TGF-beta Pathway Research Articles

Abstract 4896: Role of CDK8 in colon cancer hepatic metastasis

Abstract About half of all colon cancer patients will develop liver metastases and the 5-year survival for these patients is less than 13%, making colon cancer the second most lethal cancer worldwide. Cyclin dependent kinase 8 (CDK8), which regulates transcription but not cell cycle progression, has been identified as an oncogene amplified in many colon cancers. CDK8 acts as a positive mediator of oncogenic transcription pathways regulated by Wnt/beta-catenin and TGF-beta, both of which are strongly associated with tumor metastasis. In a colon cancer liver metastasis model based on splenic injection of CT26 murine colon carcinoma cells, treatment with Senexin B, a highly selective small-molecule inhibitor of CDK8 and its paralog CDK19, strongly inhibited metastatic growth in the liver and prolonged the survival of mice with hepatic metastases. In contrast to the effect on liver metastasis, CDK8 inhibition had little or no effect on cell growth in culture or at primary tumor sites. Hepatic metastasis was inhibited to the same extent when Senexin B was administered starting from the time of tumor injection or only during a later part of the study, suggesting that the drug affected metastatic growth in the liver rather than just initial colonization. Liver metastasis was similarly decreased by CDK8 knockdown in CT26 cells, suggesting that the anti-metastatic activity of Senexin B was due at least in part to its effect on tumor cells. Transcription profiling indicated that CDK8 inhibition by shRNA or Senexin B strongly decreased the expression of metastasis-associated metalloproteinases MMP 13, MMP10, and MMP3 and at the same time drastically increased the expression of TIMP3, a metalloproteinase inhibitor. TIMP3 overexpression in CT26 cells, like CDK8 inhibition, decreased hepatic metastasis. CDK8 inhibition also blocked TGF-beta or Wnt3a-stimulated transwell Matrigel invasion of CT26 cells in vitro. Knockdown of beta-catenin in CT26 cells decreased the expression of MMP3 and MMP13, while knockdown of SMAD4 (transcriptional mediator of TGF-beta pathway) induced TIMP3 expression. Beta-catenin knockdown decreased both the primary tumor growth in the spleen and metastatic growth in the liver, whereas SMAD4 knockdown, like that of CDK8, selectively inhibited liver metastasis. Senexin B also suppressed hepatic metastasis after splenic injection of human HCT116 colon carcinoma cells. In summary, our data identified CDK8 as a key transcriptional regulator of colon cancer metastatic growth in the liver, interacting with TGF-beta and Wnt/beta-catenin pathways and regulating the expression of MMPs and Timp3. CDK8/19 inhibitors, which are now entering clinical trials, may be effective for the treatment of hepatic metastasis of colon cancer. Citation Format: Jiaxin Liang, Mengqian Chen, Mythreye Karthikeyan, M. Marjorette Pena, Daniel Hughes, Vimala Kaza, Chang-Uk Lim, Eugenia Broude, Igor B. Roninson. Role of CDK8 in colon cancer hepatic metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2017-4896

Abstract 2530: Validation and pathway analysis of a metastasis-specific microRNA signature in primary colon cancer

Abstract Introduction: In lymph node negative (LNN) colon cancer 20% of the patients develops disease recurrence. Identification of these patients is needed. MicroRNAs (MiRNAs), a group of short non-coding RNAs, can function as tumor suppressors or oncogenes. Hur et al recently found 6 differentially expressed MiRNAs when comparing primary colorectal cancer and matched liver metastasis (MiR-320, MiR-221, MiR-30b, MiR-10b, MiR-885-5p, Let-7i). The expression of 2 MiRNAs was significantly correlated with distant metastasis (low Let-7i expression and high MiR-10b) in primary colorectal cancers. When split at the median and combined into a signature (Let-7i high and MiR-10b low [n=22] vs. Let-7i low and/or MiR-10b high[n=122]), the first group showed 100% metastasis free survival (MFS). We assessed the prognostic value of these MiRNAs and the signature in a clinically well-defined cohort of primary colon cancers. Methods: Expression of the 6 MiRNAs were measured using RT-qPCR in a cohort of 232 colon cancer patients (n=155 untreated LNN and n=77 adjuvant treated lymph node positive [LNP] patients) selected from the MATCH-cohort. Expression levels were related to microsatellite instability (MSI), and MFS, hepatic metastasis free (HFS) and overall survival (OS). Results: MiR-221, Mir-30b, Mir-10b and MiR-885-5p levels were significantly associated with MSI. In univariate Cox regression analysis, MiR-30b was significantly associated with MFS (HR=1.23 p=0.004), and MiR-30b and Let-7i were associated with HFS (HR=2.52 p=0.005 and HR=0.40 p<0.001, respectively) in the total group. In the LNN group results were similar for MiR-30b (MFS HR=2.07 p=0.008 and HFS HR=2.94 p=0.005) and Let-7i (HFS HR=0.28 p<0.001), but did not reach significance in the LNP group. These associations remained significant when correcting for MSI. Pathway analysis for MiR-30b and Let-7i with the GSEA hallmark gene sets between the 50 samples with the highest and 50 samples with lowest expression revealed a significantly higher expression of the TGF-beta pathway in the MiR-30b high group, and a significantly higher expression of the EMT pathway in the Let-7i high group. MiR-30b and Let-7i expression was split at the median level and combined into two groups (‘Let-7i high and MiR-30b low’ vs. ‘Let-7i low and/or MiR-30b high’). The ‘Let-7i high and MiR-30b low’ group in the total group (n=74) had a significantly better 5-yr HFS (100% vs 87.4% p=0.002), and had a significantly better 5-yr MFS (91.7% vs 78% p=0.036) and HFS (100% vs 87.8% p=0.01) in the LNN group (n=52). Conclusion: in our cohort and more specifically the LNN group, we confirmed Let-7i and identified MiR-30b as a prognostic factor for MFS and HFS. We did not confirm the prognostic value of Mir-10b. The combination of Let-7i and MiR-30b identified a group with a 100% HFS. Pathway analysis showed higher expression of the TGF-beta pathway in the MiR-30b high group, and higher expression of the EMT pathway in the Let-7i high group. Citation Format: Robert R. Coebergh van den Braak, Anieta M. Sieuwerts, Zarina S. Lalmahomed, Marcel Smid, Vanja de Weerd, Michelle van der Vlugt - Daane, Anne van Galen, Shanshan Xiang, Katharina Biermann, John A. Foekens, John W. Martens, Jan N. IJzermans. Validation and pathway analysis of a metastasis-specific microRNA signature in primary colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2530. doi:10.1158/1538-7445.AM2017-2530

Molecular characterization of TP53 mutations and copy number change in colorectal cancers.

e15143 Background: TP53 mutations (mut) are prevalent in colorectal cancer (CRC) patients (pts) and can result in oncogenic gain of function effect as well as loss of tumor suppressor function depending on the mut. Methods: The Cancer Genome Atlas (TCGA) data of 220 CRC pts with tumors sequenced and gene copy numbers (CN) annotated was obtained. Pts with CMS subtype data also available were included (n = 167). Chi square test was used to compare frequency of TP53 mut and CN change among CMS subtypes. TP53 mRNA and protein levels were compared by TP53 mut and CN change using independent sample t test. Results: 86 (51.5%) pts were TP53 mutant, with 35.3% missense and 16.2% loss of function (LOF) mut. LOF mut included 14 non-sense and 13 frameshift mut, and occurred mostly in codons 213 and 306. Missense mut mostly affected codons 175, 248 and 273 in 10, 9 and 8 pts respectively. TP53 mut were most frequent in CMS2 and CMS4 subtypes (62.7 and 62.5% respectively). TP53 mut in CMS2 were mostly missense and CMS 4 pts mostly had LOF mut. TP53 CN loss was seen in 74.7% CMS 2, 68.8% CMS 4, 43.5% CMS 3 and 6.9% CMS1 pts. 84.9% pts with TP53 mut had CN loss vs 28.4% of wild type pts (p < 0.001). Frequency of TP53 CN loss did not vary significantly by type of TP53 mut. Pts with TP53 missense mut had higher p53 mRNA and protein levels than those with LOF mut. Pts with TP53 CN loss had lower mRNA levels but no change in protein levels than those with normal CN. Conclusions: The higher TP53 mRNA and protein levels with missense mut suggest possible gain of function. Research on interactions of TP53 change with wnt, myc, and TGF beta pathway genes may reveal synthetic lethal treatment combinations. [Table: see text]

Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes

ABSTRACTTransforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFβ-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

Time-matched analysis of DNA adduct formation and early gene expression as predictive tool for renal carcinogenesis in methylazoxymethanol acetate treated Eker rats.

Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts not always translate into tumorigenesis, their predictive value is limited. Here we hypothesize that the combined analysis of pro-mutagenic DNA adducts along with time-matched gene expression changes could serve as a superior prediction tool for genotoxic carcinogenesis. Eker rats, heterozygous for the tuberous sclerosis (Tsc2) tumor suppressor gene and thus highly susceptible towards genotoxic renal carcinogens, were continuously treated with the DNA alkylating carcinogen methylazoxymethanol acetate (MAMAc). Two weeks of MAMAc treatment resulted in a time-dependent increase of O6-methylguanine and N7-methylguanine adducts in the kidney cortex, which was however not reflected by significant expression changes of cyto-protective genes involved in DNA repair, cell cycle arrest or apoptosis. Instead, we found a transcriptional regulation of genes involved in the tumor-related MAPK, FoxO and TGF-beta pathways. Continuous MAMAc treatment for up to 6months resulted in a mild but significant increase of cancerous lesions. In summary, the combined analysis of DNA adducts and early gene expression changes could serve as a suitable predictive tool for genotoxicant-induced carcinogenesis.

Integrated Analysis and MicroRNA Expression Profiling Identified Seven miRNAs Associated With Progression of Oral Squamous Cell Carcinoma.

MicroRNAs have been used as diagnostic and prognostic biomarkers for many cancers including oral squamous cell carcinoma (OSCC). Several studies have been shown that microRNA (miRNA) play important roles during the progression of OSCC. However, the results vary largely in different studies due to different platforms and sample sizes. In this study, we systematically evaluated a large scale of miRNA profiles from current qualified OSCC samples, and further investigated the functions of genes regulated by these key miRNAs as well as the signaling pathways through which these miRNA effect carcinogenesis. Seven key miRNAs were identified, and of which three were significantly upregulated, including hsa-miR-21, hsa-miR-31, hsa-miR-338, and four were downregulated, namely hsa-miR-125b, hsa-miR-133a, hsa-miR-133b, and hsa-miR-139. The function enrichment analysis revealed that target genes of upregulated miRNAs were associated with cellular protein metabolic process, macromolecule metabolic process, and TGF-beta pathway, while the targets of downregulated were enriched in negative regulation of macromolecule biosynthetic process and gene expression, and p53, long-term potentiation and adherens junction pathways. Transcription factor analysis revealed that there were 67 (51.1%) transcription factors influenced by both up and downregulated miRNAs. In summary, seven key miRNAs were found to play essential role in progression of OSCC, as well as the target genes and transcription factors of these miRNAs. The potential functions of these target genes identified in our study may be profitable to diagnosis and prognostic prediction of OSCC as biomarkers. J. Cell. Physiol. 232: 2178-2185, 2017. © 2016 Wiley Periodicals, Inc.

Abstract WP204: Genetic Variant in VCAM1 Mediates Acute Infarct Size in Ischemic Stroke Patients

Introduction: Even though neuroinflammation is increasingly recognized as an essential contributor to ischemic brain injury, the exact underlying mechanisms remain unclear. Higher expression of the vascular cell adhesion molecule 1 (VCAM-1) increases leukocyte-brain endothelium interaction and has been associated with larger infarct size following acute ischemic stroke (AIS). The activation of the TGF-β signaling pathways can down-regulate VCAM-1 expression and ameliorate deleterious tissue outcome during neuroinflammation. Hypothesis: We sought to investigate whether genetic variation in the TGF-beta pathway and adhesion molecule genes is associated with acute stroke lesion size. Methods: We completed genome-wide association (GWA) testing and diffusion-weighted imaging lesion volume (DWIv) analysis in a discovery cohort of 532 AIS patients of European ancestry enrolled within 48 hours of symptom onset. An independent European ancestry cohort of 724 AIS patients with GWA data and automated DWIv served as replication cohort. GWA testing per SNP was performed using linear regression modeling of natural log-transformed DWIv adjusted for age, sex and relevant principal components. We selected 42 inflammatory genes in the TGF-beta pathway for a gene-based analysis using VEGAS (Versatile Gene-based -Association Study) software. A pre-specified discovery phase Bonferroni-corrected threshold was set at p<0.001. In the replication phase, 14 SNPs overlapping were tested at the Bonferroni-corrected p-value threshold of p<0.004. Results: Of all genes in the TGF-beta pathway, VCAM1 (p=0.0006) was significantly associated with DWIv in the discovery AIS cohort (age: 66 ± 14.9 years, sex: 63.4% male, DWIv: 2.2cm 3 (IQR: 0.6-11.7cm 3 )). A single SNP within the VCAM1 gene boundaries (rs3176876 (BETA=0.2341, p=0.003)) was significantly associated with DWIv in the replication AIS cohort (age: 65 ± 14.1 years, sex: 68.6% male, DWIv: 4cm 3 (IQR: 1.3-17.2cm 3 )). Conclusion: The genetic variant rs3176876 in the VCAM1 gene is associated with larger infarct lesion size measured on brain MRI of AIS patients. These findings suggest genetic contribution to the pro-inflammatory mechanisms in acute cerebral ischemia and warrant further investigation.

TGFbeta and miRNA regulation in familial and sporadic breast cancer.

The term ‘BRCAness’ was introduced to identify sporadic malignant tumors sharing characteristics similar to those germline BRCA-related. Among all mechanisms attributable to BRCA1 expression silencing, a major role has been assigned to microRNAs. MicroRNAs role in familial and sporadic breast cancer has been explored but few data are available about microRNAs involvement in homologous recombination repair control in these breast cancer subgroups. Our aim was to seek microRNAs associated to pathways underlying DNA repair dysfunction in breast cancer according to a family history of the disease. Affymetrix GeneChip microRNA Arrays were used to perform microRNA expression analysis in familial and sporadic breast cancer. Pathway enrichment analysis and microRNA target prediction was carried out using DIANA miRPath v.3 web-based computational tool and miRWalk v.2 database. We analyzed an external gene expression dataset (E-GEOD-49481), including both familial and sporadic breast cancers. For microRNA validation, an independent set of 19 familial and 10 sporadic breast cancers was used. Microarray analysis identified a signature of 28 deregulated miRNAs. For our validation analyses by real time PCR, we focused on miR-92a-1*, miR-1184 and miR-943 because associated to TGF-β signalling pathway, ATM and BRCA1 genes expression. Our results highlighted alterations in miR-92a-1*, miR-1184 and miR-943 expression levels suggesting their involvement in repair of DNA double-strand breaks through TGF-beta pathway control.

Comprehensive analysis of the long noncoding RNA HOXA11-AS gene interaction regulatory network in NSCLC cells.

BackgroundLong noncoding RNAs (lncRNAs) are related to different biological processes in non-small cell lung cancer (NSCLC). However, the possible molecular mechanisms underlying the effects of the long noncoding RNA HOXA11-AS (HOXA11 antisense RNA) in NSCLC are unknown.MethodsHOXA11-AS was knocked down in the NSCLC A549 cell line and a high throughput microarray assay was applied to detect changes in the gene profiles of the A549 cells. Bioinformatics analyses (gene ontology (GO), pathway, Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses) were performed to investigate the potential pathways and networks of the differentially expressed genes. The molecular signatures database (MSigDB) was used to display the expression profiles of these differentially expressed genes. Furthermore, the relationships between the HOXA11-AS, de-regulated genes and clinical NSCLC parameters were verified by using NSCLC patient information from The Cancer Genome Atlas (TCGA) database. In addition, the relationship between HOXA11-AS expression and clinical diagnostic value was analyzed by receiver operating characteristic (ROC) curve.ResultsAmong the differentially expressed genes, 277 and 80 genes were upregulated and downregulated in NSCLC, respectively (fold change ≥2.0, P < 0.05 and false discovery rate (FDR) < 0.05). According to the degree of the fold change, six upregulated and three downregulated genes were selected for further investigation. Only four genes (RSPO3, ADAMTS8, DMBT1, and DOCK8) were reported to be related with the development or progression of NSCLC based on a PubMed search. Among all possible pathways, three pathways (the PI3K-Akt, TGF-beta and Hippo signaling pathways) were the most likely to be involved in NSCLC development and progression. Furthermore, we found that HOXA11-AS was highly expressed in both lung adenocarcinoma and squamous cell carcinoma based on TCGA database. The ROC curve showed that the area under curve (AUC) of HOXA11-AS was 0.727 (95% CI 0.663–0.790) for lung adenocarcinoma and 0.933 (95% CI 0.906–0.960) for squamous cell carcinoma patients. Additionally, the original data from TCGA verified that ADAMTS8, DMBT1 and DOCK8 were downregulated in both lung adenocarcinoma and squamous cell carcinoma, whereas RSPO3 expression was upregulated in lung adenocarcinoma and downregulated in lung squamous cell carcinoma. For the other five genes (STMN2, SPINK6, TUSC3, LOC100128054, and C8orf22), we found that STMN2, TUSC3 and C8orf22 were upregulated in squamous cell carcinoma and that STMN2 and USC3 were upregulated in lung adenocarcinoma. Furthermore, we compared the correlation between HOXA11-AS and de-regulated genes in NSCLC based on TCGA. The results showed that the HOXA11-AS expression was negatively correlated with DOCK8 in squamous cell carcinoma (r = −0.124, P = 0.048) and lung adenocarcinoma (r = −0.176, P = 0.005). In addition, RSPO3, ADAMTS8 and DOCK8 were related to overall survival and disease-free survival (all P < 0.05) of lung adenocarcinoma patients in TCGA.ConclusionsOur results showed that the gene profiles were significantly changed after HOXA11-AS knock-down in NSCLC cells. We speculated that HOXA11-AS may play an important role in NSCLC development and progression by regulating the expression of various pathways and genes, especially DOCK8 and TGF-beta pathway. However, the exact mechanism should be verified by functional experiments.

Identification of critical paralog groups with indispensable roles in the regulation of signaling flow.

Extensive cross-talk between signaling pathways is required to integrate the myriad of extracellular signal combinations at the cellular level. Gene duplication events may lead to the emergence of novel functions, leaving groups of similar genes - termed paralogs - in the genome. To distinguish critical paralog groups (CPGs) from other paralogs in human signaling networks, we developed a signaling network-based method using cross-talk annotation and tissue-specific signaling flow analysis. 75 CPGs were found with higher degree, betweenness centrality, closeness, and ‘bowtieness’ when compared to other paralogs or other proteins in the signaling network. CPGs had higher diversity in all these measures, with more varied biological functions and more specific post-transcriptional regulation than non-critical paralog groups (non-CPG). Using TGF-beta, Notch and MAPK pathways as examples, SMAD2/3, NOTCH1/2/3 and MEK3/6-p38 CPGs were found to regulate the signaling flow of their respective pathways. Additionally, CPGs showed a higher mutation rate in both inherited diseases and cancer, and were enriched in drug targets. In conclusion, the results revealed two distinct types of paralog groups in the signaling network: CPGs and non-CPGs. Thus highlighting the importance of CPGs as compared to non-CPGs in drug discovery and disease pathogenesis.

Smad4 in T cells plays a protective role in the development of autoimmune Sjögren's syndrome in the nonobese diabetic mouse.

We investigated the role of Smad4, a signaling molecule of the TGF-beta pathway, in T cells on the pathology of Sjögren's syndrome (SS) in nonobese diabetic (NOD) mice, an animal model of SS. T cell-specific Smad4-deleted (Smad4fl/fl,CD4-Cre; Smad4 tKO) NOD mice had accelerated development of SS compared with wild-type (Smad4+/+,CD4-Cre; WT) NOD mice, including increased lymphocyte infiltration into exocrine glands, decreased tear and saliva production, and increased levels of autoantibodies at 12 weeks of age. Activated/memory T cells and cytokine (IFN-γ, IL-17)-producing T cells were increased in Smad4 tKO NOD mice, however the proportion and function of regulatory T (Treg) cells were not different between Smad4 tKO and WT NOD mice. Effector T (Teff) cells from Smad4 tKO NOD mice were less sensitive than WT Teff cells to suppression by Treg cells. Th17 differentiation capability of Teff cells was similar between Smad4 tKO and WT NOD mice, but IL-17 expression was increased under inducible Treg skewing conditions in T cells from Smad4 tKO NOD mice. Our results demonstrate that disruption of the Smad4 pathway in T cells of NOD mice increases Teff cell activation resulting in upregulation of Th17 cells, indicating that Smad4 in T cells has a protective role in the development of SS in NOD mice.

CD51 correlates with the TGF-beta pathway and is a functional marker for colorectal cancer stem cells.

Colorectal cancer (CRC) is one of the top three most prevalent and deadly cancers. A cancer stem cell (CSC) sub-population that is characterized by the abilities of tumor initiation, self-renewal, metastasis and resistance to chemotherapy can suggest new therapeutic targets. However, no such sub-population has been conclusively identified for CRC, and we lack any marker to identify cells with all of the above characteristics. Here, we report that CD51+ CRC cells displayed greater sphere-forming and tumorigenic capacities, increased migratory and invasive potentials, and enhanced chemoresistance compared with CD51- CRC cells. CD51 knockdown reduced the side population, sphere formation, cell motility and inhibited tumor incidence and metastasis in an in vivo tumor model. Furthermore, CD51 could bind transforming growth factor beta (TGF-β) receptors, and that it upregulated TGF-β/Smad signaling. These results indicate that CD51 is a novel functional marker for colorectal CSCs which may provide an therapeutic target for the efficient elimination of colorectal CSCs.

Abstract 1878: Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape

Abstract Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumor accounting for 60-70% of all gliomas. Current achieved median patient survival ranges only 12-15 months, and improvements in survival over the last century can be measured only in weeks. A hallmark of this malignancy is the intrinsic resistance to current therapies. The Transforming Growth Factor beta (TGF-beta) signaling pathway plays a key role in GBM. Several inhibitors of different elements and regulators of the TGF-beta pathway have entered to clinical trials. Here, we analyzed the potential effect of P144, a TGF-beta pathway inhibitor peptide, over cell proliferation and apoptosis in commercial GBM cell lines (A172 and U-87 MG). We found that treatment with P144 significantly decreased cell proliferation of both cell lines analyzed. In addition, different apoptosis determinations such as ELISA and Acridine Orange/Ethidium Bromide (AO/EB) staining, confirmed a significant increase in apoptosis in both cell lines. Quantification of SMAD2 phosphorylation status and its nuclear translocation, by western blot, confirmed the inhibition of TGF-beta signaling by P144. These data support the correlation between TGF-beta pathway inhibition and the apoptotic process induction. Anoikis escape sustains invasiveness and metastatic potential in GBM, and has been established as a clear target against tumor progression. Due to P144 clear effect on apoptosis, we analyzed its influence on anoikis escape. The inhibition of anoikis escape by P144 was confirmed in A172 and U-87 MG cell lines under in vitro anchorage-independent culture conditions. The significant upregulation of BAX, Lamin A and Lamin C produced by P144 confirmed the induction of this apoptotic subtype process in the analyzed cell lines. We can conclude that P144 increases apoptosis and induces anoikis through inhibition of the TGF-beta signaling pathway. Our results highlight the therapeutic potential of P144 for the treatment against GBM. However, previous to further clinical development, additional studies are required in order to confirm the effect of P144 over GBM in the brain environment, as well as to explore the therapeutic potential of P144 in combination with current and emerging molecular based therapies. Citation Format: Gabriel Gallo-Oller, Javier Dotor, Xing Fan, Javier S. Castresana. Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1878.

Abstract 1854: The regulation of angiogenin expression and the genes regulated by angiogenin

Abstract Angiogenin (ANG) is first isolated and identified by its ability to stimulate the growth of blood vessels. As a member of the vertebrate-specific secreted ribonuclease (RNase) family, ANG is encoded by a single exon and is usually located in the middle of its family gene cluster. Particularly, ANG gene locus has a unique gene arrangement, featured by shared promoters and 5’-untranslated regions (5’-UTR) which direct two distinct exons enconding ANG and ribounclease 4 (RNASE4, the 4th member of RNase family) respectively. However, the gene structure and regulation of these genetic regions are largely unknown. Here, we have characterized the promoters, defined the transcription start site, and identified a unique mechanism of transcription regulation. We demonstrated that two Pol III elements within the promoter regulate ANG and RNASE4 expression in a position- and orientation-dependent manner. Moreover, an intragenic chromatin loop formed between the two CCCTC-binding factor (CTCF)-binding sites located in two introns flanking ANG coding exon, which preferentially enhances ANG transcription. These results suggest a multilayer transcriptional regulation of ANG and RNASE4 gene locus. The data also add more direct evidence to the notion that Pol III elements are able to directly influence Pol II gene transcription. Furthermore, our data indicate that a CTCF-dependent chromatin loop is able to differentially regulate transcription of genes that share the same promoters. On the other hand, the secreted ANG undergoes a receptor-mediated endocytosis from the cell surface to the nucleus and accumulates in the nucleoli. The nucleoli ANG can promote 47S pre-rRNA transcription by binding the ABE (Angiogenin Binding Element) and UCE (Upstream Core Element) region on the promoter of ribosomal DNA (rDNA), where ANG increases the number of actively transcribing rDNA and promotes the assembly of initiation complex by epigenetic activation through promoter methylation and histone modification. We have also shown that the surplus ANG in nucleus also related to mRNA transcription. ANG binds the first exon region of estrogen receptor-related receptor gamma (ERRγ) and inhibits the ERRγ expression. To screen and identify the mRNAs regulated by ANG at a genome-wide level, we carried out chromatin immunoprecipitation-chip assay (ChIP-on-chip) and found a total of 699 genes that may be regulated by ANG. These genes were significantly enriched to tumorigenesis, Wnt and TGF-beta pathways by the KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. Based on the findings that ANG is able to remodel the histone modification through direct binding to the histone protein, we propose that ANG might act as a chromatin remodeling activator to regulate RNA transcription. However, there is still a long way to go before fully disclosing the roles and mechanisms of ANG in gene transcription. Citation Format: Jinghao Sheng, Guo-fu Hu, Zhengping Xu. The regulation of angiogenin expression and the genes regulated by angiogenin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1854.

Epidermal growth factor-like repeats and discoidin I-like domains 3: a multifaceted oncoprotein at the crossroad of MAPK and TGF-beta pathways in human hepatocellular carcinoma

Epidermal growth factor-like repeats and discoidin I-like domains 3: a multifaceted oncoprotein at the crossroad of MAPK and TGF-beta pathways in human hepatocellular carcinoma

Epidermal growth factor-like repeats and discoidin I-like domains 3: a multifaceted oncoprotein at the crossroad of MAPK and TGF-beta pathways in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a frequent human cancer with 0.25–1 million of newly diagnosed cases each year (1-3). Major risk factors associated with the development of HCC are chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, alcoholic hepatitis, aflatoxin B1 (3-5), and some inherited diseases (6). HCC is a fatal disease, with a life expectancy of about 6 months from the time of diagnosis (6). Early liver lesions could be detected by ultrasonography and efficiently treated by resection or radiofrequency ablation (7). However, only a minority of cases is eligible to these treatment modalities due to the late diagnosis of the disease (2,7,8). In addition, therapies with pharmacological agents or alternative approaches, including percutaneous ethanol injection, trans-arterial chemo-embolization or yttrium-90 microspheres, do not improve significantly the prognosis of patients with advanced disease (2,7,8).

Colorectal cancer characterization and therapeutic target prediction based on microRNA expression profile.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer death. However, the molecular mechanisms underlying CRC initiation, growth and metastasis are poorly understood. In this study, based on our previous work for comprehensively analyzing miRNA sequencing data, we examined a series of colorectal cancer microRNAs expression profiles data. Results show that all these CRC samples share the same four pathways including TGF-beta signaling pathway, which is important in colorectal carcinogenesis. Twenty-one microRNAs that evolved in the four overlapped pathways were then discovered. Further analysis selected miR-21 as an important regulator for CRC through TGF-beta pathways. This study develops methods for discovering tumor specific miRNA cluster as biomarker and for screening new cancer therapy targets based on miRNA sequencing.

Radiomics of lung cancer

Submillisecond pulses of radiation have been shown to generate less exchange chromosomal aberrations [1, 2] and a smaller extent of delayed cell death [3, 4] than continuous irradiation delivered at conventional dose-rate. This prompted us to determine whether and how pulsed irradiation affects the response of normal lung tissue in vivo. For this purpose, C57BL/ 6J mice were given a single dose of 17 Gy of 4.5 MeV electrons in bilateral thorax exposure either at a high (> 60 Gy/s, beam-on time < 0.5 s, FLASH) or conventional dose-rate (0.03 Gy/s, beam-on time 8 min, CONV) using an experimental linear electron accelerator established in the Research Division of Institut Curie at Orsay (France). DNA damage response, apoptosis and fibrosis development were subsequently analyzed at suitable times in the two modes of irradiation. The anti-tumor efficiency was also evaluated in vivo with two xenografts (HBCx12A, HEp-2) and one syngeneic, orthotopic carcinoma (TC1-Luc). The results indicate that, in the hours following irradiation, FLASH-irradiated lungs present less DNA damages and less apoptosis than lungs irradiated at a conventional dose-rate. Furthermore, compared to the classical radiation-induced lung fibrosis observed past 16-weeks after CONV irradiation, analysis of FLASH-treated lungs did not show any histological sign of fibrosis nor activation of the TGF-beta pathway. However, FLASH irradiation was as efficient as CONV treatment in controlling tumor growth. Taken together, these results show that FLASH irradiation selectively spares normal lung tissue without any loss of the anti-tumor activity [5].

Potential of FLASH irradiation to minimize the incidence of radio-induced damage and fibrosis to normal lung in a mouse model

Submillisecond pulses of radiation have been shown to generate less exchange chromosomal aberrations [1, 2] and a smaller extent of delayed cell death [3, 4] than continuous irradiation delivered at conventional dose-rate. This prompted us to determine whether and how pulsed irradiation affects the response of normal lung tissue in vivo. For this purpose, C57BL/ 6J mice were given a single dose of 17 Gy of 4.5 MeV electrons in bilateral thorax exposure either at a high (> 60 Gy/s, beam-on time < 0.5 s, FLASH) or conventional dose-rate (0.03 Gy/s, beam-on time 8 min, CONV) using an experimental linear electron accelerator established in the Research Division of Institut Curie at Orsay (France). DNA damage response, apoptosis and fibrosis development were subsequently analyzed at suitable times in the two modes of irradiation. The anti-tumor efficiency was also evaluated in vivo with two xenografts (HBCx12A, HEp-2) and one syngeneic, orthotopic carcinoma (TC1-Luc). The results indicate that, in the hours following irradiation, FLASH-irradiated lungs present less DNA damages and less apoptosis than lungs irradiated at a conventional dose-rate. Furthermore, compared to the classical radiation-induced lung fibrosis observed past 16-weeks after CONV irradiation, analysis of FLASH-treated lungs did not show any histological sign of fibrosis nor activation of the TGF-beta pathway. However, FLASH irradiation was as efficient as CONV treatment in controlling tumor growth. Taken together, these results show that FLASH irradiation selectively spares normal lung tissue without any loss of the anti-tumor activity [5].

De Novo Assembly and Characterization of Early Embryonic Transcriptome of the Horseshoe Crab Tachypleus tridentatus.

The horseshoe crab Tachypleus tridentatus is a unique marine species and a potential model for marine invertebrate. Limited genomic and transcriptional data are currently available to understand the molecular mechanisms underlying the embryonic development of T. tridentatus. Here, we reported for the first time the de novo transcriptome assembly for T. tridentatus at embryonic developmental stage using Illumina RNA-seq platform. Approximate 38 million reads were obtained and further assembled into 133,212 unigenes. Sequence homology analysis against public databases revealed that 33,796 unigenes could be annotated with gene descriptions. Of the annotated unigenes, we identified a number of key components of several conserved metazoan signaling pathways (Hedgehog, Wnt, TGF-beta and Notch pathways) and other important regulatory genes involved in embryonic development. Targeted searching of Pax family genes which play critical roles in the formation of tissue and organ during embryonic development identified a complete set of Pax family genes. Moreover, the full length T. tridentatus Pax1/9a (TtPax1/9a) and Pax1/9b (TtPax1/9b) cDNA sequences were determined based on the transcriptome, demonstrating the immediate application of our database. Using quantitative real time PCR, we analyzed the expression patterns of TtPax1/9a and TtPax1/9b in different tissues of horseshoe crab. Taking advantage of Drosophila model, we further found that TtPax1/9b, but not TtPax1/9a, can partly rescue the Drosophila homolog Poxm dysfunction-caused lethality at the larval stage. Our study provides the embryonic transcriptome of T. tridentatus which could be immediately used for gene discovery and characterization, functional genomics studies in T. tridentatus. This transcriptome database will also facilitate the investigations of molecular mechanisms underlying embryonic development of T. tridentatus and other marine arthropods as well.