Inhibitor LY2157299 Research Articles

Abstract 1569: TGFβ signaling suppresses alternative end joining associated with genomic mutations and PARP Inhibition sensitivity

Abstract Differential use of DNA repair pathways in cancer leads to distinct clinical outcomes following genotoxic therapies. We found that deficient TGFβ signaling due to human papilloma virus (HPV) in head and neck squamous cell carcinoma (HNSCC) results in impaired DNA repair proficiency, increased use of alternative end joining (alt-EJ) and that a 50-gene TGFβ signature is negatively correlated with prognosis (Clin Ca Res 2018, 24(23): 6001). To assess the molecular consequence of TGFβ signaling in the DNA damage response (DDR), we performed a Nanostring assay on SAS HNSCC and U251 glioblastoma cell lines using the 180 gene DNA damage and repair panel. The results showed that treatment with TGFβ type 1 receptor kinase inhibitor LY2157299 or TGFβ profoundly altered DDR gene expression profiles. TGFβ inhibition for 24 hr significantly (p<0.05) increased critical alt-EJ genes, POLQ (DNA polymerase theta) and PARP1 (poly(ADP-ribose) polymerase 1), which was confirmed by qRT-PCR. To interrogate the relationship between TGFβ and alt-EJ in The Cancer Genome Atlas (TCGA), we used the literature to curate a 36 gene alt-EJ signature. Single specimen gene set enrichment analyses (ssGSEA) showed that the alt-EJ signature was significantly anti-correlated with the TGFβ signature in 15 of 17 cancer types (PCC = -0.35; p =10-16). This anti-correlation was also observed in 875 cancer cell lines in Catalogue of Somatic Mutations in Cancer (COSMIC) database, suggesting a conserved, cancer cell intrinsic mechanism by which TGFβ regulates alt-EJ. Alt-EJ is an error prone DNA repair mechanism that generates gross genomic alterations. Consistent with this, low ssGSEA score for TGFβ and high ssGSEA score for alt-EJ are correlated with increased deletions or insertions in genome sequence of patient tumors in TCGA as well as cancer cell lines in COSMIC (p<0.05). PARP1 requirement prompted analyses of drug sensitivity data in the Sanger database, which showed that cell lines with high alt-EJ and low TGFβ ssGSEA score are significantly more sensitive to PARP1 inhibitors, olaparib and ABT-888, (p < 0.0001). These data lead to the novel hypothesis that TGFβ signaling actively suppresses alt-EJ. If true, the TGFβ/alt-EJ signature may be useful as a biomarker for precision cancer medicine with genotoxic agents, TGFβ or PARP inhibitors. Citation Format: Qi Liu, Luis Palomero, Jade Moore, Ines Guix, Miquel Angel Pujana, Mary Helen Barcellos-Hoff. TGFβ signaling suppresses alternative end joining associated with genomic mutations and PARP Inhibition sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1569.

2066-P: Dyrk1a Antisense Oligonucleotides Conjugated to GLP-1R Agonist Promote Pancreatic Beta-Cell Proliferation

Loss of functional beta cell mass is a major contributor to the pathogenesis of both type 1 and type 2 diabetes. Enhancing proliferation of existing beta cells in the pancreatic islet is one of the promising approaches for restoring functional beta cell mass. Increasing evidence indicates inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a) stimulates beta cell proliferation. However, to date DYRK1a small molecule inhibitors have not been therapeutically viable due to their ability to increase proliferation in peripheral tissues. Previously we demonstrated that generation 2.5 antisense oligonucleotides (ASO) conjugated to a glucagon like peptide 1 receptor (GLP-1R) agonist can specifically target the pancreatic beta cell. When compared to control ASO treated isolated islets, islets administered a GLP-1/Dyrk1a ASO conjugate reduces DYRK1a mRNA and protein expression and significantly increases Ki67 mRNA, a cell proliferation marker, in a dose-response manner in vitro. Furthermore, combination treatment of a GLP-1/Dyrk1a ASO conjugate and the small molecule TGF-β inhibitor Ly364947, can synergistically increase Ki67 mRNA expression, cell cycle progression markers cyclin D1, cyclin E2, Cdk1, Cdk4 mRNAs and beta cell markers Pdx1 and Glut2 mRNAs when compared to a DYRK1a conjugated ASO or Ly364947 alone. Administration of a Dyrk1a conjugated ASO to wild type and db/db mice reduced Dyrk1a mRNA and increased Ki67 mRNA expression in islets when compared to a control ASO. Cell cycle markers cyclin D2, cyclin E2 and Cdk1 mRNAs are also significantly upregulated. In conclusion, a GLP-1/Dyrk1a ASO conjugate can specifically inhibit beta cell Dyrk1a mRNA expression and promote beta cell proliferation, suggesting that this may be an important approach for the treatment of type 1 and type 2 diabetes. Disclosure W. Fu: Employee; Self; Ionis Pharmaceuticals, Inc. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E.C. Pirie: Employee; Self; Ionis Pharmaceuticals, Inc. T. Prakash: None. S. Murray: Employee; Self; Ionis Pharmaceuticals. A. Powers: Employee; Self; Ionis Pharmaceuticals, Inc. Stock/Shareholder; Self; Ionis Pharmaceuticals, Inc. A.E. Mullick: None. R.M. Crooke: Employee; Self; Ionis Pharmaceuticals, Inc. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.

Mechanical strain affects collagen metabolism-related gene expression in scleral fibroblasts

We previously demonstrated that collagen metabolism affects scleral mechanical properties and scleral remodeling. Scleral remodeling changes the mechanical strain on sclera and scleral fibroblasts. We postulated that mechanical strain changes affect collagen metabolism in scleral fibroblasts. To understand the differences in collagen metabolism in scleral fibroblasts related to mechanical strain changes, scleral fibroblasts were isolated and cultured under different mechanical strains using the FX-4000 system or were treated with the TGF-β1 and TGFBR1 inhibitor LY364947. The collagen metabolism-related gene expression levels were detected. The results showed that the appropriate (lower) mechanical strain improved collagen synthesis and reduced collagen decomposition. In contrast, higher mechanical strain reduced collagen synthesis and enhanced collagen decomposition, especially a sustained higher strain. Furthermore, the effect of a transitory higher strain was recoverable, and collagen metabolism in scleral fibroblasts was regulated by TGF-β1. These results suggested that mechanical strain mediates TGF-β1 expression to regulate collagen metabolism in scleral fibroblasts, thereby affect scleral tissue remodeling.

Targeting TGR5 Ameliorates Pulmonary Fibrosis by Regulating TGF-β1 Associated Signaling Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease with high morbidity and mortality. Recently, TGR5 has been identified as a critical regulatory factor involved in inflammation and cancer. However, its function in pulmonary fibrosis remains unclear. Here, we first reported that TGR5 was overexpressed in the hyperplastic alveolar epithelial cells overlying fibroblastic foci in lung tissues of IPF patients, while mesenchymal cells in areas of fibrosis also had scattered TGR5 staining. In vitro study, TGR5 knockdown effectively inhibited TGF-β1-induced alveolar epithelial cells (AECs) activation and fibroblasts proliferation, motility and differentiation. Mechanically, TGR5 promoted EMT process in lung fibrogenesis by the Smad2/3 and Akt/Erk signaling pathways, which could be potently blocked by TGF β receptor I inhibitor LY2157299 or Akt inhibitor MK2206. Furthermore, the TGR5 expression was elevated in animal models of bleomycin (BLM)-induced pulmonary fibrosis. Our data further revealed that the degree of lung fibrosis was significantly alleviated in TGR5 knockout mice, which was accompanied by prominent decreases in TGF-β1 expression, repression of EMT process and releasing of inflammatory and fibrogenic cytokines. Collectively, this study demonstrated that TGR5 exerted a profibrogenic role in pulmonary fibrosis, thus opening up a novel therapeutic opportunity for pulmonary fibrosis. Funding Statement: This study was supported by Grants from the National Natural Science Foundation of China (Grant No. 81874314, 81903633 and 81600044). Declaration of Interests: All authors reviewed the manuscript and all declare no conflict of interest. Ethics Approval Statement: All medical research was approved by the ethics committees of Ren Ji Hospital.

Combination of Evodiamine with Berberine Reveals a Regulatory Effect on the Phenotypic Transition of Colon Epithelial Cells Induced by CCD-18Co

Objective Transdifferentiation exists between stromal cells or between stromal cells and cancer cells. Evodiamine and berberine are predominant pharmacological components of Zuojin pill, a prescription of Traditional Chinese Medicine, playing crucial functions in remolding of tumor microenvironment. This study aimed to explore the effect of combination of evodiamine with berberine (cBerEvo) on the phenotypic transition of colon epithelial cells induced by tumor-associated fibroblasts, as well as the involved mechanisms.Methods Human normal colon epithelial cell line HCoEpiC cells were treated with the prepared conditioned medium of CCD-18Co, a human colon myofibroblast line, to induce epithelial-mesenchymal transition. Phase contrast microscope was used to observe the morphological changes. Epithelial-mesenchymal transition markers including E-cadherin, vimentin and alpha-smooth muscle actin (α-SMA) were observed with immunofluorescence microscopy. Migration was assessed by wound healing assay. Western blotting was used to detect the expressions of E-cadherin, vimentin, α-SMA, Snail, ZEB1 and Smads. Results In contrast to the control, the tumor-associated fibroblasts-like CCD-18Co cells induced down-regulation of E-cadherin and up-regulation of vimentin, α-SMA, Snail and ZEB1 (P<0.05), and promoted migration of HCoEpiCs (P<0.05), with over expression of Smads including Smad2, p-Smad2, Smad3, p-Smad3 and Smad4 (P<0.05), which were abolished by a transforming growth factor-β (TGF-β) receptor inhibitor LY364947 and by cBerEvo in a concentration dependent manner. In addition, cBerEvo-inhibited ratios of p-Smad2/Smad2 and p-Smad3/Smad3 were also dose dependent.Conclusion The above results suggest that cBerEvo can regulate the differentiation of colon epithelial cells induced by CCD-18Co through suppressing activity of TGF-β/Smads signaling pathway.

Clonorchis sinensis ESPs enhance the activation of hepatic stellate cells by a cross-talk of TLR4 and TGF-β/Smads signaling pathway

Excretory/Secretory products (ESPs) from Clonorchis sinensis-a fluke dwelling on the biliary ducts-promote the activation of hepatic stellate cells (HSCs) and lead to hepatic fibrosis ultimately, although the mechanisms that are responsible for CsESPs-induced activation of HSCs are largely unknown. In the present study, we investigated the underlying mechanism of TLR4 in the regulation of the activation of HSCs caused by CsESPs. We found that the expression of TLR4 was significantly increased in the HSCs with CsESPs for 24 h, compared to the control group. However, the activation of HSCs induced by CsESPs was inhibited by interfering with TGF-β/Smad pathway using a TGF-β receptor I inhibitor LY2157299, indicating that TGF-β induced signaling pathway was involved in CsESPs-caused the activation of HSCs. In addition, the activation of HSCs caused by CsESPs was remarkably inhibited by a TLR4 specific inhibitor (VIPER), and phosphorylation of Smad2/3 was significantly attenuated but the expression of the pseudoreceptor of TGF-β-type I receptor (BAMBI) was obviously increased when TLR4 signaling pathway was blocked. The results of the present study demonstrate that activation of HSCs caused by CsESPs is mediated by a cross-talk between TLR4 and TGF-β/Smads signaling pathway, and may provide a potential treatment strategy to interrupt the process of liver fibrosis caused by C. sinensis.

DNA-PKcs Mediates An Epithelial-Mesenchymal Transition Process Promoting Cutaneous Squamous Cell Carcinoma Invasion And Metastasis By Targeting The TGF-β1/Smad Signaling Pathway.

PurposeDNA-dependent protein kinase catalytic subunit (DNA-PKcs) has attracted extensive attention in various types of malignant tumors. However, the role of DNA-PKcs in cutaneous squamous cell carcinoma (cSCC) development has not been elucidated. In this study, we investigated the role of DNA-PKcs in cSCC and the molecular mechanisms of TGF-β1-induced cSCC progression mediated by DNA-PKcs.MethodsWe performed bioinformatic analysis and RT-PCR to examine the DNA-PKcs expression level in cSCC. Then, we downregulated DNA-PKcs using a DNA-PK-specific inhibitor or small interfering RNA (siRNA) to explore the effects of DNA-PKcs on SCL-1 cell migration and invasion. To further investigate the mechanism by which DNA-PKcs promotes cSCC progression, TGF-β1 and the TGF-β receptor (TGF-βR) I/II dual inhibitor LY2109761 were used to examine whether DNA-PKcs participates in TGF-β1/Smad signaling.ResultsDNA-PKcs expression was upregulated in cSCC. DNA-PK inhibition or expression knockdown resulted in inhibited migration and invasion and altered epithelial-mesenchymal transition (EMT) marker expression patterns in SCL-1 cells. Importantly, TGF-β1 mediated EMT induction in cSCC cells, and DNA-PKcs was identified as a TGF-β1-responsive gene. TGF-β1 promoted DNA-PKcs transcription, and DNA-PKcs enhanced the TGF-β1-induced EMT program involved in cSCC invasion and metastasis by phosphorylating Smad3.ConclusionThis study is the first to show that DNA-PKcs mediates EMT to promote cSCC aggressiveness by targeting the TGF-β1/Smad signaling pathway, which provides insight into how DNA-PKcs impacts cSCC progression and identifies a new therapeutic target.

TGF-β receptor inhibitor LY2109761 enhances the radiosensitivity of gastric cancer by inactivating the TGF-β/SMAD4 signaling pathway.

Radiotherapy is used to treat gastric cancer (GC); however, radioresistance challenges the clinical outcomes of GC, and the mechanisms of radioresistance in GC remain poorly understood. Here, we report that the TGF-β receptor inhibitor, LY2109761 (LY), is a potential radiosensitizer both in vitro and in vivo. As per the Cancer Genome Atlas database, TGF-β overexpression is significantly related to poor overall survival in GC patients. We demonstrated that the TGF-β/SMAD4 signaling pathway was activated in both radioresistant GC cells and radioresistant GC patients. As a TGF-β receptor inhibitor, LY can enhance the activities of irradiation by inhibiting cell proliferation, decreasing clonogenicity and increasing apoptosis. Moreover, LY attenuated the radiation-induced migration and invasion, epithelial-mesenchymal transition (EMT), inflammatory factor activation, immunosuppression, and cancer stem cell characteristics of GC cells, thus leading to radiosensitization of the GC cells. We confirmed that LY reduced tumor growth, inhibited TGF-β/SMAD4 pathway activation and reversed irradiation-induced EMT in a tumor xenograft model. Our findings indicate that the novel TGF-β receptor inhibitor, LY, increases GC radiosensitivity by directly regulating the TGF-β/SMAD4 signaling pathway. These findings provide new insight for radiotherapy in GC patients.

Cancer-associated fibroblast regulate proliferation and migration of prostate cancer cells through TGF-β signaling pathway

AimsProstate cancer (PCa) incidence rates are rising in China currently. Cancer-associated fibroblasts (CAFs), as a major component of tumor microenvironment, are crucial for tumor progression. This study was aimed to explore the promotion effect of patient-derived CAFs on the proliferation and migration of prostate cancer cells. Main methodsCAFs were isolated from tumor tissues of PCa patients. The promotion effect of CAFs on the proliferation and migration of PC-3 and LNCaP cells were evaluated in vitro and in vivo. The concentration of TGF-β1 was measured by Luminex assay. The blocking activity of LY2109761 on the promotion effect of CAFs was also evaluated. Key findingsCAFs could significantly promote the proliferation and migration of PC-3 and LNCaP cells both in vitro and in vivo. TGF-β1 was identified as a highly increased factor in CAFs-CM compared with the normal culture medium of these two cancer cell lines. TGF-β receptor inhibitor LY2109761 could suppress the CAFs-induced cellular proliferation and migration of PC-3 cells but not LNCaP cells. SignificanceOur study suggested a crucial role for CAFs and TGF-β signaling in the progression of PCa. Zebrafish xenograft model was an ideal animal model for the study of CAFs and cancer cell interaction.

Klotho inhibits angiotensin II-induced cardiac hypertrophy, fibrosis, and dysfunction in mice through suppression of transforming growth factor-β1 signaling pathway

Recent studies have revealed critical roles of transforming growth factor-β1 (TGF-β1) and microRNA-132 (miR-132), a downstream mediator of TGF-β1, in the pathogenesis of cardiac remodeling. In this study, we tested whether the antiaging protein klotho modifies angiotensin II (Ang II)-induced cardiac remodeling through regulating TGF-β1-miR-132 axis. We found that both klotho and the TGF-β1 inhibitor LY364947 significantly inhibited cardiac hypertrophy, fibrosis, and dysfunction in Ang II-infused mice, as evidenced by the ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL), cardiomyocyte cross-sectional area, fibrotic area, and expression of prohypertrophic genes (ANP, β-MHC) and fibrotic marker genes (α-SMA, collagen I), echocardiographic parameters. Meanwhile, klotho also significantly inhibited Ang II-induced protein expression of TGF-β1 and phosphorylated Smad2/3 in the heart tissues and cultured cardiomyocytes and cardiac fibroblasts. In vitro experiments demonstrated that Ang II-induced cardiomyocyte hypertrophy and proliferation and activation of cardiac fibroblasts were markedly inhibited by klotho, LY364947 or the miR-132 inhibitor anti-miR-132. Both klotho and the TGF-β1 inhibitor LY364947 downregulated the miR-132 expression. Additionally, klotho decreased Ang II-induced protein expressions of cardiac fibroblast growth factor (FGF)23 in vivo and in vitro. The decreased protein levels of klotho in serum and renal tissues of Ang II-infused mice were elevated by klotho. Klotho downregulated the protein levels of TGF-β1 in renal tissues of Ang II-infused mice. In conclusion, our results suggest that klotho prevents Ang II-induced cardiac remodeling and dysfunction through modifying the TGF-β1-miR-132 axis, providing an experimental basis for clinical treatment on cardiac remodeling.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

TGF-β Pathway Inhibition Protects the Diaphragm From Sepsis-Induced Wasting and Weakness in Rat.

Sepsis is a frequent complication in patients in intensive care units (ICU). Diaphragm weakness, one of the most common symptoms observed, can lead to weaning problems during mechanical ventilation. Over the last couple of years, members of the transforming growth factor (TGF) β family, such as myostatin, activin A, and TGF-β1, have been reported to strongly trigger the activation of protein breakdown involved in muscle wasting. The aim of this study was to investigate the effect of TGF-β inhibitor LY364947 on the diaphragm during chronic sepsis.Rats were separated into four groups exposed to different experimental conditions: Control group, Septic group, Septic group with inhibitor from day 0 (LY D0), and Septic group with inhibitor from day 1 (LY D1). Sepsis was induced in rats by cecal ligation and puncture, and carried out for 7 days.Chronic sepsis was responsible for a decrease in body weight, food intake and diaphragm's mass. The inhibitor was able to abolish diaphragm wasting only in the LY D1 group. Similarly, LY364947 had a beneficial effect on the diaphragm contraction only for the LY D1 group. SMAD3 was over-expressed and phosphorylated within rats in the Septic group; however, this effect was reversed by LY364947. Calpain-1 and -2 as well as MAFbx were over-expressed within individuals in the Septic group. Yet, calpain-1 and MAFbx expressions were decreased by LY364947.With this work, we demonstrate for the first time that the inhibition of TGF-β pathway during chronic sepsis protects the diaphragm from wasting and weakness as early as one day post infection. This could lead to more efficient treatment and care for septic patients in ICU.

Involvement of placental growth factor upregulated via TGF-β1-ALK1-Smad1/5 signaling in prohaptoglobin-induced angiogenesis.

A potential role of haptoglobin in arterial restructuring has been suggested, and our previous study demonstrated that prohaptoglobin, the precursor of haptoglobin, stimulates endothelial angiogenesis. However, the mechanisms underlying the angiogenic effects of prohaptoglobin are still unclear. Here, we investigated angiogenic signaling induced by prohaptoglobin using human umbilical vein endothelial cells. Prohaptoglobin upregulated the expression of placental growth factor (PlGF), vascular endothelial growth factor (VEGF)-A, and VEGF receptor 1 and 2, and also induced cell migration and tube network formation. PlGF knockdown attenuated these angiogenic effects of prohaptoglobin. Furthermore, a transcription factor profiling assay indicated that Smad is involved in PlGF expression in response to prohaptoglobin. Transforming growth factor-β1 (TGF-β1) expression and Smad1/5 phosphorylation were also induced by prohaptoglobin treatment. Blockade of TGF-β1 signaling using the TGF-β receptor kinase inhibitor LY2109761 or Smad1/5 siRNA reduced the prohaptoglobin-induced PlGF expression and in vitro tube formation. Knockdown of the TGF-β receptor ALK1, but not ALK5, with a specific siRNA blocked the Smad1/5 phosphorylation and PlGF expression induced by prohaptoglobin. These findings suggest that the angiogenic effects of prohaptoglobin are dependent on PlGF and mediated via a TGF-β1-ALK1-Smad1/5–PlGF/VEGFR1–VEGF-A/VEGFR2 signaling pathway.

Difference of TGF-β/Smads signaling pathway in epithelial-mesenchymal transition of normal colonic epithelial cells induced by tumor-associated fibroblasts and colon cancer cells.

Tumor microenvironment (TME) crucially functions in tumor initiation and progression. Stroma-tumor interactions and cellular transdifferentiation are the prerequisite for tumor formation. Transforming growth factor-β (TGF-β), a major cytokine secreted by tumor-associated fibroblasts (TAFs) and cancer cells, is a crucial player involving cell transdifferentiation. Therefore, we hypothesized that these TAFs and cancer cells also affect normal colon epithelium. In our study, we found for the first time that colon cancer cells HCT116 and TAF-like CCD-18Co cells induced epithelial-mesenchymal transition (EMT)-like transdifferentiation in colon epithelial cells HCoEpiCs, with enhanced migratio. Dysfunction of TGF-β/Smads signal was also observed in the EMT-transformed HCoEpiCs. We wondered whether these phenomena were regulated by TGF-β/Smads signaling pathway. A TGFβ receptor kinase I (TβRI) inhibitor LY364947 was used. We found that the EMT induced by the HCT116- and CCD-18Co-derived CM was suppressed by the LY364947. Besides, different expression profiles for the components of TGF-β/Smads pathway were found in the EMT-like HCoEpiCs, but high expression of p-Smad2/3 and Smad4 was the common feature. Our observations suggest that the mechanisms of phenotypic transition of colon epithelial cells are cellular environment-dependent, which maybe a basis of potential therapy targeting TME.

Knockdown of KDM2A inhibits proliferation associated with TGF-β expression in HEK293T cell.

Lysine-specific demethylase 2A (KDM2A, also known as JHDM1A or FBXL11) plays an important role in regulating cell proliferation. However, the mechanisms on KDM2A controlling cell proliferation are varied among cell types, even controversial conclusions have been drawn. In order to elucidate the functions and underlying mechanisms for KDM2A controlling cell proliferation and apoptosis, we screened a KDM2A knockout HEK293T cell lines by CRISPR-Cas9 to illustrate the effects of KDM2A on both biological process. The results indicate that knocking down expression of KDM2A can significantly weaken HEK293T cell proliferation. The cell cycle analysis via flow cytometry demonstrate that knockdown expression of KDM2A will lead more cells arrested at G2/M phase. Through the RNA-seq analysis of the differential expressed genes between KDM2A knockdown HEK293T cells and wild type, we screened out that TGF-β pathway was significantly downregulated in KDM2A knockdown cells, which indicates that TGF-β signaling pathway might be the downstream target of KDM2A to regulate cell proliferation. When the KDM2A knockdown HEK293T cells were transient-transfected with KDM2A overexpression plasmid or treated by TGF-β agonist hydrochloride, the cell proliferation levels can be partial or completely rescued. However, the TGF-β inhibitor LY2109761 can significantly inhibit the KDM2A WT cells proliferation, but not the KDM2A knockdown HEK293T cells. Taken together, these findings suggested that KDM2A might be a key regulator of cell proliferation and cell cycle via impacting TGF-β signaling pathway.

Cathelicidin-related antimicrobial peptide protects against cardiac fibrosis in diabetic mice heart by regulating endothelial-mesenchymal transition

Cathelicidin-related antimicrobial peptide (CRAMP), antimicrobial peptide, was reported to protect against myocardial ischemia/reperfusion injury. In the pathology of diabetic cardiomyopathy, endothelial-to-mesenchymal transition (EndMT) results from hyperglycemia-induced endothelial injury, leading to cardiac fibrosis. This study aims to evaluate the effect of CRAMP on EndMT and cardiac fibrosis on diabetic mice heart. Mice were subjected to streptozotocin to induce diabetes. CRAMP was administered by intraperitoneal injection (1 or 8 mg/kg/d) for 4 weeks from 12 weeks till 16 weeks after final streptozotocin injection. Cardiac dysfunction was observed in diabetic mice. Only 8 mg/kg/d CRAMP treatment proved cardiac function. Increased EndMT and fibrosis level were also observed in diabetic mice heart. 8mg/kg CRAMP inhibited EndMT and fibrosis level in diabetic mice. Mouse heart endothelial cells (MHECs) were treated with CRAMP and exposed to high glucose. Hyperglycemia-induced EndMT in MHECs was also attenuated by CRAMP treatment. Activation of TGFβ/Smad signalling was increased in diabetic mice heart tissue and hyperglycemia stimulated MHECs, which was prevented following CRAMP treatment. Activation of AMPKa1/mTOR showed similar changes. AMPKa1 siRNA abrogated the effects of CRAMP in MHECs. TGFβ/Smad inhibitor LY2109761 and AMPKa agonist AIRCA mimic the effect of CRAMP. In summary, CRAMP can inhibit EndMT, cardiac fibrosis and protect against diabetic cardiomyopathy by regulating AMPKa1/TGFβ signalling.

Berberine reversed the epithelial-mesenchymal transition of normal colonic epithelial cells induced by SW480 cells through regulating the important components in the TGF-β pathway.

Stroma-tumor interactions within microenvironment play a crucial role in tumor development and growth. Cellular transdifferentiation in the stroma is a prerequisite for tumor formation. Targeting the interactions maybe a promising anticancer strategy. Berberine (BBR) has been confirmed to have anticancer and anti-inflammatory effects. We found for the first time that colon cancer cells SW480 induced spindle-like morphological changes and downregulation of E-cadherin and upregulation of vimentin and alpha-smooth muscle actin in colon epithelial cells HCoEpiCs by using transwell coculture system and conditioned medium from SW480. The conditioned medium also promoted the migration of HCoEpiCs. This transition was inhibited by a transforming growth factor-β receptor inhibitor LY364947. BBR (50 and 100 µg/ml) reversed the EMT-like transition and repressed the migration in HCoEpiCs. Further results demonstrated that downregulation of TβRII, Smad2, p-Smad3, and overexpression of Smad3 participated in the SW480-induced phenotypic transition of HCoEpiCs. In addition, BBR upregulated the expressions of TβRII, Smad2, and p-Smad3. In conclusion, our findings suggest that BBR exerts the anti-EMT and antimigration effect by mediating the expression of TβRII, Smad2, and p-Smad3.

A mediator of phosphorylated Smad2/3, evodiamine, in the reversion of TAF-induced EMT in normal colonic epithelial cells.

Purpose Transdifferentiation exists within stromal cells in the tumour microenvironment. Transforming growth factor-β (TGF-β) secreted by tumour-associated fibroblasts (TAFs) affects the differentiation states of epithelial cells, including epithelial-mesenchymal transition (EMT). Evodiamine, a natural drug, can regulate differentiation. However, the specific effects and relative mechanisms of evodiamine remain unknown. Design We used four models to observe the influence of TAF-like CCD-18Co cells on the colon epithelial cell line HCoEpiC: the 3D- and 2D-mono-culture system, Transwell and direct co-culture model. Additionally, we established conditioned medium from CCD-18Co cells. The TGF-β pathway inhibitor LY364947 and evodiamine were added. Morphological changes and classical EMT markers were observed and detected using phase contrast microscopy and immunofluorescence. Cell migration was measured by the wound-healing assay. Western blotting was performed to detect the TGF-β/Smad signalling pathway. Results CCD-18Co cells induced EMT-like changes in the 2D- and 3D-cultured epithelial cell line HCoEpiC, accompanied by high expression of ZEB1 and Snail and the enhancement of migration. Moreover, CCD-18Co-derived conditioned medium caused dysfunction of TGF-β/Smad signalling in EMT. Evodiamine inhibited these EMT-like HCoEpiC and their migration. Additionally, evodiamine down-regulated the expression of ZEB1/Snail and up-regulated the expression of phosphorylated Smad2/3 (pSmad2/3). Evodiamine also increased the ratios of pSmad2/Smad2 and pSmad3/Smad3. Conclusion Based on our observations, evodiamine can reverse the TAF-induced EMT-like phenotype in colon epithelial cells, which may be associated with its mediation of phosphorylated Smad2 and Smad3 expression.

KDM6B promotes ovarian cancer cell migration and invasion by induced transforming growth factor-β1 expression.

KDM6B, also known as JMJD3, is a member of the family of histone lysine demethylase (KDMs), which is closely related to many types of cancers. However, its role and the underlying mechanisms in ovarian cancer remain unknown. Here we show that KDM6B is elevated in epithelial ovarian cancer and its expression level is closely related with metastasis and invasion. In addition, survival analysis showed that high expression of KDM6B was associated with low overall survival in ovarian cancer patients. Overexpression of KDM6B in epithelial ovarian cancer cells promoted proliferation, epithelial-mesenchymal transition (EMT), migration and invasion in vitro, and enhanced metastatic capacities in vivo. On the contrary, silencing KDM6B in invasive and metastatic ovarian cancer cells inhibited these processes. Mechanistically, we found that KDM6B exerts its function by modulating the transforming growth factor-β1 (TGF-β1) expression, and TGF-β1 signal pathway inhibitor LY2157299 significantly inhibited KDM6B-induced proliferation, migration, metastasis, and EMT in ovarian cancer cells. Our findings, for the first time, reveal the pivotal role of KDM6B in the invasion and metastatic behavior of epithelial ovarian cancer. Thus, targeting KDM6B may be a useful strategy to interfere with these behaviors of epithelial ovarian cancer.

Abstract 2035: Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress

Abstract Background. Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer which is characterized by a poor survival rate and high incidence of metastases. The integrated stress response (ISR) is activated under stress conditions (hypoxia, nutrient deprivation, or endoplasmic reticulum stress) and reduces the protein synthesis through phospho-eIF2α/ATF4 (activating transcription factor 4) to regulate cell fate. ATF4, which is overexpressed in breast cancer including TNBC, regulates tumor growth, autophagy, drug resistance, and metastasis during ISR through PERK and GCN2 pathways. We have reported enhanced ATF4 expression in unstressed MCF10A cells treated with TGFβ1. Here, we investigate the potential TGFβ-mediated stress-independent control of ATF4 activity and its impact on the TNBC-associated metastasis. Methods. SUM159PT and BT549 TNBC cell lines were treated with human recombinant TGFβ1 (10 ng/ml) and the TGFBRI kinase inhibitor LY2157299 (5µM) for 72h. Effects of TGFβ on ATF4 expression were assessed by silencing SMAD2/3 and SMAD4 with TGFβ1 for 24h in SUM159PT and BT549 cells. Changes in ATF4 expression by IRS were assessed after PERK, GCN2, PKR, HRI, and eIF2α inhibition with TGFβ1 for 72h in SUM159PT, BT549 and MDA-MB-231 cell lines. ATF4 expression levels were determined by RT-PCR and/or western blot. Effects on TGFβ-induced metastasis were analyzed by ATF4 knockdown for 48h by using two different siRNA sequences (#1 and #2) following treatment with TGFβ1 for 24h. Migration and invasion were performed by wound healing and transwell assays, respectively. Results were compared to a scrambled siRNA as negative control (SCR). Results. Our results show that ATF4 expression (at RNA and protein levels) was abrogated by LY2157299 upon TGFβ activation, suggesting that ATF4 is active downstream of TGFBRI. This result was supported by further SMAD2/3 and SMAD4 knockdown following treatment with TGFβ1, which was correlated with decreased ATF4 expression. Additionally, we assessed whether ATF4 inhibition can reduce the TGFβ-induced metastatic properties of TNCB cell lines. We found that ATF4 depletion inhibited migration and invasiveness in the three cell lines tested. ATF4 is known to exert a pro-metastatic role downstream of ISR through PERK and GCN2 pathways. Our results demonstrate that, upon treatment with TGFβ1, knockdown of the IRS mediators PERK, PKR, GCN2, HRI, and eIF2α did not correlate with a consistent decrease of ATF4 levels in the three cell lines. Conclusion. In conclusion, our results show for the first time that ATF4 is a downstream target of the canonical TGFβ/SMAD pathway in a ISR-independent fashion, and its depletion correlates with an inhibition of the TGFβ1-mediated migration and invasion of TNBC cell lines. Therefore, ATF4 may represent a therapeutic target in TNBC patients with active TGFβ signaling pathway. Citation Format: Jenny C. Chang, Alberto Ramirez, Maria P. Molina, Francisca E. Cara, Wei Qian, Wen Chen, Anthony J. Kozielski, Roberto R. Rosato, Juan A. Marchal, Jose A. Lorente, Pedro Sanchez-Rovira, Sergio Granados-Principal. Knockdown of TGFβ-activated ATF4 inhibits triple negative breast cancer metastases independently of cellular stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2035.