Type Iγ phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with μ1B adaptin

Assembly of E-cadherin–based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIγ also interacts with the μ subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIγ or disruption of PIPKIγ binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIγ binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIγ serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate

Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol-3-kinase-dependent in breast carcinoma cells

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells

Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent <it>in situ </it>hybridization (FISH): pathologist assessment compared to quantitative image analysis

Abstract Background In breast cancer patients, HER2 overexpression is routinely assessed by immunohistochemistry (IHC) and equivocal cases are subject to fluorescent in situ hybridization (FISH). Our study compares HER2 scoring by histopathologists with automated quantitation of staining, and determines the concordance of IHC scores with FISH results. Methods A tissue microarray was constructed from 1,212 invasive breast carcinoma cases with linked treatment and outcome information. IHC slides were semi-quantitatively scored by two independent pathologists on a range of 0 to 3+, and also analyzed with an Ariol automated system by two operators. 616 cases were scorable by both IHC and FISH. Results Using data from unequivocal positive (3+) or negative (0, 1+) results, both visual and automated scores were highly consistent: there was excellent concordance between two pathologists (kappa = 1.000, 95% CI: 1-1), between two machines (kappa = 1.000, 95% CI: 1-1), and between both visual and both machine scores (kappa = 0.898, 95% CI: 0.775–0.979). Two pathologists successfully distinguished negative, positive and equivocal cases (kappa = 0.929, 95% CI: 0.909–0.946), with excellent agreement with machine 1 scores (kappa = 0.835, 95% CI: 0.806–0.862; kappa = 0.837, 95% CI: 0.81–0.862), and good agreement with machine 2 scores (kappa = 0.698, 95% CI: 0.6723–0.723; kappa = 0.709, 95% CI: 0.684–0.732), whereas the two machines showed good agreement (kappa = 0.806, 95% CI: 0.785–0.826). When comparing categorized IHC scores and FISH results, the agreement was excellent for visual 1 (kappa = 0.814, 95% CI: 0.768–0.856), good for visual 2 (kappa = 0.763, 95% CI: 0.712–0.81) and machine 1 (kappa = 0.665, 95% CI: 0.609–0.718), and moderate for machine 2 (kappa = 0.535, 95% CI: 0.485–0.584). Conclusion A fully automated image analysis system run by an experienced operator can provide results consistent with visual HER2 scoring. Further development of such systems will likely improve the accuracy of detection and categorization of membranous staining, making this technique suitable for use in quality assurance programs and eventually in clinical practice.</p

Clinicopathologic Factors and Nuclear Morphometry as Independent Prognosticators in KIT-positive Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms found in the gastrointestinal tract. The purpose of this study was to evaluate whether morphometric measurements could complement tumor size and mitotic activity in risk evaluation. Nuclear roundness and ellipse axis ratio were found to correlate with tumor size, mitotic activity, nuclear atypia, and hemorrhage. Morphometric variables in 422 GISTs were significant for overall survival in univariate analyses but did not retain independent significance in multivariate analyses incorporating mitotic count and tumor size. Traditional variables, together with sex, location of primary tumor, and nuclear atypia, seem to be the best parameters for prognostic evaluation. (J Histochem Cytochem 56:139–145, 2008