Non-canonical TGF-β Pathway Research Articles

Co-Mn Complex Oxide Nanoparticles as Potential Reactive Oxygen Species Scavenging Agents for Pulmonary Fibrosis Treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic and age-related lung disease that has few treatment options. Reactive oxygen species (ROS) play an important role in the introduction and development of IPF. In the present study, we developed multifunctional Cobalt (Co)-Manganese (Mn) complex oxide nanoparticles (Co-MnNPs), which can scavenge multiple types of ROS. Benefiting from ROS scavenging activities and good biosafety, Co-MnNPs can suppress canonical and non-canonical TGF-β pathways and, thus, inhibit the activation of fibroblasts and the productions of extracellular matrix. Furthermore, the scavenging of ROS by Co-MnNPs reduce the LPS-induced expressions of pro-inflammatory factors in macrophages, by suppressing NF-κB signaling pathway. Therefore, Co-MnNPs can reduce the excessive extracellular matrix deposition and inflammatory responses in lungs and, thus, alleviate pulmonary fibrosis induced by bleomycin (BLM) in mice. Taken together, this work offers an anti-fibrotic agent for treatment of IPF and other ROS-related diseases.

BMP1 Promotes Keloid by Inducing Fibroblast Inflammation and Fibrogenesis.

Keloid is a typical fibrotic and inflammatory skin disease with unclear mechanisms and few therapeutic targets. In this study, we found that BMP1 was significantly increased in a collagen high-expressing subtype of fibroblast by reanalyzing a public single-cell RNA-sequence data set of keloid. The number of BMP1-positive fibroblast cells was increased in keloid fibrotic loci. Increased levels of BMP1 were further validated in the skin tissues and fibroblasts from keloid patients. Additionally, a positive correlation between BMP1 and the Keloid Area and Severity Index was found in keloid patients. In vitro analysis revealed collagen production, the phosphorylation levels of p65, and the IL-1β secretion decreased in BMP1 interfered keloid fibroblasts. Besides, the knockdown of BMP1 inhibited the growth and migration of keloid fibroblast cells. Mechanistically, BMP1 inhibition downregulated the noncanonical TGF-β pathways, including p-p38 and p-ERK1/2 signaling. Furthermore, we found the delivery of BMP1 siRNAs could significantly alleviate keloid in human keloid-bearing nude mice. Collectively, our results indicated that BMP1 exhibited various pathogenic effects on keloids as promoting cell proliferation, migration, inflammation, and ECM deposition of fibroblast cells by regulating the noncanonical TGF-β/p38 MAPK, and TGF-β/ERK pathways. BMP1-lowing strategies may appear as a potential new therapeutic target for keloid.

Rhein inhibits cell proliferation of glioblastoma multiforme cells by regulating the tgfß and apoptotic signaling pathways

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a plant metabolite found in rhubarbs. It inhibits cell proliferation and stimulates apoptosis in in vivo and in vitro. However, research into the molecular mechanisms of action is insufficient for recommending it as a therapeutic agent. Therefore, this study aims to investigate the antiproliferative, apoptotic, and antimetastatic effects of rhein by targeting the TGF-β signaling pathway, and apoptotic pathway in glioblastoma cells (U87 GBM). In this study, the XTT assay was utilized to determine cell viability, the colony formation assay to measure cell survival and proliferation, RT-qPCR for the analysis of gene expressions, and ELISA for the detection of proteins. U87 GBM cells were treated with varying concentrations of rhein (5-100 µM) in a time-dependent manner (24, 48 h), after which the percentage of cell viability was calculated. The colony formation assay was performed by treating cells with the IC50 dose of rhein. According to the XTT assay, the IC50 dose of rhein was determined as 10 µM at 24 h. The ability to form colonies was significantly decreased in the cells of the treatment group. According to the gene expression analysis, rhein increased the mRNA levels of CASP3, -8, -9, BAX, and TGF-β1 genes, while a notable decrease was observed in the BCL-2, SMAD2, SMAD3, and TIMP1 genes. In conclusion, it was determined that rhein induces apoptosis via the non-canonical TGF-β pathway.

TGFβ1-driven SMAD2/3 phosphorylation and myofibroblast emergence are fully dependent on the TGFβ1 pre-activation of MAPKs and controlled by maternal leucine zipper kinase

Following wounding, endogenously secreted TGFβs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFβ effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFβ. TGFβ also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFβ1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFβ1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFβ1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFβ1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10–15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFβ pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.

Bronchial Fibroblasts from Asthmatic Patients Display Impaired Responsiveness to Direct Current Electric Fields (dcEFs).

Accumulating evidence suggests that an important role is played by electric signals in modifying cell behaviour during developmental, regenerative and pathological processes. However, their role in asthma has not yet been addressed. Bronchial fibroblasts have recently been identified having important roles in asthma development. Therefore, we adapted an experimental approach based on the lineages of human bronchial fibroblasts (HBF) derived from non-asthmatic (NA) donors and asthmatic (AS) patients to elucidate whether their reactivity to direct current electric fields (dcEF) could participate in the asthmatic process. The efficient responsiveness of NA HBF to an electric field in the range of 2-4 V/cm was illustrated based on the perpendicular orientation of long axes of the cells to the field lines and their directional movement towards the anode. These responses were related to the activity of TGF-β signalling, as the electrotaxis and re-orientation of NA HBF polarity was impaired by the inhibitors of canonical and non-canonical TGF-β-dependent pathways. A similar tendency towards perpendicular cell-dcEF orientation was observed for AS HBF. However, their motility remained insensitive to the electric field applied at 2-4 V/cm. Collectively, these observations demonstrate the sensitivity of NA HBF to dcEF, as well as the inter-relations between this parameter and the canonical and non-canonical TGF-β pathways, and the differences between the electrotactic responses of NA and AS HBF point to the possible role of their dcEFs in desensitisation in the asthmatic process. This process may impair the physiologic behaviour of AS HBF functions, including cell motility, ECM deposition, and contractility, thus promoting bronchial wall remodelling, which is a characteristic of bronchial asthma.

Multi-omic subtyping and molecular determinants of neoadjuvant therapeutic regimen for locally advanced HER2-positive and triple-negative breast cancer: Implications of novel therapy.

e12600 Background: Neoadjuvant chemotherapy is the backbone treatment modalities for BC patients for locally advanced breast cancer (BC) improves R0 resection rate. Determinants of differentially therapeutic response to anthracycline or platinum-based neoadjuvant therapy were largely unknown but were urgently needed to maximize patients’ benefit. Methods: A total of 149 local advanced (cT2-3, cN0-2, cM0-1) BC patients (HER2 positive BC, n = 54 and TNBC, n = 95) were retrospectively enrolled in the study, whole-exon sequencing (WES), mRNA sequencing and (phosphor-)proteomics were systematically conducted and comprehensively analyzed. The primary end point of the study was the pathological complete response (pCR) rate. The Cancer Genome Atlas (TCGA) BC dataset and GSE130787 set were used to validate our analysis. Cancer cell line encyclopedia (CCLE) dataset was utilized to screen putative small molecular inhibitors to BC cells with molecular features of resistance. Results: Through integrated analysis of multi-omics data, three subtypes with distinct molecular features were identified: immune-activated (IA) subtype, vesicular transport pathways activation (VTs) subtype, and kinase activation (KA) subtype. Same scenario of molecular subtypes can be repeated in TCGA dataset and prognosis of these subtypes can be differentiate ( p= 0.02). Patients with IA subtype majorly favored cisplatin-based treatment ( p= 0.005). And this phenomenon was confirmed in external dataset (GSE130787, n = 26, HER2-positive BC receiving TCbH treatment). Patients with VTs subtype, with significant enrichment of vesicular transport-related pathways, displayed anthracycline sensitivity. Anthracycline transporter, ABCB1 expression was significantly reduced in VTs subtype at both mRNA ( p < 0.001) and protein ( p < 0.05) levels. CCLE breast cancer cells with the characteristics of VTs subtype and lower ABCB1 expression also displayed the sensitivity of epirubicin ( p = 0.0037). Patient with KA subtype was insensitive to both platinum and anthracycline-based therapy. Multiple kinase pathways, especially, non-canonical TGF-β signaling pathway-related genes were overexpressed (p < 0.05), and significantly correlated with non-pCR ( p= 0.002). Phosphor-proteomic analysis indicated TGFβ1-mediated RhoA/ROCK pathway (Cofilin, RhoA kinase, MYH14 proteins) were highly activated. Eleven drugs were found in CCLE inhibiting growth of BC cell lines with upregulated non-canonical TGF-β pathway genes ( p < 0.05). Conclusions: Three multi-omic subtypes with distinct response to anthracycline or platinum-based therapy were identified. Moreover, activated non-canonical TGF-β pathway may mediate therapeutic resistance, but also indicated therapeutic vulnerabilities of several kinase inhibitors.

CRISPR activation screen identifies TGFβ-associated PEG10 as a crucial tumor suppressor in Ewing sarcoma

As the second most common pediatric bone and soft tissue tumor, Ewing sarcoma (ES) is an aggressive disease with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an “undruggable” fusion protein acting as transcriptional modulator. EWS-FLI1 rewires the protein expression in cancer cells by activating and repressing a multitude of genes. The role and contribution of most repressed genes remains unknown to date. To address this, we established a CRISPR activation system in clonal SKNMC cell lines and interrogated a custom focused library covering 871 genes repressed by EWS-FLI1. Among the hits several members of the TGFβ pathway were identified, where PEG10 emerged as prime candidate due to its strong antiproliferative effect. Mechanistic investigations revealed that PEG10 overexpression caused cellular dropout via induction of cell death. Furthermore, non-canonical TGFβ pathways such as RAF/MEK/ERK, MKK/JNK, MKK/P38, known to lead to apoptosis or autophagy, were highly activated upon PEG10 overexpression. Our study sheds new light onto the contribution of TGFβ signalling pathway repression to ES tumorigenesis and suggest that its re-activation might constitute a novel therapeutic strategy.

A Tight Control of Non-Canonical TGF-β Pathways and MicroRNAs Downregulates Nephronectin in Podocytes.

Nephronectin (NPNT) is an extracellular matrix protein in the glomerular basement membrane that is produced by podocytes and is important for the integrity of the glomerular filtration barrier. Upregulated transforming growth factor β (TGF-β) and altered NPNT are seen in different glomerular diseases. TGF-β downregulates NPNT and upregulates NPNT-targeting microRNAs (miRs). However, the pathways involved were previously unknown. By using selective inhibitors of the canonical, SMAD-dependent, and non-canonical TGF-β pathways, we investigated NPNT transcription, translation, secretion, and regulation through miRs in podocytes. TGF-β decreased NPNT mRNA and protein in cultured human podocytes. TGF-β-dependent regulation of NPNT was meditated through intracellular signaling pathways. Under baseline conditions, non-canonical pathways predominantly regulated NPNT post-transcriptionally. Podocyte NPNT secretion, however, was not dependent on canonical or non-canonical TGF-β pathways. The canonical TGF-β pathway was also dispensable for NPNT regulation after TGF-β stimulation, as TGF-β was still able to downregulate NPNT in the presence of SMAD inhibitors. In contrast, in the presence of different non-canonical pathway inhibitors, TGF-β stimulation did not further decrease NPNT expression. Moreover, distinct non-canonical TGF-β pathways mediated TGF-β-induced upregulation of NPNT-targeting miR-378a-3p. Thus, we conclude that post-transcriptional fine-tuning of NPNT expression in podocytes is mediated predominantly through non-canonical TGF-β pathways.

Nephronectin Expression Is Controlled by the Non-Canonical TGF-β Pathway in Podocytes

Nephronectin Expression Is Controlled by the Non-Canonical TGF-β Pathway in Podocytes

Potential roles of the IL-6 family in conjunctival fibrosis

Elevated intraocular pressure (IOP) is a significant risk factor for vision loss due to glaucoma, which is a major cause of blindness worldwide. Glaucoma filtration surgery (GFS) is an important method to reduce IOP by guidance of aqueous humor into a newly built filtration bleb in the conjunctiva; management of the wound healing mechanism is essential for the success of GFS. Here, we investigated the roles of interleukin (IL)-6 family members during the wound healing process after GFS. At the surgical site, the expression levels of genes encoding IL-6, oncostatin M (OSM), their receptors, and collagen I were elevated at 3 h after GFS, whereas the levels of genes encoding transforming growth factor (TGF)-β, α-smooth muscle actin (SMA), type IV collagen, and fibronectin were elevated at 3 days after GFS. IL-6 trans-signaling and OSM signaling suppressed TGF-β-induced expression of α-SMA and collagen IV, as well as activation of the non-canonical TGF-β pathway, suggesting that IL-6 and OSM may aid in controlling the phase transition from inflammation to proliferation and remodeling. The suppressive effects of OSM were accompanied by STAT3 activation, such that STAT1 function was complementary to STAT3. Taken together, these observations indicated that IL-6 family members constitute early response genes after GFS, which can suppress TGF-β-induced expression of late response genes at the surgical site after GFS.

MO011PODOCYTE NEPHRONECTIN IS REGULATED BY TRANSFORMING GROWTH FACTOR BETA VIA THE CANONICAL AND NON-CANONICAL PATHWAYS

Abstract Background and Aims The glomerular basement membrane (GBM), podocytes and glomerular endothelial cells (GEC) are composing the glomerular filtration barrier (GFB) within the glomerulus. Both podocytes and GEC are essential components for the synthesis of the extracellular matrix (ECM) of the GBM. One ECM protein of the GBM, which is mainly produced by podocytes is nephronectin (NPNT). An altered expression pattern of NPNT has been observed in different kidney diseases. While NPNT was shown to be down regulated in membranous glomerulonephropathy, its expression was elevated in diabetic glomerulopathy, compared to healthy controls. Using a morpholino-induced knockdown of npnt in zebrafish larvae, proteinuria, podocyte foot process effacement and thickening of the lamia rara interna of the GBM were observed. Mice that were intra peritoneally injected with a microRNA 378a (miR-378a) mimic showed a decrease in NPNT expression in the kidneys on both mRNA and protein level, suggesting a regulatory effect of miR-378a on NPNT. In addition, in cultured human podocytes treatment with transforming growth factor beta (TGFβ), as well as transfection with miR-378a mimic down regulated NPNT mRNA and protein expression. By blocking different parts of the TGFβ pathway, we want to further investigate the mechanisms by which TGFβ mediates the regulation of NPNT in podocytes. Method Our main model for this study are immortalized human podocytes, which are proliferating at 33°C and differentiating at 37°C due to a temperature sensitive SV40 large T antigen. Ten to 12 days differentiated podocytes were used for experiments. Differentiated cells were either transfected with a miR-192 or control miR mimic or pre-incubated with different inhibitors for components of both the canonical and the non-canonical TGFβ signaling pathways, followed by culture with or without additional TGFβ. Cell lysates were prepared to be used for Western Blot or qPCR analyses. Results Treatment of immortalized human podocytes with TGFβ decreased NPNT expression on mRNA and protein level. After transfecting immortalized human podocytes with a miR-192 mimic, a GEC-derived miR up regulated by TGFβ stimulation, we observed reduced NPNT expression, compared to control miR transfection. Blocking TGFβ receptor I signaling with the specific inhibitor SD208, caused higher NPNT protein abundance, while NPNT mRNA expression remained unchanged. Targeting downstream parts of both the canonical and non-canonical TGFβ pathways by using inhibitors for single molecules of the respective arms of the intricate TGFβ pathway showed ambiguous results. Suppression of either Smad2 and/or Smad3 tended to enhance NPNT protein levels, accompanied by mostly unaltered mRNA expression. Blockade of different parts of the non-canonical TGFβ pathway also up regulated protein expression of NPNT. However, NPNT mRNA expression was more variable. Therefore, regulation of podocyte NPNT might not be due to changes in mRNA transcription, but to modifications on posttranscriptional level. Conclusion Treating immortalized human podocytes with TGFβ or TGFβ-induced miR-192 reduced NPNT expression on both the mRNA and protein level. More detailed analysis with inhibition of different parts of the canonical and non-canonical TGFβ pathways hint that both pathways are involved in NPNT expression. Therefore, we suggest that the regulation of podocyte NPNT by TGFβ is fine-tuned via both the canonical and non-canonical pathways with additional modulation through TGFβ dependent miRs.

Physical Training Inhibits the Fibrosis Formation in Alzheimer’s Disease Kidney Influencing the TGFβ Signaling Pathways

Background:Alzheimer’s disease (AD) is a neurodegenerative illness, with several peripheral pathological signs such as accumulation of amyloid-β (Aβ) plaques in the kidney. Alterations of transforming growth factor β (TGFβ) signaling in the kidney can induce fibrosis, thus disturbing the elimination of Aβ.Objective:A protective role of increased physical activity has been proven in AD and in kidney fibrosis, but it is not clear whether TGFβ signalization is involved in this effect.Methods:The effects of long-term training on fibrosis were investigated in the kidneys of mice representing a model of AD (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) by comparing wild type and AD organs. Alterations of canonical and non-canonical TGFβ signaling pathways were followed with PCR, western blot, and immunohistochemistry.Results:Accumulation of collagen type I and interstitial fibrosis were reduced in kidneys of AD mice after long-term training. AD induced the activation of canonical and non-canonical TGFβ pathways in non-trained mice, while expression levels of signal molecules of both TGFβ pathways became normalized in trained AD mice. Decreased amounts of phosphoproteins with molecular weight corresponding to that of tau and the cleaved C-terminal of AβPP were detected upon exercising, along with a significant increase of PP2A catalytic subunit expression.Conclusion:Our data suggest that physical training has beneficial effects on fibrosis formation in kidneys of AD mice and TGFβ signaling plays a role in this phenomenon.

N-cadherin mediates the migration of bone marrow-derived mesenchymal stem cells toward breast tumor cells

Rationale: Bone marrow-derived mesenchymal stem cells (BM-MSCs) recruited into breast tumors regulate the behavior of tumor cells via various mechanisms and affect clinical outcomes. Although signaling molecules, such as transforming growth factor β (TGF-β), are known to transmit signals between BM-MSCs and breast tumor cells for recruiting BM-MSCs, it is unclear which specific intrinsic molecules involved in cell motility mediate the migration of BM-MSCs into breast tumor. It is also unclear as to how specific intrinsic molecules contribute to the migration.Methods: Conditioned medium (CM) from breast tumor cells (MCF-7 and MDA-MB-231) that simulates breast tumor secreting TGF-β was used to examine the migration of BM-MSCs into breast tumors. A three-dimensional migration assay was performed to investigate the collective migration of BM-MSCs, maintaining cell-cell adhesion, toward breast tumor cells.Results: N-cadherin formed adherens junction-like structures on the intercellular borders of BM-MSCs, and TGF-β increased the expression of N-cadherin on these borders. Knockdown of Smad4 impaired the TGF-β-mediated increase in N-cadherin expression in BM-MSCs, but inhibitors of non-canonical TGF-β pathways, such as extracellular signal‐regulated kinases, Akt, and p38, did not affect it. siRNA-mediated knockdown of N-cadherin and Smad4 impaired the migration of BM-MSCs in response to TGF-β. Conditioned medium from breast tumor cells also enhanced the expression of N-cadherin in BM-MSCs, but inactivation of TGF-β type 1 receptor (TGFBR1) with SB505124 and TGFBR1 knockdown abolished the increase in N-cadherin expression. BM-MSCs collectively migrated toward CM from MDA-MB-231 in vitro while maintaining cell-cell adhesion through N-cadherin. Knockdown of N-cadherin abolished the migration of BM-MSCs toward the CM from breast tumor cells.Conclusion: In the present study, we identified N-cadherin, an intrinsic transmembrane molecule in adherens junction-like structures, on BM-MSCs as a mediator for the migration of these cells toward breast tumor. The expression of N-cadherin increases on the intercellular borders of BM-MSCs through the TGF-β canonical signaling and they collectively migrate in response to breast tumor cells expressing TGF-β via N-cadherin-dependent cell-cell adhesion. We, herein, introduce a novel promising strategy for controlling and re-engineering the breast tumor microenvironment.

Canonical and noncanonical TGF-\u03b2 signaling regulate fibrous tissue differentiation in the axial skeleton

Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-β regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-β stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-β did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-β-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-β-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-β treatment. Next, the role of several noncanonical TGF-β pathways were tested. We found that ERK1/2 was activated by TGF-β and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-β regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFβ-mediated fibrous differentiation in sclerotome.

Bone marrow stromal cells induce an ALDH+ stem cell-like phenotype and enhance therapy resistance in AML through a TGF-β-p38-ALDH2 pathway.

The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) consists of various cell types that support the growth of AML cells and protect them from chemotherapy. Mesenchymal stromal cells (MSCs) in the BME have been shown to contribute immensely to leukemogenesis and chemotherapy resistance in AML cells. However, the mechanism of stroma-induced chemotherapy resistance is not known. Here, we hypothesized that stromal cells promote a stem-like phenotype in AML cells, thereby inducing tumorigenecity and therapy resistance. To test our hypothesis, we co-cultured AML cell lines and patient samples with BM-derived MSCs and determined aldehyde dehydrogenase (ALDH) activity and performed gene expression profiling by RNA sequencing. We found that the percentage of ALDH+ cells increased dramatically when AML cells were co-cultured with MSCs. However, among the 19 ALDH isoforms, ALDH2 and ALDH1L2 were the only two that were significantly upregulated in AML cells co-cultured with stromal cells compared to cells cultured alone. Mechanistic studies revealed that the transforming growth factor-β1 (TGF-β1)-regulated gene signature is activated in AML cells co-cultured with MSCs. Knockdown of TGF-β1 in BM-MSCs inhibited stroma-induced ALDH activity and ALDH2 expression in AML cells, whereas treatment with recombinant TGF-β1 induced the ALDH+ phenotype in AML cells. We also found that TGF-β1-induced ALDH2 expression in AML cells is mediated by the non-canonical pathway through the activation of p38. Interestingly, inhibition of ALDH2 with diadzin and CVT-10216 significantly inhibited MSC-induced ALDH activity in AML cells and sensitized them to chemotherapy, even in the presence of MSCs. Collectively, BM stroma induces ALDH2 activity in AML cells through the non-canonical TGF-β pathway. Inhibition of ALDH2 sensitizes AML cells to chemotherapy.

Abstract 15530: Single-cell Analysis in Aortic Aneurysmal Tissue From Patients With Marfan Syndrome Reveals Increased Tgf-beta Production but Downregulation of Downstream Canonical Tgf-beta Signaling Pathways

Background: Marfan syndrome (MFS) is caused by mutations in the gene for fibrillin-1 ( FBN1 ); however, the mechanisms by which these mutations cause aortic aneurysms are poorly understood. Although it was hypothesized previously that dysregulation of the complex TGF-β signaling pathway leads to aortic aneurysm formation, FBN1 mutations appear to have a paradoxical effect on TGF-β signaling in MFS. In this study, we evaluated cell-specific TGF-β expression in non-immune cells in MFS aortic tissue. Methods: We performed single-cell RNA sequencing of ascending aortic aneurysm tissues from MFS patients (n=3) undergoing aneurysm repair and age-matched, non-aneurysmal control tissue from cardiac transplant donors and recipients (n=4). Non-immune cells were separated out from the data and analyzed using the Seurat package in R. Differentially expressed genes were identified using edgeR. Results: Conserved gene expression was used to identify populations of smooth muscle cells (SMCs; n=6), fibroblasts (n=3), and endothelial cells (ECs; n=3). We found that TGFB1 was significantly upregulated in quiescent fibroblasts (identified by increased expression of DCN , LUM , and complement factors) with log2FC of 1.30 and FDR 8.25x10 -8 , as well as in activated fibroblasts (identified by increased expression of genes involved in blood vessel repair and healing including ACTA2 , NOTCH3 , THBS2 , and PDGFRB ) with log2FC of 1.25 and FDR 6.15x10 -22 . Despite this increase in TGFB1 , expression of TGF-β receptor genes (predominately TGFBR2 ) as well as downstream SMAD genes was downregulated significantly in the SMC, fibroblast, and endothelial cell clusters. Finally, genes involved in the non-canonical TGF-β pathway, including ERK , JNK, and p38, were not differentially expressed in non-immune cells in MFS compared with control tissues. Conclusion: Increased expression of TGFB1 in non-immune cells in MFS was driven by two clusters of fibroblasts. Despite this, our data do not support associated upregulation of other genes in the canonical or non-canonical TGF-β pathways and in fact support downregulation of canonical TGF-β signaling in non-immune cells of aneurysmal tissues from MFS patients with advanced aortic disease.

Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

Silencing COX-2 blocks PDK1/TRAF4-induced AKT activation to inhibit fibrogenesis during skeletal muscle atrophy

Skeletal muscle atrophy with high prevalence can induce weakness and fatigability and place huge burden on both health and quality of life. During skeletal muscle degeneration, excessive fibroblasts and extracellular matrix (ECM) accumulated to replace and impair the resident muscle fiber and led to loss of muscle mass. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in synthesis of prostaglandin, has been identified as a positive regulator in pathophysiological process like inflammation and oxidative stress. In our study, we found injured muscles of human subjects and mouse model overexpressed COX-2 compared to the non-damaged region and COX-2 was also upregulated in fibroblasts following TGF-β stimulation. Then we detected the effect of selective COX-2 inhibitor celecoxib on fibrogenesis. Celecoxib mediated anti-fibrotic effect by inhibiting fibroblast differentiation, proliferation and migration as well as inactivating TGF-β-dependent signaling pathway, non-canonical TGF-β pathways and suppressing generation of reactive oxygen species (ROS) and oxidative stress. In vivo pharmacological inhibition of COX-2 by celecoxib decreased tissue fibrosis and increased skeletal muscle fiber preservation reflected by less ECM formation and myofibroblast accumulation with decreased p-ERK1/2, p-Smad2/3, TGF-βR1, VEGF, NOX2 and NOX4 expression. Expression profiling further found that celecoxib could suppress PDK1 expression. The interaction between COX-2 and PDK1/AKT signaling remained unclear, here we found that COX-2 could bind to PDK1/AKT to form compound. Knockdown of COX-2 in fibroblasts by pharmacological inactivation or by siRNA restrained PDK1 expression and AKT phosphorylation induced by TGF-β treatment. Besides, si-COX-2 prevented TGF-β-induced K63-ubiquitination of AKT by blocking the interaction between AKT and E3 ubiquitin ligase TRAF4. In summary, we found blocking COX-2 inhibited fibrogenesis after muscle atrophy induced by injury and suppressed AKT signaling pathway by inhibiting upstream PDK1 expression and preventing the recruitment of TRAF4 to AKT, indicating that COX-2/PDK1/AKT signaling pathway promised to be target for treating muscle atrophy in the future.

AML-241: Bone Marrow Stromal Cells Induce Chemotherapy Resistance in AML Cells through TGF-β-p38-ALDH2 Pathway

Context Chemotherapy resistance is one of the leading causes of disease progression and relapse in patients with acute myeloid leukemia (AML). The interaction between mesenchymal stromal cells (MSCs) and AML cells in the bone marrow (BM) is critical for leukemogenesis and chemotherapy resistance. Objective As tumor initiation and chemotherapy resistance are characteristic features of leukemia stem cells, we hypothesize that BM-MSCs induce a stem-like phenotype in AML cells. The objectives of this study are to determine the effect of stromal cells on the stem cell function of AML cells and to identify signaling pathways that support AML-stroma interactions in the BM microenvironment. Design We cultured AML cell lines and patient samples with or without BM-MSCs and measured aldehyde dehydrogenase (ALDH) activity in AML cells by ALDEFLUOR™ assay. In addition, we performed genomic and proteomic analyses to identify signaling pathways that support AML-stroma interactions. Finally, we used specific inhibitors that target ALDH activity to sensitize AML cells to chemotherapeutic agents. Results Co-culture of AML cells with BM-MSCs significantly induced ALDH activity in AML cells, mainly through upregulation of ALDH2 and ALDH1L2 isoforms. TCGA data analysis revealed that ALDH2 is a prognostic factor in AML. Mechanistic studies revealed that TGF-β1-regulated gene signature is activated in AML cells co-cultured with stromal cells. Knockdown of TGF-β1 in BM-MSCs inhibited stroma-induced ALDH activity and ALDH2 expression in AML cells, whereas treatment with recombinant TGF-β1 induced an ALDH+ phenotype in AML cells. We also found that the non-canonical TGF-β pathway, through p38 activation, regulates ALDH2 expression in AML cells. Pharmacologic inhibition of ALDH2 significantly inhibited BM-MSC-induced ALDH activity and sensitized AML cells to chemotherapy. Conclusion BM-MSCs induce ALDH+ stem cell phenotype in AML cells through the non-canonical TGF-β pathway. ALDH2 is the crucial isoform regulating ALDH activity in AML cells and is a prognostic factor. Pharmacologic inhibition of ALDH2 sensitizes AML cells to standard chemotherapy. Grant Acknowledgement This study is partially supported by Leukemia SPORE CDA, IRG from MDACC, Cure Sonia Foundation and Golfer's Against Cancer to VLB. NIH (CA055164), MDACC Support Grant (CA016672), CPRIT-MIRA, and Paul and Mary Haas Chair in Genetics to MA.

Human Skin Keratinocytes on Sustained TGF-β Stimulation Reveal Partial EMT Features and Weaken Growth Arrest Responses.

Defects in wound closure can be related to the failure of keratinocytes to re-epithelize. Potential mechanisms driving this impairment comprise unbalanced cytokine signaling, including Transforming Growth Factor-β (TFG-β). Although the etiologies of chronic wound development are known, the relevant molecular events are poorly understood. This lack of insight is a consequence of ethical issues, which limit the available evidence to humans. In this work, we have used an in vitro model validated for the study of epidermal physiology and function, the HaCaT cells to provide a description of the impact of sustained exposure to TGF-β. Long term TGF-β1 treatment led to evident changes, HaCaT cells became spindle-shaped and increased in size. This phenotype change involved conformational re-arrangements for actin filaments and E-Cadherin cell-adhesion structures. Surprisingly, the signs of consolidated epithelial-to-mesenchymal transition were absent. At the molecular level, modified gene expression and altered protein contents were found. Non-canonical TGF-β pathway elements did not show relevant changes. However, R-Smads experienced alterations best characterized by decreased Smad3 levels. Functionally, HaCaT cells exposed to TGF-β1 for long periods showed cell-cycle arrest. Yet, the strength of this restraint weakens the longer the treatment, as revealed when challenged by pro-mitogenic factors. The proposed setting might offer a useful framework for future research on the mechanisms driving wound chronification.