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1.
J Transl Med ; 22(1): 440, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38720358

ABSTRACT

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.


Subject(s)
Fibroblasts , Fibrosis , Filtering Surgery , Glaucoma , MicroRNAs , Rats, Sprague-Dawley , Animals , MicroRNAs/metabolism , MicroRNAs/genetics , Glaucoma/pathology , Glaucoma/genetics , Filtering Surgery/adverse effects , Fibroblasts/metabolism , Male , Tenon Capsule/metabolism , Tenon Capsule/pathology , Cell Proliferation/drug effects , Transforming Growth Factor beta1/metabolism , Rats , Smad4 Protein/metabolism , Smad4 Protein/genetics , NF-kappa B/metabolism , Mitomycin/pharmacology , Mitomycin/therapeutic use , Gene Expression Regulation
2.
Int J Mol Sci ; 25(9)2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38731813

ABSTRACT

Increased expression and nuclear translocation of ß-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting ß-CATENIN are not as efficient as desired. Therefore, detailed understanding of ß-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing ß-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of ß-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce ß-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces ß-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on ß-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced ß-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding ß-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.


Subject(s)
Breast Neoplasms , Protein Stability , beta Catenin , Humans , beta Catenin/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Phosphorylation , Female , Cell Line, Tumor , Smad4 Protein/metabolism , Smad4 Protein/genetics , Gene Expression Regulation, Neoplastic , MCF-7 Cells , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Protein 2/metabolism
3.
Oncol Res ; 32(5): 999-1009, 2024.
Article in English | MEDLINE | ID: mdl-38686046

ABSTRACT

Background: EBV-miR-BARTs exhibit significant relevance in epithelial tumors, particularly in EBV-associated gastric and nasopharyngeal cancers. However, their specific mechanisms in the initiation and progression of gastric cancer remain insufficiently explored. Material and Methods: Initially, EBV-miRNA-BART6-5p and its target gene SMAD4 expression were assessed in EBV-associated gastric cancer tissues and cell lines. Subsequent transfection induced overexpression of EBV-miRNA-BART6-5p in AGS and MKN-45, and downregulation in EBV-positive cells (SUN-719). The subsequent evaluation aimed to observe their impact on gastric cancer cell proliferation, migration, and glycolytic processes, with the TGF-ß/SMAD4 signaling pathway value clarified using a TGF-ß inhibitor. Results: EBV-miRNA-BART6-5p exhibits pronounced upregulation in EBV-associated gastric cancer tissues and EBV-positive cells, while its target gene SMAD4 demonstrates downregulated expression. Upregulation of it can promote the proliferation and migration of gastric cancer cells. Additionally, We found EBV-miRNA-BART6-5p promotes glycolysis of gastric cancer cells. Inhibition of the TGF-ß/SMAD4 signaling pathway resulted in suppressed proliferation and migration of gastric cancer cells, concomitant with a diminished glycolytic capacity. Conclusion: In this study, we found that EBV-miRNA-BART6-5p can target SMAD4, effectively increasing glycolysis in gastric cancer cells by regulating the TGF-ß/SMAD4 signaling pathway, thereby enhancing the proliferation and metastasis of gastric cancer cells. Our findings may offer new insights into the metabolic aspects of gastric cancer.


Subject(s)
Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glycolysis , Herpesvirus 4, Human , MicroRNAs , Signal Transduction , Smad4 Protein , Stomach Neoplasms , Transforming Growth Factor beta , Humans , Stomach Neoplasms/pathology , Stomach Neoplasms/virology , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Smad4 Protein/genetics , Smad4 Protein/metabolism , MicroRNAs/genetics , Glycolysis/genetics , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , Herpesvirus 4, Human/genetics , Cell Line, Tumor , Cell Movement/genetics , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/pathology , Neoplasm Metastasis , RNA, Viral/genetics
4.
Cell Commun Signal ; 22(1): 248, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38689334

ABSTRACT

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.


Subject(s)
Bone Morphogenetic Protein 4 , Breast Neoplasms , Neoplasm Metastasis , Signal Transduction , Smad4 Protein , Smad4 Protein/genetics , Smad4 Protein/metabolism , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Humans , Animals , Female , Cell Line, Tumor , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Mice , Cell Proliferation/genetics
5.
Biochem Biophys Res Commun ; 715: 150007, 2024 Jun 30.
Article in English | MEDLINE | ID: mdl-38678783

ABSTRACT

Smad4, a critical mediator of TGF-ß signaling, plays a pivotal role in regulating various cellular functions, including immune responses. In this study, we investigated the impact of Smad4 knockout specifically in macrophages on anti-tumor immunity, focusing on lung metastasis of B16 melanoma cells. Using a mouse model with Smad4 knockout in macrophages established via Lyz2-cre mice and Smad4 flox/flox mice, we demonstrated a significant inhibition of B16 metastasis in the lungs. Interestingly, the inhibition of tumor growth was found to be independent of adaptive immunity, as no significant changes were observed in the numbers or activities of T cells, B cells, or NK cells. Instead, Smad4 knockout led to the emergence of an MCHIIlow CD206high subset of lung interstitial macrophages, characterized by enhanced phagocytosis function. Our findings highlight the crucial role of Smad4 in modulating the innate immune response against tumors and provide insights into potential therapeutic strategies targeting lung interstitial macrophages to enhance anti-tumor immunity.


Subject(s)
Lung Neoplasms , Melanoma, Experimental , Mice, Inbred C57BL , Mice, Knockout , Phagocytosis , Smad4 Protein , Animals , Smad4 Protein/deficiency , Smad4 Protein/genetics , Smad4 Protein/metabolism , Lung Neoplasms/secondary , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Melanoma, Experimental/pathology , Melanoma, Experimental/immunology , Mice , Macrophages/immunology , Macrophages/metabolism , Lung/pathology , Lung/immunology , Lung/metabolism , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/immunology , Macrophages, Alveolar/pathology , Cell Line, Tumor
6.
Stem Cell Reports ; 19(5): 618-628, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38579708

ABSTRACT

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.


Subject(s)
Enhancer Elements, Genetic , Mesoderm , Neural Stem Cells , SOXB1 Transcription Factors , SOXB1 Transcription Factors/metabolism , SOXB1 Transcription Factors/genetics , Animals , Mice , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Mesoderm/cytology , Mesoderm/metabolism , Neurogenesis , Gene Expression Regulation, Developmental , Octamer Transcription Factor-3/metabolism , Octamer Transcription Factor-3/genetics , Cell Differentiation/genetics , Nanog Homeobox Protein/metabolism , Nanog Homeobox Protein/genetics , Cell Lineage/genetics , Smad4 Protein/metabolism , Smad4 Protein/genetics , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , Chromatin/metabolism , Protein Binding
7.
Genomics ; 116(3): 110809, 2024 May.
Article in English | MEDLINE | ID: mdl-38492821

ABSTRACT

Colorectal cancer (CRC) is the third most common cancer and the prevalence rate of CRC is increasing in the China. In this study, whole-exome sequencing (WES) was performed on primary tissues of 47 CRC Chinese patients including 22 metastatic and 25 non-metastatic patients. By comparison with data from western colorectal cancer patients in the Cancer Genome Atlas (TCGA), we identified a number of genes that are more likely to be mutated in Chinese colorectal cancer patients, such as MUC12, MUC12, MUC2, MUC4, HYDIN and KMT2C. Interestingly, MUC family genes including MUC12, MUC2 and MUC4, have mutation rates of >20%, while the mutation frequency was extremely low in western colorectal cancer patients, which were <3% in TCGA and 0% in Memorial Sloan Kettering Cancer Center (MSKCC). We detected metastasis-specific mutated genes including TCF7L2, MST1L, HRNR and SMAD4, while MUC4, NEB, FLG and RFPL4A alteration is more prevalent in the non-metastasis group. Further analysis reveals mutation genes in metastasis group are more focus in the Wnt and Hippo signaling pathway. APC, SMAD4 and TCF7L2 accounted for the major genetic abnormalities in this pathway. In conclusion, this study identified the unique characteristics of gene mutations in Chinese patients with colorectal cancer, and is a valuable reference for personalized treatment in Chinese CRC patients.


Subject(s)
Colorectal Neoplasms , Exome Sequencing , Mutation , Neoplasm Metastasis , Humans , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Male , Female , Middle Aged , Aged , Adult , Hippo Signaling Pathway , Mucin-4/genetics , Mucin-4/metabolism , China , Mucin-2/genetics , Mucin-2/metabolism , Smad4 Protein/genetics , Smad4 Protein/metabolism , Filaggrin Proteins , Protein Serine-Threonine Kinases/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Wnt Signaling Pathway , Asian People/genetics , East Asian People , DNA-Binding Proteins
8.
Biol Direct ; 19(1): 24, 2024 Mar 19.
Article in English | MEDLINE | ID: mdl-38504385

ABSTRACT

BACKGROUND: Pancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis. METHODS: The expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People's Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied. RESULTS: LINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models. CONCLUSION: We demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Animals , Humans , Mice , Carcinogenesis/genetics , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Pancreatic Neoplasms/genetics , Phenotype , Smad4 Protein/genetics , Smad4 Protein/metabolism , RNA, Untranslated/genetics
9.
Sci Rep ; 14(1): 6769, 2024 03 21.
Article in English | MEDLINE | ID: mdl-38514720

ABSTRACT

Breast cancer is a serious threat to human health. The transforming growth factor-ß signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-ß signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-ß signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.


Subject(s)
Breast Neoplasms , Trans-Activators , Humans , Animals , Mice , Female , Trans-Activators/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Signal Transduction , Smad4 Protein/genetics , Smad4 Protein/metabolism , Smad2 Protein/genetics , Smad2 Protein/metabolism , Transforming Growth Factor beta/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism , Smad Proteins/genetics , Smad Proteins/metabolism
10.
Cell Rep ; 43(2): 113714, 2024 Feb 27.
Article in English | MEDLINE | ID: mdl-38306271

ABSTRACT

Drug resistance is the leading problem in non-small-cell lung cancer (NSCLC) therapy. The contribution of histone methylation in mediating malignant phenotypes of NSCLC is well known. However, the role of histone methylation in NSCLC drug-resistance mechanisms remains unclear. Here, our data show that EZH2 and G9a, two histone methyltransferases, are involved in the drug resistance of NSCLC. Gene manipulation results indicate that the combination of EZH2 and G9a promotes tumor growth and mediates drug resistance in a complementary manner. Importantly, clinical study demonstrates that co-expression of both enzymes predicts a poor outcome in patients with NSCLC. Mechanistically, G9a and EZH2 interact and promote the silencing of the tumor-suppressor gene SMAD4, activating the ERK/c-Myc signaling pathway. Finally, SU08, a compound targeting both EZH2 and G9a, is demonstrated to sensitize resistant cells to therapeutic drugs by regulating the SMAD4/ERK/c-Myc signaling axis. These findings uncover the resistance mechanism and a strategy for reversing NSCLC drug resistance.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Signal Transduction , Proto-Oncogene Proteins c-myc/genetics , Histones , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Smad4 Protein/genetics , Enhancer of Zeste Homolog 2 Protein
11.
Nucleic Acids Res ; 52(9): 4935-4949, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38421638

ABSTRACT

TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.


Subject(s)
Cell Differentiation , Histone Acetyltransferases , Mesoderm , Signal Transduction , Smad4 Protein , Humans , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Protein 4/genetics , Cell Line , Chromatin/metabolism , Endoderm/cytology , Endoderm/metabolism , Histone Acetyltransferases/metabolism , Histone Acetyltransferases/genetics , Histones/metabolism , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Mesoderm/metabolism , Mesoderm/cytology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Smad4 Protein/metabolism , Smad4 Protein/genetics , Transforming Growth Factor beta/metabolism
12.
Int J Cancer ; 155(1): 40-53, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38376070

ABSTRACT

Rectal cancer poses challenges in preoperative treatment response, with up to 30% achieving a complete response (CR). Personalized treatment relies on accurate identification of responders at diagnosis. This study aimed to unravel CR determinants, overall survival (OS), and time to recurrence (TTR) using clinical and targeted sequencing data. Analyzing 402 patients undergoing preoperative treatment, tumor stage, size, and treatment emerged as robust response predictors. CR rates were higher in smaller, early-stage, and intensively treated tumors. Targeted sequencing analyzed 216 cases, while 120 patients provided hotspot mutation data. KRAS mutation dramatically reduced CR odds by over 50% (odds ratio [OR] = 0.3 in the targeted sequencing and OR = 0.4 hotspot cohorts, respectively). In contrast, SMAD4 and SYNE1 mutations were associated with higher CR rates (OR = 6.0 and 6.8, respectively). Favorable OS was linked to younger age, CR, and low baseline carcinoembryonic antigen levels. Notably, CR and an APC mutation increased TTR, while a BRAF mutation negatively affected TTR. Beyond tumor burden, SMAD4 and SYNE1 mutations significantly influenced CR. KRAS mutations independently correlated with radiotherapy resistance, and BRAF mutations heightened recurrence risk. Intriguingly, non-responding tumors with initially small sizes carried a higher risk of recurrence. The findings, even if limited in addition to the imperfect clinical factors, offer insights into rectal cancer treatment response, guiding personalized therapeutic strategies. By uncovering factors impacting CR, OS, and TTR, this study underscores the importance of tailored approaches for rectal cancer patients. These findings, based on extensive analysis and mutation data, pave the way for personalized interventions, optimizing outcomes in the challenges of rectal cancer preoperative treatment.


Subject(s)
Mutation , Neoadjuvant Therapy , Neoplasm Recurrence, Local , Rectal Neoplasms , Smad4 Protein , Humans , Rectal Neoplasms/genetics , Rectal Neoplasms/therapy , Rectal Neoplasms/pathology , Rectal Neoplasms/mortality , Male , Female , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoadjuvant Therapy/methods , Aged , Smad4 Protein/genetics , Adult , Proto-Oncogene Proteins p21(ras)/genetics , Nerve Tissue Proteins/genetics , Chemoradiotherapy/methods , Aged, 80 and over , Treatment Outcome , Biomarkers, Tumor/genetics , Cytoskeletal Proteins/genetics , Nuclear Proteins/genetics
13.
Cell Commun Signal ; 22(1): 45, 2024 01 17.
Article in English | MEDLINE | ID: mdl-38233864

ABSTRACT

OBJECTIVES: Histological transformation to small cell lung cancer (SCLC) has been identified as a mechanism of TKIs resistance in EGFR-mutant non-small cell lung cancer (NSCLC). We aim to explore the prevalence of transformation in EGFR-wildtype NSCLC and the mechanism of SCLC transformation, which are rarely understood. METHODS: We reviewed 1474 NSCLC patients to investigate the NSCLC-to-SCLC transformed cases and the basic clinical characteristics, driver gene status and disease course of them. To explore the potential functional genes in SCLC transformation, we obtained pre- and post-transformation specimens and subjected them to a multigene NGS panel involving 416 cancer-related genes. To validate the putative gene function, we established knocked-out models by CRISPR-Cas 9 in HCC827 and A549-TP53-/- cells and investigated the effects on tumor growth, drug sensitivity and neuroendocrine phenotype in vitro and in vivo. We also detected the expression level of protein and mRNA to explore the molecular mechanism involved. RESULTS: We firstly reported an incidence rate of 9.73% (11/113) of SCLC transformation in EGFR-wildtype NSCLC and demonstrated that SCLC transformation is irrespective of EGFR mutation status (P = 0.16). We sequenced 8 paired tumors and identified a series of mutant genes specially in transformed SCLC such as SMAD4, RICTOR and RET. We firstly demonstrated that SMAD4 deficiency can accelerate SCLC transition by inducing neuroendocrine phenotype regardless of RB1 status in TP53-deficient NSCLC cells. Further mechanical experiments identified the SMAD4 can regulate ASCL1 transcription competitively with Myc in NSCLC cells and Myc inhibitor acts as a potential subsequent treatment agent. CONCLUSIONS: Transformation to SCLC is irrespective of EFGR status and can be accelerated by SMAD4 in non-small cell lung cancer. Myc inhibitor acts as a potential therapeutic drug for SMAD4-mediated resistant lung cancer. Video Abstract.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Small Cell Lung Carcinoma , Humans , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/therapeutic use , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , ErbB Receptors/genetics , Lung Neoplasms/pathology , Mutation/genetics , Protein Kinase Inhibitors/pharmacology , Retinoblastoma Binding Proteins/genetics , Smad4 Protein/genetics , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/pathology , Ubiquitin-Protein Ligases/genetics
14.
Transl Res ; 268: 13-27, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38286358

ABSTRACT

Inflammation is a crucial pathophysiological mechanism in atherosclerosis (AS). This study aims to investigate the impact of sulfotransferase family 2b member 1 (SULT2B1) on the inflammatory response of macrophages and the progression of AS. Here, we reported that SULT2B1 expression increased with the progression of AS. In AS model mice, knockdown of Sult2b1 led to remission of AS and reduced inflammation levels. Further exploration of the downstream molecular mechanisms of SULT2B1 revealed that suppressing Sult2b1 in macrophages resulted in decreased levels of 25HC3S in the nucleus, elevated expression of Lxr, and increased the transcription of Lncgga3-204. In vivo, knockdown of Lncgga3-204 aggravated the inflammatory response and AS progression, while the simultaneous knockdown of both Sult2b1 and Lncgga3-204 exacerbated AS and the inflammatory response compared with knockdown of Sult2b1 alone. Increased binding of Lncgga3-204 to SMAD4 in response to oxidized-low density lipoprotein (ox-LDL) stimulation facilitated SMAD4 entry into the nucleus and regulated Smad7 transcription, which elevated SMAD7 expression, suppressed NF-κB entry into the nucleus, and ultimately attenuated the macrophage inflammatory response. Finally, we identified the presence of a single nucleotide polymorphism (SNP), rs2665580, in the SULT2B1 promoter region in monocytes from coronary artery disease (CAD) patients. The predominant GG/AG/AA genotypes were observed in the Asian population. Elevated SULT2B1 expression in monocytes with GG corresponded to elevated inflammatory factor levels and more unstable coronary plaques. To summarize, our study demonstrated that the critical role of SULT2B1/Lncgga3-204/SMAD4/NF-κB in AS progression. SULT2B1 serves as a novel biomarker indicating inflammatory status, thereby offering insights into potential therapeutic strategies for AS.


Subject(s)
Atherosclerosis , Disease Progression , Inflammation , Macrophages , Smad4 Protein , Sulfotransferases , Atherosclerosis/metabolism , Atherosclerosis/genetics , Atherosclerosis/pathology , Sulfotransferases/genetics , Sulfotransferases/metabolism , Animals , Mice , Macrophages/metabolism , Humans , Inflammation/metabolism , Inflammation/pathology , Smad4 Protein/metabolism , Smad4 Protein/genetics , Male , Mice, Inbred C57BL , Female
15.
Acta Pharmacol Sin ; 45(4): 844-856, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38057506

ABSTRACT

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 ß (TGF-ß) pathway, which affects the TGF-ß-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 ß1 (TGF-ß1)-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-ß1-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.


Subject(s)
Carcinoma, Pancreatic Ductal , Ferroptosis , Pancreatic Neoplasms , Smad4 Protein , Transforming Growth Factor beta1 , Humans , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Smad4 Protein/genetics , Smad4 Protein/metabolism , Transforming Growth Factor beta1/metabolism
16.
Clin J Gastroenterol ; 17(1): 23-28, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37950802

ABSTRACT

A 50-year-old female was diagnosed with gastric hyperplastic polyps 7 years before and was followed up at another hospital. She was referred to our hospital because of the growth of gastric polyps and progression of anemia. She had no family history of polyposis. The polyps were observed only in the stomach, increased in size and number, and the erythematous edema got worse. Endoscopic mucosal resection (EMR) of the gastric polyp was performed. Pathologically, the gastric polyp was hamartomatous polyp, and the intervening mucosa between polyps showed no atypical structure without inflammation. Given that gastric juvenile polyposis (GJP) was clinically suspected, a genetic test using peripheral blood was performed. Target resequencing and Sanger sequencing analysis revealed a nonsense mutation in the SMAD4 gene at codon 169. The mutation was detected at a low frequency of 11%, and considered a mosaic mutation. Therefore, she was diagnosed with a sporadic GJP, and total gastrectomy was performed. Immunostaining of SMAD4 for the resected specimen showed a mixture of stained and unstained area in the epithelium of the polyp, indicating partial loss of SMAD4 expression. To our knowledge, this is the first reported case of GJP with a nonsense SMAD4 mutation at codon 169 in a mosaic pattern.


Subject(s)
Adenomatous Polyps , Intestinal Polyposis , Neoplastic Syndromes, Hereditary , Polyps , Stomach Neoplasms , Female , Humans , Middle Aged , Codon, Nonsense , Stomach Neoplasms/genetics , Stomach Neoplasms/surgery , Stomach Neoplasms/diagnosis , Smad4 Protein/genetics , Smad4 Protein/metabolism
18.
Gastroenterology ; 166(2): 298-312.e14, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37913894

ABSTRACT

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Gemcitabine , Cell Line, Tumor , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Smad4 Protein/genetics , Smad4 Protein/metabolism , Mitogen-Activated Protein Kinases/metabolism , Smad3 Protein/metabolism
19.
Cancer ; 130(3): 476-484, 2024 02 01.
Article in English | MEDLINE | ID: mdl-37823514

ABSTRACT

BACKGROUND: In select patients, pancreatic adenocarcinoma remains a local disease, yet there are no validated biomarkers to predict this behavior and who may benefit from aggressive local treatments. This study sought to determine if SMAD4 (mothers against decapentaplegic homolog 4) messenger RNA-sequencing (RNA-seq) expression is a robust method for predicting overall survival (OS) and distant metastasis-free survival (DMFS) in patients with resected pancreatic adenocarcinoma. METHODS: Utilizing The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), 322 patients with resected stage I-III pancreatic adenocarcinoma were identified. In TCGA, multivariable proportional hazards models were used to determine the association of SMAD4 genomic aberrations and RNA-seq expression with OS and DMFS. In the ICGC, analysis sought to confirm the predictive performance of RNA-seq via multivariable models and receiver operator characteristic curves. RESULTS: In TCGA, the presence of SMAD4 genomic aberrations was associated with worse OS (hazard ratio [HR], 1.55; 95% CI, 1.00-2.40; p = .048) but not DMFS (HR, 1.33; 95% CI, .87-2.03; p = .19). Low SMAD4 RNA-seq expression was associated with worse OS (HR, 1.83; 95% CI, 1.17-2.86; p = .008) and DMFS (HR, 1.70; 95% CI, 1.14-2.54; p = .009). In the ICGC, increased SMAD4 RNA-seq expression correlated with improved OS (area under the curve [AUC], .92; 95% CI, .86-.94) and DMFS (AUC, .84; 95% CI, .82-.87). CONCLUSIONS: In patients with resected pancreatic adenocarcinoma, SMAD4 genomic aberrations are associated with worse OS but do not predict for DMFS. Increased SMAD4 RNA-seq expression is associated with improved OS and DMFS in patients with resected pancreatic adenocarcinoma. This reproducible finding suggests SMAD4 RNA-seq expression may be a useful marker to predict metastatic spread.


Subject(s)
Adenocarcinoma , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/surgery , Pancreatic Neoplasms/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/surgery , Smad4 Protein/genetics , Proportional Hazards Models , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Prognosis
20.
Dev Cell ; 59(1): 48-63.e8, 2024 Jan 08.
Article in English | MEDLINE | ID: mdl-38103553

ABSTRACT

Loss of TGF-ß growth-inhibitory responses is a hallmark of human cancer. However, the molecular mechanisms underlying the TGF-ß resistance of cancer cells remain to be fully elucidated. Splicing factor proline- and glutamine-rich (SFPQ) is a prion-like RNA-binding protein that is frequently upregulated in human cancers. In this study, we identified SFPQ as a potent suppressor of TGF-ß signaling. The ability of SFPQ to suppress TGF-ß responses depends on its prion-like domain (PrLD) that drives liquid-liquid phase separation (LLPS). Mechanistically, SFPQ physically restrained Smad4 in its condensates, which excluded Smad4 from the Smad complex and chromatin occupancy and thus functionally dampened Smad-dependent transcriptional responses. Accordingly, SFPQ deficiency or loss of phase separation activities rendered human cells hypersensitive to TGF-ß responses. Together, our data identify an important function of SFPQ through LLPS that suppresses Smad transcriptional activation and TGF-ß tumor-suppressive activity.


Subject(s)
Neoplasms , Prions , Humans , Transcriptional Activation , Smad4 Protein/genetics , Smad4 Protein/metabolism , Transforming Growth Factor beta/metabolism , RNA-Binding Proteins
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