BACKGROUND

Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression.

RESULTS

We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer.

CONCLUSIONS

Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex-activating and filamentous (F)-actin-binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 39 (28%) HNSCC cases, cortactin overexpression determined by immunohistochemistry correlates with lymph node metastasis and CTTN gene amplification. HNSCC cells containing cortactin gene amplification and protein overexpression display increased binding and activation of Arp2/3 complex, and were more motile and invasive than HNSCC cells lacking CTTN amplification. Down-regulation of cortactin expression in CTTN-amplified HNSCC cells by small interfering RNA impairs HNSCC motility and invasion. Treatment of HNSCC cells with the epidermal growth factor receptor inhibitor gefitinib inhibits HNSCC motility and down-regulates cortactin tyrosine phosphorylation. These data suggest that cortactin may be a valid prognostic and therapeutic marker for invasive and metastatic HNSCC and other carcinomas with 11q13 amplification.

Gain of chromosome 11q13 is a common event in esophageal squamous cell carcinoma (ESCC). The cortactin gene (CTTN, also EMS1), located at 11q13, plays a pivotal role in coupling membrane dynamics to cortical actin assembly. This gene has been implicated in the motility of several types of cells. In the present study, we found that the amplification and overexpression of the CTTN gene was associated with lymph node metastasis in ESCC. Functional analysis by small interfering RNA-mediated silencing of CTTN revealed that in addition to the effect on cell migration, CTTN influenced cell invasiveness by anoikis resistance. In vivo assay showed that inhibition of CTTN expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, we showed for the first time that the protective role of CTTN in anoikis resistance was correlated with the activation of the phosphatidylinositol 3-kinase/Akt pathway. Overall, the data suggest that CTTN is an oncogene in the 11q13 amplicon and exerts functions on tumor metastasis in ESCC.

Regional lymph node metastasis is a critical event in oral tongue squamous cell carcinoma (OTSCC) progression. The identification of biomarkers associated with the metastatic process would provide critical prognostic information to facilitate clinical decision making for improved management of OTSCC patients. Global expressional profiles were obtained for 25 primary OTSCCs, where 11 cases showed lymph node metastasis (pN+) histologically and 14 cases were nonmetastatic (pN-). Seven of pN+ cases also exhibited extracapsular spread (ECS) of metastatic nodes. Multiple expression indices were used to generate signature gene sets for pN+/- and ECS+/- cases. Selected genes from signature gene sets were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The classification powers of these genes were then evaluated using a logistic model, receiver operating characteristic curve analysis, and leave-one-out cross-validation. qRT-PCR validation data showed that differences at RNA levels are either statistically significant (P < .05) or suggestive (P < .1) for six of eight genes tested (BMP2, CTTN, EEF1A1, GTSE1, MMP9, and EGFR) for pN+/- cases, and for five of eight genes tested (BMP2, CTTN, EEF1A1, MMP9, and EGFR) for ECS+/- cases. Logistic models with specific combinations of genes (CTTN+MMP9+EGFR for pN and CTTN+EEF1A1+MMP9 for ECS) achieved perfect specificity and sensitivity. Leave-one-out cross-validation showed overall accuracy rates of 85% for both pN and ECS prediction models. Our results demonstrated that the pN and the ECS of OTSCCs can be predicted by gene expression analyses of primary tumors.

Tubulobulbar complexes are actin filament-rich plasma membrane protrusions that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. Tubulobulbar complexes morphologically resemble podosomes found at cell/substrate attachments in other systems. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat testis for two key actin-related components found at podosomes, and for the endocytosis-related protein clathrin. N-WASP and cortactin, two regulators of actin network assembly known to be components of podosomes, are concentrated at tubulobulbar complexes. Clathrin-positive structures occur in Sertoli cell regions containing tubulobulbar complexes when analyzed by immunofluorescence microscopy and occur at the ends of the complexes when evaluated by immunoelectron microscopy. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures. We propose that the formation of tubulobulbar complexes may be clathrin initiated and that their growth is due to the dendritic assembly of a membrane-related actin network.

Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis.

Lung cancer is one of the main causes of cancer death worldwide. The cortactin gene, CTTN, may play a pivotal role in the proliferation and invasion of tumors. A microRNA (miR-182) was cloned and used to study the expression of CTTN and its regulatory effects on the proliferation and invasion of the lung cancer cell line, A549. Cortactin protein and CTTN mRNA expression decreased in A549 cells that were transfected with the miR-182 expression plasmid. A cell proliferation assay indicated that miR-182 expression affected cell cycle regulation and suppressed proliferation of lung cancer cells in vitro. In addition, xenograft experiments confirmed the suppression of tumor growth in vivo, which was due to the promotion of apoptosis. In conclusion, endogenous mature miR-182 expression may have an important role in the pathogenesis of lung cancer through its interference with the target gene CTTN by epigenetic modification.

The epithelial-mesenchymal transition (EMT), a prerequisite for cancer progression and metastasis formation, is regulated not only at the transcriptional but also at the post-transcriptional level, including at the level of alternative pre-mRNA splicing. Several recent studies have highlighted the involvement of splicing factors, including epithelial splicing regulatory proteins (Esrps) and RNA-binding Fox protein 2 (Rbfox2), in this process. Esrps regulate epithelial-specific splicing, and their expression is downregulated during EMT. By contrast, the role of Rbfox2 is controversial because Rbfox2 regulates epithelial as well as mesenchymal splicing events. Here, we have used several established cell culture models to investigate the functions of Rbfox2 during EMT. We demonstrate that induction of an EMT upregulates the expression of Rbfox2, which correlates with an increase in Rbfox2-regulated splicing events in the cortactin (Cttn), Pard3 and dynamin 2 (Dnm2) transcripts. At the same time, however, the epithelial-specific ability to splice the Enah, Slk and Tsc2 transcripts is either reduced or lost completely by Rbfox2, which might be due, in part, to downregulation of the expression of the Esrps cooperative factors. Depletion of Rbfox2 during EMT did not prevent the activation of transforming growth factor-β signaling, the upregulation of mesenchymal markers or changes in cell morphology toward a mesenchymal phenotype. In addition, this depletion did not influence cell migration. However, depletion of Rbfox2 in cells that have completed an EMT significantly reduced their invasive potential. Taken together, our results suggest that during an EMT, Rbfox2-regulated splicing shifts from epithelial-to mesenchymal-specific events, leading to a higher degree of tissue invasiveness.

11q13 amplification is a late-stage event in several cancers that is often associated with poor prognosis. Among 11q13-amplified genes, the actin assembly protein cortactin/CTTN is considered a likely candidate for direct involvement in tumor progression because of its cell motility-enhancing functions. We modulated cortactin expression in head and neck squamous cell carcinoma (HNSCC) cell lines. Cortactin expression levels directly correlated with tumor size, vascularization and cell proliferation in an orthotopic HNSCC in vivo model. In contrast, under normal in vitro culture conditions, cortactin expression levels had no effect on cell proliferation. However, cell lines in which cortactin expression was reduced by knockdown (KD) grew poorly in vitro under harsh conditions of growth factor deprivation, anchorage independence and space constraint. In contrast, overexpression of cortactin enhanced in vitro growth under the same harsh conditions. Surprisingly, defects in growth factor-independent proliferation of cortactin-KD cells were rescued by coculture with cortactin-expressing cells. As the cocultured cells are separated by permeable filters, cortactin-expressing cells must secrete growth-supporting autocrine factors to rescue the cortactin-KD cells. Overall, cortactin expression modulates multiple cellular traits that may allow survival in a tumor environment, suggesting that the frequent overexpression of cortactin in tumors is not an epiphenomenon but rather promotes tumor aggressiveness.

Gene amplifications and deletions are frequent in head and neck squamous cell carcinomas (SCC) but the association of these alterations with gene expression is mostly unknown. Here, we characterized genome-wide copy number and gene expression changes on microarrays for 18 oral tongue SCC (OTSCC) cell lines. We identified a number of altered regions including nine high-level amplifications such as 6q12-q14 (CD109, MYO6), 9p24 (JAK2, CD274, SLC1A1, RLN1), 11p12-p13 (TRAF6, COMMD9, TRIM44, FJX1, CD44, PDHX, APIP), 11q13 (FADD, PPFIA1, CTTN), and 14q24 (ABCD4, HBLD1, LTBP2, ZNF410, COQ6, ACYP1, JDP2) where 9% to 64% of genes showed overexpression. Across the whole genome, 26% of the amplified genes had associated overexpression in OTSCC. Furthermore, our data implicated that OTSCC cell lines harbored similar genomic alterations as laryngeal SCC cell lines We have previously analyzed, suggesting that despite differences in clinicopathological features there are no marked differences in molecular genetic alterations of these two HNSCC sites. To identify genes whose expression was associated with copy number increase in head and neck SCC, a statistical analysis for oral tongue and laryngeal SCC cell line data were performed. We pinpointed 1,192 genes that had a statistically significant association between copy number and gene expression. These results suggest that genomic alterations with associated gene expression changes play an important role in the malignant behavior of head and neck SCC. The identified genes provide a basis for further functional validation and may lead to the identification of novel candidates for targeted therapies. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

The CTTN gene (formerly designated EMS1), encodes cortactin, a key regulator of dynamic actin networks. Both CTTN and CCND1, the latter encoding the cell cycle regulator cyclin D1, reside at chromosomal locus 11q13, a region commonly amplified in breast cancers and head and neck squamous cell carcinoma (HNSCC). Previously, we identified a novel role for cortactin in cancer cells, whereby cortactin overexpression attenuated ligand-induced down-regulation of the epidermal growth factor (EGF) receptor (EGFR), leading to sustained signaling. However, how this affected growth factor-induced cellular responses was unclear. Here, by modulation of cortactin expression in a panel of HNSCC cell lines, we show that cortactin overexpression enhances serum- and EGF-stimulated proliferation under both anchorage-dependent and anchorage-independent conditions and also increases resistance to anoikis (detachment-induced apoptosis). These effects are associated with increased activation of extracellular signal-regulated kinase and/or AKT. Furthermore, we report that cortactin stabilizes the c-MET receptor tyrosine kinase and enhances hepatocyte growth factor-induced mitogenesis and cell scattering. Therefore, cortactin may modulate signaling by a broader range of receptors than originally proposed and thereby affect a variety of responses. Finally, we have determined that cortactin overexpression, either alone or in combination with cyclin D1 up-regulation, promotes resistance to the EGFR kinase inhibitor gefitinib. These findings indicate that cortactin may play multiple roles in progression of HNSCC and should be evaluated as a marker of prognosis, disease progression, and therapeutic responsiveness, particularly to EGFR-directed agents.

Amplification of the 11q13 region is a prevalent genetic alteration in head and neck squamous cell carcinoma (HNSCC). We investigated the clinical significance of cortactin (CTTN) and cyclin D1 (CCND1) amplification in both malignant transformation and tumour progression. CTTN and CCND1 amplification was analysed by differential and real-time PCR in a prospective series of laryngeal/pharyngeal carcinomas and archival premalignant tissues. CTTN mRNA and protein expression were respectively determined by real-time RT-PCR and immunohistochemistry, and correlated with gene status. Molecular alterations were associated with clinicopathological parameters and disease outcome. CTTN and CCND1 amplifications were respectively found in 75 (37%) and 90 (45%) tumours. Both correlated with advanced disease; however, only CTTN amplification was associated with recurrence and reduced disease-specific survival (p = 0.0022). Strikingly, CTTN amplification differentially influenced survival depending on tumour site (p = 0.0001 larynx versus p = 0.68 pharynx) and was an independent predictor of reduced survival in the larynx (p = 0.04). CCND1 amplification was detected in early tumourigenesis and increased with the severity of dysplasia. Importantly, CTTN amplification was only found in high-grade dysplasias that progressed to invasive carcinoma. CTTN gene status strongly correlated with mRNA and protein expression. Furthermore, CTTN overexpression correlated significantly with reduced disease-specific survival (p = 0.018). Taken together, these data indicate that CTTN may serve as a valuable biomarker to identify patients with laryngeal tumours at high risk of recurrence and poor outcome.

We ascertained three different families affected with oto-dental syndrome, a rare but severe autosomal-dominant craniofacial anomaly. All affected patients had the unique phenotype of grossly enlarged molar teeth (globodontia) segregating with a high-frequency sensorineural hearing loss. In addition, ocular coloboma segregated with disease in one family (oculo-oto-dental syndrome). A genome-wide scan was performed using the Affymetrix GeneChip10K 2.0 Array. Parametric linkage analysis gave a single LOD score peak of 3.9 identifying linkage to chromosome 11q13. Haplotype analysis revealed three obligatory recombination events defining a 4.8 Mb linked interval between D11S1889 and SNP rs2077955. Higher resolution mapping and Southern blot analysis in each family identified overlapping hemizygous microdeletions. SNP expression analysis and real-time quantitative RT-PCR in patient lymphoblast cell lines excluded a positional effect on the flanking genes ORAOV1, PPFIA1 and CTTN. The smallest 43 kb deletion resulted in the loss of only one gene, FGF3, which was also deleted in all other otodental families. These data suggest that FGF3 haploinsufficiency is likely to be the cause of otodental syndrome. In addition, the Fas-associated death domain (FADD) gene was also deleted in the one family segregating ocular coloboma. Spatiotemporal in situ hybridization in zebrafish embryos established for the first time that fadd is expressed during eye development. We therefore propose that FADD haploinsufficiency is likely to be responsible for ocular coloboma in this family. This study therefore implicates FGF3 and FADD in human craniofacial disease.

We have shown earlier that overexpression of Calreticulin (CRT) contributed to a poor prognosis for patients with esophageal squamous cell carcinoma (ESCC). Here, we have shown an important role of CRT in tumorigenesis through enhancing cell motility and anoikis resistance. SiRNA-mediated knockdown of CRT caused impaired cell migration, invasion and resistance to anoikis. Notably, CRT downregulation decreased the expression of Cortactin (CTTN), which has been previously reported as a candidate oncogene associated with anoikis through the PI3K-Akt pathway. In addition, Akt phosphorylation was abolished after CRT downregulation and its activation can be refreshed by CRT upregulation, suggesting that CRT-enhanced cell resistance to anoikis through the CRT-CTTN-PI3K-Akt pathway. Moreover, the CTTN mRNA level was decreased in CRT-siRNA cells, coupled with the inactivation of STAT3. Expression of both CTTN and p-STAT3 was reduced in tumor cells following incubation with the JAK-specific inhibitor, AG490. Chromatin immunoprecipitation assay showed direct binding of p-STAT3 to the conservative STAT3-binding sequences in CTTN promoter. Furthermore, overexpression of CTTN in CRT-downregulated ESCC cells restored its motility and resistance to anoikis. This study not only reveals a role of CRT in motility promotion and anoikis resistance in ESCC cells, but also identifies CRT as an upstream regulator in the CRT-STAT3-CTTN-Akt pathway.

The large-conductance Ca(2+)-activated K(+) (BK) channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS) in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP), stathmin (STMN), cortactin (CTTN), and prohibitin (PHB), of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR) and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca(2+) binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt), glycogen synthase kinase-3β (GSK3β) and phosphoinositide-dependent kinase-1 (PDK1). Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite effect. This comparative systems approach suggests conservation in BK function across different species in addition to novel functions that may include the initiation of signals relevant to cell death/survival.

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.

Malignant mesothelioma (MM) is a highly aggressive cancer that is refractory to all current chemotherapeutic regimens. Therefore, uncovering new rational therapeutic targets is imperative in the field. Tyrosine kinase signaling pathways are aberrantly activated in many human cancers and are currently being targeted for chemotherapeutic intervention. Thus, we sought to identify tyrosine kinases hyperactivated in MM. An unbiased phosphotyrosine proteomic screen was employed to identify tyrosine kinases activated in human MM cell lines. From this screen, we have identified novel signaling molecules, such as JAK1, STAT1, cortactin (CTTN), FER, p130Cas (BCAR1), SRC and FYN as tyrosine phosphorylated in human MM cell lines. Additionally, STAT1 and SRC family kinases (SFK) were confirmed to be active in primary MM specimens. We also confirmed that known signal transduction pathways previously implicated in MM, such as EGFR and MET signaling axes, are co-activated in the majority of human MM specimens and cell lines tested. EGFR, MET, and SFK appear to be co-activated in a significant proportion of MM cell lines, and dual inhibition of these kinases was demonstrated to be more efficacious for inhibiting MM cell viability and downstream effector signaling than inhibition of a single tyrosine kinase. Consequently, these data suggest that TKI mono-therapy may not represent an efficacious strategy for the treatment of MM, due to multiple tyrosine kinases potentially signaling redundantly to cellular pathways involved in tumor cell survival and proliferation.

Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.

To better understand the molecular basis of radiation-induced osteosarcoma (OS), we performed global gene expression profiling of rat OS tumors induced by the bone-seeking alpha emitter (238)Pu, and the expression profiles were compared with those of normal osteoblasts (OB). The expressions of 72 genes were significantly differentially expressed in the tumors related to OB. These included genes involved in the cell adhesion (e.g., Podxl, Col18a1, Cd93, Emcn and Vcl), differentiation, developmental processes (e.g., Hhex, Gata2, P2ry6, P2rx5, Cited2, Osmr and Igsf10), tumor-suppressor function (e.g., Nme3, Blcap and Rrm1), Src tyrosine kinase signaling (e.g., Hck, Shf, Arhgap29, Cttn and Akap12), and Wnt/beta-catenin signaling (e.g., Fzd6, Lzic, Dkk3 and Ctnna1) pathways. Expression changes of several genes were validated by quantitative real-time RT-PCR analysis. Notably, all of the identified genes involved in the Wnt/beta-catenin signaling pathway were known or proposed to be negative regulators of this pathway and were downregulated in the tumors, suggesting the activation of beta-catenin in radiation-induced OS. By using immunohistochemical and immunoblot analyses, constitutive activation of the Wnt/beta-catenin signaling pathway in the tumors was confirmed by observing nuclear and/or cytoplasmic localization of beta-catenin and a decrease in its inactive (phosphorylated) form. Furthermore, we found a significant reduction in the levels of glycogen synthase kinase 3beta (GSK-3beta) protein in the tumors relative to OB. Taken together, these findings provide new insights into the molecular basis of radiation-induced OS.

BACKGROUND

Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1-5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1-5 cells to a dcEF, microarray analysis was performed in this study.

RESULTS

A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1-5 cells were treated with the EF strength of 0 mV/mm (the control group) and 300 mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction.

CONCLUSIONS

In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.

OBJECTIVE

The identification of genetic markers associated with oral cancer is considered essential to improve the diagnosis, prognosis, early tumor and relapse detection and, ultimately, to delineate individualized therapeutic approaches. Here, we aimed at identifying such markers.

METHODS

Multiplex Ligation-dependent Probe Amplification (MLPA) analyses encompassing 133 cancer-related genes were performed on a panel of primary oral tumor samples and its corresponding resection margins (macroscopically tumor-free tissue) allowing, in both types of tissue, the detection of a wide arrange of copy number imbalances on various human chromosomes.

RESULTS

We found that in tumor tissue, from the 133 cancer-related genes included in this study, those that most frequently exhibited copy number gains were located on chromosomal arms 3q, 6p, 8q, 11q, 16p, 16q, 17p, 17q and 19q, whereas those most frequently exhibiting copy number losses were located on chromosomal arms 2q, 3p, 4q, 5q, 8p, 9p, 11q and 18q. Several imbalances were highlighted, i.e., losses of ERBB4, CTNNB1, NFKB1, IL2, IL12B, TUSC3, CDKN2A, CASP1, and gains of MME, BCL6, VEGF, PTK2, PTP4A3, RNF139, CCND1, FGF3, CTTN, MVP, CDH1, BRCA1, CDKN2D, BAX, as well as exon 4 of TP53. Comparisons between tumor and matched macroscopically tumor-free tissues allowed us to build a logistic regression model to predict the tissue type (benign versus malignant). In this model, the TUSC3 gene showed statistical significance, indicating that loss of this gene may serve as a good indicator of malignancy.

CONCLUSIONS

Our results point towards relevance of the above mentioned cancer-related genes as putative genetic markers for oral cancer. For practical clinical purposes, these genetic markers should be validated in additional studies.

Adenoid cystic carcinoma (ACC) of the salivary glands exhibits persistent growth, invasion and metastasis. Chromosome 11q13 amplification is a frequent event associated with tumor progression in a number of carcinomas and is associated with poor prognosis. Two genes within the 11q13 amplicon that are overexpressed as a result of 11q13 amplification are the cell cycle regulatory protein cyclin D1 (CCND1) and cortactin (CTTN), a protein involved cell motility and invasion. To determine the expression and gene status of cyclin D1 and cortactin in ACC, we evaluated 39 ACC cases by immunohistochemistry (IHC) for cyclin D1 and cortactin expression. Amplification of CCND1 and CTTN was determined by fluorescent in situ hybridization (FISH). Cyclin D1 overexpression was present in 90% (35/39) and cortactin expression in 62% (24/39) of evaluated cases, although CCND1 and CTTN levels were elevated in only two cases (5%) as determined by FISH. Our results indicate that chromosome 11q13 amplification is uncommon in ACC, but that cyclin D1 and cortactin are frequently overexpressed and may therefore contribute to the growth and invasive potential of ACC.

Development of computational approaches and tools to effectively integrate multidomain data is urgently needed for the development of newly targeted cancer therapeutics.

We proposed an integrative network-based infrastructure to identify new druggable targets and anticancer indications for existing drugs through targeting significantly mutated genes (SMGs) discovered in the human cancer genomes. The underlying assumption is that a drug would have a high potential for anticancer indication if its up-/down-regulated genes from the Connectivity Map tended to be SMGs or their neighbors in the human protein interaction network.

We assembled and curated 693 SMGs in 29 cancer types and found 121 proteins currently targeted by known anticancer or noncancer (repurposed) drugs. We found that the approved or experimental cancer drugs could potentially target these SMGs in 33.3% of the mutated cancer samples, and this number increased to 68.0% by drug repositioning through surveying exome-sequencing data in approximately 5000 normal-tumor pairs from The Cancer Genome Atlas. Furthermore, we identified 284 potential new indications connecting 28 cancer types and 48 existing drugs (adjusted P < .05), with a 66.7% success rate validated by literature data. Several existing drugs (e.g., niclosamide, valproic acid, captopril, and resveratrol) were predicted to have potential indications for multiple cancer types. Finally, we used integrative analysis to showcase a potential mechanism-of-action for resveratrol in breast and lung cancer treatment whereby it targets several SMGs (ARNTL, ASPM, CTTN, EIF4G1, FOXP1, and STIP1).

In summary, we demonstrated that our integrative network-based infrastructure is a promising strategy to identify potential druggable targets and uncover new indications for existing drugs to speed up molecularly targeted cancer therapeutics.

BACKGROUND

Surgical management of primary melanoma is curative for most patients with clinically localized disease at diagnosis; however, a substantial number of patients recur and progress to advanced disease. Understanding molecular alterations that influence differential tumor progression of histopathologically similar lesions may lead to improved prognosis and therapies to slow or prevent metastasis.

METHODS

We examined microRNA dysregulation by expression profiling of primary melanoma tumors from 92 patients. We screened candidate microRNAs selected by differential expression between recurrent and nonrecurrent tumors or associated with primary tumor thickness (Student's t test, Benjamini-Hochberg False Discovery Rate [FDR] < 0.05), in in vitro invasion assays. We performed in vivo metastasis assays, matrix remodeling experiments, and molecular studies to identify metastasis-regulating microRNAs and their cellular and molecular mechanisms. All statistical tests were two-sided.

RESULTS

We identified two microRNAs (hsa-miR-382, hsa-miR-516b) whose expression was lower in aggressive vs nonaggressive primary tumors, which suppressed invasion in vitro and metastasis in vivo (mean metastatic foci: control: 37.9, 95% confidence interval [CI] = 25.6 to 50.2; miR-382: 19.5, 95% CI = 12.2 to 26.9, P = .009; miR-516b: 12.5, 95% CI = 7.7 to 17.4, P < .001, Student's t test). Mechanistically, miR-382 overexpression inhibits extracellular matrix degradation by melanoma cells. Moreover, we identified actin regulators CTTN, RAC1, and ARPC2 as direct targets of miR-382. Depletion of CTTN partially recapitulates miR-382 effects on matrix remodeling, invasion, and metastasis. Inhibition of miR-382 in a weakly tumorigenic melanoma cell line increased tumor progression and metastasis in vivo.

CONCLUSIONS

Aberrant expression of specific microRNAs that can functionally impact progression of primary melanoma occurs as an early event of melanomagenesis.