TGF-β Pathway Genes Research Articles

Vitamin D as an effective treatment in human uterine leiomyomas independent of mediator complex subunit 12 mutation

To study whether vitamin D (VitD) inhibits cell proliferation and Wnt/β-catenin and transforming growth factor-β (TGFβ) signaling pathways in uterine leiomyomas independent of mediator complex subunit 12 (MED12) mutation status. Prospective study comparing leiomyoma vs. myometrial tissues and human uterine leiomyoma primary (HULP) cells treated with or without VitD and analyzed by MED12 mutation status. Hospital and university laboratories. Women with uterine leiomyoma without any treatment (n = 37). Uterine leiomyoma and myometrium samples were collected from women undergoing surgery because of symptomatic leiomyoma pathology. Analysis of Wnt/β-catenin and TGFβ pathways and proliferation by quantitative real-time polymerase chain reaction in leiomyoma and myometrial tissue as well as in VitD-treated HULP cells analyzed by Sanger sequencing. Sequencing data showed that 46% of leiomyomas presented MED12 mutation, whereas no mutations were detected in adjacent myometrium. Expression of Wnt/β-catenin and TGFβ pathway genes was significantly increased in MED12-mutated leiomyomas compared to matched myometrium; no significant differences were found in wild-type (WT) leiomyomas. In HULP cells, VitD significantly decreased PCNA expression of both MED12-mutated and WT groups. VitD treatment decreased WNT4 and β-catenin expression in both groups compared to controls, with significance for WNT4 expression in MED12-mutated samples. Similarly, VitD significantly inhibited TGFβ3 expression in cells from both groups. MMP9 expression also decreased. Despite molecular differences between MED12-mutated and WT leiomyomas, VitD inhibited Wnt/β-catenin and TGFβ pathways in HULP cells, suggesting VitD as an effective treatment to reduce proliferation and extracellular matrix formation in different molecular subtypes of uterine leiomyomas.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-β pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Alteration of Transforming Growth Factor β Signaling Pathway Predicts Worse Prognosis in Pancreatic Ductal Adenocarcinoma.

Transforming growth factor β (TGF-β) signaling pathway is one of the core pathways in pancreatic ductal adenocarcinoma (PDAC). Prognostic value of TGF-β pathway genes as a functionally related group in PDAC is rarely studied. Seventy-two PDAC patients who underwent surgery between November 30, 2015, and September 13, 2017, in West China Hospital, Sichuan University, were identified and included in this study. Whole-exome sequencing or targeted next-generation sequencing was performed with tumor tissue. Clinicopathologic characteristics and survival data were retrospectively collected and analyzed. Genetic alterations were detected in 71 patients (98.6%). Although 1 patient (1.4%) had one genetic alteration, 33 patients (45.8%) had 2 to 4 alterations and 37 patients (51.4%) had 5 or more alterations. Twenty-five patients with TGF-β pathway alteration were identified as TGF-βm+ group. Other 47 patients were TGF-βm- group. Mutation of TGF-β pathway was independently associated with inferior survival (hazard ratio, 2.22, 95% confidence interval, 1.05-4.70, P = 0.04), especially in patients accepting radical surgery (hazard ratio, 3.25, 95% confidence interval, 1.01-10.49, P = 0.04). Inferior prognosis was observed in PDACs with mutations of TGF-β pathway. Genomic information could help screen out patients at risk after surgery, and adjuvant therapy might benefit this subgroup of PDACs.

Pim1 maintains telomere length in mouse cardiomyocytes by inhibiting TGFβ signalling.

Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFβ) signalling pathway where inhibiting TGFβ signalling maintains telomere length. The relationship between Pim1 and TGFβ has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFβ signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFβ signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFβ signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFβ pathway genes. Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFβ pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

A Positive Feedback Loop of TET3 and TGF-β1 Promotes Liver Fibrosis

SUMMARYPathological activation of TGF-β signaling is universal in fibrosis. Aberrant TGF-β signaling in conjunction with transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a central role in liver fibrosis. Here we report that the DNA demethylase TET3 is anomalously upregulated in fibrotic livers in both humans and mice. We demonstrate that in human HSCs, TET3 promotes profibrotic gene expression by upregulation of multiple key TGF-β pathway genes, including TGFB1. TET3 binds to target gene promoters, inducing demethylation, which in turn facilitates chromatin remodeling and transcription. We also reveal a positive feedback loop between TGF-β1 and TET3 in both HSCs and hepatocytes. Furthermore, TET3 knockdown ameliorates liver fibrosis in mice. Our results uncover a TET3/TGF-β1 positive feedback loop as a crucial determinant of liver fibrosis and suggest that inhibiting TET3 may represent a therapeutic strategy for liver fibrosis and perhaps other fibrotic diseases.

Identification of an Endoglin Variant Associated With HCV-Related Liver Fibrosis Progression by Next-Generation Sequencing.

Despite the astonishing progress in treating chronic hepatitis C virus (HCV) infection with direct-acting antiviral agents, liver fibrosis remains a major health concern in HCV infected patients, in particular due to the treatment cost and insufficient HCV screening in many countries. Only a fraction of patients with chronic HCV infection develop liver fibrosis. While there is evidence that host genetic factors are involved in the development of liver fibrosis, the common variants identified so far, in particular by genome-wide association studies, were found to have limited effects. Here, we conducted an exome association study in 88 highly selected HCV-infected patients with and without fibrosis. A strategy focusing on TGF-β pathway genes revealed an enrichment in rare variants of the endoglin gene (ENG) in fibrosis patients. Replication studies in additional cohorts (617 patients) identified one specific ENG variant, Thr5Met, with an overall odds ratio for fibrosis development in carriers of 3.04 (1.39–6.69). Our results suggest that endoglin, a key player in TGF-β signaling, is involved in HCV-related liver fibrogenesis.

Abstract 2636: Tumor associated MUC1 mediates TGF-β in pancreatic cancer

Abstract Pancreatic Cancer (PC) is the third leading cause of cancer-related deaths in the United States, with a five-year survival rate at 9%. Over 90% of PC is categorized as Pancreatic Ductal Adenocarcinoma (PDA). 85% of PDA overexpress tumor associated Mucin-1 (tMUC1), a membrane bound glycoprotein that is aberrantly glycosylated. Overexpression of tMUC1 in PDA plays a critical role in tumor progression and metastasis by promoting oncogenic signaling through its cytoplasmic tail (tMUC1-CT). Transforming growth factor-β (TGF-β) plays pleiotropic roles during cancer development and progression. Within normal cells and early carcinogenesis, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical SMAD pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However, during later stages of cancer, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition (EMT), migration, and invasion thus enhancing metastasis through the non-canonical Erk1/2 pathway. We have shown that high tMUC1 expressing PDA cells respond to TGF-β via activation of the non-canonical pathway and undergoes EMT whereas, low tMUC1 expressing PDA cells respond to TGF-β via activation of the canonical SMAD pathway and undergoes apoptosis. tMUC1 plays a key role in regulating TGF-β’s dual function in PDA. We hypothesize that signaling via tMUC1-CT enables TGF-β function to switch from a tumor suppressor to a tumor promoter. The effects of TGF-β1 to induce apoptosis versus proliferation and invasiveness was determined in a panel of tMUC1 high and low PDA cell lines. Analysis of apoptosis and cell cycle was conducted using flow cytometry, while invasiveness was determined using the Boyden chamber assay. Proteins levels were determined using western blot. TGF-β pathway genes were studied in patient data collected from GTEx (healthy, n=171) and TCGA (PDA, n=178). It was compared using Pearson's correlation coefficient. Finally, we determined the anti-tumor efficacy of neutralizing TGF-β1 in vivo in high and low tMUC1-expressing PDA tumors. In response to TGF-β, tMUC1 high PDA cells resisted apoptosis, enhanced invasiveness, and activated the Erk pathway, while tMUC1 low cells became apoptotic and activated the SMAD pathway. Cell cycle analysis confirms a significant increase in apoptosis in tMUC1 low PDA cells, however tMUC1 high PDA cells resist TGF-β induced apoptosis. Signal transduction was dependent upon tyrosine phosphorylation of tMUC1-CT domain by Src. RNA-Seq analysis in TCGA PDA tissue reveal eight genes that show significant correlation (low p-value OR p-value < 0.05) while it was insignificant in the GTEx healthy tissue. Finally, in mice, tMUC1-high PDA tumors responded to the neutralization of TGF-β1 by significantly reducing tumor growth, while having no effect on tMUC1-low PDA tumors. tMUC1 expression levels in PDA may serve as a marker for eligibility for potential anti-TGF-β therapies. Citation Format: Priyanka Grover, Ru Zhou, Mahboubeh Yazdanifar, Mohammad Ahmad, Angat Puri, Kajal Grover, Xinghua Shi, Pinku Mukherjee. Tumor associated MUC1 mediates TGF-β in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2636.

Abstract 3382: A pan-cancer analysis reveals high frequency genetic alterations in mediators of signaling by the TGF-β superfamily

Abstract Background: TGF-β/SMAD signaling is a crucial, often contradictory regulator in multiple stages of liver disease that include inflammation, cirrhosis and development of HCC as well as other cancers. The context-specific role of this pathway in treatment strategies has yet to be clarified. Therefore, understanding the multiple context-specific roles of the pathway across broad cancer types is critical towards deciphering the complexities of the pathway. Methods: We followed our previous analysis of HCCs, by extending and examining TGF-β pathway across 33 TCGA tumor types and 9125 samples to address this question. We focused on 43 core genes that encode components that regulate signaling by the TGF-β superfamily with 50 target genes collectively identified through a consensus among TCGA network members. In addition, we extended our analyses to functional studies in mouse mutants and human cell lines with alterations of TGF-β signaling. Results: Focusing on 43 core TGF-β pathway genes, we found at least one of them was genomically altered in 39% of samples (mutations: 24%, homozygous deletions: 10%, or amplifications: 14%). We observed the highest alteration frequencies with hotspot mutations, 65% of which were in liver and GI cancers. We identified hotspots in 6 genes, with new discoveries in TGFBR2 and BMP5. Interestingly, with all 6 hotspot mutations we observed increased expression of TERT, HMGA2, IL6, MMP9, COL1A1/1A2/3A1, MYC, and FOXP3. Surprisingly, CDH2, and ALDH1A1expression levels were markedly reduced in liver and GI cancers. Alterations in the core genes correlated positively with expression of metastasis-associated genes, and poor patient survival. Epigenetic silencing and miRNA expression were associated with limited activity of the pathway in a cancer dependent manner. Using proteomics data, elevated TGF-β pathway activity showed positive correlation activity of DNA damage repair and EMT pathways (R=0.24, p < 0.0001), while the cell cycle and apoptosis pathways showed strong negative correlation (R= -0.3, and -0.15, p < 0.0001). Functional analyses reveal that disruption of TGF-β leads to increased sensitivity to cisplatin and other DNA cross linking agents as well as radiation. Conclusions: Our data suggest that TGF-β superfamily indices when combined with specific genes, such as HMGA2 and TERT, may represent strong prognostic markers, and targets in some cancer types such as HCC. This study provides a rich resource and broad molecular perspective that could guide future functional and therapeutic studies of the diverse set of cancer pathways mediated by TGF-β superfamily. In addition, when the pathway is disrupted, epithelial cells are more susceptible to transformation and invasion, potentially identifying specific populations that are more sensitive to chemotherapy such as cisplatin and 5FU, as well as radiation therapy. Citation Format: Kazufumi Ohshiro, Sobia Zaidi, Anil Korkut, Jian Chen, Shuyun Rao, Shoujun Gu, Wilma Jogunoori, Bibhuti Mishra, Rehan Akbani, Lopa Mishra. A pan-cancer analysis reveals high frequency genetic alterations in mediators of signaling by the TGF-β superfamily [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3382.

Alteration of TGF-β signaling pathway predicts worse prognosis in pancreatic ductal adenocarcinoma.

e13006 Background: Transforming growth factor beta (TGF-β) signaling pathway is one of the core pathways involved in tumor initiation and progression. The prognostic value of TGF-β pathway genes as a functionally related group in pancreatic ductal adenocarcinoma (PDAC) is rarely studied. Methods: 72 PDAC patients who underwent surgery between November 30, 2015 and September 13, 2017 in West China Hospital, Sichuan University were identified and included in this study. Whole-exome sequencing (WES) or targeted next-generation sequencing was performed with tumor tissue from all the participants. Clinical data was retrospectively collected. Clinicopathologic characteristics and overall survival (OS) were analyzed. Results: Genetic alterations were detected in 71 patients (98.6%). While 1 patient (1.4%) had only one genetic alteration, 33 patients (45.8%) had 2-4 genetic alterations and 37 patients (51.4%) had ≥5 genetic alterations. Overall, 25 patients (34.7%) with alteration of TGF-β pathway and 47 patients (65.3%) with wild type of TGF-β pathway were identified as TGF-βm+ group and TGF-βm- group respectively. Mutation of TGF-β pathway was independently associated with inferior OS in present cohort (HR, 2.22; 95% CI, 1.05-4.70; P = 0.04), adjusted for tumor stage, metastasis, surgery method and comorbidity. Subgroup analysis showed that patients with mutation of TGF-β pathway had shorter OS after radical surgery (HR, 3.25, 95% CI: 1.01–10.49; P = 0.04). While in the subgroup of patients who underwent palliative surgery, OS was not statistically different (HR, 0.93, 95% CI: 0.41–2.09; P = 0.86). Conclusions: Inferior prognosis was observed in PDACs with mutations of TGF-β pathway. Genomic information could help screen out patients at risk after surgery and adjuvant therapy might benefit this subgroup of PDACs.

The role of different TGFβ signatures in predicting outcome in high grade serous ovarian carcinoma.

e14262 Background: Ovarian cancer (OV) is the most lethal gynecologic cancer in the US. Despite initial responsiveness to chemotherapy, > 50% experience recurrence. Recurrent disease is characterized by low response to chemotherapy and poor prognosis, especially platinum-resistant disease. Thus, there is an urgent need to identify new targeted therapy to improve the outcome of this aggressive cancer. The Transforming Growth Factor (TGFβ) family controls different cellular responses in development and cell homeostasis. Disruption of TGFβ signaling has been implicated in many cancers, including OV, but with paradox effects. It is unclear if TGFβ family genes can serve as a predictive marker and be selected for future therapy target in OV. Methods: We used quantified RNAseq data from 374 OV samples in The Cancer Genomic Atlas (TCGA) to extract 4 different sets of TGFβ pathway genes including: 3-gene ( TGFB1, TGFB2, TGFB3), 6-gene ( TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3), 43-genes from cBioPortal, or 173-genes from Calon et al, 2012 to predict colorectal cancer relapse. Expression counts for these genes were normalized and log transformed to generate a TGFβ score based on the quartiles of the average. Samples were categorized in to low (lowest quartile), medium (middle 2 quartiles), and high (highest quartile) expression groups. Overall (OS) and progression-free (PFS) survivals were compared among groups using Kaplan-Meier method. Results: Interestingly, the minimum TGFβ 6-gene set can predict high grade serous ovarian cancer patient survival among all 4 gene panels using TCGA data, with average age of 60 years (range: 38 –81). Majority of the patients were stage IIIIV. OV patients with highest quartile, TGFβ signature expression have in general less somatic mutations and copy number alterations (average mutation count = 41, average CNA fraction = 0.5) compared to lowest quartile TGFβ expression group (average mutation count = 52, average CNA fraction = 0.6). Patients with TGFB 6-gene expression scored high showed worse OS (36.2 months vs 63.5 months, log-rank p = 0.001) and worse PFS (18.6 months vs 31.4 months, log-rank p = 0.09). Details of outcome by each specified gene signature will be outlined later. Conclusions: Our analysis suggested that the narrowed 6-gene TGFβ family expression profile could potential serve as a prognosis biomarker for OV. With proper validation and mechanistic investigation, existing TGFβ inhibitors may hold promise for targeted or combination therapies for OV. Targeting both TGFβ and PD1 might be of therapeutic value.

Mutations in DNA repair genes are associated with increased neoantigen burden and a distinct immunophenotype in lung squamous cell carcinoma

Deficiencies in DNA repair pathways, including mismatch repair (MMR), have been linked to higher tumor mutation burden and improved response to immune checkpoint inhibitors. However, the significance of MMR mutations in lung cancer has not been well characterized, and the relevance of other processes, including homologous recombination (HR) and polymerase epsilon (POLE) activity, remains unclear. Here, we analyzed a dataset of lung squamous cell carcinoma samples from The Cancer Genome Atlas. Variants in DNA repair genes were associated with increased tumor mutation and neoantigen burden, which in turn were linked with greater tumor infiltration by activated T cells. The subset of tumors with DNA repair gene variants but without T cell infiltration exhibited upregulation of TGF-β and Wnt pathway genes, and a combined score incorporating these genes and DNA repair status accurately predicted immune cell infiltration. Finally, high neoantigen burden was positively associated with genes related to cytolytic activity and immune checkpoints. These findings provide evidence that DNA repair pathway defects and immunomodulatory genes together lead to specific immunophenotypes in lung squamous cell carcinoma and could potentially serve as biomarkers for immunotherapy.

ASXL1 and SETBP1 mutations promote leukaemogenesis by repressing TGF\u03b2 pathway genes through histone deacetylation

Mutations in ASXL1 and SETBP1 genes have been frequently detected and often coexist in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We previously showed that coexpression of mutant ASXL1 and SETBP1 in hematopoietic progenitor cells induced downregulation of TGFβ pathway genes and promoted the development of MDS/AML in a mouse model of bone marrow transplantation. However, whether the repression of TGFβ pathway in fact contributes to leukaemogenesis remains unclear. Moreover, mechanisms for the repression of TGFβ pathway genes in ASXL1/SETBP1-mutated MDS/AML cells have not been fully understood. In this study, we showed that expression of a constitutively active TGFβ type I receptor (ALK5-TD) inhibited leukaemic proliferation of MDS/AML cells expressing mutant ASXL1/SETBP1. We also found aberrantly reduced acetylation of several lysine residues on histone H3 and H4 around the promoter regions of multiple TGFβ pathway genes. The histone deacetylase (HDAC) inhibitor vorinostat reversed histone acetylation at these promoter regions, and induced transcriptional derepression of the TGFβ pathway genes. Furthermore, vorinostat showed robust growth-inhibitory effect in cells expressing mutant ASXL1, whereas it showed only a marginal effect in normal bone marrow cells. These data indicate that HDAC inhibitors will be promising therapeutic drugs for MDS and AML with ASXL1 and SETBP1 mutations.

Of pleiotropy and trajectories: Does the TGF-β pathway link childhood asthma and chronic obstructive pulmonary disease?

The study of developmental trajectories is where epigenetics truly shines. The "epi" in epigenetics captures the fact that although epigenetic processes also preside over the maintenance and termination of gene expression, the unfolding and remodeling of chromatin architecture are especially critical to prepare genes for regulated transcription. These properties imply being on a path, a trajectory to events that will occur later thanks to epigenetic programming. Thus epigenetics is about timed and timely events. In this article we discuss epigenetic and genetic evidence from several independent studies of asthma, chronic obstructive pulmonary disease, and lung function, which converge to highlight a potential role of the TGF-β gene pathway in these processes. These results raise the possibility that at least in a subset of subjects, these conditions might be functionally connected in ways that need to be further defined but that likely reflect the uniquely pleiotropic nature of TGF-β pathway genes, particularly their ability to control both lung development and immune responses essential for regulation and inflammation. Further characterization of this pathway in longitudinally phenotyped populations might unmask novel trajectories to lung disease that begin in utero and unfold into old age.

Hyperoxia induces epigenetic changes in newborn mice lungs

Supplemental oxygen exposure is a risk factor for the development of bronchopulmonary dysplasia (BPD). Reactive oxygen species may damage lung tissue, but hyperoxia also has the potential to alter genome activity via changes in DNA methylation. Understanding the epigenetic potential of hyperoxia would enable further improvement of the therapeutic strategies for BPD. Here we aimed to identify hyperoxia-related alterations in DNA methylation, which could affect the activity of crucial genetic pathways involved in the development of hyperoxic lung injury. Newborn mice (n = 24) were randomized to hyperoxia (85% O2) or normoxia groups for 14 days, followed by normoxia for the subsequent 14 days. The mice were sacrificed on day 28, and lung tissue was analyzed using microarrays developed for the assessment of genome methylation and expression profiles. The mean DNA methylation level was higher in the hyperoxia group than the normoxia group. The analysis of specific DNA fragments revealed hypermethylation of > 1000 gene promoters in the hyperoxia group, confirming the presence of the DNA-hypermethylation effect of hyperoxia. Further analysis showed significant enrichment of the TGF-β signaling pathway (p = 0.0013). The hypermethylated genes included Tgfbr1, Crebbp, and Creb1, which play central roles in the TGF-β signaling pathway and cell cycle regulation. Genome expression analysis revealed in the hyperoxia group complementary downregulation of genes that are crucial for cell cycle regulation (Crebbp, Smad2, and Smad3). These results suggest the involvement of the methylation of TGF-β pathway genes in lung tissue reaction to hyperoxia. The data also suggest that hyperoxia may be a programming factor in newborn mice.

Dysregulation of fibrosis related genes in HCV induced liver disease

BackgroundLiver fibrosis results from a wound healing response to chronic injury, which leads to excessive matrix deposition. Genome wide association studies have showen transcriptional dysregulation in mild and severe liver fibrosis. Recent studies suggested that genetic markers may be able to define the exact stage of liver fibrosis. AimTo define genes or genetic pathways that could serve as markers for staging or as therapeutic targets to halt progression of liver fibrosis. MethodsThe study was performed on 105 treatment naïve HCV genotype 4 infected patients [F0–F2, n = 56; F3–F4, n = 49] and 16 healthy subjects. The study included PCR array on 84 fibrosis related genes followed by customization of a smaller array consisting of 11 genes that were designed on the bases of results obtained from the larger array. Genes that displayed significant dysregulation at mRNA levels were validated at protein levels. Results and discussionTwo major pathways exhibited high dysregulation in early fibrosis as compared with controls or when compared with late fibrosis, these were the TGFβ - related pathway genes and Matrix - deposition associated genes. Hepatic stellate cell (HSC) activators i.e. TGFβ pathway genes [TGFβ1, 2 and 3, their receptors TGFβR1 and 2, signaling molecules SMAD genes and PDGF growth factors] were considerably over-expressed at transcriptional levels as early as F0, whereas expression of their inhibitor TGIF1 was simultaneously down regulated. Matrix proteins including collagen and MMPs were upregulated in early fibrosis whereas tissue inhibitors TIMPs 1 and 2 began over expression in late fibrosis. Expression at protein levels was concordant with RNA data excluding dysregulation at post transcriptional levels. ConclusionSince these 2 gene sets are closely interrelated regarding HSC activation and proliferation, we assume that the current findings suggest that they are favorable targets to further search for stage specific markers.

Identification and Evolution of TGF-β Signaling Pathway Members in Twenty-Four Animal Species and Expression in Tilapia.

Transforming growth factor β (TGF-β) signaling controls diverse cellular processes during embryogenesis as well as in mature tissues of multicellular animals. Here we carried out a comprehensive analysis of TGF-β pathway members in 24 representative animal species. The appearance of the TGF-β pathway was intrinsically linked to the emergence of metazoan. The total number of TGF-β ligands, receptors, and smads changed slightly in all invertebrates and jawless vertebrates analyzed. In contrast, expansion of the pathway members, especially ligands, was observed in jawed vertebrates most likely due to the second round of whole genome duplication (2R) and additional rounds in teleosts. Duplications of TGFB2, TGFBR2, ACVR1, SMAD4 and SMAD6, which were resulted from 2R, were first isolated. Type II receptors may be originated from the ACVR2-like ancestor. Interestingly, AMHR2 was not identified in Chimaeriformes and Cypriniformes even though they had the ligand AMH. Based on transcriptome data, TGF-β ligands exhibited a tissue-specific expression especially in the heart and gonads. However, most receptors and smads were expressed in multiple tissues indicating they were shared by different ligands. Spatial and temporal expression profiles of 8 genes in gonads of different developmental stages provided a fundamental clue for understanding their important roles in sex determination and reproduction. Taken together, our findings provided a global insight into the phylogeny and expression patterns of the TGF-β pathway genes, and hence contribute to the greater understanding of their biological roles in the organism especially in teleosts.

Abstract 5330: Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases

Abstract Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer deaths worldwide, and rising in the United States at an alarming rate. Multiple E3 ubiquitin ligases such as the SMURFs and RINGH2 proteins have been identified as negative regulators of the TGF-β pathway. However, to our knowledge, there remains a gap in the integration between genomics, underlying mechanisms and the development of targeted therapeutics harnessing these TGF-β-associated E3 ligases for HCC. The aim of this study is to elucidate the role of E3 ligases in HCC, through TCGA analyses and provide mechanistic insight into these as therapeutic targets for HCC. We first analyzed the 488 hepatocellular cancers and screened for alterations in The Cancer Genome Atlas (TCGA). Immunohistochemistry (IHC), Q-PCR, Western blot analysis were used to validate the expression levels of two of the most highly altered E3 ligases, PRAJA and Keap1 in hepatocellular cancer tissues and cell lines in human and in TGF-β-deficient β2SP+/- mouse models. Inhibition studies of PRAJA and Keap1 were performed by lentivirus shRNA in HCC cell lines, and xenograft studies. From the TCGA data, we observe two different signatures (activated and inactivated) for 18 TGF-β pathway genes. While increased levels of TGF-β-related transcripts were associated with activation of hepatic fibrosis/immune microenvironment pathways, decreased levels of TGF-β members were associated with loss of TGF-β tumor suppressor function. HCCs characterized by the “inactivated” TGF-β signature were associated with a significantly poorer survival, compared to HCCs with the “activated” TGF-β signature (p=0.0027). We next analyzed 29 TGF-β-related E3 ligases, and observed raised expression of the following: PRAJA1 (12.7% of HCCs), KEAP1 (6.4%), UCHL5 (16.4%), WWP2 (11.8%), WWP1 (10%), Smurf2 (9.1%), Skp2 (9.1%), and Smurf1 (8.2%). Interestingly, expression patterns corresponded with a few TGF-β signaling members regulated by some of these E3 ligases, namely Smad3 (altered in 54%) and β2SP (27%). We identified that PRAJA1 targets Smad3 and β2SP for ubiquitination and degradation. We further observe raised levels of PRAJA (25%) and KEAP1 (70%) in 176 human liver cancers, by IHC, compared to normal controls. Depletion of PRAJA and KEAP1 with either shRNAs or E3 ligase inhibitors, substantially inhibited growth and induced apoptosis through PRAJA/Smad3/β2SP and KEAP1/Nrf signaling in HCC cell lines and xenografts. These results suggest that E3 ligases such as PRAJA1 and KEAP1 may be valuable therapeutic targets for liver cancer in the context of TGF-β signaling, an important approach given that few effective targeted therapeutics are available for this cancer with poor prognosis. Citation Format: Shuyun Rao, Heather Levin, Jian Chen, Rehan Akbani, Jon White, Wilma Jogunoori, Shoujun Gu, Kazufumi Ohshiro, Sobia Zaidi, Bibhuti Mishra, Asif Rashid, Shulin Li, Lopa Mishra. Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2017-5330

TMPRSS2:ERG gene fusion variants induce TGF-β signaling and epithelial to mesenchymal transition in human prostate cancer cells.

TMPRSS2:ERG (T/E) gene fusions are present in approximately 50% of all prostate cancer (PCa) cases. The expression of fusion mRNAs from distinct T/E variants is associated with clinicopathological parameters, while the underlying molecular processes remain unclear. We characterized the molecular mechanisms and functional implications caused by doxycycline (Dox)-inducible overexpression of the frequent T/E III and VI fusion variants in LNCaP cells. Induction of T/E expression resulted in increased cellular migratory and invasive potential, and reduced proliferation and accumulation in G1 phase. T/E overexpressing cells showed epithelial-to-mesenchymal transition (EMT), as demonstrated by upregulation of TGF-β and WNT pathway genes, mesenchymal markers, and increased phosphorylation of the p38 MAPK. Augmented secretion of TGF-β1 and –β2, and T/E-mediated regulation of ALK1, a member of the TGF-β receptor family, was detected. ALK1 inhibition in T/E overexpressing cells blocked p38 phosphorylation and reduced the expression of the TGF-β target genes VIM, MMP1, CDH2, and SNAI2. We found a T/E variant VI-specific induction of miR-503 associated with reduced expression of SMAD7 and CDH1. Overexpression of miR-503 led to increased levels of VIM and MMP1. Our findings indicate that TGF-β signaling is a major determinant of EMT in T/E overexpressing LNCaP cells. We provide evidence that T/E VI-specific transcriptional modulation by miR-503 accounts for differences in the activation of EMT pathway genes, promoting the aggressive phenotype of tumors expressing T/E variant VI. We suggest that ALK1-mediated TGF-β signaling is a novel oncogenic mechanism in T/E positive PCa.

TGFbeta and miRNA regulation in familial and sporadic breast cancer.

The term ‘BRCAness’ was introduced to identify sporadic malignant tumors sharing characteristics similar to those germline BRCA-related. Among all mechanisms attributable to BRCA1 expression silencing, a major role has been assigned to microRNAs. MicroRNAs role in familial and sporadic breast cancer has been explored but few data are available about microRNAs involvement in homologous recombination repair control in these breast cancer subgroups. Our aim was to seek microRNAs associated to pathways underlying DNA repair dysfunction in breast cancer according to a family history of the disease. Affymetrix GeneChip microRNA Arrays were used to perform microRNA expression analysis in familial and sporadic breast cancer. Pathway enrichment analysis and microRNA target prediction was carried out using DIANA miRPath v.3 web-based computational tool and miRWalk v.2 database. We analyzed an external gene expression dataset (E-GEOD-49481), including both familial and sporadic breast cancers. For microRNA validation, an independent set of 19 familial and 10 sporadic breast cancers was used. Microarray analysis identified a signature of 28 deregulated miRNAs. For our validation analyses by real time PCR, we focused on miR-92a-1*, miR-1184 and miR-943 because associated to TGF-β signalling pathway, ATM and BRCA1 genes expression. Our results highlighted alterations in miR-92a-1*, miR-1184 and miR-943 expression levels suggesting their involvement in repair of DNA double-strand breaks through TGF-beta pathway control.

Abstract 4425: Comprehensive study of TGF-β pathway-driven functional molecular characterization of human hepatocellular cancer

Abstract Background: TGF-β plays a complex role in cancer- from tumor suppression to immune modulation to tumor promotion that are unclearly defined to date. Recent clinical studies show that targeting TGF-β improves survival up to 21 months, yet prognostic significances are undefined. Moreover, the relationships between patterns of mutations and transcriptomic phenotypes for the TGF-β pathway are unclear. Methods: 1. We analyzed the transcriptome of 488 hepatocellular cancers and screened for mutations in the TGF-b pathway in 202 HCCs from The Cancer Genome Atlas (TCGA). 2. Next we correlated mouse models of HCC with a functional analysis of drivers. Results: 1. Transcriptomic analyses revealed aberrant TGF-β superfamily profiles in 72% of hepatocellular cancers, with mutations in 38% of patients. 2. Significantly, HCCs characterized by the “inactivated” TGF-β signature were associated with a significantly poorer survival particularly in early stage HCCs, compared to HCCs with the “activated” TGF-β signature (p = 0.0027). 3. We observed the greatest number of functional mutations in the SPTBN1 gene (6%), which encodes a tumor suppressor TGF-β/Smad3 adaptor protein. 4. We found a strong association between DNA damage response genes and the TGF-β pathway at both transcriptomic and genomic levels. 5. We also observed a strong correlation between VD related genes and TGF-β pathway genes in the TCGA genomic analysis. 6. subsequent VD deprivation synergistically with TGF-β inactivation promotes liver tumor development. 7. Through a transcriptomic and functional analysis we observed that TLR7 mRNA levels are increased 4-fold in liver tissues from Smad3+/- mice, and TLR7 is a direct target of Smad3. Conclusions: The TGF-β pathway plays a pivotal role in liver tumorigenesis and the molecular signatures we characterize here have prognostic significance. In specific populations, VD deficiency and TGF-β disruption synergistically promote liver tumor growth, possibly through regulating TLR7 expression. The additional association with the DNA repair pathway supports new approaches to biomarker driven targeting of TGF-β, improving survival of liver cancer. Citation Format: Jian Chen, Jiun-Sheng Chen, Jianping Zhang, YoungJin Gi, Lior Katz, Ji-Hyun Shin, YunSeong Jeong, Mitch Belkin, Wilma Jogunoori, Bibhuti Mishra, Jon White, Shoujun Gu, Milind Javle, Xiaoping Su, John Stroehlein, Marta Davila, Xuemei Wang, Jeffrey Morris, Patrizia Farci, Rehan Akbani, Lopa Mishra. Comprehensive study of TGF-β pathway-driven functional molecular characterization of human hepatocellular cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4425.