Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Carcinoma of the ampulla of Vater: morphological and immunophenotypical classification predicts overall survival.

OBJECTIVES: The objective of the study was to verify if histopathological differentiation of ampullary carcinoma after surgical resection may be related to survival. METHODS: The prognostic role of an accurate histological and immunohistochemical classification has been investigated in a multicentric series of carcinoma of the ampulla of Vater. Immunohistochemical expression of cytokeratin 7 (CK7) and CK20 were analyzed in the different morphological histotypes of ampullary cancers, and results were compared with overall survival. RESULTS: Of 72 ampullary cancers, 48.6% were classified as pancreaticobiliary-type carcinomas, 43.1% were classified as intestinal-type carcinomas, and 8.3% were classified as "unusual"-type carcinomas. Cytokeratin 20 was expressed in 28 (90.3%) of the 31 intestinal-type carcinomas, whereas it was always negative in the pancreaticobiliary histotype; CK7 was expressed in 32 (91.4%) of the 35 pancreaticobiliary-type carcinomas and in 18 (58.1%) of the 31 intestinal-type carcinomas. By univariate analysis, overall survival was influenced significantly by pathological T factor, lymph node involvement, and histological/immunohistochemical subtyping. Furthermore, using a multivariate Cox regression model, lymph node metastasis and CK20 were identified as significant independent factors related to prognosis. CONCLUSION: Our results prove the clinical use of ampullary cancer subclassification based on different histotypes and indicate the useful role of the CK7/CK20 expression profile for consistent histopathological classification and prognostic relevance.

Human equilibrative nucleoside transporter 1 (hENT1) levels predict response to gemcitabine in patients with biliary tract cancer (BTC).

BACKGROUND AND AIM: Translational data suggest that nucleoside transporters, in particular human equilibrative nucleoside transporter 1 (hENT1), play an important role in predicting clinical outcome after gemcitabine chemotherapy for several types of cancer. The aim of this study was to retrospectively determine patients' outcome according to the expression of hENT1 in tumoral cells of patients receiving gemcitabine-based therapy. MATERIALS AND METHODS: The immunohistochemistry analysis was performed on samples from thirty-one patients with unresectable biliary tract cancer (BTC) consecutively treated with first line gemcitabine-based regimens. RESULTS: Positive hENT1 staining patients were 21 (67.7%); negative hENT1 staining patients were 10 (32.3%). Statistical analysis revealed no association between baseline characteristics, toxicities and tumor response to gemcitabine and hENT1 levels. In the univariate analysis, HENT1 expression was significantly correlated with time to progression (TTP) (p=0.0394; HR 2.902, 95%CI 1.053-7.996). The median TTP was 6.33 versus 2.83 months, respectively in patients with positive versus negative hENT1 staining. Moreover, patients with positive hENT1 expression showed a longer median overall survival when compared with patients with low hENT1 expression (14 versus 7 months, respectively), but this difference did not reach the statistical significance (p=0.128). CONCLUSIONS: Therefore, hENT1 may be a relevant predictive marker of benefit from gemcitabine-based therapies in patients with advanced BTC.

The anaplastic lymphoma kinase controls cell shape and growth of anaplastic large cell lymphoma through Cdc42 activation.

Anaplastic large cell lymphoma (ALCL) is a non-Hodgkin's lymphoma that originates from T cells and frequently expresses oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. The proliferation and survival of ALCL cells are determined by the ALK activity. Here we show that the kinase activity of the nucleophosmin (NPM)-ALK fusion regulated the shape of ALCL cells and F-actin filament assembly in a pattern similar to T-cell receptor-stimulated cells. NPM-ALK formed a complex with the guanine exchange factor VAV1, enhancing its activation through phosphorylation. VAV1 increased Cdc42 activity, and in turn, Cdc42 regulated the shape and migration of ALCL cells. In vitro knockdown of VAV1 or Cdc42 by short hairpin RNA, as well as pharmacologic inhibition of Cdc42 activity by secramine, resulted in a cell cycle arrest and apoptosis of ALCL cells. Importantly, the concomitant inhibition of Cdc42 and NPM-ALK kinase acted synergistically to induce apoptosis of ALCL cells. Finally, Cdc42 was necessary for the growth as well as for the maintenance of already established lymphomas in vivo. Thus, our data open perspectives for new therapeutic strategies by revealing a mechanism of regulation of ALCL cell growth through Cdc42.

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase gamma.

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.

Inhibition of PI3K induces Rac activation and membrane ruffling in proto-Dbl expressing cells.

Proto-Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is tightly regulated by a combination of mechanisms that involve intra- and intermolecular interaction and N- and C-terminal domain-dependent turnover of the protein. Moreover, the interaction of the PH domain of proto-Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3-kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto-Dbl-induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto-Dbl-induced cell transformation and motility and observed that inhibition of PI3K in proto-Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto-Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.

Effect of the intracellular localization of a Gd-based imaging probe on the relaxation enhancement of water protons.

Gd-HPDO3A has been internalized into rat hepatocarcinoma cells in the cytoplasm (by electroporation) or in intracellular vesicles (by pinocytosis), respectively. In the former case, the observed relaxation rates are likely dependent upon the amount of internalized paramagnetic complex, whereas in the latter case the relaxation enhancement is "quenched" to a plateau value (about 3 s(-1)) when the entrapped amount of Gd-chelate is higher than 1 x 10(10) Gd/cell. The observed behavior has been accounted in terms of a theoretical treatment based on equations formally derived by Labadie et al. (J Magn Reson B 1994;105:99-102). On this basis, entrapment into intracellular vesicles has been treated as a three-site water exchange (extracellular/cytoplasm/vesicle compartments), whereas the cell pellets containing the paramagnetic agent spread out in the cytoplasm can be analyzed by a two-site exchange system.

Ablation of oncogenic ALK is a viable therapeutic approach for anaplastic large-cell lymphomas.

Anaplastic large-cell lymphomas (ALCLs) carry chromosome translocations in which the anaplastic lymphoma kinase (ALK) gene is fused to several partners, most frequently, the NPM1 gene. We have demonstrated that the constitutive activation of ALK fusion proteins results in cellular transformation and lymphoid neoplasia. Herein, we specifically down-regulated ALK protein expression by using small hairpin RNA (shRNA) targeting a sequence coding for the catalytic domain of ALK. The ablation of ALK leads to the down-modulation of known ALK downstream effectors, cell growth arrest, and reversion of the transformed phenotype of ALK(+) mouse embryonic fibroblasts in vitro and in vivo. In human ALCL cells lentiviral-mediated ALK knock-down leads to G(1) cell-cycle arrest and apoptosis in vitro and tumor growth inhibition and regression in vivo. Using a specific approach we have demonstrated that the survival and growth of ALK(+) ALCLs are strictly dependent on ALK activation and signaling. Therefore, ALK is a viable target for therapeutic intervention and its inactivation might represent a pivotal approach for the treatment of ALK lymphomas and other ALK-dependent human tumors.

p130Cas mediates the transforming properties of the anaplastic lymphoma kinase.

Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)-ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry-based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor-bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALK(K210R) or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.

Follicular origin of a subset of CD5+ diffuse large B-cell lymphomas.

Most follicular lymphomas (FLs) have a phenotype consistent with the origin from CD5-, CD10+, bcl-6+ follicular center cells and can progress to diffuse large B-cell lymphoma (DLBCL). CD5 is expressed in about 10% of DLBCLs, showing prognostic value, whereas expression is rare in FL. We present 6 cases with coexisting features of CD5+ FL and CD5+ DLBCL, supporting a follicular origin for some CD5+ DLBCLs. The follicular areas showed a meshwork of CD21+ follicular dendritic cells that were lacking in the DLBCL areas. All cases showed a clonal CD19+, CD20+, CD5+, and CD10+ population in both follicular and diffuse areas. Molecularly, 4 of 6 cases demonstrated immunoglobulin heavy chain rearrangements and 1 case, a bcl-2/immunoglobulin heavy chain gene rearrangement. Somatic hypermutations were high in all 4 cases, in keeping with their germinal center origin. Four of five patients died of disease within 42 months, consistent with the proposed prognostic value of CD5 expression in DLBCL. Our data describe an aggressive variant of CD5+ FL suggesting the follicular origin of some CD5+ DLBCLs.

Confocal microscope analysis and tridimensional reconstruction of papillary thyroid carcinoma nuclei.

Nuclei of papillary thyroid carcinoma (PTC) are characterised by diagnostic morphological features, which include optically clear nuclei, irregular nuclear profile, pseudoinclusions and grooves. In the present study, such nuclear features were analysed by means of confocal microscopy using anti-lamin B antibodies to outline the nuclear membrane. Parallel sections of the nucleus, produced by confocal microscope analysis, showed that the nuclear shape is markedly irregular with profound invaginations, clefts and tunnel-like structures, which correspond to the "grooves" and "holes" detectable using light microscopy, respectively. A tridimensional (3-D) model of the nuclei, obtained by a computer-based reconstruction of confocal microscope images, showed, in the vast majority of PTC cells, nuclei with crateriform areas, clefts and even tunnel-like structures "piercing" the whole nuclear thickness. By rotating these models in space, it became evident that the holes and grooves seen in light microscopy correspond to invaginations and tunnels, depending on the viewpoint. In conclusion, this is the first application of confocal microscopy and tridimensional reconstruction to the study of nuclear morphology of PTC and of tumours in general. The light microscopic appearance of PTC nuclei, so familiar to pathologists, is, therefore, due to profound remodelling of the nuclear shape with invaginations and tunnels, which appear as either grooves or holes, according to the viewpoint.