knk-437 and Carcinoma--Non-Small-Cell-Lung

Cell surface nucleolin (NCL) plays fundamental roles in tumor angiogenesis. However, the mechanism underlying its surface translocation remains obscure. The present study discovered that heat shock cognate 70 (Hsc70) is essential in both the surface translocation and the angiogenic function of NCL.. We identified that Hsc70 interacted with NCL in endothelial cells via the peptide-binding domain of Hsc70 and the RNA-binding domain of NCL. Functional knockdown of Hsc70 remarkably inhibited the expression of surface NCL, which was rescued by wild-type Hsc70 rather than its truncations. Phosphorylation of NCL by either protein kinase C-ξ or casein kinase 2 mediated its interaction with Hsc70 and the surface expression. Hsc70 regulated NCL translocation via stabilizing NCL and enhancing its interaction with nonmuscle myosin heavy chain 9. Moreover, Hsc70 was associated with NCL-induced endothelial cell migration and tubule formation in vitro and angiogenesis in both matrigel plugs and xenograft tumors. Tissue array analysis revealed that the expression levels of NCL and Hsc70 were intimately correlated in human lung adenocarcinomas.. Our study demonstrates that Hsc70 is a prerequisite for the surface translocation and angiogenic function of NCL, which suggests strategies to target both Hsc70 and NCL for more effective antiangiogenic therapies.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Benzhydryl Compounds; Binding Sites; Carcinoma, Non-Small-Cell Lung; Casein Kinase II; Cell Line, Tumor; Cell Movement; HSC70 Heat-Shock Proteins; Human Umbilical Vein Endothelial Cells; Humans; Lung Neoplasms; Mice; Mice, Nude; Myosin Heavy Chains; Neovascularization, Pathologic; Neovascularization, Physiologic; Nucleolin; Phosphoproteins; Phosphorylation; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Kinase C-epsilon; Protein Stability; Protein Transport; Pyrrolidinones; RNA Interference; RNA-Binding Proteins; Tissue Array Analysis; Transfection; Xenograft Model Antitumor Assays

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat-induced DNA double-strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat-labile repair proteins such as DNA polymerase-beta (Pol beta). Here the authors show that thermotolerance was partially suppressed in Pol beta(-/-) mouse embryonic fibroblasts (MEF) when compared to the wild-type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat-induced gamma H2AX was suppressed in the thermotolerant cells. These results suggest that Pol beta at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat-induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat-labile proteins besides Pol beta. The present novel findings provide strong evidence that Pol beta functions as a critical element involved in thermotolerance and exerts an important role in heat-induced DSB.

Topics: Animals; Benzhydryl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Breaks, Double-Stranded; DNA Polymerase beta; Fever; Fibroblasts; Flow Cytometry; Heat-Shock Proteins; Hot Temperature; Humans; Lung Neoplasms; Mice; Models, Biological; Pyrrolidinones