Pancreatic Adenocarcinoma Therapeutics Targeting RTK and TGF Beta Receptor.

Despite the improved overall survival rates in most cancers, pancreatic cancer remains one of the deadliest cancers in this decade. The rigid microenvironment, which majorly comprises cancer-associated fibroblasts (CAFs), plays an important role in the obstruction of pancreatic cancer therapy. To overcome this predicament, the signaling of receptor tyrosine kinases (RTKs) and TGF beta receptor (TGFßR) in both pancreatic cancer cell and supporting CAF should be considered as the therapeutic target. The activation of receptors has been reported to be aberrant to cell cycle regulation, and signal transduction pathways, such as growth-factor induced proliferation, and can also influence the apoptotic sensitivity of tumor cells. In this article, the regulation of RTKs/TGFßR between pancreatic ductal adenocarcinoma (PDAC) and CAFs, as well as the RTKs/TGFßR inhibitor-based clinical trials on pancreatic cancer are reviewed.

Synthesis, Molecular Docking and Preliminary Antileukemic Activity of 4-Methoxybenzyl Derivatives Bearing Imidazo[2,1-b][1,3,4]thiadiazole.

In this study, we synthesized 22 compounds in a series with various substitution on imidazo[2,1-b][1,3,4]thiadiazole. The potential cytotoxic activity of these compounds investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identified two compounds, 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate and 6-(4-chlorophenyl)-2-(4-methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde, exhibited the most cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa) with IC50 values ranging between 0.79 and 1.6 µM. The results indicate that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-ß) type I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.

Lysophosphatidic acid receptor 5 transactivation of TGFBR1 stimulates the mRNA expression of proteoglycan synthesizing genes XYLT1 and CHST3.

Lysophosphatidic acid (LPA) via transactivation dependent signalling pathways contributes to a plethora of physiological and pathophysiological responses. In the vasculature, hyperelongation of glycosaminoglycan (GAG) chains on proteoglycans leads to lipid retention in the intima resulting in the early pathogenesis of atherosclerosis. Therefore, we investigated and defined the contribution of transactivation dependent signalling in LPA mediated GAG chain hyperelongation in human vascular smooth muscle cells (VSMCs). LPA acting via the LPA receptor 5 (LPAR5) transactivates the TGFBR1 to stimulate the mRNA expression of GAG initiation and elongation genes xylosyltransferase-1 (XYLT1) and chondroitin 6-sulfotransferase-1 (CHST3), respectively. We found that LPA stimulates ROS and Akt signalling in VSMCs, however they are not associated in LPAR5 transactivation of the TGFBR1. We observed that LPA via ROCK dependent pathways transactivates the TGFBR1 to stimulate genes associated with GAG chain elongation. We demonstrate that GPCR transactivation of the TGFBR1 occurs via a universal biochemical mechanism and the identified effectors represent potential therapeutic targets to inhibit pathophysiological effects of GPCR transactivation of the TGFBR1.

Dysregulated epidermal growth factor and tumor growth factor-beta receptor signaling through GFAP-ACTA2 protein interaction in liver fibrosis.

OBJECTIVE: Viral hepatitis is associated with high morbidity and mortality. Identification of biological pathways involved in hepatic fibrosis resulting from chronic hepatitis C are essential for better management of patients. Constructing the HCV-human protein interaction network through bioinformatics may enable us to discover diagnostic biological pathways. We investigated to identify dysregulated pathways and gene enrichment based on actin alpha 2 (ACTA2) and glial fibrillar acidic protein (GFAP) interaction network analysis in hepatic fibrosis. METHODS: This is an in-silico study conducted at Ziauddin University from March,2019 to September 2019. Enrichment and protein-protein interaction (PPI) network analysis of the identified proteins: GFAP and ACTA2 along with their mapped gene data sets was performed using FunRich version 3.1.3. RESULTS: Biological pathway grouping showed enrichment of proteins (85.7%) in signalling pathway by epidermal growth factor receptor (EGFR) and Tumor growth factor (TGF)-beta Receptor followed by signaling by PDGF, FGFR and NGF (71.4%) (p < 0.001). SRC, PRKACA, PRKCA and PRKCD were enriched in both EGFR and TGF-beta Signalling pathways. CONCLUSION: EGFR and TGF-beta signalling pathways were enriched in liver fibrosis. SRC, PRKACA, PRKCA and PRKCD were enriched and differentially expressed in both EGFR and TGF-beta signalling pathways.

Expression of TGF-beta receptor 1 and Smads in the tissues of primary spontaneous pneumothorax.

BACKGROUND: Primary spontaneous pneumothorax (PSP) is a common disease which is often caused by the rupture of bullae in the lungs. The underlying pathogenesis of PSP remains unclear. Some molecules may be involved in the development of PSP potentially. The aim of this study was to investigate the expression of TGF-beta receptor 1 (TßR1), Smad2, Smad3 and Smad4 in the resected bullae of patients with PSP. METHODS: From May 2015 to May 2016, 34 patients with PSP underwent video-assisted thoracoscopic surgery (VATS) bullectomy. Immunohistochemistry was performed to identify the expression of TßR1, Smad2, Smad3 and Smad4 in the resected pulmonary bullae tissues. The levels of these cytokines were calculated by immunoreactivity scoring system (IRS). Ten patients without pneumothorax associated disease were selected as the control group. RESULTS: The analysis showed that the expression levels of TßR1, Smad2 and Smad4 were significantly higher in bullae tissues of patients with PSP than that in normal lung tissues (P=0.012, 0.031, 0.000 respectively). There was no significant difference between the expression level of Smad3 in bullae tissue of PSP patients and that in normal lung tissues of the control group (P=0.140). However, the absolute quantity of Smad3 expression in PSP bullae tissues was (4.2529±1.7193), scored by the IRS, which is higher than that in the control lung tissues (3.2600±2.2132). Also, the expression of TßR1, Smad2, Smad3 and Smad4 were not showed correlation with the clinical characteristics of PSP patients, such as age, sex, body mass index (BMI), recurrence and side of pneumothorax. CONCLUSIONS: TßR1, Smad2 and Smad4 highly expressed in bullae tissues of PSP patients. Our findings suggested that TßR1, Smad2 and Smad4 may be related to the development of PSP bullae.

A genotypic difference in primary root length is associated with the inhibitory role of transforming growth factor-beta receptor-interacting protein-1 on root meristem size in wheat.

Previously we identified a major quantitative trait locus (QTL) qTaLRO-B1 for primary root length (PRL) in wheat. Here we compare proteomics in the roots of the qTaLRO-B1 QTL isolines 178A, with short PRL and small meristem size, and 178B, with long PRL and large meristem size. A total of 16 differentially expressed proteins were identified: one, transforming growth factor (TGF)-beta receptor-interacting protein-1 (TaTRIP1), was enriched in 178A, while various peroxidases (PODs) were more abundantly expressed in 178B. The 178A roots showed higher TaTRIP1 expression and lower levels of the unphosphorylated form of the brassinosteroid (BR) signaling component BZR1, lower expression of POD genes and reduced POD activity and accumulation of the superoxide anion O2(-) in the root elongation zone compared with the 178B roots. Low levels of 24-epibrassinolide increased POD gene expression and root meristem size, and rescued the short PRL phenotype of 178A. TaTRIP1 directly interacted with the BR receptor TaBRI1 of wheat. Moreover, overexpressing TaTRIP1 in Arabidopsis reduced the abundance of unphosphorylated BZR1 protein, altered the expression of BR-responsive genes, inhibited POD activity and accumulation of the O2(-) in the root tip and inhibited root meristem size. Our data suggested that TaTRIP1 is involved in BR signaling and inhibited root meristem size, possibly by reducing POD activity and accumulation of O2(-) in the root tip. We further demonstrated a negative correlation between the level of TaTRIP1 mRNA and PRL of landraces and modern wheat varieties, providing a valuable insight for better understanding of the molecular mechanism underlying the genotypic differences in root morphology of wheat in the future.

The Prognostic Significance of Chromosome 18 Monosomy in the Colon Cancer: Correlations with the Expressions of Smad4 and TGF-beta Receptor II Proteins

PURPOSE: Chromosomal instability of chromosome 18 and inhibition of the transforming growth factor beta (TGF-beta) signaling pathway, which is mediated through Smad4, play important roles in the tumorigenesis of colon cancer. This study evaluated the value of the expression of chromosome 18 monosomy in colon cancer as a prognostic factor and its correlations with the expressions of Smad4 and TGF-beta receptor II proteins. METHODS: We analyzed the rate of the expression of chromosome 18 monosomy in 66 colon cancers with using chromogenic in situ hybridization (CISH) and we evaluated its value as a prognostic factor by determining its correlation with the pathologic factors and immunohistochemical expressions of Smad4 and TGF-beta receptor II proteins. RESULTS: Of the 66 colon cancers, monosomy of chromosome 18, as determined by CISH, was observed in 18 cases (27.3%), and the decreased expression of Smad4 and TGF-beta receptor II proteins was observed in 30 cases (45.5%) and 25 cases (37.9%), respectively. The monosomy of chromosome 18 and the decreased expression of Smad4 proteins showed statistically significant correlations with the histologic differentiation, the presence of tumor emboli, the nodal status and the stage. The decreased expression of TGF-beta receptor II proteins had statistically significant correlations with the histologic differentiation, the T-stage, the nodal status and the stage. The monosomy of chromosome 18 showed a statistically significant correlation with the decreased expression of Smad4 and TGF-beta receptor II proteins. CONCLUSION: These results suggested that chromosome 18 monosomy may have prognostic value for colon cancer.

Effects of Transforming Growth Factor-beta1 and Its Receptor on the Development, Recurrence and Progression of Human Bladder Cancer / 대한비뇨기과학회지

PURPOSE: We investigated whether the expression levels of Transforming growth factor beta1 (TGF-beta1) and its receptors were related to the development, recurrence, progression and disease-free survival in the patients with bladder cancer. MATERIALS AND METHODS: The mRNA levels of TGF-beta1 and its receptors were examined in 102 tumor specimens from patients with primary bladder cancer, 29 corresponding normal bladder mucosae specimens surrounding these tumors and 15 normal bladder mucosae specimens by performing quantitative competitive PCR (QC-PCR). The protein levels of TGF-beta1 and its receptors were investigated by performing immunohistochemical staining on sections cut from 86 archival bladder tissue paraffin blocks. RESULTS: QC-PCR analysis showed that expressions of TGF-beta1, TGF-beta receptor I (TGF-betaRI) and receptor II (TGF-betaRII) in the superficial and low-grade bladder cancers were significantly higher than those in both the corresponding normal bladder mucosae surrounding the cancer (p= 0.0069, 0.0022 and 0.0046, respectively) and the control's normal bladder mucosae (p=0.0014, 0.0125 and 0.0089, respectively). Expressions of TGF-beta1 and its receptors were enhanced in the non-recurred and non-progressed patients compared to the recurred cases (p=0.0022, 0.0003 and 0.0001, respectively) and the progressed cases (p=0.0002, <0.0001 and <0.0001, respectively). Patients with high expression of TGF-beta and its receptors had a significantly higher disease-free survival rate than those patients with low expressions (p=0.0129, 0.0121 and 0.0132, respectively). CONCLUSIONS: The enhanced expression of TGF-beta1 and its receptors was correlated not only with superficial and low-grade bladder cancer, but also with enhanced patient survival. In conclusion, our findings suggest that the expressions of TGF-beta1 and its receptors are useful prognostic markers for a patient's resistance to disease recurrence and/or progression.

Role of TGF-beta signaling and Ski/SnoN mRNA expression in cervical carcinomas / 대한산부인과학회잡지

OBJECTIVE: TGF-beta signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaR-II) with the type I TGF-beta receptor (TbetaR-I). which mediate intracellular signals through Smad proteins. Whereas physiologic concentrations of SnoN and Ski allow a feedback regulation of TGF-beta signaling, deregulation of SnoN or Ski expression leads to total inhibition of TGF-beta signaling and of the tumor suppressors Smad2 and Smad4, which can explain the role of SnoN and Ski as oncogenes. In order to identify possible molecular mechanisms responsible for TGF-beta resistance, the author investigated the mutation and expression of TGF-beta1, its receptors, Ski/SnoN in cervical carcinomas. METHODS: From December 1995 to December 1999, 45 carcinomas and 7 normal cervical tissue specimens were obtained by surgical resection in the Kyung Hee University Medical Center. Tissue specimens were snap-forzen in liquid N2 and stored at -70 degrees C until used. Total RNA was extracted from specimens and evaluated the expression levels using densitometric analysis of quantitative RT-PCR products (TGF-beta1, Tbeta1R-I, Tbeta1R-II, Ski/SnoN), and the mutations were investigated by quantitative genomic-PCR followed by nonisotopic RT-PCR-SSCP analysis (Tbeta1R-II, Tbeta1R-I, Ski/SnoN). The abnorally expressed levels of RT-PCR products (TGF-beta1, Tbeta1R-II) were analysed for the clinicopathologic characteristics. RESULTS: Quantitative RT-PCR analysis demonstrated variable expression of TGF-beta1 mRNA (0.05-0.89) in tumors and significantly increased TGF-beta1 expression level (>0.48) in 15 of 45 samples (33.3%). There is no significant reduction of Tbeta1R-I expression (1.36) in 2 of 45 samples (4.4%), and there is no amplification of Ski/SnoN gene by quantitative genomic-PCR analysis. CONCLUSIONS: The overexpression of TGF-beta1 mRNA and the reduced or absent expression of Tbeta1R-II may be an important contributing factors, and the abnormally low genomic levels and no mutational alterations of Tbeta1R-II is caused by monoallelic deletion suggesting that Tbeta1R-II might play as a tumor suppressor of haloinsufficiency in cervical carcinomas. We could not show that high levels of Ski/SnoN expression could produce a disruption of TGF-beta signaling in cervical carcinomas.

Expression of Transforming Growth Factor-beta Receptors in Food Protein-Induced Enterocolitis Syndrome in Infancy / 소아알레르기및호흡기학회지

PURPOSE: Food protein-induced enterocolitis syndrome (FPIES) is a symptom complex of vomiting and diarrhea caused by non-IgE mediated allergy to cow's milk and/or soy in young infants. Transforming growth factor (TGF)-beta has been reported to protect the epithelial barrier of the gut from foreign antigens. We studied the expression of type 1 and 2 TGF-beta receptors in the mucosa of small intestine to investigate their roles in the pathogenesis of FPIES. METHODS: Twenty-eight patients, aged 7 to 120 days (mean 49 days) who were diagnosed with FPIES by clinical criteria and challenge tests were included. Immunohistochemical stainings for type 1 and 2 TGF-beta receptors were performed on endoscopic duodenal biopsy specimens. RESULTS: Type 1 and 2 TGF-beta receptors were expressed in the villous and crypt epithelial cells but nearly absent in the lamina propria in both patients and controls. Type 1 TGF-beta receptor expression was significantly lower in the patients who had villous atrophy than in the patients who had not and in controls. The expression of type 1 TGF-beta receptor was negatively correlated with the severity of villous atrophy. Type 2 TGF-beta receptor expression showed no significant difference between the patients and controls. CONCLUSION: Our results suggests that the decreased activity of type 1 TGF-beta receptor is implicated in the pathogenesis of FPIES in young infants.

Immunohistochemical Study of TGF- type I and type II receptor Expression in Psoriatic Epidermis / 대한피부과학회지

BACKGROUND: Previous studies have demonstrated the pathogenetic role and expression of TGF-beta in psoriatic lesion. Transforming growth factor s are a family of growth factors with inhibitory effects on epithelial cell proliferation. Their effects are mediated by two interacting receptors, of which type I receptor mediates signal transduction after interaction with type II receptor carrying the TGF ligand. OBJECTIVE: The purpose of this study was to investigate the relationship between development of psoriasis and expression of TGF-beta receptors in psoriatic lesion. METHODS: We have studied the expression of TGF-beta type I and type II receptors in psoriatic lesions of 30 psoriatic patients who had not been treated for 1 month, 5 non-lesional psoriatic skin, and 3 normal human skin by immunohistochemical staining using polyclonal rabit antisera. RESULTS: 1. Immunohistochemical analysis revealed an intense immunoreactivity for TGF-beta type I and type II receptors in the basal and also suprabasal layer of normal epidermis and non-lesional psoriatic skin. 2. Almost all psoriatic lesions studied lacked detectable immunoreactivity of either receptor in the epidermis. CONCLUSION: We suggest the lack of TGF-beta - mediated growth inhibition by down regulation of TGF-beta receptor expression may play an important role in the pathogenesis of psoriasis.

Correlation between the Expression Levels of Transforming Growth Factor-beta (TGF-beta) Receptors and Responsiveness to TGF-beta1-Induced Growth Inhibitory Effects in Leukemic Cell Lines / 대한혈액학회지

BACKGROUND: Defects in TGF-beta receptors have been found in a variety of malignant cells, we therefore investigated whether these defects could be also demonstrated in leukemic cells. In addition, we analyzed the relation between TGF-beta receptor expression and responsiveness to TGF-beta-induced growth inhibitory effects. METHODS: Eleven human leukemic cell lines and two normal cell lines were recruited for the study. To evaluate the expression of TGF-beta receptor type I (RI) and type II (RII), Western blotting analysis was conducted utilizing two kinds of primary antibodies against both RI and RII. Band strength was quantitated with densitometry. Moreover, specific peptides against primary antibodies were employed for competitive inhibition assay. Responsiveness to TGF-beta1 was assessed by [3H] thymidine uptake. RESULTS: Bands of same molecular size were demonstrated by two different primary antibodies. Peptides against RI or RII antibodies successfully blocked the emergence of RI or RII message, respectively, verifying that former bands represented specific RI or RII. Relative RI levels in leukemic cells except HL-60 compared with CCL-64, normal lung epithelial cells, were in the range of 0.48~1.14. In contrast, relative RII levels, although variable between individual cells, were less than 0.25 in all the leukemic cells. Percents [3H]thymidine uptake of leukemic cells at 10 ng/mL of TGF-beta1 compared with untreated control were widely distributed in the range of 7.7~62.9%. Positive correlation between RII levels and TGF-beta responsiveness was observed (P=0.025). CONCLUSION: Defective RI expression seems to be rare, however, defective RII expression appears to be rather common in leukemic cells. Positive correlation between RII levels and TGF-beta responsiveness suggests role of defective RII expression in the acquisition of resistance to TGF-beta in leukemic cells.

The loss of expression of transforming growth factor-beta receptors correlates with the histopathologic tumor grade in bladder transitional cell carcinoma patients

Transforming growth factor-beta (TGF-beta), a pleiotropic growth factor, is a potent inhibitor of cellular proliferation in cells of epithelial origin. Recently, it has been suggested that a loss of sensitivity to TGF-beta through a loss of expression of TGF-beta receptors T beta R-I and T beta R-II--is associated with tumor initiation and progression. Therefore, to investigate the relationship between TGF-beta receptors expression and carcinogenesis of bladder TCC, this study examined the expression of T beta R-I and T beta R-II in 46 bladder TCC patients using immunohistochemistry. Since histopathological grade is a widely accepted marker of prognosis, the results were compared in relation to the three grades of bladder TCC. The results demonstrated that the loss of TGF-beta receptors expression is associated with increasing histopathological grades of bladder TCC. Specifically, both T beta R-I and T beta R-II were readily detected in all 10 normal bladder mucosa specimens. Likewise, all 6 specimens of grade I TCC samples expressed high levels of both TGF-beta receptors. However, among grade II TCC samples, T beta R-I and T beta R-II were detected in 78% and 89%, respectively: among grade III TCC samples, T beta R-I and T beta R-II were detected in 45% and 41%, respectively. These results suggested that loss of sensitivity to TGF-beta may play a role in the progression of TCC from low to high grade disease.

Immunohistochemical Analysis of Transforming Growth Factor (TGF)-beta1 and TGF-beta Receptor II and Quantitative Analysis of TGF-beta1 mRNA during Multistep Hepatocarcinogenesis Induced by Diethylnitrosamine in Sprague-Dawley Rats

Transforming growth factor (TGF)-beta1 plays an important role in hepatocarcinogenesis and has been described as a useful tumor marker and one of the poor prognostic indicators in patients with hepatocellular carcinoma (HCC). To investigate the role and cellular localization of TGF-beta1 during multistep hepatocarcinogenesis we performed a quantitative analysis of TGF-beta1 mRNA and immunohistochemical expression of TGF-beta1 and TGF-beta receptor II (TGF-betarII) in female Sprague-Dawley rats. The experimental groups included neoplastic lesions produced by Solt-Farber's protocol, regenerating liver after partial hepatectomy, and normal control. Quantitative change of TGF-beta1 mRNA was analysed by competitive reverse-transcription polymerase chain reaction (RT-PCR). TGF-beta1 protein and TGF-betarII expression were evaluated by immunohistochemical stain. The discrete tumor nodules were detected on 14th day and then increased in number and size. Three HCCs were induced on 8th or 9th month. RT-PCR demonstrated TGF-beta1 mRNA band in all examples of the normal and regenerating liver, nodules and HCCs. Competitive RT-PCR displayed higher TGF-beta1 mRNA in nodules, HCCs and regenerating liver than in normal controls. Hepatocytes from control and regenerating livers showed weak immunoreactivity for TGF-beta1. In contrast, the cytoplasm of hepatocytes of nodules in 7th, 8th and 9th month and HCCs were intensely stained for TGF-beta1. Some sinusoidal cells showed immunoreactivity for TGF-beta1 in all experimental groups. In early phase of carcinogenesis, the cytoplasm of hepatocytes in liver of 12h, 1d and 3d showed transiently increased immunoreactivity for TGF-beta1 and The immunoreactivity decreased thereafter. TGF-beta1 mRNA was also detected in the neoplastic hepatocytes by in-situ hybridization. Although TGF-betarII expression was correlated with TGF-beta1 immunoreactivity during early phase of carcinogenesis, hepatocytes in most nodules in 7th, 8th, 9th month and carcinomas showed decreased or little immunoreactivity for TGF-betarII. Based on the above results, it is concluded that TGF-beta1 expression increases not only in precancerous nodules but also in HCCs and its increase seems to be correlated with decrease or loss of TGF-betarII expression although its mechanism remains unclear. Hepatocytes may be a major cellular source of TGF-beta1 during hepatocarcinogenesis.