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

NPC2 protein expression in cultured H460 cells.

<p>Immunohistochemical staining showed abundant NPC2 protein in wild and empty vector cells and markedly reduced NPC2 protein in <i>NPC2-shRNA</i>-containing cells.</p

NPC2 protein expression in human papillary tissues.

<p>Immunohistochemical stain showed the presence of cytoplasmic NPC2 protein (brown color) in surgical samples of human papillary tissues. All papillary tissues have open spaces outside of papillary projections. The numbers indicate the degree of the magnification.</p

NPC2 protein expression in CHO cells.

<p>Immunohistochemical stain of NPC2 protein in wild CHO cell (a), CHO cells stably transfected with <i>NPC2</i> (b) and xenograft of CHO cells transfected with <i>NPC2</i> (c). No papilla was seen in the xenograft, despite abundant NPC2 protein expression.</p

Presence of apoptotic cells in human papillary tissues.

<p>TUNEL assay showed the presence of apoptotic cells in representative samples of human papillary tissues; arrows indicate examples of apoptotic cells.</p

Characterization of three types of cultured H460 cells.

<p>Cell morphology: Wild and empty vector cells showed the same cell morphology, whereas cells containing <i>NPC2-shRNA</i> displayed clearly separated individual cells. Cell proliferation activity in NPC2-rich and-depleted H460 cells: Cells (1000 cells/well) were plated into a 96-well plate. Cell proliferation activity on each day was measured by incubating cells with the reagents for 2 hours in a CO2 incubator. The results are mean ± SD of quadruplicate samples of one of the three experiments. *<i>p</i><0.01 when compared with the empty vector cells.</p

Caspase 3/7 activities in cultured H460 cells.

<p>Caspase activity of empty vector cell is considered as 100%. The results are mean ± SD of three different experiments performed in duplicates. *<i>p</i><0.0004.</p

TUNEL assay in xenografts NPC2 protein-rich and-depleted cells.

<p>Apoptotic ell death was seen between papillary structures (brown colored areas) in wild H460 cells. The transition from epithelial cells (blue colored cells) to apoptotic cells (brown colored cells) was seen in the outer border of epithelium (middle panel). NPC2 protein-depleted H460 cells showed only small areas of apoptotic cell death.</p

Papillae formation in xenografts of three types of H460 cells.

<p>A: Hematoxylin-eosin stain. B: Immunohistochemical stain for NPC2 protein. Distinct papillae formation was seen in wild and empty vector cells. No papilla was seen in <i>NPC2-shRNA</i> containing cells (A). NPC2 protein was localized in the epithelium of papillae (B).</p

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