Amplification of 11q13 is found in approximately 15% of breast cancers. Cyclin D1 (CCND1) has been reported to be the 'driver' of this amplicon, however, multiple genes map to the smallest region of amplification of 11q13. Out of these genes, cortactin (CTTN) has been shown to be consistently overexpressed at the mRNA level in tumours harbouring 11q13 amplification. The aims of this study are to define whether CTTN is consistently co-amplified with the main core of the 11q13 amplicon, whether it is consistently overexpressed when amplified and to determine correlations between CTTN amplification and overexpression with clinicopathological features of breast cancers and survival of breast cancer patients. CTTN and CCND1 chromogenic in situ hybridisation (CISH) probes and a validated monoclonal antibody against CTTN were applied to a tissue microarray of a cohort of breast cancers from patients treated with anthracycline-based chemotherapy. CTTN and CCND1 amplifications were found in 12.3 and 12.4% of cases, respectively. All cases harbouring CTTN amplification also displayed CCND1 amplification. High expression of CTTN was found in 10.8% of cases and was associated with CTTN amplification, expression of 'basal' markers and topoisomerase IIα. Exploratory subgroup analysis of tumours devoid of 11q13 amplification revealed that high expression of CTTN in the absence of CTTN gene amplification was associated with lymph node negative disease, lack of hormone receptors and FOXA1, expression of 'basal' markers, high Ki-67 indices, p53 nuclear expression, and basal-like and triple negative phenotypes. CTTN expression and CTTN gene amplification were not associated with disease-, metastasis-free and overall survival. In conclusion, CTTN is consistently co-amplified with CCND1 and expressed at higher levels in breast cancers harbouring 11q13 amplification, suggesting that CTTN may also constitute one of the drivers of this amplicon. CTTN expression is not associated with the outcome of breast cancer patients treated with anthracycline-based chemotherapy.

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. High-throughput sequencing data have laid a solid foundation for identifying genes related to cancer prognosis, but a gene marker is needed to predict clinical outcomes in HNSCC. In our study, we downloaded RNA Seq, single nucleotide polymorphism, copy number variation, and clinical follow-up data from TCGA. The samples were randomly divided into training and test. In the training set, we screened genes and used random forests for feature selection. Gene-related prognostic models were established and validated in a test set and GEO verification set. Six genes (PEX11A, NLRP2, SERPINE1, UPK, CTTN, D2HGDH) were ultimately obtained through random forest feature selection. Cox regression analysis confirmed the 6-gene signature is an independent prognostic factor in HNSCC patients. This signature effectively stratified samples in the training, test, and external verification sets (P < 0.01). The 5-year survival AUC in the training and verification sets was greater than 0.74. Thus, we have constructed a 6-gene signature as a new prognostic marker for predicting survival of HNSCC patients.

We investigated the prognostic significance and post-transcriptional acetylation-modification of cortactin (CTTN) via the nucleus accumbens-associated 1 (NACC1)-histone deacetylase 6 (HDAC6) deacetylation system in primary melanomas and melanoma cell lines. Overexpression of CTTN protein was observed in 56 (73%) of 77 stage I-IV melanomas, and was significantly correlated with tumor thickness, lymph node metastasis, distant metastasis, and disease outcome. The patients whose tumors exhibited CTTN overexpression had a poorer outcome than patients without this feature (P=0.028, log-rank test). NACC1 and CTTN proteins, but not HDAC6, were overexpressed in four melanoma cell lines in comparison with a primary culture of normal human epidermal melanocytes. Knockdown of both NACC1 and HDAC6 markedly downregulated the migration activity of all melanoma cell lines (P<0.05), and induced a gain of CTTN protein acetylation status. Confocal microscopy showed that hyperacetylation of CTTN modulated by depletion of both NACC1 and HDAC6 induced disappearance of CTTN protein at the leading edge of migrating cells, resulting in stabilization of the focal adhesion structure and development of actin stress fibers. These data suggest that the acetylation status of CTTN modulated by the NACC1-HDAC6 deacetylation system induces acceleration of melanoma cell migration activity via an actin-dependent cellular process, possibly contributing to aggressive behavior (invasion/metastasis) of the melanoma cells.

Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20-2.43; p=0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40-6.13, p=0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants.

Gastric cancer is an aggressive disease that continues to have a daunting impact on global health. Fra-1 (FOSL1) plays important roles in oncogenesis in various malignancies. We investigated the expression of Fra-1 in gastric cancer (GC) tissues by qPCR, immunohistochemistry (IHC) and western blot technologies. The results showed that Fra-1 was overexpressed in gastric cancer tissues compared with the adjacent non‑cancerous tissues. To explore the possible mechanism of Fra-1 in GC, we elucidated the effect of Fra-1 in the apoptosis and cell cycle of gastric cancer cells, AGS, and found that a considerable decrease in apoptotic cells and increase of S phase rate were observed for AGS cells with Fra-1 overexpession. We identified and confirmed that Fra-1 affected the expression level of CTTN and EZR in vitro through LC-MS/MS analyses and western blot technology. Furthermore, we found that Fra-1 was correlated with dysregulation PI3K/Akt and p53 signaling pathway in gastric cancer tissues in vitro. Moreover, we found that Fra-1 overexpression affected the expression of PI3K, Akt, MDM2 and p53 in vivo. In summary, our results suggest that Fra-1 is upregulated in gastric cancer tissues and plays its function by affecting the PI3K/Akt and p53 signaling pathway in gastric cancer.

The adhesion of osteoclasts (OCs) to bone and bone resorption require the assembly of specific F-actin adhesion structures, the podosomes, and their dense packing into a sealing zone. The OC-specific formation of the sealing zone requires the interaction of microtubule (MT) + ends with podosomes. Here, we deleted cofilin, a cortactin (CTTN)- and actin-binding protein highly expressed in OCs, to determine if it acts downstream of the MT-CTTN axis to regulate actin polymerization in podosomes. Conditional deletion of cofilin in OCs in mice, driven by the cathepsin K promoter (Ctsk-Cre), impaired bone resorption in vivo, increasing bone density. In vitro, OCs were not able to organize podosomes into peripheral belts. The MT network was disorganized, MT stability was decreased, and cell migration impaired. Active cofilin stabilizes MTs and allows podosome belt formation, whereas MT disruption deactivates cofilin via phosphorylation. Cofilin interacts with CTTN in podosomes and phosphorylation of either protein disrupts this interaction, which is critical for belt stabilization and for the maintenance of MT dynamic instability. Accordingly, active cofilin was required to rescue the OC cytoskeletal phenotype in vitro. These findings suggest that the patterning of podosomes into a sealing zone involves the dynamic interaction between cofilin, CTTN, and the MTs + ends. This interaction is critical for the functional organization of OCs and for bone resorption. © 2016 American Society for Bone and Mineral Research.

BACKGROUND

Vascular endothelial growth factor-C (VEGF-C) plays an important role during cancer progression and metastasis through activation of VEGF receptors. However, the role of VEGF-C in esophageal squamous cell carcinoma (ESCC) remains unclear.

METHODS

The expression of VEGF-C in advanced stages of esophageal cancer was examined by immunohistochemistry and its expression was correlated with the protein level of cortactin (CTTN) by Western blot. Knockdown and overexpression of the CTTN protein were respectively performed to investigate the effects on VEGF-C-enhanced ESCC migration/invasion by in vitro transwell assay, cell tracing assay, and tumor growth/experimental metastasis in animal models.

RESULTS

The expression of VEGF-C was positively correlated with tumor status and poor clinical prognosis in patient with esophageal cancer. VEGF-C-upregulated CTTN expression contributed the migration/invasive abilities of ESCC cell lines through Src-mediated downregulation of miR-326. Moreover, knockdown of CTTN expression significantly abolished VEGF-C-induced tumor growth and experimental lung metastasis in vivo.

CONCLUSIONS

Upregulation of CTTN is critical for VEGF-C-mediated tumor growth and metastasis of ESCC. These finding suggest that VEGF-C upregulated CTTN expression through Src-mediated downregulation of miR-326. CTTN may be a crucial mediator of VEGF-C-involved ESCC metastasis, which provides a potential target for diagnosis and individualized treatment in clinical practice.

BACKGROUND

The gene encoding cortactin, CTTN (locus 11q13), an actin-binding substrate of Src kinases, is frequently amplified in breast and head and neck squamous cell carcinomas (HNSCC) and cortactin overexpression is thought to contribute in a significant way to the invasive phenotype of these tumors. Elevated Epidermal Growth Factor receptor (EGFR) expression is also commonly observed in HNSCC and has been associated with poor prognosis and resistance to cytotoxic agents, including ionizing radiation. It has been suggested that cortactin overexpression may increase EGFR levels in these tumors by affecting receptor downregulation, however we recently found by multivariate analysis, that cortactin expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival.

METHODS

To examine the potential link between cortactin overexpression and EGFR status, we compared cortactin and EGFR levels in a series of tumor lines derived from HNSCC. RNAi-mediated silencing was performed in cortactin overexpressing cells and in vivo tumoral potential with respect to cortactin and EGFR status was analyzed.

CONCLUSIONS

Cortactin and EGFR levels were not strictly coupled in these lines and cortactin depletion did not decrease steady state receptor levels, although it did affect the epithelial to mesenchymal phenotypic conversion of cells. These results, together with clinical findings point to the existence of an EGFR-independent role of cortactin in HNSCC that may have important implications regarding the design of targeted therapies to combat tumor spread.

Head and neck squamous cell carcinoma (HNSCC) is a solid malignant neoplasm exhibiting aggressive phenotypes and high recurrence rates. To improve its clinical management, understanding the molecular basis of HNSCC development is of critical importance. For the investigation of tumor-associated genes, functional analyses in well-characterized tumor cell systems are required. To establish an experimental platform, a set of 20 HNSCC cell lines was screened for genetic imbalances by chromosomal comparative genomic hybridization (cCGH). Frequent DNA copy number gains were detected on 3q26.3-qter, 5p, 7p11-p13, 8q23-qter, 9p11-p13, 9q31-qter, 11q13 and 20q13.1, whereas copy number losses were found on 3p, 4p, 4q32.1-qter, 8p11-p12 and 18q22 in agreement with previous observations on genetic aberrations detected in primary HNSCC specimens. Subsequent mRNA expression analysis of 11q13 candidate genes CCND1 and CTTN revealed that HNSCC cell lines exhibiting a DNA copy number gain on 11q13 had a higher transcript level of CCND1 and CTTN compared with HNSCC cell lines without 11q13 copy number gain (P = 0.014 and P = 0.009, respectively). Furthermore, CCND1 and CTTN amplification as detected by fluorescence in situ hybridization correlated with protein expression as assessed by immunocytochemistry. In summary, the cytogenetic characterization illustrates that this set of HNSCC cell lines is representative for the HNSCC genome and provides tumor model systems for detailed analysis of genes with a possible role in the pathomechanism of head and neck tumors.

Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and in cell motility and invasion. CTTN gene is amplified and its protein is overexpressed in several types of cancer. The phosphorylated form of cortactin (pTyr(421)) is required for cancer cell motility and invasion. In this study, we demonstrate that a majority of the tested primary colorectal tumor specimens show greatly enhanced expression of pTyr(421)-CTTN, but no change at the mRNA level as compared to healthy subjects, thus suggesting post-translational activation rather than gene amplification in these tumors. Curcumin (diferulolylmethane), a natural compound with promising chemopreventive and chemosensitizing effects, reduced the indirect association of cortactin with the plasma membrane protein fraction in colon adenocarcinoma cells as measured by surface biotinylation, mass spectrometry, and Western blotting. Curcumin significantly decreased the pTyr(421)-CTTN in HCT116 cells and SW480 cells, but was ineffective in HT-29 cells. Curcumin physically interacted with PTPN1 tyrosine phosphatases to increase its activity and lead to dephosphorylation of pTyr(421)-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr(421)-CTTN. Transduction with adenovirally-encoded CTTN increased migration of HCT116, SW480, and HT-29. Curcumin decreased migration of HCT116 and SW480 cells which highly express PTPN1, but not of HT-29 cells with significantly reduced endogenous expression of PTPN1. Curcumin significantly reduced the physical interaction of CTTN and pTyr(421)-CTTN with p120 catenin (CTNND1). Collectively, these data suggest that curcumin is an activator of PTPN1 and can reduce cell motility in colon cancer via dephosphorylation of pTyr(421)-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr(421)-CTTN expression.

Esophageal squamous cell carcinoma (ESCC) is a genetically complex tumor type and is a major cause of cancer-related mortality. The combination of genetics, diet, behavior, and environment plays an important role in the carcinogenesis of ESCC. To characterize the genomic aberrations of this disease, we investigated the genomic imbalances in 19 primary ESCC cases using high-resolution array comparative genomic hybridization (CGH). All cases showed either loss or gain of whole chromosomes or segments of chromosome(s) with variable genomic sizes. The copy number alterations per case affected the median 34% (~ 1,034Mb/3,000Mb) of the whole genome. Recurrent gains were 1q21.3-qter, 3q13.11-qter, 5pter-p11, 7pter-p15.3, 7p12.1-p11.2, 7q11-q11.2, 8p12-qter, 11q13.2-q13.3, 12pter-p13.31, 17q24.2, 20q11.21-qter, and 22q11.21-q11.22 whereas the recurrent losses were 3pter-p11.1, 4pter-p12, 4q28.3-q31.22, 4q31.3-q32.1, 9pter-p12, 11q22.3-qter and 13q12.11-q22.1. Amplification of 11q13 resulting in overexpression of CTTN/CCND1 was the most prominent finding, which was observed in 13 of 19 ESCC cases. These unique profiles of copy number alteration should be validated by further studies and need to be taken into consideration when developing biomarkers for early detection of ESCC.

TMEM16A gene is located within the CCND1-ORAOV1-FGF19-FGF4-FGF3-TMEM16A-FADD-PPFIA1-CTTN (cortactin or EMS1) amplicon at human chromosome 11q13.3. TMEM16A, TMEM16B, TMEM16C, TMEM16D, TMEM16E (GDD1), TMEM16F, TMEM16G and TP53I5 are TMEM16 family proteins with TM16H1, TM16H2 and TM16H3 domains. Here, we identified and characterized TMEM16H gene by using bioinformatics. Human TMEM16H gene, consisting of 18 exons, was located at human chromosome 19p13.11. AB046843.2 cDNA corresponded to TMEM16H isoform 1 without intron 17, while CR616031.1 cDNA corresponded to TMEM16H isoform 2 with retained intron 17. TMEM16H isoforms were eight-transmembrane proteins with TM16H3 domain. The last exon of isoforms 1 and 2 corresponded to C-terminal 122 and 227 aa, respectively. Human TMEM16H isoform 1 was the major transcript, while isoform 2 was the minor transcript prone to non-sense mediated mRNA decay (NMD). TMEM16H mRNA was expressed in embryonic stem (ES) cells, fetal brain, and neural tissues. Mouse Tmem16h representative cDNA was determined by assembling BC027735.1 and BC059855.1 cDNAs. Mouse and rat Tmem16h genes encoded Tmem16h proteins similar to human TMEM16H isoform 2. The regions corresponding to exons 17 and 18 of human TMEM16H gene were not separated by intron within mouse and rat Tmem16h genes. Insertion of intron 17 into human TMEM16H gene during evolution resulted in the generation of TMEM16H isoform 1. This is the first report on identification and characterization of the TMEM16H gene as well as on the molecular evolution of TMEM16H.

Thyroid cancer is the fastest growing cancer among all solid tumors in recent decades. Papillary thyroid carcinoma (PTC) is the most predominant type of thyroid cancer. Around 30% of PTC patients with distant metastases and local invasion receive poor prognosis. Thus, the identification of new druggable biological targets is of great importance. Accumulating evidence indicates that solute carrier family numbers have emerged as obligate effectors during the progression of multiple malignancies. Here, we uncovered the functional significance, molecular mechanisms, and clinical impact of solute carrier family 34 member A2 (SLC34A2) in PTC. SLC34A2 was markedly overexpressed in PTC tissues at both mRNA and protein levels compared with matched adjacent normal tissues due to promoter hypomethylation mediated by the DNA methyltransferase 3 beta (DNMT3B). Furthermore, a series of in vivo and in vitro gain- or loss-of-functional assays elucidated the role of SLC34A2 in boosting cell proliferation, cell cycle progression, migration, invasion, and adhesion of PTC cells. Using immunoprecipitation and mass spectrometry, we discovered that SLC34A2 bound to the actin-binding repeats domain of Cortactin (CTTN), thereby inducing the invadopodia formation of PTC cells to promote the metastasis potential of PTC cells. Besides, our mechanistic studies, as well as gene set enrichment analysis (GSEA), have pinpointed the PTEN/AKT/FOXO3a pathway as a major signaling functioning downstream of SLC34A2 regulated cell growth. Taken together, our results highlighted that SLC34A2 plays a pivotal oncogenic role during carcinogenesis and metastasis through distinct mechanisms in PTC.

The cortactin gene, mapped at 11q13, has been associated with an aggressive clinical course in many cancers because of its function of invasiveness. This study evaluated CTTN protein and its prognostic value in the deep invasive front and superficial areas of laryngeal squamous cell carcinomas. The transcript expression levels were evaluated in a subset of cases. Overexpression of CTTN cytoplasmatic protein (80% of cases in both the deep invasive front and superficial areas) and transcript (30% of samples) was detected in a significant number of cases. In more than 20% of cases, observation verified membrane immunostaining in the deep invasive front and superficial areas. Perineural invasion was significantly associated with N stage and recurrence (P = .0058 and P = .0037, respectively). Higher protein expression levels were correlated with perineural invasion (P = .004) in deep invasive front cells, suggesting that this area should be considered a prognostic tool in laryngeal carcinomas. Although most cases had moderate to strong CTTN expression on the tumor surface, 2 sets of cases revealed a differential expression pattern in the deep invasive front. A group of cases with absent to weak expression of CTTN in the deep invasive front showed good prognosis parameters, and a second group with moderate to strong expression of CTTN were associated with an unfavorable prognosis, suggesting an association with worse outcome. Taken together, these results suggest that the deep invasive front might be considered a grading system in laryngeal carcinomas and that cortactin is a putative marker of worse outcome in the deep invasive front of laryngeal carcinomas.

The CCND1-ORAOV1-FGF19-FGF4-FGF3-TMEM16A-FADD-PPFIA1-CTTN (EMS1) locus at human chromosome 11q13.3 is amplified in head and neck tumors, esophageal cancer, Kaposi's sarcoma, bladder tumors, breast cancer, and liver cancer. Fgf4 mRNA is expressed in embryonic stem (ES) cells depending on Sox2 and Pou5f1 (Oct3/Oct4) transcription factors, and in myotomes and limb bud AER depending on MyoD (or Myf5) and GATA transcription factors. Here, rat Fgf3 and Fgf4 complete coding sequences were determined by using bioinformatics. Multiple errors, including one-base insertion and 22-base deletion, were identified within the coding region of rat Fgf4 RefSeq (NM_053809.1 or AB079673.1). Rat Fgf3 and Fgf4 genes, consisting of three exons, were clustered in tail-to-head manner with an interval of about 16 kb. CUTL1 (CCAAT-displacement protein, CDP) and NKX2-5 binding sites and TATA box within 5'-flanking promoter region were conserved among human, rat and mouse Fgf3 orthologs. MYOD and MYOG (Myogenin) binding sites and TATA box within 5'-flanking promoter region as well as GATA, MYOD, SOX2 and POU5F1 binding sites within exon 3 were conserved among mammalian Fgf4 orthologs. Human FGF3 and FGF4 genes were clustered in tail-to-head manner with an interval of about 35 kb. Major repetitive sequence (FGF34Rep1) and minor repetitive sequence (FGF34Rep2) were identified within human FGF3-FGF4 gene cluster. FGF34Rep1 were clustered within the FGF3-FGF4 locus as well as around the IL28RA locus (1p36.11) and the NFAM1 locus (22q13.2). FGF34Rep2 was characterized by the CCA(T/C) repeats. This is the first report on comparative genomics analyses on the Fgf3-Fgf4 locus within human, rat and mouse genomes.

Macroautophagy (autophagy) is a multistep intracellular degradation system. Autophagosomes form, mature, and ultimately fuse with lysosomes, where their sequestered cargo molecules are digested. In contrast to autophagosome formation, our knowledge of autophagosome-lysosome fusion is limited. In a recent study, we identified a novel regulator of autophagy, INPP5E (inositol polyphosphate-5-phosphatase E), which is essential for autophagosome-lysosome fusion. INPP5E primarily functions in neuronal cells, and knockdown of the corresponding gene causes accumulation of autophagosomes by impairing fusion with lysosomes. Some INPP5E molecules localize at the lysosome, and both lysosomal localization and INPP5E enzymatic activity are crucial for autophagy. In addition, INPP5E decreases PtdIns(3,5)P2 levels on lysosomes, leading to activation of CTTN (cortactin) and stabilization of actin filaments, which are also essential for autophagosome-lysosome fusion. Mutations in INPP5E are causative for Joubert syndrome, a rare brain abnormality, and our results indicate that defects in autophagy play a critical role in pathogenesis.

Colorectal cancer is one of the most common malignancies. Previous studies have reported that cortactin (CTTN) is often overexpressed in tumors and is associated with metastasis and poor prognosis of patients. The abnormal expression of microRNAs (miRNAs or miRs) is closely related to the development and progression of various types of cancer, including colorectal cancer. However, little is known about the miRNAs targeting cortactin. In the present study, prediction using biological software revealed that cortactin has binding sites for miR-542-3p. Transfection with miR-542-3p mimic demonstrated that miR‑542-3p reduced the expression of cortactin in colorectal cancer cells. Dual luciferase reporter assays further demonstrated that miR-542-3p regulated cortactin in a targeted manner and that miR-542-3p expression was significantly downregulated in colorectal cancer cells. A cell proliferation assay and Transwell migration assay were undertaken: we noted that miR‑542-3p inhibited the proliferation and invasion of colorectal cancer cells while promoting their apoptosis. By contrast, cortactin acted antagonistically. When co-transfected with miR-542-3p mimic and CTTN overexpression vector, the inhibitory effect of miR-542-3p was blocked. This indicates that miR-542-3p regulates CTTN in a targeted manner to modulate the growth and invasion of colorectal cancer cells. The present study thus provides new targets for the prevention and treatment of colorectal cancer.

This study was aimed to evaluate the regulation mechanism of cortactin (CTTN) on matrix metalloproteinases 9 (MMP-9) and its relations with Exo70 in invasion of hepatoma carcinoma (HCC) cells. The expression levels of CTTN, Exo70 and MMP-9 were detected in normal hepatocytes and various HCC cells by real-time PCR. Then the migration and invasion ability of these cells was revealed by scratch and invasion assay. The effects of CTTN on MMP-9 and the ability of migration and invasion were evaluated by silence and overexpress CTTN. During this process, the expression of CTTN was detected by Western blot, the activity and concentration of MMP-9 in supernatant of culture medium was detected by zymography and ELISA assay. Besides, Exo70 was also inhibited to reveal its effects on MMP-9 and the migration and invasion ability of LM3. Increased expression of CTTN, MMP-9, Exo70, reduced scratch area and increased puncture cell numbers were found in HCC cells (p < 0.05). The expression of CTTN was significantly correlated with Exo70 and the migration and invasion ability of HCC (p < 0.05). In addition, the activity and concentration of MMP-9 were significantly affected by the expression level of CTTN, while the expression of MMP-9 was not influenced. Besides, Exo70-si also exhibited significantly inhibition effects on the activity and concentration of MMP-9 and puncture cell numbers (p < 0.05). A synergistic reaction may exhibited on CTTN and Exo70, which could mediate the secretion of MMPs thereby regulate tumor invasion.

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Characterizing the factors that regulate the growth and development of muscle is central to animal production. Skeletal muscle satellite cells (SMSCs) provide an important material for simulating the proliferation and differentiation of muscle cells. YAP1, which can promote muscle growth, is closely related to the proliferation of SMSCs in Hu sheep (Ovis aries). In addition, some miRNAs, such as miR-541-3p, miR-142-5p, and miR-29a, can play critical roles in muscle growth by specifically binding with their target mRNAs. Meanwhile, lncRNA can competitively bind these miRNAs and reduce the regulatory effect of miRNAs on their target genes and thus play critical roles themselves in muscle growth. However, the regulatory molecular mechanism of miRNA and lncRNA on SMSC proliferation through YAP1 remains unclear. Here, we characterized the regulatory network among YAP1 and its targeted miRNAs and lncRNAs in Hu sheep SMSCs. The potential ncRNAs that regulate YAP1 (miR-29a and CTTN-IT1) were predicted through multilevel bioinformatics analysis. Dual-luciferase assays, RT-qPCR, and western blots revealed that miR-29a can significantly reduce the mRNA and protein expression level by binding to a specific 3'-UTR of YAP1 (P < 0.05), while CTTN-IT1 can restore the expression of YAP1 through competitive binding to miR-29a. Furthermore, the mRNA and protein expression levels of MyoG, MyoD, and MyHC showed that miR-29a can inhibit the expression of genes related to the differentiation of SMSCs, and CTTN-IT1 can increase the expression of these same genes. Thus, miR-29a may inhibit the differentiation of SMSCs and CTTN-IT1 can restore this inhibition. The EdU staining assay indicated that excessive miR-29a can significantly reduce the proliferation ability of SMSCs (P < 0.05), while overexpression of CTTN-IT1 can significantly increase the proliferation of SMSCs (P < 0.01). CTTN-IT1 is a novel lncRNA that is a competing endogenous RNA (ceRNA) of miR-29a and can promote SMSC proliferation and differentiation by restoring the expression of YAP1 when it is inhibited by miR-29a in Hu sheep. Overall, our findings construct a CTTN-IT1-miR-29a-YAP1 regulatory network that will help contribute new insight into improving the muscle development of Hu sheep.

Keywords: Hu sheep; YAP1; lncRNA; miR-29a; satellite cell; skeletal muscle.

BACKGROUND

In the new pathologic classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011, lepidic type adenocarcinomas are constituted by three subtypes; adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPIA). Although these subtypes are speculated to show sequential progression from preinvasive lesion to invasive lung cancer, changes of protein expressions during these processes have not been fully studied yet. This study aims to glimpse a proteomic view of the early lepidic type lung adenocarcinomas.

METHODS

A total of nine formalin-fixed and paraffin-embedded (FFPE) lepidic type lung adenocarcinoma tissues were selected from our archives, three tissues each in AIS, MIA and LPIA. The tumor and peripheral non-tumor cells in these FFPE tissues were collected with laser microdissection (LMD). Using liquid chromatography-tandem mass spectrometry (MS/MS), protein compositions were compared with respect to the peptide separation profiles among tumors collected from three types of tissues, AIS, MIA and LPIA. Proteins identified were semi-quantified by spectral counting-based or identification-based protein-based approach, and statistical evaluation was performed by pairwise G-tests.

RESULTS

A total of 840 proteins were identified. Spectral counting-based semi-quantitative comparisons of all identified proteins through AIS to LPIA have revealed that the protein expression profile of LPIA was significantly differentiated from other subtypes. 70 proteins including HPX, CTTN, CDH1, EGFR, MUC1 were found as LPIA-type marker candidates, 15 protein candidates for MIA-type marker included CRABP2, LMO7, and NPEP, and 26 protein candidates for AIS-type marker included LTA4H and SOD2. The STRING gene set enrichment resulted from the protein-protein interaction (PPI) network analysis suggested that AIS was rather associated with pathways of focal adhesion, adherens junction, tight junction, that MIA had a strong association predominantly with pathways of proteoglycans in cancer and with PI3K-Akt. In contrast, LPIA was associated broadly with numerous tumor-progression pathways including ErbB, Ras, Rap1 and HIF-1 signalings.

CONCLUSIONS

The proteomic profiles obtained in this study demonstrated the technical feasibility to elucidate protein candidates differentially expressed in FFPE tissues of LPIA. Our results may provide candidates of disease-oriented proteins which may be related to mechanisms of the early-stage progression of lung adenocarcinoma.

Hepatitis B virus-encoded X protein (HBx) acts as a tumor promoter during hepatocellular carcinoma (HCC) development, probably by regulating the expression of host proteins through protein-protein interaction. A proteomics approach was used to identify HBx-interacting proteins involved in HBx-induced hepatocarcinogenesis. We validated the proteomics findings by co-immunoprecipitation and confocal microscopy. We performed cell proliferation, migration assays and cell cycle analyses in HCC cells. Finally, we confirmed the clinical significance of our findings in samples from patients. We found that cortactin (CTTN) is a novel HBx-interacting protein, and HBx regulates the expression of CTTN in the HCC cell lines MHCC-LM3 and HepG2. Mechanistically, by upregulating the expression of cAMP response element-binding protein (CREB1) and its downstream targets, such as cyclin D1 and MMP-9, the effects of the HBx-CTTN interaction on the enhancement of cellular proliferation and migration were maintained by inhibiting cell cycle arrest. In addition, we demonstrated that the levels of CTTN and CREB1 were closely correlated in clinical samples from HBV-infected patients with HCC. Overall, our data suggests that HBx contributes to cell migration and proliferation of HCC cells by interacting with CTTN and regulating the expression of CTTN and CREB1. Therefore, the HBx/CTTN/CREB1 axis is a potential novel therapeutic target in HCC.

Wilms tumor is one of the most common solid tumors in children. We evaluated expression and amplification of a number of genes and their prognostic significance in 45 patients with Wilms tumor, using tissue microarray technology. The expression of EGFR, ERBB2, MDM2, CCND1, MLH1, MSH2, TP53, and ABCB1 (alias MDR1) was studied by immunohistochemistry. Amplification of the EGFR, ERBB2, MDM2, CCND1, CTTN (previously EMS1), RAF1, MYC, FGF3 (previously INT2), WNT1, GLI1, CDK4, and NCOA3 (alias AIB1) genes was assessed by fluorescence in situ hybridization. Expression of EGFR was seen in 17 of the 45 cases (38%) but was not associated with gene amplification. The ERBB2 gene was neither overexpressed nor amplified in any case. Tissue microarray and immunohistochemistry analyses for ERBB2 in whole-tumor sections were also negative in all cases. Strong p53 reactivity was noted in blastemal cells in two cases with an unfavorable outcome. ABCB1 reactivity was seen in five cases with favorable histology and outcome. Only one case showed nuclear cyclin D1 positivity. All tumors showed MLH1 and MSH2 expression. All examined genes showed normal copy numbers. Unfavorable histology correlated with poor prognosis (P=0.03). There was no significant association between gene expression and prognosis. Overexpression of the EGFR gene in many Wilms tumor cases warrants further study to determine the therapeutic benefit of EGFR inhibitors in combination with other therapies in Wilms tumor patients.