Tumor MK2 transcript levels are associated with improved response to chemotherapy and patient survival in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.

MK2 nonenzymatically promotes nuclear translocation of caspase-3 and resultant apoptosis.

We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.

Upregulation of Aquaporin 1 Mediates Increased Migration and Proliferation in Pulmonary Vascular Cells From the Rat SU5416/Hypoxia Model of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by exuberant vascular remodeling leading to elevated pulmonary arterial pressure, maladaptive right ventricular remodeling, and eventual death. The factors controlling pulmonary arterial smooth muscle cell (PASMC) and endothelial cell hyperplasia and migration, hallmark features of the vascular remodeling observed in PAH, remain poorly understood. We previously demonstrated that hypoxia upregulates the expression of aquaporin 1 (AQP1), a water channel, in PASMCs, and that this upregulation was required for hypoxia-induced migration and proliferation. However, whether the same is true in a model of severe PAH and in pulmonary microvascular endothelial cells (MVECs) is unknown. In this study, we used the SU5416 plus hypoxia (SuHx) rat model of severe pulmonary hypertension, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in PASMCs and MVECs and contributed to a hyperproliferative/hypermigratory phenotype. Rats received a single injection of SU5416 (20 mg/kg) and then were placed in 10% O2 for 3 weeks, followed by a return to normoxic conditions for an additional 2 weeks. We found that AQP1 protein levels were increased in both PASMCs and MVECs from SuHx rats, even in the absence of sustained hypoxic exposure, and that in MVECs, the increase in protein expression was associated with upregulation of AQP1 mRNA levels. Silencing of AQP1 had no significant effect on PASMCs from control animals but normalized enhanced migration and proliferation observed in cells from SuHx rats. Loss of AQP1 also reduced migration and proliferation in MVECs from SuHx rats. Finally, augmenting AQP1 levels in MVECs from control rats using forced expression was sufficient to increase migration and proliferation. These results demonstrate a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in pulmonary vascular cells from a rodent model that reflects many of the features of human PAH.