Localization Of Β-catenin Research Articles

EHMT2 promotes the pathogenesis of hepatocellular carcinoma by epigenetically silencing APC expression

BackgroundHepatocellular carcinoma (HCC), the second leading cause of cancer death worldwide, alone accounts for over half (466,100) of new cancer cases and 422,100 deaths based on the average year incidence rates of 2009 to 2011 in China. Due to unclear and complex underlying mechanisms for HCC development, effective therapy for HCC is still unavailable. The Wnt–β-catenin pathway is a critical contributor of HCC pathogenesis: 40–70% of HCCs from patients harbor the nuclear accumulation of β-catenin protein. However, the mechanisms for β-catenin activation are not fully understood.MethodsThe deletion of EHMT2 in Hep3B and Huh1 cells was achieved by transiently transfecting cells with pX459 plasmids, which carry EHMT2 specific small guide RNA (sgRNA) sequences for Cas9 protein. All experiments were performed in triplicate and repeated more than three times.ResultsIn the present study, we observed that EHMT2 (but not EHMT1) mRNA and protein levels were significantly elevated in HCC compared with normal controls. Next, the results of Ki67 staining, as well as MTT, soft-agar and xenograft assays, in wild-type and EHMT2−/− Hep3B and Huh1 cancer stem cells collectively revealed that the elevation of EHMT2 expression is required for the tumorigenesis of HCC. Meanwhile, we found that elevated EHMT2 expression contributes to the activation of Wnt–β-catenin signaling: deletion of EHMT2 in Hep3B or Huh1 cells promoted the cytoplasmic localization of β-catenin and restrained the expression of Wnt–β-catenin signaling targets such as Myc, CCND1, MMP-7, etc. We demonstrated that EMHT2 directly mediates the H3K9me2 methylation of the APC promoter to epigenetically silence its expression. More intriguingly, our findings also showed that UNC0642, a specific inhibitor of EHMT2, exhibits anti-tumorigenesis effects in HCC both in vitro and in vivo, which were largely abolished by deletion of EHMT2 or overexpression of APC in Hep3B and Huh1 cells.ConclusionAltogether, our observations emphasize that the EHMT2–APC axis is a critical contributor to Wnt–β-catenin pathway activation in HCC, and UNC0642 may be a potential candidate for target drug treatment of HCC.

Membrane Localization of β-Catenin in Prostate Cancer PC3 Cells Treated with Teucrium persicum Boiss. Extract

Teucrium persicum Boiss. is an Iranian endemic plant which belongs to the Lamiaceae family and has been used to relieve pains in traditional Iranian medicine. We have previously found that treatment of prostate cancer PC3 cells with Teucrium persicum extract leads to the formation of small populations of epithelial cells. β-Catenin is a component of cell adherens junctions in epithelial cells and therefore, in this study, we have investigated the effect of Teucrium persicum extract on expression, cellular localization, and transcriptional activity of β-Catenin protein in PC-3 cells. Indirect immunofluorescence microscopy results showed that the cells treated with T. persicum extract had higher levels of β-Catenin protein at the cell membrane. Western blotting experiments produced consistent results. Gene expression studies by using a few β-Catenin-target genes including c-MYC, CYCLIN D1, and a reporter Luciferase gene under the control of several β-Catenin/TCF binding elements showed that treatment of PC3 cells with the methanolic extract of T. persicum decreases the transcriptional activities of β-Catenin. The results of this study provide further support for the anticancer properties of T. persicum. Definitely, more detailed molecular investigations are needed to find the mechanism(s) behind these effects. Highlights β-Catenin protein is a main component of Wnt signaling pathway and adherens junction. Activation of Wnt signaling pathway affects translocation of β-Catenin. Teucrium persicum extract induces β-Catenin localization at cell membrane. Teucrium persicum affects the transcriptional activity of β-Catenin. It stabilizes E-cadherin/β-Catenin protein complex and adherens junction.

Extracellular domain shedding of the ALK receptor mediates neuroblastoma cell migration.

SUMMARYAlthough activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in ~10% of neuroblastoma (NB) tumors, the role of the wild-type (WT) receptor, which is aberrantly expressed in most non-mutated cases, is unclear. Both WT and mutant proteins undergo extracellular domain (ECD) cleavage. Here, we map the cleavage site to Asn654-Leu655 and demonstrate that cleavage inhibition of WT ALK significantly impedes NB cell migration with subsequent prolongation of survival in mouse models. Cleavage inhibition results in the downregulation of an epithelial-to-mesenchymal transition (EMT) gene signature, with decreased nuclear localization and occupancy of β-catenin at EMT gene promoters. We further show that cleavage is mediated by matrix metalloproteinase 9, whose genetic and pharmacologic inactivation inhibits cleavage and decreases NB cell migration. Together, our results indicate a pivotal role for WT ALK ECD cleavage in NB pathogenesis, which may be harnessed for therapeutic benefit.

Targeted Therapy with BET Protein and β Catenin Antagonists Against Ruxolitinib Resistant Post-Myeloproliferative Neoplasm (MPN) Secondary (s) AML Cells

Hematopoietic stem/progenitor cells (HPCs) in myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in the JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, secondary AML (sAML) occurs in up to 20% of patients. Ruxolitinib (Rux), a type I, ATP-competitive, dual JAK1/2 inhibitor (JAKi), confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Because standard chemotherapy is also relatively ineffective, there is an urgent need to develop and test novel agents for the treatment of sAML. We previously reported that, although they lack additional JAK2 mutations compared to their sensitive parental counterparts, in vitro generated Rux-resistant, JAKi-persister/resistant HEL-92.1.7 and SET2 cells (JAKi-P/R) cells exhibit 3 to 10-fold lower sensitivity to Rux, and display reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TKs). We hypothesized that the relative resistance to JAKi in sAML cells would be governed by a dysregulated transcriptome and dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against JAKi-P/R sAML cells. ARV-771 recruits the E3 ligase VHL (Von Hippel-Lindau) to polyubiquitylate and mediate multiple rounds of sub-stoichiometric catalysis, leading to efficient and prolonged degradation and depletion of BETPs, including BRD4 (> 90%) in the sAML cells. Treatment with BETP-PROTAC (50 to 250 nM) markedly depleted BRD4 and BRD2, as well as caused robust attenuation of the levels of MYC, p-STAT5, Bcl-xL, PIM1 and CDK6 in JAKi-P/R sAML cells (Leukemia, 2017, PMID: 28042144). Concomitantly, BETP-PROTAC treatment mediated profound growth inhibition and apoptosis of Rux-sensitive and JAKi-P/R cultured and patient-derived CD34+ sAML cells. This translated into a significantly reduced sAML burden and improved survival, without toxicity, in the luciferase-transduced immune depleted (NSG) mice treated with ARV-771 (30 mg/kg/day for 3 weeks) versus vehicle control. In myeloid malignancies, canonical WNT-β catenin signaling is essential for survival, growth and self-renewal of leukemia stem and blast progenitor cells (BPCs). Notably, we discovered that, as compared to their sensitive controls, JAKi-P/R sAML cells exhibited higher levels of p-AKT, p-GSK3-β and increased nuclear localization of β-catenin (by confocal microscopy), associated with increased levels of β-catenin-TCF4 targets, MYC and survivin. In a previous report, we had demonstrated that treatment with the β-catenin antagonist BC-2059 that degrades and depletes nuclear levels of β-catenin and inhibits β-catenin/TCF4 transcriptional targets, resulted in apoptosis of AML stem progenitor cells (Leukemia, 2015;29:1267-78). Consistent with this, we determined that BC-2059 (50 to 100 nM) also markedly attenuated nuclear β-catenin levels, reduced expression of the β-catenin/TCF4 targets MYC and survivin, and induced in vitro apoptosis of JAKi-P/R sAML and PD CD34+ sAML cells. Co-treatment with BC-2059 and Rux synergistically induced in vitro apoptosis (combination indices < 1.0) of sAML cells, and combined therapy for 3 weeks with BC-2059 (30 mg/kg/day) and Rux (30 mg/kg/day) significantly reduced sAML burden and improved survival of NSG mice engrafted with sAML cells (p < 0.01). Recently, resistance to BETP inhibitors that evict BETPs from chromatin was shown to be at least in part due to increased activity of WNT-β-catenin/TCF4 and the increased c-Myc expression. Based on this, we also determined the in vitro and in vivo effects of co-treatment with BETP-PROTAC and BC-2059 against sAML cells. Compared to each drug alone, co-treatment with BETP-PROTAC exerted synergistic in vitro lethality against JAKi sensitive and JAKi-P/R sAML cells. These findings strongly support further in vivo development and testing of BETP-PROTAC and β-catenin antagonist-based combinations against post-MPN sAML. DisclosuresQian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Verstovsek:Celgene: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Lilly Oncology: Research Funding; Pfizer: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Astrazeneca: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Bristol Myers Squibb: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Incyte: Research Funding; Lilly Oncology: Research Funding; Promedior: Research Funding; Roche: Research Funding; Roche: Research Funding; Astrazeneca: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Bristol Myers Squibb: Research Funding; NS Pharma: Research Funding.

Asymmetric distribution of dynamin-2 and β-catenin relative to tight junction spikes in alveolar epithelial cells

ABSTRACT Tight junctions between lung alveolar epithelial cells maintain an air–liquid barrier necessary for healthy lung function. Previously, we found that rearrangement of tight junctions from a linear, cortical orientation into perpendicular protrusions (tight junction spikes) is associated with a decrease in alveolar barrier function, especially in alcoholic lung syndrome. Using quantitative super-resolution microscopy, we found that spikes in control cells were enriched for claudin-18 as compared with alcohol-exposed cells. Moreover, using an in situ method to measure barrier function, tight junction spikes were not associated with localized increases in permeability. This suggests that tight junction spikes have a regulatory role as opposed to causing a physical weakening of the epithelial barrier. We found that tight junction spikes form at cell–cell junctions oriented away from pools of β-catenin associated with actin filaments, suggesting that adherens junctions determine the directionality of tight junction spikes. Dynamin-2 was associated with junctional claudin-18 and ZO-1, but showed little localization with β-catenin and tight junction spikes. Treatment with Dynasore decreased the number of tight junction spikes/cell, increased tight junction spike length, and stimulated actin to redistribute to cortical tight junctions. By contrast, Dynole 34–2 and MiTMAB altered β-catenin localization, and reduced tight junction spike length. These data suggest a novel role for dynamin-2 in tight junction spike formation by reorienting junction-associated actin. Moreover, the greater spatial separation of adherens and tight junctions in squamous alveolar epithelial cells as compared with columnar epithelial cells facilitates analysis of molecular regulation of the apical junctional complex.

Does Loss of Immunohistochemical Expression of Glypican 3 in Oral Squamous Cell Carcinoma Play a Role in the Wnt/β-catenin Signaling Pathway?

Glypican 3 (GPC3) is a cell membrane protein and plays a dual role, as a tumor suppressor and oncogene, depending on its structure. It is known to regulate the Wnt/β-catenin signaling pathway and affect cell growth and proliferation. β-catenin plays a major oncogenic role in progression of oral squamous cell carcinoma (OSCC); thus, this study aimed to explore the relationship between β-catenin and GPC3 in OSCC. Immunoexpression of GPC3 and β-catenin was evaluated semiquantitatively in tumor tissue (n=80) and normal oral mucosa tissue (n=20). For GPC3, the percentage of stained cells and the staining intensity were assessed. For β-catenin, the percentage of stained cells, localization, and intensity of staining were assessed at the tumor-invasive front. The Pearson correlation was used to determine the correlation between the GPC3 and β-catenin immunoreactivity. Significantly decreased expression of GPC3 (P=0.008) and a highly significant difference in the case of localization of β-catenin (P=0.0001) were observed in OSCC when compared with normal oral mucosa. Cytoplasmic expression with a shift of β-catenin expression to the nucleus was seen in OSCC in comparison with primarily membranous and membranous and cytoplasmic staining in normal mucosa. A significant difference was observed with respect to localization of stain, with β-catenin staining moving to the nuclear compartment with an increase in the tumor grade (P=0.011). No correlation was observed between β-catenin and GPC3 expression in OSCC cases. It is concluded that loss of expression of GPC3 in OSCC compared with normal oral mucosa indicates that it plays the role of a tumor suppressor gene in OSCC and its expression is therefore silenced in OSCC.

Dehydrated human amniotic membrane modulates canonical Wnt signaling in multiple cell types in vitro

Canonical Wnt signaling is a major pathway known to regulate diverse physiological processes in multicellular organisms. Signaling is tightly regulated by feedback mechanisms; however, persistent dysregulation of this pathway is implicated in the progression of multiple disease states. In this study, proteomic analysis identified endogenous Wnt antagonists in micronized dehydrated human amnion/chorion membrane (μdHACM); thereby, prompting a study to further characterize the intrinsic properties of μdHACM as it relates to Wnt activity, in vitro. A TCF/LEF reporter cell line demonstrated the general ability of μdHACM to inhibit β-catenin induced transcription activity. Furthermore, in vitro systems, modeling elevated Wnt signaling, were developed in relevant cell types including tenocytes, synoviocytes, and human dermal fibroblasts (HDFs). Stimulation of these cells with Wnt3A resulted in translocation of β-catenin to the nucleus and increased expression of Wnt related genes. The subsequent addition of μdHACM, in the continued presence of Wnt-stimulus, mitigated the downstream effects of Wnt3A in tenocytes, synoviocytes, and HDFs. Nuclear localization of β-catenin was abated with corresponding reduction of Wnt related gene expression. These data demonstrate the in vitro regulation of canonical Wnt signaling as an inherent property of μdHACM and a novel mechanism of action.

Effects of Tunneling Nanotubes on the Mitochondrial Regulation of the Amount and Subcellular Localization of Β-Catenin During Osteogenesis of MSC/HUVEC Spheroids

Tunneling nanotubular expressways (TNTs), which allow direct cell-to-cell transfer of intracellular organelles, have been widely identified in various cell types. However, the precise functions of TNTs in intercellular communication and their practical application in tissue regeneration is still uncertain. Mesenchymal stem cells (MSCs) are commonly employed as seed cells in tissue engineering. The differentiation of MSCs requires sufficient energy, which can be regulated by the fusion and fission of mitochondria. The phenomenon of mitochondrial shuttle between cells has been observed, and has led to the hypothesis that applying TNTs to deliver mitochondria into MSCs might be a promising approach to stimulate osteogenic differentiation. In proliferating endothelial cells (ECs), cellular dynamics including the fusion and fission of mitochondria is increased, and thus ECs are considered as ideal candidates for mitochondria donors. In order to exploit the application of TNT-mediated mitochondria transfer, we employed mesenchymal stem cell/ human umbilical vein endothelial cell (MSC/HUVEC) spheroids as a research model, and investigated the transfer among them, as well as the underlying mechanism. Fluorescence staining showed that directional transfer of mitochondria between MSC-HUVEC pairs, especially from HUVEC to MSC. Through TNT-mediated mitochondrial transfer, osteogenesis markers were up-regulated, accompanied by an increased amount of β-catenin in MSCs. Moreover, the improved generation of pre-vascular network has also been observed in the spheroids, as a result of β-catenin translocation to the periphery of HUVECs. However, all of these effects are abolished by the destruction of TNTs. Collectively, our results indicate that the TNT strategy can be applied widely to various aspects of biological research, such as but not limited to tissue regeneration and targeted drug delivery.

Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate.

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of β-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.

LDL receptor related protein 2 to promote colorectal cancer metastasis via enhancing GSK3β/β-catenin signaling.

e15507 Background: LDL receptor related protein 2 (LRP2), a multi-ligand endocytic receptor, has been implicated in the tumourigenesis of various human cancers. However, the biological roles and underlying regulatory mechanism of LRP2 in CRC remain unclear. Methods: Real-time PCR (RT-PCR), western blot, and immunohistochemistry were used to examine LRP2 expression. Gene silencing and overexpressing efficiencies of LRP2 were confirmed by RT-PCR and western blot. Then wound-healing, migration assay and invasion assay were used to investigate the function of LRP2 in CRC cells. The protein expression and cellular localization of LRP2 and β-catenin were characterized by immunofluorescence staining. The nude mice tail vein metastasis model was established to observe the effect of LRP2 on the lung metastasis of CRC cells in vivo. In addition, gene set enrichment analysis was carried out to explore the potential mechanism of LRP2 in CRC. Results: LRP2 expression was highly upregulated in CRC compared with matched adjacent normal tissue. LRP2 overexpression was positively correlated with shorter overall survival in CRC patients. The biological function observation indicated that LRP2 promoted CRC metastasis in vitro and in vivo. Further molecular mechanism investigation demonstrated that LRP2 protein interacted with glycogen synthase kinase-3β (GSK-3β) and led to decreased expression of GSK3β at ser9, followed by enhanced β-catenin signaling pathway in CRC cells. Conclusions: Our study demonstrated that LRP2 could enhance the metastatic abilities of CRC cells in vitro and in vivo by inhibiting the expression of GSK3β and enhancing the activity of β-catenin signaling pathway, providing that LRP2 might be a novel therapeutic target for metastasis in CRC.

Nanoarchitecture and Molecular Interactions of Epithelial Cell Junction Proteins Revealed by Super‐Resolution Microscopy

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin-matrix interactions. However, it is increasingly recognized that their characteristic resident proteins can be found in each other's territories and demonstrate previously unrecognized functions. This study presents the nanoarchitecture and nano-colocalization of cell junction proteins in culture and in intestinal tissue by Stochastic Optical Reconstruction Microscopy (STORM). STORM allows for resolution of approximately 20 nm in the xy plane and 50 nm in the z plane. The Z-axial view of non-cancerous MDCK-II cell AJ proteins, β-catenin and p120ctn, showed greatest expression density towards the apical milieu of AJ. This is accompanied by the distinctive displacement of p120ctn being apical to β-catenin. Towards the basal direction, p120ctn and β-catenin become increasingly colocalized. Interestingly, this topography is lost in metastatic prostate cancer PC3 cells, in that p120ctn localization becomes basal to β-catenin. The deletion of TJ protein claudin-7 is sufficient in altering the polarity of p120ctn to basal β-catenin localization, like that seen in PC3 cells compared to MDCK-II. Loss of claudin-7 also significantly altered localization and distribution of many cell junction proteins. Therefore, STORM revealed the regional nanoarchitecture of cellular junctions that were previously unavailable, providing new insights in their potential trans-compartmental modulation of protein functions.

Clinicopathological features of 70 desmoid-type fibromatoses confirmed by β-catenin immunohistochemical staining and CTNNB1 mutation analysis.

Desmoid-type fibromatosis (DF) is a locally aggressive neoplasm characterized by mutations in the CTNNB1 gene, which encodes the β-catenin protein. We reviewed 85 cases of DF and performed Sanger sequencing for detecting mutations in CTNNB1 and immunostaining for detecting β-catenin localization. We included 70 DF samples, of which 56 cases demonstrated nuclear β-catenin localization and 43 cases harboured CTNNB1 mutations. CTNNB1-mutant DF samples consistently displayed nuclear β-catenin expression and were derived from larger-sized tumours compared to samples with wild-type CTNNB1. When we further classified DF cases into 2 subgroups based on the type of specimen, excised specimens with nuclear β-catenin expression frequently displayed CTNNB1 mutation and no statistical correlation between nuclear β-catenin expression and CTNNB1 mutation was observed in biopsies. When we classified CTNNB1 mutation cases into 2 subgroups (DF with T41A or T41I, and DF with S45F or S45P), T41A or T41I mutations were observed more frequently in males than in females. Additionally, DF tumours harbouring S45F or S45P mutations were located more frequently in the abdominal wall than tumours with T41A or T41I mutations. In conclusion, CTNNB1 mutation correlates with nuclear β-catenin expression in larger or excised DF tumours, and DF harbouring CTNNB1 mutations manifest variable clinical presentations.

Fisetin inhibits lipopolysaccharide-induced inflammatory response by activating \u03b2-catenin, leading to a decrease in endotoxic shock

Fisetin is a naturally occurring flavonoid that possesses several pharmacological benefits including anti-inflammatory activity. However, its precise anti-inflammatory mechanism is not clear. In the present study, we found that fisetin significantly inhibited the expression of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Additionally, fisetin attenuated LPS-induced mortality and abnormalities in zebrafish larvae and normalized the heart rate. Fisetin decreased the recruitment of macrophages and neutrophils to the LPS-microinjected inflammatory site in zebrafish larvae, concomitant with a significant downregulation of proinflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase-2a (COX-2a), IL-6, and TNF-α. Fisetin inhibited the nuclear localization of nuclear factor-kappa B (NF-κB), which reduced the expression of pro-inflammatory genes. Further, fisetin inactivated glycogen synthase kinase 3β (GSK-3β) via phosphorylation at Ser9, and inhibited the degradation of β-catenin, which consequently promoted the localization of β-catenin into the nucleus. The pharmacological inhibition of β-catenin with FH535 reversed the fisetin-induced anti-inflammatory activity and restored NF-κB activity, which indicated that fisetin-mediated activation of β-catenin results in the inhibition of LPS-induced NF-κB activity. In LPS-microinjected zebrafish larvae, FH535 promoted the migration of macrophages to the yolk sac and decreased resident neutrophil counts in the posterior blood island and induced high expression of iNOS and COX-2a, which was accompanied by the inhibition of fisetin-induced anti-inflammatory activity. Altogether, the current study confirmed that the dietary flavonoid, fisetin, inhibited LPS-induced inflammation and endotoxic shock through crosstalk between GSK-3β/β-catenin and the NF-κB signaling pathways.

Dual activation of Hedgehog and Wnt/β-catenin signaling pathway caused by downregulation of SUFU targeted by miRNA-150 in human gastric cancer.

Mounting evidence has shown that miRNA-150 expression is upregulated in gastric cancer (GC) and is associated with gastric carcinogenesis, but the underlying oncogenic mechanism remains elusive. Here, we discovered that miRNA-150 targets the tumor suppressor SUFU to promote cell proliferation, migration, and the epithelial–mesenchymal transition (EMT) via the dual activation of Hedgehog (Hh) and Wnt signaling. MiRNA-150 was highly expressed in GC tissues and cell lines, and the level of this miRNA was negatively related to that of SUFU. In addition, both the miRNA-150 and SUFU levels were associated with tumor differentiation. Furthermore, miRNA-150 activated GC cell proliferation and migration in vitro. We found that miRNA-150 inhibitors repressed not only Wnt signaling by promoting cytoplasmic β-catenin localization, but also repressed Hh signaling and EMT. MiRNA-150 inhibition also resulted in significant tumor volume reductions in vivo, suggesting the potential application of miRNA-150 inhibitors in GC therapy. The expression of genes downstream of Hh and Wnt signaling was also reduced in tumors treated with miRNA-150 inhibitors. Notably, anti-SUFU siRNAs rescued the inhibitory effects of miRNA-150 inhibitors on Wnt signaling, Hh activation, EMT, cell proliferation, cell migration, and colony formation. Taken together, these findings indicate that miRNA-150 is oncogenic and promotes GC cell proliferation, migration, and EMT by activating Wnt and Hh signaling via the suppression of SUFU expression.

Selected Articles from This Issue

Ductal carcinoma in situ (DCIS) breast cancers are non-invasive but can progress to invasive ductal carcinoma (IDC) through tumor intrinsic and -extrinsic factors that are poorly understood. In this study, Trinh and colleagues model interactions between DCIS/IDC tumors and their respective immune cell infiltrates using integrated molecular analyses. The authors find that IDC tumors are often genetically related to their DCIS precursors despite long periods of latency between the two stages, and that features of the tumor immune microenvironment are established early in tumor development and could shap. the progression to IDC. In particular, recurrent TP53 and PIK3CA mutations were associated with sustained inflammatory immune cell activity across the DCIS-IDC transition. The authors argue that, despite their status as neoantigens, these mutations and others could be retained through immune editing of the tumor. While in-depth study in a diverse cohort is needed, these data suggest a key role for the immune system in shaping the progression of certain DCIS tumors to IDC based on their constellation of genomic alterations.Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) face a dismal prognosis, and clinical management of PDAC is limited to cytotoxic regimens. 5-fluorouracil is a key component of these regimens, but its use is hampered by systemic toxicity due to the off-target effects of its metabolites and inefficient conversion to the active metabolite, FdUMP. Here, Haber and colleagues report a novel fluoropyrimidine agent, CF10. Consisting of a capped polymeric chain of FdUMP molecules, CF10 displayed enhanced efficacy, increased stability, and reduced toxicity in preclinical models compared to precursor compounds such as 5-fluorouracil. While CF10 was shown to be effective as a single agent in preclinical models of PDAC, combination with an experimental Poly (ADP-ribose) Glycohydrolase (PARG) inhibitor or PARG ablation resulted in synergistic induction of DNA damage and PDAC cell death. Taken together, the data nominate CF10 as a promising candidate that could significantly improve the efficacy of standard-of-care regimens for PDAC.Mutations in the PI3K/AKT pathway are common in cancer and have significant implications for cell metabolism, proliferation, and survival. Despite its central role in cell biology, pharmacological agents targeting this pathway have met with limited success in the clinic. Now, using CRISp. to model specific genetic lesions, Gao and colleagues have uncovered differential functions of mutations in AKT isoforms: E17K mutations in AKT1 and AKT2 both conferred survival and proliferation advantages, but AKT1 E17K had the additional effect of suppressing invasion. Specifically, AKT1 mutation prevented nuclear localization of β-catenin, thereby repressing ZEB1 expression and impeding migratory capacity. This effect on cell migration was not observed in the context of AKT2 activation nor in the context PIK3CA or PTEN mutations, which activate both AKT1 and AKT2. The authors therefore suggest that the use of pharmacological inhibitors of the AKT pathway may paradoxically drive disease progression in certain genetic backgrounds by releasing an AKT1-mediated brake on cell migration and epithelial-mesenchymal transition.BH3-mimetic compounds such as venetoclax have potent pro-apoptotic effects in leukemic B cells, but tumors frequently amplify their expression of anti-apoptotic BH2 domain-containing proteins, such as MCL-1, to circumvent pro-apoptotic therapeutics. In this study, Smith and colleagues employ a genome-wide CRISPR screen to identify pathways involved in regulating MCL-1 expression, and thereby identify the heme-regulated inhibitor (HRI) as a key target. The authors show that, in the absence of heme or when the interaction between heme and HRI are pharmacologically disrupted, HRI initiates the cellular stress response and suppresses MCL-1 expression. These effects were not observed when HRI expression was genetically ablated, suggesting that HRI represents a control node for the modulation of MCL-1 expression in leukemic cells. These data nominate HRI and the heme-sensing pathway as a potential candidate for development of combinatorial regimens to enhance the efficacy of BH3-mimetics in certain subsets of leukemia patients.

Microtubule-targeting agents impair kinesin-2-dependent nuclear transport of β-catenin: Evidence of inhibition of Wnt/β-catenin signaling as an important antitumor mechanism of microtubule-targeting agents.

An aberrant accumulation of nuclear β-catenin is closely associated with the augmentation of cancer malignancy. In this work, we report that several microtubule-targeting agents (MTAs) such as vinblastine, taxol, and C12 (combretastatin-2-aminoimidazole analog) inhibit Wnt/β-catenin signaling in oral squamous cell carcinoma (OSCC). We showed that the inhibition of microtubule dynamics by MTAs decreased the level of β-catenin by increasing Axin and adenomatous polyposis coli levels and reducing the level of dishevelled. Furthermore, MTAs strongly reduced the localization of β-catenin in the nucleus. The reduction in the level of nuclear β-catenin was neither due to the degradation of β-catenin in the nucleus nor due to an increase in the export of nuclear β-catenin from the nucleus. A motor protein kinesin-2 was found to assist the nuclear transportation of β-catenin. Interestingly, Wnt/β-catenin signaling antagonist treatment synergized with MTAs and the activators of Wnt/β-catenin signaling antagonized with the MTAs. C12 potently suppressed the growth of 4-Nitroquinoline 1-oxide-induced OSCC in the tongue of C57 black 6 mice and also abrogated Wnt/β-catenin signaling pathway in the tumor. Our results provide evidence that the decrease in Wnt/β-catenin signaling is an important antitumor effect of MTAs and the combined use of MTAs with Wnt/β-catenin signaling antagonists could be a promising strategy for cancer chemotherapy.

Endometrial Adenocarcinomas With No Specific Molecular Profile: Morphologic Features and Molecular Alterations of "Copy-number Low" Tumors.

The study evaluated morphologic patterns, mutational profiles, and β-catenin immunohistochemistry (IHC) in copy-number low (CNL) endometrial adenocarcinomas (EAs). CNL EAs (n=19) with next-generation or whole genome sequencing results and available tissue for IHC were identified from our institutional database. Clinical data and histologic slides were reviewed. IHC for β-catenin was performed and correlated with mutation status. Images of digital slides of CNL EAs from The Cancer Genome Atlas (TCGA) database (n=90) were blindly reviewed by 4 pathologists, and morphology was correlated with mutation status. Categorical variables were analyzed using the Fisher exact test, and agreement was assessed using Fleiss κ. CTNNB1 mutations were present in 63% (12/19) of CNL EAs. β-catenin nuclear localization was present in 83% of CTNNB1-mutated tumors (10/12) and in 0% (0/7) of CTNNB1-wildtype tumors (sensitivity 0.83, specificity 1.00). Squamous differentiation (SD) was present in 47% (9/19) and was more often observed in CTNNB1-mutated tumors (P=0.02). Mucinous differentiation (MD) was associated with KRAS mutations (P<0.01). Digital image review of TCGA CNL EAs revealed that pathologist agreement on SD was strong (κ=0.82), whereas agreement on MD was weak (κ=0.48). Pathologists identified SD in 22% (20/90), which was significantly associated with the presence of CTNNB1 mutations (P<0.01). CNL EAs demonstrate several morphologies with divergent molecular profiles. SD was significantly associated with CTNNB1 mutations and nuclear localization of β-catenin in these tumors. Nuclear expression of β-catenin is a sensitive and specific IHC marker for CTNNB1 mutations in CNL EAs. CNL EAs with KRAS mutations often displayed MD.

Low-Dose Exposure to Endocrine Disruptor Dichlorodiphenyltrichloroethane (DDT) Affects Transcriptional Regulation of Adrenal Zona Reticularis in Male Rats

The study examined transcriptional regulation of adrenal zona reticularis development in male Wistar rats exposed to low doses of endocrine disruptor dichlorodiphenyltrichloroethane (DDT) prenatally and postnatally. At pubertal age, zona reticularis demonstrated a retarded growth with a low proliferative activity of its cells. At this age, zona reticularis was characterized with overexpression of β-catenin by steroid-producing cells; a high percentage of cells with membrane and cytoplasmic localization of β-catenin, and reduced number of cells with nuclear β-catenin attesting to insufficient activation of Wnt signaling. Expression of transcriptional factor Oct4 by reticularis cells was down-regulated indicating their diminished proliferative potency. No significant alterations in Sonic Hedgehog expression were observed. Thus, DDT-provoked disorders of transcriptional regulation result in abnormal development of zona reticularis thereby disturbing sexual maturation in males.

Human Epidermal Growth Factor Receptor-3 Expression Is Regulated at Transcriptional Level in Breast Cancer Settings by Junctional Adhesion Molecule-A via a Pathway Involving Beta-Catenin and FOXA1

Simple SummarySignaling from the human epidermal growth factor receptor (HER) family of proteins increases in many cancers, including breast. HER2-high breast cancers are successfully treated with anti-HER2 therapies, but these drugs are limited by the fact that patients frequently develop resistance to them. One common mechanism by which resistance develops is when tumors acquire high levels of a family member called HER3. We had previously shown that a protein called JAM-A regulates the level of HER2 in breast cancer cells, and is associated with the development of resistance to HER2-targeted therapies. In this study we show for the first time that JAM-A levels also regulate those of HER3. Using breast cancer cell and tissue models and culminating in patient tissue material, we provide evidence that JAM-A regulates HER3 expression via a pathway involving the transcription factors β-catenin and FOXA1. We suggest that JAM-A merits future investigation as a novel drug target for its potential to reduce HER3 tumorigenic signaling and to offset the development of resistance to HER2-targeted therapies. The success of breast cancer therapies targeting the human epidermal growth factor receptor-2 (HER2) is limited by the development of drug resistance by mechanisms including upregulation of HER3. Having reported that HER2 expression and resistance to HER2-targeted therapies can be regulated by Junctional Adhesion Molecule-A (JAM-A), this study investigated if JAM-A regulates HER3 expression. Expressional alteration of JAM-A in breast cancer cells was used to test expressional effects on HER3 and its effectors, alongside associated functional behaviors, in vitro and semi-in vivo. HER3 transcription factors were identified and tested for regulation by JAM-A. Finally a patient tissue microarray was used to interrogate connections between putative pathway components connecting JAM-A and HER3. This study reveals for the first time that HER3 and its effectors are regulated at gene/protein expression level by JAM-A in breast cancer cell lines; with functional consequences in in vitro and semi-in vivo models. In bioinformatic, cellular and patient tissue models, this was associated with regulation of the HER3 transcription factor FOXA1 by JAM-A via a pathway involving β-catenin. Our data suggest a novel model whereby JAM-A expression regulates β-catenin localization, in turn regulating FOXA1 expression, which could drive HER3 gene transcription. JAM-A merits investigation as a novel target to prevent upregulation of HER3 during the development of resistance to HER2-targeted therapies, or to reduce HER3-dependent tumorigenic signaling.

Abstract PR005: CD73 sequestering mutant β-catenin at the membrane explains recurrence in CTNNB1 mutant endometrial cancer

Abstract Somatic missense mutations in exon 3 of CTNNB1 are associated with significantly worse recurrence-free survival in patients with low grade, early stage endometrioid-type endometrial carcinoma (EEC). CTNNB1 encodes for β-catenin, an integral protein in Wnt signaling and core member of E-cadherin-catenin cell-cell adhesions. Although CTNNB1 mutation identifies patients at higher risk for recurrence, not all patients will recur. Nuclear expression of β-catenin protein in CTNNB1-mutated EEC is not robust as is normally seen in other CTNNB1-mutated cancers such as desmoid tumor and colon cancer. This suggests cancer-specific molecular mechanisms may regulate mutant β-catenin in EEC. Previously, we identified a cancer-specific role for CD73, a cell surface 5’nuclotidase, in EEC. CD73 is downregulated and its loss associates with poor survival. Notably, CD73 inhibits tumor progression by increasing the localization of wild-type β-catenin to the membrane. Through fractionation and co-immunoprecipitation studies, using a highly homologous (97.4% identical; 100% identical in exon 3 (UniPort)) Xenopus exon 3 CTNNB1 mutant expressed in HEC-1-A cells, and human tumor data we provide evidence that CD73 sequesters mutant β-catenin at the membrane with E-cadherin, subsequently limiting its nuclear translocation. EEC patients with CTNNB1 mutation and low CD73 expression have increased incidence of recurrence (n = 29). CD73 levels were not significantly different with clinical stage or lymphatic and vascular space invasion, suggesting that CD73 expression independently predicts disease recurrence in patients with CTNNB1 mutant tumors. CTNNB1 mutations in EEC rarely occur in the region of the β-catenin protein that binds to E-cadherin. Thus, mechanisms regulating the movement of wild-type β-catenin to the membrane would be expected to be able to localize mutant β-catenin to the membrane regardless of exon 3 mutation. Immunohistochemistry studies of CTNNB1 mutant tumors (n = 11) show a strong association between CD73 expression and β-catenin localization in cancer cells. Cancer cells expressing membrane CD73 largely express β-catenin at the membrane. Whereas, in cancer cells with CD73 loss or cytoplasmic CD73 expression, β-catenin nuclear staining is increased. While these data are preliminary, CD73 sequestering mutant β-catenin to the membrane suggests a molecular mechanism for why certain patients with CTNNB1 mutant tumors recur. Accordingly, CD73 may serve as a biomarker for identifying patients for more aggressive clinical management to prevent recurrence. Citation Format: Jessica L. Bowser, Katherine C. Kurnit, Luan H. Phan, Jerry B. Harvey, Jiping Feng, SuSu Xie, Andrew B. Gladden, Pierre D. McCrea, Russell R. Broaddus. CD73 sequestering mutant β-catenin at the membrane explains recurrence in CTNNB1 mutant endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR005.