We conducted an RNA sequencing study to identify novel gene fusions in 80 discovery dataset tumors collected from young patients with diffuse gastric cancer (DGC). Twenty-five in-frame fusions are associated with DGC, three of which (CLDN18-ARHGAP26, CTNND1-ARHGAP26, and ANXA2-MYO9A) are recurrent in 384 DGCs based on RT-PCR. All three fusions contain a RhoGAP domain in their 3' partner genes. Patients with one of these three fusions have a significantly worse prognosis than those without. Ectopic expression of CLDN18-ARHGAP26 promotes the migration and invasion capacities of DGC cells. Parallel targeted RNA sequencing analysis additionally identifies TACC2-PPAPDC1A as a recurrent and poor prognostic in-frame fusion. Overall, PPAPDC1A fusions and in-frame fusions containing a RhoGAP domain clearly define the aggressive subset (7.5%) of DGCs, and their prognostic impact is greater than, and independent of, chromosomal instability and CDH1 mutations. Our study may provide novel genomic insights guiding future strategies for managing DGCs.

MicroRNAs (miRNAs) are solid factors involved in the initiation and progression of hepatocellular carcinoma (HCC). Recently, miR-298 is recognized as a cancer-associated miRNA in breast, gastric and ovarian cancer. However, the functional role of miR-298 and its underlying mechanism are rarely reported in HCC. Herein, we found that the expression of miR-298 was down-regulated in HCC tissues and cell lines. The in vitro experiments showed that miR-298 overexpression inhibited cell proliferation, migration and invasion, and induced G1 arrest and apoptosis of HCC cells. miR-298 knockdown exerted an opposite effect on these cellular behaviors of HCC cells. Moreover, miR-298 restoration suppressed HCC tumor growth and metastasis in vivo. Additionally, catenin delta 1 (CTNND1) was demonstrated to be a direct target of miR-298 in HCC cells. CTNND1 knockdown led to similar effects with miR-298 overexpression on HCC cell proliferation, cell cycle progression, apoptosis and mobility. CTNND1 restoration reversed miR-298-induced inhibitory effects on HCC cells. Mechanistically, both miR-298 overexpression and CTNND1 knockdown repressed Wnt/β-catenin signaling and resulted in reduced expression of β-catenin, WNT11, Cyclin D1 and MMP7 in HCCLM3 cells. While, CTNND1 restoration abolished miR-298-induced inactivation of Wnt/β-catenin signaling. In conclusion, our findings provide the first evidence that miR-298 suppresses HCC progression at least partially by targeting CTNND1-mediated Wnt/β-catenin signaling. MiR-298 may be a target for new therapies in HCC patients.

BACKGROUND: Despite recent advances in the diagnosis and treatment of breast cancer, metastasis remains the main cause of death. Since migration of tumor cells is considered a prerequisite for tumor cell invasion and metastasis, a pressing goal in tumor biology has been to elucidate factors regulating their migratory activity. Protein kinase C alpha (PKCα) is a serine-threonine protein kinase implicated in cancer metastasis and associated with poor prognosis in breast cancer patients. In this study, we set out to define the signaling axis mediated by PKCα to promote breast cancer cell migration.
METHODS: Oncomine™ overexpression analysis was used to probe for PRKCA (PKCα) and FOXC2 expression in mRNA datasets. The heat map of PRKCA, FOXC2, and CTNND1 were obtained from the UC Santa Cruz platform. Survival data were obtained by PROGgene and available at http://www.compbio.iupui.edu/proggene . Markers for EMT and adherens junction were assessed by Western blotting and quantitative polymerase chain reaction. Effects of PKCα and FOXC2 on migration and invasion were assessed in vitro by transwell migration and invasion assays respectively. Cellular localization of E-cadherin and p120-catenin was determined by immunofluorescent staining. Promoter activity of p120-catenin was determined by dual luciferase assay using a previously validated p120-catenin reporter construct. Interaction between FOXC2 and p120-catenin promoter was verified by chromatin immunoprecipitation assay.
RESULTS: We determined that PKCα expression is necessary to maintain the migratory and invasive phenotype of both endocrine resistant and triple negative breast cancer cell lines. FOXC2 acts as a transcriptional repressor downstream of PKCα, and represses p120-catenin expression. Consequently, loss of p120-catenin leads to destabilization of E-cadherin at the adherens junction. Inhibition of either PKCα or FOXC2 is sufficient to rescue p120-catenin expression and trigger relocalization of p120-catenin and E-cadherin to the cell membrane, resulting in reduced tumor cell migration and invasion.
CONCLUSIONS: Taken together, these results suggest that breast cancer metastasis may partially be controlled through PKCα/FOXC2-dependent repression of p120-catenin and highlight the potential for PKCα signal transduction networks to be targeted for the treatment of endocrine resistant and triple negative breast cancer.

BACKGROUND: It is possible that the relative lack of progress in treatment outcomes among adolescent and young adult (AYA) patients with cancer is caused by a difference in disease biology compared with the corresponding diseases in younger and older individuals. There is evidence that colon cancer is more aggressive and has a poorer prognosis in AYA patients than in older adult patients.
METHODS: To further understand the molecular basis for this difference, whole-exome sequencing was conducted on a cohort of 30 adult, 30 AYA, and 2 pediatric colon cancers.
RESULTS: A statistically significant difference in mutational frequency was observed between AYA and adult samples in 43 genes, including ROBO1, MYC binding protein 2 (MYCBP2), breast cancer 2 (early onset) (BRCA2), MAP3K3, MCPH1, RASGRP3, PTCH1, RAD9B, CTNND1, ATM, NF1; KIT, PTEN, and FBXW7. Many of these mutations were nonsynonymous, missense, stop-gain, or frameshift mutations that were damaging. Next, RNA sequencing was performed on a subset of the samples to confirm the mutations identified by exome sequencing. This confirmation study verified the presence of a significantly greater frequency of damaging mutations in AYA compared with adult colon cancers for 5 of the 43 genes (MYCBP2, BRCA2, PHLPP1, TOPORS, and ATR).
CONCLUSIONS: The current results provide the rationale for a more comprehensive study with a larger sample set and experimental validation of the functional impact of the identified variants along with their contribution to the biologic and clinical characteristics of AYA colon cancer. Cancer 2018;124:1070-82. © 2017 American Cancer Society.

Retinoic acid-related orphan receptor α (RORα) regulates diverse physiological processes, including inflammatory responses, lipid metabolism, circadian rhythm, and cancer biology. RORα has four different isoforms which have distinct N-terminal domains but share identical DNA binding domain and ligand binding domain in human. However, lack of specific antibody against each RORα isoform makes biochemical studies on each RORα isoform remain unclear. Here, we generate RORα2-specific antibody and characterize the role of RORα2 in promoting tumor progression in breast cancer. RORα2 requires lysine specific demethylase 1 (LSD1/KDM1A) as a coactivator for transcriptional activation of RORα2 target genes, exemplified by CTNND1. Intriguingly, RORα2 and LSD1 protein levels are dramatically elevated in human breast cancer specimens compared to normal counterparts. Taken together, our studies indicate that LSD1-mediated RORα2 transcriptional activity is important to promote tumor cell migration in human breast cancer as well as breast cancer cell lines. Therefore, our data establish that suppression of LSD1-mediated RORα2 transcriptional activity may be potent therapeutic strategy to attenuate tumor cell migration in human breast cancer.

Gastric cancer continues to be the second most frequent cause of cancer deaths worldwide. However, the exact molecular mechanisms are still unclear. Further research to find potential targets for therapy is critical and urgent. In this study, we found that ARPC2 promoted cell proliferation and invasion in the human cancer cell line MKN-28 using a cell total number assay, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, cell colony formation assay, migration assay, invasion assay, and wound healing assay. For downstream pathways, CTNND1, EZH2, BCL2L2, CDH2, VIM, and EGFR were upregulated by ARPC2, whereas PTEN, BAK, and CDH1 were downregulated by ARPC2. In a clinical study, we examined the expression of ARPC2 in 110 cases of normal human gastric tissues and 110 cases of human gastric cancer tissues. ARPC2 showed higher expression in gastric cancer tissues than in normal gastric tissues. In the association analysis of 110 gastric cancer tissues, ARPC2 showed significant associations with large tumor size, lymph node invasion, and high tumor stage. In addition, ARPC2-positive patients exhibited lower RFS and OS rates compared with ARPC2-negative patients. We thus identify that ARPC2 plays an aneretic role in human gastric cancer and provided a new target for gastric cancer therapy.

Despite recent improvements in treatment for head and neck squamous cell carcinoma (HNSCC), half of all patients with a regional or advanced disease will die within 5 years from diagnosis. Therefore, identification of mechanisms driving the aggressive behavior of HNSCC is of utmost importance. Because p120-catenin (CTNND1/P120CTN) downregulation and

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) targets multiple organs for tumorigenesis in the rat, including the colon and the skin. PhIP-induced skin tumors were subjected to mutation screening, which identified genetic changes in Hras (7/40, 17.5%) and Tp53 (2/40, 5%), but not in Ctnnb1, a commonly mutated gene in PhIP-induced colon tumors. Despite the absence of Ctnnb1 mutations, β-catenin was overexpressed in nuclear and plasma membrane fractions from PhIP-induced skin tumors, coinciding with loss of p120-catenin from the plasma membrane, and the appearance of multiple p120-catenin-associated bands in the nuclear extracts. Real-time RT-PCR revealed that p120-catenin isoforms 1 and 4 were upregulated in PhIP-induced skin tumors, whereas p120-catenin isoform 3 was expressed uniformly, compared with adjacent normal-looking tissue. In human epidermoid carcinoma and colon cancer cells, transient transfection of p120-catenin isoform 1A enhanced the viability and cell invasion index, whereas transient transfection of p120-catenin isoform 4A increased cell viability and cell proliferation. Knockdown of p120-catenin revealed a corresponding reduction in the expression of β-catenin and a transcriptionally regulated target, Ccnd1/Cyclin D1. Co-immunoprecipitation experiments identified associations of β-catenin with p120-catenin isoforms in PhIP-induced skin tumors and human cancer cell lines. The results are discussed in the context of therapeutic strategies that might target different p120-catenin isoforms, providing an avenue to circumvent constitutively active β-catenin arising via distinct mechanisms in skin and colon cancer.

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (

Genetic alterations disrupting the transcription factor

ESRPs are master splice regulators implicated in alternative mRNA splicing programs important for epithelial-mesenchymal transition (EMT) and tumor progression. ESRP1 was identified in some tumors as good or worse predictor of outcome, but in colorectal cancer (CRC) the prognostic value of ESRPs and relation with mesenchymal splice variants is not clear. Here, we studied 68 CRC cases, compared tissue expression of ESRPs with clinical data and with EMT gene splice patterns of conditional CRC cells with deficient ESRP1 expression.Around 72% of patients showed global decreased transcript expression of both ESRPs in tumor as compared to matched non-neoplastic colorectal epithelium. Reduction of ESRP1 in tumor cells was evaluated by immunohistochemistry, associated with microsatellite stability and switch to mesenchymal splice signatures of FGFRs, CD44, ENAH and CTNND1(p120-catenin). Expression of ESRPs was significantly associated with favorable overall survival (log-rank test, P=0.0186 and 0.0408), better than prognostic stratification by tumor staging; and for ESRP1 confirmed with second TCGA cohort (log-rank test, P=0.0435). Prognostic value is independent of the pathological stage and microsatellite instability (ESRP1: HR=0.36, 95%CI 0.15-0.91, P=0.032; ESRP2: HR=0.23, 95%CI 0.08-0.65, P=0.006).Our study supports the role of ESRP1 as tumor suppressor and strongly suggests that ESRPs are candidate markers for early detection, diagnosis, and prognosis of CRC.

OBJECTIVE: To investigate the relationship between 755 T>G polymorphisms in the CTNND
METHODS: CTNND
RESULTS: In control subjects, the frequency of T/T and G/T genotypes, and T and G alleles was 79.4%, 17.2%, 88.1%, and 11.9%, respectively. The distribution of genotypes and allelotypes in the pancreatic carcinoma patients was significantly different from that in the controls (P = 0.007, P = 0.012). Combined GG and GT genotypes were found to have a higher OR in male pancreatic carcinoma patients and the group under the age of 70 years (males: OR, 1.409; 95%CI, 0.912~1.921; under 70 years: OR 1.626; 95% CI, 0.878~2.312). This study also showed a distinct difference in the distribution of P120ctn and single nucleotide polymorphisms (SNPs) between Chinese and Canadian (11.9% vs. 3.9%, P = 0.008).
CONCLUSION: CTNND

BACKGROUND: Increasing evidence supports the association of CTNND1 with tumor development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in hepatocellular carcinoma (HCC) remains unknown. In this study, we aim to investigate the role of CTNND1 in HCC.
METHODS: qRT-PCR and immunohistochemical analyses were used to measure the levels of CTNND1 in HCC specimens and HCC cell lines. CTNND1 and shCTNND1 were transfected into HCC cell lines to investigate its role in HCC. Cell migration and invasion were measured by Transwell and Matrigel analyses in vitro. In vivo metastasis assays were performed in SCID mice.
RESULTS: In clinical HCC samples, we found that CTNND1 expression was significantly up-regulated in cancer lesions compared with paired normal liver tissues. By silencing or overexpressing CTNND1 in HCC cells, we found that CTNND1 could promote cell proliferation, migration, and invasion in vitro. An in-vivo assay showed that CTNND1 dramatically promoted HCC cell tumor formation and metastasis. Moreover, CTNND1 promoted HCC metastasis, at least in part, by indirectly enhancing Wnt/β-catenin signaling. Consistent with these results, the expression of CTNND1 was positively correlated with β-catenin, WNT11, Cyclin D1, and BMP7 expression in human HCC specimens.
CONCLUSIONS: Our study provides evidence that CTNND1 functions as a novel tumor oncogene in HCC, and may be a potential therapeutic target for HCC management.

Aberrant regulation of cellular extrusion can promote invasion and metastasis. Here, we identify molecular requirements for early cellular invasion using a premalignant mouse model of pancreatic cancer with conditional knockout of p120 catenin (Ctnnd1). Mice with biallelic loss of p120 catenin progressively develop high-grade pancreatic intraepithelial neoplasia (PanIN) lesions and neoplasia accompanied by prominent acute and chronic inflammatory processes, which is mediated, in part, through NF-κB signaling. Loss of p120 catenin in the context of oncogenic Kras also promotes remarkable apical and basal epithelial cell extrusion. Abundant single epithelial cells exit PanIN epithelium basally, retain epithelial morphology, survive, and display features of malignancy. Similar extrusion defects are observed following p120 catenin knockdown in vitro, and these effects are completely abrogated by the activation of S1P/S1pr2 signaling. In the context of oncogenic Kras, p120 catenin loss significantly reduces expression of genes mediating S1P/S1pr2 signaling in vivo and in vitro, and this effect is mediated at least, in part, through activation of NF-κB. These results provide insight into mechanisms controlling early events in the metastatic process and suggest that p120 catenin and S1P/S1pr2 signaling enhance cancer progression by regulating epithelial cell invasion. Cancer Res; 76(11); 3351-63. ©2016 AACR.

Osteosarcoma is the most common primary bone cancer which is associated with early metastatic potential and poor prognosis. However, the molecular mechanisms underlying osteosarcoma progression are not well characterized. Here, we investigated the role of miR-409-3p in osteosarcoma metastasis. Osteosarcoma tissue showed decreased expression of miR-409-3p compared to adjacent non-tumorous tissue. The expression level of miR-409-3p was negatively correlated with osteosarcoma metastasis. Overexpression of miR-409-3p in osteosarcoma cells (U2OS) inhibited cell migration and invasion. Bioinformatics analysis showed that catenin-δ1 (CTNND1, p120-catenin) is a direct target of miR-409-3p. Overexpression of miR-409-3p repressed the expression of catenin-δ1 in U2OS cells at both mRNA and protein levels. Meanwhile, miR-409-3p repressed the activity of luciferase reporter containing the 3'-untranslated region (3'UTR) of CTNND1 gene. Furthermore, expression of catenin-δ1 rescued the inhibitory effect of miR-409-3p on cell migration and invasion. Altogether, these results indicated that miR-409-3p targets catenin-δ1 to repress osteosarcoma metastasis.

BACKGROUND: Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. While previous genomic-based studies demonstrated the expression of a handful of tumor-specific isoforms, genome-wide alterations in the balance between isoforms and cancer subtypes is understudied.
RESULT: We systematically analyzed the isoform-level expression patterns and isoform switching events of 819 breast tumor and normal samples assayed by mRNA-seq from TCGA project. On average, 2.2 isoforms per gene were detected and 67.5 % of detected genes (i.e. expressed) showed 1-2 isoforms only. While the majority of isoforms for a given gene were positively correlated with each other and the overall gene level, 470 pairs of isoforms displayed an inverse correlation suggesting a switching event. Most of the isoform switching events were associated with molecular subtypes, including a Basal-like-associated switching in CTNND1. 88 genes showed switching independent of subtypes, among which the isoform pattern of PRICKLE1 was associated with a large genomic signature of biological significance.
CONCLUSION: Our results reveal that the majority of genes do not undergo complex mRNA splicing within breast cancers, and that there is a general concordance in isoform and gene expression levels in breast tumors. We identified hundreds of isoform switching events across breast tumors, most of which were associated with differences in tumor subtypes. As exemplified by the detailed analysis of CTNND1 and PRICKLE1, these isoform switching events potentially provide new insights into the post-transcriptional regulatory mechanisms of tumor subtypes and cancer biology.

Chemotherapy has improved the survival of patients with gastric cancer by unknown mechanisms. In this study, we showed that cisplatin and docetaxel used in gastric cancer treatment increase the expression of miRNA-29 (miR-29) family members and decrease the expression of their oncogenic targets, mediating a significant part of the efficacious benefits of these chemotherapeutic agents. In particular, patients with gastric cancer who experienced recurrences after chemotherapy tended to exhibit low levels of miR-29c expression in their tumors, suggesting that miR-29c activation may contribute to the chemotherapeutic efficacy. Enforced expression of miR-29s in gastric cancer cells inhibited cell invasion in vitro and in vivo by directly targeting catenin-δ (CTNND1). Drug treatment suppressed gastric cancer cell invasion by restoring miR-29c-mediated suppression of catenin-δ and RhoA signaling. In parallel, drug treatment also activated several tumor-suppressive miRNAs, thereby decreasing expression of their oncogenic effector targets. Overall, our findings defined a global mechanism for understanding the efficacious effects of cytotoxic chemotherapy in gastric cancer.

Increasing evidence supports the association of catenin-δ1 (CTNND1, p120ctn) with tumour development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in gastric cancer remain unknown. The expression level and cellular localization of CTNND1 were determined by immunohistochemistry in 126 human gastric cancer and 50 non-tumourous tissues. The cellular localization of CTNND1 and epithelial cadherin (E-cadherin) were detected by immunofluorescence. Cell proliferation, apoptosis, migration and invasion assays were performed to assess the effect of CTNND1 cDNA or CTNND1 siRNA transfection on gastric cancer cells. Luciferase assay, western blot analysis and in vivo assays were used to determine whether CTNND1 could be regulated by miR-145. The results demonstrate that the cytoplasmic localization of CTNND1 protein, rather than expression level, was indicative of higher clinical stage, positive lymph node metastasis and poorer prognosis in gastric cancers. CTNND1 could promote gastric cancer cell migration and invasion with little effect on cellular proliferation and apoptosis. CTNND1 was proved to be a direct target gene for miR-145. Besides suppressing cytoplasmic CTNND1 expression, miR-145 could recover the membranous localization of CTNND1 and E-cadherin. We conclude that cytoplasmic CTNND1 can serve as an independent prognostic factor for patients with gastric cancers. MiR-145 inhibits invasion of gastric cancer cells not only by down-regulating cytoplasmic CTNND1 expression but also by inducing the translocation of CTNND1 and E-cadherin from the cytoplasm to the cell membrane through down-regulating N-cadherin.

BACKGROUND: Ikaros, the product of IKZF1, is a regulator of lymphoid development and polymorphisms in the gene have been associated with the acute lymphoblastic leukemia (ALL). Additionally, IKZF1 deletions and mutations identify high-risk biological subsets of childhood ALL [Georgopoulos et al. Cell 1995;83(2):289-299; Mullighan et al. N Engl J Md 2009;360(5):470-480].
PROCEDURES: To discover the underlying pathways modulated by Ikaros we performed gene expression and gene ontology analysis in IKZF1 deleted primary B-ALL pediatric patient samples. To validate downstream targets we performed qPCR on individual patient samples. We also created IKZF1 knockdown B-ALL cell lines with over 50% reduction of Ikaros, mimicking haplosufficient Ikaros deletions, and again performed qPCR to investigate the downstream targets. Finally, to understand the association of Ikaros deletion with a poor prognosis we challenged our IKZF1 knockdown cell lines with chemotherapy and compared responses to IKZF1 wild-type controls.
RESULTS: We report a specific gene expression signature of 735 up-regulated and 473 down-regulated genes in IKZF1 deleted primary B-ALL pediatric patient samples. Gene ontology studies revealed an up-regulation of genes associated with cell adhesion, cytoskeletal regulation, and motility in IKZF deleted patient samples. Validated up-regulated target genes in IKZF1 deleted patient samples included CTNND1 and PVRL2 (P = 0.0003 and P = 0.001), and RAB3IP and SPIB (P = 0.005 and P = 0.032) were down-regulated. In further studies in IKZF1 knockdown cell lines, apoptosis assays showed no significant chemoresistance.
CONCLUSION: IKZF1 knockdown alone does not impart intrinsic chemotherapy resistance suggesting that the association with a poor prognosis may be due to additional lesions, microenvironmental interactions with the bone marrow niche, or other factors.

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.

The epithelial adherens junction is an E-cadherin-based complex that controls tissue integrity and is stabilized at the plasma membrane by p120-catenin (p120, also known as CTNND1). Mutational and epigenetic inactivation of E-cadherin has been strongly implicated in the development and progression of cancer. In this setting, p120 translocates to the cytosol where it exerts oncogenic properties through aberrant regulation of Rho GTPases, growth factor receptor signaling and derepression of Kaiso (also known as ZBTB33) target genes. In contrast, indirect inactivation of the adherens junction through conditional knockout of p120 in mice was recently linked to tumor formation, indicating that p120 can also function as a tumor suppressor. Supporting these opposing functions are findings in human cancer, which show that either loss or cytoplasmic localization of p120 is a common feature in the progression of several types of carcinoma. Underlying this dual biological phenomenon might be the context-dependent regulation of Rho GTPases in the cytosol and the derepression of Kaiso target genes. Here, we discuss past and present findings that implicate p120 in the regulation of cancer progression and highlight opportunities for clinical intervention.

BACKGROUND: Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.
METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1.
CONCLUSIONS: Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.

In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with reads per kilobase per million mapped reads (RPKM) values for ERBB2 (14.4, 400, and 300 for SUM149, SUM190, and SKBR3, respectively) and for EGFR (60.1, not detected, and 1.4 for the same 3 cell lines). We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g., 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used bioinformatics sites GeneGo, PathwayCommons and NCI receptor signaling networks to identify pathways that contained the four main oncogenes and had good coverage in the transcriptomic and proteomic data sets as well as a significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling; caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings; branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 subpathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.

BACKGROUND: Approximately one third of the hereditary diffuse gastric cancer (HDGC) families carry germline mutations in the E-cadherin gene (CDH1). Risk prediction in members of families with this rare but deadly cancer could be improved by the identification of additional HDGC genes in non-CDH1 families.
METHODS: Affected individuals from 22 CDH1 mutation-negative families were screened for germline mutations in four catenin genes: CTNNA1, CTNNB1, JUP, and CTNND1. Catenins interact closely with E-cadherin molecules in cells, and are therefore logical candidate genes for mutation screening in HDGC families.
RESULTS: No nonsynonymous variants were seen in CTNNA1, CTNNB1, or CTNND1; only JUP contained nonsynonymous variants, of which only two rare variants were predicted to be deleterious.
CONCLUSION: Catenin genes are not commonly mutated in non-CDH1 HDGC families.
IMPACT: Germline mutations in CTNNA1, CTNNB1, JUP, or CTNND1 are unlikely to play a major role in HDGC.

Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.

PURPOSE: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer.
EXPERIMENTAL DESIGN: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N(0)) and 19 with positive lymph nodes (N(+)), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways.
RESULTS: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N(0), and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N(+) group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N(0) and N(+) tumors (P < 0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N(0) (OR: 0.20, 95% CI: 0.06-0.66) and the β-catenin pathway (p120 positivity) to N(+) (OR: 1.79, 95%CI: 1.05-3.05).
CONCLUSIONS: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated noncanonical β-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer.

Stable E-cadherin-based adherens junctions are pivotal in maintaining epithelial tissue integrity and are the major barrier for epithelial cancer metastasis. Proteins of the p120(ctn) subfamily have emerged recently as critical players for supporting this stability. The identification of the unique juxtamembrane domain (JMD) in E-cadherin that binds directly to delta-catenin/NPRAP/neurojungin (CTNND2) and p120(ctn) (CTNND1) provides a common motif for their interactions. Recently, crystallographic resolution of the JMD of p120(ctn) further highlighted possibilities of intervening between interactions of p120(ctn) subfamily proteins and E-cadherin for designing anti-cancer therapeutics. For most epithelial cancers, studies have demonstrated a reduction of p120(ctn) expression or alteration of its subcellular distribution. On the other hand, delta-catenin, a primarily neural-enriched protein in the brain of healthy individuals, is up-regulated in all cancer types that have been studied to date. Two research articles in the September 2010 issue of The Journal of Pathology increase our understanding of the involvement of these proteins in lung cancer. One reports the identification of rare p120(ctn) (CTNND1) gene amplification in lung cancer. One mechanism by which delta-catenin and p120(ctn) may play a role in carcinogenesis is their competitive binding to E-cadherin through the JMD. The other presents the first vigorous characterization of delta-catenin overexpression in lung cancer. Unexpectedly, the authors observed that delta-catenin promotes malignant phenotypes of non-small cell lung cancer by non-competitive binding to E-cadherin with p120(ctn) in the cytoplasm. Looking towards the future, the understanding of delta-catenin and p120(ctn) with and beyond their localization at the cell-cell junction should provide further insight into their roles in cancer pathogenesis.

The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.

PURPOSE: To identify gene expression patterns and interaction networks related to BCR-ABL status and clinical outcome in adults with acute lymphoblastic leukemia (ALL).
PATIENTS AND METHODS: DNA microarrays were used to profile a set of 54 adult ALL specimens from the Medical Research Council UKALL XII/Eastern Cooperative Oncology Group E2993 trial (21 p185BCR-ABL-positive, 16 p210BCR-ABL-positive and 17 BCR-ABL-negative specimens).
RESULTS: Using supervised and unsupervised analysis tools, we detected significant transcriptomic changes in BCR-ABL-positive versus -negative specimens, and assessed their validity in an independent cohort of 128 adult ALL specimens. This set of 271 differentially expressed genes (including GAB1, CIITA, XBP1, CD83, SERPINB9, PTP4A3, NOV, LOX, CTNND1, BAALC, and RAB21) is enriched for genes involved in cell death, cellular growth and proliferation, and hematologic system development and function. Network analysis demonstrated complex interaction patterns of these genes, and identified FYN and IL15 as the hubs of the top-scoring network. Within the BCR-ABL-positive subgroups, we identified genes overexpressed (PILRB, STS-1, SPRY1) or underexpressed (TSPAN16, ADAMTSL4) in p185BCR-ABL-positive ALL relative to p210BCR-ABL-positive ALL. Finally, we constructed a gene expression- and interaction-based outcome predictor consisting of 27 genes (including GRB2, GAB1, GLI1, IRS1, RUNX2, and SPP1), which correlated with overall survival in BCR-ABL-positive adult ALL (P = .0001), independent of age (P = .25) and WBC count at presentation (P = .003).
CONCLUSION: We identified prominent molecular features of BCR-ABL-positive adult ALL, which may be useful for developing novel therapeutic targets and prognostic markers in this disease.