Overcoming cellular mechanisms of de novo and acquired resistance to drug therapy remains a central challenge in the clinical management of many cancers, including non-small cell lung cancer (NSCLC). While much work has linked the epithelial-mesenchymal transition (EMT) in cancer cells to the emergence of drug resistance, it is less clear where tractable routes may exist to reverse or inhibit EMT as a strategy for drug sensitization. Here, we demonstrate that ERK1/2 (MAPK3/1) signaling plays a key role in directing the mesenchymal character of NSCLC cells, and that blocking ERK signaling is sufficient to heighten therapeutic responses to EGFR inhibitors. MEK1/2 (MAPKK1/2) inhibition promoted an epithelial phenotype in NSCLC cells, preventing induction of EMT by exogenous TGFβ. Moreover, in cells exhibiting de novo or acquired resistance to the EGFR inhibitor gefitinib, MEK inhibition enhanced sensitivity to gefitinib and slowed cell migration. These effects only occurred, however, if MEK was inhibited for a period sufficient to trigger changes in EMT marker expression. Consistent with these findings, changes in EMT phenotypes and markers were also induced by expression of mutant KRAS in a MEK-dependent manner. Our results suggest that prolonged exposure to MEK or ERK inhibitors may not only restrain EMT but overcome naïve or acquired resistance of NSCLC to EGFR-targeted therapy in the clinic.

Complexes of signaling proteins that are nucleated upon activation of receptor tyrosine kinases are dynamic macromolecular assemblies held together by interactions, such as the recognition of phosphotyrosines by Src homology 2 (SH2) domains. We predicted that reversible binding and phosphatase activity enable dynamic regulation of these protein complexes, which could affect signal transduction. We explored how dynamics in the interactions among the epidermal growth factor (EGF) receptor (EGFR), GRB2-associated binder protein 1 (GAB1), and SH2 domain-containing phosphatase 2 (SHP2) affected EGFR signaling output, specifically SHP2 binding to tyrosine-phosphorylated GAB1, which relieves the autoinhibition of SHP2. Among the effects of activated SHP2 is increased extracellular signal-regulated kinase (ERK) activity. We found that in H1666 lung adenocarcinoma cells, EGFR-activated Src family kinases (SFKs) counteracted repeated GAB1 dephosphorylation events and maintained the association of SHP2 with phosphorylated GAB1 at a cytosolic site distal from EGFR. A computational model predicted that an experimentally verified delay in SFK inactivation after EGFR inactivation, combined with an amplification of GAB1 phosphorylation in cells with proteins in a specific range of concentrations, enabled GAB1 phosphorylation and GAB1-SHP2 complexes to persist longer than EGFR phosphorylation persisted in response to EGF. This SFK-dependent mechanism was specific to EGFR and did not occur in response to activation of the receptor tyrosine kinase c-MET. Thus, our results quantitatively describe a regulatory mechanism used by some receptor tyrosine kinases to remotely control the duration of a signal by regulating the persistence of a signaling protein complex.

In many epithelial cells, epidermal growth factor (EGF) augments the epithelial-mesenchymal transition (EMT) that occurs when cells are treated with transforming growth factor β (TGFβ). We demonstrate that this augmentation requires activation of SH2 domain-containing phosphatase-2 (SHP2; also known as PTPN11), a proto-oncogene. In lung and pancreatic cancer cell lines, reductions in E-cadherin expression, increases in vimentin expression and increases in cell scatter rates were larger when cells were treated with TGFβ and EGF versus TGFβ or EGF alone. SHP2 knockdown promoted epithelial characteristics basally and antagonized EMT in response to TGFβ alone or in combination with EGF. Whereas EGF promoted SHP2 binding to tyrosine phosphorylated GAB1, which promotes SHP2 activity, TGFβ did not induce SHP2 association with phosphotyrosine-containing proteins. Knockdown of endogenous SHP2 and reconstitution with an SHP2 mutant with impaired phosphotyrosine binding ability eliminated the EGF-mediated EMT augmentation that was otherwise restored with wild-type SHP2 reconstitution. These results demonstrate roles for basal and ligandinduced SHP2 activity in EMT and further motivate efforts to identify specific ways to inhibit SHP2, given the role of EMT in tumor dissemination and chemoresistance.

Glioblastoma multiforme (GBM) is notoriously resistant to therapy, and the development of a durable cure will require the identification of broadly relevant regulators of GBM cell tumorigenicity and survival. Here, we identify Sprouty2 (SPRY2), a known regulator of receptor tyrosine kinases (RTKs), as one such regulator. SPRY2 knockdown reduced proliferation and anchorage-independent growth in GBM cells and slowed xenograft tumor growth in mice. SPRY2 knockdown also promoted cell death in response to co-inhibition of the epidermal growth factor receptor (EGFR) and the c-MET receptor in GBM cells, an effect that involved regulation of the ability of the p38 mitogen activated protein kinase (MAPK) to drive cell death in response to inhibitors. Analysis of clinical tumor specimens further demonstrated that SPRY2 protein is definitively expressed in GBM tissue, that SPRY2 expression is elevated in GBM tumors expressing EGFR variant III (EGFRvIII), and that elevated SPRY2 mRNA expression portends reduced GBM patient survival. Overall, these results identify SPRY2 and the pathways it regulates as novel candidate biomarkers and therapeutic targets in GBM. Implications: SPRY2, counter to its roles in other cancer settings, promotes glioma cell and tumor growth and cellular resistance to targeted inhibitors of oncogenic RTKs; thus, making SPRY2 and the cell signaling processes it regulates potential novel therapeutic targets in glioma.

Information from multiple signaling axes is integrated in the determination of cellular phenotypes. Here, we demonstrate this aspect of cellular decision making in glioblastoma multiforme (GBM) cells by investigating the multivariate signaling regulatory functions of the protein tyrosine phosphatase SHP2. Specifically, we demonstrate that SHP2's ability to simultaneously drive ERK and antagonize STAT3 pathway activities produces qualitatively different effects on the phenotypes of proliferation and resistance to EGFR and c-MET co-inhibition. While the ERK and STAT3 pathways independently promote proliferation and resistance to EGFR and c-MET co-inhibition, SHP2-driven ERK activity is dominant in driving cellular proliferation, and SHP2's antagonism of STAT3 phosphorylation prevails in promoting GBM cell death in response to EGFR and c-MET co-inhibition. Interestingly, the extent of these SHP2 signaling regulatory functions is diminished in glioblastoma cells expressing sufficiently high levels of the EGFR variant III (EGFRvIII) mutant, which is commonly expressed in GBM. In cells and tumors expressing EGFRvIII, SHP2 also antagonizes EGFRvIII and c-MET phosphorylation and drives expression of HIF-1/2α, adding complexity to the evolving understanding of SHP2's regulatory functions in GBM.