Β-catenin Activation Research Articles

Oncogenic KRAS Mutations Confer a Unique Mechanotransduction Response to Peristalsis in Colorectal Cancer Cells.

Colorectal cancer (CRC) tumors start as polyps on the inner lining of the colorectum, where they are exposed to the mechanics of peristalsis. Our previous work leveraged a custom-built peristalsis bioreactor to demonstrate that colonic peristalsis led to cancer stem cell enrichment in CRC cells. However, this malignant mechanotransductive response was confined to select CRC lines that harbored an oncogenic mutation in the KRAS gene. Here, we explored the involvement of activating KRAS mutations on peristalsis-associated mechanotransduction in CRC. Peristalsis enriched cancer stem cell marker LGR5 in KRAS mutant lines, in a Wnt-ligand-independent manner. Conversely, LGR5 enrichment in wild type KRAS lines exposed to peristalsis were minimal. LGR5 enrichment downstream of peristalsis translated to increased tumorigenicity in vivo. Differences in mechanotransduction was apparent via unbiased gene set enrichment analysis, where many unique pathways were enriched in wild type vs. mutant lines. Peristalsis also triggered β-catenin nuclear localization independent of Wnt-ligands, particularly in KRAS mutant lines. The involvement of KRAS was validated via gain and loss of function strategies. Peristalsis induced β-catenin activation and LGR5 enrichment depended on the activation of the MEK/ERK cascade. Taken together, our results demonstrated that oncogenic KRAS mutations conferred a unique peristalsis-associated mechanotransduction response to colorectal cancer cells, resulting in cancer stem cell enrichment and increased tumorigenicity. These mechanosensory connections can be leveraged in improving the sensitivity of emerging therapies that target oncogenic KRAS. Implications: Oncogenic KRAS empowers colorectal cancer cells to harness the mechanics of colonic peristalsis for malignant gain, independent of other cooperating signals. .

Cardiac secreted-HSP90alpha in cardiomyocyte hypertrophy by N-Cadherin mediated signaling under pressure overload

Abstract Background Left ventricular hypertrophy (LVH) in response to pressure overload is strongly associated with adverse cardiovascular outcomes. Heat shock protein 90 (HSP90) has been reported to be involved in cardiac remodeling under stress. Aims Here, we evaluate whether serum HSP90α (an isoform of HSP90) increases and associates with cardiac hypertrophy in patients with hypertension or severe aortic stenosis (AS), and explore the potential mechanisms in experimental pressure overload mouse model. Methods Serum HSP90α levels in patients with hypertension or aortic stenosis (AS) were determined by ELISA. Pressure overload mouse model was built by a transverse aortic constriction (TAC). Cardiac function was assessed by echocardiography. Cardiac hypertrophy was examined by histology, western blot and RNA analysis. Results Serum levels of HSP90α were higher in patients with hypertension or AS than in the control group, respectively, and the levels positively correlated with LVH. HSP90α levels also increased in heart tissues of patients with obstructive hypertrophic cardiomyopathy (HCM), and mice after TAC, in parallel with the hypertrophic markers. TAC induced the enhanced expression and secretion of HSP90α from cardiomyocytes and cardiac fibroblasts. Knockdown of HSP90α or blockade of extracellular HSP90α (eHSP90α) prevent the development of LVH under pressure overload in vivo and in vitro. By Nano-HPLC-MS/MS analysis, we identified eHSP90α interacted with N-cadherin in cardiomyocyte surface, which induced the activation of β-catenin and promoted β-catenin to bind TCF7 (a member of TCF/Lef family) in nucleus, enhanced the transcription of hypertrophic genes, contributing to cardiac hypertrophy and dysfunction under pressure overload. Conclusions Our data demonstrated that cardiac secreted-HSP90α promoted myocardial hypertrophy by N-Cadherin mediated β-catenin/TCF7 signaling under pressure overload. It indicates the therapeutic potential of targeting HSP90α-initiated signaling against cardiac hypertrophy and dysfunction under pressure overload.

Chlorpyrifos and dimethoate exposure impairs female fertility by deregulating WNT signaling pathway & uterine receptivity

The study assessed histological, biochemical, oxidative stress, and molecular parameters to evaluate the consequences of Chlorpyrifos and Dimethoate exposure on uterine health in female rats. Despite showing no obvious signs of toxicity apart from minor clinical symptoms in DM-exposed rats, both pesticides caused degenerative changes in uterine tissue. This study demonstrates that pesticides induce inflammatory responses and oxidative stress in rats, by NF-κB activation and altering antioxidant enzyme levels. Besides, CPF and DM exposure disrupted gene expression of HOXA10, HOXA11, and WNT and reduced activation of β-catenin in the uterus, which is crucial for implantation and reproductive function. These findings suggest that pesticide exposure may impair reproductive health and fertility in females, highlighting potential implications for human health.

ELAVL1 regulates glycolysis in nasopharyngeal carcinoma cells through the HMGB3/β-catenin axis

BackgroundThe role of ELAVL1 in the progression of various tumors has been demonstrated. Our research aims to investigate how ELAVL1 controls the glycolytic process in nasopharyngeal carcinoma cells through the HMGB3/β-catenin pathway.MethodsThe expression of ELAVL1 was detected in clinical tumor samples and nasopharyngeal carcinoma cell lines. A subcutaneous tumor model was established in nude mice to investigate the role of ELAVL1 in tumor progression. The relationship between HMGB3 and ELAVL1 was validated by RNA pull down and RIP assays. TOPFlash/FOPFlash reporter assay was used to detect β-catenin activity. Assay kits were utilized to measure glucose consumption, lactate production, and G6PD activity in nasopharyngeal carcinoma cells. Western blot was conducted to detect the expression of glycolysis-related proteins. The glycolytic capacity was analyzed through extracellular acidification rate (ECAR).ResultsIn both clinical samples and nasopharyngeal carcinoma cell lines, the expression levels of ELAVL1 mRNA and protein were found to be upregulated. Knockdown of ELAVL1 significantly inhibited the in vivo proliferation of nasopharyngeal carcinoma and suppressed the glycolytic capacity of nasopharyngeal carcinoma cells. ELAVL1 interacts with HMGB3, leading to an increase in the stability of HMGB3 mRNA. Overexpression of HMGB3 elevated the reduced β-catenin activity caused by sh-ELAVL1 and reversed the inhibitory effect of sh-ELAVL1 on cellular glycolytic capacity. Treatment with β-catenin inhibitor (FH535) effectively suppressed the promotion of glycolytic capacity induced by HMGB3 overexpression.ConclusionsELAVL1 promotes glycolysis in nasopharyngeal carcinoma cells by interacting with HMGB3 to stabilize HMGB3 mRNA, thereby activating β-catenin pathway. Therefore, targeting the ELAVL1-HMGB3-β-catenin axis has the potential to be a novel approach for treating nasopharyngeal carcinoma.

Upregulation of Fatty Acid Synthase Increases Activity of β-Catenin and Expression of NOTUM to Enhance Stem-like Properties of Colorectal Cancer Cells.

Dysregulated fatty acid metabolism is an attractive therapeutic target for colorectal cancer (CRC). We previously reported that fatty acid synthase (FASN), a key enzyme of de novo synthesis, promotes the initiation and progression of CRC. However, the mechanisms of how upregulation of FASN promotes the initiation and progression of CRC are not completely understood. Here, using Apc/VillinCre and ApcMin mouse models, we show that upregulation of FASN is associated with an increase in activity of β-catenin and expression of multiple stem cell markers, including Notum. Genetic and pharmacological downregulation of FASN in mouse adenoma organoids decreases the activation of β-catenin and expression of Notum and significantly inhibits organoid formation and growth. Consistently, we demonstrate that NOTUM is highly expressed in human CRC and its expression positively correlates with the expression of FASN in tumor tissues. Utilizing overexpression and shRNA-mediated knockdown of FASN, we demonstrate that upregulation of FASN increases β-catenin transcriptional activity, NOTUM expression and secretion, and enhances stem-like properties of human CRC cells. Pharmacological inhibition of NOTUM decreases adenoma organoids growth and proliferation of cancer cells. In summary, upregulation of FASN enhances β-catenin signaling, increases NOTUM expression and stem-like properties of CRC cells, thus suggesting that targeting FASN upstream of the β-catenin/NOTUM axis may be an effective preventative therapeutic strategy for CRC.

8773 CD44 Variant Isoforms Expressed Within PAX7/SOX2-Positive Pituitary Neuroendocrine Tumors Attribute To Therapy Resistance And Recurrence Of Cushing’s Disease

Abstract Disclosure: J. Chakrabarti: None. J. Eschbacher: None. S. Mallick: None. E. Ferris: None. B. Hermes: None. A. Little: None. Y. Zavros: None. Background: Cushing’s disease (CD) is a serious rare endocrine disease caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that subsequently stimulates the adrenal glands to overproduce cortisol. Approximately 56% of patients will experience disease recurrence during the 2 to 10-year follow-up period. Surgical failures and late recurrences of CD are common, and despite multiple treatments, biochemical control is achieved in only 50% of patients. Therefore, there is a need to advance targeted therapies that prevent recurrence and therapy resistance. The Cluster of Differentiation (CD) 44 transmembrane glycoprotein (CD44) is a prominent WNT signaling target, and deregulation of the Wnt pathway is associated with constitutively active βcatenin in SOX2-positive cells. Although a similar mechanism has been proposed in PitNETs, it has not been fully tested. The alternative mRNA splice variant, CD44v9 is expressed on cancer stem cells promotes resistance to ROS through the stabilization of the SLC7A11 (xCT) cystine glutamate transporter. Proteolytic cleavage of CD44 releases the CD44 intracellular domain which translocates to the nucleus activating cell stemness genes including SOX2. Although CD44 is a known cancer stem cell marker that plays multiple key roles including drug resistance and tumor cell invasiveness, the mechanisms underlying human PitNET tumorigenesis are unclear. Hypothesis: CD44 expressed on PAX7/SOX2-positive corticotroph PitNET cells define a cell population that attributes to therapy resistance and disease recurrence. Methods: CD patient PitNET tissues and matched PitNET-generated organoids (PitNETOs) were used for CosMx™ Spatial Molecular Imaging (SMI) and scRNAseq analyses respectively. Resistance to drug- and radiation-induced apoptosis was analyzed using PitNETOs generated. Results: SMI and scRNAseq data revealed a cell population co-expressing PAX7 and SOX2, CD44 and EpCAM that was abundant in invasive and silent corticotroph PitNET subtypes with Knosp grades 3-4. Invasive and silent corticotroph PitNETs also expressed an increased cell population of CD163+ macrophages, myofibroblast cancer associated fibroblasts (myCAFs), inflammatory CAFs. While cells isolated from the dura revealed growth of PitNETOs, adherent cells expressed increased proliferation (Ki67), and expression of somatostatin receptor subtype 2, cancer stem markers (CD44, SOX2, NESTIN, FUT4, PROM1), PAX7 progenitor cell population expressing stem cell markers NESTIN, SOX2, CD44 and EpCAM. Increased CD44/SLCA7A11 expression in CD44/PAX7/SOX2-positive PitNETOs rendered tumor cells resistant to radiation-induced apoptosis. Conclusions: Drugs targeting the CD44 variant isoforms may increase the efficacy of medical therapies and stereotactic radiosurgery to prevent residual and recurrent PitNETs in CD patients. Presentation: 6/3/2024

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

MiR-27a-3p regulates intestinal cell proliferation and differentiation through Wnt/β-catenin signalling.

Intestinal stem cells differentiate into absorptive enterocytes, characterised by increased brush border enzymes such as intestinal alkaline phosphatase (IAP), making up the majority (95%) of the terminally differentiated cells in the villus. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here, we show that the intestinal microRNA (miR)-27a-3p is an important regulator of intestinal epithelial cell proliferation and enterocyte differentiation. Repression of endogenous miR-27a-3p leads to increased enterocyte differentiation and decreased intestinal epithelial cell proliferation in mouse and human small intestinal organoids. Mechanistically, miR-27a-3p regulates intestinal cell differentiation and proliferation at least in part through the regulation of retinoic acid receptor α (RXRα), a modulator of Wnt/β-catenin signalling. Repression of miR-27a-3p increases the expression of RXRα and concomitantly, decreases the expression of active β-catenin and cyclin D1. In contrast, overexpression of miR-27a-3p mimic decreases the expression of RXRα and increases the expression of active β-catenin and cyclin D1. Moreover, overexpression of the miR-27a-3p mimic results in impaired enterocyte differentiation and increases intestinal epithelial cell proliferation. These alterations were attenuated or blocked by Wnt inhibition. Our study demonstrates an miR-27a-3p/RXRα/Wnt/β-catenin pathway that is important for the maintenance of enterocyte homeostasis in the small intestine.

PRR promotes hypertensive renal injury by activating Wnt/β-catenin signaling and inflammation infiltration in mice

Hypertension stands out as a substantial independent risk factor in the progression of chronic kidney disease; however, the exact pathological mechanisms remain elusive. Our preliminary studies find that Wnt/β-catenin control renin-angiotensin system (RAS) expression, thus playing an important role in the pathogenesis of hypertension and renal fibrosis. As an integral component of the RAS, the (pro)renin receptor (PRR) plays a crucial role in the activation of the RAS and hypertension. Recent studies suggest a reciprocal relationship between PRR and Wnt/β-catenin signaling, potentially contributing to hypertensive renal fibrosis development. To assess the role of PRR in mediating hypertensive nephropathy, we manipulated this signaling by over expression of PRR ligand or blockade of PRR by siPRR. In vivo, PRR induction promoted hypertension, proteinuria, renal fibrosis, inflammatory response and β-catenin activation in Ang II induced hypertension mice. Conversely, blockade of PRR inhibited Ang II mediated hypertension, renal fibrosis and inflammation. In vitro, PRR over expression renal tubular cells exacerbated the Ang II induced fibrotic response and inflammation. Moreover, PRR was upregulated in hypertensive nephropathy patients, and correlated with renal function and renal fibrosis. These results indicate that PRR interact with Wnt/β-catenin signaling promote the progression of hypertensive nephropathy. PRR could be served as a biomarker for the diagnosis and treatment of hypertensive renal fibrosis.

A novel liquid plasma derivative inhibits melanogenesis through upregulation of Nrf2

Non-thermal plasma (NTP) is an emerging technology with extensive applications in biomedicine, including treatment of abnormal pigmentation. However, very few studies have investigated how plasma induces anti-melanogenesis. Here, liquid plasma was prepared by treating an NTP jet with helium and oxygen (as carrier gases) for 15 min in serum-free culture media. In the zebrafish model, pigmentation ratio was observed with or without liquid plasma. The anti-melanogenic effect of liquid plasma was evaluated in human melanocytes by assessing the expression of melanogenesis-related genes using western blotting, RT-PCR, and immunohistochemistry. Liquid plasma reduced pigmentation in the zebrafish model and inhibited melanin synthesis in primary human melanocytes. Intracellular reactive oxygen species levels decreased and Nrf2 expression increased in liquid plasma–treated melanocytes. Liquid plasma affected microphthalmia-associated transcription factor (MITF) and tyrosinase mRNA and protein levels, tyrosinase activity, and melanin content. Considering the role of Wnt/β-catenin and PI3K/Akt pathways in melanogenesis, the effect of liquid plasma on this pathway was determined; liquid plasma decreased active β-catenin, LEF1/TCF4, MITF, and tyrosinase levels in a time-dependent manner and inhibited the nuclear translocation of β-catenin. This inhibition subsequently suppressed melanogenesis by downregulating MITF and tyrosinase. These results suggest that liquid plasma may be used for treating pigmentary disorders.

The RNA helicase DDX21 activates YAP to promote tumorigenesis and is transcriptionally upregulated by β-catenin in colorectal cancer

The RNA helicase DDX21 is vital for ribosome biogenesis and is upregulated in CRC, but the mechanism by which DDX21 is dysregulated and by which DDX21 promotes tumorigenesis in CRC remains poorly understood. Here, we showed that DDX21 is a direct transcriptional target gene of β-catenin and mediates the protumorigenic function of β-catenin in CRC. DDX21 expression is correlated with the expression and activity of β-catenin, and high DDX21 expression is associated with a poor prognosis in CRC patients. Loss of DDX21 leads to cytoplasmic translocation and decreased transcriptional activity of YAP and suppresses the proliferation and migration of CRC cells, which can be partially rescued by YAP reactivation. Importantly, by using translation elongation inhibitors and DNA intercalators, we showed that ribosomal stress upregulates DDX21 expression and induces the downregulation of LATS and the activation of YAP, probably through the ZAKα-MKK4/7-JNK axis. Overall, our study revealed the transcriptional activation mechanism of DDX21 in CRC and the activation of YAP in the ribosomal stress response, indicating the potential of combination therapy involving the induction of ribosomal stress and YAP inhibition.

Pyrimidine-triazole-tethered tert-butyl-piperazine-carboxylate suppresses breast cancer by targeting estrogen receptor signaling and β-catenin activation.

Several chemotherapeutics against breast cancer are constrained by their adverse effects and chemoresistance. The development of novel chemotherapeutics to target metastatic breast cancer can bring effective clinical outcomes. Many breast cancer patients present with tumors that are positive for estrogen receptors (ERs), highlighting the importance of targeting the ER pathway in this particular subtype. Although selective estrogen receptor modulators (SERMs) are commonly used, their side effects and resistance issues necessitate the development of new ER-targeting agents. In this study, we report that a newly synthesized compound, TTP-5, a hybrid of pyrimidine, triazole, and tert-butyl-piperazine-carboxylate, effectively binds to estrogen receptor alpha (ERα) and suppresses breast cancer cell growth. We assessed the impact of TTP-5 on cell proliferation using MTT and colony formation assays and evaluated its effect on cell motility through wound healing and invasion assays. We further explored the mechanism of action of this novel compound by detecting protein expression changes using Western blotting. Molecular docking was used to confirm the interaction of TTP-5 with ERα. The results indicated that TTP-5 significantly reduced the proliferation of MCF-7 cells by blocking the ERα signaling pathway. Conversely, although it did not influence the growth of MDA-MB-231 cells, TTP-5 hindered their motility by modulating the expression of proteins associated with epithelial-mesenchymal transition (EMT), possibly via the Wnt/β-catenin pathway.

GSK-3β in Dendritic Cells Exerts Opposite Functions in Regulating Cross-Priming and Memory CD8 T Cell Responses Independent of β-Catenin.

GSK-3β plays a critical role in regulating the Wnt/β-catenin signaling pathway, and manipulating GSK-3β in dendritic cells (DCs) has been shown to improve the antitumor efficacy of DC vaccines. Since the inhibition of GSK-3β leads to the activation of β-catenin, we hypothesize that blocking GSK-3β in DCs negatively regulates DC-mediated CD8 T cell immunity and antitumor immunity. Using CD11c-GSK-3β-/- conditional knockout mice in which GSK-3β is genetically deleted in CD11c-expressing DCs, we surprisingly found that the deletion of GSK-3β in DCs resulted in increased antitumor immunity, which contradicted our initial expectation of reduced antitumor immunity due to the presumed upregulation of β-catenin in DCs. Indeed, we found by both Western blot and flow cytometry that the deletion of GSK-3β in DCs did not lead to augmented expression of β-catenin protein, suggesting that GSK-3β exerts its function independent of β-catenin. Supporting this notion, our single-cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3β-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated β-catenin. This suggests that the deletion of GSK-3β in DCs is unlikely to lead to upregulation of β-catenin at the transcriptional level. Consistent with enhanced antitumor immunity, we also found that CD11c-GSK-3β-/- mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that the deletion of GSK-3β in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3β in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although the deletion of GSK-3β in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3β-/- mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3β in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3β exerts its functions independent of its regulation of β-catenin. These novel insights suggest that targeting GSK-3β in cancer immunotherapies must consider its dual role in CD8 T cell responses.

Dose-dependent responses to canonical Wnt transcriptional complexes in the regulation of mammalian nephron progenitors.

In vivo and in vitro studies argue that concentration-dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of β-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA-binding partners. Using the GSK3β inhibitor CHIR99021 (CHIR) to block GSK3β-dependent destruction of β-catenin, we examined dose-dependent responses to β-catenin in mouse NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on β-catenin removal, with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following β-catenin removal, mRNA-seq identified low CHIR and β-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and β-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated stabilized form of β-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together, these studies provide evidence for concentration-dependent Wnt signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.

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.

Equol exerts anti-tumor effects on choriocarcinoma cells by promoting TRIM21-mediated ubiquitination of ANXA2

Choriocarcinoma is a malignant cancer that belongs to gestational trophoblastic neoplasia (GTN). Herein, serum metabolomic analysis was performed on 29 GTN patients and 30 healthy individuals to characterize the metabolic variations during GTN progression. Ultimately 24 differential metabolites (DMs) were identified, of which, Equol was down-regulated in GTN patients, whose VIP score is the 3rd highest among the 24 DMs. As an intestinal metabolite of daidzein, the anticancer potential of Equol has been demonstrated in multiple cancers, but not choriocarcinoma. Hence, human choriocarcinoma cell lines JEG-3 and Bewo were used and JEG-3-derived subcutaneous xenograft models were developed to assess the effect of Equol on choriocarcinoma. The results suggested that Equol treatment effectively suppressed choriocarcinoma cell proliferation, induced cell apoptosis, and reduced tumorigenesis. Label-free quantitative proteomics showed that 136 proteins were significantly affected by Equol and 20 proteins were enriched in Gene Ontology terms linked to protein degradation. Tripartite motif containing 21 (TRIM21), a E3 ubiquitin ligase, was up-regulated by Equol. Equol-induced effects on choriocarcinoma cells could be reversed by TRIM21 inhibition. Annexin A2 (ANXA2) interacted with TRIM21 and its ubiquitination was modulated by TRIM21. We found that TRIM21 was responsible for proteasome-mediated degradation of ANXA2 induced by Equol, and the inhibitory effects of Equol on the malignant behaviors of choriocarcinoma cells were realized by TRIM21-mediated down-regulation of ANXA2. Moreover, β-catenin activation was inhibited by Equol, which also depended on TRIM21-mediated down-regulation of ANXA2. Taken together, Equol may be a novel candidate for the treatment for choriocarcinoma.Graphical abstract

A Boolean model explains phenotypic plasticity changes underlying hepatic cancer stem cells emergence

In several carcinomas, including hepatocellular carcinoma, it has been demonstrated that cancer stem cells (CSCs) have enhanced invasiveness and therapy resistance compared to differentiated cancer cells. Mathematical-computational tools could be valuable for integrating experimental results and understanding the phenotypic plasticity mechanisms for CSCs emergence. Based on the literature review, we constructed a Boolean model that recovers eight stable states (attractors) corresponding to the gene expression profile of hepatocytes and mesenchymal cells in senescent, quiescent, proliferative, and stem-like states. The epigenetic landscape associated with the regulatory network was analyzed. We observed that the loss of p53, p16, RB, or the constitutive activation of β-catenin and YAP1 increases the robustness of the proliferative stem-like phenotypes. Additionally, we found that p53 inactivation facilitates the transition of proliferative hepatocytes into stem-like mesenchymal phenotype. Thus, phenotypic plasticity may be altered, and stem-like phenotypes related to CSCs may be easier to attain following the mutation acquisition.

SLC13A3 is a major effector downstream of activated β-catenin in liver cancer pathogenesis

Activated Wnt/β-catenin pathway is a key genetic event in liver cancer development. Solute carrier (SLC) transporters are promising drug targets. Here, we identify SLC13A3 as a drug-targetable effector downstream of β-catenin in liver cancer. SLC13A3 expression is elevated in human liver cancer samples with gain of function (GOF) mutant CTNNB1, the gene encoding β-catenin. Activation of β-catenin up-regulates SLC13A3, leading to intracellular accumulation of endogenous SLC13A3 substrates. SLC13A3 is identified as a low-affinity transporter for glutathione (GSH). Silencing of SLC13A3 downregulates the leucine transporter SLC7A5 via c-MYC signaling, leading to leucine depletion and mTOR inactivation. Furthermore, silencing of SLC13A3 depletes GSH and induces autophagic ferroptosis in β-catenin-activated liver cancer cells. Importantly, both genetic inhibition of SLC13A3 and a small molecule SLC13A3 inhibitor suppress β-catenin-driven hepatocarcinogenesis in mice. Altogether, our study suggests that SLC13A3 could be a promising therapeutic target for treating human liver cancers with GOF CTNNB1 mutations.

DNA Methylation in Recurrent Glioblastomas: Increased TEM8 Expression Activates the Src/PI3K/AKT/GSK-3β/B-Catenin Pathway.

Glioblastomas (GBM) are infiltrative malignant brain tumors which mostly recur within a year's time following surgical resection and chemo-radiation therapy. Studies on glioblastoma cells following radio-chemotherapy, have been demonstrated to induce trans-differentiation, cellular plasticity, activation of DNA damage response and stemness. As glioblastomas are heterogenous tumors that develop treatment resistance and plasticity, we investigated if there exist genome-wide DNA methylation changes in recurrent tumors. Utilizing genome-wide DNA methylation arrays, we compared the DNA methylation profile of 11 primary (first occurrence) tumors with 13 recurrent (relapsed) GBM, to delineate the contribution of epigenetic changes associated with therapy exposure, therapy resistance, and relapse of disease. Our data revealed 1,224 hypermethylated- and 526 hypomethylated-probes in recurrent glioblastomas compared to primary disease. We found differential methylation of solute carrier and ion channel genes, interleukin receptor/ligand genes, tumor-suppressor genes and genes associated with metastasis. We functionally characterized one such hypomethylated-up-regulated gene, namely anthrax toxin receptor 1/tumor endothelial marker 8 (ANTXR1/TEM8), whose expression was validated to be significantly up-regulated in recurrent glioblastomas compared to primary tumors and confirmed by immunohistochemistry. Using overexpression and knockdown approaches, we showed that TEM8 induces proliferation, invasion, migration, and chemo-radioresistance in glioblastoma cells. Additionally, we demonstrated a novel mechanism of β-catenin stabilization and activation of the β-catenin transcriptional program due to TEM8 overexpression via a Src/PI3K/AKT/GSK3β/β-catenin pathway. We report genome-wide DNA methylation changes in recurrent GBM and suggest involvement of the TEM8 gene in GBM recurrence and progression.

Targeting Galectin-1 Overcomes Paclitaxel Resistance in Esophageal Squamous Cell Carcinoma.

Resistance to paclitaxel poses a major obstacle in esophageal squamous cell carcinoma (ESCC) treatment. A better understanding of the mechanisms underlying paclitaxel resistance could help identify prognostic biomarkers and improved therapeutic strategies. In this study, we established a patient-derived xenograft (PDX) model of acquired paclitaxel resistance and used RNA-sequencing to identify galectin-1, encoded by LGALS1, as a key mediator of resistance. Integrative analysis of clinical data and physiological studies indicated that serum galectin-1 levels were elevated in resistant patients and correlated with treatment outcomes before and during taxane therapy. Importantly, exposing cells to serum from resistant patients resulted in increased paclitaxel resistance compared to serum from sensitive patients, which was closely associated with galectin-1 concentrations in the serum. The specific clearance of galectin-1 from resistant patient serum significantly restored paclitaxel sensitivity, and inhibiting galectin-1, through knockdown or the pharmacologic inhibitor OTX008, increased sensitivity to paclitaxel. Galectin-1 inhibition reduced the activity of β-catenin, thereby inhibiting stem cell properties induced by the Wnt/β-catenin pathway. Furthermore, galectin-1 regulated MDR1 transcription through increased nuclear accumulation of β-catenin, thus increasing resistance to paclitaxel. Combining OTX008 with clinical taxane formulations effectively reversed paclitaxel resistance in vitro and in vivo. Elevated galectin-1 levels thus serve as an indicator of response to paclitaxel therapy in ESCC, offering a therapeutic intervention strategy to overcome drug resistance.