Nuclear Localization Of Β-catenin Research Articles

Nkd1 functions downstream of Axin2 to attenuate Wnt signaling.

Wnt signaling is a crucial developmental pathway involved in early development as well as stem cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic β-catenin, and Nkd1 functions to inhibit the nuclear localization of β-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2gh1/gh1, nkd1gh2/gh2 single mutants and axin2gh1/gh1;nkd1gh2/gh2 double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2gh1/gh1;nkd1gh2/gh2 double mutants. Further, Wnt target gene expression by qRT-PCR, and RNA-seq analysis and protein expression by mass spectrometry demonstrated that the double axin2gh1/gh1;nkd1gh2/gh2 mutant resembled the nkd1gh2/gh2 phenotype demonstrating that Nkd1 functions downstream of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of β-catenin-dependent transcription having novel readouts of Wnt activity compared to nkd1gh2/gh2 or the axin2gh1/gh1;nkd1gh2/gh2 double mutant. We also tested the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/β-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.

Abstract 4671: Efficacy and mechanism of mebendazole for ovarian cancer therapy

Abstract Introduction: Ovarian cancer (OvCa) is the most lethal gynecological malignancy. Although most OvCa patients initially respond to frontline platinum-based therapies with approximately 80% of patients experiencing remission, approximately 75% of those women experience a recurrence with only approximately 50% of patients alive 5 years following diagnosis. There is a critical need to develop novel therapeutic agents or methods to overcome chemoresistance and toxicities experienced with conventional chemotherapy regimens used for OvCa patients. Repurposing drugs is a promising strategy to safely and more rapidly evaluate potentially effective treatment regimens. Over the past two decades, Mebendazole (MBZ), an anti-parasitic drug, has gained much attention in oncology due to its favorable biosafety profile and potent anti-cancerous activity seen in several human malignancies. Therefore, we examined the efficiency and mechanism of MBZ against OvCa. Methods: The in vitro efficacy of MBZ was evaluated in cisplatin-resistant (CPR) and parent (WT/sensitive) OVCAR-8 cell lines as well as in ascites samples collected from chemo-resistant OvCa patients by using 2D and/or 3D cell viability assay and invasion assay. The in vivo efficacy of MBZ was assessed in orthotopic animal models, utilizing CPR-OVCAR8 spheroids, and patient-derived xenograft (PDX) animal models with specimens from chemo-resistant ovarian cancer patients. Molecular mechanisms were investigated through western blot and immunofluorescence analysis. Result: Our results confirmed that MBZ efficiently decreased cell viability, spheroid size, and the invasion ability of OvCa cell lines and ascites samples. OvCa cells treated with MBZ demonstrated downregulation of proliferation markers and upregulation of apoptosis markers indicating inhibition of cell proliferation and induction of apoptosis. Mechanistically, MBZ inhibited the Wnt/β-catenin signaling pathway by downregulating the expression and nuclear localization of β-catenin and epithelial-mesenchymal transition (EMT) markers. Preclinical studies showed that MBZ at 50 mg/kg/day dose for 28 days significantly reduced tumor growth in PDXs and orthotopic tumor models without any evidence of toxicity. Conclusion: Collectively, our study strongly supports the therapeutic potential of MBZ against OvCa particularly, which warrants further clinical studies. Citation Format: Rajani Rai, Dhanamjai Penta, Debasish Dey, Doris M Benbrook, Lin Wang, Magdalena Bieniasz, Lauren E Dockery. Efficacy and mechanism of mebendazole for ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4671.

Abstract 5366: ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression

Abstract The stiffening of the extracellular matrix (ECM), a phenomenon that often promotes metastasis and immunosuppression in many cancers, is a process that is not yet fully understood. Our study reveals that ATR, a molecule primarily recognized for its role in maintaining the genome, plays a crucial role in ECM stiffness-induced immune evasion and metastasis in triple negative breast cancer (TNBC) through DNA repair independent manner. In response to ECM stiffness, ATR activates SUN2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. This activation enhances the interaction of SUN2 with outer nuclear membrane proteins KASH1 and KASH2. The interaction between SUN2 and KASH1/2 facilitates the nuclear localization of β-catenin, which in turn promotes epithelial mesenchymal transition (EMT) and reduces the expression of E-cadherin. E-cadherin is essential for attracting CD103+ immune cells that perform anti-tumor immunity. Interestingly, inhibiting ATR reduces EMT in tumor cells, leading to an increase in CD103+ dendritic cell infiltration while decreasing neutrophil and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration. Further, we found that ATR levels are inversely correlated with immunotherapy response rates in patients. The alteration in immune cell composition sensitizes tumors to immunotherapy, thereby suppressing tumor growth and metastasis. In conclusion, the role of ATR in remodeling the immune environment could be crucial for optimizing the clinical use of ATR inhibitors. This makes ATR a promising target for overcoming immunotherapy resistance. We suggest early or prolonged treatment with an ATR inhibitor to potentially reduce metastasis rates and improve the prognosis of TNBC patients. Furthermore, we suggest using ATR levels as a marker to predict response rates and prognosis in TNBC patients. For patients with higher ATR expression levels, combination treatment with ATR inhibitors and immunotherapy may yield better outcomes. Enhanced anti-tumor immunity could increase the chances for patients to eliminate cancer cells and help end breast cancer. Finally, our research suggests the combined treatment of ATR inhibition and immunotherapy for patients without necessarily applying DNA damage stimulation. This approach could potentially reduce toxicity while still benefiting patients. Citation Format: Xinyi Tu, Zhenkun Lou, Robert Mutter. ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5366.

Abstract PR008: Location, location, location: Why cellular localization of mutant β-catenin matters in endometrial cancer

Abstract The present study aims to understand the mechanisms of mutant β-catenin in endometrial cancer (EC) patients with exon 3 CTNNB1 mutations. Endometrial cancer patients with exon 3 CTNNB1 mutations tend to be younger in age and have low cancer grades, but they exhibit worse recurrence-free survival than patients with similar clinical characteristics. It is unknown why EC patients with CTNNB1 mutations have worse survival. CTNNB1 encodes the protein β-catenin, which functions primarily at the cell membrane in the adherens junction complex and in the Wnt signaling pathway, where it localizes in the cytoplasm or nucleus. Exon 3 CTNNB1 mutations in EC are classically thought to promote nuclear localization of β-catenin. In this study, we conducted immunohistochemistry for β-catenin localization on 53 EC cases with an exon 3 CTNNB1 mutation from the UNCseq project. Surprisingly, 32 cases had primarily non-nuclear, either cytoplasmic or membranous, localization and 21 cases had primarily nuclear localization. Additionally, we identified 16 cases with intratumoral heterogeneity containing both nuclear and non-nuclear β-catenin localization. To investigate the functional consequences of this differential localization, we performed spatial transcriptomics using the Nanostring GeoMx Whole Transcriptome Analysis. Nuclear and non-nuclear regions of interest (ROI) were selected from 16 CTNNB1 mutant EC cases exhibiting heterogenous β-catenin localization within the same tissue (n=95 total ROIs). We hypothesize that β-catenin nuclear and non-nuclear ROIs will have differing transcriptional outputs in CTNNB1 mutant EC. Differential expression analysis of all ROIs yielded distinct transcriptional profiles based on localization of mutant β-catenin. Gene Set Enrichment Analysis revealed enrichment for Wnt-signaling and the epithelial-to-mesenchymal transition pathways in nuclear ROIs and enrichment for estrogen and androgen signaling pathways in non-nuclear ROIs. Hierarchical clustering of all ROIs yielded two genetically distinct clusters comprised of entirely nuclear or non-nuclear ROIs, with the exception of four ROIs. Higher expression of TACSTD2, encoding the protein Trop2, was enriched in the nuclear ROI cluster. Trop2 is the target of Sacituzumab Govitecan (SG), a drug conjugate with recent success in metastatic triple negative breast cancer patients. Additionally, studies have shown Trop2 to induce nuclear localization of β-catenin in other cancer types. Therefore, we hypothesize that CTNNB1 mutant EC patients with nuclear localization of β-catenin will respond better to SG treatment. To conclude, we established gene expression profiles for areas of nuclear and non-nuclear β-catenin in CTNNB1 mutant EC tissues and identified a novel gene with implications in mechanisms of β-catenin localization which may allow therapeutic targeting of advanced disease. Citation Format: Molly L. Parrish, Andrew B. Gladden, Russell R. Broaddus. Location, location, location: Why cellular localization of mutant β-catenin matters in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR008.

Abstract B004: Investigating loss of ARID1A protein on endometrial cancer progression through the destabilization of adherens junctions

Abstract The purpose of this work is to test the hypothesis that loss of ARID1A promotes endometrial cancer progression by destabilizing tumor cell adherens junctions proteins. ARID1A, an important structural subunit of the SWI/SNF chromatin remodeling complex, is mutated in 26-39% of endometrioid tumors. In endometrial cancer loss of ARID1A has been classically linked to loss of ARID1A expression and has been shown to correlate with increasing tumor grade. However, little is known about the functional impact of ARID1A loss on endometrial cancer progression. A common hallmark of aggressive endometrial cancer is decreased membranous expression of adherens junction proteins, E-Cadherin and β-Catenin. In colorectal and gastric cancer loss of ARID1A expression has been shown to directly downregulate E-Cadherin expression. To determine how loss of ARID1A affects adherens junctions and its impact on endometrial cancer progression we utilized human endometrial cancers and human endometrial cancer cell lines. Using immunohistochemistry, we determined the expression and localization of ARID1A and the adherens junction protein β-Catenin in forty-six human endometrioid type endometrial cancers. Intratumoral heterogeneity of ARID1A expression allowed for quantification of membranous E-Cadherin in relation to presence or absence of ARID1A protein within the same human endometrial tumor. To quantify membranous E-Cadherin we performed immunofluorescence and used line intensity measurements in intratumoral areas of ARID1A presence and ARID1A absence. To assess effects of ARID1A loss on adherens junction in vitro we used short-hairpin RNA to acutely knockdown ARID1A in the endometrial cancer cell lines Ishikawa and ECC-1. Protein expression of ARID1A and E-Cadherin in ARID1A knockdown cells was assessed by Western Blot. Within our cohort of forty-six human endometrial tumors, 75% of tumors with wild-type ARID1A and 45% of tumors with mutated ARID1A had absence of ARID1A protein expression. Due to the discordance between presence of an ARID1A gene mutation and the expression of ARID1A protein, we hypothesize that there are alternative mechanisms of ARID1A protein regulation in endometrial cancer. Additionally, tumors with loss of ARID1A show a trend towards nuclear localization of β-Catenin and have a significant decrease in membranous E-Cadherin expression (P<0.001). However, acute knockdown of ARID1A in Ishikawa and ECC-1 human endometrial cancer cells does not affect total E-Cadherin protein. This suggests that loss of ARID1A may decrease adherens junction stability, as decreased membranous expression of E-Cadherin without total protein change is a common phenotype of destabilized adherens junctions. Thus, we hypothesize that loss of ARID1A destabilizes adherens junctions leading to increased endometrial tumor aggression. Citation Format: Savannah E. Wright, Isurika Weerasinghe, Russell R. Broaddus, Andrew B. Gladden. Investigating loss of ARID1A protein on endometrial cancer progression through the destabilization of adherens junctions [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B004.

SATB1 mediated tumor colonization and β-catenin nuclear localization are associated with colorectal cancer progression

ABSTRACT Colorectal cancer (CRC) is a malignancy with high incidence and poor prognosis. It is urgent to identify valuable biomarkers for early diagnosis and potent therapeutic targets. It has been reported that SATB1 is associated with the malignant progression in CRC. To explore the role of SATB1 in CRC progression and the underlying mechanism, we evaluated the expression of SATB1 in the paired CRC tissues with immunohistochemistry. The results showed that the expression of SATB1 in lymph node metastasis was higher than that in primary lesion, and that in distant organ metastasis was higher than that in primary lesion. The retrospective analysis showed that patients with high expression of SATB1 had a significantly worse prognosis than those with negative and moderate expression. In vitro experiments that employing SATB1 over-expressing and depleted CRC cell lines confirmed that SATB1 contributes to cell proliferation and colonization, while inhibiting cell motility. Furthermore, the tissue immunofluorescence assay, Co-IP and Western blot were conducted to reveal that SATB1 induced translocation of β-catenin and formed a protein complex with it in the nuclei. In conclusion, SATB1 mediated tumor colonization and β-catenin nuclear localization are associated with the malignant progression and poor prognosis of CRC.

LAIR1-mediated resistance of hepatocellular carcinoma cells to T cells through a GSK-3β/β-catenin/MYC/PD-L1 pathway

BackgroundAn increasing number of studies have reported the involvement of oncogenes in the regulation of the immune system. LAIR1 is an immunosuppressive molecule and its role in immune-related diseases has been mainly reported. To date, it is unclear whether LAIR1 in tumor cells is involved in immune regulation. Therefore, the aim of this study was to investigate the role of LAIR1 in the immune microenvironment of hepatocellular carcinoma (HCC) to seek the novel therapeutic discoveries. MethodsTumor Immune Dysfunction and Exclusion database was used to predict the response of LAIR1 expression to immune checkpoint blockade. CD8+ T cells were co-cultured with HCC cells, and the killing efficiency of leukocytes on HCC cells was detected by flow cytometry. Flow cytometry was also used to detect the expression of inhibitory receptors. In addition, Western blot, immunofluorescence, and nucleus/cytoplasm fractionation experiments were performed to explore the molecular mechanisms by which LAIR1 created a suppressive tumor microenvironment. ResultsLAIR1 expression in HCC was associated with worse immune prognosis and T-cell dysfunction. HCC cells overexpressing LAIR1 co-cultured with CD8+ T cells induced exhaustion of latter. Mechanism studies indicated that LAIR1 in HCC cells up-regulated the phosphorylation of β-catenin by inducing the phosphorylation of GSK-3β, leading to the impairment of the expression and the nuclear localization signal of β-catenin. Low β-catenin expression and nuclear localization signal inhibited MYC-mediated PD-L1 expression. Therefore, PD-L1 up-regulated by LAIR1 caused the exhaustion of infiltrating CD8+ T cells in HCC, which aggravated the malignant progression of HCC. ConclusionLAIR1 increased PD-L1 expression through the GSK-3β/β-catenin/MYC/PD-L1 pathway and promoted immune evasion of HCC cells. Targeted inhibition of LAIR1 helped to enhance the immune killing effect of CD8+ T cells in HCC.

Ring Finger 43 Hot-spot Frameshift Mutation G659V in Colorectal Cancer Patients: Report from a Tertiary Cancer Care Hospital in North India.

The Ring Finger 43 (RNF43) is a tumor suppressor gene that negatively regulates the Wnt/β-catenin signaling. The p.G659fs is a recurrent RNF43 C-terminal truncating variant frequent in colorectal cancer (CRC) patients. We aimed to identify this hotspot variant in CRC patients and assessed the relationship between the mutation, clinical characteristics, and tumor β-catenin localization. Formalin-fixed, paraffin-embedded tissue samples of upfront, surgically resected, sporadic colorectal adenocarcinoma cases were selected. The p.G659fs mutation was determined by capillary sequencing with sequence-specific primers. Tissue microarray and immunohistochemistry were employed to analyze nuclear β-catenin expression and the expression of mismatch repair (MMR) proteins, respectively. In addition, clinical details were retrieved from the hospital medical records and data were analyzed. The RNF43 p.G659fs mutation was observed in 8% of CRC patients. In total, 25% of tumors showed a loss of immunostaining for one or more MMR proteins and 14.6% of tumors showed positive nuclear β-catenin staining. The p.G659fs variant was significantly enriched in MMR-deficient tumors (P = 0.04). Importantly, no correlation was observed between the variant and nuclear β-catenin localization (P = 0.48), indicating a Wnt-independent role of this variant in CRC tumors. To the best of our knowledge, this is the first study from North India to show the involvement of RNF43 p.G659fs variant in CRC patients. The mutation correlated with MMR protein deficiency and seems to be conferring tumorigenicity independent of the Wnt pathway.

Kindlin-2 regulates colonic cancer stem-like cells survival and self-renewal via Wnt/β-catenin mediated pathway

BackgroundCancer Stem Cells (CSCs) have emerged as a critical mediator in recurrence and resistance in cancers. Kindlin-isoform (1 and 2) binds with cytoplasmic β-tail of integrin and are essential co-activators of integrin function. Given their important function in regulating cancer hallmarks such as cell proliferation, invasion, migration, and metastasis, we hypothesize that it might play a critical role in CSC growth, survival, and self-renewal of colon cancer. Materials and methodsUsing knockdown approaches, we inhibited Kindlin-2 expression in HCT116 and HT29 colon cancer cells. Extreme limiting dilution and self-renewal assay were performed to measure the role of Kindlin in colonic CSC. Standard methods such as qRT-PCR and western blotting were carried out to understand the signaling cascade by which Kindlin regulates CSC marker expression and downstream targets. ResultsOur data show isoform-specific upregulation of Kindlin-2 in colonic CSCs. The silencing of Kindlin-2 reduces colonosphere formation, decreases CSC size, and self-renewal marker genes such as CD-133, CXCR-4, LGR-5, and C-MYC. Kindlin-2 silencing reduces colonosphere proliferation, invasion, and migration of colonic CSCs. Mechanistically, Kindlin-2 silencing reduces the expression, and nuclear localization of β-catenin, and decreases β-catenin target genes such as C-MYC, cyclin D1, DKK-1, and Snail-1. ConclusionOur study delineates the isoform-specific activity of Kindlin-2 in regulating Colonic CSC. Isoform-specific targeting of Kindlin-2 may be a novel strategy to tackle this devastating disease.

Synthesis and Antitumor Activity of Brominated-Ormeloxifene (Br-ORM) against Cervical Cancer.

Aberrant regulation of β-catenin signaling is strongly linked with cancer proliferation, invasion, migration, and metastasis, thus, small molecules that can inhibit this pathway might have great clinical significance. Our molecular modeling studies suggest that ormeloxifene (ORM), a triphenylethylene molecule that docks with β-catenin, and its brominated analogue (Br-ORM) bind more effectively with relatively less energy (-7.6 kcal/mol) to the active site of β-catenin as compared to parent ORM. Herein, we report the synthesis and characterization of a Br-ORM by NMR and FTIR, as well as its anticancer activity in cervical cancer models. Br-ORM treatment effectively inhibited tumorigenic features (cell proliferation and colony-forming ability, etc.) and induced apoptotic death, as evident by pronounced PARP cleavage. Furthermore, Br-ORM treatment caused cell cycle arrest at the G1-S phase. Mechanistic investigation revealed that Br-ORM targets the key proteins involved in promoting epithelial-mesenchymal transition (EMT), as demonstrated by upregulation of E-cadherin and repression of N-cadherin, Vimentin, Snail, MMP-2, and MMP-9 expression. Br-ORM also represses the expression and nuclear subcellular localization of β-catenin. Consequently, Br-ORM treatment effectively inhibited tumor growth in an orthotopic cervical cancer xenograft mouse model along with EMT associated changes as compared to vehicle control-treated mice. Altogether, experimental findings suggest that Br-ORM is a novel, promising β-catenin inhibitor and therefore can be harnessed as a potent anticancer small molecule for cervical cancer treatment.

Evaluation of potential targets to enhance the sensitivity of cholangiocarcinoma cells to anticancer drugs

BackgroundCholangiocarcinoma (CCA) is a highly lethal cancer originated in the biliary tree. Available treatments for CCA are scarcely effective, partly due to mechanisms of chemoresistance, such as aberrant activation of Wnt/β-catenin pathway and dysfunctional p53. AimTo evaluate the impact of enhancing the expression of negative regulators of the Wnt/β-catenin pathway (AXIN1, AXIN2, and GSK3B) and the tumor suppressor gene TP53. MethodsGene expression in paired samples of CCA and adjacent non-tumor liver tissue was determined by RT-qPCR and immunohistochemistry (IHC). Using lentiviral vectors, CCA cells were transduced with genes of interest to assess their impact on the resistome (TLDA), apoptosis (annexin V/propidium iodide), and decreased cell viability (MTT). ResultsIHC revealed marked nuclear localization of β-catenin, consistent with Wnt/β-catenin pathway activation. In silico analysis with data from TCGA showed heterogeneous down-regulation of AXIN1, AXIN2, and GSK3B in CCA. Enhancing the expression of AXIN1, AXIN2, and GSK3B in CCA cells was not enough to block the activity of this signaling pathway or significantly modify resistance to 5-FU, gemcitabine, and platinated drugs. Consistent with impaired p53 function, CDKN1A was down-regulated in CCA. Forced TP53 expression induced p21 up-regulation and reduced cell proliferation. Moreover, the resistome was modified (FAS, BAX, TYMP, and CES2 up-regulation along with DHFR, RRM1, and BIRC5 down-regulation), which was accompanied by enhanced sensitivity to some antitumor drugs, mainly platinated drugs. ConclusionEnhancing TP53 expression, but not that of AXIN1, AXIN2, and GSK3B, in CCA cells may be a useful strategy to sensitize CCA to antitumor drugs.

FAT4 overexpression promotes antitumor immunity by regulating the β-catenin/STT3/PD-L1 axis in cervical cancer

BackgroundFAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation and metastasis. The Wnt/β-catenin pathway activation is highly associated with PD-L1-associated tumor immune escape. Here, we report the mechanism by which FAT4 overexpression regulates anti-tumor immunity in cervical cancer by inhibiting PD-L1 N-glycosylation and cell membrane localization in a β-catenin-dependent manner.MethodsFAT4 expression was first detected in cervical cancer tissues and cell lines. Cell proliferation, clone formation, and immunofluorescence were used to determine the tumor suppressive impact of FAT4 overexpression in vitro, and the findings were confirmed in immunodeficient and immunocomplete mice xenografts. Through functional and mechanistic experiments in vivo and in vitro, we investigated how FAT4 overexpression affects the antitumor immunity via the β-catenin/STT3/PD-L1 axis.ResultsFAT4 is downregulated in cervical cancer tissues and cell lines. We determined that FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1). FAT4 overexpression decreases programmed death-ligand 1 (PD-L1) mRNA expression at the transcriptional level, and causes aberrant glycosylation of PD-L1 via STT3A at the post-translational modifications (PTMs) level, leading to its endoplasmic reticulum (ER) accumulation and polyubiquitination-dependent degradation. We found that FAT4 overexpression promotes aberrant PD-L1 glycosylation and degradation in a β-catenin-dependent manner, thereby increasing cytotoxic T lymphocyte (CTL) activity in immunoreactive mouse models.ConclusionsThese findings address the basis of Wnt/β-catenin pathway activation in cervical cancer and provide combination immunotherapy options for targeting the FAT4/β-catenin/STT3/PD-L1 axis.Graphical Schematic cartoons showing the antitumor immunity mechanism of FAT4. (left) when Wnts bind to their receptors, which are made up of Frizzled proteins and LRP5/6, the cytoplasmic protein DVL is activated, inducing the aggregation of degradation complexes (AXIN, GSK3β, CK1, APC) to the receptor. Subsequently, stable β-catenin translocates into the nucleus and binds to TCF/LEF and TCF7L2 transcription factors, leading to target genes transcription. The catalytically active subunit of oligosaccharyltransferase, STT3A, enhances PD-L1 glycosylation, and N-glycosylated PD-L1 translocates to the cell membrane via the ER-to-Golgi pathway, resulting in immune evasion. (Right) FAT4 exerts antitumor immunity mainly through following mechanisms: (i) FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1); (ii) FAT4 inhibits PD-L1 and STT3A transcription in a β-catenin-dependent manner and induces aberrant PD-L1 glycosylation and ubiquitination-dependent degradation; (iii) Promotes activation of cytotoxic T lymphocytes (CTL) and infiltration into the tumor microenvironment.

CTNNB1 and APC Mutations in Sinonasal Myxoma : Expanding the Spectrum of Tumors Driven By WNT/β-catenin Pathway.

Sinonasal myxoma (SNM) is a rare, benign mesenchymal neoplasm with distinct clinicopathologic features and aberrant nuclear localization of β-catenin by immunohistochemistry. The molecular underpinnings have been linked to that of a "myxoid variant" of desmoid fibromatosis. Herein, we describe a series of 8 cases of SNM and propose clinical and biologic differences compared with desmoid fibromatosis. Our patient cohort is comprised of 5 males and 3 females (age range: 10mo to 12y), 6 of whom are aged less than or equal to 24 months. All presented with facial swelling, reflecting lesions involving the maxillary bone, and all underwent resection. All tumors were variably cellular and comprised of bland spindled to stellate cells in a profusely myxoid background with diffuse nuclear β-catenin expression. All cases of SNM were analyzed by next-generation sequencing using the Oncopanel assay. Three cases failed sequencing, 2 of 5 successful cases exhibited exon 3 CTNNB1 alterations involving the ubiquitin recognition motif, and 3 had adenomatous polyposis coli ( APC ) deletions. One patient had APC germline testing which was negative. No germline testing was available for the remaining 7 patients. Follow-up data over a range of 1 month to 23 years was available for 7 of the 8 SNMs. One case patient had local recurrence, and all were alive without evidence of disease. This is in contrast to the high recurrence rate typically seen in desmoid fibromatosis, particularly after resection. Our findings expand the spectrum of tumors with underlying WNT/β-catenin pathway and highlight the histologic, clinical, and genetic differences of SNM compared with desmoid fibromatosis. APC deletion raises the possibility of underlying germline alteration and familial adenomatous polyposis.

LncRNA DGCR5-encoded polypeptide RIP aggravates SONFH by repressing nuclear localization of β-catenin in BMSCs

The differentiation fate of bone marrow mesenchymal stem cells (BMSCs) affects the progression of steroid-induced osteonecrosis of the femoral head (SONFH). We find that lncRNA DGCR5 encodes a 102-amino acid polypeptide, RIP (Rac1 inactivated peptide), which promotes the adipogenic differentiation of BMSCs and aggravates the progression of SONFH. RIP, instead of lncRNA DGCR5, binds to the N-terminal motif of RAC1, and inactivates the RAC1/PAK1 cascade, resulting in decreased Ser675 phosphorylation of β-catenin. Ultimately, the nuclear localization of β-catenin decreases, and the differentiation balance of BMSCs tilts toward the adipogenesis lineage. In the femoral head of rats, overexpression of RIP causes trabecular bone disorder and adipocyte accumulation, which can be rescued by overexpressing RAC1. This finding expands the regulatory role of lncRNAs in BMSCs and suggests RIP as a potential therapeutic target.

Neuropilin-1 promotes mitochondrial structural repair and functional recovery in rats with cerebral ischemia

ObjectivesAvailable literature documents that ischemic stroke can disrupt the morphology and function of mitochondria and that the latter in other disease models can be preserved by neuropilin-1 (NRP-1) via oxidative stress suppression. However, whether NRP-1 can repair mitochondrial structure and promote functional recovery after cerebral ischemia is still unknown. This study tackled this very issue and explored the underlying mechanism.MethodsAdeno-associated viral (AAV)-NRP-1 was stereotaxically inoculated into the cortex and ipsilateral striatum posterior of adult male Sprague-Dawley (SD) rats before a 90-min transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion. Lentivirus (LV)-NRP-1 was transfected into rat primary cortical neuronal cultures before a 2-h oxygen-glucose deprivation and reoxygenation (OGD/R) injury to neurons. The expression and function of NRP-1 and its specific protective mechanism were investigated by Western Blot, immunofluorescence staining, flow cytometry, magnetic resonance imaging, transmission electron microscopy, etc. The binding was detected by molecular docking and molecular dynamics simulation.ResultsBoth in vitro and in vivo models of cerebral ischemia/reperfusion (I/R) injury presented a sharp increase in NRP-1 expression. The expression of AAV-NRP-1 markedly ameliorated the cerebral I/R-induced damage to the motor function and restored the mitochondrial morphology. The expression of LV-NRP-1 alleviated mitochondrial oxidative stress and bioenergetic deficits. AAV-NRP-1 and LV-NRP-1 treatments increased the wingless integration (Wnt)-associated signals and β-catenin nuclear localization. The protective effects of NRP-1 were reversed by the administration of XAV-939.ConclusionsNRP-1 can produce neuroprotective effects against I/R injury to the brain by activating the Wnt/β-catenin signaling pathway and promoting mitochondrial structural repair and functional recovery, which may serve as a promising candidate target in treating ischemic stroke.

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance.

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Abstract LB016: Immunotherapeutic potential of ST316, a peptide antagonist of β-catenin

Abstract The transcription factor β-catenin is a key player in many cellular processes, including stem cell renewal, cellular homeostasis and inflammation. Deregulation of β-catenin occurs frequently in several cancer types by mutation or overexpression of the β-catenin gene or mutation of negative regulators such as Adenomatous Polyposis Coli (APC). ST316 is a novel peptide antagonist that specifically interferes with the association of β-catenin with its co-activator BCL9, disrupting β-catenin nuclear localization and attenuating target gene expression. In an orthotopic triple-negative breast cancer (TNBC) model, ST316 demonstrates potent anti-tumor activity, resulting in 84% tumor growth inhibition (TGI) (p<0.001 vs. vehicle control). In addition to its cell-autonomous mechanisms, deregulated Wnt/β-catenin signaling can promote tumorigenesis by impacting the tumor microenvironment. Reprogramming of immunosuppressive M2-like Tumor Associated Macrophages (TAMs) toward an immune-promoting program (M1-like) is an attractive cancer immunotherapeutic strategy. Here we explored ST316 potential for macrophage repolarization toward the M1-like phenotype, activation of cytotoxic T-cells in macrophage/T-cell co-culture assays and cooperation of ST316 with anti-PD-1 to enhance anti-tumor activity in vivo. Initial studies demonstrate that human macrophages derived from Peripheral Blood Mononuclear Cells (hPBMCs) and subsequently committed to the M2-like identity are reprogrammed toward an M1-like phenotype upon ST316 exposure. ST316 treatment dose-dependently suppressed expression of the M2 marker CD163 by flow cytometry and quantitative PCR, resulting in 100-fold increase in the M1/M2 ratio without substantial impact on cell viability. Importantly, T-cell viability and activation markers associated with M1-state (CD80, CD86) were not affected by ST316 at the concentrations used in these studies. Further, in co-cultures of M2 macrophage with T cells, ST316 exposure resulted in a three-fold increase in T-cell activation compared to control M2/T cell co-cultures, as measured by intracellular IFN-γ staining. Finally, in an orthotopic TNBC model in vivo, subtherapeutic ST316 enhanced the anti-tumor activity of anti-PD-1 [85% TGI with combination, compared to 51% TGI with anti-PD1 alone (p<0.01) and -9% TGI with subtherapeutic ST316 alone (p<0.001)]. Anti-tumor activity was accompanied by an increase the M1/M2 ratio. Overall, these results support the immuno-therapeutic potential of ST316 and extend the application range of ST316 to include Wnt-driven cancers with poor clinical response to immune checkpoint blockade and other immunotherapeutic agents. Citation Format: Claudio Scuoppo, Lila Ghamsari, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein-Haras, Jim Rotolo. Immunotherapeutic potential of ST316, a peptide antagonist of β-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB016.

Suppression of preadipocyte determination by SOX4 limits white adipocyte hyperplasia in obesity.

Preadipocyte determination expanding the pool of preadipocytes is a vital process in adipocyte hyperplasia, but the molecular mechanisms underlying this process are yet to be elucidated. Herein, SRY-related HMG box transcription factor 4 (SOX4) was identified as a critical target in response to BMP4- and TGFβ-regulated preadipocyte determination. SOX4 deficiency is sufficient to promote preadipocyte determination in mesenchymal stem cells (MSCs) and acquisition of preadipocyte properties in nonadipogenic lineages, while its overexpression impairs the adipogenic capacity of preadipocytes and converts them into nonadipogenic lineages. Mechanism studies indicated that SOX4 activates and cooperates with LEF1 to retain the nuclear localization of β-catenin, thus mediating the crosstalk between TGFβ/BMP4 signaling pathway and Wnt signaling pathway to regulate the preadipocyte determination. Invivo studies demonstrated that SOX4 promotes the adipogenic-nonadipogenic conversion and suppresses the adipocyte hyperplasia. Together, our findings highlight the importance of SOX4 in regulating the adipocyte hyperplasia in obesity.

The endocannabinoid system promotes hepatocyte progenitor cell proliferation and maturation by modulating cellular energetics

The proliferation and differentiation of hepatic progenitor cells (HPCs) drive the homeostatic renewal of the liver under diverse conditions. Liver regeneration is associated with an increase in Axin2+Cnr1+ HPCs, along with a marked increase in the levels of the endocannabinoid anandamide (AEA). But the molecular mechanism linking AEA signaling to HPC proliferation and/or differentiation has not been explored. Here, we show that in vitro exposure of HPCs to AEA triggers both cell cycling and differentiation along with increased expression of Cnr1, Krt19, and Axin2. Mechanistically, we found that AEA promotes the nuclear localization of the transcription factor β-catenin, with subsequent induction of its downstream targets. Systemic analyses of cells after CRISPR-mediated knockout of the β-catenin-regulated transcriptome revealed that AEA modulates β-catenin-dependent cell cycling and differentiation, as well as interleukin pathways. Further, we found that AEA promotes OXPHOS in HPCs when amino acids and glucose are readily available as substrates, but AEA inhibits it when the cells rely primarily on fatty acid oxidation. Thus, the endocannabinoid system promotes hepatocyte renewal and maturation by stimulating the proliferation of Axin2+Cnr1+ HPCs via the β-catenin pathways while modulating the metabolic activity of their precursor cells.

The role of β-catenin in cardiac diseases.

The Wnt/β-catenin signaling pathway is a classical Wnt pathway that regulates the stability and nuclear localization of β-catenin and plays an important role in adult heart development and cardiac tissue homeostasis. In recent years, an increasing number of researchers have implicated the dysregulation of this signaling pathway in a variety of cardiac diseases, such as myocardial infarction, arrhythmias, arrhythmogenic cardiomyopathy, diabetic cardiomyopathies, and myocardial hypertrophy. The morbidity and mortality of cardiac diseases are increasing, which brings great challenges to clinical treatment and seriously affects patient health. Thus, understanding the biological roles of the Wnt/β-catenin pathway in these diseases may be essential for cardiac disease treatment and diagnosis to improve patient quality of life. In this review, we summarize current research on the roles of β-catenin in human cardiac diseases and potential inhibitors of Wnt/β-catenin, which may provide new strategies for cardiac disease therapies.