BACKGROUNDThe 14‐3‐3ζ protein has been identified as a putative oncoprotein in several cancers, including non–small cell lung cancer (NSCLC). However, the mechanisms underlying its functions have not been well defined.METHODSProteins that interact with 14‐3‐3ζ were identified through coimmunoprecipitation and mass spectrometry in NSCLC cells. The interaction of 14‐3‐3ζ with these molecular partners and their roles in the invasiveness and metastasis of NSCLC cells were assayed through specific disruptions in the 14‐3‐3ζ signaling network. In addition, the clinical implications of this 14‐3‐3ζ complex were examined in samples from patients with NSCLC.RESULTSAmong the identified proteins that interacted with 14‐3‐3ζ, there were 230 proteins in 95‐D cells, 181 proteins in 95‐C cells, and 203 proteins in A549 cells; and 16 interacting proteins were identified that overlapped between all cell lines. Further studies revealed 14‐3‐3ζ complexes within the heat shock protein 27 (Hsp27) protein and demonstrated that the interference of Hsp27 or 14‐3‐3ζ inhibited the invasion and metastasis of NSCLC cells. The invasive and metastatic capabilities of cells with both Hsp27 and 14‐3‐3ζ interference could be completely restored only by Hsp27 and 14‐3‐3ζ complementary DNA transfection and not by either agent alone. Clinically, the postoperative 5‐year overall survival (OS) in patients who had high expression of both 14‐3‐3ζ and Hsp27 was significantly lower than the 5‐year OS in patients who had low expression of both 14‐3‐3ζ and Hsp27 (26.5% vs 59.7%, respectively). Multivariate analysis revealed that the combined expression of 14‐3‐3ζ and Hsp27 was an independent prognostic indicator of OS (P = .036).CONCLUSIONSThe current data suggest that the combined expression of 14‐3‐3ζ and Hsp27 may be a biomarker for predicting survival in patients with NSCLC, and this combination may have potential as a therapeutic target for NSCLC. Cancer 2014;120:652–663. © 2013 American Cancer Society.