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BMP/SMAD signaling is a crucial regulator of intestinal differentiation 1 – 4 . However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other’s expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4–SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity 5 . The authors show that the transcription factors HNF4A and HNF4G regulate the transcriptome of the intestinal epithelium. HNF4 factors cooperate with BMP/SMAD signaling to promote enterocyte identity.

BackgroundHereditary haemorrhagic telangiectasia (HHT) and juvenile polyposis syndrome (JPS) can be caused by SMAD4 pathogenic variants. SMAD4 is a common transcription factor of the BMP/TGFβ signalling pathway. In this study, we developed a cell-based functional assay to address the pathogenicity of SMAD4 variants identified in the French HHT cohort.MethodsSMAD4 variants were generated by site-directed mutagenesis. A functional assay was developed in a cell line that does not express SMAD4, and the different SMAD4 variants were tested for their capacity to activate the BMP and TGFβ response using luciferase reporter assays.ResultsTwelve SMAD4 variants were identified and studied. We were able to develop a robust functional assay for these variants. All the expressed variants resulted in loss of function (LOF) in response to BMP9 or TGFβ1 stimulation. SMAD4 variants within the MH2 domain expressed SMAD4 mutated proteins that were unable to hetero-oligomerise with other SMADs, which could explain their LOF. Finally, we tested primary human endothelial cells isolated from patients with HHT carrying SMAD4 heterozygous pathogenic variants and observed that they behaved like the control cells at rest or when stimulated with BMP9.ConclusionWe developed a SMAD4 functional assay that allows discrimination between benign and pathogenic SMAD4 variants. We demonstrated that the underlying molecular mechanism of this pathogenicity is due mostly to a loss of hetero-oligomerisation. This assay will be transferable to clinical genetic laboratories and will improve the diagnosis of patients with HHT–JPS.

Background: Recently several microRNAs (miRNAs) have been found to be regulated by genistein in cancer cells. In this study, we focused on the gene regulatory effect of genistein on microRNA and its target genes in prostate cancer (PC). Methods: Initially, we investigated the effect of genistein on prostate cancer cells and identified that the expression of miRNA-1260b was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA-1260b expression and prostate cancer patient outcomes. Two target genes ( sFRP1 and Smad4 ) of miR-1260b were identified based on computer algorithm and 3′UTR luciferase assay was carried out to determine direct miRNA regulation of the genes. Results: Genistein promoted apoptosis while inhibiting prostate cancer cell proliferation, invasion and TCF reporter activity in PC cells. MiR-1260b was highly expressed in prostate cancer tissues and significantly downregulated by genistein in PC cells. After knocking down miR-1260b, cell proliferation, invasion, migration and TCF reporter activity were decreased in PC cells. Western analysis and 3′UTR luciferase assay showed that the two target genes ( sFRP1 and Smad4 ) were directly regulated by miR-1260b. The expression of sFRP1 and Smad4 was significantly decreased in prostate cancer tissues. Genistein also increased expression of these two genes via DNA demethylation and histone modifications. Conclusions: Our data suggest that genistein exerts its anti-tumour effect via downregulation of miR-1260b that targeted sRRP1 and Smad4 genes in prostate cancer cells. The expression of sFRP1 and Smad4 was also modulated by genistein via DNA methylation or histone modifications in PC cell lines.

Perturbations in transforming growth factor-β (TGF-β) signaling can lead to a plethora of diseases, including cancer. Mutations and posttranslational modifications (PTMs) of the partner of SMAD complexes contribute to the dysregulation of TGF-β signaling. Here, we reported a PTM of SMAD4, R361 methylation, that was critical for SMAD complexes formation and TGF-β signaling activation. Through mass spectrometric, co-immunoprecipitation (Co-IP) and immunofluorescent (IF) assays, we found that oncogene protein arginine methyltransferase 5 (PRMT5) interacted with SMAD4 under TGF-β1 treatment. Mechanically, PRMT5 triggered SMAD4 methylation at R361 and induced SMAD complexes formation and nuclear import. Furthermore, we emphasized that PRMT5 interacting and methylating SMAD4 was required for TGF-β1-induced epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis, and SMAD4 R361 mutation diminished PRMT5 and TGF-β1-induced metastasis. In addition, highly expressed PRMT5 or high level of SMAD4 R361 methylation indicated worse outcomes in clinical specimens analysis. Collectively, our study highlights the critical interaction of PRMT5 and SMAD4 and the roles of SMAD4 R361 methylation for controlling TGF-β signaling during metastasis. We provided a new insight for SMAD4 activation. And this study indicated that blocking PRMT5-SMAD4 signaling might be an effective targeting strategy in SMAD4 wild-type CRC.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of GI tumour, and it possesses deregulated cellular energetics. Although recent advances in PDAC biology have led to the discovery of recurrent genetic mutations in Kras, TP53 and SMAD4, which are related to this disease, clinical application of the molecular phenotype of PDAC remains challenging.DesignWe combined molecular imaging technology (positron emission tomography/CT) and immunohistochemistry to evaluate the correlation between the maximum standardised uptake value and SMAD4 expression and examined the effect of SMAD4 on glycolysis through in vitro and in vivo experiments. Furthermore, we identified the effect of SMAD4 on metabolic reprogramming by metabolomics and glucose metabolism gene expression analyses. Dual luciferase reporter assays and chromatin immunoprecipitation were performed to identify whether SMAD4 functioned as a transcription factor for phosphoglycerate kinase 1 (PGK1) in PDAC cells. Proliferative and metastatic assays were performed to examine the effect of PGK1 on the malignant behaviour of PDAC.ResultsWe provide compelling evidence that the glycolytic enzyme PGK1 is repressed by transforming growth factor-β/SMAD4. Loss of SMAD4 induces PGK1 upregulation in PDAC, which enhances glycolysis and aggressive tumour behaviour. Notably, in SMAD4-negative PDAC, nuclear PGK1 preferentially drives cell metastasis via mitochondrial oxidative phosphorylation induction, whereas cytoplasmic PGK1 preferentially supports proliferation by functioning as a glycolytic enzyme. The PDAC progression pattern and distinct PGK1 localisation combine to predict overall survival and disease-free survival.ConclusionPGK1 is a decisive oncogene in patients with SMAD4-negative PDAC and can be a target for the development of a therapeutic strategy for SMAD4-negative PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a minimal difference between its incidence rate and mortality rate. Advances in oncology over the past several decades have dramatically improved the overall survival of patients with multiple cancers due to the implementation of new techniques in early diagnosis, therapeutic drugs, and personalized therapy. However, pancreatic cancers remain recalcitrant, with a 5-year relative survival rate of <9%. The lack of measures for early diagnosis, strong resistance to chemotherapy, ineffective adjuvant chemotherapy and the unavailability of molecularly targeted therapy are responsible for the high mortality rate of this notorious disease. Genetically, PDAC progresses as a complex result of the activation of oncogenes and inactivation of tumor suppressors. Although next-generation sequencing has identified numerous new genetic alterations, their clinical implications remain unknown. Classically, oncogenic mutations in genes such as KRAS and loss-of-function mutations in tumor suppressors, such as TP53 , CDNK2A , DPC4 / SMAD4, and BRCA2 , are frequently observed in PDAC. Currently, research on these key driver genes is still the main focus. Therefore, studies assessing the functions of these genes and their potential clinical implications are of paramount importance. In this review, we summarize the biological function of key driver genes and pharmaceutical targets in PDAC. In addition, we conclude the results of molecularly targeted therapies in clinical trials and discuss how to utilize these genetic alterations in further clinical practice.

Cell division cycle associated 7 (CDCA7) is a copy number amplification gene that contributes to the metastasis and invasion of tumors, including esophageal squamous cell carcinoma (ESCC). This present study aimed at clarifying whether high expression of CDCA7 promotes the metastasis and invasion of ESCC cell lines and exploring the underlying mechanisms implicated in epithelial–mesenchymal transition (EMT) of ESCC. The role of CDCA7 in the regulation of ESCC metastasis and invasion was evaluated using ESCC cell lines. Expression of EMT‐related markers including E‐cadherin, N‐cadherin, Vimentin, Snail, and Slug, transforming growth factor β (TGF‐β) signaling pathway including Smad2/3, p‐Smad2/3, Smad4, and Smad7 were detected in CDCA7 knockdown and overexpressed cell lines. Dual‐luciferase reporter assay and rescue assay were used to explore the underlying mechanisms that CDCA7 contributed to the metastasis and invasion of ESCC. High CDCA7 expression significantly promoted the metastasis and invasion of ESCC cell lines both in vivo and in vitro. Additionally, the expression of CDCA7 positively correlated with the expression of N‐cadherin, Vimentin, Snail, Slug, TGF‐β signaling pathway and negatively correlated with the expression of E‐cadherin. Furthermore, CDCA7 transcriptionally regulated the expression of Smad4 and Smad7. Knockdown of CDCA7 inhibited the TGF‐β signaling pathway and therefore inhibited EMT. Our data indicated that CDCA7 was heavily involved in EMT by regulating the expression of Smad4 and Smad7 in TGF‐β signaling pathway. CDCA7 might be a new therapeutic target in the suppression of metastasis and invasion of ESCC. High cell division cycle associated 7 (CDCA7) expression significantly promoted the metastasis and invasion of esophageal squamous cell carcinoma cell lines both in vivo and in vitro. The expression of CDCA7 positively correlated with the expression of N‐cadherin, Vimentin, Snail, Slug, transforming growth factor β signaling pathway and negatively correlated with the expression of E‐cadherin. CDCA7 transcriptionally regulated the expression of Smad4 and Smad7

Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis due to limited therapeutic options. This study examines the roles of genome-wide association study identified PDAC-associated genes as therapeutic targets. We have identified HNF4G gene whose silencing most effectively repressed PDAC cell invasiveness. HNF4G overexpression is induced by the deficiency of transcriptional factor and tumor suppressor SMAD4. Increased HNF4G are correlated with SMAD4 deficiency in PDAC tumor samples and associated with metastasis and poor survival time in xenograft animal model and in patients with PDAC (log-rank P = 0.036; HR= 1.60, 95% CI= 1.03-2.47). We have found that Metformin suppresses HNF4G activity via AMPK-mediated phosphorylation-coupled ubiquitination degradation and inhibits in vitro invasion and in vivo metastasis of PDAC cells with SMAD4 deficiency. Furthermore, Metformin treatment significantly improve clinical outcomes and survival in patients with SMAD4-deficient PDAC (log-rank P = 0.022; HR= 0.31, 95% CI= 0.14-0.68) but not in patients with SMAD4-normal PDAC. Pathway analysis shows that HNF4G may act in PDAC through the cell-cell junction pathway. These results indicate that SMAD4 deficiency-induced overexpression of HNF4G plays a critical oncogenic role in PDAC progression and metastasis but may form a druggable target for Metformin treatment.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy prone to recurrence and metastasis. Studies show that tumor cells with increased invasive and metastatic potential are more likely to undergo ferroptosis. SMAD4 is a critical molecule in the transforming growth factor β (TGF-β) pathway, which affects the TGF-β-induced epithelial-mesenchymal transition (EMT) status. SMAD4 loss is observed in more than half of patients with PDAC. In this study, we investigated whether SMAD4-positive PDAC cells were prone to ferroptosis because of their high invasiveness. We showed that SMAD4 status almost determined the orientation of transforming growth factor β1 (TGF-β1)-induced EMT via the SMAD4-dependent canonical pathway in PDAC, which altered ferroptosis vulnerability. We identified glutathione peroxidase 4 (GPX4), which inhibited ferroptosis, as a SMAD4 down-regulated gene by RNA sequencing. We found that SMAD4 bound to the promoter of GPX4 and decreased GPX4 transcription in PDAC. Furthermore, TGF-β1-induced high invasiveness enhanced sensitivity of SMAD4-positive organoids and pancreas xenograft models to the ferroptosis inducer RAS-selective lethal 3 (RSL3). Moreover, SMAD4 enhanced the cytotoxic effect of gemcitabine combined with RSL3 in highly invasive PDAC cells. This study provides new ideas for the treatment of PDAC, especially SMAD4-positive PDAC.