Deletion Of Smad4 Research Articles

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling

BackgroundBone 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.MethodsTo 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.ResultsIn 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.ConclusionsBMP4 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.

Abstract 2896: Elucidating the role of transglutaminase 2 (TGM2) in pancreatic ductal adenocarcinoma pathogenesis and its therapeutic implications

Abstract Pancreatic cancer remains one of the deadliest malignancies, with limited therapeutic options and a poor prognosis due to delayed symptom presentation and disease detection. Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 8%, predominantly attributed to oncogenic KRAS mutations and loss-of-function mutations in TP53, SMAD4, and P16. Our lab is interested in studying Transglutaminase 2 (TGM2), a multifunctional enzyme involved in protein cross-linking and cellular signaling, because it is highly correlated with cancer cell survival, malignancy, metastasis, and treatment resistance. Clinically, overexpression of TGM2 is associated with poorer survival in patients with Pancreatic Ductal Adenocarcinoma so it is imperative to understand the mechanism of its action in disease progression. To elucidate the role of TGM2 in PDAC, we studied the targeted deletion of TGM2 in the context of a mouse model of PDAC and saw a significant reduction in PDAC incidence and precursor lesion formation, suggesting that TGM2 has a role in the tumor microenvironment. Using single cell sequencing, we found TGM2 levels to be elevated in certain cell populations, specifically endothelial cells and cancer cells. We discovered high TGM2 levels in TIE2-GFP mouse vessels and subsequently found high TGM2 levels in patient tumor vasculature. Further studies aim to conditionally ablate TGM2 in endothelial cells to assess its mechanistic involvement in PDAC initiation and progression. Additionally, we are studying concurrent conditional TGM2 and SMAD4 deletions in mice to investigate pancreatic cancer precursor lesion formation and are further evaluating the protective mechanisms of a TGM2 deletion in a more aggressive mouse model of PDAC. Our preliminary findings substantiate TGM2 as a critical player in PDAC pathogenesis, opening avenues for targeted therapeutics to ultimately improve patient prognosis. Citation Format: Polina Wright, Azeddine Atfi. Elucidating the role of transglutaminase 2 (TGM2) in pancreatic ductal adenocarcinoma pathogenesis and its therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2896.

Abstract B036: Mutational landscape and genetic feature of gastric adenocarcinoma in multiple Chinese ethnicities

Abstract Gastric cancer is a heterogeneous malignancy and the fifth most common cancer worldwide, with 44% of cases occurring in China. Elucidation of genetic subtypes may improve gastric cancer treatment planning. Matched tumour and peritumoural (non-malignant gastric mucosa) tissue samples were collected from 306 Chinese patients and subjected to whole-exome sequencing (WES). The results were used to produce a Chinese Gastric Cancer Genome Atlas (CGCGA). Patients from nine Chinese ethnic groups (Han, Zhuang, Hui, Uygur, Yi, Tibetan, Mongolian, Bai, and Kazakh), representing 95% of the Chinese population were included. Systematic comparison of CGCGA data with TCGA (The Cancer Genome Atlas) data, in which 437 gastric cancer cases are represented, showed that genomic alteration signatures in the present cohort differed markedly from those reported for Western cohorts. TP53 and FAT1 deletions were more frequent (35% vs. 0%) while ARID1A deletions (13% vs. 25%), MYC oncogene amplifications (53% vs. 70%), and SMAD4 deletions (34% vs. 46%) were less frequent in our CGCGA data than in the TCGA data. TRIM49B mutation was associated with worse survival. Thus, TRIM49B was identified as a novel oncogene. The Zhuang ethnic group had a much higher tumour mutation burden (TMB) than the other ethnic groups. Typically, tumours from Han group had microsatellite stability, whereas those from the other groups had microsatellite instability (MSI). We further propose a molecular classification of four gastric cancer subtypes that are associated with distinct survival outcomes: TP53 mutation positive (TP53+)/TTN_missense+; TP53+/TTN_missense–; TP53–/TTN_missense+, and TP53–/TTN_missense–. Our study established a comprehensive gastric cancer genome atlas in China and underscored the importance of considering population context in genomic mapping and rational treatment options for the disease. Citation Format: Liang Zong. Mutational landscape and genetic feature of gastric adenocarcinoma in multiple Chinese ethnicities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B036.

Abstract A117: Molecular stratification of therapeutic targets in pancreatic cancer

Abstract Precision medicine approaches using molecular tumor profiling have resulted in striking clinical benefits in certain patient populations. However, targeted therapy in pancreatic cancer has been unable to displace multi-agent chemotherapy, in part due to lack of actionable biomarkers to anchor stratification. To address this need, we have conducted a biomarker focused analysis of cancer dependencies and cell surface targets to understand specific vulnerabilities present in molecular subgroups. We started by interrogating genome scale CRISPR screening and expression data from The Cancer Dependency Map to identify lineage-enriched, selective dependencies in pancreatic cancer. Our results reveal synthetic lethal interactions such as paralog pairs (e.g., VPS4A/B), dependencies predictable by overexpression (e.g., KLF5), and dependencies predictable by genetic alterations (e.g., RAB10 with SMAD4 deletion). Our results reaffirm the importance of KRAS signaling but also highlight a diversity of downstream essential mechanisms including DOCK5 and RAF1. We also identified vulnerabilities specific to transcriptional subtypes, finding ITGA3 to be a selective dependency of not only the basal-like state in pancreas cancer but also across the spectrum of epithelial malignancies. Given the central role of KRAS mutations in tumorigenesis and the emergence of KRAS inhibitors, we studied predictors of KRAS dependency. GSEA analysis of genes overexpressed in cell lines with lower dependency on KRAS identified gene sets related to focal adhesion, EMT, and neuron development, while lines with high KRAS dependency were enriched in genes related to epithelial differentiation/development and the canonical Hallmark KRAS up signature. These gene signatures also predict resistance and sensitivity to small molecule inhibition of KRAS with a high degree of accuracy (Pearson r = 0.68, 0.94 respectively). In addition to vulnerabilities from intracellular target inhibition, cancer cells may be targeted selectively based on surface proteins using cellular and antibody based therapeutic strategies. We used bulk gene expression data from TCGA and GTEx as well as single cell expression data from The Human Cell Atlas, to calculate enriched genes in pancreas cancer versus critical normal tissues. Our approach captures targets including MSLN, TROP2, MUC1, and CLDN18, which have established tumor specificity, but also identifies PSCA, GPRC5A, and CEACAM5 as additional emerging pancreas tumor specific antigens. We charted interpatient and intratumoral heterogeneity in spatial transcriptomics data using a cohort of 21 treated and untreated pancreatic cancer tissue specimens, identifying MUC1 and GPRC5A as both expressed homogenously across patients and throughout individual tumors while scarcely expressed in normal tissues. Ultimately, these findings may prompt prioritization of new molecularly defined dependencies, and construction of rational combinatorial regimens. Citation Format: Dennis Gong, Jimmy A. Guo, Carina Shiau, Ananya D. Jambhale, Steven Wang, Scott Ginebaugh, Patrick Z. Yu, Kevin S. Kapner, Seema Chugh, Laleh Abbassi, Daniel Zhao, Westley W. Wu, Peter Chen, Harshabad Singh, William L. Hwang, Andrew J. Aguirre. Molecular stratification of therapeutic targets in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A117.

Hepatocyte-specific Smad4 deficiency inhibits hepatocarcinogenesis by promoting CXCL10/CXCR3-dependent CD8+- T cell-mediated anti-tumor immunity.

Rationale: Sma mothers against decapentaplegic homologue 4 (Smad4) is a key mediator of the transforming growth factor β (TGF-β) pathway and plays complex and contradictory roles in hepatocellular carcinoma (HCC). However, the specific role of Smad4 in hepatocytes in regulating hepatocarcinogenesis remains poorly elucidated. Methods: A diethylnitrosamine/carbon tetrachloride-induced HCC model was established in mice with hepatocyte-specific Smad4 deletion (AlbSmad4-/-) and liver tumorigenesis was monitored. Immune cell infiltration was examined by immunofluorescence and fluorescence activated cell sorting (FACS). Cytokine secretion, glycolysis, signal pathway, and single-cell RNA sequencing were analysed for mechanism. Results: AlbSmad4-/- mice exhibited significantly fewer and smaller liver tumor nodules, less fibrosis, reduced myeloid-derived suppressor cell infiltration and increased CD8+ T cell infiltration. Smad4 deletion in hepatocytes enhanced C-X-C motif ligand 10 (CXCL10) secretion, promoting tumor necrosis factor-α (TNF-α) production in CD8+ T cells. The loss of Smad4 activated the CXCL10/mammalian target of rapamycin (mTOR)/lactate dehydrogenase A (LDHA) pathway, which increased glycolytic activity in CD8+ T cells. HCC patients with high Smad4 expression exhibited decreased CD8+ T cell infiltration and altered glycolysis. Conclusion: Our results demonstrate that Smad4 in hepatocytes promotes hepatocarcinogenesis and is a potential and candidate target for the prevention and therapy of HCC.

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations.

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression-mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer.

e16022 Background: Leptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was under-investigated in GCLM. Methods: We retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes. Results: CSF had higher mutation allele frequency ( P=0.015), more somatic mutations ( P=0.032), and more copy-number variations ( P<0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival ( P=0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation ( P=0.014), IGF1R mutation ( P=0.034), AR mutation ( P=0.038), SMARCB1 deletion ( P<0.001), SMAD4 deletion ( P=0.0034), and TGF-beta pathway aberration ( P=0.0038). Additionally, improvement in intracranial pressure ( P<0.001), improvement in CSF cytology ( P=0.0038), and relatively low levels of CSF ctDNA ( P=0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment. Conclusions: CSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Transforming Growth Factor BetaPromotes Inflammation and Tumorigenesis in Smad4-Deficient Intestinal Epithelium in a YAP-Dependent Manner.

Transforming growth factor beta (TGF-β), a multifunctional cytokine, plays critical roles in immune responses. However, the precise role of TGF-β in colitis and colitis-associated cancer remains poorly defined. Here, it is demonstrated that TGF-β promotes the colonic inflammation and related tumorigenesis in the absence of Smad family member 4 (Smad4). Smad4 loss in intestinal epithelium aggravates colitis and colitis-associated neoplasia induced by dextran sulfate sodium (DSS) and azoxymethane/dextran sulfate sodium (AOM/DSS), leading to over-activated immune responses and increased TGF-β1 levels. In Smad4-deficient organoids, TGF-β1 stimulates spheroid formation and impairs intestinal stem cell proliferation and lineage specification. YAP, whose expression is directly upregulated by TGF-β1 after Smad4 deletion, mediates the effect of TGF-β1 by interacting with Smad2/3. Attenuation of YAP/TAZ prevents TGF-β1-induced spheroid formation in Smad4-/- organoids and alleviates colitis and colitis-associated cancer in Smad4-deficient mice. Collectively, these results highlight an integral role of the TGF-β/Smad4 axis in restraining intestinal inflammation and tumorigenesis and suggest TGF-β or YAP signaling as therapeutic targets for these gastrointestinal diseases intervention.

Genome-wide CRISPR Screen Reveals RAB10 as a Synthetic Lethal Gene in Colorectal and Pancreatic Cancers Carrying SMAD4 Loss.

This study identified and validated RAB10 as new synthetic lethal gene with SMAD4. This was achieved by conducting a whole-genome CRISPR screens in different colorectal and pancreatic cell lines. A future RAB10 inhibitors could correspond to a new therapeutic solution for patients with cancer with SMAD4 deletion.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer

BackgroundLeptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was underinvestigated in GCLM.MethodsWe retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes.ResultsCSF had higher mutation allele frequency (P = 0.015), more somatic mutations (P = 0.032), and more copy-number variations (P < 0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival (P = 0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation (P = 0.014), IGF1R mutation (P = 0.034), AR mutation (P = 0.038), SMARCB1 deletion (P < 0.001), SMAD4 deletion (P = 0.0034), and TGF-beta pathway aberration (P = 0.0038). Additionally, improvement in intracranial pressure (P < 0.001), improvement in CSF cytology (P = 0.0038), and relatively low levels of CSF ctDNA (P = 0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment.ConclusionsCSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Novel strategy for oncogenic alteration-induced lipid metabolism reprogramming in pancreatic cancer.

The pathogenesis of pancreatic cancer involves substantial metabolic reprogramming, resulting in abnormal proliferation of tumor cells. This tumorigenic reprogramming is often driven by genetic mutations, such as activating mutations of the KRAS oncogene and inactivating or deletions of the tumor suppressor genes SMAD4, CDKN2A, and TP53, which play a critical role in the initiation and development of pancreatic cancer. As a normal cell gradually develops into a cancer cell, a series of signature characteristics are acquired: activation of signaling pathways that sustain proliferation; an ability to resist growth inhibitory signals and evade apoptosis; and an ability to generate new blood vessels and invade and metastasize. In addition to these features, recent research has revealed that metabolic reprogramming and immune escape are two other novel characteristics of tumor cells. The effect of the interactions between tumor and immune cells on metabolic reprogramming is a key factor determining the antitumor immunotherapy response. Lipid metabolism reprogramming, a feature of many malignancies, not only plays a role in maintaining tumor cell proliferation but also alters the tumor microenvironment by inducing the release of metabolites that in turn affect the metabolism of normal immune cells, ultimately leading to the attenuation of the antitumor immune response and resistance to immunotherapy. Pancreatic cancer has been found to have substantial lipid metabolism reprogramming, but the mechanisms remain elusive. Therefore, this review focuses on the mechanisms regulating lipid metabolism reprogramming in pancreatic cancer cells to provide new therapeutic targets and aid the development of new therapeutic strategies for pancreatic cancer.

A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

Brain arteriovenous malformation in hereditary hemorrhagic telangiectasia: Recent advances in cellular and molecular mechanisms.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by vessel dilatation, such as telangiectasia in skin and mucosa and arteriovenous malformations (AVM) in internal organs such as the gastrointestinal tract, lungs, and brain. AVMs are fragile and tortuous vascular anomalies that directly connect arteries and veins, bypassing healthy capillaries. Mutations in transforming growth factor β (TGFβ) signaling pathway components, such as ENG (ENDOGLIN), ACVRL1 (ALK1), and SMAD4 (SMAD4) genes, account for most of HHT cases. 10-20% of HHT patients develop brain AVMs (bAVMs), which can lead to vessel wall rupture and intracranial hemorrhages. Though the main mutations are known, mechanisms leading to AVM formation are unclear, partially due to lack of animal models. Recent mouse models allowed significant advances in our understanding of AVMs. Endothelial-specific deletion of either Acvrl1, Eng or Smad4 is sufficient to induce AVMs, identifying endothelial cells (ECs) as primary targets of BMP signaling to promote vascular integrity. Loss of ALK1/ENG/SMAD4 signaling is associated with NOTCH signaling defects and abnormal arteriovenous EC differentiation. Moreover, cumulative evidence suggests that AVMs originate from venous ECs with defective flow-migration coupling and excessive proliferation. Mutant ECs show an increase of PI3K/AKT signaling and inhibitors of this signaling pathway rescue AVMs in HHT mouse models, revealing new therapeutic avenues. In this review, we will summarize recent advances and current knowledge of mechanisms controlling the pathogenesis of bAVMs, and discuss unresolved questions.

Abstract C063: Smad4 deletion alters the composition of the tumor microenvironment in pancreatic cancer

Abstract Rationale: Pancreatic ductal adenocarcinoma (PDAC) tumors are notorious for their extensive non-malignant stroma in which the most abundant cells are cancer-associated fibroblasts (CAFs). CAFs are comprised of distinct subtypes with seemingly diverse roles within the PDAC tumor microenvironment (TME) and have been shown to influence disease progression and therapy response. However, most insights into PDAC CAF heterogeneity have been based on KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) mouse models, which recapitulate the pathophysiology of human PDAC but not its genetic complexity, representing only ~5% of patient tumor mutation profiles. We hypothesized that further understanding of CAF heterogeneity in PDAC tumors with commonly occurring mutations is essential for improving patient stratification and treatment strategies, and consequently, survival of this disease. To start exploring this, we developed and analyzed new PDAC models with deletion of Smad4, which is inactivated in ~30% of PDAC cases, in the context of Kras and Trp53 mutations. Methods: Smad4 deletion was performed using CRISPR-Cas9 technology in pancreatic cancer organoids derived from KPC tumors to generate matched KPC Smad4 knockout organoids. To assess whether this mutation profile modulates the PDAC TME, we cultured pancreatic stellate cells (PSCs), a precursor of CAFs, in media conditioned from KPC and KPC Smad4 knockout organoids and assessed the activation of inflammatory CAF (iCAF) and myofibroblastic CAF (myCAF) markers by RT-qPCR. We also established co-cultures of PSCs and organoids and performed bulk RNA-sequencing on flow-sorted CAFs and cancer cells. Finally, orthotopically-grafted organoid (OGO) transplantation mouse models were generated and pancreatic tumors were evaluated by ultrasound-based imaging, single-cell RNA-sequencing, immunohistochemistry, and flow cytometry. Results: We found that Smad4 deletion in PDAC cells, in the context of Kras and Trp53 mutations, promotes PDAC progression, as expected, and alters the PDAC TME. In particular, Smad4 deletion leads to a decrease in matrix-producing myCAFs, CD105-positive CAFs and antigen-presenting apCAFs, while increasing potentially immunosuppressive iCAFs. Additionally, pancreatic tumors with Smad4 deletion are depleted for macrophages, while enriched for neutrophils. Conclusions and Significance: This study demonstrates how distinct cancer genetics differentially shape the PDAC TME and establishes a workflow for ongoing investigation of additional relevant mutation profiles. This approach could lead to better PDAC patient stratification based on the profile of both cancer and stromal cells for more effective and targeted therapies. Citation Format: Eloise G. Lloyd, Gianluca Mucciolo, Muntadher Jihad, Judhell Sandoval Manansala, Sara Pinto Teles, Giulia Biffi. Smad4 deletion alters the composition of the tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C063.

Deletion of Smad7 Ameliorates Intestinal Inflammation and Contributes to Fibrosis.

Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of transforming growth factor (TGF)-β compared with non-IBD controls. SMAD7 negatively regulates TGF-β signaling. An earlier study aiming to target Smad7 showed a lack of clinical benefit. It remains unknown whether inhibition of SMAD7 is beneficial in specific settings of IBD. We evaluated the effect of Smad7 deficiency on inflammation, fibrogenesis, and wound healing. For the initiation of fibrosis in Smad7-/- (Smad7Δex-I) CD-1 mice, the dextran sodium sulfate-induced chronic colitis model and the heterotopic transplantation model of fibrosis were used. Wound closure of fibroblasts from Smad7-/- mice was determined using culture inserts and electric cell-substrate impedance sensing in vitro. In dextran sodium sulfate-induced chronic colitis, Smad7 deficiency was associated with ameliorated inflammation, as evidenced by decreased clinical score, histological score, and myeloperoxidase activity. Absence of SMAD7 decreased T-cell accumulation in colonic tissue and tumor necrosis factor (TNF) mRNA expression levels. Smad7-/- mice showed a significant increase in hydroxyproline and collagen content, as well as ColIVa1 mRNA expression. Wild type mice transplanted with terminal ileum from Smad7-/- mice in the heterotopic animal model for intestinal fibrosis showed a significant increase in collagen content and protein expression of α-smooth muscle actin. Smad7 deficiency is associated with a decrease in intestinal inflammation and an increase in fibrosis. Targeting SMAD7 constitutes a potential new treatment option for IBD; progression of disease-associated fibrosis should be considered.

Hepatocyte-Specific Smad4 Deficiency Alleviates Liver Fibrosis via the p38/p65 Pathway.

Liver fibrosis is a wound-healing response caused by the abnormal accumulation of extracellular matrix, which is produced by activated hepatic stellate cells (HSCs). Most studies have focused on the activated HSCs themselves in liver fibrosis, and whether hepatocytes can modulate the process of fibrosis is still unclear. Sma mothers against decapentaplegic homologue 4 (Smad4) is a key intracellular transcription mediator of transforming growth factor-β (TGF-β) during the development and progression of liver fibrosis. However, the role of hepatocyte Smad4 in the development of fibrosis is poorly elucidated. Here, to explore the functional role of hepatocyte Smad4 and the molecular mechanism in liver fibrosis, a CCl4-induced liver fibrosis model was established in mice with hepatocyte-specific Smad4 deletion (Smad4Δhep). We found that hepatocyte-specific Smad4 deficiency reduced liver inflammation and fibrosis, alleviated epithelial-mesenchymal transition, and inhibited hepatocyte proliferation and migration. Molecularly, Smad4 deletion in hepatocytes suppressed the expression of inhibitor of differentiation 1 (ID1) and the secretion of connective tissue growth factor (CTGF) of hepatocytes, which subsequently activated the p38 and p65 signaling pathways of HSCs in an epidermal growth factor receptor-dependent manner. Taken together, our results clearly demonstrate that the Smad4 expression in hepatocytes plays an important role in promoting liver fibrosis and could therefore be a promising target for future anti-fibrotic therapy.

The Smad3-dependent microRNA let-7i-5p promoted renal fibrosis in mice with unilateral ureteral obstruction.

Renal fibrosis is a common feature of all types of chronic kidney disease (CKD) and is tightly regulated by the TGF-β/Smad3 pathway. Let-7i-5p belongs to the let-7 microRNA family with diverse biological functions. It has been reported that let-7i-5p suppresses fibrotic disease in the heart, lungs, and blood vessels, while the role of let-7i-5p in renal fibrosis remains limited. In this study, we aimed to investigate the role of let-7i-5p in renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and TGF-β1–stimulated renal tubular cell line TCMK1. The RNA-targeting CRISPR/Cas13d system was used to knock down let-7i-5p. Renal injury and fibrosis were determined by histological analysis, RT-PCR, Western blot, and immunostaining. Our results have shown that in the kidneys after UUO, the expression of let-7i-5p was significantly increased along with notable tubular injury and interstitial fibrosis. Electroporation of let-7i–targeting Cas13d plasmid efficiently knocked down let-7i-5p in kidneys after UUO with reduced tubular injury, fibrotic area, and expression of fibrotic marker genes α-SMA, fibronectin, and Col1a1. In TGF-β1–stimulated TCMK1 cells, knockdown of let-7i-5p by Cas13d plasmid transfection also blunted the expression of fibrotic marker genes. Most importantly, the genomic locus of let-7i showed enriched binding of Smad3 as revealed by chromatin immunoprecipitation. In TCMK1 cells, the overexpression of Smad3 can directly induce the expression of let-7i-5p. However, the deletion of Smad3 abolished TGF-β1–stimulated let-7i-5p expression. Collectively, these findings suggest that let-7i-5p is a Smad3-dependent microRNA that plays a pathogenic role in renal fibrosis. Let-7i-5p could be a promising target for the treatment of CKD-associated renal fibrosis.

Smad3 regulates smooth muscle cell fate and mediates adverse remodeling and calcification of the atherosclerotic plaque.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of SMAD3 in SMC. However, the mechanism by which this gene modifies CAD risk remains poorly understood. Here we show that SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more outward remodelling and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a SMC phenotype that governs both vascular remodelling and recruitment of inflammatory cells, as well as a chondromyocyte fate. Together, the findings reveal that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including outward remodelling, monocyte recruitment, and vascular calcification.

Role of TGF-Beta Signaling in Beta Cell Proliferation and Function in Diabetes.

Beta (β) cell dysfunction or loss is the common pathological feature in all types of diabetes mellitus (diabetes). Resolving the underlying mechanism may facilitate the treatment of diabetes by preserving the β cell population and function. It is known that TGF-β signaling plays diverse roles in β cell development, function, proliferation, apoptosis, and dedifferentiation. Inhibition of TGF-β signaling expands β cell lineage in the development. However, deletion of Tgfbr1 has no influence on insulin demand-induced but abolishes inflammation-induced β cell proliferation. Among canonical TGF-β signaling, Smad3 but not Smad2 is the predominant repressor of β cell proliferation in response to systemic insulin demand. Deletion of Smad3 simultaneously improves β cell function, apoptosis, and systemic insulin resistance with the consequence of eliminated overt diabetes in diabetic mouse models, revealing Smad3 as a key mediator and ideal therapeutic target for type-2 diabetes. However, Smad7 shows controversial effects on β cell proliferation and glucose homeostasis in animal studies. On the other hand, overexpression of Tgfb1 prevents β cells from autoimmune destruction without influence on β cell function. All these findings reveal the diverse regulatory roles of TGF-β signaling in β cell biology.