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Supplementary Figure S2. Correlation between plasma RTK changes with MAPKi, PFS, and initial tumor shrinkage (using RECIST) in melanoma patients.

Supplementary Methods, Figure Legends

MenaINV, an actin-regulatory protein known to promote metastasis, has roles in driving cross-talk between extracellular matrix, growth factor cues, and their downstream pathways during tumor cell invasion. MenaINV is a shared component of the signaling pathway driving both EGF chemotaxis and fibronectin haptotaxis., Directed cell migration, a key process in metastasis, arises from the combined influence of multiple processes, including chemotaxis—the directional movement of cells to soluble cues—and haptotaxis—the migration of cells on gradients of substrate-bound factors. However, it is unclear how chemotactic and haptotactic pathways integrate with each other to drive overall cell behavior. MenaINV has been implicated in metastasis by driving chemotaxis via dysregulation of phosphatase PTP1B and more recently in haptotaxis via interaction with integrin α5β1. Here we find that MenaINV-driven haptotaxis on fibronectin (FN) gradients requires intact signaling between α5β1 integrin and the epidermal growth factor receptor (EGFR), which is influenced by PTP1B. Furthermore, we show that MenaINV-driven haptotaxis and ECM reorganization both require the Rab-coupling protein RCP, which mediates α5β1 and EGFR recycling. Finally, MenaINV promotes synergistic migratory response to combined EGF and FN in vitro and in vivo, leading to hyperinvasive phenotypes. Together our data demonstrate that MenaINV is a shared component of multiple prometastatic pathways that amplifies their combined effects, promoting synergistic cross-talk between RTKs and integrins.

Kinase inhibitor resistance often involves upregulation of poorly understood “bypass” signaling pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling and drug resistance. Proteolytic shedding of surface receptors, which can provide negative feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine kinases (RTK), including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate, thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of patients with melanoma treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding reduction following treatment, as measured noninvasively in blood plasma. Disrupting protease inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether, extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism for bypass signaling in cancer drug resistance. Significance: Genetic, epigenetic, and gene expression alterations often fail to explain adaptive drug resistance in cancer. This work presents a novel post-translational mechanism of such resistance: Kinase inhibitors, particularly targeting MAPK signaling, increase tumor cell surface receptor levels due to widely reduced proteolysis, allowing tumor signaling to circumvent intended drug action. Cancer Discov; 6(4); 382–99. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 331

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes., Virginia and D.K. Ludwig Fund for Cancer Research (Graduate Student Fellowship), National Institutes of Health (U.S.) (GM58801), Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology, David H. Koch Institute for Integrative Cancer Research at MIT (NCI Core Grant P30-CA14051)

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell–cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue “LERER” repeats. In fibroblasts, the Mena–α5 complex was required for “outside-in” α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins., National Institutes of Health (U.S.) (Grant GM58801), National Cancer Institute (U.S.) (Grant U54-CA112967), Howard Hughes Medical Institute

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena[superscript INV], which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5′ inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena[superscript INV] is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor–induced signaling. Disruption of this attenuation by Mena[superscript INV] sensitizes tumor cells to low–growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes., Breast Cancer Research Program (U.S.) (Grant W81XWH-10-1-0040), Breast Cancer Research Program (U.S.) (Grant W81XWH-13-1-0031), National Institutes of Health (U.S.) (Grant U54-CA112967), National Institutes of Health (U.S.) (Grant GM58801), Virginia and D.K. Ludwig Fund for Cancer Research