Roles of XB130, a novel adaptor protein, in cancer

Adaptor proteins, with multi-modular structures, can participate in the regulation of various cellular functions. During molecular cloning process of actin filament associated protein, we have discovered a novel adaptor protein, referred to as XB130. The human xb130 gene is localized on chromosome 10q25.3, and encodes an 818 amino acid protein. The N-terminal region of XB130 includes several tyrosine phosphorylation sites and a proline-rich sequence that might interact with Src homology 2 and 3 domain-containing proteins, respectively. Our studies have indeed implicated XB130 as a likely substrate and regulator of tyrosine kinase-mediated signaling. Down-regulation of endogenous XB130 with small interfering RNA reduced c-Src activity, IL-8 production and phosphorylation of Akt in human lung epithelial cells. Further, XB130 binds the p85α subunit of phosphatidyl-inositol-3-kinase and subsequently mediates signaling through RET/PTC in thyroid cancer cells. Knockdown of XB130 using small interfering RNA inhibited G1-S phase progression, induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death in human lung and thyroid cancer cells. Growth of tumors in nude mice formed from XB130 short hairpin RNA stably transfected human thyroid cancer cells were significantly reduced, with decreased cell proliferation and increased apoptosis. Further, XB130 has a high affinity to lamellipodial F-actin meshwork and is involved in the motility and invasiveness of cancer cells. Gene expression profiling identified 246 genes significantly changed in XB130 short hairpin RNA transfected thyroid cancer cells. Among them, 57 genes are related to cell proliferation or survival, including many transcription regulators. Pathway analysis showed that the top ranked disease related to XB130 is Cancer, and the top molecular and cellular functions are Cellular Growth and Proliferation, and Cell Cycle. These observations suggest that the expression of XB130 may affect cell proliferation, survival, motility and invasion in various cancer cells. A deeper understanding of these mechanisms may lead to the discovery of XB130 as an important mediator in tumor development and as a novel therapeutic target for cancer

AFAP1L2 (actin filament associated protein 1-like 2)

Review on AFAP1L2, with data on DNA/RNA, on the protein encoded and where the gene is implicated

Claudin 1 Mediates TNFα-Induced Gene Expression and Cell Migration in Human Lung Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies

A New Member of the AFAP Family, AFAP1L1, Binds to Cortactin and Localizes to Invadosomes

Cellular motility and invasion in normal cellular processes and disease states such as cancer are dependent upon the ability of a cell to efficiently interact with its microenvironment, rearrange its cytoskeleton and degrade tissue barriers for purposes of cell movement. The AFAP family of adaptor proteins, AFAP1, AFAP1L1 and AFAP1L2, integrates signals received from the microenvironment into coordinated cytoskeletal changes. While there have been many reports on the functions and binding partners of AFAP1 and AFAP1L2, this work aimed to determine the cellular location and function of newly discovered AFAP1L1. The overall amino acid and protein structures of AFAP family members were compared so as to determine similarities and differences as well as to propose an evolutionary link between all three family members. As AFAP1 and AFAP1L1 have been shown to be more closely related, studies focused on a detailed comparison of these two family members. AFAP1 and AFAP1L1 were shown to have similar cellular localization in the cell by associating with stress filaments and cortical actin and also showing localization to invadosomes. Immunohistochemistry demonstrated differential expression of AFAP1L1 in the brain, particularly surrounding the Purkinje neurons, granular cells and neurons of the dentate nucleus. Although AFAP1 is a well known cSrc binding partner and activator, AFAP1L1 was determined to be a binding partner for cortactin, possibly through the SH3 domain. As other AFAP family members have been shown to be increased in various cancers, AFAP1L1 expression levels are upregulated in a number of cancers, particularly neuroblastoma and glioblastoma. While the similar amino acid sequence and modular domain identifies AFAP1L1 as a previously undescribed member of the AFAP family, the ability of AFAP1L1 to interact with cortactin and localize to distinct areas of the brain implies that AFAP1L1 has unique functions separate from AFAP1 and AFAP1L2

Odin (ANKS1A) is a Src family kinase target in colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>Src family kinases (SFK) are implicated in the development of some colorectal cancers (CRC). One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised.</p> <p>Results</p> <p>64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY) proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM), GIT1, vimentin and AFAP1L2 (XB130). Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells.</p> <p>Conclusion</p> <p>Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.</p

Genome-wide interaction study of smoking behavior and non-small cell lung cancer risk in Caucasian population.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Both environmental and genetic risk factors contribute to lung carcinogenesis. We conducted a genome-wide interaction analysis between SNPs and smoking status (never vs ever smokers) in a European-descent population. We adopted a two-step analysis strategy in the discovery stage: we first conducted a case-only interaction analysis to assess the relationship between SNPs and smoking behavior using 13,336 NSCLC cases. Candidate SNPs with p-value less than 0.001 were further analyzed using a standard case-control interaction analysis including 13970 controls. The significant SNPs with p-value less than 3.5x10-5 (correcting for multiple tests) from the case-control analysis in the discovery stage were further validated using an independent replication dataset comprising 5377 controls and 3054 NSCLC cases. We further stratified the analysis by histological subtypes. Two novel SNPs, rs6441286 and rs17723637, were identified for overall lung cancer risk. The interaction odds ratio and meta-analysis p-value for these two SNPs were 1.24 with 6.96x10-7 and 1.37 with 3.49x10-7, respectively. Additionally, interaction of smoking with rs4751674 was identified in squamous cell lung carcinoma with an odds ratio of 0.58 and p-value of 8.12x10-7. This study is by far the largest genome-wide SNP-smoking interaction analysis reported for lung cancer. The three identified novel SNPs provide potential candidate biomarkers for lung cancer risk screening and intervention. The results from our study reinforce that gene-smoking interactions play important roles in the etiology of lung cancer and account for part of the missing heritability of this disease

The Role Of TKS5 SH3 Domains In Invadopodia Development And Activity

One mechanism by which cancer cells metastasize is through the formation of actin-rich structures called invadopodia. Tks5 is a Src tyrosine kinase substrate and scaffolding protein necessary for invadopodia formation and associated extracellular matrix-remodeling activity. The purpose of this study is to appreciate how the five, protein binding SH3 domains of Tks5 impact its function. Here, Tks5 SH3 domain mutants were introduced into cancer cells to study invadopodia development and activity. Mutant Tks5 constructs were introduced into LNCaP cells to study effects on extracellular matrix degradation based on a microscopic in situ zymography assay. Then, mutant Tks5 constructs were introduced into invadopodia-competent Src-transformed fibroblasts in order to observe their localization by immunofluorescent microscopy. Tks5 constructs with mutations in any of the first three SH3 domains result in an enhancement of invadopodia activity in LNCaP cells while mis-localizing to lipid-rich endosomes in Src-transformed cells and inhibiting their ability to form invadopodia. The loss of invadopodia formation was attributed to a retention of Src kinase in perinuclear endosomes. Current experimentation is focused on elucidating the mechanisms behind the observed results with particular focus on the putative intra and intermolecular Tks5 interactions that would drive the invasive/metastatic behavior of cancer cells

Insulin receptor substrate-1 (IRS-1) forms a ribonucleoprotein complex associated with polysomes

AbstractInsulin receptor substrates (IRSs) are known to play important roles in mediating intracellular insulin-like growth factors (IGFs)/insulin signaling. In this study, we identified components of messenger ribonucleoprotein (mRNP) as IRS-1-associated proteins. IRS-1 complex formation analysis revealed that IRS-1 is incorporated into the complexes of molecular mass more than 1000kDa, which were disrupted by treatment with RNase. Furthermore, oligo(dT) beads precipitated IRS-1 from cell lysates, showing that the IRS-1 complexes contained messenger RNA. Taken together with the data that IRS-1 was fractionated into the polysome-containing high-density fractions, we concluded that IRS-1 forms the novel complexes with mRNPs