cells and associated endothelial cells in vitro at high spatio-temporal resolution not easily achieved in conventional transmigration assays. Dynamic imaging revealed that β1-depleted cells lacked the ability to sustain protrusions into the subendothelial matrix in contrast with control cells. Specifically, adhesion via α3β1 and α6β1 to subendothelial laminin was a critical prerequisite for successful transmigration. β1 was required to invade past the endothelial basement membrane, whereas its attenuation in a syngeneic tumor model resulted in reduced metastatic colonization of the lung, an effect not observed upon depletion of other integrin alpha and beta subunits. Collectively, our findings in this novel model of the extravasation microenvironment revealed a critical requirement for β1 in several steps of extravasation, providing new insights into the mechanisms underlying metastasis. Cancer Res; 76(9); 2513–24. ©2016 AACR.