Β-catenin Target Research Articles

Strategic targeting of miR-183 and β-catenin to enhance BMSC stemness in age-related osteoporosis therapy

Age-related osteoporosis is a prevalent bone metabolic disorder distinguished by an aberration in the equilibrium between bone formation and resorption. The reduction in the stemness of Bone Marrow Mesenchymal Stem Cells (BMSCs) plays a pivotal role in the onset of this ailment. Comprehending the molecular pathways that govern BMSCs stemness is imperative for delineating the etiology of age-related osteoporosis and devising efficacious treatment modalities. The study utilized single-cell RNA sequencing and miRNA sequencing to investigate the cellular heterogeneity and stemness of BMSCs. Through dual-luciferase reporter assays and functional experiments, the regulatory effect of miR-183 on CTNNB1 (β-catenin) was confirmed. Overexpression and knockdown studies were conducted to explore the impact of miR-183 and β-catenin on stemness-related transcription factors Oct4, Nanog, and Sox2. Cell proliferation assays and osteogenic differentiation experiments were carried out to validate the influence of miR-183 and β-catenin on the stemness properties of BMSCs. Single-cell analysis revealed that β-catenin is highly expressed in both high stemness clusters and terminal differentiation clusters of BMSCs. Overexpression of β-catenin upregulated stemness transcription factors, while its suppression had the opposite effect, indicating a dual regulatory role of β-catenin in maintaining BMSCs stemness and promoting bone differentiation. Furthermore, the confluence of miRNA sequencing analyses and predictions from online databases revealed miR-183 as a potential modulator of BMSCs stemness and a novel upstream regulator of β-catenin. The overexpression of miR-183 effectively diminished the stemness characteristics of BMSCs by suppressing β-catenin, whereas the inhibition of miR-183 augmented stemness. These outcomes align with the observed alterations in the expression levels and functional assessments of transcription factors associated with stemness. This study provides evidence for the essential involvement of β-catenin in preserving the stemness of BMSCs, as well as elucidating the molecular mechanism through which miR-183 selectively targets β-catenin to modulate stemness. These results underscore the potential of miR-183 and β-catenin as molecular targets for augmenting the stemness of BMSCs. This strategy is anticipated to facilitate the restoration of bone microarchitecture and facilitate bone tissue regeneration by addressing potential cellular dysfunctions, thereby presenting novel targets and perspectives for the management of age-related osteoporosis.

Deciphering the cellular and molecular landscapes of Wnt/β-catenin signaling in mouse embryonic kidney development

BackgroundThe Wnt/β-catenin signaling pathway is critical in kidney development, yet its specific effects on gene expression in different embryonic kidney cell types are not fully understood. MethodsWnt/β-catenin signaling was activated in mouse E12.5 kidneys in vitro using CHIR99021, with RNA sequencing performed afterward, and the results were compared to DMSO controls (dataset GSE131240). Differential gene expression in ureteric buds and cap mesenchyme following pathway activation (datasets GSE20325 and GSE39583) was analyzed. Single-cell RNA-seq data from the Mouse Cell Atlas was used to link differentially expressed genes (DEGs) with kidney cell types. β-catenin ChIP-seq data (GSE39837) identified direct transcriptional targets. ResultsActivation of Wnt/β-catenin signaling led to 917 significant DEGs, including the upregulation of Notum and Apcdd1 and the downregulation of Crym and Six2. These DEGs were involved in kidney development and immune response. Single-cell analysis identified 787 DEGs across nineteen cell subtypes, with Macrophage_Apoe high cells showing the most pronounced enrichment of Wnt/β-catenin-activated genes. Gene expression profiles in ureteric buds and cap mesenchyme differed significantly upon β-catenin manipulation, with cap mesenchyme showing a unique set of DEGs. Analysis of β-catenin ChIP-seq data revealed 221 potential direct targets, including Dpp6 and Fgf12. ConclusionThis study maps the complex gene expression driven by Wnt/β-catenin signaling in embryonic kidney cell types. The identified DEGs and β-catenin targets elucidate the molecular details of kidney development and the pathway's role in immune processes, providing a foundation for further research into Wnt/β-catenin signaling in kidney development and disease.

Basic Fibroblast Growth Factor Supports the Function of Limbal Niche Cells via the Wnt/β-Catenin Pathway.

Purpose: To test the effects and underlying mechanisms of basic fibroblast growth factor (bFGF) on the limbal niche cell (LNC) function ex vivo. Methods: By using different concentrations of bFGF (0, 4, 8, 12, and 16 ng/mL) and fibroblast growth factor receptor (FGFR) inhibitors, the effects of bFGF on LNC proliferation, expression of stem cell markers, and transcription levels of the β-catenin were investigated. Single-cell RNA sequencing (scRNA-seq) was used to analyze the action and mechanisms of FGFR subtypes and the Wnt/β-catenin pathway during LNC culture. An mature corneal epithelial cell (MCEC)/LNC three-dimensional model was constructed to verify whether bFGF activates the Wnt/β-catenin pathway in LNC by inhibiting FGFR or β-catenin targets. Results: scRNA-seq showed that FGFR1 is the main receptor in LNC, along with the molecules in the Wnt pathway, including WNT2, FZD7, LRP5, LRP6, and β-catenin. The 12 ng/mL bFGF treatment group showed higher LNC proliferation rate and transcription levels of OCT4, SOX2, NANOG, and β-catenin than any other groups (P < 0.001). In the MCEC/LNC co-culture model, MCEC/LNC treated with 12 ng/mL bFGF promoted the aggregation of the spheres than other groups, associated with increased transcription levels of P63α, WNT2, β-catenin, and a decreased transcription level of CK12 (P < 0.001). Wnt/β-catenin inhibitor LF3 treatment reversed the abovementioned effect of bFGF. Conclusions: bFGF could maintain and promote the stemness of LNC via the FGFR1/Wnt2/FZD7/LRP6 axis in a concentration-dependent manner.

Decoding the Genetic Threads of Disc Degeneration

Abstract Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder characterized by the progressive degeneration of intervertebral discs, often leading to chronic low back pain and disability. While the etiology of DDD is multifactorial, genetic factors play a significant role in disease susceptibility and progression. This review provides a comprehensive overview of the genetic aspects of DDD, summarizing previously reported genes and variations associated with the disease. Through an analysis of animal studies and molecular pathways implicated in disc degeneration, including the lipid kinase phoshoinositide-3-kinase signaling pathway (PI3K-Akt), mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway (MAPK-ERK), Wingless-related integration (Wnt)/β-catenin, Sonic Hedgehog (Shh), and mammalian target of rapamycin (mTOR) pathways, this review elucidates the intricate interplay between genetic factors and disc pathology. Several candidate genes have been identified in association with DDD, including those involved in extracellular matrix regulation, inflammation, and cell signaling. Genome-wide association studies have further expanded our understanding of the genetic architecture underlying DDD, revealing novel susceptibility loci and pathways. Animal studies utilizing genetically modified models have provided valuable insights into the molecular mechanisms driving disc degeneration and have validated the relevance of specific genetic pathways in disease pathogenesis. Understanding the genetic basis of DDD holds promise for identifying individuals at risk, developing predictive biomarkers, and informing personalized treatment approaches. Furthermore, elucidating the molecular pathways involved in disc degeneration may lead to the identification of novel therapeutic targets for DDD management. Overall, this review consolidates current knowledge on DDD genetics and pathways, providing a foundation for future research endeavors aimed at unraveling the intricate genetic mechanisms underlying this prevalent musculoskeletal disorder.

Abstract B013: Deep learning-based prediction of synthetic essentialities in CTNNB1-mutated hepatocellular carcinoma

Abstract Background: Synthetic essentiality represents a promising therapeutic approach by identifying genes that are necessary for the proliferation and survival of tumors harboring hard-to-target gene alterations. Understanding and accurately predicting synthetic essential genes, through genetic dependencies, may reveal therapeutically effective drug targets in a specific molecular context. Deep learning, as exemplified by our published DeepDEP model, has the potential to capture intricate multi-omic profiles for such prediction tasks. However, the validity of such tools in specific biological contexts remains to be fully examined and presents a major obstacle to adoption by researchers. Materials and Methods: To address this gap, we conducted a case study in which we screened for synthetic essential genes for one of the most frequently mutated and yet undruggable genes, CTNNB1, in hepatocellular carcinoma (HCC). Specifically, we predicted the genetic dependencies of each HCC patient in The Cancer Genome Atlas (TCGA; n=346) by DeepDEP and identified potential dependencies that were intensified with the presence of CTNNB1 mutations. The top 10 genes, ranked by p-value of differential gene-effect scores for CTNNB1-mutated (n=92) versus CTNNB1-WT HCC (n=254), were reviewed in the literature to validate their essentiality in CTNNB1-mutated HCC as well as their potential for pharmacologic inhibition. Survival analysis was performed using published data from the IMBrave150 trial to validate one of the findings. Results: Experimental evidence in the literature supported the essentiality of many of the top 10 predicted genes for CTNNB1-mutated HCC, including one gene with mechanistic evidence of being a transcriptional co-activator of β-catenin target genes. Furthermore, several of these genes have known pharmacologic inhibitors which are either natural compounds or FDA-approved drugs. One example was PDGFB, which encodes a ligand activating the PDGF signaling pathway. PDGF signaling is targeted by sorafenib, an FDA-approved first line drug for HCC. Survival analysis of the sorafenib-treated arm of the IMBrave150 trial showed that patients with mutated CTNNB1 had improved progression-free survival compared to those with wild-type CTNNB1 (p = 0.044). Conclusions: Our study illustrates a potential application of deep learning to identify synthetic essential genes, including genes with readily available pharmacologic inhibitors, for targeting challenging gene alterations. Remarkably, our tool demonstrates the ability to predict cancer dependencies with molecular subtype specificity, suggesting a potential for in silico screening of gene dependencies to facilitate drug discovery and personalized medicine approaches. Our current efforts are focused on optimizing this computational pipeline and making it publicly available for cancer researchers. Citation Format: Tyler M. Yasaka, Michael Kasper, Li-Ju Wang, Michael Ning, Yufei Huang, Satdarshan P Monga, Yu-Chiao Chiu. Deep learning-based prediction of synthetic essentialities in CTNNB1-mutated hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B013.

MEF2D facilitates liver metastasis of gastric cancer cells through directly inducing H1X under IL-13 stimulation

Liver metastasis is the most common metastatic occurrence in gastric cancer patients, although the precise mechanism behind it remains unclear. Through a combination of proteomics and quantitative RT-PCR, our study has revealed a significant correlation between the upregulation of myocyte enhancer factor-2D (MEF2D) and both distant metastasis and poor prognosis in gastric cancer patients. In mouse models, we observed that overexpressing or knocking down MEF2D in gastric cancer cells respectively promoted or inhibited liver metastasis. Furthermore, our research has demonstrated that MEF2D regulates the transcriptional activation of H1X by binding to the H1X promoter. This regulation leads to the upregulation of H1X, which, in turn, promotes the in vivo metastasis of gastric cancer cells along with the upregulation of the downstream gene β-CATENIN. Additionally, we found that the expression of MEF2D and H1X at both mRNA and protein levels can be induced by the inflammatory factor IL-13, and this induction exhibits a time gradient dependence. In human gastric cancer tissues, the expression of IL13RA1, the receptor for IL-13, positively correlates with the expression of MEF2D and H1X. IL13RA1 has been identified as an intermediate receptor through which IL-13 regulates MEF2D. In conclusion, our findings suggest that MEF2D plays a crucial role in promoting liver metastasis of gastric cancer by upregulating H1X and downstream target β-CATENIN in response to IL-13 stimulation. Targeting MEF2D could therefore be a promising therapeutic strategy for the clinical management of gastric cancer. Statement of significanceMEF2D promotes its transcriptional activation in gastric cancer cells by binding to the H1X promoter and is upregulated by IL-13-IL13RA1, thereby promoting distant metastasis of gastric cancer.

Vilazodone Alleviates Neurogenesis-Induced Anxiety in the Chronic Unpredictable Mild Stress Female Rat Model: Role of Wnt/β-Catenin Signaling

Defective β-catenin signaling is accompanied with compensatory neurogenesis process that may pave to anxiety. β-Catenin has a distinct role in alleviating anxiety in adolescence; however, it undergoes degradation by the degradation complex Axin and APC. Vilazodone (VZ) is a fast, effective antidepressant with SSRI activity and 5-HT1A partial agonism that amends somatic and/or psychic symptoms of anxiety. Yet, there is no data about anxiolytic effect of VZ on anxiety-related neurogenesis provoked by stress-reduced β-catenin signaling. Furthermore, females have specific susceptibility toward psychopathology. The aim of the present study is to uncover the molecular mechanism of VZ relative to Wnt/β-catenin signaling in female rats. Stress-induced anxiety was conducted by subjecting the rats to different stressful stimuli for 21 days. On the 15th day, stressed rats were treated with VZ(10 mg/kg, p.o.) alone or concomitant with the Wnt inhibitor: XAV939 (0.1 mg/kg, i.p.). Anxious rats showed low β-catenin level turned over by Axin-1 with unanticipated reduction of APC pursued with elevated protein levels of neurogenesis-stimulating proteins: c-Myc and pThr183-Erk likewise gene expressions of miR-17-5p and miR-18. Two weeks of VZ treatment showed anxiolytic effect figured by alleviation of hippocampal histological examination. VZ protected β-catenin signal via reduction in Axin-1 and elevation of APC conjugated with modulation of β-catenin downstream targets. The cytoplasmic β-catenin turnover by Axin-1 was restored by XAV939. Herein, VZ showed anti-anxiety effect, which may be in part through regaining the balance of the reduced β-catenin and its subsequent exaggerated response of p-Erk, c-Myc, Dicer-1, miR-17-5p, and miR-18.Graphical

Abstract 1810: Development of new phthalic hydrazide scaffold for potent TNKS inhibitors

Abstract Tankyrase 1 and 2 (TNKS1/2) catalyze post-translational modifications of target proteins through poly-ADP-ribosylation. Inhibition of TNKS stabilizes AXIN and induces β-catenin degradation in adenomatous polyposis coli (APC)-mutated colorectal cancer (CRC) cells. Therefore, TNKS emerges as a therapeutic target for APC mutant CRC, and there is a critical need for drug development against it. Tankyrase 1 and 2 (TNKS1/2) catalyze the post-translational modification of target proteins through poly-ADP-ribosylation. Their inhibition stabilizes AXIN and induces β-catenin degradation. Therefore, TNKS is a therapeutic target for APC mutant colorectal cancer (CRC), and there is a need for drug development against it. However, reported TNKS inhibitors have serious limitations in that they cause intestinal toxicity. To address these issues and enhance efficacy, we synthesized 60 compounds based on the tetrahydrophthalazine scaffold using protein structure modeling-Al technology. Using an in vitro TNKS1 and TNKS2 enzyme assay and derivatizing Hit compounds, we confirmed that TI-61987 significantly inhibits TNKS enzyme activity at low nanomolar levels. TI-61987 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM, APC-mutated CRC cells. Additionally, TI-61987 exhibited excellent anticancer effects not only in COLO320DM cells but also in in vivo xenograft mouse models. Our findings demonstrate that TI-61987 is a therapeutic candidate targeting TNKS for the treatment of APC-mutated CRCs, and this anticancer drug may represent an effective therapy regimen for APC-mutated CRCs. Citation Format: Hwani Ryu, Hye-Ran Seo, Ju Han Lee, Kyu Myung Lee, Jiyeon Ahn. Development of new phthalic hydrazide scaffold for potent TNKS inhibitors [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 1810.

Exhaustive identification of genome-wide binding events of transcriptional regulators.

Genome-wide binding assays aspire to map the complete binding pattern of gene regulators. Common practice relies on replication-duplicates or triplicates-and high stringency statistics to favor false negatives over false positives. Here we show that duplicates and triplicates of CUT&RUN are not sufficient to discover the entire activity of transcriptional regulators. We introduce ICEBERG (Increased Capture of Enrichment By Exhaustive Replicate aGgregation), a pipeline that harnesses large numbers of CUT&RUN replicates to discover the full set of binding events and chart the line between false positives and false negatives. We employed ICEBERG to map the full set of H3K4me3-marked regions, the targets of the co-factor β-catenin, and those of the transcription factor TBX3, in human colorectal cancer cells. The ICEBERG datasets allow benchmarking of individual replicates, comparing the performance of peak calling and replication approaches, and expose the arbitrary nature of strategies to identify reproducible peaks. Instead of a static view of genomic targets, ICEBERG establishes a spectrum of detection probabilities across the genome for a given factor, underlying the intrinsic dynamicity of its mechanism of action, and permitting to distinguish frequent from rare regulation events. Finally, ICEBERG discovered instances, undetectable with other approaches, that underlie novel mechanisms of colorectal cancer progression.

The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis.

Canonical Wnt and sphingosine-1-phosphate (S1P) signaling pathways are highly conserved systems that contribute to normal vertebrate development, with key consequences for immune, nervous, and cardiovascular system function; despite these functional overlaps, little is known about Wnt/β-catenin-S1P cross-talk. In the vascular system, both Wnt/β-catenin and S1P signals affect vessel maturation, stability, and barrier function, but information regarding their potential coordination is scant. We report an instance of functional interaction between the two pathways, including evidence that S1P receptor 1 (S1PR1) is a transcriptional target of β-catenin. By studying vascular smooth muscle cells and arterial injury response, we find a specific requirement for the β-catenin carboxyl terminus, which acts to induce S1PR1, and show that this interaction is essential for vascular remodeling. We also report that pharmacological inhibition of the β-catenin carboxyl terminus reduces S1PR1 expression, neointima formation, and atherosclerosis. These findings provide mechanistic understanding of how Wnt/β-catenin and S1P systems collaborate during vascular remodeling and inform strategies for therapeutic manipulation.

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.

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P&amp;lt;0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

HuR-positive stress granules: Potential targets for age-related osteoporosis.

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target β-catenin by binding and recruiting β-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of β-catenin into HuR-positive SGs, facilitated β-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.

A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor β-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as β-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because β-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

Wnt signaling in cell adhesion, development, and colon cancer.

Wnt signaling is essential for embryonic development, influencing processes such as axis formation, cell proliferation and differentiation, cell fate decisions, and axon guidance. It also plays a role in maintaining tissue homeostasis in adult organisms. The loss of normal cell polarity and adhesion caused by Wnt signaling activation is a fundamental step for tumor progression and metastasis. Activating the canonical Wnt pathway is a driving force in many human cancers, especially colorectal, hepatocellular, and mammary carcinomas. Wnt causes the stabilization and nuclear transport of newly synthesized transcriptional regulator β-catenin. The generally accepted view is that the canonical effects of Wnt growth factors are caused by the transcription of β-catenin target genes. Here, we review recent findings that indicate Wnt is a regulator of many other cellular physiological activities, such as macropinocytosis, endosome trafficking, protein stability, focal adhesions, and lysosomal activity. Some of these regulatory responses occur within minutes and do not require new protein synthesis, indicating that there is much more to Wnt beyond the well-established transcriptional role of β-catenin. The main conclusion that emerges from these studies is that in basal cell conditions, the activity of the key protein kinase GSK3, which is inhibited by Wnt pathway activation, normally represses the actin machinery that orchestrates macropinocytosis with implications in cancer. These contributions expand our understanding of the multifaceted roles of Wnt signaling in cellular processes, development, and cancer, providing insights into potential therapeutic targets and strategies.

UCHL3 inhibits ferroptosis by stabilizing β-catenin and maintains stem-like properties of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic liver cancer. Dysregulated Wnt/β-catenin activation is closely related to the progression of cancer. Nevertheless, the mechanism that sustains the abnormal expression of β-catenin in HCC has yet to be identified. In this study, we find that UCHL3 is overexpressed in HCC tissues and correlated with β-catenin protein level. High expression of UCHL3 is associated with poor prognosis. UCHL3 knockdown markedly reduces the protein level of β-catenin in HCC cells. TOP-luciferase activity and β-catenin target genes expression are also decreased upon UCHL3 depletion. We find that the ARM domain of β-catenin is required for the interaction with UCHL3. UCHL3 increases β-catenin protein stability via removing K48-specific poly-ubiquitin chains from β-catenin protein. Furthermore, the depletion of UCHL3 induces ferroptosis and hinders the growth, invasion, and stem cell properties of HCC cells. These impacts could be restored by the overexpression of β-catenin. In addition, the UCHL3 inhibitor TCID inhibits the aggressive phenotype of HCC through the degradation of β-catenin. In general, our results indicates that UCHL3 increases the stability of β-catenin, which in turn facilitates tumorigenesis of HCC, suggesting that targeting UCHL3 may be a promising approach for the treatment of HCC.

PDGFRβ Signaling Cooperates with β-Catenin to Modulate c-Abl and Biologic Behavior of Desmoid-Type Fibromatosis.

This study sought to identify β-catenin targets that regulate desmoid oncogenesis and determine whether external signaling pathways, particularly those inhibited by sorafenib (e.g., PDGFRβ), affect these targets to alter natural history or treatment response in patients. In vitro experiments utilized primary desmoid cell lines to examine regulation of β-catenin targets. Relevance of results was assessed in vivo using Alliance trial A091105 correlative biopsies. CTNNB1 knockdown inhibited hypoxia-regulated gene expression in vitro and reduced levels of HIF1α protein. ChIP-seq identified ABL1 as a β-catenin transcriptional target that modulated HIF1α and desmoid cell proliferation. Abrogation of either CTNNB1 or HIF1A inhibited desmoid cell-induced VEGFR2 phosphorylation and tube formation in endothelial cell co-cultures. Sorafenib inhibited this activity directly but also reduced HIF1α protein expression and c-Abl activity while inhibiting PDGFRβ signaling in desmoid cells. Conversely, c-Abl activity and desmoid cell proliferation were positively regulated by PDGF-BB. Reduction in PDGFRβ and c-Abl phosphorylation was commonly observed in biopsy samples from patients after treatment with sorafenib; markers of PDGFRβ/c-Abl pathway activation in baseline samples were associated with tumor progression in patients on the placebo arm and response to sorafenib in patients receiving treatment. The β-catenin transcriptional target ABL1 is necessary for proliferation and maintenance of HIF1α in desmoid cells. Regulation of c-Abl activity by PDGF signaling and targeted therapies modulates desmoid cell proliferation, thereby suggesting a reason for variable biologic behavior between tumors, a mechanism for sorafenib activity in desmoids, and markers predictive of outcome in patients.

PYGO2-MDR1 Axis in Multiple Myeloma Patients with 1q21 Amplification As Promising Target to Overcome Carfilzomib Resistance

Multiple myeloma (MM) is a hematological malignancy characterized by an accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Gain and/or amplification of 1q21 (1q21+) is one of the most frequent secondary cytogenetic abnormalities present in MM patients and in smoldering MM (SMM). The incidence and copy number alteration (CNA) of the 1q21 region increase during the progression and relapse of the disease; in fact, additional copies of 1q21 can be detected in around 40% of newly diagnosed MM (NDMM) and 70% of relapse-refractory MM (RRMM). Recently, 1q21+ has been identified as an adverse prognostic factor in MM patients and its presence has been correlated with a shorter duration of the response in MM patients treated with carfilzomib (CFZ)-based regimens. Thus, the identification of possible target genes in 1q21 region is an emerging unmet medical need in MM patients. PYGOPUS2 (PYGO2) is a gene located in the 1q21 chromosomal region and it is a downstream target of β-catenin, and it has been shown to promote the transcription of gene target of the Wnt-signaling, forming a nuclear complex with β-catenin/BCL9. Different studies have reported that upregulation of PYGO2 is involved in tumorigenesis in breast and lung cancers, showing that overexpression of PYGO2 leads to drug resistance by promoting the activation of the multidrug resistance 1 polypeptide ( MDR1). The expression profile of PYGO2 in1q21+ MM patients and its possible role in CFZ resistance is still unknown and have been investigated in this study. Firstly, we purified CD138 + PCs from BM samples from 18 NDMM and from 11 SMM patients. Fluorescent in situ hybridization (FISH) analysis was performed on purified CD138 + PCs for all patients to access CNA in the region 1q21. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. 14/29 (48%) patients presented CNA in the 1q21 region. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA) . The sam package was used to identify differentially expressed genes between 1q21+ and control samples. Our analysis identified PYGO2 to be significantly upregulated in patients with 1q21+ when compared with controls (p= 0.0008). Additionally, we found a positive correlation between gene expression and the 1q21 copy number (p= &amp;lt;0.0001, r= 0.6738). Secondly, we evaluated PYGO2 mRNA expression levels in several human myeloma cell lines (HMCLs) with 1q21 CNA. Our results showed that PYGO2 is overexpressed in HMCLs with 1q21 CNA. Previous studies in solid tumor have shown that PYGO2 is upregulated in drug resistant cancer cells, and this upregulation results in overexpression of MDR1; indeed, we evaluated the mRNA expression of PYGO2 in CFZ-resistant HMCLs. Interestingly our results showed that both PYGO2 and MDR1 expression are upregulated in CFZ-R cells. We next examined the relationship between PYGO2 and MDR1. We generated a PYGO2-knockdown in JJN3 MM cell line using short hairpin RNA (shRNA) lentivectors. Knockdown of PYGO2 resulted in a significant decrease in expression PYGO2, a decrease in cell viability, and a reduction in MDR1 expression. In conclusion, our results show that PYGO2 expression is significantly upregulated in MM patients carrying 1q21+ and that its expression is correlated with 1q21 copy number. Moreover, we found that the PYGO2-MDR1 axis is involved in the CFZ resistance in HMCLs MM cells suggesting that PYGO2 could be a possible future druggable target in 1q21+ MM patients.

14-3-3ζ Targets β-Catenin Nuclear Translocation to Maintain Mitochondrial Homeostasis, Promoting Balance of Proliferation and Apoptosis in Cisplatin-Induced AKI

14-3-3ζ Targets β-Catenin Nuclear Translocation to Maintain Mitochondrial Homeostasis, Promoting Balance of Proliferation and Apoptosis in Cisplatin-Induced AKI

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.