The simultaneous miR-155-5p overexpression and miR-223-3p inhibition can activate pEMT in oral squamous cell carcinoma.

OBJECTIVE: This study aims to explore the effects of miR-223-3p and miR-155-5p on epithelial-mesenchymal transition (EMT) and migration in oral squamous cell carcinoma (OSCC). METHODOLOGY: EMT markers (E-cadherin, N-cadherin, P120 catenin (P120ctn), and vimentin) expression was determined by qRT-PCR and western blot analysis in SCC-9 cells which overexpress miR-155-5p and/or not express miR-223-3p. Scratch assays and Transwell migration assays were conducted to evaluate cell migration ability. RESULTS: When miR-223-3p was inhibited in OSCC cells, P120ctn and E-cadherin mRNA levels were dramatically downregulated (P<0.05), while N-cadherin levels were significantly upregulated, and the migration ability of OSCC cells increased. The overexpression of miR-155-5p in OSCC cells upregulated miR-223-3p significantly (34-fold) compared to the control group. It also led to significant downregulation of the mRNA of P120ctn and E-cadherin and significant upregulation of the mRNA of N-cadherin and Vimentin (P<0.05). Meanwhile, the migratory ability of OSCC cells significantly increased. When miR-155-5p was overexpressed while miR-223-3p was inhibited, the highest expression of E-cadherin and P120ctn mRNA and the lowest expression of N-cadherin(P<0.05) was observed. Simultaneously, tumor cell migration was significantly facilitated. CONCLUSION: miR-223-3p inhibits the migration of OSCC cells, while miR-155-5p can elevate the miR-223-3p mRNA expression. The simultaneous miR-155-5p overexpression and miR-223-3p inhibition can activate pEMT, increasing OSCC migration in vitro. This provides a novel approach and potential target for the effective treatment of OSCC.

CTNND1 is involved in germline predisposition to early-onset gastric cancer by affecting cell-to-cell interactions.

BACKGROUND: CDH1 and CTNNA1 remain as the main genes for hereditary gastric cancer. However, they only explain a small fraction of gastric cancer cases with suspected inherited basis. In this study, we aimed to identify new hereditary genes for early-onset gastric cancer patients (EOGC; < 50 years old). METHODS: After germline exome sequencing in 20 EOGC patients and replication of relevant findings by gene-panel sequencing in an independent cohort of 152 patients, CTNND1 stood out as an interesting candidate gene, since its protein product (p120ctn) directly interacts with E-cadherin. We proceeded with functional characterization by generating two knockout CTNND1 cellular models by gene editing and introducing the detected genetic variants using a lentiviral delivery system. We assessed ß-catenin and E-cadherin levels, cell detachment, as well as E-cadherin localization and cell-to-cell interaction by spheroid modeling. RESULTS: Three CTNND1 germline variants [c.28_29delinsCT, p.(Ala10Leu); c.1105C > T, p.(Pro369Ser); c.1537A > G, p.(Asn513Asp)] were identified in our EOGC cohorts. Cells encoding CTNND1 variants displayed altered E-cadherin levels and intercellular interactions. In addition, the p.(Pro369Ser) variant, located in a key region in the E-cadherin/p120ctn binding domain, showed E-cadherin mislocalization. CONCLUSIONS: Defects in CTNND1 could be involved in germline predisposition to gastric cancer by altering E-cadherin and, consequently, cell-to-cell interactions. In the present study, CTNND1 germline variants explained 2% (3/172) of the cases, although further studies in larger external cohorts are needed.

Inhibition of δ-catenin palmitoylation slows the progression of prostate cancer.

Prostate cancer (PCa) is the second leading cause of death in males. It has been reported that δ-catenin expression is upregulated during the late stage of prostate cancer. Palmitoylation promotes protein transport to the cytomembrane and regulates protein localization and function. However, the effect of δ-catenin palmitoylation on the regulation of cancer remains unknown. In this study, we utilized prostate cancer cells overexpressing mutant δ-catenin (J6A cells) to induce a depalmitoylation phenotype and investigate its effect on prostate cancer. Our results indicated that depalmitoylation of δ-catenin not only reduced its membrane expression but also promoted its degradation in the cytoplasm, resulting in a decrease in the effect of EGFR and E-cadherin signaling. Consequently, depalmitoylation of δ-catenin reduced the proliferation and metastasis of prostate cancer cells. Our findings provide novel insights into potential therapeutic strategies for controlling the progression of prostate cancer through palmitoylation-based targeting of δ-catenin.

DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Dinaciclib exerts a tumor-suppressing effect via ß-catenin/YAP axis in pancreatic ductal adenocarcinoma.

Dinaciclib, a cyclin-dependent kinase-5 (CDK5) inhibitor, has significant anti-tumor properties. However, the precise mechanism of dinaciclib requires further investigation. Herein, we investigated the anti-tumor functions and molecular basis of dinaciclib in pancreatic ductal adenocarcinoma (PDAC). PDAC and matched para-carcinoma specimens were collected from the patients who underwent radical resection. Immunohistochemistry was performed to assess CDK5 expression. Cell proliferation ability, migration, and invasion were measured using Cell Counting Kit-8, wound healing, and transwell assay, respectively. The cell cycle and apoptosis were assessed using flow cytometry. Gene expression was examined using RNA-seq and quantitative real-time PCR. Protein expression of proteins was measured by western blot analysis and immunofluorescence microscopy. Tumor-bearing mice were intraperitoneally injected with dinaciclib. CDK5 is highly expressed in PDAC. The expression level of CDK5 was significantly related to tumor size, T stage, and the American Joint Committee on Cancer stage. High CDK5 expression can predict poor survival in PDAC patients. In addition, the expression level of CDK5 might be an independent prognostic factor for PDAC patients. Dinaciclib inhibits the growth and motility of PDAC cells and induces apoptosis and cell cycle arrest in the G2/M phase. Mechanistically, dinaciclib down-regulated yes-associated protein (YAP) mRNA and protein expression by reducing ß-catenin expression. Moreover, dinaciclib significantly inhibited PDAC cell growth in vivo . Our findings reveal a novel anti-tumor mechanism of dinaciclib in which it decreases YAP expression by down-regulating ß-catenin at the transcriptional level rather than by activating Hippo pathway-mediated phosphorylation-dependent degradation.

MicroRNA-34c-5p Reduces Malignant Properties of Lung Cancer Cells through Regulation of TBL1XR1/Wnt/ß-catenin Signaling.

INTRODUCTION: Lung cancer is common cancer with high mortality. A growing number of studies have focused on investigating the regulatory effects of microRNAs (miRs/miRNAs) during cancer progression. Nevertheless, the biological function of miR- 34c-5p in lung cancer and the underlying mechanism have not been determined. This study explored the effect of miR-34c-5p on the malignant behaviors of lung cancer cells. METHODS: In this study, we utilized diverse public databases to obtain differentially expressed miRNAs. Then, qRT-PCR and western blot were conducted to determine miR-34c-5p and transducin ß-like 1 X-linked receptor 1 (TBL1XR1) expression. Next, H1299 and H460 cells were transfected with miR-34c-5p-mimic and pcDNA3.1- TBL1XR1. To examine the anticancer effects of miR-34c-5p, CCK-8, scratch, and Matrigel-Transwell assays were conducted to test cell viability, migration, and invasion, respectively. The StarBase database and dual-luciferase reporter gene assay were used to predict and verify the relationship between miR-34c-5p and TBL1XR1. RESULTS: Finally, Wnt/ß-catenin signaling- and epithelial-mesenchymal transition (EMT)- related protein levels were detected using western blot. The results demonstrated that miR-34c-5p was poorly expressed in lung cancer cells, while TBL1XR1 was highly expressed. The findings also confirmed the direct interaction between miR-34c-5p and TBL1XR1. In H1299 and H460 cells, miR-34c-5p overexpression inhibited cell proliferation, migration, and invasion, Wnt/ß-catenin signaling activity, and EMT, while TBL1XR1 upregulation reversed these effects of miR-34c-5p overexpression. CONCLUSION: These findings illustrated that miR-34c-5p might repress the malignant behaviors of lung cancer cells via TBL1XR1, providing evidence for miR-34c-5p-based lung cancer therapy.

LINC01226 promotes gastric cancer progression through enhancing cytoplasm-to-nucleus translocation of STIP1 and stabilizing ß-catenin protein.

Gastric cancer (GC) remains one of the most common malignances and the leading cause of cancer-related mortality worldwide. Although the critical role of several long non-coding RNAs (lncRNAs) transcribed from several GC-risk loci has been established, we still know little about the biological significance of these lncRNAs at most gene loci and how they play in cell signaling. In the present study, we identified a novel oncogenic lncRNA LINC01226 transcribed from the 1p35.2 GC-risk locus. LINC01226 shows markedly higher expression levels in GC specimens compared with those in normal tissues. High expression of LINC01226 is evidently correlated with worse prognosis of GC cases. In line with these, oncogenic LINC01226 promotes proliferation, migration and metastasis of GC cells ex vivo and in vivo. Importantly, LINC01226 binds to STIP1 protein, leads to disassembly of the STIP1-HSP90 complex, elevates interactions between HSP90 and ß-catenin, stabilizes ß-catenin protein, activates the Wnt/ß-catenin signaling and, thereby, promote GC progression. Together, our findings uncovered a novel layer regulating the Wnt signaling in cancers and uncovers a new epigenetic mode of GC tumorigenesis. These discoveries also shed new light on the importance of functional lncRNAs as innovative therapeutic targets through precisely controlling protein-protein interactions in cancers.

APC and P53 mutations synergise to create a therapeutic vulnerability to NOTUM inhibition in advanced colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with the majority of cases initiated by inactivation of the APC tumour suppressor. This results in the constitutive activation of canonical WNT pathway transcriptional effector ß-catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM which antagonises WNT-FZD receptor-ligand interactions. Here, we sought to evaluate the effects of NOTUM activity on CRC as a function of driver mutation landscape. DESIGN: Mouse and human colon organoids engineered with combinations of CRC driver mutations were used for Notum genetic gain-of-function and loss-of-function studies. In vitro assays, in vivo endoscope-guided orthotopic organoid implantation assays and transcriptomic profiling were employed to characterise the effects of Notum activity. Small molecule inhibitors of Notum activity were used in preclinical therapeutic proof-of-principle studies targeting oncogenic Notum activity. RESULTS: NOTUM retains tumour suppressive activity in APC-null adenomas despite constitutive ß-catenin activity. Strikingly, on progression to adenocarcinoma with P53 loss, NOTUM becomes an obligate oncogene. These phenotypes are Wnt-independent, resulting from differential activity of NOTUM on glypican 1 and 4 in early-stage versus late-stage disease, respectively. Ultimately, preclinical mouse models and human organoid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonisation of distal organs. CONCLUSIONS: Our findings that a single agent targeting the extracellular enzyme NOTUM is effective in treating highly aggressive, metastatic adenocarcinomas in preclinical mouse models and human organoids make NOTUM and its glypican targets therapeutic vulnerabilities in advanced CRC.

Identification of a Yorkie homolog from Litopenaeus vannamei as a negative regulator in anti-WSSV immune response.

Hippo signaling pathway is a serine threonine kinase cascade that is evolutionary conserved with well-established roles in organ size control, development, tumorigenesis and immunity. As its core molecule, Yorkie also plays an important role against pathogen. In this study, we cloned and characterized a Yorkie homolog from Litopenaeus vannamei, designed as LvYKI, which has a 1650 bp open reading frame. It has the characterized domains of Yokie family, and displayed to be close to the insects and crustacean. Quantitative Real-time PCR showed that LvYKI had different regulatory mechanisms in different tissues. The transcriptional level of Lvyki was down-regulated in gill, while up-regulated in hepatopancreas post white spot syndrome virus (WSSV) infection. Moreover, the expression and phosphorylation of LvYKI was reduced upon WSSV infection, which indicated that LvYKI was involved in WSSV infection. Furthermore, RNAi was performed to evaluate the role of LvYKI in shrimp immune responses. Knocking down of Lvyki resulted in inhibition of the transcription of WSSV gene ie1 and vp28, and delayed mortality of shrimp post WSSV infection. Meanwhile, the apoptosis of hemocyte was increased as well. All results suggested that shrimp can promote apoptosis to resist WSSV infection mediated by down-regulation of LvYKI. In addition, it was found that LvYKI could interact with Lvß-catenin, which cross-linked the Wnt and Hippo signaling pathway in innate immunity. Conclusively, our study provided clues that LvYKI plays an important role in the interaction between shrimp and virus. It will promote our understanding of the molecular mechanism in innate immunity.

Downregulated circular RNA hsa_circ_0005797 inhibits endometrial cancer by modulating microRNA-298/Catenin delta 1 signaling.

Abnormal expression of circular RNA (circRNA) expression has been implicated in endometrial cancer (EC) progression. Thus, investigation of the mechanism of hsa_circ_0005797 during EC etiology may provide new insight into the treatment of EC. In the present study, we found that hsa_circ_0005797 expression was significantly increased in EC biological samples and cell lines, whereas its downregulation inhibited in vitro tumor cells proliferation and invasion phenotypes and suppressed tumor formation in nude mice. In mechanism, we characterized hsa_circ_0005797 as an miR-298 sponge, with CTNND1 identified as a target of miR-298. Our rescue assay data further revealed that hsa_circ_0005797 silencing inhibited EC cells proliferation and invasion via miR-298/CTNND1 signaling. In conclusion, our study confirmed hsa_circ_0005797 is a poor prognostic factor for EC and modulates EC phenotypes by regulating the hsa_circ_000579/miR-298/CTNND1 signaling, which provides potential treatment targets for EC.

A ß-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer.

BACKGROUND & AIMS: Sporadic colorectal cancers arise from initiating mutations in APC, producing oncogenic ß-catenin/TCF-dependent transcriptional reprogramming. Similarly, the tumor suppressor axis regulated by the intestinal epithelial receptor GUCY2C is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by ß-catenin/TCF signaling. METHODS: We performed RNA sequencing analysis of 4 unique conditional human colon cancer cell models of ß-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, chromatin immunoprecipitation sequencing, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) knockout, and CRISPR epigenome editing, which were cross-validated with human tissue chromatin immunoprecipitation sequencing datasets, to identify functional gene enhancers mediating GUCY2C ligand loss. RESULTS: RNA sequencing analyses reveal the GUCY2C hormones as 2 of the most sensitive targets of ß-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant ß-catenin/TCF signaling. CONCLUSIONS: These studies reveal DNA elements regulating corepression of GUCY2C ligand transcription by ß-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could reestablish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUCY2C axis and oppose transformation.

N6-Isopentenyladenosine Hinders the Vasculogenic Mimicry in Human Glioblastoma Cells through Src-120 Catenin Pathway Modulation and RhoA Activity Inhibition.

BACKGROUND: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. METHODS: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. RESULTS: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. CONCLUSIONS: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.

Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission.

Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression­enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

An N-Cadherin 2 expressing epithelial cell subpopulation predicts response to surgery, chemotherapy and immunotherapy in bladder cancer.

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.

PLEKHA7, an Apical Adherens Junction Protein, Suppresses Inflammatory Breast Cancer in the Context of High E-Cadherin and p120-Catenin Expression.

Inflammatory breast cancer is a highly aggressive form of breast cancer that forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. These cancers express high levels of E-cadherin, the major mediator of adherens junctions, which enhances formation of tumor emboli. Previous studies suggest that E-cadherin promotes cancer when the balance between apical and basolateral cadherin complexes is disrupted. Here, we used immunohistochemistry of inflammatory breast cancer patient samples and analysis of cell lines to determine the expression of PLEKHA7, an apical adherens junction protein. We used viral transduction to re-express PLEKHA7 in inflammatory breast cancer cells and examined their aggressiveness in 2D and 3D cultures and in vivo. We determined that PLEKHA7 was deregulated in inflammatory breast cancer, demonstrating improper localization or lost expression in most patient samples and very low expression in cell lines. Re-expressing PLEKHA7 suppressed proliferation, anchorage independent growth, spheroid viability, and tumor growth in vivo. The data indicate that PLEKHA7 is frequently deregulated and acts to suppress inflammatory breast cancer. The data also promote the need for future inquiry into the imbalance between apical and basolateral cadherin complexes as driving forces in inflammatory breast cancer.

Identification of New Genes Involved in Germline Predisposition to Early-Onset Gastric Cancer.

The genetic cause for several families with gastric cancer (GC) aggregation is unclear, with marked relevance in early-onset patients. We aimed to identify new candidate genes involved in GC germline predisposition. Whole-exome sequencing (WES) of germline samples was performed in 20 early-onset GC patients without previous germline mutation identified. WES was also performed in nine tumor samples to analyze the somatic profile using SigProfilerExtractor tool. Sequencing germline data were filtered to select those variants with plausible pathogenicity, rare frequency and previously involved in cancer. Then, a manual filtering was performed to prioritize genes according to current knowledge and function. These genetic variants were prevalidated with Integrative Genomics Viewer 2.8.2 (IGV). Subsequently, a further selection step was carried out according to function and information obtained from tumor samples. After IGV and selection step, 58 genetic variants in 52 different candidate genes were validated by Sanger sequencing. Among them, APC, FAT4, CTNND1 and TLR2 seem to be the most promising genes because of their role in hereditary cancer syndromes, tumor suppression, cell adhesion and Helicobacter pylori recognition, respectively. These encouraging results represent the open door to the identification of new genes involved in GC germline predisposition.

A requirement for p120-catenin in the metastasis of invasive ductal breast cancer.

We report here the effects of targeted p120-catenin (encoded by CTNND1; hereafter denoted p120) knockout (KO) in a PyMT mouse model of invasive ductal (mammary) cancer (IDC). Mosaic p120 ablation had little effect on primary tumor growth but caused significant pro-metastatic alterations in the tumor microenvironment, ultimately leading to a marked increase in the number and size of pulmonary metastases. Surprisingly, although early effects of p120-ablation included decreased cell-cell adhesion and increased invasiveness, cells lacking p120 were almost entirely unable to colonized distant metastatic sites in vivo The relevance of this observation to human IDC was established by analysis of a large clinical dataset of 1126 IDCs. As reported by others, p120 downregulation in primary IDC predicted worse overall survival. However, as in the mice, distant metastases were almost invariably p120 positive, even in matched cases where the primary tumors were p120 negative. Collectively, our results demonstrate a strong positive role for p120 (and presumably E-cadherin) during metastatic colonization of distant sites. On the other hand, downregulation of p120 in the primary tumor enhanced metastatic dissemination indirectly via pro-metastatic conditioning of the tumor microenvironment.

Twist2 is NFkB-responsive when p120-catenin is inactivated and EGFR is overexpressed in esophageal keratinocytes.

Esophageal squamous cell carcinoma (ESCC) is among the most aggressive and fatal cancer types. ESCC classically progresses rapidly and frequently causes mortality in four out of five patients within two years of diagnosis. Yet, little is known about the mechanisms that make ESCC so aggressive. In a previous study we demonstrated that p120-catenin (p120ctn) and EGFR, two genes associated with poor prognosis in ESCC, work together to cause invasion. Specifically, inactivation of p120ctn combined with overexpression of EGFR induces a signaling cascade that leads to hyperactivation of NFkB and a resultant aggressive cell type. The purpose of this present study was to identify targets that are responsive to NFkB when p120ctn and EGFR are modified. Using human esophageal keratinocytes, we have identified Twist2 as an NFkB-responsive gene. Interestingly, we found that when NFkB is hyperactivated in cells with EGFR overexpression and p120ctn inactivation, Twist2 is significantly upregulated. Inhibition of NFkB activity results in nearly complete loss of Twist2 expression, suggesting that this potential EMT-inducing gene, is a responsive target of NFkB. There exists a paucity of research on Twist2 in any cancer type; as such, these findings are important in ESCC as well as in other cancer types.

Neural defects caused by total and Wnt1-Cre mediated ablation of p120ctn in mice.

BACKGROUND: p120 catenin (p120ctn) is an important component in the cadherin-catenin cell adhesion complex because it stabilizes cadherin-mediated intercellular junctions. Outside these junctions, p120ctn is actively involved in the regulation of small GTPases of the Rho family, in actomyosin dynamics and in transcription regulation. We and others reported that loss of p120ctn in mouse embryos results in an embryonic lethal phenotype, but the exact developmental role of p120ctn during brain formation has not been reported. RESULTS: We combined floxed p120ctn mice with Del-Cre or Wnt1-Cre mice to deplete p120ctn from either all cells or specific brain and neural crest cells. Complete loss of p120ctn in mid-gestation embryos resulted in an aberrant morphology, including growth retardation, failure to switch from lordotic to fetal posture, and defective neural tube formation and neurogenesis. By expressing a wild-type p120ctn from the ROSA26 locus in p120ctn-null mouse embryonic stem cells, we could partially rescue neurogenesis. To further investigate the developmental role of p120ctn in neural tube formation, we generated conditional p120ctnfl/fl;Wnt1Cre knockout mice. p120ctn deletion in Wnt1-expressing cells resulted in neural tube closure defects (NTDs) and craniofacial abnormalities. These defects could not be correlated with misregulation of brain marker genes or cell proliferation. In contrast, we found that p120ctn is required for proper expression of the cell adhesion components N-cadherin, E-cadherin and ß-catenin, and of actin-binding proteins cortactin and Shroom3 at the apical side of neural folds. This region is of critical importance for closure of neural folds. Surprisingly, the lateral side of mutant neural folds showed loss of p120ctn, but not of N-cadherin, ß-catenin or cortactin. CONCLUSIONS: These results indicate that p120ctn is required for neurogenesis and neurulation. Elimination of p120ctn in cells expressing Wnt1 affects neural tube closure by hampering correct formation of specific adhesion and actomyosin complexes at the apical side of neural folds. Collectively, our results demonstrate the crucial role of p120ctn during brain morphogenesis.

p120-catenin phosphorylation status alters E-cadherin mediated cell adhesion and ability of tumor cells to metastasize.

p120-catenin is considered to be a tumor suppressor because it stabilizes E-cadherin levels at the cell surface. p120-catenin phosphorylation is increased in several types of cancer, but the role of phosphorylation in cancer is unknown. The phosphorylation state of p120-catenin is important in controlling E-cadherin homophilic binding strength which maintains epithelial junctions. Because decreased cell-cell adhesion is associated with increased cancer metastasis we hypothesize that p120-catenin phosphorylation at specific Serine and Threonine residues alters the E-cadherin binding strength between tumor cells and thereby affect the ability of tumor cells to leave the primary tumor and metastasize to distant sites. In this study we show that expression of the p120-catenin phosphorylation dead mutant, by converting six Serine and Threonine sites to Alanine, leads to enhanced E-cadherin adhesive binding strength in tumor cells. We observed a decrease in the ability of tumor cells expressing the p120-catenin phosphorylation mutant to migrate and invade using in-vitro models of cancer progression. Further, tumor cells expressing the phosphorylation mutant form of p120-catenin demonstrated a decrease in ability to metastasize to the lungs using an in-vivo orthotopic mammary fat pad injection model of breast cancer development and metastasis. This suggests that regulation of p120-catenin phosphorylation at the cell surface is important in mediating cell-adhesion, thereby impacting cancer progression and metastasis.