D. Easty

Requirement for focal adhesion kinase in tumor cell adhesion

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase and a major phosphotyrosine-containing protein. FAK is found in cell-matrix attachment sites (focal adhesions), and is activated on integrin-ligand binding and by other signaling pathways. Several roles have been proposed for FAK; here we report a novel function. We observed abundant FAK protein in all human melanoma cell lines tested except COLO839, a line that grows predominantly in suspension and was derived from peripheral blood. Five adherent lines, isolated from solid metastases in the same patient as COLO839, did express FAK. We derived four adherent sublines from COLO839. These did express FAK, even when plated on bacteriological plastic, to which they did not adhere. Thus, substrate attachment was not required for FAK expression. Three of the adherent sublines were then grown in the presence of antisense oligonucleotides to the initial FAK coding sequence. All showed substantially reduced FAK expression and, interestingly, the cells largely detached from the substrate while continuing to grow. Similar results were obtained with an independent melanoma line, DX3. Thus, FAK expression appears to be required by melanoma cells for substrate adhesion.

Characterization and mapping of the human SOX4 gene

The SOX genes comprise a large family related by homology to the HMG-box region of the testis-determining gene SRY. We have cloned and sequenced the human SOX4 gene. The open reading frame encodes a 474 amino acid protein, which includes an HMG-box. The non-box sequence is particularly rich in serine residues and has several polyglycine and polyalanine stretches. With somatic cell hybrids, human SOX4 has been mapped to Chromosome (Chr) 6p distal to the MHC region. There is no evidence for clustering of other members of the SOX1,-2, and-3 or SOX4 gene families around the SOX4 locus.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural‐crest‐related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest‐like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage‐gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest‐like stem cells. FASEB J. 23, 3179–3192 (2009). www.fasebj.org

Abstract 4114: IGF-1R and EGFR expression and IGF-1R mutational status in non-small cell lung cancer (NSCLC)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Lung cancer is the commonest cause of cancer related deaths worldwide and has a very poor prognosis Recent evidence indicates that epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF-1R) interact in the pathogenesis of malignant epithelial tumours including lung cancer. IGF-1R suppresses apoptosis primarily through the phosphoinositide 3-kinase (PI3K) pathway. Currently, several different approaches are being investigated for targeting the IGF-1R. EGFR targeted therapies are now established in the first and second line treatment of NSCLC. Somatic mutations in the tyrosine kinase domain of a receptor can alter its activity and impact on the ability of an inhibitor to bind efficiently. To date there is limited knowledge of mutations in the IGF-1R tyrosine kinase domain. The aims of this study are (i)to examine EGFR and IGF-1R expression and (ii)to screen for mutations in the tyrosine kinase domain of the IGF-1R in a cohort of 184 NSCLC patients and to correlate the results to patient survival/pathological data. Methods: The expression of IGF-1R and EGFR were examined in a panel of cell lines and 20 resected normal/tumour matched NSCLC patient tissues(10 Adenocarcinomas (ADC) and 10 Squamous (SCC)) using Western Blot analysis. IGF-1R and EGFR expression was evaluated in 184 patients using immunohistochemistry analysis and the results were scored by a pathologist. The DNA from the same cohort of patients is being examined for mutations in the tyrosine kinase domain of the IGF1R using sequencing analysis. Results: A panel of NSCLC cell lines showed differences in IGF1R expression. In the fresh frozen resected NSCLC tumours IGF-1R and EGFR were overexpressed relative to matched normal tissues. SCC had higher levels of expression than ADC. IGF-1R 3+/2+ expression was observed in 53.8% of NSCLC with SCC having higher expression than non-SCC(62.6% versus 37.3%,p=0.0004).EGFR 3+ expression was detected in 51% of NSCLC and SCC had higher EGFR expression than non-SCC (57.4% versus 42.5%,p=0.028).A significant association was shown between IGF-1R 3+/2+ and EGFR 3+ protein expression(p G) in exon 16 of IGF1R in a significantly higher proportion of NSCLC patients and than in an age/race/gender-matched disease-free control population. Conclusions: Our findings indicate a close inter-relationship between IGFR and EGFR. EGFR and IGF-1R expression alone are not independent prognostic markers in NSCLC. In contrast co-expression correlates with a poor survival. This subset of patients may benefit from treatments co-targeting IGF-1R and EGFR. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4114.

Abstract 702: RON receptor tyrosine kinase: A potential therapeutic target in malignant pleural mesothelioma

BACKGROUND: Expression of receptor tyrosine kinases (RTK) and their cognate ligands has been described in mesothelioma, leading to the hypothesis that autocrine growth pathways could be therapeutic targets of tyrosine kinase inhibitors (TKIs). METHODS: Phospho-RTK arrays were used to evaluate phosphorylation of 42 RTKs in 4 mesothelioma cell lines and 11 mesothelioma surgical specimens. Western Blot analysis, IHC, cellular migration and invasion assays were performed. RESULTS: Tumours contained 14 phospho-RTKs. RON emerged as a frequently identifed kinase which has not been previously reported in mesothelioma. An extended panel of sixteen mesothelioma tumour and five benign pleural tissue samples were characterised by western blot analysis. The presence of RON message was confirmed by RT-PCR with specific primers in mesothelioma cell lines Western blot analysis detected RON in 16 tumours and 4 cell lines but not in the SV-40 transformed normal mesothelial MET-5A cells. Mesothelioma expressed different isoforms of RON compared to benign pleural plaques: both benign pleural plaques and mesotheliomas expressed the shortform of RON (sf-RON), whereas, the larger RON variants, Δ160 and Δ165, were seen in mesothelioma samples. IHC was subsequently carried out on a large series of FFPE samples (n>400), with clinical followup to determine if RON expression correlates with prognosis or response to therapy. Scratch assays using antibodies directed against RON prevented MPM cell line migration CONCLUSIONS: Mesothelioma contains multiple activated RTKs including RON. Previously, RON was shown to mediate epithelial mesenchymal transition (EMT). This may be significant given the epithelioid to sarcomatoid spectrum of mesothelioma. Expression of Δ160 and Δ165 was previously reported in gastric and colorectal cancers and may be associated with oncogenesis. RON has been identified as a therapeutic target in pancreatic cancer and this may also apply in mesothelioma. Functional studies on targeting RON in mesothelioma are ongoing. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 702.