Non-small cell lung cancer (NSCLC) cells with activating epidermal growth factor receptor (EGFR) somatic mutations have unique biological properties, including high expression of the ErbB ligand epiregulin; however, the biological role of epiregulin in these cells has not been elucidated. To examine its role, we used an immunohistochemical approach to detect epiregulin expression in NSCLC biopsy samples and pharmacologic and genetic approaches to inhibit epiregulin in cultured NSCLC cells. In NSCLC biopsy samples, epiregulin was detected in 237 of 366 (64.7%) tumors, which correlated with nodal metastasis and a shorter duration of survival. In EGFR-mutant NSCLC cell lines, treatment with a small-molecule EGFR tyrosine kinase inhibitor diminished mRNA levels of the gene encoding epiregulin (EREG). The ability of EGFR-mutant NSCLC cells to invade through Matrigel in vitro was inhibited by treatment with an anti-epiregulin neutralizing antibody or by transfection with an EREG short hairpin RNA. Collectively, these findings show that epiregulin expression correlated with advanced disease, was EGFR dependent, and conferred invasive properties on NSCLC cells. Additional studies are warranted in NSCLC patients to evaluate whether epiregulin expression predicts the metastatic potential of primary tumors and whether anti-epiregulin treatment strategies are efficacious in the prevention of metastasis.Treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors leads to rapid and sustained tumor shrinkage in a subset of patients with non-small cell lung cancer (NSCLC; refs. 1-3). The tumor cells in these patients have somatic mutations in the EGFR kinase domain that constitutively activate EGFR (1-3). Mouse models constructed to investigate the oncogenicity of mutant EGFR develop invasive lung adenocarcinomas that regress after treatment of the mice with EGFR tyrosine kinase inhibitors (4, 5). Similarly, immortalized human bronchial epithelial cells acquire malignant properties after transfection with mutant EGFR (6). Treatment with EGFR tyrosine kinase inhibitors induces apoptosis of these EGFR-transfected cells and NSCLC cells with somatic mutations in EGFR (7, 8). Thus, evidence from human, murine, and cellular models indicates that mutant EGFR is oncogenic and confers EGFR dependence on NSCLC for cell survival.Some of the downstream mediators of mutant EGFR that confer oncogenic properties on NSCLC cells have been identified. For example, EGFR forms a heterodimeric complex with ErbB3, which binds to and directly activates phosphatidylinositol 3-kinase and maintains cell survival through AKT-dependent mechanisms (9, 10). Other prosurvival signals in EGFR-mutant NSCLC cells are mediated through Src family kinases, which are constitutively activated (11,12), and by the proapoptotic BH3-only BCL2 family member BIM, which is transcriptionally suppressed (13-16). Anchorage-independent growth of these cells is maintained by a different set of mediators, including cyclooxygenase-2, EphA2 receptor t...

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP] 2 -InsP 4 ), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP] 2 -InsP 4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP] 2 -InsP 4 synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP] 2 -InsP 4 levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP] 2 -InsP 4 synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2Ј-amino-3Ј-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP] 2 -InsP 4 is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.

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