TGF-β signaling regulates cellular processes such as proliferation, differentiation and apoptosis through activation of SMAD transcription factors that are in turn modulated by members of the Ski-SnoN family. In this process, Ski has been shown to negatively modulate TGF-β signaling by disrupting active R-SMAD/Co-SMAD heteromers. Here, we show that the related regulator SnoN forms a stable complex with the R-SMAD (SMAD3) and the Co-SMAD (SMAD4). To rationalize this stabilization at the molecular level, we determined the crystal structure of a complex between the SAND domain of SnoN and the MH2-domain of SMAD4. This structure shows a binding mode that is compatible with simultaneous coordination of R-SMADs. Our results show that SnoN, and SMAD heteromers can form a joint structural core for the binding of other transcription modulators. The results are of fundamental importance for our understanding of the molecular mechanisms behind the modulation of TGF-β signaling.

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.

Hepatocellular carcinoma (HCC) has emerged as one of the most prevalent life-threatening cancers, and the high mortality rate is largely due to the metastasis. The sustained activation of Smad4 and transforming growth factor-β (TGF-β) is closely associated with advanced HCC metastasis. However, the regulatory mechanism underlying the aberrant activation of Smad4 and TGF-β pathway remains elusive. In this study, using a functional screen of USPs siRNA library, we identified ubiquitin-specific proteases USP10 as a deubiquitinating enzyme (DUB) that sustains the protein level of Smad4 and activates TGF-β signaling. Further analysis showed that USP10 directly interacts with Smad4 and stabilizes it through the cleavage of its proteolytic ubiquitination, thus promoting HCC metastasis. The suppression of USP10 by either shRNAs or catalytic inhibitor Spautin-1 significantly inhibited the migration of HCC cells, whereas the reconstitution of Smad4 was able to efficiently rescue this defect. Overall, our study not only uncovers the regulatory effect of USP10 on the protein abundance of Smad4, but also indicates that USP10 could be regarded as a potential intervention target for the metastatic HCC in Smad4-positive patients.

Loss of muscle mass and strength are important sequelae of chronic disease, but the response of individuals is remarkably variable, suggesting important genetic and epigenetic modulators of muscle homeostasis. Such factors are likely to modify the activity of pathways that regulate wasting, but to date, few such factors have been identified.

Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-of-function tumor suppressors remains challenging. To address this gap, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4 as an example. SMAD4, a central mediator of the TGF-β pathway, is recurrently mutated in many tumors. Here, we report the development of a TR-FRET technology that recapitulated the dynamic differential interaction of SMAD4 and SMAD4R361H with SMAD3 and identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4R361H/SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4R361H-mediated transcriptional activity and restored TGF-β-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer illustrates a general strategy to reverse the lost function of tumor suppressors with hypomorph mutations and supports a systematic approach to develop small-molecule protein-protein interaction (PPI) molecular glues for biological insights and therapeutic discovery.

Colorectal cancer is a common health-threatening tumor within the gastrointestinal tract. The aim of this study was to test the biological role of DUSP4 in colorectal cancer cells. In our study, DUSP4 overexpression-treated HCT116 cells and DUSP4 knockdown-treated SW480 cells were selected to perform study. Quantitative real-time PCR test (qRT-PCR) and western blot were used to detect DUSP4 abundance in clinical tissues and six cell lines, as well as ubiquitin-related Smad4 degradation. Western blot, migration and invasion. were used to assess the relationships between DUSP4 and Smad4. Higher DUSP4 expression of functional significance was observed in colorectal cancer tissues and cells. The results showed that both treatments could affect the proliferation, colony formation, migration, invasion of tumor cells, and the expression of epithelial mesenchymal transformation (EMT)-associated biomarkers. Moreover, in colorectal cancer cells, DUSP4 could promote the Smad4 degradation by regulating ubiquitin-related Smad4 degradation, and promote the cell proliferation, migration and invasion by regulating Smad4 degradation via Smad4 gene. Meanwhile, DUSP4 can directly deubiquitinate and stabilize Smad4 protein, hence further promote proliferation and metastasis of colorectal cancer cells.

Bone morphogenetic proteins (BMP), a member of the TGF-beta superfamily, have broad activities in regulating various kinds of cellular behaviors. Previously, we have demonstrated that BMP-4 is up-regulated in human colonic adenocarcinoma and promotes the invasive phenotypes of human colorectal cancer HCT116 cells. Smad4 is a central mediator in BMP signaling pathway and it is frequently mutated in metastatic colorectal cancers. To address whether Smad4 was required for enhancing metastatic potentials by BMP-4 in colorectal cancer, we generated BMP-4 overexpressing clones from Smad4-deficient SW480 cells. The growth rate of BMP-4 overexpressing cells was slightly higher than that of empty-vector controls. Overexpression of BMP-4 resulted in an increased expression of vimentin, a marker of epithelial-mesenchymal transition, and caused the changes of cell morphology, spreading and formation of focal adhesions. BMP-4 overexpressing cells increased cell adhesion on fibronectin and collagen, and augmented cell migration and invasion potentials in comparison to empty-vector controls. The induction of cell migration by BMP-4 overexpression was inhibited by BMP-4 siRNA. To further identify the target genes of the elevated BMP-4 signaling in SW480 cells, an oligonucleotide microarray was performed. Among 46,000 genes, 91 genes (65 up-regulated and 26 down-regulated) with 2-fold difference have been identified between BMP-4 overexpressing and empty-vector cells. This study demonstrates that Smad4 is dispensable for enhanced invasiveness of human colorectal cancer cells due to BMP-4 overexpression.

Growth and metastasis of colorectal cancer is closely connected to the biosynthetic capacity of tumor cells, and colorectal cancer stem cells that reside at the top of the intratumoral hierarchy are especially dependent on this feature. By performing disease modeling on patient-derived tumor organoids, we found that elevated expression of the ribosome biogenesis factor NLE1 occurs upon SMAD4 loss in TGFβ1-exposed colorectal cancer organoids. TGFβ signaling-mediated downregulation of NLE1 was prevented by ectopic expression of c-MYC, which occupied an E-box-containing region within the NLE1 promoter. Elevated levels of NLE1 were found in colorectal cancer cohorts compared with normal tissues and in colorectal cancer subtypes characterized by Wnt/MYC and intestinal stem cell gene expression. In colorectal cancer cells and organoids, NLE1 was limiting for de novo protein biosynthesis. Upon NLE1 ablation, colorectal cancer cell lines activated p38/MAPK signaling, accumulated p62- and LC3-positive structures indicative of impaired autophagy, and displayed more reactive oxygen species. Phenotypically, knockout of NLE1 inhibit.ed proliferation, migration and invasion, clonogenicity, and anchorage-independent growth. NLE1 loss also increased the fraction of apoptotic tumor cells, and deletion of TP53 further sensitized NLE1-deficient colorectal cancer cells to apoptosis. In an endoscopy-guided orthotopic mouse transplantation model, ablation of NLE1 impaired tumor growth in the colon and reduced primary tumor-derived liver metastasis. In patients with colorectal cancer, NLE1 mRNA levels predicted overall and relapse-free survival. Taken together, these data reveal a critical role of NLE1 in colorectal cancer growth and progression and suggest that NLE1 represents a potential therapeutic target in colorectal cancer patients.

Pokemon, also known as ZBTB7A, belongs to the POZ and Krüppel (POK) family of transcription repressors and is implicated in tumor progression as a key proto-oncogene. This present study aimed at determining the mechanism by which Pokemon inhibits transforming growth factor β (TGFβ)-Smad4 pathway-dependent proliferation arrest of breast cancer cells via specificity protein 1 (SP1).

SMAD4 is frequently inactivated and associated with a poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Abnormal SMAD4 expression also plays an important role in the malignant progression of PDAC.

Smad4 protein, whose gene is coded at chromosome 18q21.1, is an important tumour suppressor that mediates transforming growth factor-beta. It has been reported that inactivation of the Smad4 gene and allelic loss of chromosome 18q correlate with liver metastasis and poorer prognosis in colorectal cancers. Utilising a recently developed method of immunohistochemical staining for Smad4 protein, we focused on the specific impact of Smad4 protein expression on liver metastasis in colorectal cancer. We also evaluated the association between chromosome18q deletion and liver metastasis. We selected 20 colorectal cancers with liver metastasis for the experimental group, and 20 cases without liver metastasis for the control. In order to exclude the influence of lymph node metastasis, all cases were lymph node negative. In addition, the two groups were matched for tumour depth, tumour differentiation and tumour location. We compared the expression level of Smad4 protein immunohistochemically in these 20 matched pairs. We also compared the loss of heterozygosity status at chromosome 18q in these 20 matched pairs. Immunohistochemical staining revealed a significant difference (P = 0.024) in the level of Smad4 protein between the two groups. We also observed a significantly different (P=0.0054) ratio of allelic deletion at chromosome 18q21. Smad4 protein expression level and allelic loss at 18q21 are associated with the process of liver metastasis in colorectal cancers evaluated when excluding clinical and pathological features except for liver metastasis.

Androgens and androgen-related molecules exert a plethora of functions across different tissues, mainly through binding to the transcription factor androgen receptor (AR). Despite widespread therapeutic use and misuse of androgens as potent anabolic agents, the molecular mechanisms of this effect on skeletal muscle are currently unknown. Muscle mass in adulthood is mainly regulated by the bone morphogenetic protein (BMP) axis of the transforming growth factor (TGF)-β pathway via recruitment of mothers against decapentaplegic homolog 4 (SMAD4) protein. Here we show that, upon activation, AR forms a transcriptional complex with SMAD4 to orchestrate a muscle hypertrophy programme by modulating SMAD4 chromatin binding dynamics and enhancing its transactivation activity. We challenged this mechanism of action using spinal and bulbar muscular atrophy (SBMA) as a model of study. This adult-onset neuromuscular disease is caused by a polyglutamine expansion (polyQ) in AR and is characterized by progressive muscle weakness and atrophy secondary to a combination of lower motor neuron degeneration and primary muscle atrophy. Here we found that the presence of an elongated polyQ tract impairs AR cooperativity with SMAD4, leading to an inability to mount an effective anti-atrophy gene expression programme in skeletal muscle in response to denervation. Furthermore, adeno-associated virus, serotype 9 (AAV9)-mediated muscle-restricted delivery of BMP7 is able to rescue the muscle atrophy in SBMA mice, supporting the development of treatments able to fine-tune AR-SMAD4 transcriptional cooperativity as a promising target for SBMA and other conditions associated with muscle loss.

The TGF-β and Wnt signaling pathways are interrelated in many cell types and tissues, and control cell functions in coordination. Here, we report that SMAD4, a downstream effector of the TGF-β signaling pathway, induces FZD4, a receptor of the Wnt signaling pathway, establishing a novel route of communication between these two pathways in granulosa cells (GCs). We found that SMAD4 is a strong inducer of FZD4, not only initiating FZD4 transcription but also activating FZD4-dependent Wnt signaling and GC apoptosis. Furthermore, we identified the direct and indirect mechanisms by which SMAD4 promotes expression of FZD4 in GCs. First, SMAD4 functions as a transcription factor to directly bind to the FZD4 promoter region to increase its transcriptional activity. Second, SMAD4 promotes production of SDNOR, a novel lncRNA that acts as a sponge for miR-29c, providing another mean to block miR-29c from degenerating FZD4 mRNA. Overall, our findings not only reveal a new channel of crosstalk between the TGF-β and Wnt signaling pathways, SMAD4-FZD4 axis, but also provide new insights into the regulatory network of GC apoptosis and follicular atresia. These RNA molecules, such as miR-29c and lnc-SDNOR, represent potential targets for treatment of reproductive diseases and improvement of female fertility.

BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5' RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region -405 to -200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5' regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.

Bcl-2-modifying factor (Bmf) functions to mediate follicular atresia and oocyte growth in mice. It has been proven that TGF-β can induce Bmf expression via the Smad4 pathway in a variety of cells, and then induce cell apoptosis. Based on this, we hypothesized that Smad4 and Bmf may play important roles in the apoptosis of granulosa cells (GCs) in domestic animals. This study used small-tailed Han sheep follicular GCs cultured in vitro as a model system, and overexpression or interference experiments, to explore the biological roles of Bmf and reveal the preliminary regulatory mechanisms between Smad4 and Bmf in the process of GCs' apoptosis. We found that the proliferation rate of sheep GCs was significantly increased after the knockdown of Bmf, whereas overexpressing Bmf increased the apoptosis rate of GCs, results also verified by the expression patterns of PCNA, Bcl-2, and Bax genes. After the Smad4 knockdown, the apoptosis rate of GCs was increased, while the mRNA and protein expression of Bmf was significantly up-regulated. A rescue experiment verified that the Bmf knockdown could alleviate GCs' apoptosis induced by Smad4 knockdown. In conclusion, our study not only elucidated an important role for Bmf in the apoptosis of sheep GCs but also revealed a new regulatory pathway between Smad4 and Bmf in this process.

Small mothers against decapentaplegic (SMAD)4 and SMAD7 are key regulatory components in the immunosuppressive transforming growth factor- (TGF-) β signaling pathway, which is defective in inflammatory bowel disease (IBD). SMAD4 may play an important role in the pathogenesis of IBD as indicated in experimental models of colitis.

Variants in SMAD4 or BMPR1A cause juvenile polyposis syndrome, a rare autosomal dominant condition characterized by multiple gastrointestinal hamartomatous polyps. A phenotype of attenuated adenomatous polyposis without hamartomatous polyps is rare.

TGF-β family signaling pathways, including TGF-β and BMP pathways, are widely involved in the regulation of health and diseases through downstream SMADs, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. However, it is still unclear whether R-SMADs or Co-SMAD can feedback regulate the TGF-β family signaling pathways in granulosa cells (GCs). In this study, we report a novel mechanism underlying the feedback regulation of TGF-β family signaling pathways, i.e., SMAD4, the only Co-SMAD, positive feedback activates the TGF-β family signaling pathways in GCs with a basal level of TGF-β ligands by interacting with the core promoters of its upstream receptors. Mechanistically, SMAD4 acts as a transcription factor, and feedback activates the transcription of its upstream receptors, including ACVR1B, BMPR2, and TGFBR2, of the canonical TGF-β signaling pathways by interacting with three coactivators (c-JUN, CREB1, and SP1), respectively. Notably, three different interaction modes between SMAD4 and coactivators were identified in SMAD4-mediated feedback regulation of upstream receptors through reciprocal ChIP assays. Our findings in the present study indicate for the first time that SMAD4 feedback activates the canonical TGF-β family signaling pathways in GCs, which improves and expands the regulatory mechanism, especially the feedback regulation modes of TGF-β family signaling pathways in ovarian GCs.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-β (TGF-β). TGF-β is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3β which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-β-responsive SMAD binding element (SBE). This study's findings imply that cellular O-GlcNAc may regulate the TGF-β/SMAD signaling pathway by stabilizing SMAD4.

Transcripts with alternative 5'-untranslated regions (UTRs) result from the activity of alternative promoters and they can determine gene expression by influencing its stability and translational efficiency, thus executing complex regulation of developmental, physiological and pathological processes. Transcriptional regulation of human SMAD4, a key tumor suppressor deregulated in most gastrointestinal cancers, entails four alternative promoters. These promoters and alternative transcripts they generate remain unexplored as contributors to the SMAD4 deregulation in cancer. The aim of this study was to investigate the relative abundance of the transcript SMAD4-201 in colorectal cell lines and tissues in order to establish if its fluctuations may be associated with colorectal cancer (CRC).