Phosphorylation Of Β-catenin Research Articles

Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell-types. NDRG1 overexpression decreased β-catenin and down-regulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 that are classically induced by GSK-3β were significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression up-regulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. In fact, PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. In fact, the association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the down-regulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), that is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that up-regulate PKCα and inhibit WNT signaling.

WITHDRAWN: Recombinant osteopontin stabilizes the blood brain barrier following subarachnoid hemorrhage in rats

Background and PurposeRecombinant osteopontin (rOPN) decreases neuronal cell death and brain edema and improves the neurological scores following subarachnoid hemorrhage (SAH) in rats. The molecular mechanisms of the preservation of blood-brain barrier of rOPN following SAH in rats were examined in this study. MethodsAdult rats were divided into 3 groups: sham, vehicle, and treatment groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the brains was harvested at 24 hours for analysis. For the mechanism part of the study two additional groups were operated and treated with Fib-14 and Wortmannin additional to rOPN. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 hours after SAH induction. The blood brain barrier (BBB) permeability was evaluated using Evans Blue dye extravasation. The expressions of tight junction proteins claudin-3, claudin-5, occludin and the phosphorylation of β-catenin were examined using Western blot analysis. ResultsrOPN improved neurological scores and reduced brain edema at 24 hours after SAH. The BBB preservation action of rOPN is mediated in part through stabilizing β-catenin and increasing expression of tight junction proteins, claudin-3, claudin-5, and occludin. rOPN is thought to exert its effects through FAK activated PI3K-Akt pathway. ConclusionSince BBB disruption is one of the major pathophysiology of EBI after SAH, rOPN may be a promising drug to maintain BBB integrity and functions in SAH treatment.

β-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells

β-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular β-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of β-catenin, suggestive of a Nek10 role in the control of β-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for β-catenin degradation, where it phosphorylates β-catenin at Tyr30, located within the regulatory region governing β-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of β-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.

BMP Stimulation Differentially Affects Phosphorylation and Protein Stability of β-Catenin in Breast Cancer Cell Lines.

Increased expression and nuclear translocation of β-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting β-CATENIN are not as efficient as desired. Therefore, detailed understanding of β-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing β-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of β-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce β-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces β-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on β-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced β-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding β-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.

Abstract LB188: PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC

Abstract Triple negative breast cancer (TNBC) is a subtype of breast cancer that is defined by a lack of estrogen receptor, progesterone receptor, and HER2-neu gene product expression. Previously, we found that TNBC cells with phosphorylated PEA-15 adaptor protein exhibited decreased clonogenic and anchorage-independent growth in vitro, and decreased growth and lung metastasis in vivo. Further, we found lower levels of phosphorylated β-catenin at S675 in PEA-15-AA (non-phosphorylatable) mutants than in PEA-15-DD (phosphomimetic) mutants. We therefore hypothesized that phosphorylated PEA-15 enhances the aggressiveness of TNBC by regulating the phosphorylation of β-catenin at S675. We first screened a panel of TNBC cells for β-catenin levels via western blot. Next to assess the biological role of β-catenin we knocked down its expression in TNBC cells (SUM149, BT549). In the β-catenin knockouts, proliferation decreased by 20%(P<0.0001) mammosphere formation by 69% (P<0.001), and clonogenic growth by 65% (P<0.0001), when compared to the vector control. To determine if PEA-15 mediates pro-tumor effects by phosphorylating β-catenin at S675, we created a nonphosphorylatable (S675A) and a phosphomimetic (S675D) form of β-catenin. We transfected BT549 cells with S675A and observed a decrease of 26% (P=0.011) in proliferation, 65% (P<0.001) in mammospheres, and 54% (P=0.0001) in clonogenic formation when compared with WT β-catenin transfected cells. Next, we transfected SUM149 cells with S675D and found an increase of 58% (P<0.001) in proliferation, 79% (P=0.001) in mammospheres, and 23% (P=0.0303) in clonogenic formation when compared with WT β-catenin transfected cells. To test if PEA-15 phosphorylation mediates β-catenin activity, we transfected the S675A and S675D form of β-catenin into MDA-MB-468 cell lines which stably express PEA-15-AA and PEA-15-DD. When we performed a rescue experiment with PEA-15-AA cells transfected with S675D we found a 78% (P=0.0008) increase in mammospheres when compared to vector transfected cells. Conversely, when PEA-15-DD cells were transfected with S675A we found a 53% (P=0.0004) decrease in mammospheres when compared to vector transfected cells. Next, we did a multiplex staining of a tissuemicroarray (n= 102 human TNBC tumors). We observed that in patients with grade 3 tumors, the percentages of cells expressing total PEA-15(P=0.005), P-PEA-15-S116(P=0.038), and p-β-catenin (P=0.0005) were significantly higher than those in patients with grade 2 tumors. Together, these results show that PEA-15 phosphorylation mediates tumor aggressiveness in TNBC by promoting β-catenin activity. Future studies will evaluate the clinical significance of PEA-15 and β-catenin and their phosphorylated forms in TNBC patients. These results will establish β-catenin and PEA-15 phosphorylation as potential markers of, and targets against TNBC progression. Citation Format: Alex W. Tan, Mohd Mughees, Wendy A. Woodward, Debu Tripathy, Natalie Fowlkes, Chandra Bartholomeusz. PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB188.

FZD7-Targeted Nanoparticles to Enhance Doxorubicin Treatment of Triple-Negative Breast Cancer.

Doxorubicin (DOX) is a chemotherapy agent commonly used to treat triple-negative breast cancer (TNBC), but it has insufficient efficacy against the disease and considerable toxicity due to its off-target delivery. To improve the specificity of DOX for TNBC, we encapsulated it in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) coated with antibodies against Frizzled7 (FZD7), a receptor that is overexpressed on TNBC cells and which is a key activator of the Wnt signaling pathway. In vitro studies show that DOX encapsulation does not hinder its ability to localize to the nucleus in human TNBC cell cultures and that DOX delivered via NPs induces apoptosis and DNA damage via H2A.X phosphorylation to the same degree as freely delivered DOX. FZD7-targeted NPs delivering DOX caused significantly greater inhibition of metabolic activity and led to a smaller cell population following treatment when compared to freely delivered DOX or DOX-loaded NPs coated only with poly(ethylene glycol) (PEG). The FZD7 antibodies additionally provided significant levels of Wnt pathway inhibition, as demonstrated by an increase in β-catenin phosphorylation, indicative of β-catenin destruction and downregulation. These results show that FZD7-targeted platforms have great promise for improving the therapeutic window of otherwise toxic chemotherapies like DOX in TNBC and other cancers that display the overexpression of FZD7 receptors.

Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR/HDAC9

BackgroundDiabetic angiogenesis is closely associated with disabilities and death caused by diabetic microvascular complications. Advanced glycation end products (AGEs) are abnormally accumulated in diabetic patients and are a key pathogenic factor for diabetic angiogenesis. The present study focuses on understanding the mechanisms underlying diabetic angiogenesis and identifying therapeutic targets based on these mechanisms.MethodsIn this study, AGE-induced angiogenesis serves as a model to investigate the mechanisms underlying diabetic angiogensis. Mouse aortic rings, matrigel plugs, and HUVECs or 293T cells were employed as research objects to explore this pathological process by using transcriptomics, gene promoter reporter assays, virtual screening and so on.ResultsHere, we found that AGEs activated Wnt/β-catenin signaling pathway and enhanced the β-catenin protein level by affecting the expression of β-catenin degradation-related genes, such as FZDs (Frizzled receptors), LRPs (LDL Receptor Related Proteins), and AXIN1. AGEs could also mediate β-catenin Y142 phosphorylation through VEGFR1 isoform5. These dual effects of AGEs elevated the nuclear translocation of β-catenin and sequentially induced the expression of KDR (Kinase Insert Domain Receptor) and HDAC9 (Histone Deacetylase 9) by POU5F1 and NANOG, respectively, thus mediating angiogenesis. Finally, through virtual screening, Bioymifi, an inhibitor that blocks VEGFR1 isoform5-β-catenin complex interaction and alleviates AGE-induced angiogenesis, was identified.ConclusionCollectively, this study offers insight into the pathophysiological functions of β-catenin in diabetic angiogenesis.

SAA1 regulated by S1P/S1PR1 promotes the progression of ESCC via β-catenin activation

Serum amyloid A1 (SAA1), an inflammation-related molecule, is associated with the malignant progression of many tumors. This study aimed to investigate the role of SAA1 in the progression of esophageal squamous cell carcinoma (ESCC) and its molecular mechanisms. The expression of SAA1 in ESCC tissues and cell lines was analyzed using bioinformatics analysis, western blotting, and reverse transcription-quantitative PCR (RT‒qPCR). SAA1-overexpressing or SAA1-knockdown ESCC cells were used to assess the effects of SAA1 on the proliferation, migration, apoptosis of cancer cells and the growth of xenograft tumors in nude mice. Western blotting, immunofluorescence and RT‒qPCR were used to investigate the relationship between SAA1 and β-catenin and SAA1 and sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 1 (S1PR1). SAA1 was highly expressed in ESCC tissues and cell lines. Overexpression of SAA1 significantly promoted the proliferation, migration and the growth of tumors in nude mice. Knockdown of SAA1 had the opposite effects and promoted the apoptosis of ESCC cells. Moreover, SAA1 overexpression promoted the phosphorylation of β-catenin at Ser675 and increased the expression levels of the β-catenin target genes MYC and MMP9. Knockdown of SAA1 had the opposite effects. S1P/S1PR1 upregulated SAA1 expression and β-catenin phosphorylation at Ser675 in ESCC cells. In conclusion, SAA1 promotes the progression of ESCC by increasing β-catenin phosphorylation at Ser675, and the S1P/S1PR1 pathway plays an important role in its upstream regulation.

Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/β-catenin pathway, with significant downregulation of both Ser 675-phosphorylated β-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and β-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/β-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

GTP binding protein 2 maintains the quiescence, self-renewal, and chemoresistance of mouse colorectal cancer stem cells via promoting Wnt signaling activation

Colorectal cancer (CRC) is one of the most common cancers and the second most deadly cancer across the globe. Colorectal cancer stem cells (CCSCs) fuel CRC growth, metastasis, relapse, and chemoresistance. A complete understanding of the modulatory mechanisms of CCSC biology is essential for developing efficacious CRC treatment. In the current study, we characterized the expression and function of GTP binding protein 2 (GTPBP2) in a chemical-induced mouse CRC model. We found that GTPBP2 was expressed at a higher level in CD133+CD44+ CCSCs compared with other CRC cells. Using a lentivirus-based Cas9/sgRNA system, GTPBP2 expression was ablated in CRC cells in vitro. GTPBP2 deficiency caused the following effects on CCSCs: 1) Significantly accelerating proliferation and increasing the proportions of cells at G1, S, and G2/M phase; 2) Impairing resistance to 5-Fluorouracil; 3) Weakening self-renewal but not impacting cell migration. In addition, GTPBP2 deficiency remarkably decreased β-catenin expression while increasing β-catenin phosphorylation in CCSCs. These effects of GTPBP2 were present in CCSCs but not in other CRC cell populations. The Wnt agonist SKL2001 completely abolished these changes in GTPBP2-deficient CCSCs. When GTPBP2-deficient CCSCs were implanted in nude mice, they exhibited consistent changes compared with GTPBP2-expressing CCSCs. Collectively, this study indicates that GTPBP2 positively modulates Wnt signaling to reinforce the quiescence, self-renewal, and chemoresistance of mouse CCSCs. Therefore, we disclose a novel mechanism underlying CCSC biology and GTPBP2 could be a therapeutic target in future CRC treatment.

Dual-inhibition of NAMPT and PAK4 induces anti-tumor effects in 3D-spheroids model of platinum-resistant ovarian cancer

Ovarian cancer follows a characteristic progression pattern, forming multiple tumor masses enriched with cancer stem cells (CSCs) within the abdomen. Most patients develop resistance to standard platinum-based drugs, necessitating better treatment approaches. Targeting CSCs by inhibiting NAD+ synthesis has been previously explored. Nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway for NAD+ synthesis is an attractive drug target in this pathway. KPT-9274 is an innovative drug targeting both NAMPT and p21 activated kinase 4 (PAK4). However, its effectiveness against ovarian cancer has not been validated. Here, we show the efficacy and mechanisms of KPT-9274 in treating 3D-cultured spheroids that are resistant to platinum-based drugs. In these spheroids, KPT-9274 not only inhibited NAD+ production in NAMPT-dependent cell lines, but also suppressed NADPH and ATP production, indicating reduced mitochondrial function. It also downregulated of inflammation and DNA repair-related genes. Moreover, the compound reduced PAK4 activity by altering its mostly cytoplasmic localization, leading to NAD+-dependent decreases in phosphorylation of S6 Ribosomal protein, AKT, and β-Catenin in the cytoplasm. These findings suggest that KPT-9274 could be a promising treatment for ovarian cancer patients who are resistant to platinum drugs, emphasizing the need for precision medicine to identify the specific NAD+ producing pathway that a tumor relies upon before treatment.

The effect of novel nitrogen-based chalcone analogs on colorectal cancer cells: Insight into the molecular pathways

In colorectal cancer (CRC), aberrations in KRAS are associated with aggressive tumorigenesis and an overall low survival rate because of chemoresistance and adverse effects. Ergo, complementary, and integrative medicines are being considered for CRC treatment. Among which is the use of natural chalcones that are known to exhibit anti-tumor activities in KRAS mutant CRC subtypes treatment regimens. Consequently, we examine the effect of two novel compounds (DK13 and DK14) having chalcones with nitrogen mustard moiety on CRC cell lines (HCT-116 and LoVo) with KRAS mutation. These compounds were synthesized in our lab and previously reported to exhibit potent activity against breast cancer cells. Our data revealed that DK13 and DK14 treatment suppress cell growth, disturb the progression of cell cycle, and trigger apoptosis in CRC cell lines. Besides, treatment with both compounds impedes cell invasion and colony formation in both cell lines as compared to 5-FU; this is accompanied by up and down regulations of E-cadherin and Vimentin, respectively. At the molecular level, both compounds deregulate the expression and phosphorylation of β-catenin, Akt and mTOR, which are the main likely molecular mechanisms underlying these biological occurrences. Our findings present DK13 and DK14 as novel chemotherapies against CRC, through β-catenin/Akt/mTOR signaling pathways.

MiR-557 inhibits hepatocellular carcinoma progression through Wnt/β-catenin signaling pathway by targeting RAB10.

Accumulating evidence suggests that aberrant miRNAs participate in carcinogenesis and progression of hepatocellular carcinoma (HCC). Abnormal miR-557 expression is reported to interfere with the progression of several human cancers. However, the potential roles of miR-557 in HCC remain largely unknown. In the current study, we found that miR-557 was down-regulated in HCC tissues and cell lines, and was closely related to recurrence and metastasis of HCC. Notably, overexpression of miR-557 inhibited proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT) progression, blocked cells in G0/G1 phase of MHCC-97H cells in vitro, and suppressed tumor growth in vivo. However, loss of miR-557 facilitated these parameters in Huh7 cells both in vitro and in vivo. Moreover, RAB10 was identified as a direct downstream target of miR-557 through its 3'-UTR. Furthermore, RAB10 re-expression or knockdown partially abolished the effects of miR-557 on proliferation, migration, invasion, and EMT progression of HCC cells. Mechanistically, overexpression of miR-557 suppressed Wnt/β-catenin signaling by inhibiting GSK-3β phosphorylation, increasing β-catenin phosphorylation, and decreasing β-catenin transport to the nucleus, while knockdown of miR-557 activated Wnt/β-catenin signaling. Moreover, the TOP/FOP-Flash reporter assays showed that miR-557 overexpression or knockdown significantly suppressed or activated Wnt signaling activity, respectively. Additionally, low expression of miR-557 and high expression of RAB10 in HCC tissues was closely associated with tumor size, degree of differentiation, TNM stage and poor prognosis in HCC patients. Taken together, these results demonstrate that miR-557 blocks the progression of HCC via the Wnt/β-catenin pathway by targeting RAB10.

Tmem119 is involved in bone anabolic effects of PTH through enhanced osteoblastic bone formation in mice

The intermittent administration of parathyroid hormone (PTH) exerts potent bone anabolic effects, which increase bone mineral density (BMD) and reduce fracture risk in osteoporotic patients. However, the underlying mechanisms remain unclear. Tmem119 has been proposed as a factor that is closely linked to the osteoblast phenotype, and we previously reported that PTH enhanced the expression of Tmem119 in mouse osteoblastic cells. However, roles of Tmem119 in the bone anabolic effects of PTH in vivo remain unknown. We herein investigated the roles of Tmem119 in bone anabolic effects of PTH using Tmem119-deficient mice. Tmem119 deficiency significantly reduced PTH-induced increases in trabecular bone volume and cortical BMD of femurs. Effects of Tmem119 deficiency on bone mass seemed predominant in female mice. Histomorphometric analyses with calcein labeling showed that Tmem119 deficiency significantly attenuated PTH-induced increases in the rates of bone formation and mineralization as well as numbers of osteoblasts. Moreover, Tmem119 deficiency significantly blunted PTH-induced decreases in phosphorylation of β-catenin and increases in alkaline phosphatase activity in osteoblasts. In conclusion, the present results indicate that Tmem119 is involved in bone anabolic effects of PTH through osteoblastic bone formation partly related to canonical Wnt-β-catenin signaling in mice.

APC mutations disrupt β-catenin destruction complex condensates organized by Axin phase separation

The Wnt/β-catenin pathway is critical to maintaining cell fate decisions. Recent study showed that liquid–liquid-phase separation (LLPS) of Axin organized the β-catenin destruction complex condensates in a normal cellular state. Mutations inactivating the APC gene are found in approximately 80% of all human colorectal cancer (CRC). However, the molecular mechanism of the formation of β-catenin destruction complex condensates organized by Axin phase separation and how APC mutations impact the condensates are still unclear. Here, we report that the β-catenin destruction complex, which is constructed by Axin, was assembled condensates via a phase separation process in CRC cells. The key role of wild-type APC is to stabilize destruction complex condensates. Surprisingly, truncated APC did not affect the formation of condensates, and GSK 3β and CK1α were unsuccessfully recruited, preventing β-catenin phosphorylation and resulting in accumulation in the cytoplasm of CRCs. Besides, we propose that the phase separation ability of Axin participates in the nucleus translocation of β-catenin and be incorporated and concentrated into transcriptional condensates, affecting the transcriptional activity of Wnt signaling pathway.

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.

A mosaic variant in CTNNB1/β-catenin as a novel cause for osteopathia striata with cranial sclerosis.

Osteopathia striata with cranial sclerosis (OSCS) is a rare bone disorder with X-linked dominant inheritance, characterized by a generalized hyperostosis in the skull and long bones and typical metaphyseal striations in the long bones. So far, loss-of-function variants in AMER1 (also known as WTX or FAM123B), encoding the APC membrane recruitment protein 1 (AMER1), have been described as the only molecular cause for OSCS. AMER1 promotes the degradation of β-catenin via AXIN stabilization, acting as a negative regulator of the WNT/β-catenin signaling pathway, a central pathway in bone formation. In this study, we describe a Dutch adult woman with an OSCS-like phenotype, i.e. generalized high bone mass and characteristic metaphyseal striations, but no genetic variant affecting AMER1. Whole exome sequencing led to the identification of a mosaic missense variant (c.876A>C; p.Lys292Asn) in CTNNB1, coding for β-catenin. The variant disrupts an amino acid known to be crucial for interaction with AXIN, a key factor in the β-catenin destruction complex. Western blotting experiments demonstrate that the p.Lys292Asn variant does not significantly affect the β-catenin phosphorylation status, and hence stability in the cytoplasm. Additionally, luciferase reporter assays were performed to investigate the effect of p.Lys292Asn β-catenin on canonical WNT signaling. These studies indicate an average 70-fold increase in canonical WNT signaling activity by p.Lys292Asn β-catenin. In conclusion, this study indicates that somatic variants in the CTNNB1 gene could explain the pathogenesis of unsolved cases of osteopathia striata.

ISX-9 Promotes KGF Secretion From MSCs to Alleviate ALI Through NGFR-ERK-TAU-β-Catenin Signaling Axis.

Mesenchymal stem cells (MSCs) have been widely studied to alleviate acute lung injury (ALI) due to their paracrine function. However, the microenvironment of inflammatory outbreaks significantly restricted the factors secreted from MSCs like keratinocyte growth factor (KGF). KGF is a growth factor with tissue-repaired ability. Is there a better therapeutic prospect for MSCs in combination with compounds that promote their paracrine function? Through compound screening, we screened out isoxazole-9 (ISX-9) to promote MSCs derived KGF secretion and investigated the underlying mechanisms of action. Compounds that promote KGF secretion were screened by a dual-luciferase reporter gene assay. The TMT isotope labeling quantitative technique was used to detect the differential proteins upon ISX-9 administrated to MSCs. The expressions of NGFR, ERK, TAU, and β-catenin were detected by Western blot. In the ALI model, we measured the inflammatory changes by HE staining, SOD content detection, RT-qPCR, immunofluorescence, etc. The influence of ISX-9 on the residence time of MSCs transplantation was explored by optical in vivo imaging. We found out that ISX-9 can promote the expression of KGF in MSCs. ISX-9 acted on the membrane receptor protein NGFR, upregulated phosphorylation of downstream signaling proteins ERK and TAU, downregulated phosphorylation of β-catenin, and accelerated β-catenin into the nucleus to further increase the expression of KGF. In the ALI model, combined ISX-9 with MSCs treatments upgraded the expression of KGF in the lung, and enhanced the effect of MSCs in reducing inflammation and repairing lung damage compared with MSCs alone. ISX-9 facilitated the secretion of KGF from MSCs both in vivo and in vitro. The combination of ISX-9 with MSCs enhanced the paracrine function and anti-inflammatory effect of MSCs compared with MSCs applied alone in ALI. ISX-9 played a contributive role in the transplantation of MSCs for the treatment of ALI.

Metadherin orchestrates PKA and PKM2 to activate β-catenin signaling in podocytes during proteinuric chronic kidney disease

Podocyte damage is the major cause of glomerular injury and proteinuria in multiple chronic kidney diseases. Metadherin (MTDH) is involved in podocyte apoptosis and promotes renal tubular injury in mouse models of diabetic nephropathy and renal fibrosis; however, its role in podocyte injury and proteinuria needs further exploration. Here, we show that MTDH was induced in the glomerular podocytes of patients with proteinuric chronic kidney disease and correlated with proteinuria. Podocyte-specific knockout of MTDH in mice reversed proteinuria, attenuated podocyte injury, and prevented glomerulosclerosis after advanced oxidation protein products challenge or adriamycin injury. Furthermore, specific knockout of MTDH in podocytes repressed β-catenin phosphorylation at the Ser675 site and inhibited its downstream target gene transcription. Mechanistically, on the one hand, MTDH increased cAMP and then activated protein kinase A (PKA) to induce β-catenin phosphorylation at the Ser675 site, facilitating the nuclear translocation of MTDH and β-catenin; on the other hand, MTDH induced the deaggregation of pyruvate kinase M2 (PKM2) tetramers and promoted PKM2 monomers to enter the nucleus. This cascade of events leads to the formation of the MTDH/PKM2/β-catenin/CBP/TCF4 transcription complex, thus triggering TCF4-dependent gene transcription. Inhibition of PKA activity by H-89 or blockade of PKM2 deaggregation by TEPP-46 abolished this cascade of events and disrupted transcription complex formation. These results suggest that MTDH induces podocyte injury and proteinuria by assembling the β-catenin-mediated transcription complex by regulating PKA and PKM2 function.

TP53INP2 modulates the malignant progression of colorectal cancer by reducing the inactive form of β-catenin

TP53INP2 (tumor protein p53-inducible nuclear protein 2), known as an autophagy protein, is essential for regulating transcription and starvation-induced autophagy, which plays a crucial role in the oncogenesis and progression of various cancers. The present study aims to investigate the expression pattern, function and prognostic value of TP53INP2 in colorectal cancer (CRC). Here, we report that low expression of TP53INP2 correlates with poor survival in CRC patients. TP53INP2 was significantly downregulated in CRC tissues compared with adjacent tissues. As the malignancy of CRC progresses, the expression level of TP53INP2 gradually decreased. Knockdown of TP53INP2 promoted CRC cell proliferation and tumor growth in mice. Mechanistically, TP53INP2 deficiency decreased phosphorylation of β-catenin on S33, S37, and T41, resulting in increased accumulation of β-catenin and enhanced nuclear translocation and transcriptional activity. Moreover, we further demonstrated that TP53INP2 sequestered TIM50, thereby inhibiting its activation of β-catenin. Taken together, our findings indicate that the downregulation of TP53INP2 promotes CRC progression by activating β-catenin and suggest that TP53INP2 may be a candidate therapeutic target for CRC.