Publications by authors named "Mohammad Mahdi Forghanifard"

65 Publications

Elucidated tumorigenic role of MAML1 and TWIST1 in gastric cancer is associated with Helicobacter pylori infection.

Microb Pathog 2021 Nov 21:105304. Epub 2021 Nov 21.

Medical Genetics Research Center, Faculty of Medical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Background: Epithelial-mesenchymal transition (EMT) has a fundamental role in tumor initiation, progression, and metastasis. Helicobacter pylori (HP) induces EMT and thus causes gastric cancer (GC) by deregulating multiple signaling pathways involved in EMT. TWIST1 and MAML1 have been confirmed to be critical inducers of EMT via diverse signaling pathways such as Notch signaling. This study aimed to investigate for the first time possible associations between TWIST1/MAML1 mRNA expression levels, HP infection, and clinicopathological characteristics in GC patients.

Method: TWIST1 and MAML1 mRNA expression levels were evaluated in tumoral and adjacent normal tissues in 73 GC patients using the quantitative reverse transcription PCR (RT-qPCR) method. PCR technique was also applied to examine the infection with HP in GC samples.

Results: Upregulation of TWIST1 and MAML1 expression was observed in 35 (48%) and 34 (46.6%) of 73 tumor samples, respectively. Co-overexpression of these genes was found in 26 of 73 (35.6%) tumor samples; meanwhile, there was a significant positive correlation between MAML1 and TWIST1 mRNA expression levels (P < 0.001). MAML1 overexpression exhibited meaningful associations with advanced tumor stages (P = 0.006) and nodal metastases (P ˂ 0.001). 34 of 73 (46.6%) tumors tested positive for HP, and meanwhile, MAML1 expression was positively related with T (P = 0.05) and grade (P = 0.0001) in these HP-positive samples. Increased TWIST1 expression was correlated with patient sex (P = 0.035) and advanced tumor grade (P = 0.017) in HP-infected tumors. Furthermore, TWIST1 and MAML1 expression levels were inversely linked with histologic grade in HP-negative tumor samples (P = 0.021 and P = 0.048, respectively).

Conclusion: We propose TWIST1 and MAML1 as potential biomarkers of advanced-stage GC that determine the characteristics and aggressiveness of the disease. Based on accumulating evidence and our findings, they can be introduced as promising therapeutic targets to modify functional abnormalities in cells that promote GC progression. Moreover, HP may enhance GC growth and metastasis by disrupting TWIS1/MAML1 expression patterns and related pathways.
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http://dx.doi.org/10.1016/j.micpath.2021.105304DOI Listing
November 2021

A new gene panel as a marker for ESCC poor prognosis; INPP5A, TWIST1, MMP2, and EGFR.

Adv Med Sci 2021 Sep 30;66(2):231-236. Epub 2021 Mar 30.

Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran. Electronic address:

Purpose: Esophageal squamous cell carcinoma (ESCC) is categorized among ten common aggressive malignancies, with a higher incidence and mortality rates in the developing than in developed countries. The inositol polyphosphate 5-phosphatase (INPP5A), as an intracellular-calcium mobilizer and modifier enzyme, facilitates cell responses to various stimuli. Epithelial-mesenchymal transition (EMT), a transformation procedure, has a vital role in cancer progression and metastasis when epithelial cells lose their traits in favor of obtaining mesenchymal features. In this study, we analyzed the correlation between the expression of INPP5A and the involved genes in EMT pathway through the progression and development of the ESCCs.

Materials And Methods: The gene expression analyses of INPP5A, TWIST1, MMP-2, and EGFR were performed using relative comparative real-time PCR in 58 ESCCs patients compared to corresponding margin-normal esophageal tissues.

Results: A significant inverse correlation between INPP5A and EGFR/MMP-2 mRNA expression was observed in tumor samples. Underexpression of INPP5A was significantly correlated with overexpression of TWIST1, MMP-2, and EGFR in different invasiveness and aggressiveness pathological features of the ESCCs (P ​< ​0.05).

Conclusions: The results propose a tumor suppressor role for INPP5A and oncogenic function for concomitant expression of the other genes in ESCC invasion and metastasis. The current study is the first report elucidating the correlation between the downregulation of INPP5A and upregulation of TWIST1, MMP-2, and EGFR in ESCC and introduces this panel of the genes as a marker for poor prognosis of the disease.
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http://dx.doi.org/10.1016/j.advms.2021.03.004DOI Listing
September 2021

Interaction between LINC-ROR and Stemness State in Gastric Cancer Cells with Helicobacter pylori Infection.

Iran Biomed J 2021 Mar 1;25(3):157-68. Epub 2021 Mar 1.

Medical Genetics Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Large intergenic non-coding RNA regulator of reprogramming (LINC-ROR), as a cancer-related Long non-coding RNA, has vital roles in stem cell survival, pluripotency, differentiation, and self-renewal in human embryonic stem cell. However, cancer-related molecular mech¬anisms, its functional roles, and clinical value of LINC-ROR in gastric cancer (GC) remain unclear. In this study, we aimed to investigate probable interplay between LINC-ROR with SALL4 stemness regulator and their role with the development of the disease.

Methods: The mRNA expression profile of LINC-ROR and SALL4 was assessed in tumoral and adjacent non-cancerous tissues of GC patients, using quantitative real-time PCR.

Results: Significant LINC-ROR underexpression and SALL4 overexpression were observed in 55.81% and 75.58% (p < 0.0001) of samples, respectively. The expression of LINC-ROR and SALL4 were significantly correlated with each other (p = 0.044). There was an association between the underexpression of LINC-ROR and sex, stage of tumor progression, tumor type, and location of tumor (p < 0.05), and Helicobacter pylori infection with SALL4 expression (p = 0.036). There were also significant correlations between concomitant mRNA expression of SALL4 and LINC-ROR in tumors located at distal noncardiac, positive for H. pylori infection, tumors with invasion into the muscle layer of the stomach, and grade II tumor (p < 0.05).

Conclusion: The clinical results of the SALL4-LINC-ROR association propose a probable functional interaction between these markers in tumor maintenance and aggressiveness. Our study can help to understand one of the mechanisms involved in the progression of gastric cancer through the function of these regulators.
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http://dx.doi.org/10.29252/ibj.25.3.157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183384PMC
March 2021

Crosstalk between MMP-13, CD44, and TWIST1 and its role in regulation of EMT in patients with esophageal squamous cell carcinoma.

Mol Cell Biochem 2021 Jun 19;476(6):2465-2478. Epub 2021 Feb 19.

Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Matrix metalloproteinases (MMPs) play key roles in epithelial-mesenchymal transition (EMT) for the development of cancer cell invasion and metastasis. MMP-13 is an extracellular matrix (ECM)-degrading enzyme that plays crucial roles in angiogenesis, cell cycle regulation, niche maintenance, and transforming squamous epithelial cells in various tissues. CD44, a transmembrane glycoprotein expressed on esophageal tumor cells, is required for EMT induction and invasion in esophageal squamous cell carcinoma (ESCC). The transcription factor TWIST1, as EMT and stemness marker, regulates MMPs expression and is identified as the downstream target of CD44. In this study, we aimed to investigate the probable interplay between the expression of key genes contributing to ESCC development, including MMP-13, TWIST1, and CD44 with clinical features for introducing novel diagnostic and therapeutic targets in the disease. The gene expression profiling of MMP-13, TWIST1, and CD44 was performed using quantitative real-time PCR in tumor tissues from 50 ESCC patients compared to corresponding margin non-tumoral tissues. Significant overexpression of MMP-13, CD44S, CD44V3, CD44V6, and TWIST1 were observed in 74%, 36%, 44%, 44%, and 52% of ESCC tumor samples, respectively. Overexpression of MMP-13 was associated with stage of tumor progression, metastasis, and tumor location (P < 0.05). There was a significant correlation between TWIST1 overexpression and grade (P < 0.05). Furthermore, overexpression of CD44 variants was associated with stage of tumor progression, grade, tumor invasion, and location (P < 0.05). The results indicated the significant correlation between concomitant expression of MMP-13/TWIST1, TWIST1/CD44, and CD44/MMP-13 with each other in a variety of clinicopathological traits, including depth of tumor invasion, tumor location, stage of tumor, and lymph node involvement in ESCC tissue samples (P < 0.05). Collectively, our results indicate that the TWIST1-CD44-MMP-13 axis is involved in tumor aggressiveness, proposing these genes as regulators of EMT, diagnostic markers, and therapeutic targets in ESCC.
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http://dx.doi.org/10.1007/s11010-021-04089-2DOI Listing
June 2021

SOX2/SALL4 stemness axis modulates Notch signaling genes to maintain self-renewal capacity of esophageal squamous cell carcinoma.

Mol Cell Biochem 2021 Feb 24;476(2):921-929. Epub 2020 Oct 24.

Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Stemness phenotype is considered as the centerpiece of cancer biology due to its potential in conventional chemo-radiotherapy resistance and tumor recurrence after clinical intervention. This feature in tumor mass belongs to activation of core regulatory stemness factors and different cell signaling pathways in cancer stem cells. We aimed in this study to elucidate contribution of Notch signaling pathway in stemness state of esophageal squamous cell carcinoma (ESCC) through their relevance with stem cell markers SOX2 and SALL4. 50 ESCC tumor and related margin normal tissues were considered and categorized based on SOX2/SALL4 expression pattern, and mRNA levels of Notch signaling genes including ligands, receptors, target genes, and transcriptional coactivator were analyzed in the selected groups using qRT-PCR. Concomitant overexpression of stem cell markers SOX2 and SALL4 in ESCCs upregulated the involved genes in Notch signaling pathway. Upregulation of Notch pathway genes associated with depth of tumor invasion and lymph node metastasis of ESCC. Based on biological function of SOX2 and SALL4 axis in stemness state potential, our results may suggest contribution of Notch signaling pathway in self-renewal capacity of ESCCs, as well as invasion and metastasis of the disease. To the best of our knowledge, this is the first report elucidating the crosstalk between SOX2/SALL4 stemness factors and Notch signaling pathway in cancer research.
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http://dx.doi.org/10.1007/s11010-020-03956-8DOI Listing
February 2021

MEIS1 promotes expression of stem cell markers in esophageal squamous cell carcinoma.

BMC Cancer 2020 Aug 20;20(1):789. Epub 2020 Aug 20.

Department of Biology, Damghan branch, Islamic Azad University, P.O.Box: 3671639998, Cheshmeh-Ali Boulevard, Sa'dei Square, Damghan, Islamic Republic of Iran.

Background: MEIS1 (Myeloid ecotropic viral integration site 1) as a homeobox (HOX) transcription factor plays regulatory roles in a variety of cellular processes including development, differentiation, survival, apoptosis and hematopoiesis, as well as stem cell regulation. Few studies have established pluripotency and self-renewal regulatory roles for MEIS1 in human esophageal squamous cell carcinoma (ESCC), and our aim in this study was to evaluate the functional correlation between MEIS1 and the stemness markers in ESCC patients and cell line KYSE-30.

Methods: Expression pattern of MEIS1 and SALL4 gene expression was analyzed in different pathological features of ESCC patients. shRNA in retroviral vector was used for constantly silencing of MEIS1 mRNA in ESCC line (KYSE-30). Knockdown of MEIS1 gene and the expression pattern of selected stemness markers including SALL4, OCT4, BMI-1, HIWI, NANOG, PLK1, and KLF4 were evaluated using real-time PCR.

Results: Significant correlations were observed between MEIS1 and stemness marker SALL4 in different early pathological features of ESCC including non-invaded tumors, and the tumors with primary stages of progression. Retroviral knockdown of MEIS1 in KYSE-30 cells caused a noteworthy underexpression of both MEIS1 and major involved markers in stemness state of the cells including SALL4, OCT4, BMI-1, HIWI and KLF4.

Conclusions: The results highlight the important potential role of MEIS1 in modulating stemness properties of ESCCs and cells KYSE-30. These findings may confirm the linkage between MEIS1 and self-renewal capacity in ESCC and support probable oncogenic role for MEIS1 in the disease.
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http://dx.doi.org/10.1186/s12885-020-07307-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441725PMC
August 2020

TWIST1 correlates with Notch signaling pathway to develop esophageal squamous cell carcinoma.

Mol Cell Biochem 2020 Nov 25;474(1-2):181-188. Epub 2020 Jul 25.

Department of Biology, Damghan Branch, Islamic Azad University, Cheshmeh-Ali Boulevard, Sa'dei Square, Damghan, Iran.

Notch signaling pathway mediates different biological processes including stem cell self-renewal, progenitor cell fate decision, and terminal differentiation. TWIST1 plays a key role in tumor development and metastasis through inducing epithelial-mesenchymal transition (EMT). Expression of the core transcriptional complex of Notch pathway and its target genes, as well as TWIST1 overexpression, are closely related to the aggressive clinicopathological variables of esophageal squamous cell carcinoma (ESCC). Here we aimed to functionally elucidate probable crosstalk between TWIST1 and Notch pathway in ESCCs. Correlation between TWIST1 and Notch target genes was analyzed in 50 ESCCs and corresponding normal tissues. Using retroviral system, enforced expression of TWIST1 was established in ESCC line KYSE-30 cells and expression of Notch signaling genes was assessed. Significant correlation between TWIST1 and HEY1/HEY2 expression was found in different pathological variable of ESCC poor prognosis. Induced expression of TWIST1 in KYSE-30 cells caused a noteworthy increase of Notch pathway genes expression revealing regulatory role of TWIST1 on Notch signaling genes in the cells. Based on existed correlations between expression of TWIST1 and Notch pathway genes in different pathological features of ESCC patients, as well as KYSE-30 cell line, we may extrapolate that TWIST1 is involved in aggressiveness of the disease through regulation of Notch signaling genes. To the best of knowledge, this is the first report describing the impact of TWIST1 on Notch cascade genes in ESCC.
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http://dx.doi.org/10.1007/s11010-020-03843-2DOI Listing
November 2020

TWIST1 upregulates matrix metalloproteinase (MMP) genes family in esophageal squamous carcinoma cells.

Gene Expr Patterns 2020 09 22;37:119127. Epub 2020 Jul 22.

Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran. Electronic address:

Twist-related protein 1 (TWIST1), a highly conserved basic helix-loop-helix transcription factor, stimulates epithelial-mesenchymal transition (EMT) and plays a crucial role in the regulation of the extracellular matrix (ECM) and cell-cell adhesion. Our aim in this study was to evaluate the functional correlation between TWIST1 and MMP genes in human ESCC cell lines, KYSE-30 and YM-1. To generate recombinant retroviral particles, the Pruf-IRES-GFP-hTWIST1 was co-transfected into HEK293T along with pGP and pMD2. G as well as Pruf-IRES-GFP control plasmid. Stably transduced high-expressing GFP-hTWIST1 and GFP-control KYSE-30 cells were generated. The produced retroviral particles were transduced into the KYSE-30 and YM-1 ESCC cells. Ectopic expression of TWIST1 mRNA and expression of the MMP genes (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-10) were examined by relative comparative real-time PCR. In silico analysis of the MMP markers and their promoter elements was explored. Moreover, the scratch wound assay was used to evaluate the migration of TWIST1-induced cells. TWIST1 level was up-regulated by nearly 5-fold and 7.4-fold in GFP-hTWIST1 KYSE-30 and YM-1 cells compared to GFP control cells, respectively. Interestingly, this enforced expression of TWIST1 subsequently caused significant overexpression of transcripts for selected MMP genes in GFP-hTWIST1 in comparison with GFP control cells in both ESCC cell lines. Also, the scratch assay indicated that TWIST1 expression effectively increased the migration of GFP-TWIST1 KYSE-30 cells against GFP KYSE-30 control cells in vitro. The present findings illuminate that TWIST1 may contribute broadly to ESCC development in concert with up-regulation of MMPs expression and further suggest the potential advantage of exerting TWIST1/MMPs signaling axis as a framework from which to expand our understanding about the mechanisms of ESCC tumorigenesis.
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http://dx.doi.org/10.1016/j.gep.2020.119127DOI Listing
September 2020

GSTs polymorphisms are associated with epigenetic silencing of CDKN2A gene in esophageal squamous cell carcinoma.

Environ Sci Pollut Res Int 2020 Sep 1;27(25):31269-31277. Epub 2020 Jun 1.

Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Esophageal cancer is the eighth most common cancer and the sixth most frequent cause of cancer mortality worldwide. Exposure to polycyclic aromatic hydrocarbons formed by incomplete combustion of organic matter is an important risk factor. Genetic polymorphisms in genes encoding PAH-metabolizing enzymes like glutathione S-transferases (GSTM1, GSTP1, GSTT1) which conjugate glutathione to PAHs for reduction of oxidative stress may affect an individual's response to PAH exposure. Genomic DNA from 50 esophageal squamous cell carcinoma patients extracted from peripheral blood. PCR-RFLP technique was employed to determine GSTM1, GSTT1, and GSTP1 polymorphisms. Aberrant promoter methylation of CDKN2A was applied by methylation-specific PCR technique. Concentration of urinary 1-hydroxypyrene was determined using a HPLC system. About 38.7% showed the null GSTM1 genotype (54% cases and 13% controls), 23.7% showed GSTT1 null genotype (30% cases and 13% controls), and 62.5% were GSTP1 A/A genotype (66% cases and 56% controls). Polymorphic variants of GSTM1 and GSTT1 were significantly associated with aberrant methylation of CDKN2A gene. The null state of GSTT1 was significantly associated with high concentrations of 1-OHP in urea (p < 0.01). There was significant association between methylated states of CDKN2A and high concentrations of 1-OHP in urine (p < 0.01). We identified significant association between polymorphism of GSTs genes and epigenetic silencing of tumor suppressor gene CDKN2A in esophageal squamous cell carcinoma.
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http://dx.doi.org/10.1007/s11356-020-09408-6DOI Listing
September 2020

Crosstalk between MEIS1 and markers of different cell signaling pathways in esophageal squamous cell carcinoma.

Mol Biol Rep 2020 May 5;47(5):3439-3448. Epub 2020 May 5.

Department of Biology, Damghan branch, Islamic Azad University, Cheshmeh-Ali boulevard, Sa'dei square, Damghan, Islamic Republic of Iran.

The homeobox transcription factor MEIS1 is involved in cell fate decision, stem cells properties, gastrointestinal (GI) tract development, and progression of several malignancies such as esophageal squamous cell carcinoma (ESCC). Increasing evidences suggest the crosstalk between MEIS1 and cell signaling pathways. Therefore, our aim in present study was to investigate the probable linkage of MEIS1 expression with key genes of different cell signaling pathways in ESCC tumorigenesis, and their correlation with clinicopathological feature of the patients. The gene expression profiling of MEIS1 and different cell signaling genes including SALL4, SIZN1, and HEY1 (stemness state, BMP, and NOTCH signaling pathways, respectively) was performed using quantitative real-time reverse transcription polymerase chain reaction (PCR) in fresh tumoral compared to margin normal tissues of 50 treatment-naive ESCC samples. The mRNA expression of MEIS1/SIZN1, SIZN1/HEY1, and SIZN1/SALL4 were significantly associated to each other (P < 0.05). There were remarkable correlations between concomitant mRNA expression of MEIS1 and SIZN1 in tumors with invasion to adventitia, early stages of tumor progression and poorly differentiated tumors. Moreover, expression of MEIS1 and HEY1 was correlated to each other in primary stages of tumor progression and non-invaded tumors. Expression of MEIS1 was significantly associated with SALL4 in poorly differentiated tumors. Our results indicated that correlation between different cell signaling pathway-related genes may lead to esophageal tumorigenesis. It is illustrated that MEIS1 as a HOX gene has a significant correlation with stemness state, BMP, and NOTCH signaling pathways via the SIZN1.
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http://dx.doi.org/10.1007/s11033-020-05423-5DOI Listing
May 2020

MAML1 promotes ESCC aggressiveness through upregulation of EMT marker TWIST1.

Mol Biol Rep 2020 Apr 16;47(4):2659-2668. Epub 2020 Mar 16.

Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Mastermind-like 1 (MAML1) is the main transcriptional co-activator of Notch signaling pathway. It plays essential roles in several pathways including MEF2C, p53, Nf-кB and Wnt/β-catenin. TWIST1 is known as a regulator of epithelial mesenchymal transition (EMT), which is considered as a primary step in promotion of tumor cell metastasis. Since concomitant expression of these genes was observed in tumors, our aim in this study was to elucidate the linkage between MAML1 and TWIST1 co-overexpression in esophageal squamous cell carcinoma (ESCC).

Results: While MAML1 silencing significantly down-regulated TWIST1, its ectopic expression up-regulated TWIST1 expression in both mRNA and protein levels in KYSE-30 cells. Expression of mesenchymal markers was increased significantly after MAML1 and TWIST1 ectopic expression, while epithelial markers expression was significantly decreased after silencing of both genes. Concomitant protein expression of MAML1 and TWIST1 was significantly observed in ESCC patients. Enforced expression of TWIST1 had no impact on MAML1 gene expression in KYSE-30 cells.

Conclusion: The results clearly suggest transcriptional regulation of TWIST1 by MAML1 transcription factor in ESCC cells KYSE-30. Since TWIST1 is known as an EMT inducing marker, our results may revealed the mastermind behind TWIST1 function and introduced MAML1 as an upstream master regulator of TWIST1 and EMT in KYSE-30 cells.
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http://dx.doi.org/10.1007/s11033-020-05356-zDOI Listing
April 2020

PYGO2 as an independent diagnostic marker expressed in a majority of colorectal cancers.

J Histotechnol 2019 09 13;42(3):98-103. Epub 2019 May 13.

Department of Biology, Damghan Branch, Islamic Azad University , Damghan , Iran.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide. Detection of CRC at the early stages of disease can play an important role in decrease of associated mortality rates. The Wnt signaling pathway is crucial for the progression of different cellular and developmental processes and Wnt pathway deregulation has been well characterized in a variety of cancers, particularly in CRC. The aim of this study was to analyze protein expression of Pygopus2 (PYGO2), the main transcription factor of Wnt pathway, in CRC tissues and evaluate its probable correlation with clinicopathological features of the patients. The expression pattern of PYGO2 was evaluated by immunohistochemistry in tumor tissues and their margin normal which is the piece of normal, or unaffected tissue excised from the surrounding the visible tumors in 46 CRC patients. A defined scoring system was applied to analyze immunostaining results. The expression of PYGO2 protein was detected in all tumor tissues. Furthermore, this expression was significantly higher in CRC samples than in normal tissues. There was a significant association between PYGO2 protein expression in CRC and tumor cell metastasis to the lymph nodes. Considering the significant expression of PYGO2 protein in colorectal tumor cells and its correlation with lymph node metastasis, this protein may be used as a biomarker for metastatic CRC as well as a putative therapeutic target to inhibit aggressiveness and metastasis of CRC.
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http://dx.doi.org/10.1080/01478885.2019.1610214DOI Listing
September 2019

Linkage between EMT and stemness state through molecular association between TWIST1 and NY-ESO1 in esophageal squamous cell carcinoma.

Biochimie 2019 Aug 31;163:84-93. Epub 2019 May 31.

Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran. Electronic address:

Aberrant expression of cancer testis antigens (CTAs) is reported in tumors, especially those with stemness properties. A number of CTAs can induce epithelial mesenchymal transition (EMT) process and promote cancer stem cells (CSCs) characteristics. We aimed in this study to analyze the correlation between NY-ESO1 and TWIST1 in esophageal squamous cell carcinoma (ESCC), as well as their impact on EMT process. Gene expression profiling of NY-ESO1 and TWIST1 was performed in 43 esophageal tumors compared to their margin normal tissues of using qRT-PCR, and their correlation with clinicopathological variables of the patients was evaluated. In silico analysis of the NY-ESO1, epithelial and mesenchymal cell markers and also their promoter sequences was executed. ESCC cell lines KYSE-30 and YM-1 were transduced to ectopically express TWIST1 using a retroviral system, followed by qRT-PCR mRNA expression analysis to reveal the probable correlation among TWIST1, NY-ESO1 and EMT markers gene expression. Scratch assay was performed to estimate migration of TWIST1-induced cells. Overexpression of TWIST1 and NY-ESO1 mRNA was observed in 42% and 39.5% (P ˂ 0.05) of tumors, respectively. Expression of the genes was significantly correlated with each other (p = 0.005). TWIST1 and NY-ESO1 overexpression was significantly associated with stage of progression and size of tumors, respectively. A direct association between TWIST1 and NY-ESO1 mRNA expression was confirmed by induced ectopic expression of TWIST1 in ESCC cell lines KYSE-30 and YM-1. TWIST1-induced cells led to increase migration in ESCC cell line. Furthermore, significant up-regulation of EMT markers was observed following ectopic expression of TWIST1 in these cells. Based on our findings, it may be proposed that a vital association is exist between the EMT and the acquisition of cancer stemness state in tumor cells through the TWIST1/NY-ESO1 axis and it can be a critical hallmark in ESCC tumorigenesis.
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http://dx.doi.org/10.1016/j.biochi.2019.05.016DOI Listing
August 2019

MEIS1 knockdown may promote differentiation of esophageal squamous carcinoma cell line KYSE-30.

Mol Genet Genomic Med 2019 07 14;7(7):e00746. Epub 2019 May 14.

Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

Background: MEIS1 (Myeloid ecotropic viral integration site 1), as a homeobox (HOX) transcription factor, has a dual function in different types of cancer. Although numerous roles are proposed for MEIS1 in differentiation, stem cell function, gastrointestinal development and tumorigenesis, the involved molecular mechanisms are poor understood. Our aim in this study was to elucidate the functional correlation between MEIS1, as regulator of differentiation process, and the involved genes in cell differentiation in human esophageal squamous carcinoma (ESC) cell line KYSE-30.

Methods: The KYSE-30 cells were transduced using recombinant retroviral particles containing specific shRNA sequence against MEIS1 to knockdown MEIS1 gene expression. Following RNA extraction and cDNA synthesis, mRNA expression of MEIS1 and the selected genes including TWIST1, EGF, CDX2, and KRT4 was examined using relative comparative real-time PCR.

Results: Retroviral transduction caused a significant underexpression of MEIS1 in GFP-hMEIS1 compared to control GFP cells approximately 5.5-fold. While knockdown of MEIS1 expression caused a significant decrease in EGF and TWIST1 mRNA expression, nearly -8- and -12-fold respectively, it caused a significant increase in mRNA expression of differentiation markers including KRT4 and CDX2, approximately 34- and 1.14-fold, correspondingly.

Conclusion: MEIS1 gene silencing in KYSE-30 cells increased expression of epithelial markers and decreased expression of epithelial-mesenchymal transition (EMT) marker TWIST1. It may highlight the role of MEIS1 in differentiation process of KYSE-30 cells. These results may confirm that MEIS1 silencing promotes differentiation and decreases EMT capability of ESC cell line KYSE-30.
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http://dx.doi.org/10.1002/mgg3.746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625128PMC
July 2019

TWIST1, MMP-21, and HLAG-1 co-overexpression is associated with ESCC aggressiveness.

J Cell Biochem 2019 09 23;120(9):14838-14846. Epub 2019 Apr 23.

Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive types of cancer, requiring reliable biomarkers for prognosis and therapeutic responsiveness. TWIST1, as an important factor responsible for metastasis of several cancers, is involved in tumor invasion and metastasis through indirectly regulation of MMP-21 expression. On the other hand, NF-ĸβ which is a regulator of HLAG-1 has direct interaction with TWIST1 protein. In this retrospective study we investigated the clinical significance of TWIST1, MMP-21, and HLAG-1 expression in ESCC, and the possible correlation between these genes and progression of the disease. The gene expression analyses of TWIST1, MMP-21, and HLA-G1 were performed by relative comparative real-time polymerase chain reaction in 58 ESCCs compared with corresponding margin-normal esophageal tissues. Significant overexpression of HLAG-1, TWIST1, and MMP-21 messenger RNA was observed in 22.4%, 41.4%, and 60.3% of tumor samples, respectively. Concomitant overexpression of TWIST1/MMP-21 and TWIST1/HLAG-1 were significantly correlated to each other in various clinicopathological features, including depth of tumor invasion, stage of tumor progression, lymphatic invasion, and grade of tumor cell differentiation ( P < 0.05). The current study is the first report of coexpression of TWIST1, MMP-21, and HLAG-1 in ESCC. Such findings suggest an oncogenic role for concomitant expression of these genes in ESCC invasion and metastasis.
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http://dx.doi.org/10.1002/jcb.28745DOI Listing
September 2019

Role of MAML1 in targeted therapy against the esophageal cancer stem cells.

J Transl Med 2019 04 16;17(1):126. Epub 2019 Apr 16.

Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Esophageal cancer is the sixth-leading cause of cancer-related deaths worldwide. Cancer stem cells (CSCs) are the main reason for tumor relapse in esophageal squamous cell carcinoma (ESCC). The NOTCH pathway is important in preservation of CSCs, therefore it is possible to target such cells by targeting MAML1 as the main component of the NOTCH transcription machinery.

Methods: In present study we isolated the CD44+ ESCC CSCs and designed a MAML1-targeted therapy to inhibit the NOTCH signaling pathway. CSCs were isolated using magnetic cell sorting utilizing the CD44 cell surface marker. Several stem cell markers were analyzed in the levels of protein and mRNA expression. The isolated CSCs were characterized in vivo in NUDE mice. Biological role of MAML1 was assessed in isolated CD44+ CSCs. A drug resistance assay was also performed to assess the role of MAML1 in CD44+ CSCs with 5FU resistance.

Results: The CD44+ CSCs had ability to form tumors in NUDE mice. MAML1 silencing caused a significant decrease (p = 0.019) and ectopic expression caused a significant increase in migration of CD44+ CSCs (p = 0.012). Moreover, MAML1 silencing and ectopic expression significantly increased and decreased 5FU resistance, respectively (p < 0.05). MAML1 silencing significantly increased the number of cells in G1 phase (p = 0.008), and its ectopic expression significantly increased the number of CD44+ CSCS in S phase (p = 0.037).

Conclusions: MAML1 may be utilized for targeted therapy with a low side effect to eliminate the CD44+ CSCs through inhibition of canonical NOTCH pathway in ESCC patients.
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http://dx.doi.org/10.1186/s12967-019-1876-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469193PMC
April 2019

Whole Exome Sequencing Reveals a Novel Damaging Mutation in Human Fibroblast Activation Protein in a Family with Esophageal Squamous Cell Carcinoma.

J Gastrointest Cancer 2020 Mar;51(1):179-188

Immunology research center, Mashhad university of Medical Sciences, Mashhad, Iran.

Purpose: Esophageal squamous cancer cell (ESCC), with late diagnosis and poor rate of survival, is a significant cause of mortality in the developing countries. The hypothesis of rare high penetrance with mutations in new genes may explain the underlying predisposition in some of these familial cases.

Methods: Exome sequencing was performed in the patients with ESCC with strong disease aggregation, two sisters with ESCC cancer, and one with breast cancer. Data analysis selected only very rare variants (0-0.1%) located in genes with a role compatible with cancer. In addition, the homology modeling of the novel mutation (A459D) discovered in FAP gene was performed by using the online Swiss-Prot server for automated modeling and the resulted structure has been modified and analyzed by using bioinformatics software to thoroughly study the structural deficiencies caused by the novel mutation.

Results: Ten final candidate variants were selected and six genes validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in FAP, BOD1L, RAD51, Gasdermin D, LGR5, and CERS4. A novel, human mutation C1367A encoding Ala459 Asp (accession number: KT988039), occurring in the blade of the β propeller domain, was identified in two sisters with ESCC.

Conclusions: We identified novel mutations in three drug delivery genes, a tumor suppressor and also a stem cell marker of esophageal that may have a role in cancer treatment and are involved in cellular pathways, which supports their putative involvement in germ-line predisposition to this neoplasm.
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http://dx.doi.org/10.1007/s12029-019-00224-xDOI Listing
March 2020

Role of DIDO1 in Progression of Esophageal Squamous Cell Carcinoma.

J Gastrointest Cancer 2020 Mar;51(1):83-87

Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Purpose: Apoptosis is one of the main involved processes during development and organogenesis and its aberration may result in tumorigenesis. In the present study, due to the role of death inducer-obliterator 1 (DIDO1) in activation of caspases 9 and 3 during the apoptosis process, the role of DIDO1 as the shortest splicing variant of the DIDO gene was assessed for the first time in esophageal squamous cell carcinoma (ESCC) patients.

Methods: DIDO1 mRNA expression in tumor tissues from 50 ESCC patients was compared to their corresponding margin normal tissues using the real-time polymerase chain reaction (RT-PCR).

Results: Nine out of 50 (18%) and 13 out of 50 (26%) cases had DIDO1 under- and overexpression, respectively. There was a significant correlation between DIDO1 mRNA expression and tumor depth of invasion (p = 0.050). Also, there was a significant correlation between age of patients and levels of DIDO1 mRNA expression (p = 0.039).

Conclusions: This study is the first report that assessed the DIDO1 expression in ESCC patients and revealed its probable role in the early steps of tumor progression and metastasis. Therefore, DIDO1 can be suggested as a marker for the primary ESCCs.
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http://dx.doi.org/10.1007/s12029-019-00212-1DOI Listing
March 2020

MAML1 regulates EMT markers expression through NOTCH-independent pathway in breast cancer cell line MCF7.

Biochem Biophys Res Commun 2019 03 4;510(3):376-382. Epub 2019 Feb 4.

Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Tumor relapse is the main cause of breast cancer related deaths and metastasis due to epithelial-mesenchymal transition (EMT) having a critical role in this process. MAML1 is the main co activator of NOTCH signaling pathway and its role in EMT remains unknown. In this study, this role was evaluated through overexpression and knockdown study of MAML1 in MCF7 and MDA-MB-231 cells. MAML1 overexpression up regulated the epithelial and down regulated the mesenchymal markers. In addition, MAML1 silencing decreased epithelial and increased mesenchymal markers. Notch inhibition using γ-secretase inhibitor resulted in increased E-cadherin expression. MAML1 ectopic expression, further increased E-cadherin expression with inhibition of NOTCH signaling. Wound healing assay showed that MAML1 overexpression decreases the rate of migration, while MAML1 silencing increases this rate significantly. In conclusion, our data indicated that MAML1 negatively regulates EMT markers expression in breast cancer cells.
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http://dx.doi.org/10.1016/j.bbrc.2019.01.101DOI Listing
March 2019

Correlation between SALL4 stemness marker and bone morphogenetic protein signaling genes in esophageal squamous cell carcinoma.

J Biochem Mol Toxicol 2019 Mar 15;33(3):e22262. Epub 2018 Nov 15.

Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.

SALL4, as a stemness marker, plays a key role in the maintenance of pluripotency and self-renewal of cancer stem cells. To elucidate probable linkage between SALL4 stemness marker and bone morphogenetic protein (BMP) cell signaling pathway, we aimed to analyze the expression levels of the related genes in esophageal squamous cell carcinoma (ESCC) patients. Tumoral and corresponding margin normal tissues from 50 treatment-naive ESCC patients were subjected for expression analysis using relative comparative real-time reverse transcription polymerase chain reaction. There were significant correlations between SALL4 mRNA and BMP signaling target genes expression including SIZN1, VENTX, and DIDO1 (P < 0.01). Tight associations of gene expression were observed in primary stages of tumor progression (stages I/II), and the invaded tumors to the adventitia (T3/T4). Furthermore, significant correlations between the expression of BMP signaling target genes were observed (P < 0.01). SALL4 may play role in tumorigenesis and tumor cell invasiveness of ESCC through correlation with BMP signaling genes.
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http://dx.doi.org/10.1002/jbt.22262DOI Listing
March 2019

WNT and NOTCH signaling pathways as activators for epidermal growth factor receptor in esophageal squamous cell carcinoma.

Cell Mol Biol Lett 2018 3;23:42. Epub 2018 Sep 3.

4Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Esophageal squamous cell carcinoma (ESCC) is the most common histological type of esophageal cancer, with a poor prognosis. Deregulation of WNT and NOTCH signaling pathways is important in ESCC progression, which can be due to either malfunction of their components or crosstalk with other pathways. Therefore, identification of new crosstalk between such pathways may be effective to introduce new strategies for targeted therapy of cancer. A correlation study was performed to assess the probable interaction between growth factor receptors and WNT/NOTCH pathways via the epidermal growth factor receptor (EGFR) and Musashi1 (MSI1), respectively.

Methods: Levels of MSI1/EGFR mRNA expression in tumor tissues from 48 ESCC patients were compared to their corresponding normal tissues using real-time polymerase chain reaction.

Results: There was a significant correlation between EGFR and MSI1 expression ( = 0.05). Moreover, there was a significant correlation between EGFR/MSI1 expression and grade of tumor differentiation ( = 0.02).

Conclusion: This study confirms a direct correlation between MSI1 and EGFR and may support the important role of MSI1 in activation of EGFR through NOTCH/WNT pathways in ESCC.
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http://dx.doi.org/10.1186/s11658-018-0109-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122622PMC
October 2018

Investigation of melanoma-associated antigen A4 cancer/testis antigen clinical relevance in esophageal squamous cell carcinoma.

J Cancer Res Ther 2018 Jul-Sep;14(5):1059-1064

Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Esophageal squamous cell carcinoma (ESCC) is considered as the seventh most common cancer worldwide, and the second prevalent malignancy in the north of Iran. A subfamily group of tumor-specific antigens, commonly specified as cancer/testis antigens (CTAs), are expressed restrictedly in testis, ovary, and placenta. Melanoma-associated antigen A4 (MAGEA4) as a CTA is overexpressed in a variety of cancers. Expressional analysis of MAGEA4 protein in ESCC may be useful to investigate its clinical relevance leading to effective improvements in ESCC diagnosis and treatment.

Materials And Methods: Fifty-six ESCC patients with no preoperative therapeutic circumstance such as radiotherapy or chemotherapy were analyzed to explore the protein expression level of MAGEA4 using immunohistochemistry assay.

Results: MAGEA4 overexpression was detected in 66% of ESCC samples showing strong nuclear and cytoplasmic staining compared to the normal epithelium. There were significant correlations between MAGEA4 protein expression and depth of tumor invasion (P = 0.019), and the number of involved lymph nodes (P = 0.045).

Conclusion: Because of the significant correlation of MAGEA4 and indices of poor prognosis, the role of this CTA may be confirmed in ESCC aggressiveness and metastasis. Therefore, MAGEA4 may be a promising therapeutic candidate for suppressing ESCC aggressiveness.
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http://dx.doi.org/10.4103/0973-1482.183180DOI Listing
October 2018

Telomere shortening associated with increased levels of oxidative stress in sulfur mustard-exposed Iranian veterans.

Mutat Res Genet Toxicol Environ Mutagen 2018 Oct 20;834:1-5. Epub 2018 Jun 20.

Medical Toxicology Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Sulfur Mustard (SM) is the most widely used chemical weapon. It was used in World War 1 and in the more recent Iran-Iraq conflict. Genetic toxicity and DNA alkylation effects of SM in molecular and animal experiments are well documented. In this study, lymphocytic telomere lengths and serum levels of isoprostane F2α were measured using q-PCR and enzyme immunoassay-based methods in 40 Iranian veterans who had been exposed to SM between 1983-88 and 40 non-exposed healthy volunteers. The relative telomere length in SM-exposed individuals was found to be significantly shorter than the non-exposed individuals. In addition, the level of 8-isoprostane F2α was significantly higher in the SM-exposed group compared to controls. Oxidative stress can be caused by defective antioxidant responses following gene mutations or altered activities of antioxidant enzymes. Chronic respiratory diseases and infections may also increaseoxidative stress. The novel finding of this study was a the identification of 'premature ageing phenotype'. More specifically, telomere shortening which occurs naturally with aging is accelerated in SM-exposed individuals. Oxidative stress, mutations in DNA repair genes and epimutaions may be among the major mechanisms of telomere attrition. These findings may help for a novel therapeutic strategy by telomere elongation or for validation of an exposure biomarker for SM toxicity.
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http://dx.doi.org/10.1016/j.mrgentox.2018.06.017DOI Listing
October 2018

Ectopic Expression of Human Gene in ESCC Cell Line Using Retroviral System.

Avicenna J Med Biotechnol 2018 Apr-Jun;10(2):75-82

Human Genetic Division, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Cancer/Testis Antigens (CTAs) are a sub-group of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of in developmental events and cancer, preparing a suitable platform to analyze roles in the cells seems to be necessary.

Methods: In this study, the coding sequence of gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of gene by real-time PCR.

Results: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression.

Conclusion: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells leads to an understanding of the biological importance of in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of existence in any biological fluid through ELISA system.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960063PMC
June 2018

Association of Two CD44 Polymorphisms with Clinical Outcomes of Gastric Cancer Patients

Asian Pac J Cancer Prev 2018 May 26;19(5):1313-1318. Epub 2018 May 26.

Department of Cellular and Molecular Biology, University of Science and Culture, Tehran, Iran.

Objective: CD44 is an important cell adhesion molecule that plays a key role in growth, invasion, proliferation and metastasis of cancer cells. CD44 protein over-expression is associated with a poor prognosis of gastric cancer (GC) and previous studies have shown that CD44 gene polymorphisms could affect survival and recurrence. In this study, we tested the hypothesis that polymorphisms impacting on the CD44 signaling pathway may predict clinical outcomes in patients with GC. Materials and Methods: DNA was extracted from blood of 150 healthy individuals and formalin-fixed paraffin-embedded (FFPE) tumor tissue of 150 patients. The two polymorphisms rs187116 and rs7116432 were studied by RFLP-PCR and sequencing techniques. Results: There was a strong significant correlation between single nucleotide polymorphisms (SNPs) in the CD44 gene, tumor recurrence, and overall survival (p <0.0001). The existence of a significant relationship between tumor recurrence and overall survival was proved in this study, with at least one allele G for the polymorphism rs187116 and at least one allele A for polymorphism rs7116432. Conclusion: These results provide evidence of a relationship between CD44 gene polymorphisms and clinical outcomes in our GC patients. This result could help identify individuals with GC who have a high risk of tumor recurrence.
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http://dx.doi.org/10.22034/APJCP.2018.19.5.1313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031830PMC
May 2018

Molecular Signaling in Tumorigenesis of Gastric Cancer

Iran Biomed J 2018 07 30;22(4):217-30. Epub 2018 Apr 30.

Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Gastric cancer (GC) is regarded as the fifth most common cancer and the third cause of cancer-related deaths worldwide. Mechanism of GC pathogenesis is still unclear and relies on multiple factors, including environmental and genetic characteristics. One of the most important environmental factors of GC occurrence is infection with Helicobacter pylori that is classified as class one carcinogens. Dysregulation of several genes and pathways play an essential role during gastric carcinogenesis. Dysregulation of developmental pathways such as Wnt/β-catenin signaling, Hedgehog signaling, Hippo pathway, Notch signaling, nuclear factor-kB, and epidermal growth factor receptor have been found in GC. Epithelial-mesenchymal transition, as an important process during embryogenesis and tumorigenesis, is supposed to play a role in initiation, invasion, metastasis, and progression of GC. Although surgery is the main therapeutic modality of the disease, the understanding of biological processes of cell signaling pathways may help to develop new therapeutic targets for GC.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949124PMC
http://dx.doi.org/10.22034/ibj.22.4.217DOI Listing
July 2018

MAML1 and TWIST1 co-overexpression promote invasion of head and neck squamous cell carcinoma.

Asia Pac J Clin Oncol 2018 Oct 15;14(5):e434-e441. Epub 2018 Jan 15.

Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.

Aims: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with considerable morbidity and mortality. Invasion and metastasis of HNSCC is a complex process involving multiple molecules and signaling pathways. Twist Family BHLH Transcription Factor 1 (TWIST1) and Mastermind-like 1 (MAML1) are essential in induction of epithelial-mesenchymal transition through direct regulation of implicated molecules in cellular adhesion, migration and invasion. Our aim in this study was to assess the clinical significance of MAML1 and TWIST1 expression in HNSCC, and elucidate the probable correlation between these genes to exhibit their possible associations with progression and metastasis of the disease.

Methods: The gene expression profile of MAML1 and TWIST1 was assessed in fresh tumoral compared to distant tumor-free tissues of 55 HNSCC patients using quantitative real-time Polymerase chain reaction (PCR).

Results: Significant overexpression of MAML1 and TWIST1 mRNA was observed in 49.1% and 38.2% (P ˂ 0.05) of tumor specimens, respectively. Overexpression of MAML1 was associated with vascular invasion (P = 0.048). Concomitant overexpression of MAML1 and TWIST1 was significantly correlated to each other (P = 0.004). Co-overexpression of the genes was significantly correlated to the various clinicopathological indices of poor prognosis including depth of tumor invasion (P < 0.01), lymphatic invasion and grade of tumor cell differentiation (P < 0.05).

Conclusions: Significant correlation between MAML1 and TWIST1 in HNSCC was revealed. This study was the first report elucidating MAML1 clinical relevance in HNSCC. These new findings suggest an oncogenic role for concomitant expression of MAML1 and TWIST1 genes in HNSCC invasion and metastasis.
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http://dx.doi.org/10.1111/ajco.12843DOI Listing
October 2018

Ectopic expression of TWIST1 upregulates the stemness marker OCT4 in the esophageal squamous cell carcinoma cell line KYSE30.

Cell Mol Biol Lett 2017 29;22:33. Epub 2017 Dec 29.

Department of Biology, Damghan Branch, Islamic Azad University, P.O.Box: 3671639998, Cheshmeh-Ali Boulevard, Sa'dei square, Damghan, Islamic Republic of Iran.

Background: The transcription factor TWIST1 plays an important role in the epithelial-mesenchymal transition (EMT) process and in the migration, invasion and metastasis of cancer cells. OCT4, which is a homeobox transcription factor, has an important role in the self-renewal potential of cancer cells. Our aim here is to elucidate impact of ectopic expression of TWIST1 on OCT4 gene expression in esophageal squamous cell carcinoma (ESCC).

Methods: The ESCC line was KYSE30. GP293T cells were transfected with purf-IRES-GFP and pGP plasmids to produce recombinant viral particles. A semi-confluent KYSE30 culture was transduced with the prepared retroviral particles. mRNA extraction and cDNA synthesis were performed from normal KYSE30 cells and those ectopically expressing . Expressional analysis of and were performed with relative comparative real-time PCR.

Results: Ectopic expression of in KYSE30 cells was related to its significant overexpression: nearly nine-fold higher in GFP-h KYSE-30 cells than in control GFP cells. This induced expression of caused significant upregulation of in GFP-h KYSE-30 cells: nearly eight-fold higher. In silico analysis predicted the correlation of and through .

Conclusions: Overexpressed can be correlated with upregulation of the cancer stem cell marker and the protein may play critical regulatory role in gene expression. Since OCT4 is involved in the self-renewal process, the results may suggest a new linkage between and in the cell biology of ESCC, highlighting the probable role of TWIST1 in inducing self-renewal.
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http://dx.doi.org/10.1186/s11658-017-0065-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747156PMC
July 2018

Predicting the Correlation of EZH2 and Cancer Stem Cell Markers in Esophageal Squamous Cell Carcinoma.

J Gastrointest Cancer 2018 Dec;49(4):437-441

Department of Biology, Damghan Branch, Islamic Azad University, Cheshmeh-Ali Boulevard, Sa'dei Square, P.O. Box: 3671639998, Damghan, Iran.

Background: Enhancer of zeste homolog 2 (EZH2), a stemness factor, plays roles in regulation of cell differentiation and embryonic development as well as cancer progression. Deregulation of EZH2 in cancers is correlated with tumor cell invasiveness, metastasis, and the patients' poor outcome. However, the mechanistic role of EZH2 in cancer is ambiguous. In this study, we aimed to inhibit the expression of EZH2 in a cancer cell line, and evaluate consequence changes in gene expression pattern.

Materials And Methods: Using specific retroviral shRNA-EZH2, EZH2 gene was silenced in the KYSE30 cell line. Relative comparative real-time PCR was used to confirm silencing of EZH2 and evaluate expression pattern of selected markers.

Results: Inhibition of EZH2 expression in KYSE30 cells caused significant changes in different genes. Indeed, HIWI and HEY1 genes were over- and underexpressed in KYSE30 cells, respectively, following EZH2 silencing. Other selected cancer stem cell markers were not changed significantly.

Conclusion: To the best our knowledge, there are variety of small molecule inhibitors to target EZH2 in cancer cells as a treatment candidate; therefore, our data in this study helps the researchers to select EZH2 for cancer therapy based on its mechanism and correlation with other markers.
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http://dx.doi.org/10.1007/s12029-017-9985-yDOI Listing
December 2018

Biological and Clinicopathological Significance of Cripto-1 Expression in the Progression of Human ESCC.

Rep Biochem Mol Biol 2017 Apr;5(2):83-90

Immunology Research Center, BuAli Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Human Cripto-1, a member of the EGF-CFC family, is involved in embryonic development, embryonic stem cell maintenance, and tumor progression. It also participates in multiple cell signaling pathways including Wnt, Notch, and TGF-β. Remarkably, it is expressed in cancer stem cell (CSC) compartments, boosting tumor cell migration, invasion, and angiogenesis. Although Cripto-1 is overexpressed in a variety of human malignant tumors, its expression in esophageal squamous cell carcinoma (ESCC) remains unclear. Our aim in this study was to evaluate the possible oncogenic role of Cripto-1 in ESCC progression and elucidate its association with clinicopathological parameters in patients.

Methods: In this study, Cripto-1 expression in 50 ESCC tissue samples was analyzed and compared to corresponding margin-normal esophageal tissues using quantitative real-time PCR.

Results: Cripto-1 was overexpressed in nearly 40% of ESCC samples compared with normal tissue samples. Significant correlations were observed between Cripto-1 expression and tumor differentiation grade, progression stage, and location (p < 0.05).

Conclusions: Our results indicate that overexpression of Cripto-1 is involved in the development of ESCC. Further assessment will be necessary to determine the role of Cripto-1 cross talk in ESCC tumorigenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346274PMC
April 2017
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