Β-catenin Degradation Research Articles

USP10 drives cancer stemness and enables super-competitor signalling in colorectal cancer.

The contribution of deubiquitylating enzymes (DUBs) to β-Catenin stabilization in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, and by using an unbiassed screen, we discovered that the DUB USP10 stabilizes β-Catenin specifically in APC-truncated CRC in vitro and in vivo. Mechanistic studies, including in vitro binding together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and is outcompeted by intact APC, favouring β-catenin degradation. However, in APC-truncated cancer cells USP10 binds to β-catenin, increasing its stability which is critical for maintaining an undifferentiated tumour identity. Elimination of USP10 reduces the expression of WNT and stem cell signatures and induces the expression of differentiation genes. Remarkably, silencing of USP10 in murine and patient-derived CRC organoids established that it is essential for NOTUM signalling and the APC super competitor-phenotype, reducing tumorigenic properties of APC-truncated CRC. These findings are clinically relevant as patient-derived organoids are highly dependent on USP10, and abundance of USP10 correlates with poorer prognosis of CRC patients. Our findings reveal, therefore, a role for USP10 in CRC cell identity, stemness, and tumorigenic growth by stabilising β-Catenin, leading to aberrant WNT signalling and degradation resistant tumours. Thus, USP10 emerges as a unique therapeutic target in APC truncated CRC.

Yes-associated protein indispensably mediates hirsutine-induced inhibition on cell growth and Wnt/β-catenin signaling in colorectal cancer

Background and purpose: Targeting Wnt/β-catenin signaling emerges as one of the promising strategies for colorectal cancer (CRC) treatment, as this signaling is highly activated in CRC progression. Despite reports on the cytotoxic effects of hirsutine (HT), an indole alkaloid found in herbal medicines from the genus Uncaria, its therapeutic potential for CRC and the involved mechanisms are poorly understood. This study investigates the anticancer efficacy and the probable mechanisms of HT against CRC. Methods: To evaluate in vitro anticancer activity of HT, cell growth examined by MTT and colony formation assay, and apoptosis examined by flow cytometry were performed. To explore the mechanisms, RNA-sequencing, western blotting, dual-luciferase reporter assays, immunofluorescence, and co-immunoprecipitation were performed. Mouse model of azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colon cancer was utilized to assess HT's in vivo anticancer efficacy. Results: HT significantly inhibited CRC cell proliferation with IC50 values of 22.25 ± 3.27 μM for SW620 cells and 22.24 ± 2.36 μM for HCT116 cells, and induced apoptosis. HT decreased protein levels of Wnt3a and β-catenin dose- and time-dependently, and inhibited TOP/FOP FLASH reporter activity, nuclear travel of β-catenin, and downstream targets like c-Myc, Cyclin D1, VEGF. HT reduced β-catenin protein half-life, and the reversal of this effect by MG132 indicated that HT facilitated proteasome-dependent degradation of β-catenin in these two cell lines. HT also increased β-catenin ubiquitination without affecting Axin and β-TrCP levels. HT treatment for 24 h induced YAP cytoplasmic retention, enhanced YAP interacting with β-catenin and β-TrCP, triggering destruction complex formation and β-catenin ubiquitination and degradation, while YAP siRNA impaired these effects. Additionally, β-catenin overexpression and LiCl treatment counteracted HT-induced inhibition on cell growth and Wnt/β-catenin cascade. In model of AOM/DSS-induced mouse colon cancer, compared with AOM/DSS treatment group, HT recovered colon length, reduced tumor numbers and radius, and downregulated β-catenin and Ki-67, while upregulated cleaved PARP in the colorectal tissue with tumors. Conclusion: HT exhibits anticancer activity against CRC probably by inhibiting Wnt/β-catenin signaling, with YAP playing an indispensible role during the process, highlighting HT as a potential novel candidate drug for CRC therapy.

Self-assembled PROTACs enable protein degradation to reprogram the tumor microenvironment for synergistically enhanced colorectal cancer immunotherapy

Both β-catenin and STAT3 drive colorectal cancer (CRC) growth, progression, and immune evasion, and their co-overexpression is strongly associated with a poor prognosis. However, current small molecule inhibitors have limited efficacy due to the reciprocal feedback activation between STAT3 and β-catenin. Inspired by the PROteolysis TArgeting Chimera (PROTAC), a promising pharmacological modality for the selective degradation of proteins, we developed a strategy of nanoengineered peptide PROTACs (NP-PROTACs) to degrade both β-catenin and STAT3 effectively. The NP-PROTACs were engineered by coupling the peptide PROTACs with DSPE-PEG via disulfide bonds and self-assembled into nanoparticles. Notably, the dual degradation of β-catenin and STAT3 mediated by NP-PROTACs led to a synergistic antitumor effect compared to single-target treatment. Moreover, NP-PROTACs treatment enhanced CD103+ dendritic cell infiltration and T-cell cytotoxicity, alleviating the immunosuppressive microenvironment induced by β-catenin/STAT3 in CRC. These results highlight the potential of NP-PROTACs in facilitating the simultaneous degradation of two pathogenic proteins, thereby providing a novel avenue for cancer therapy.

Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.

Characteristic cerebral pathological changes of Alzheimer's disease (AD) such as glucose hypometabolism or the accumulation of cleavage products of the amyloid precursor protein (APP), known as Aβ peptides, lead to sustained endoplasmic reticulum (ER) stress and neurodegeneration. To preserve ER homeostasis, cells activate their unfolded protein response (UPR). The rhomboid-like-protease 4 (RHBDL4) is an enzyme that participates in the UPR by targeting proteins for proteasomal degradation. We demonstrated previously that RHBLD4 cleaves APP in HEK293T cells, leading to decreased total APP and Aβ. More recently, we showed that RHBDL4 processes APP in mouse primary mixed cortical cultures as well. Here, we aim to examine the physiological relevance of RHBDL4 in the brain. We first found that brain samples from AD patients and an AD mouse model (APPtg) showed increased RHBDL4 mRNA and protein expression. To determine the effects of RHBDL4's absence on APP physiology in vivo, we crossed APPtg mice to a RHBDL4 knockout (R4-/-) model. RHBDL4 deficiency in APPtg mice led to increased total cerebral APP and amyloidogenic processing when compared to APPtg controls. Contrary to expectations, as assessed by cognitive tests, RHBDL4 absence rescued cognition in 5-month-old female APPtg mice. Informed by unbiased RNAseq data, we demonstrated in vitro and in vivo that RHBDL4 absence leads to greater levels of active β-catenin due to decreased proteasomal clearance. Decreased β-catenin activity is known to underlie cognitive defects in APPtg mice and AD. Our work suggests that RHBDL4's increased expression in AD, in addition to regulating APP levels, leads to aberrant degradation of β-catenin, contributing to cognitive impairment.

E3 ubiquitin ligase BTBD3 inhibits tumorigenesis of colorectal cancer by regulating the TYRO3/Wnt/β-catenin signaling axis

Clinical trials and studies have implicated that E3 ubiquitin ligase BTBD3 (BTB Domain Containing 3) is a cancer-associated gene. However, the role and underlying mechanism of BTBD3 in colorectal cancer (CRC) is not fully understood yet. Herein, our study demonstrated that the mRNA and protein levels of BTBD3 were decreased in CRC tissues and associated with TYPO3 and Wnt/β-catenin pathway. Our results showed that circRAE1 knockdown and TYRO3 overexpression activated Wnt/β-catenin signaling pathway and the EMT process-associated markers, indicating that circRAE1/miR-388-3p/TYRO3 axis exacerbated tumorigenesis of CRC by activating Wnt/β-catenin signaling pathway. In addition, overexpression of BTBD3 reduced CRC cell migration and invasion in vitro and inhibited tumor growth in vivo. Our data demonstrated that BTBD3 suppressed CRC progression through negative regulation of the circRAE1/miR-388-3p/TYRO3 axis and the Wnt/β-catenin pathway. Our data further confirmed that BTBD3 bound and ubiquitinated β-catenin and led to β-catenin degradation, therefore blocked the Wnt/β-catenin pathway and suppressed the CRC tumorigenesis. This study explored the mechanism of BTBD3 involved in CRC tumorigenesis and provided a new theoretical basis for the prevention and treatment of CRC.

MEN1 Deficiency-Driven Activation of the β-Catenin-MGMT Axis Promotes Pancreatic Neuroendocrine Tumor Growth and Confers Temozolomide Resistance.

O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the guanine O6 position (O6-MG) and repairs DNA damage. High MGMT expression results in poor response to temozolomide (TMZ). However, the biological importance of MGMT and the mechanism underlying its high expression in pancreatic neuroendocrine tumors (PanNETs) remain elusive. Here, it is found that MGMT expression is highly elevated in PanNET tissues compared with paired normal tissues and negatively associated with progression-free survival (PFS) time in patients with PanNETs. Knocking out MGMT inhibits cancer cell growth in vitro and in vivo. Ectopic MEN1 expression suppresses MGMT transcription in a manner that depends on β-Catenin nuclear export and degradation. The Leucine 267 residue of MEN1 is crucial for regulating β-Catenin-MGMT axis activation and chemosensitivity to TMZ. Interference with β-Catenin re-sensitizes tumor cells to TMZ and significantly reduces the cytotoxic effects of high-dose TMZ treatment, and MGMT overexpression counteracts the effects of β-Catenin deficiency. This study reveals the biological importance of MGMT and a new mechanism by which MEN1 deficiency regulates its expression, thus providing a potential combinational strategy for treating patients with TMZ-resistant PanNETs.

CRYAB Promotes Colorectal Cancer Progression by Inhibiting Ferroptosis Through Blocking TRIM55-Mediated β-Catenin Ubiquitination and Degradation.

α-Crystallin B (CRYAB) is a chaperone member of the HSPs family that protects proteins with which it interacts from degradation. This study aims to investigate the effect of CRYAB on the progression of colorectal cancer (CRC) and its underlying mechanism. CRYAB expression was evaluated in CRC tissues. Cell growth was tested by CCK-8 kit. Lipid reactive oxygen species (ROS) assays, lipid peroxidation assays, glutathione assays were used to assess the degree of cellular lipid peroxidation of CRC cells. The potential signal pathways of CRYAB were analyzed and verified by Western blot (WB) and immunoprecipitation (Co-IP). CRYAB expression was elevated in CRC tissues and exhibited sensitivity and specificity in predicting CRC. Functionally, knockdown of CRYAB induced ferroptosis in CRC cells. Mechanistically, CRYAB binding prevented from β-catenin interacting with TRIM55, leading to an increase in β-catenin protein stability, which desensitized CRC cells to ferroptosis and ultimately accelerated cancer progression. Targeting CRYAB might be a promising strategy to enhance ferroptosis and improve the efficacy of CRC therapy.

Accumulated β-catenin is associated with human atrial fibrosis and atrial fibrillation

BackgroundAtrial fibrillation (AF) is associated with increased risk of stroke and mortality. It has been reported that the process of atrial fibrosis was regulated by β-catenin in rats with AF. However, pathophysiological mechanisms of this process in human with AF remain unclear. This study aims to investigate the possible mechanisms of β-catenin in participating in the atrial fibrosis using human right atrial appendage (hRAA) tissues .MethodsWe compared the difference of β-catenin expression in hRAA tissues between the patients with AF and sinus rhythm (SR). The possible function of β-catenin in the development of AF was also explored in mice and primary cells.ResultsFirstly, the space between the membrane of the gap junctions of cardiomyocytes was wider in the AF group. Secondly, the expression of the gap junction function related proteins, Connexin40 and Connexin43, was decreased, while the expression of β-catenin and its binding partner E-cadherin was increased in hRAA and cardiomyocytes of the AF group. Thirdly, β-catenin colocalized with E-cadherin on the plasma membrane of cardiomyocytes in the SR group, while they were dissociated and accumulated intracellularly in the AF group. Furthermore, the expression of glycogen synthase kinase 3β (GSK-3β) and Adenomatous Polyposis Coli (APC), which participated in the degradation of β-catenin, was decreased in hRAA tissues and cardiomyocytes of the AF group. Finally, the development of atrial fibrosis and AF were proved to be prevented after inhibiting β-catenin expression in the AF model mice.ConclusionsBased on human atrial pathological and molecular analyses, our findings provided evidence that β-catenin was associated with atrial fibrosis and AF progression.

Screening and identification of miRNAs negatively regulating FAM83A/Wnt/β-catenin signaling pathway in non-small cell lung cancer

The prevalence of non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers, with the Wnt/β-catenin signaling pathway exhibiting robust activation in this particular subtype. The expression of FAM83A (family with sequence similarity 83, member A) has been found to be significantly upregulated in lung cancer, leading to the stabilization of β-catenin and activation of the Wnt signaling pathway. In this study, we conducted a screening of down-regulated miRNAs in lung cancer with FAM83A as the target. Ultimately, we identified miR-1 as a negative regulator of FAM83A and confirmed that FAM83A is a direct target gene of miR-1 through dual luciferase reporter assays. The overexpression of miR-1 significantly attenuated the expression level of FAM83A and suppressed the Wnt signaling pathway, leading to a reduction in the expression levels of downstream target genes AXIN2, CyclinD1, and C-MYC. Additionally, it decreased the nuclear translocation of β-catenin. In addition, overexpression of miR-1 accelerated the degradation of β-catenin by inhibiting FAM83A, promoted the assembly of β-catenin degradation complex, and inhibited the proliferation, migration and invasion of NSCLC cells. In summary, miR-1 may be a potential candidate miRNA for the treatment of NSCLC.

Increased oxygen stimulation promotes chemoresistance and phenotype shifting through PLCB1 in gliomas

Gliomas, the most common CNS (central nerve system) tumors, face poor survival due to severe chemoresistance exacerbated by hypoxia. However, studies on whether altered hypoxic conditions benefit for chemo-sensitivity and how gliomas react to increased oxygen stimulation are limited. In this study, we demonstrated that increased oxygen stimulation promotes glioma growth and chemoresistance. Mechanically, increased oxygen stimulation upregulates miR-1290 levels. miR-1290, in turn, downregulates PLCB1, while PLCB1 facilitates the proteasomal degradation of β-catenin and active-β-catenin by increasing the proportion of ubiquitinated β-catenin in a destruction complex-independent mechanism. This process inhibits PLCB1 expression, leads to the accumulation of active-β-catenin, boosting Wnt signaling through an independent mechanism and ultimately promoting chemoresistance in glioma cells. Pharmacological inhibition of Wnt by WNT974 could partially inhibit glioma volume growth and prolong the shortened survival caused by increased oxygen stimulation in a glioma-bearing mouse model. Moreover, PLCB1, a key molecule regulated by increased oxygen stimulation, shows promising predictive power in survival analysis and has great potential to be a biomarker for grading and prognosis in glioma patients. These results provide preliminary insights into clinical scenarios associated with altered hypoxic conditions in gliomas, and introduce a novel perspective on the role of the hypoxic microenvironment in glioma progression. Furthermore, the outcomes reveal the potential risks of utilizing hyperbaric oxygen treatment (HBOT) in glioma patients, particularly when considering HBOT as a standalone option to ameliorate neuro-dysfunctions or when combining HBOT with a single chemotherapy agent without radiotherapy.

Isoleucyl-tRNA synthetase 2 promotes pancreatic ductal adenocarcinoma proliferation and metastasis by stabilizing β-catenin

Isoleucyl-tRNA synthetase 2 (IARS2), originally regarded as an enzyme ligating isoleucine to the corresponding tRNA, has been identified as an oncogene recently. However, its function in pancreatic ductal adenocarcinoma (PDAC) remains to be discovered. Here we explored the biological role of IARS2 in PDAC. Up-regulated IARS2 was found in PDAC tissues and cell lines. Kaplan–Meier survival analysis indicated a worse prognosis in patients with high IARS2 expression. CCK-8, EdU, and colony formation assays showed IARS2 overexpression enhanced PDAC proliferation, which was reduced by IARS2 knockdown. Meanwhile, IARS2 down-regulation inhibited PDAC metastasis by impeding epithelial–mesenchymal transition. These results were also supported by subcutaneous xenograft and metastasis assays in vivo. To figure out underlying mechanisms, differential and enrichment analyses were conducted and the WNT signaling pathway was discovered. Our results demonstrated that there was no significant relationship between the WNT signaling pathway key factor CTNNB1 and IARS2 at the transcription level. However, cycloheximide assays showed that IARS2 reduced the β-catenin degradation rate. IARS2 inhibited the phosphorylation of β-catenin at the Ser33/37 site and regulated downstream targets of WNT signaling including c-MYC, c-JUN, and MMP7. The enhancement of proliferation and metastasis caused by IARS2 could be reversed by MSAB, an agent that promotes β-catenin degradation. In summary, IARS2 facilitates PDAC proliferation and metastasis by stabilizing β-catenin, which leads to WNT/β-catenin activation. IARS2 serves as an underlying prognosis marker and a potential therapeutic target for PDAC.

TRIM26 deficiency enhancing liver regeneration through macrophage polarization and β-catenin pathway activation

Liver regeneration is a complex process involving the crosstalk between parenchymal and non-parenchymal cells, especially macrophages. However, the underlying mechanisms remain incompletely understood. Here, we identify the E3 ubiquitin ligase TRIM26 as a crucial regulator of liver regeneration. Following partial hepatectomy or acute liver injury induced by carbon tetrachloride, Trim26 knockout mice exhibit enhanced hepatocyte proliferation compared to wild-type controls, while adeno-associated virus (AAV)-mediated overexpression of Trim26 reverses the promotional effects. Mechanistically, Trim26 deficiency promotes the recruitment of macrophages to the liver and their polarization towards pro-inflammatory M1 phenotype. These M1 macrophages secrete Wnts, including Wnt2, which subsequently stimulate hepatocyte proliferation through the activation of Wnt/β-catenin signaling. In hepatocytes, Trim26 knockdown reduces the ubiquitination and degradation of β-catenin, thereby further enhancing Wnt/β-catenin signaling. Pharmacological inhibition of Wnt/β-catenin pathway by ICG-001 or depletion of macrophages by clodronate liposomes diminishes the pro-regenerative effects of Trim26 deficiency. Moreover, bone marrow transplantation experiments provide evidence that Trim26 knockout in myeloid cells alone can also promote liver regeneration, highlighting the critical role of macrophage Trim26 in this process. Taken together, our study uncovers TRIM26 as a negative regulator of liver regeneration by modulating macrophage polarization and Wnt/β-catenin signaling in hepatocytes, providing a potential therapeutic target for promoting liver regeneration in clinical settings.

Targeting histone deacetylase 6 (HDAC6) to enhance radiation therapy in meningiomas in a 2D and 3D in vitro study

External radiation therapy (RT) is often a primary treatment for inoperable meningiomas in the absence of established chemotherapy. Histone deacetylase 6 (HDAC6) overexpression, commonly found in cancer, is acknowledged as a driver of cellular growth, and inhibiting HDACs holds promise in improving radiotherapeutic efficacy. Downregulation of HDAC6 facilitates the degradation of β-catenin. This protein is a key element in the Wnt/β-catenin signalling pathway, contributing to the progression of meningiomas. In order to elucidate the associations and therapeutic potential of HDAC6 inhibitors (HDAC6i) in conjunction with RT, we administered Cay10603, HDAC6i, to both immortalised and patient-derived meningioma cells prior to RT in this study. Our findings reveal an increase in HDAC6 expression following exposure to RT, which is effectively mitigated with pre-treated Cay10603. The combination of Cay10603 with RT resulted in a synergistic augmentation of cytotoxic effects, as demonstrated through a range of functional assays conducted in both 2D as well as 3D settings; the latter containing syngeneic tumour microenvironment (TME). Radiation-induced DNA damage was augmented by pre-treatment with Cay10603, concomitant with the inhibition of β-catenin and minichromosome maintenance complex component 2 (MCM2) accumulation within the nucleus. This subsequently inhibited c-myc oncogene expression. Our findings demonstrate the therapeutic potential of Cay10603 to improve the radiosensitisation and provide rationale for combining HDAC6i with RT for the treatment of meningioma. This work was funded by Brain Tumour Research Centre of Excellence award to C Oliver Hanemann.

Dicerandrol C Suppresses Proliferation and Induces Apoptosis of HepG2 and Hela Cancer Cells by Inhibiting Wnt/β-Catenin Signaling Pathway.

Overwhelming evidence points to an aberrant Wnt/β-catenin signaling as a critical factor in hepatocellular carcinoma (HCC) and cervical cancer (CC) pathogenesis. Dicerandrol C (DD-9), a dimeric tetrahydroxanthenone isolated from the endophytic fungus Phomopsis asparagi DHS-48 obtained from mangrove plant Rhizophora mangle via chemical epigenetic manipulation of the culture, has demonstrated effective anti-tumor properties, with an obscure action mechanism. The objective of the current study was to explore the efficacy of DD-9 on HepG2 and HeLa cancer cells and its functional mechanism amid the Wnt/β catenin signaling cascade. Isolation of DD-9 was carried out using various column chromatographic methods, and its structure was elucidated with 1D NMR. The cytotoxicity of DD-9 on HepG2 and HeLa cells was observed with respect to the proliferation, clonality, migration, invasion, apoptosis, cell cycle, and Wnt/β-catenin signaling cascade. We found that DD-9 treatment significantly reduced tumor cell proliferation in dose- and time-dependent manners in HepG2 and HeLa cells. The subsequent experiments in vitro implied that DD-63 could significantly suppress the tumor clonality, metastases, and induced apoptosis, and that it arrested the cell cycle at the G0/G1 phase of HepG2 and HeLa cells. Dual luciferase assay, Western blot, and immunofluorescence assay showed that DD-9 could dose-dependently attenuate the Wnt/β-catenin signaling by inhibiting β-catenin transcriptional activity and abrogating β-catenin translocated to the nucleus; down-regulating the transcription level of β-catenin-stimulated Wnt target gene and the expression of related proteins including p-GSK3-β, β-catenin, LEF1, Axin1, c-Myc, and CyclinD1; and up-regulating GSK3-β expression, which indicates that DD-9 stabilized the β-catenin degradation complex, thereby inducing β-catenin degradation and inactivation of the Wnt/β-catenin pathway. The possible interaction between DD-9 and β-catenin and GSK3-β protein was further confirmed by molecular docking studies. Collectively, DD-9 may suppress proliferation and induce apoptosis of liver and cervical cancer cells, possibly at least in part via GSK3-β-mediated crosstalk with the Wnt/β-catenin signaling axis, providing insights into the mechanism for the potency of DD-9 on hepatocellular and cervical cancer.

Clinicopathological characteristics and survival in metastatic colorectal cancer with short-form APC mutations.

3555 Background: Wnt/β-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli ( APC) gene and plays a key role in tumorigenesis in colorectal cancer (CRC). Tankyrase inhibitors, which cause degradation of β-catenin, are expected to have a promising therapeutic effect for CRC, and the short-form APC mutations predict the sensitivity of CRC cells to tankyrase inhibitors (Tanaka et al. Mol Cancer Ther 2017; Chen et al. Br J Cancer, in press).However, clinicopathological characteristics and survival in metastatic CRC (mCRC) with short-form APCmutations remain unclear. Methods: Patients with mCRC who were treated with chemotherapy between 2010 and 2021 and have tissue or circulating tumor DNA-based next-generation sequencing results were enrolled. Short-form APC mutations were defined as those truncated at amino acid 1,256 or shorter, lacking all seven 20-amino acid repeat (20-AAR) regions. Non-short-form APC mutations were defined as those possess more than one 20-AAR. Short-form and non-short-form heterozygous mutations were classified as non-short-form APC mutations. We confirmed incidence of mCRC with short-form APC mutations and compared clinicopathological characteristics and overall survival (OS) between mCRC patients with short-form and non-short-form APCmutations. Results: Of 177 patients (median age, 56.0 years) included in this study, 44 (25%) and 79 (45%) had short-form and non-short-form APC mutations, respectively. Patients with short-form APC mutations were not likely to have lung metastasis (P = 0.15). Logistic regression multivariate analysis, lung metastasis was related to mCRC patients with short-form APC mutations (Odds ratio: 0.46, P = 0.047). Patients with short-form APC mutations had significantly shorter overall survival (OS) than did those without (n = 133), with a median OS of 34.7 vs 50.5 months ( P Log-rank= 0.035, hazard ratio (HR), 1.59; 95% confidence interval (CI), 1.03-2.45). In multivariate analysis, short-form APC mutation was independent factor related to OS (hazard ratio (HR): 1.68, P = 0.02), as well as the BRAF V600E mutation, liver metastasis and peritoneal metastasis. Conclusions: Short-form APC mutation is more common in mCRC patients without lung metastases and is independently associated with a short prognosis.

A phase 1/2 study of the TBL1 inhibitor, tegavivint (BC2059), in patients (pts) with advanced hepatocellular carcinoma (aHCC) with β-catenin activating mutations.

TPS4192 Background: β-catenin mutations are present in up to 40% of aHCC pts. Elevated nuclear β-catenin expression levels correlate with poor responses to standard of care and β-catenin regulates both tumor metabolism and the immune microenvironment. Tegavivint is a first-in-class small molecule inhibitor of TBL1, a novel downstream Wnt-signaling pathway target. Tegavivint binds to TBL1 in the β-catenin pocket, disrupting the formation of the activation complex necessary for oncogenic activity, and enabling degradation of free nuclear β-catenin. The safety, clinical activity, and PK/PD of tegavivint was demonstrated through a proof-of-concept study in pts with desmoid tumors and is being studied through multiple Investigator Initiated Trials in AML (NCT04874480), pediatric solid tumors (NCT04851119), NSCLC (NCT04780568), and lymphoma (NCT05755087). Tegavivint was also studied in preclinical mouse models of β-cateninexon3 mutant HCC and the H22 syngeneic HCC model. In these models tegavivint decreased Wnt target gene expression and enhanced CD3+ T-cell infiltration in liver tumors. Tegavivint treatment of established β-cateninexon3 activated tumors resulted in reduced tumor growth and burden. Based on these promising preclinical results, the following phase 1/2 exploratory study was designed. Methods: This is a phase 1/2 study of tegavivint in pts with aHCC to characterize safety, PK/PD, and preliminary antitumor activity (NCT05797805). Eligibility includes aHCC pts ≥18 years old, with AXIN1or CTNNB1 mutation for all pts, except those in the single agent dose escalation; have BCLC Stage C or Stage B disease not amendable to local therapy or curative approaches, have Child-Pugh class A or B7 liver score, and must have received at least one prior line of systemic therapy. This study will be conducted in 2 parts. First, tegavivint will be administered as a single agent in a dose escalation/optimization and subsequent dose expansion cohort. Upon completion of the dose escalation via a 3+3 design, two dose levels will be selected for the dose selection optimization to determine the recommended phase 2 dose (RP2D) for use in the dose expansion. If sufficient clinical benefit is observed, the combination of tegavivint plus pembrolizumab will be explored in the second part of the study in aHCC pts previously treated with a PD-1/PD-L1 inhibitor. Tegavivint will be administered intravenously weekly. Primary objective is safety and secondary objectives are preliminary efficacy and PK/PD. The first patient began treatment in October of 2023 and the first dose escalation cohort was completed in January. Three institutions are open for enrollment; 5 other sites are pending site activation; all sites are in the United States and Canada. Clinical trial information: NCT05797805 .

Splice variants of CK1α and CK1α-like: Comparative analysis of subcellular localization, kinase activity, and function in the Wnt signaling pathway

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the invitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.

Smoking-induced CCNA2 expression promotes lung adenocarcinoma tumorigenesis by boosting AT2/AT2-like cell differentiation

Lung adenocarcinoma (LUAD), a type of non-small cell lung cancer (NSCLC), originates from not only bronchial epithelial cells but also alveolar type 2 (AT2) cells, which could differentiate into AT2-like cells. AT2-like cells function as cancer stem cells (CSCs) of LUAD tumorigenesis to give rise to adenocarcinoma. However, the mechanism underlying AT2 cell differentiation into AT2-like cells in LUAD remains unknown. We analyze genes differentially expressed and genes with significantly different survival curves in LUAD, and the combination of these two analyses yields 147 differential genes, in which 14 differentially expressed genes were enriched in cell cycle pathway. We next analyze the protein levels of these genes in LUAD and find that Cyclin-A2 (CCNA2) is closely associated with LUAD tumorigenesis. Unexpectedly, high CCNA2 expression in LUAD is restrictedly associated with smoking and independent of other driver mutations. Single-cell sequencing analyses reveal that CCNA2 is predominantly involved in AT2-like cell differentiation, while inhibition of CCNA2 significantly reverses smoking-induced AT2-like cell differentiation. Mechanistically, CCNA2 binding to CDK2 phosphorylates the AXIN1 complex, which in turn induces ubiquitination-dependent degradation of β-catenin and inhibits the WNT signaling pathway, thereby failing AT2 cell maintenance. These results uncover smoking-induced CCNA2 overexpression and subsequent WNT/β-catenin signaling inactivation as a hitherto uncharacterized mechanism controlling AT2 cell differentiation and LUAD tumorigenesis.

Sesamolin serves as an MYH14 inhibitor to sensitize endometrial cancer to chemotherapy and endocrine therapy via suppressing MYH9/GSK3β/β-catenin signaling

BackgroundEndometrial cancer (EC) is one of the most common gynecological cancers. Herein, we aimed to define the role of specific myosin family members in EC because this protein family is involved in the progression of various cancers.MethodsBioinformatics analyses were performed to reveal EC patients’ prognosis-associated genes in patients with EC. Furthermore, colony formation, immunofluorescence, cell counting kit 8, wound healing, and transwell assays as well as coimmunoprecipitation, cycloheximide chase, luciferase reporter, and cellular thermal shift assays were performed to functionally and mechanistically analyze human EC samples, cell lines, and a mouse model, respectively.ResultsMachine learning techniques identified MYH14, a member of the myosin family, as the prognosis-associated gene in patients with EC. Furthermore, bioinformatics analyses based on public databases showed that MYH14 was associated with EC chemoresistance. Moreover, immunohistochemistry validated MYH14 upregulation in EC cases compared with that in normal controls and confirmed that MYH14 was an independent and unfavorable prognostic indicator of EC. MYH14 impaired cell sensitivity to carboplatin, paclitaxel, and progesterone, and increased cell proliferation and metastasis in EC. The mechanistic study showed that MYH14 interacted with MYH9 and impaired GSK3β-mediated β-catenin ubiquitination and degradation, thus facilitating the Wnt/β-catenin signaling pathway and epithelial–mesenchymal transition. Sesamolin, a natural compound extracted from Sesamum indicum (L.), directly targeted MYH14 and attenuated EC progression. Additionally, the compound disrupted the interplay between MYH14 and MYH9 and repressed MYH9-regulated Wnt/β-catenin signaling. The in vivo study further verified sesamolin as a therapeutic drug without side effects.ConclusionsHerein, we identified that EC prognosis-associated MYH14 was independently responsible for poor overall survival time of patients, and it augmented EC progression by activating Wnt/β-catenin signaling. Targeting MYH14 by sesamolin, a cytotoxicity-based approach, can be applied synergistically with chemotherapy and endocrine therapy to eventually mitigate EC development. This study emphasizes MYH14 as a potential target and sesamolin as a valuable natural drug for EC therapy.Graphical

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.