TGF-β Signaling Components Research Articles

The Role of SMAD7 in the Epigenetic Regulation of TGF-β Targets in the Metastasis of Ovarian Cancer.

The role of TGF-β signaling in the epigenetic modifications involved in ovarian cancer is not fully understood. This study investigated the relationship between TGF-β signaling, epigenetic modifications, and cellular behaviors in ovarian cancer. We found that E-cadherin, a key cell adhesion molecule, underwent epigenetic silencing via promoter DNA hypermethylation in ovarian cancer cell lines and that this was accompanied by the upregulation of vimentin, which is indicative of a mesenchymal and invasive phenotype. DNA-demethylating agents restored E-cadherin expression, which suggests that TGF-β signaling mediates this epigenetic silencing. Overexpression of SMAD7, an inhibitory component of TGF-β signaling, reversed E-cadherin silencing, which suggests a role of SMAD7 in modulating the epigenetic status. Functionally, SMAD7 overexpression inhibited the migration and invasion in ovarian cancer cells, which suggests its therapeutic potential for suppressing metastasis. Clinically, ovarian cancer patients with high SMAD7 expression had significantly longer disease-free survival. Mechanistically, SMAD7 overexpression decreased the acetylation of H3K9 and the binding of the transcriptional repressor TWIST1 at the E-cadherin promoter, which promoted its demethylation and reactivation. Disruption of TGF-β signaling upregulated SMAD4 target genes, which are silenced by epigenetic mechanisms, a finding that suggests broader therapeutic implications. Overall, our results provide insights into the role of TGF-β-mediated epigenetic regulation in ovarian cancer metastasis and underscore the therapeutic potential of targeting TGF-β signaling and its downstream effectors. Further research is needed to elucidate the underlying mechanisms and validate these therapeutic strategies.

Transforming growth factor β-2 is rhythmically expressed in both WT and BMAL1-deficient hypothalamic neurons and regulates neuropeptide Y: Disruption by palmitate.

The hypothalamus contains neuropeptide Y (NPY)-expressing neurons that control food intake and regulate energy homeostasis. During the development of obesity, neuroinflammation occurs in the hypothalamus before peripheral tissues, but the cytokines involved have not been thoroughly studied. Among them is the transforming growth factor beta (TGF-β) family of cytokines. Herein we demonstrate that Tgfb 1-3, as well as its receptors Tgfbr1 and Tgfbr2, exhibit high levels of expression in the whole hypothalamus, primary hypothalamic culture, and immortalized hypothalamic neurons. Of interest, only Tgfb2 mRNA displays circadian expression in the immortalized hypothalamic neurons and maintains this rhythmicity in BMAL1-KO-derived hypothalamic neurons that are deficient of inherent clock gene rhythmicity. Although BMAL2 may serve as an alternative rhythm generation mechanism in the absence of BMAL1, its knockdown did not affect Tgfb2 expression. Treatment of immortalized NPY-expressing neurons with TGF-β2 upregulates the core circadian oscillators Bmal1 and Nr1d1, and importantly, also Npy mRNA expression. With obesity, the hypothalamus is exposed to elevated levels of palmitate, a saturated fatty acid that promotes neuroinflammation by upregulating pro-inflammatory cytokines. Palmitate treatment disrupts the expression of TGF-β signaling components, increases BMAL1 binding to the Tgfb2 5’ regulatory region, and upregulates Npy mRNA, whereas antagonizing TGFBRI attenuates the upregulation of Npy. These results suggest that hypothalamic neuronal TGF-β2 lies at the intersection of circadian rhythms, feeding neuropeptide control, and neuroinflammation. A better understanding of the underlying mechanisms that link nutrient excess to hypothalamic dysfunction is critical for the development of effective prevention and treatment strategies.

376 (PB364): Targeting TGF-β signaling components to ameliorate Palbociclib resistance in breast cancer

376 (PB364): Targeting TGF-β signaling components to ameliorate Palbociclib resistance in breast cancer

The paracaspase MALT1 is a downstream target of Smad3 and potentiates the crosstalk between TGF-β and NF-kB signaling pathways in cancer cells

TGF-β signaling mediates its biological effects by engaging canonical Smad proteins and crosstalking extensively with other signaling networks, including the NF-kB pathway. The paracaspase MALT1 is an intracellular signaling molecule essential for NF-kB activation downstream of several key cell surface receptors. Despite intensive research on TGF-β and NF-kB interactions, the significance of MALT1 in this context remains undecoded. Here we provide experimental evidence supporting that MALT1 functions to converge these pathways. Using A549 and Huh7 cancer cell line models, we report that TGF-β stimulation enhances MALT1 protein and transcript levels in a time- and dose-dependent manner. Systematic and selective perturbation of TGF-β signaling components identifies MALT1 as a downstream target of Smad3. Rescue experiments in SMAD3 knockout cells confirm that C-terminal phosphorylation of Smad3 is central to MALT1 induction. Corroborating these data, we document that the expression of SMAD3 and MALT1 genes are positively correlated in TCGA cohorts, and we trace the molecular basis of MALT1 elevation to promoter activation. Functional studies in parental as well as NF-kB p65 signaling reporter engineered cells conclusively reveal that MALT1 is paramount for TGF-β-stimulated nuclear translocation and transcriptional activation of NF-kB p65. Furthermore, we find that BCL10 is also implicated in TGF-β activation of NF-kB target genes, potentially coupling the TGF-β-MALT1-NF-kB signaling axis to the CARMA-BCL10-MALT1 (CBM) signalosome. The novel findings of this study indicate that MALT1 is a downstream target of the canonical TGF-β/Smad3 pathway and plays a critical role in modulating TGF-β and NF-kB crosstalk in cancer.

Downregulated miR-495-3p in colorectal cancer targets TGFβR1, TGFβR2, SMAD4 and BUB1 genes and induces cell cycle arrest

BackgroundHsa-miR-495 (miR-495) has been extensively investigated in cancer initiation and progression. On the other hand, our bioinformatics analysis suggested that miR-495 exerts its effects through targeting of TGFβ signaling components. Methods & ResultsIn order to investigate such an effect, miR-495 precursor was overexpressed in HEK293T, SW480, and HCT116 cells, which was followed by downregulation of TGFβR1, TGFβR2, SMAD4, and BUB1 putative target genes, detected by RT-qPCR. Also, luciferase assay supported the direct interaction of miR-495 with 3′UTR sequences of TGFβR1, TGFβR2, SMAD4, and BUB1 genes. Furthermore, a negative correlation of expression between miR-495-3p and some of these target genes was deduced in a set of colorectal and breast cancer cell lines. Then, flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase. Consistently, western blotting analysis showed a significant reduction of the Cyclin D1 protein in the cells overexpressing miR-495, pointing to downregulation of the TGFβ signaling pathway and cell cycle arrest. Finally, microarray data analysis showed that miR-495-3p is significantly downregulated in colorectal tumors, compared to the normal pairs. ConclusionsOverall, the results of the current study introduced miR-495-3p as a cell cycle progression suppressor, which may negatively regulate TGFβR1, TGFβR2, SMAD4, and BUB1 genes. This finding suggests miR-495-3p as a tumor suppressor candidate for further evaluation.

Role of TGF-β Signaling in Coronavirus Disease 2019

Abstract Coronavirus disease 2019 (COVID-19) has a broad spectrum of clinical manifestations involving the respiratory, cardiovascular, renal, neuropsychiatric, gastrointestinal, and dermatological systems. Some patients with COVID-19 experience acute infection and post-COVID-19 syndrome. There is increasing evidence that TGF-β signaling plays an important role in the pathogenesis of both acute and chronic COVID-19 infection. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein was reported to interact with Smad3, a key downstream mediator of TGF-β signaling, thereby promoting TGF-β1/Smad3 signaling and causing cell death during the acute phase of COVID-19 infection. Because activation of TGF-β/Smad3 signaling has an essential role in multiple organ fibrosis, it is possible that overreactive TGF-β/Smad3 signaling may cause tissue fibrosis in the lung, heart, and kidney after SARS-CoV-2 infection. Thus, not only administration of antiviral drugs and traditional Chinese medicines, but also targeting of TGF-β signaling components, particularly Smad3, with various therapeutic strategies involving OT-101, pirfenidone, and specific Smad3 inhibitors, such as SIS3, may provide novel and specific therapies for COVID-19 patients.

Endothelial cilia dysfunction in pathogenesis of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is associated with defective capillary network, leading to dilated superficial vessels and arteriovenous malformations (AVMs) in which arteries connect directly to the veins. Loss or haploinsufficiency of components of TGF-β signaling, ALK1, ENG, SMAD4, and BMP9, have been implicated in the pathogenesis AVMs. Emerging evidence suggests that the inability of endothelial cells to detect, transduce and respond to blood flow, during early development, is an underpinning of AVM pathogenesis. Therefore, components of endothelial flow detection may be instrumental in potentiating TGF-β signaling in perfused blood vessels. Here, we argue that endothelial cilium, a microtubule-based and flow-sensitive organelle, serves as a signaling hub by coupling early flow detection with potentiation of the canonical TGF-β signaling in nascent endothelial cells. Emerging evidence from animal models suggest a role for primary cilia in mediating vascular development. We reason, on recent observations, that endothelial cilia are crucial for vascular development and that embryonic loss of endothelial cilia will curtail TGF-β signaling, leading to associated defects in arteriovenous development and impaired vascular stability. Loss or dysfunction of endothelial primary cilia may be implicated in the genesis of AVMs due, in part, to inhibition of ALK1/SMAD4 signaling. We speculate that AVMs constitute part of the increasing spectrum of ciliopathy-associated vascular defects.

Protein Tyrosine Phosphatase 1B Deficiency in Vascular Smooth Muscle Cells Promotes Perivascular Fibrosis following Arterial Injury.

Background Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury. Methods Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ER T2 recombinase under the Myh11 promoter with PTP1B flox/flox mice and subjected to FeCl 3 carotid artery injury. Results Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA + and PDGFRβ + myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11 -Cre reporter cells in the remodeling adventitia, and SCA1 + CD45 - vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor β (TGFβ) signaling components or enzymes involved in extracellular matrix remodeling and TGFβ liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFRβ and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion. Conclusion Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription.

Single-cell transcriptomic signatures and gene regulatory networks modulated by Wls in mammalian midline facial formation and clefts.

Formation of highly unique and complex facial structures is controlled by genetic programs that are responsible for the precise coordination of three-dimensional tissue morphogenesis. However, the underlying mechanisms governing these processes remain poorly understood. We combined mouse genetic and genomic approaches to define the mechanisms underlying normal and defective midfacial morphogenesis. Conditional inactivation of the Wnt secretion protein Wls in Pax3-expressing lineage cells disrupted frontonasal primordial patterning, cell survival and directional outgrowth, resulting in altered facial structures, including midfacial hypoplasia and midline facial clefts. Single-cell RNA sequencing revealed unique transcriptomic atlases of mesenchymal subpopulations in the midfacial primordia, which are disrupted in the conditional Wls mutants. Differentially expressed genes and cis-regulatory sequence analyses uncovered that Wls modulates and integrates a core gene regulatory network, consisting of key midfacial regulatory transcription factors (including Msx1, Pax3 and Pax7) and their downstream targets (including Wnt, Shh, Tgfβ and retinoic acid signaling components), in a mesenchymal subpopulation of the medial nasal prominences that is responsible for midline facial formation and fusion. These results reveal fundamental mechanisms underlying mammalian midfacial morphogenesis and related defects at single-cell resolution.

MiR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFβR3

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFβR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFβR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFβR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFβ2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm invivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFβR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines. Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway. We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes. Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.

A human importin-β-related disorder: Syndromic thoracic aortic aneurysm caused by bi-allelic loss-of-function variants in IPO8

Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-β protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-β signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm (TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8-/- mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-β signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8-/- mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-β signaling pathway in TAA development. Because importin 8 is the most downstream TGF-β-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.

Dissecting the Functional Selectivity of TGFβ Signaling Pathway Components Using Genome Engineered Human Organotypic Skin Models

Transforming growth factor β (TGFβ) signaling is essential for cell growth and differentiation. Yet, the role of the individual TGFβ signaling components in human tissue homeostasis and transformation is still not completely understood. Here we applied a combined strategy of genetic engineering, a human organotypic 3D skin model, and global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGFβ signaling pathway. Our results show that loss of Smad4-dependent TGFβ signaling affected skin homeostasis through regulation of ECM expression and cell cycle control. In contrast, deletion of TGFβRII resulting in the combined loss of Smad4-dependent and independent TGFβ signaling induced invasive growth through activation of p38 and ERK signaling. The study provides a framework for exploration of signaling pathways with genetic engineering, human 3D tissue models, and with global proteomics and phosphoproteomics.

Crosstalk between 17β-Estradiol and TGF-β Signaling Modulates Glioblastoma Progression.

Epithelial–mesenchymal transition (EMT) is an essential mechanism contributing to glioblastoma multiforme (GBM) progression, the most common and malignant brain tumor. EMT is induced by signaling pathways that crosstalk and regulate an intricate regulatory network of transcription factors. It has been shown that downstream components of 17β-estradiol (E2) and transforming growth factor β (TGF-β) signaling pathways crosstalk in estrogen-sensitive tumors. However, little is known about the interaction between the E2 and TGF-β signaling components in brain tumors. We have investigated the relationship between E2 and TGF-β signaling pathways and their effects on EMT induction in human GBM-derived cells. Here, we showed that E2 and TGF-β negatively regulated the expression of estrogen receptor α (ER-α) and Smad2/3. TGF-β induced Smad2 phosphorylation and its subsequent nuclear translocation, which E2 inhibited. Both TGF-β and E2 induced cellular processes related to EMT, such as morphological changes, actin filament reorganization, and mesenchymal markers (N-cadherin and vimentin) expression. Interestingly, we found that the co-treatment of E2 and TGF-β blocked EMT activation. Our results suggest that E2 and TGF-β signaling pathways interact through ER-α and Smad2/3 mediators in cells derived from human GBM and inhibit EMT activation induced by both factors alone.

MicroRNAs regulating TGFβ and BMP signaling in the osteoblast lineage.

This review showcases miRNAs contributing to the regulation of bone forming osteoblasts through their effects on the TGFβ and BMP pathways, with a focus on ligands, receptors and SMAD-mediated signaling. The goal of this work is to provide a basis for broadly understanding the contribution of miRNAs to the modulation of TGFβ and BMP signaling in the osteoblast lineage, which may provide a rationale for potential therapeutic strategies. Therefore, the search strategy for this review was restricted to validated miRNA-target interactions within the canonical TGFβ and BMP signaling pathways; miRNA-target interactions based only bioinformatics are not presented. Specifically, this review discusses miRNAs targeting each of the TGFβ isoforms, as well as BMP2 and BMP7. Further, miRNAs targeting the signaling receptors TGFβR1 and TGFβR2, and those targeting the type 1 BMP receptors and BMPR2 are described. Lastly, miRNAs targeting the receptor SMADs, the common SMAD4 and the inhibitory SMAD7 are considered. Of these miRNAs, the miR-140 family plays a prominent role in inhibiting TGFβ signaling, targeting both ligand and receptor. Similarly, the miR-106 isoforms target both BMP2 and SMAD5 to inhibit osteoblastic differentiation. Many of the miRNAs targeting TGFβ and BMP signaling components are induced during fracture, mechanical unloading or estrogen deprivation. Localized delivery of miRNA-based therapeutics that modulate the BMP signaling pathway could promote bone formation.

Endoplasmic Reticulum Associated Protein Degradation (ERAD) in the Pathology of Diseases Related to TGFβ Signaling Pathway: Future Therapeutic Perspectives.

The transforming growth factor signaling pathway (TGFβ) controls a wide range of cellular activities in adulthood as well as during embryogenesis including cell growth, differentiation, apoptosis, immunological responses and other cellular functions. Therefore, germline mutations in components of the pathway have given rise to a heterogeneous spectrum of hereditary diseases with variable phenotypes associated with malformations in the cardiovascular, muscular and skeletal systems. Our extensive literature and database searches revealed 47 monogenic diseases associated with germline mutations in 24 out of 41 gene variant encoding for TGFβ components. Most of the TGFβ components are membrane or secretory proteins and they are therefore expected to pass through the endoplasmic reticulum (ER), where fidelity of proteins folding is stringently monitored via the ER quality control machineries. Elucidation of the molecular mechanisms of mutant proteins’ folding and trafficking showed the implication of ER associated protein degradation (ERAD) in the pathogenesis of some of the diseases. For example, hereditary hemorrhagic telangiectasia types 1 and 2 (HHT1 and HHT2) and familial pulmonary arterial hypertension (FPAH) associated with mutations in Endoglin, ALK1 and BMPR2 components of the signaling pathway, respectively, have all exhibited loss of function phenotype as a result of ER retention of some of their disease-causing variants. In some cases, this has led to premature protein degradation through the proteasomal pathway. We anticipate that ERAD will be involved in the mechanisms of other TGFβ signaling components and therefore warrants further research. In this review, we highlight advances in ER quality control mechanisms and their modulation as a potential therapeutic target in general with particular focus on prospect of their implementation in the treatment of monogenic diseases associated with TGFβ components including HHT1, HHT2, and PAH. In particular, we emphasis the need to establish disease mechanisms and to implement such novel approaches in modulating the molecular pathway of mutant TGFβ components in the quest for restoring protein folding and trafficking as a therapeutic approach.

MicroRNA-148a-3p inhibits progression of hepatocelluar carcimoma by repressing SMAD2 expression in an Ago2 dependent manner

BackgroundHepatocellular carcinoma (HCC) is one of the most prevalent common cancer worldwide with high mortality. Transforming growth factor-β (TGF-β) signaling pathway was reported dysregulated during liver cancer formation and progression. As a key component of TGF-β signaling, the role of SMAD2 and its regulatory mechanisms in HCC remain unclear.MethodsSMAD2 expression in paired HCC specimens were determined by western blot and immunohistochemistry (IHC). quantitative real-time PCR (qRT-PCR) was used to measure mRNA and microRNA (miRNA) expression level. Cell migration, invasion and proliferation ability were evaluated by transwell, CCK8 and EdU assay. In silico websites were used to manifest overall survival rates of HCC patients or to predict miRNAs targeting SMAD2. Dual luciferase reporter assay and anti-Ago2 immunoprecipitation assay were performed to confirm the binding between SMAD2 mRNA and miRNA-148a-3p (miR-148a). Tumorigenesis and lung metastasis mouse model were used to explore the role of miR-148a in vivo. In situ hybridization (ISH) was conducted to determine the expression of miR-148a in liver tissues.ResultsIn this study, we found that SMAD2 was highly expressed in HCC and elevated SMAD2 expression predicted shorter overall survival (OS) time for HCC patients. SMAD2 promoted mobility and proliferation of HCC cells in vitro. We further revealed that the expression of miR-148a was negatively correlated with SMAD2 and found that miR-148a repressed SMAD2 expression by downregulating its mRNA through binding with Argonaute 2 (Ago2) in HCC. Transwell, CCK8 and animal experiments exhibited miR-148a inhibited metastasis and proliferation of HCC in vitro and in vivo. Moreover, the phenotype changes caused by miR-148a manipulation were recovered by rescuing SMAD2 expression in HCC cells. ISH assay indicated miR-148a was downregulated in HCC and low expression of miR-148a associated with more aggressive clinic features and poor prognosis.ConclusionmiR-148a was identified as a repressor of HCC progression by downregulating SMAD2 in an Ago2 dependent manner.

The evolutionarily conserved deubiquitinase UBH1/UCH-L1 augments DAF7/TGF-β signaling, inhibits dauer larva formation, and enhances lung tumorigenesis

Modification of the transforming growth factor β (TGF-β) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-β signaling, suggesting that this mode of regulation of TGF-β signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-β signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-βRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-β signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-β signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-β/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-β/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-β/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike WT UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-β signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.

Abstract A07: Improved prognosis of HPV-positive oropharyngeal cancers is associated with a suppression of TGF-β-mediated immune evasion

Abstract Among head and neck squamous carcinoma (HNSC), human papilloma virus (HPV)-driven tumors are known to enjoy more favorable prognostic outcomes compared to non-HPV-driven disease. This is especially true of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC), which is associated with significantly improved outcomes compared to HPV-positive non-OPSCC and HPV-negative HNSC. Previous reports have suggested that increased immune infiltration and expression of the estrogen receptor α (ERα) may play a role in conferring improved prognosis to HPV-positive OPSCC. However, the underlying signaling pathways specifically driven by HPV in the oropharynx have remained elusive. Here we analyzed HPV-positive OPSCC and non-OPSCC tumors from The Cancer Genome Atlas (TCGA) and found that HPV-positive OPSCC is associated with significantly improved overall and disease-specific survival and progression-free interval compared to HPV-positive non-OPSCC. Among HPV-positive HNSC, we observed a distinct separation of OPSCC from non-OPSCC tumors based on unsupervised clustering of gene expression, microRNA, and methylation profiling data. Separation based on anatomic site was not observed among HPV-negative HNSC, suggesting that common biology drives the carcinogenesis of HPV-negative HNSC across subsites. In line with a recent report that ERα mRNA expression is elevated HPV-positive OPSCC, we found significantly higher ERα mRNA levels in HPV-positive OPSCC compared to HPV-positive non-OPSCC, where ERα mRNA expression mirrored that in HPV-negative HNSC. We also found that HPV induces downregulation of TGF-β signaling and extracellular matrix (ECM)-associated genes while upregulating a T-cell and dendritic cell-associated immune response signature in an OPSCC-specific manner. Our analyses suggest that HPV induces downregulation of TGF-β signaling and ECM components in OPSCC to improve immune access to these tumors, facilitating tumor clearance and improved patient survival. These findings are in line with our recent report establishing TGF-β-mediated immunosuppression through ECM deregulation and its negative effect on patient prognosis. Citation Format: Anthony Bolyos, Pinaki Bose. Improved prognosis of HPV-positive oropharyngeal cancers is associated with a suppression of TGF-β-mediated immune evasion [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A07.

Clinical Implications

Clinical Implications