Regulation of E-cadherin/catenin complex patterns by epidermal growth factor receptor modulation in human lung cancer cells

We previously demonstrated that a ligand-blocking monoclonal antibody (mAb) against the epidermal growth factor-receptor (EGF-R), LA1, induced morphological conversion from epithelial-like to epithelial of the human lung cancer cell line, H322. This was accompanied by an up-regulation of epithelial cadherin (E-cadherin) expression (Clin. Cancer Res. 5 (1999) 681). In the present paper, we show that mAb LA1 induces the epithelial-like to epithelial conversion of the human lung cancer cell line, A549. In A549 and H322 cells, which express a detectable amount of EGF-R (ErbB-1), ErbB-2, ErbB-3, and ErbB-4 receptors, the LA1 mAb induces up-regulation of the E-cadherin/catenin complex (alpha-, beta-, and gamma-catenins). This is associated with re-localization of E-cadherin, alpha-catenin, (and to a lesser extent beta-catenin), but not gamma-catenin. Additionally, we report that mAb LA1 inhibits cell motility. In contrast, epidermal growth factor (EGF) or heparin-binding EGF-like growth factor (HB-EGF) induces the epithelial-like to fibroblastoid conversion of A549 and H322 cell lines, slightly reduces the expression of E-cadherin and beta-catenin, but not alpha- and gamma-catenins, and stimulates cell motility. These studies demonstrate that EGF-R modulation regulates the E-cadherin/catenin complex and cell motility in human lung epithelial carcinoma cells. Our results may have important therapeutic implications for the treatment of invasive human lung carcinomas via the restoration of the cadherin/catenin complex using inhibitors of EGF-R.     

Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation.