Expression Of Twist Protein Research Articles

Abstract 1234: Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way

Abstract The immune checkpoint molecule programmed cell death-ligand 1 (PD-L1, CD274) suppresses immunity when the cell surface PD-L1 is engaged by programmed death-1 (PD-1) on antitumor immune cells. Although PD-L1 has been established and targeted in cancer immunotherapy, the tumor-intrinsic role of PD-L1 is less appreciated in tumor biology and therapeutic development. In this study, we showed that PD-L1 facilitates epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells by upregulating EMT transcription factors and mesenchymal markers, and downregulating epithelial marker. RNA-sequencing was performed on paired exogenously PD-L1-overexpressed A549 cells and wild-type cells, and PD-L1-depleted H460 cells and wild-type cells, and transcriptomic features associated with PD-L1 status were identified using Gene Set Enrichment Analysis (GSEA). In both the paired samples, PD-L1 expression was associated with EMT-related pathways. Subsequent in vitro experiment showed that exogenous overexpression of PD-L1 in A549 and H522 cells increased both mRNA and protein expression of Twist, Zeb1, Snail, Slug, Vimentin, Fibronectin, and N-Cadherin. Correspondingly, the transcription factors and mesenchymal markers were decreased in PD-L1-depleted H460 and H596 cells. Recombinant PD-1 treatment also increased the expression of EMT transcription factors and markers on A549 and H460 cells. Moreover, interferon-γ stimulation on tumor cells increased expression of EMT transcription factors and markers, which was suppressed by PD-L1-knockdown, suggesting that interferon-γ-induced PD-L1 contributes to EMT. Our findings suggest that PD-L1 facilitates NSCLC progression by promoting the EMT in interferon-γ-independent and dependent way, and this intrinsic PD-L1 function could be targeted for better clinical management of PD-L1-overexpressing NSCLCs. Citation Format: Hyein Jeong, Hyun Kyung Ahn, Sehui Kim, Yoon Kyung Jeon. Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1234.

The Transcription Factor Twist1 Has a Significant Role in Mycosis Fungoides (MF) Cell Biology: An RNA Sequencing Study of 40 MF Cases.

The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I-IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF.

The ADAM9/WISP-1 axis cooperates with osteoblasts to stimulate primary prostate tumor growth and metastasis.

Background: Metastatic prostate cancer (PCa) predicts a poor prognosis and lower likelihood of survival. Osteoblasts (OBs) are known to be responsible for the synthesis and mineralization of bone, although it is unclear as to whether PCa in the prostate gland cooperates with OBs in bone to promote PCa malignant transformation. We aimed to elucidate how primary PCa cells cooperate with distal OBs and contribute to the vicious cycle that leads to metastatic PCa. Methods: N-cadherin, E-cadherin, and Twist protein expression were measured by Western blot. Twist translocation into the nucleus was detected by the immunofluorescence (IF) assay. Enzyme-linked immunosorbent assay (ELISA) detected protein levels in human serum samples. Levels of candidate protein expression were examined by the human cytokine array. Prostate tumor growth and metastasis were analyzed by orthotopic and metastatic prostate cancer models, respectively. Immunohistochemistry (IHC) staining was used to observe ADAM metallopeptidase domain 9 (ADAM9) and WNT1 inducible signaling pathway protein 1 (WISP-1) expression in tissue. Results: Our in vitro and in vivo analyses have now discovered that primary PCa expressing ADAM9 protein enables the transformation of OBs into PCa-associated osteoblasts (PCa-OBs), inducing WISP-1 secretion from PCa-OBs in the bone microenvironment. The upregulation of WISP-1 in bone provided feedback to primary PCa and promoted PCa cell aggressiveness via epithelial-mesenchymal transition (EMT) activity. Elevated levels of WISP-1 expression were detected in the serum of patients with PCa. ADAM9 levels were overexpressed in tumor tissue from PCa patients; ADAM9 blockade interrupted OB-induced release of WISP-1 and also suppressed primary tumor growth and distal metastasis in orthotopic PCa mouse models. Conclusion: Our study suggests that the ADAM9/WISP-1 axis assists with metastatic PCa progression. Thus, targeting the ADAM9/WISP-1 axis may help to prevent the malignant phenotypes of PCa cells.

INVERSE EXPRESSION BETWEEN TGF-β1 AND TWIST IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE AFTER CHOLESTEROL DEPLETION

Objectives To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). Study Design SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. Results The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Conclusions Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology. To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology.

Aristolactam I inhibits cell migration and invasion through regulation of Twist1 in MDA‐MB‐231 breast cancer cells

AbstractTriple‐negative breast cancer (TNBC) is one of the most difficult cancer types to treat. Because three key receptors are absent in these cancer cells, the cells bypass the human immune system and chemotherapy does not work well. Aristolactam I (AL‐I) is a natural compound in the plant genus Aristolochia. Previous studies have shown that aristolactam variants exhibit an inhibitory effect on several bioactive cancer developments. However, the regulatory mechanism and biological activity of AL‐I in TNBC cell lines have not been identified. In this study, AL‐I affected an increase in E‐cadherin expression in MDA‐MB‐231 cancer cells. AL‐I increased E‐cadherin mRNA expression by down‐regulating Twist1 protein expression. The novel anticancer effect of AL‐I was confirmed to be inhibition of the development of breast cancer cells through reduced cell mobility.

The Expression Profile of miRNA in Glioma and the Role of miR-339-5p in Glioma.

Objective To reveal the expression profile of miRNA in glioma and the effects of microRNA-339-5p (miR-339-5p) on glioma. Methods The glioma and normal tissues were randomly selected for miRNA gene chip detection and qRT-PCR verification. The U87 cells were separated into miR-NC, miR-339-5p mimic, and miR-339-5p suppressor group. Clonogenesis test, flow cell technique, Transwell, and cell scratch assay were utilized to verify the roles of miR-339-5p in cell proliferation, cell apoptosis, cell invasion, and cell migration. The epithelial-meso-transformation-associated proteins was verified by Western blot. Results A total of 49 miRNAs (16 upregulated and 33 downregulated) were differentially expressed in glioma tissues, and miR-339-5p was the most downregulated. The clone number, invasion number, and healing rate of cells in miR-339-5p mimic group were decreased compared with miR-NC group (P < 0.05); the clone quantity, invasion number, and healing rate of cells in miR-339-5p inhibitor group were increased compared with miR-NC group (P < 0.05). The apoptosis rate of human glioma U87 cells in miR-339-5P mimic group was compared with miR-NC group (P < 0.05); the apoptosis rate of human glioma U87 cells in miR-339-5p suppressor group decreased compared with miR-NC group (P < 0.05). Compared with miR-NC group, the protein expression of Twist, Snsnail, N-cadherin, and Vimentin in miR-339-5p mimic group was considerably decreased, whereas E-cadherin was elevated (P < 0.05). Compared with miR-NC group, the protein expression of Twist, Snsnail, N-cadherin, and Vimentin in miR-339-5p suppressor group was considerably increased, whereas E-cadherin was considerably decreased (P < 0.05). Conclusion Forty-nine glioma-related miRNAs were screened out, and miRNA expression was significantly different between glioma and normal tissues. The downregulated miR-339-5p in glioma can regulate the proliferative, apoptotic, invasive, and migratory abilities of glioma U87 cells and might suppress the occurrence and development of glioma.

Splice-Variant Knock-Out of TGFβ Receptors Perturbates the Proteome of Ovarian Carcinoma Cells.

The aim of this study was to analyze the biological role of different transforming growth factor-β (TGFβ) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TβRI variant 1 (TβRIv1) KO in ES-2 cells and TβRII variant 1 (TβRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial–mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TβRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TβRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and MALAT1 mRNA levels were observed in the TβRIIv1 KO compared to control. A significant decrease in JNK1 and JNK2 activation was found in the TβRIv1 KO compared to control cells. These findings provide new insight regarding the biological role of the TGFβ receptor variants in the biology and potentially the progression of OC.

Harmine-inspired design and synthesis of benzo[d]imidazo[2,1-b]thiazole derivatives bearing 1,3,4-oxadiazole moiety as potential tumor suppressors

Standard chemotherapy and personalized target therapies are commonly used in patients with advanced non-small cell lung cancer (NSCLC). However, multidrug resistance (MDR) and tumor metastasis lead to the decline of therapeutic efficacy, which are closely related to epithelial-mesenchymal transition (EMT). Twist1, an EMT transcription factor, plays an essential role in promoting EMT, MDR and tumor metastasis. In view of the essential role of Twist1 in the tumorigenesis of NSCLC, developing antitumor small molecules that can suppress the expression of Twist1 is of far-reaching significance for the treatment of NSCLC. A series of novel benzo[d]imidazo[2,1-b]thiazole derivatives possessing 1,3,4-oxadiazole moiety were designed based on the structure of the first-in-class Twist1 inhibitor harmine. Among the synthetic twenty-two compounds, the compound containing 2-(piperidine-1-yl) ethyl exhibited remarkable anti-proliferative activity with IC50 value of 2.03 μM and 9.80 μM against A549 and H2228 cell lines superior to harmine (IC50 = 17.12 μM against A549, IC50 = 31.06 μM against H2228). Meanwhile, western blot assay showed that the optimal compound significantly down-regulated Twist1 protein expression in a dose-dependent manner and reduced Twist1 level better than harmine. Collectively, the promising compound was identified a potential antineoplastic lead with the ability of down-regulating Twist1 level.

MBRS-45. TWIST1 AND ABCB1 ARE FUNCTIONAL DETERMINANTS OF METASTASIS IN MEDULLOBLASTOMA

Paediatric medulloblastomas (MB) are frequently metastatic, resulting in a poor prognosis for the patient. Of the four MB subgroups, group 3 patients present with the highest rates of metastasis and worst outcomes. The mechanisms behind the metastatic process are poorly understood, limiting our ability to develop novel therapeutic treatments. We hypothesised that the epithelial-mesenchymal transition (EMT) transcription factor TWIST1 and the multidrug efflux pump ABCB1 (ATP-binding cassette subfamily B member 1) synergistically drive MB metastasis. TWIST1 protein expression was analysed in patient tissue microarrays by immunohistochemistry. High TWIST1 expression was associated with metastatic patients (p=0.041). Physical and functional interactions between TWIST1 and ABCB1 were investigated using chromatin immunoprecipitation (ChIP) and a 3D migration and invasion model. ChIP analysis confirmed TWIST1 binding to the ABCB1 promoter in SHH (ONS-76) and group 3 (D283MED and HD-MB03) metastatic cell lines. TWIST1 and ABCB1 were inhibited in HDMB03 cells with harmine and vardenafil respectively, resulting in attenuated cell migration in the 3D model. Western blot and qRT-PCR analysis of harmine treated cells confirmed a reduction in ABCB1 protein and gene expression. Overall our data reveals TWIST1 and ABCB1 to be key targets for MB metastatic disease. Using bioinformatics analysis and ChIP sequencing, additional TWIST1 downstream targets are now being identified and compared across the metastatic cell lines (ONS-76, D283MED and HD-MB03). This data will provide a deeper insight into the pathways associated with MB metastases, enabling personalised treatment approaches for patients with metastatic disease.

Inhibition of p53/p21 by TWIST alleviates TNF-α induced nucleus pulposus cell senescence in vitro.

This study aims to reveal the TWIST protein expression in the degenerated nucleus pulposus (NP), its effect on the TNF-α treated NP cells, and to explore its specific mechanism of anti-senescence. NP tissues from spine fracture patients without intervertebral disc degeneration (IDD) and the IDD patients were collected to detect the TWIST1/2 protein expression by Western blot (WB). NP cells isolated from the healthy tissue was treated with TNF-α to induce senescence, and the TWIST1/2 protein expression was also analyzed. We transfected NP cells with the plasmid coding TWIST to upregulate its expression, which was also cultured in the TNF-α condition. Besides, the TNF-α pretreated NP cells were further stimulated with the recombinant human TWIST1/2 protein. The collagen II and senescent marker β-galactosidase (β-gal) were determined by immunofluorescence (IF); the MMP-13, TIMP-3, IL-10, IL-1β mRNA expression level was detected by quantitative Real Time PCR; the cell proliferation was analyzed by CCK8 assay; the cell cycle was measured by flow cytometry. TWIST1/2 protein was decreased both in the degenerated NP tissue, and TNF-α treated NP cells. The overexpression of TWIST1/2 could prevent the p53, p21, β-gal, MMP-13, and IL-1β expression, moreover, it protected the collagen II, TIMP-3, and IL-10 expression in the TNF-α treated NP cells. Additionally, TWIST overexpression also promoted cell proliferation by ensuring the process of the cell cycle. Furthermore, the supplement of TWIST protein was functional to reverse these senescent phenotypes caused by TNF-α partly. TWIST alleviates the TNF-αinduced NP cells senescence via the inhibition of the p53/p21 pathway.

Effects of LncRNA UNC5B-AS1 on adhesion, invasion and migration of lung cancer cells and its mechanism

Objective: To investigate the effects of long-chain non-coding RNA (lncRNA) UNC5B-AS1 on the adhesion, invasion and migration of lung cancer cells by regulating the expression of miR-218-5p. Methods: Twenty specimens of lung cancer patients and corresponding paracancerous tissues were surgically removed and collected from the oncology department of Chongqing Three Gorges Central Hospital from June 2017 to June 2019. Real-time quantitative PCR (qRT-PCR) was used to detect the expressions of UNC5B-AS1 in human bronchial epithelial cells HBE and different lung cancer cells of A549, H1437, H1975, H1299 and H460. UNC5B-AS1 siRNA was transfected into lung cancer A549 cells. Adhesion assay, transwell invasion assay and scratch assay were used to detect the effect of UNC5B-AS1 on adhesion, invasion and migration of A549 cells. qRT-PCR and dual luciferase reporter gene were used for the detection and identification of UNC5B-AS1 targeting miR-218-5p. The expression of epithelial-mesenchymal transition (EMT)-related protein was detected by Western blot. Results: The expression of UNC5B-AS1 in lung cancer tissues and cells was significantly higher than that in adjacent tissues and bronchial epithelial cells (P＜0.05). The expression of UNC5B-AS1 in lung cancer A549 cells was the highest (P＜0.05). Down-regulation of UNC5B-AS1 expression inhibited adhesion, invasion and migration of A549 cells (P＜0.05). qRT-PCR and dual luciferase reporter assay experiments showed that UNC5B-AS1 targeted the regulation of miR-218-5p expression. Down-regulation of UNC5B-AS1 inhibited E-cadherin protein expression and promoted Vimentin and Twist protein expression. Conclusion: lncRNA UNC5B-AS1 promotes adhesion, invasion and migration of lung cancer cells through targeted regulation of miR-218-5p expression, and its mechanism may be related to the promotion of EMT.

LncRNA DANCR affected cell growth, EMT and angiogenesis by sponging miR-345-5p through modulating Twist1 in cholangiocarcinoma.

LncRNA DANCR has been reported to play an important role in various cancers. Therefore, this study aimed at exploring the function and regulatory mechanism of DANCR in Cholangiocarcinoma (CCA). qRT-PCR was used to measure the expression of DANCR, miR-345-5p in tissues and cells. Western blot was applied to measure the protein expression of Twist, N-cadherin, Vimentin, E-cadherin, VEGF-A, VEGF-C, PCNA and C-caspase 3. The relationship between DANCR and miR-345-5p was determined by luciferase reporter assay. MTT assay and flow cytometry were used to assess cell proliferation and apoptosis, respectively. Transwell assay was performed to detect cell invasion and migration. We found that the expression of DANCR was significantly induced in CCA tissues and cells. Inhibition of DANCR remarkably suppressed CCA cell proliferation, migration, invasion, EMT and angiogenesis as well as induced cell apoptosis in vitro and in vivo. Luciferase reporter assay determined that DANCR directly targeted miR-345-5p and Twist1 was a target mRNA of miR-345-5p. Otherwise, miR-345-5p down-expression partially reversed the effect induced by the suppression of DANCR in CCA. Moreover, the suppressive effects of high miR-345-5p expression on CCA cells were reversed by improving Twist1 expression. In this study, we verified that LncRNA DANCR affected cell proliferation, migration, invasion, angiogenesis, epithelial-mesenchymal transition (EMT) and induced apoptosis through modulating miR-345-5p/Twist1 axis in Cholangiocarcinoma.

Abstract P1-01-08: Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells

Abstract About 90% of the patients diagnosed with breast cancer succumb due to metastases and not from primary tumor. There are currently few methods that can distinguish indolent and aggressive breast tumors. Recent studies have shown differences in the metabolic profiles of primary, circulating, and metastatic breast cancer cells. One of the key regulators of metastases in breast cancer is the transcription factor Twist. The goal of our study was to determine the relationship between Twist expression and cellular metabolism, and the effect of this relationship on metastatic potential. Multiphoton microscopy can provide non-invasive, label-free and reproducible measurements of cellular metabolism. An optical redox ratio of endogenous fluorescence from nicotinamide adenine dicnucleotide (NADH) and flavin adenine dinucleotide (FAD) (calculated as FAD/NADH+FAD) can reflect the balance between mitochondrial oxidative phosphorylation and glucose catabolism in the cell. Because hypoxia is known to promote metastasis, we studied the effect of both intermittent and acute hypoxia on cellular metabolism in 4T1 murine breast cancer cells. We generated a 4T1-Twist− cell line using CRISPR-Cas9 technology. Western blots of Twist protein expression confirmed that Twist expression was significantly lower in the selected cell population compared with the parental 4T1 line. Both cell lines were exposed to 0.5% oxygen for 3 hours and one hour of reoxygenation (acute hypoxia), and 3 cycles of one-hour hypoxia followed by one-hour reoxygenation (intermittent hypoxia). Multiphoton imaging of endogenous fluorescence as well as metabolic flux analysis (Seahorse Biosciences) was performed for both hypoxic exposures in both cell lines. Our results indicate a significant increase in the optical redox ratio in 4T1 cells when exposed to acute hypoxia, indicating a possible increase in oxidative metabolism. There were no signfiicant differences in the 4T1-Twist− line. On the other hand, we observed a decrease in the optical redox ratio in response to intermittent hypoxia in the 4T1 cells and the opposite effect in the 4T1-Twist− line. In addition to these cell lines, we will also present data from sibling cell lines of the 4T1 with varying Twist levels and hence metastatic potential. These data indicate indicate potentially measurable metabolic differences between indolent and aggressive breast cancer cells when subject to a hypoxia-reoxygenation regimen. Citation Format: Lisa Rebello, Narasimhan Rajaram. Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-01-08.

Involvement of epithelial-mesenchymal transition-inducing transcription factors in the mechanism of Helicobacter pylori-induced fibroblasts activation.

Gastric cancer (GC) originating from the lining of the stomach is one of the most frequent malignancies in humans. The most efficient method giving hope of full recovery from GC is gastric resection combined with adjuvant chemotherapy and radiotherapy. However, over 50% of patients after treatment suffer from recurrence and peritoneal metastasis. The bacteria Helicobacter pylori (Hp) is nowadays considered as the major pathogen capable of colonizing gastric mucosa. This bug causes deregulation of multiple signaling pathways including the activation of nuclear factor kappaB (NFκB) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) responsible for inflammation and development of carcinogenic cascade. The pathomechanism of these changes remains little understood, but the Hp virulence factors affecting mainly gastric epithelium have been postulated. Nevertheless, the changes associated with inflammation progressing to cancer are not only limited to epithelial cells. The cells surrounding the tumor, mainly activated fibroblasts (CAFs - cancer-associated fibroblasts) create molecular microenvironment promoting tumorigenesis and cancer invasion. The downstream targets of STAT3, epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) are expressed in activated fibroblasts providing them with additional properties. Thus, our aim was to determine if the Hp strain expressing CagA and VacA cytotoxins may result in the activation/differentiation of rat gastric fibroblasts resulting in NFκB and STAT3 signaling, which could lead to EMT-TFs expression and secretome responsible for inflammatory and EMT inducing microenvironment. In this study, gastric samples were harvested from 8-week-old Spraque-Dowley rats and the primary and secondary fibroblast cultures were established. The 70% confluent secondary fibroblast cultures were infected with 1 x 109 of live Hp expressing cytotoxins CagA VacA per dish and incubated in humidified atmosphere for 3, 24, 48 and 72 hours, before the conditioned media or the cells were used for endpoint experiments. As the control, fibroblast culture in DMEM with 10% FBS and antibiotics, free from Hp infection was used. The expression of mRNA for 18S (control), toll-like receptors: TLR2 and TLR4, STAT3, NFκB p65/Rel A, inhibitor of NF-κB (Iκβ), Snail and Twist was determined by RT-PCR. The protein expression of Snail and Twist was assessed by Western blot technique. The fibroblast supernatant was collected at 72 hours from non-infected and Hp (cagA+ vacA+)-infected culture and the concentrations of interleukin 8 (IL-8), hepatocyte growth factor (HGF) and stromal derived factor-1 (SDF-1) were measured by ELISA. In fibroblasts infected with Hp (cagA+ vacA+), the significant increase of mRNA expression for both, TLR2 and TLR4, as well as STAT3, NFκB/RelA subunit was observed already after 3 hours of fibroblasts infection with Hp strain compared with control non-infected fibroblasts. Simultaneously, the significant decrease of Iκβ mRNA has been noticed starting from 48 hours after the Hp infection of fibroblasts was carried out. The strong increase in the expression of Snail1 and Twist mRNA was recorded already at 3 hours in Hp-infected fibroblasts comparing to control non-infected fibroblasts and this increase persisted at 24 and 48 hours being the most pronounced at 72 hours post incubation with Hp. The expression of Snail1 protein was observed after 3 hours post Hp infection and this increase persisted throughout entire time periods upon Hp infection. In contrast, no detectable level of Twist protein expression was observed up to 72 hours post-infection neither in control conditions nor in fibroblasts co-infected with Hp. These changes in fibroblasts were accompanied by a significant increase in the release of HGF, SDF-1 and IL-8 determined in cell supernatants collected from Hp-infected fibroblasts. These data indicate that the activation/differentiation of rat gastric fibroblasts can occur directly by Hp releasing CagA and indirectly through TLR2 and TLR4 and these effects can be mediated by transcription factors NFκB and STAT3 signaling leading to rapid Snail1 protein expression. We conclude that NFκB and STAT3 signaling together with Snail1 protein expression may activate the secretome responsible for fibroblasts inflammatory and EMT-inducing microenvironment likely serving as prerequisite for GC development.

Experimental study of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells

Objective To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells. Methods Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups: blank control group, negative control group and experimental group. The blank control group was the untreated cell group, while the negative control group was the lentivirus transfected cell group by the blank vector. The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist. The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot to detect the expression of Twist. Transwell kit was used to detect cell invasion. Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation. Results ⑴ The titer of lentivirus was detected. The transfection titer of lentivirus vector: shRNA-Twist vector was 3×108 TU/ml. ⑵ The results of qRT-PCR test showed that compared with the negative control group, the mRNA expression of Twist in the experimental group was decreased (q=3.177, P=0.0234). ⑶ The results of Western blot showed that compared with the negative control group, the protein expression of Twist in the experimental group was decreased (q=4.071, P=0.0304). ⑷ The results of Transwell test showed that there was significant statistical difference among the three groups (F=19.472, P=0.000). Compared with the negative control group, the number of cell imigration in the experimental group was decreased (q=3.567, P=0.0318). ⑸ The results of CCK-8 showed that there was significant statistical difference among the three groups (F=20.983, P=0.000). Compared with the negative control group, the proliferation rate in the experimental group was decreased (q=5.272, P=0.0157). Conclusions ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells. Key words: Cell line, tumor; Adenocarcinoma; Lung neoplasms; RNA, small interfering; Cell proliferation; Cell migration assays

Abstract 2374: Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression

Abstract Introduction: While the genomic and proteomic characterization of pancreatic ductal adenocarcinoma (PDAC) is extraordinarily complex, the transforming growth factor-beta (TGF-β) pathway is critically important. TGF-β has a context dependent role- it is tumor suppressive in early PDAC and tumor promotive in metastatic PDAC. Our hypothesis is that non-canonical TGF-β signaling induces metastatic PDAC. Non-canonical signaling may help explain the paradoxical nature of TGF-β in PDAC. Methods: Two cell lines were utilized- Panc-1, a cell line derived from primary PDAC, was compared to Capan-1, a cell line derived from metastatic PDAC- in order to model an early PDAC (Panc-1) and a more aggressive PDAC (Capan-1). We investigated the gene and protein expression of markers of epithelial to mesenchymal transition (Snail and Twist), Akt, β-catenin, and PD-L1 upon treatment with TGF-β and the Akt inhibitor MK-2206. Results: While TGF-β increased Snail gene and protein expression in Panc-1 cells, it had nil effect on Capan-1 cells where TWIST1 protein expression increased with TGF-β. TGF-β increased pAkt expression in both cell lines. TGF-β increased active and total β-catenin expression in Capan-1 but not Panc-1 cell lines. We then found that PD-L1 expression was generally diminished by Akt inhibition in both cell lines but this was reversed with TGF-β receptor inhibition and gemcitabine treatment in the Capan-1 cells. Conclusions: Our data suggests that modulation of the non-canonical TGF-β pathway may lead to increased epithelial to mesenchymal transition through multiple mechanisms. PD-L1 expression is a response to chemotherapy in metastatic PDAC when multiple proteins in that pathway are inhibited. Our data suggests that (1) personalized therapy based on the degree of non-canonical TGF-β pathway activation will be critical, (2) patients with uncontrolled non-canonical TGF-β pathway activation may have lower survival, and (3) there is an opportunity to utilize anti-PD-L1 based therapy in combination with other therapy in selected patients based on their response to chemotherapy. Citation Format: S. Mazher Hussain, Gustavo Miranda-Carboni, Marcus A. Alvarez, Abul Elahi, Leah E. Hedrick, David Shibata, Evan S. Glazer. Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression [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 2374.

Translational downregulation of Twist1 expression by antiproliferative gene, B-cell translocation gene 2, in the triple negative breast cancer cells

Twist1, a key transcription factor regulating epithelial–mesenchymal transition and cancer metastasis, is highly expressed in invasive cancers in contrast to the loss of BTG2/TIS21 expression. Based on our observation that forced expression of BTG2/TIS21 downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2/TIS21-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2/TIS21-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2/TIS21 interacted with each other, which abrogated Twist1 activity. (2) BTG2/TIS21 inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2/TIS21 maintained p-eIF2α that downregulates initiation of protein translation, confirmed by eIF2α-AA mutant expression and BTG2/TIS21 knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2/TIS21-KO mouse than that in the wild type. (5) BTG2/TIS21-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2/TIS21 expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2/TIS21 expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2/TIS21-mediated Twist1 loss was not due to the protein degradation by ubiquitination and autophagy activation. (8) Twist1 protein level was significantly higher in various organs of TIS21-KO mice compared with that in the control, indicating the in vivo role of BTG2/TIS21 gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2/TIS21 is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2/TIS21 expression.

Chrysin inhibits sphere formation in SMMC-7721 cells via modulation of SHP-1/STAT3 signaling pathway.

Background: Chrysin is a natural flavonoid which has been identified as a candidate anti-cancer agent due to its inhibitory effect on a variety of cancer cells, including targeted inhibition of sphere formation in hepatocellular carcinoma (HCC) cell lines. However, the mechanism by which chrysin modulates HCC spheres remains unclear.Materials and methods: In this study, we investigate the effect of chrysin on the regulation of SHP-1 and its downstream signal molecule STAT3 to explain the mechanism by which chrysin inhibits sphere formation of HCC cell lines.Results: Here, we found that SHP-1 protein expression was markedly down-regulated in the spheres from both SMMC-7721 and MHCC97H cells. Chrysin significantly inhibited sphere formation and upregulated the expression of SHP-1 protein in both SMMC-7721 and MHCC97H cells, as well as reduced p-STAT3 and Twist1 expressions in SMMC-7721 cells. Furthermore, knockdown of SHP-1 in SMMC-7721 cells resulted in the induction of p-STAT3 and Twist1 protein expression and antagonizing the inhibitory effect of chrysin on sphere formation in SMMC-7721 cells.Conclusion: Overall, the study findings demonstrated that chrysin acts as a candidate for the treatment of HCC through modulating SHP-1/STAT3 signaling pathway.

Correlations of Twist Expression with Pathological and Computed Tomography (CT) Characteristics and Prognosis of Non-Small Cell Lung Cancer (NSCLC).

BackgroundThe aim of this study was to assess the correlations of Twist expression with pathological and computed tomography (CT) characteristics and prognosis of non-small cell lung cancer (NSCLC).Material/MethodsWe enrolled 120 patients with lung cancer who underwent CT examination. The Twist protein expression level was detected in 120 cases of cancer tissues and a control group using immunohistochemical method. The survival curve was plotted using the Kaplan-Meier method and analyzed via log-rank test.ResultsThe Twist expression was associated with tumor stage, differentiation degree, and presence or absence of lymph node metastasis, but had no correlations with sex, age, or histological type. Grade-3 bronchial involvement, pleural indentation, and hilar and mediastinal lymph node enlargement occurred more frequently in the high-expression Twist group compared with the low-expression Twist group. The overall survival rate of patients with Twist overexpression was significantly lower than that of patients with normal Twist expression. The mean survival time was 69.8 months in Twist protein expression-negative patients and 45.8 months in Twist protein expression-positive patients. Finally, the positive expression of Twist protein was significantly correlated with the long-term survival and prognosis of patients.ConclusionsThe Twist gene might be involved in the occurrence and development of NSCLC, which is correlated with patient prognosis.

Knockdown of TWIST enhances the cytotoxicity of chemotherapeutic drugs in doxorubicin-resistant HepG2 cells by suppressing MDR1 and EMT.

The transcription factor twist family bHLH transcription factor1 (TWIST), which is a member of the basic helix-loop-helix class of proteins, is known to induce epithelial-mesenchymal transition (EMT) and promote cancer metastasis. TWIST has previously been reported to be associated with multidrug resistance (MDR), since its depletion increases drug sensitivity. Although these previous studies have established a strong association between EMT and MDR, the molecular mechanism remains obscure. The present study demonstrated that TWIST protein expression was elevated in liver cancer, and was positively correlated with multidrug resistance protein1 (MDR1) expression. Conversely, MDR1 was negatively correlated with E‑cadherin expression in liver cancer samples. In addition, the present study indicated that doxorubicin-resistant HepG2 (R‑HepG2) cells acquired an EMT phenotype. TWIST was also more highly expressed in R‑HepG2 cells compared with in parental HepG2 cells. Knockdown of TWIST increased the sensitivity of R‑HepG2 cells to 5-fluroracil, cisplatin and doxorubicin through a reduction in MDR1 expression and drug efflux ability. Furthermore, knockdown of TWIST in R‑HepG2 cells inhibited the migratory ability of cells and suppressed the EMT phenotype. These findings demonstrated that targeting TWIST may be considered a novel strategy to overcome drug resistance in liver cancer.