CRK SH3N Domain Diminishes Cell Invasiveness of Non-Small Cell Lung Cancer.

CRK (c-Crk) as an adaptor protein is involved in several oncogenic signal transduction pathways, conveying oncogenic signals to its downstream effectors and thereby affecting multiple cellular processes including proliferation, differentiation, and migration. For example, we have observed that CRK expression and phosphorylation influence the invasiveness of non-small cell lung cancer (NSCLC) cells. To intervene in CRK signaling pathway, we examined whether CRK protein domains can be used as therapeutic tools to interrupt CRK signaling, thus influencing the biological behavior of NSCLC cells. For this purpose, Src Homology domains of CRK-I (i.e., SH2 and SH3N domains) were overexpressed in H157, Rh2, and A549 cells. CRK-SH3N domain expression induced epithelial morphology in H157 cells and enhanced epithelial morphology of A549 and Rh2 cells as compared to cells transfected with CRK-SH2 domain or empty vector. In addition, CRK-SH3N domain expression significantly decreased the motility and invasiveness of A549 and H157 cells. Furthermore, CRK-SH3N domain expression disrupted the interaction of CRK-II with DOCK180. In summary, these data provide evidence that the CRK-SH3N domain can be used to influence the malignant phenotype of NSCLC cells and also reduce the metastatic potential of these cells.

A novel role for Niemann-Pick disease type 2C protein in papillae formation.

BACKGROUND: Despite the presence of papillary structures and papillary tumors in humans, the mechanism of papillae formation is unknown. We describe herein a novel role for Niemann-Pick disease type 2C (NPC2) protein, a cholesterol binding protein in the lysosome, in papillae formation. METHODOLOGY/PRINCIPAL FINDING: We examined NPC2 protein expression in surgical samples of papillary tissues by immunohistochemical stain, and all papillary tissues expressed NPC2 protein in the epithelium. To examine our hypothesis of NPC2 protein-mediated papillae formation, we carried out xenograft experiments using wild H460 cells (large cell lung carcinoma cell line) that constitutively expressed abundant NPC2 protein and NPC2 protein-depleted H460 cells by NPC2 shRNA. The xenografts of wild H460 cells and empty shRNA vector cells showed distinct papillae formation, whereas NPC2 protein-depleted H460 cells displayed markedly reduced or no papillae. Since all papillary tissues have open spaces we examined whether NPC2 protein might also contribute to the creation of open spaces. The TUNEL assay in the xenografts of wild and empty shRNA vector H460 cells showed massive cell death, and NPC2 protein-depleted cells displayed minimal cell death. Measurement of caspase 3/7 activities in cultured H460 cells supported NPC2 protein-mediated apoptotic cell death. The presence of excess NPC2 protein, however, did not always produce papillae as seen in the xenografts of CHO cells that were stably transfected with NPC2. CONCLUSIONS/SIGNIFICANCE: The NPC2 protein of certain cells forms papillae coupled with apoptosis that creates open space. This protein may have future applications to modulate papillae formation and papillary growth in tumor tissues.

Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor.

Although preclinical work with rapalogs suggests potential in treatment of multiple myeloma (MM), they have been less successful clinically. These drugs allostearically inhibit the mammalian target of rapamycin kinase primarily curtailing activity of the target of rapamycin complex (TORC)1. To assess if the mammalian target of rapamycin within the TORC2 complex could be a better target in MM, we tested a new agent, pp242, which prevents activation of TORC2 as well as TORC1. Although comparable to rapamycin against phosphorylation of the TORC1 substrates p70S6kinase and 4E-BP-1, pp242 could also inhibit phosphorylation of AKT on serine 473, a TORC2 substrate, while rapamycin was ineffective. pp242 was also more effective than rapamycin in achieving cytoreduction and apoptosis in MM cells. In addition, pp242 was an effective agent against primary MM cells in vitro and growth of 8226 cells in mice. Knockdown of the TORC2 complex protein, rictor, was deleterious to MM cells further supporting TORC2 as the critical target for pp242. TORC2 activation was frequently identified in primary specimens by immunostaining for AKT phosphorylation on serine 473. Potential mechanisms of up-regulated TORC2 activity in MM were stimulation with interleukin-6 or insulin-like growth factor 1, and phosphatase and tensin homolog or RAS alterations. Combining pp242 with bortezomib led to synergistic anti-MM effects. These results support TORC2 as a therapeutic target in MM.

Pathophysiology of tethered cord syndrome and similar complex disorders.

Tethered cord syndrome (TCS) is a stretch-induced functional disorder of the spinal cord due to the fact that its caudal portion is anchored by an inelastic structure. The functional lesion of TCS is generally situated in the lumbosacral cord, and many authors have shown that the syndrome is reversible via surgery to untether the cord. To clarify the expressions relevant to TCS, such as "cord tethering" and "tethered cord," the authors have formulated three categories. These categories include cases that show the anatomical appearance of spinal cord stretching. Among them, Category 1 is isolated to represent the "true TCS." The authors focus their discussion of the pathophysiology of TCS on Category 1 to explain the impaired oxidative metabolism and electrophysiological derangements within the tethered spinal cord, which is the primary intrinsic cause of the dysfunction. Furthermore, they extend the discussion to the extrinsic (outside the spinal cord) factors and other complex conditions that mimic TCS.