ESCC Metastasis Research Articles

Chimeric RNA ASTN2-PAPPAas aggravates tumor progression and metastasis in human esophageal cancer.

Transcription-induced chimeric RNAs are an emerging area of research into molecular signatures for disease biomarker and therapeutic target development. Despite their importance, little is known for chimeric RNAs-relevant roles and the underlying mechanisms for cancer pathogenesis and progression. Here we describe a unique ASTN2-PAPPAantisense chimeric RNA (A-PaschiRNA) that could be the first reported chimeric RNA derived from the splicing of exons and intron antisense of two neighboring genes, respectively. Aberrant A-PaschiRNA level in ESCC tissues was associated with tumor progression and patients' outcome. In vitro and in vivo studies demonstrated that A-PaschiRNA aggravated ESCC metastasis and enhanced stemness through modulating OCT4. Mechanistic studies demonstrated that ERK5-mediated non-canonical PAF1 activity was required for A-PaschiRNA-induced cancer malignancy. The study defined an undocumented function of chimeric RNAs in aggravating cancer stemness and metastasis.

Integrating transcriptome and metabolome variability to reveal pathogenesis of esophageal squamous cell carcinoma

BackgroundEsophageal Squamous Cell Carcinoma (ESCC) is an aggressive malignancy, leading to more than 250,000 deaths in China every year. However, the pathogenesis of ESCC remains unclear, which hinders the diagnosis and treatment of the disease in clinic. MethodTo elucidate underlying mechanism and identify potential biomarkers, an integrative strategy of combining transcriptome and metabolome has been implemented to find potential causal genes and metabolites for ESCC. ResultsAt the transcriptional level, dysregulated genes in ESCC patients were identified and pathway enrichment analysis discovered tyrosine metabolic pathway as a promising target. Subsequently, up- and down-stream metabolites of tyrosine pathway were explored through targeted metabolome approach. Five metabolites, i.e. phenylalanine, 4-hydroxyphenyllactic acid, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid and tyrosine were identified as diagnosis biomarkers for ESCC and metastatic ESCC patients. A biological model incorporating both transcriptional and metabolic dysregulation was also established to illustrate the potential mechanism of tumorigenesis and metastasis for ESCC. ConclusionIntegrative transcriptomics and metabolomics analysis suggested that tyrosine pathway was essential for the tumorigenesis and metastasis of ESCC primarily through altering immune response and regulating tumor microenvironment. This research sheds light on the pathogenesis of ESCC and discovers potential biomarkers for the diagnosis of the disease.

3,3\u2032-Diindolylmethane modulates aryl hydrocarbon receptor of esophageal squamous cell carcinoma to reverse epithelial-mesenchymal transition through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most aggressive tumors in the world. Aryl hydrocarbon receptor (AHR) has been reported to promote tumor metastasis and epithelial-mesenchymal transition (EMT) is a vital process of conferring cancer cells capabilities of migration and invasion. However, the mechanism by which modulation of AHR can inhibit tumor metastasis remains unknown. Thus, we aim to investigate the underlying mechanism regarding reversing EMT process of ESCC through modulation of AHR.MethodsWe used AHR selective modulator 3,3′-diindolylmethane (DIM) to treat ESCC cell lines TE1 and KYSE150 so as to examine alterations of migration and invasion by wound healing and Transwell assay. Western blotting (WB) and qPCR were performed to detect relative genes and proteins changes regarding EMT process. Cell transfection was utilized for confirming pathways involved in DIM-induced reversal of EMT and in vivo assay was conducted for verification of the underlying mechanism. Co-IP assay was conducted for detecting protein-protein interactions.ResultsAHR was overexpressed in ESCC and modulation of AHR by DIM could inhibit migration and invasion as well as downregulate mesenchymal cell markers β-Catenin, Vimentin and Slug and upregulate epithelial cell marker Claudin-1. Meanwhile, synergically overexpression of AHR, RhoA and ROCK1 correlated with poor clinical outcomes. DIM could inhibit COX2/PGE2 pathway by targeting AHR, and COX2 selective inhibitor Celecoxib could suppress EMT and metastasis. Results of PGE2 treatment were opposite to that of Celecoxib. Meanwhile, blockade of RhoA/ROCK1 pathway also exerted prohibitive effects on EMT and metastasis. WB results showed COX2/PGE2 pathway could be regulated by RhoA/ROCK1 pathway and DIM could inhibit RhoA/ROCK1 pathway through modulation of AHR. In vivo assay verified the results in vitro. Co-IP results showed DIM could modulate AHR to reverse EMT directly through inhibition of interaction between AHR and EGFR (epidermal growth factor receptor) so as to block RhoA/ROCK1-mediated COX2/PGE2 pathway which was connected by NF-κB.ConclusionsIn brief, modulation of AHR by DIM can reverse EMT process and inhibit metastasis of ESCC through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway.

Ivermectin suppresses tumour growth and metastasis through degradation of PAK1 in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC), the most common form of oesophageal malignancies in the Asia‐Pacific region, remains a major clinical challenge. In this study, we found that ivermectin, an effective antiparasitic drug that has been approved for patients to orally treat onchocerciasis for over 30 years, displayed potent antitumour activity against ESCC cells in vitro and in nude mice. We demonstrated that ivermectin significantly inhibited cell viability and colony formation, and induced apoptosis through a mitochondrial‐dependent manner in ESCC cells. Ivermectin also abrogated ESCC cell migration, invasion, as well as the protein levels of MMP‐2 and MMP‐9. Mechanistically, ivermectin strongly inhibited the expression of PAK1; by further gain‐ and loss‐of‐function experiments, we confirmed that PAK1 played a crucial role in ivermectin‐mediated inhibitory effects on ESCC cells. In addition, the data indicated that ivermectin promoted PAK1 degradation through the proteasome‐dependent pathway. Additionally, ivermectin synergized with chemotherapeutic drugs including cisplatin and 5‐fluorouracil to induce apoptosis of ESCC cells. Interestingly, the in vivo experiments also confirmed that ivermectin effectively suppressed tumour growth and lung metastasis of ESCC. Collectively, these results indicate that ivermectin exerts a potent antitumour activity against ESCC and is a promising therapeutic candidate drug for ESCC patients, even those carrying metastasis.

Decreased plasma riboflavin is associated with poor prognosis, invasion, and metastasis in esophageal squamous cell carcinoma.

Riboflavin deficiency confers a predisposition for esophageal cancer. The role of plasma riboflavin levels in development and prognosis of individuals with digestive tract inflammation and ulcer (DTIU), digestive tract polyps (DTPs), and ESCC is not well understood. We performed a cross-sectional study, including 177 DTIU, 80 DTP, and 324 ESCC cases, to measure the plasma riboflavin levels among the three populations. Correlation between plasma riboflavin levels (categorized as ≥31.8, 6.5-31.8 and ≤6.5 nmol/L groups) and clinical characteristics, as well as survival of ESCC patients (556 cases) was analyzed. There was no difference in plasma riboflavin levels between DTIU, DTP, and ESCC cases (P > 0.05). Plasma riboflavin levels were inversely correlated with invasive depth (correlation coefficient = -0.09, P = 0.026) and lymph node metastasis (correlation coefficient = -0.11, P = 0.010) of ESCC, and ESCC patients with low riboflavin levels had poor recurrence-free survival (P = 0.035) and overall survival (P = 0.003). Decreased riboflavin was a prognostic factor for poor overall survival (HR = 1.91, 95% CI = 1.19-3.07, P = 0.007). Plasma riboflavin levels in DTIU, DTP, and ESCC patients are similar. Plasma riboflavin levels are associated with the development and prognosis of ESCC.

Fbxw8 protein promotes the proliferation and metastasis of ESCC

Objective: This article plans to investigate Fbxw8 protein expression in esophageal squamous cell carcinoma tissues. And its effect on the proliferation ability of esophageal squamous cancer KYSE70 cells is analyzed. Methods: We can detect Fbxw8 protein expression in esophageal squamous cell carcinoma tissues with IHC and analysis the expression with the sex, age, lymph node metastasis and tumor stage. We alsodetect the proliferation of KYSE70 cells with CCK 8. Result: Most esophageal squamous carcinoma tissues highly expressed Fbxw8 protein, and the expression was positively correlated with lymph node metastasis and tumor staging (P < 0.05). Fbxw8 siRNAs can significantly cut FBXW8 protein expression, while lowering the cell cycle related protein expression of Cyclin A, Cyclin B1, and inhibiting KYSE70 cell proliferation. Conclusion: Fbxw8 protein highly expressed in esophageal squamous carcinoma tissues, and closely related to tumor staging. Targeted inhibition Fbxw8 protein expression could significantly inhibit the proliferation of esophageal squamous carcinoma cells.

TOPK promotes metastasis of esophageal squamous cell carcinoma by activating the Src/GSK3\u03b2/STAT3 signaling pathway via \u03b3-catenin

BackgroundEsophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis. The predominant reason for ESCC-related death is distal metastasis. A comprehensive understanding of the molecular mechanism underlying metastasis is needed for improving patient prognosis. T-LAK cell-originated protein kinase (TOPK) is a MAPKK-like kinase, which plays a vital role in various physiological and pathophysiological processes. However, the role of TOPK in ESCC metastasis is unclear.MethodsTissue array was used to evaluate the correlation between TOPK expression and ESCC lymph node metastasis. Wound healing assay, transwell assay, and lung metastasis mice model were used to examine the role of TOPK in the migration of ESCC cells in vitro and in vivo. Protein kinase array, mass spectrometry (MS), and molecular modeling were used to examine the pathways and direct target proteins of TOPK that are involved in ESCC metastasis. Additionally, immunofluorescence and western blotting analyses were performed to verify these findings.ResultsThe enhanced expression of TOPK was correlated with lymph node metastasis in the ESCC tissues. TOPK knockdown or treatment with the TOPK inhibitor (HI-TOPK-032) decreased the invasion and migration of ESCC cells in vitro. HI-TOPK-032 also inhibited the lung metastasis in ESCC cell xenograft in vivo model. Moreover, TOPK promoted the invasion of ESCC cells by activating the Src/GSK3β/STAT3 and ERK signaling pathways via γ-catenin.ConclusionThe findings of this study reveal that TOPK is involved in ESCC metastasis and promoted the ESCC cell mobility by activating the Src/GSK3β/STAT3 and ERK signaling pathways. This indicated that TOPK may be a potential molecular therapeutic target for ESCC metastasis.

P11 KRT17 PROMOTES PROLIFERATION AND METASTASIS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA LEADING TO POOR PROGNOSIS

Abstract Background Keratin17(KRT17), as a multifunction cytoskeletal protein, associated with a multitudinous of biological processes, including cell proliferation, migration, and invasion. Previously, we have found overexpression of KRT17 genes in ESCC tissues. Currently, published kinds of researches claimed KRT17 is engaged in the tumorigenesis and progression of multiple cancers. However, the prognostic significance of KRT17 in ESCC patients and its effects in ESCC progression remain indistinct. Methods We verified the expression of KRT17 in ESCC tissues by Western blotting and q-PCR. And then the immunochemistry was performed to investigate the KRT17 expression in 64 ESCC tissue specimens. KRT17 upregulated, and knockdown EC9706 and Eca109 cells were constructed by Lentivirus overexpression and CRISPR-Cas9. The function of KRT17 in ESCC proliferation and metastasis were studied by CCK-8 assays, plate colony formation assays, trans-well and wound healing in vitro. Results overexpression of KRT17 was verified in 12 pairs of fresh ESCC tissues. We found that the expression of KRT17 was remarkably positively correlated with the depth of invasion (T) (P<0.05), lymph node metastasis (N) (P<0.05), tumor node metastasis (TNM) stage (P<0.0001). High expression of KRT17 predicted a lower 5-year survival rate(P<0.001) of ESCC patients. We verified the KRT17 malignant behavior of ESCC in vitro. We conformed proliferation and migration capacity of ESCC were significantly enhanced by overexpression of KRT17 but compromised when KRT17 was knocked down. Collectively, it supported that the increased expression of KRT17 promoted proliferation and migratory phenotype of ESCC. Conclusion The breakthrough indicated that the upregulation of KRT17 in ESCC is closely related to malignant progression, our findings revealed that KRT17 plays a vital role in facilitating proliferation and metastasis, and could be used as a novel predictive biomarker, thus providing a novel diagnosis and therapeutic target for ESCC patients. Keywords: Molecular target ESCC Keratin17 Therapeutic target

P8 KERATIN 17 PROMOTES CELLS PROLIFERATION AND METASTASIS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Keratin17(KRT17), as a multifunction cytoskeletal protein, associated with a multitudinous of biological processes, including cell proliferation, migration, and invasion. Previously, we have found up-expression of KRT17 genes in ESCC tissues. Currently, published kinds of researches claimed KRT17 is engaged in the tumorigenesis and progression of multiple cancers. However, the prognostic significance of KRT17 in ESCC patients and its effects in ESCC progression remains indistinct. Methods We verified the expression level of KRT17 in ESCC tissues by Western blotting and q-PCR and constructed KRT17 upregulated and knockdown EC9706 and Eca109 cells by Lentivirus overexpression and CAS9. The function of KRT17 in ESCC proliferation and metastasis were studied in vitro. We performed CCK-8 assays and plate colony formation assays and the plate colony formation assays to explore the effect of KRT17 on the proliferation, Moreover, the trans-well migration chamber experiment and wound healing test was taken to reveal the impact of KRT17 on migration capabilities. Results We verified that KRT17 is overexpressed in ESCC tissues and the role of KRT17 in the malignant behavior of ESCC in vitro and vivo . In CCK-8 assays and plate colony formation assays. We conformed proliferation capacity was notably enhanced by overexpression of KRT17 but compromised when KRT17 was knocked out. On the other hand, we used the plate colony formation assays to explore the effect of KRT17 on the proliferation of ESCC cells. KRT17 up-regulated significantly strengthened the proliferation while the KRT17 knockdown was weakened. To reveal the impact of KRT17 on migration, the trans-well migration chamber experiment was used to compare the role of down-regulation and up-regulation of KRT17 in migration; the results demonstrated that reducing expression of KRT17 significantly blocked the movement of ESCC cells as compared with the empty vector control. Otherwise, it has been confirmed that the cell migration ability employs a wound healing/scratch test because wound closure is generally a measure of cell motility. The results showed KRT17 overexpression notably reinforced the mobility of ESCC cells compared with the empty groups, whereas KRT17 knockdown cells prolonged the time to close the injury compared to blank groups. Collectively, these discoveries support that the increased expression of KRT17 in ESCC cells promoted a more proliferation and migratory phenotype in vitro. Conclusion The breakthrough indicated that the up-regulation of KRT17 in ESCC is closely related to malignant progression. Our data confirmed that KRT17 plays an essential role in reinforcing proliferation and metastasis in ESCC. Thence, KRT17 may serve as a significant molecular target for the diagnosis and therapy of ESCC.

Cancer‐associated fibroblasts (CAFs) promote the lymph node metastasis of esophageal squamous cell carcinoma

Lymph node metastasis is a pathognomonic feature of spreading tumors, and overcoming metastasis is a challenge in attaining more favorable clinical outcomes. Esophageal cancer is an aggressive tumor for which lymph node metastasis is a strong poor prognostic factor, and the tumor microenvironment (TME), and cancer-associated fibroblasts (CAFs) in particular, has been implicated in esophageal cancer progression. CAFs play a central role in the TME and have been reported to provide suitable conditions for the progression of esophageal cancer, similar to their role in other malignancies. However, little is known concerning the relevance of CAFs to the lymph node metastasis of esophageal cancer. Here, we used clinical samples of esophageal cancer to reveal that CAFs promote lymph node metastasis and subsequently verified the intercellular relationships in vitro and in vivo using an orthotopic metastatic mouse model. In the analysis of clinical samples, FAP+ CAFs were strongly associated with lymph node metastasis rather than with other prognostic factors. Furthermore, CAFs affected the ability of esophageal cancer cells to acquire metastatic phenotypes in vitro; this finding was confirmed by data from an in vivo orthotopic metastatic mouse model showing that the number of lymph node metastases increased upon injection of cocultured cancer cells and CAFs. In summary, we verified in vitro and in vivo that the accumulation of CAFs enhances the lymph node metastasis of ESCC. Our data suggest that CAF targeted therapy can reduce lymph node metastasis and improve the prognosis of patients with esophageal cancer in the future.

Infiltrated M2 tumour-associated macrophages in the stroma promote metastasis and poor survival in oesophageal squamous cell carcinoma.

Although M2 tumour-associated macrophages (M2 TAMs) have been shown to be associated with the progression and metastasis of breast cancer, their role in oesophageal squamous cell carcinoma (ESCC) remains less well understood. Therefore, to understand the clinicopathological significance of infiltrated M2 TAMs in ESCC, statistical analysis was performed after immunohistochemical evaluation of CD163 expression, a well-accepted surface marker of M2 TAMs in ESCC. To gain insight into the effect of M2 TAMs, ESCC cell lines Eca109 and KYSE150 cells were co-cultured with M2 TAMs artificially induced from THP-1 cells. The variations in the proliferation, migration and invasion were assessed using the MTT, wound-healing and Transwell assays, respectively. The variation in the typical biomarkers of the epithelial-mesenchymal transition (EMT) was evaluated using western blotting. Infiltrated M2 TAMs were confirmed to predominate in the stroma of ESCC relative to normal controls. Moreover, it turned out that M2 TAMs were shown to promote the migration and invasion of ESCC cells but not proliferation. Furthermore, M2 TAMs were observed to induce EMT in ESCC cells. Together, our results showed that infiltrated M2 TAMs in the stroma is a feature accompanying ESCC metastasis and that M2 TAMs can promote the migration and invasion, but not proliferation, of ESCC cells, thereby inducing EMT. Thus, M2 TAMs could be an alternative therapeutic target in ESCC.

IL-1β from M2 macrophages promotes migration and invasion of ESCC cells enhancing epithelial-mesenchymal transition and activating NF-κB signaling pathway.

Despite the phenotype has been established that M2 macrophages promotes the metastasis of ESCC, question still remains as to how the M2 macrophages facilitated metastasis of ESCC cells. To begin with, immunohistochemistry was performed to detect the expression of CD163, one typical surface marker of M2 macrophages in 90 paired ESCC and its normal controls after meta-analyzing the relevant studies regarding M2 macrophages in ESCC, confirming that infiltration of M2 macrophages was significantly linked with lymph node metastasis, T classification, and inferior overall survival of ESCC. To explore the mechanism behind, protein factors secreted by M2 macrophages were identified using antibody microarray. Six different significantly differential protein factors were screened out, including IL-1β, TIMP1, IL-1α, MDC, TGF-β1, and TGF-β2. Among which, IL-1β was picked up as cytokine as interest based on previous reports and its absolute fold. Functional analysis of IL-1β showed that IL-1β was able to promote migration and invasion of ESCC cells, enhancing epithelial-mesenchymal transition, and activating NF-κB pathway. Our study supports the promoting role of M2 macrophages in metastasis of ESCC cells, enriching the profile of protein factors released from M2 macrophages.

FSTL1 Promotes Metastasis and Chemoresistance in Esophageal Squamous Cell Carcinoma through NFκB-BMP Signaling Cross-talk.

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886-99. ©2017 AACR.

Whole-exome sequencing reveals critical genes underlying metastasis in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers, owing to a high frequency of metastasis. However, little is known about the genomic landscape of metastatic ESCC. To identify the genetic alterations that underlie ESCC metastasis, whole-exome sequencing was performed for 41 primary tumours and 15 lymph nodes (LNs) with metastatic ESCCs. Eleven cases included matched primary tumours, synchronous LN metastases, and non-neoplastic mucosa. Approximately 50-76% of the mutations identified in primary tumours appeared in the synchronous LN metastases. Metastatic ESCCs harbour frequent mutations of TP53, KMT2D, ZNF750, and IRF5. Importantly, ZNF750 was recurrently mutated in metastatic ESCC. Combined analysis from current and previous genomic ESCC studies indicated more frequent ZNF750 mutation in diagnosed cases with LN metastasis than in those without metastasis (14% versus 3.4%, n = 629, P = 1.78 × 10-5 ). The Cancer Genome Atlas data further showed that ZNF750 genetic alterations were associated with early disease relapse. Previous ESCC studies have demonstrated that ZNF750 knockdown strongly promotes proliferation, migration, and invasion. Collectively, these results suggest a role for ZNF750 as a metastasis suppressor. TP53 is highly mutated in ESCC, and missense mutations are associated with poor overall survival, independently of pathological stage, suggesting that these missense mutations have important functional impacts on tumour progression, and are thus likely to be gain-of-function (GOF) mutations. Additionally, mutations of epigenetic regulators, including KMT2D, TET2, and KAT2A, and chromosomal 6p22 and 11q23 deletions of histone variants, which are important for nucleosome assembly, were detected in 80% of LN metastases. Our study highlights the important role of critical genetic events including ZNF750 mutations, TP53 putative GOF mutations and nucleosome disorganization caused by genetic lesions seen with ESCC metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1) facilitates progression of oesophageal squamous cell carcinoma through MAPK/MEK/ERK/FRA-1 activation

BackgroundOesophageal cancer is one of the most common malignancies worldwide,and oesophageal squamous cell carcinoma (ESCC) is the predominant histological type both globally and in China. Collagen triple helix repeat containing 1 (CTHRC1) has been found to be upregulated in ESCC. However, its role in tumourigenesis and progression of ESCC remains unclear.MethodsUsing our previous ESCC mRNA profiling data, we screened upregulated genes to identify those required for proliferation. Immunohistochemistry was performed to determine the level of CTHRC1 protein expression in 204 ESCC patients. Correlations between CTHRC1 expression and clinicopathological characteristics were assessed. In addition, pyrosequencing and 5-aza-dC treatment were performed to evaluate methylation status of CTHRC1 promoter. In vitro and in vivo analyses were also conducted to determine the role of CTHRC1 in ESCC cell proliferation, migration and invasion, and RNA sequencing and molecular experiments were performed to study the underlying mechanisms.ResultsBased on mRNA profiling data, CTHRC1 was identified as one of the most significantly upregulated genes in ESCC tissues (n = 119, fold change = 20.5, P = 2.12E-66). RNA interference screening also showed that CTHRC1 was required for cell proliferation. Immunohistochemistry confirmed markedly high CTHRC1 protein expression in tumour tissues, and high CTHRC1 expression was positively correlated with advanced T stage (P = 0.043), lymph node metastasis (P = 0.023), TNM stage (P = 0.024) and poor overall survival (P = 0.020). Promoter hypomethylation at cg07757887 may contribute to increased CTHRC1 expression in ESCC cells and tumours. Forced overexpression of CTHRC1 significantly enhanced cell proliferation, migration and invasion, whereas depletion of CTHRC1 suppressed these cellular functions in three ESCC cell lines and xenografts. CTHRC1 was found to activate FRA-1 (Fos-related antigen 1, also known as FOSL1) through the MAPK/MEK/ERK cascade, which led to upregulation of cyclin D1 and thus promoted cell proliferation. FRA-1 also induced snail1-mediated MMP14 (matrix metallopeptidase 14, also known as MT1-MMP) expression to facilitate ESCC cell invasion, migration, and metastasis.ConclusionsOur data suggest that CTHRC1 may act as an oncogenic driver in progression and metastasis of ESCC, and may serve as a potential biomarker for prognosis and personalized therapy.

Dual cyclin-dependent kinase 4/6 inhibition by PD-0332991 induces apoptosis and senescence in oesophageal squamous cell carcinoma cells.

Aberrant activation of the cyclin D1-cyclin-dependent kinase 4/6 (CDK4/6)-Rb signalling pathway is common in oesophageal squamous cell carcinoma (ESCC). PD-0332991, a highly specific inhibitor of CDK4/6, has potent antitumour activity against many types of cancer. The purpose of this study was to examine the in vitro and in vivo antineoplastic effect of PD-0332991 against the growth and metastasis of ESCC cells. Cell viability and any synergy between PD-0332991 and 5-fluorouracil or cisplatin were measured by MTS assay and CalcuSyn software respectively. Cell migration and invasion were detected by wound healing and transwell assays. Apoptosis was evaluated by flow cytometry after staining annexin V-FITC/PI. Cellular senescence was assessed by measuring SA-β-gal activity. Nude mouse xenograft models of ESCC were employed to determine the in vivo activity of PD-0332991 against tumour growth and lung metastasis. PD-0332991 inhibited cellular growth and induced mitochondrial-dependent apoptosis in ESCC cells. PD-0332991 also suppressed migration, invasion and the expression of MMP-2 in ESCC cells. Furthermore, PD-0332991 treatment caused cell senescence in a FOXM1-dependent manner. In addition, there was synergy between PD-0332991 and cisplatin or 5-fluorouracil. Importantly, the xenografted tumour experiments demonstrated that PD-0332991 potently inhibits ESCC cell growth and lung metastasis. PD-0332991 can elicit a strong antitumour activity against ESCC growth and metastasis and may be a promising candidate drug for the treatment of patients with ESCC. Our results warrant a clinical trial to further evaluate the efficacy of PD-0332991 in ESCC patients, even those with metastasis.

Cripto-1 acts as a functional marker of cancer stem-like cells and predicts prognosis of the patients in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is highly malignant with highly invasive and metastatic capabilities and poor prognosis. It is believed that the ESCC cancer stem-like cells (ECSLCs) are critical for tumorigenicity, invasion and metastasis of ESCC. However, the properties of ECSLCs vary with different markers used in isolation, so that new and more effective markers of ECSLCs need to be identified. This study aimed to estimate the potentiality of Cripto-1 (CR-1) as an ECSLC surface marker and investigate the clinical significance of CR-1 expression in ESCC.MethodsESCC cells with CR-1 high or CR-1low were obtained by flow cytometry then their self-renewal capability and tumorigenicity were compared by colony and limiting dilution sphere formation analysis in vitro and xenograft in nude mice in vivo, respectively. Knockdown of CR-1 expression in ESCC cells was conducted with short hairpin RNA. Cell migration and invasion were examined by scratch test and matrigel transwell assay, respectively. Metastatic capability of ESCC cells was assayed by a mouse tail vein metastasis model. The levels of CR-1 expression in cancerous and paired adjacent normal tissues were assessed by IHC and qRT-RCR.ResultsCR-1high subpopulation of ESCC cells isolated by FACS expressed high level of genes related to stemness and epithelial-mesenchymal transition (EMT), and possessed high capacities of self-renewal, tumorigenesis, invasion and metastasis. Suppression of CR-1 expression significantly reduced the expression of stemness- and EMT-related genes and the capabilities of self-renewal in vitro, tumorigenicity and metastasis in vivo in ESCC cells. In the clinical ESCC specimens, the expression levels of CR-1 in cancerous tissues were positively correlated to TNM stage, invasive depth, and lymph node metastasis. Cox regression analysis indicated that CR-1 was an independent indicator of prognosis. The expression of CR-1 was found overlapping with aldehyde dehydrogenase 1A1 (ALDH1A1), an intracellular marker for ESCLCs, in ESCC cell lines and specimens.ConclusionsCR-1 is a functional and cell surface ECSLC marker, and an independent prognostic indicator as well as a potential therapeutic target for ESCC.

MACC1 induces autophagy to regulate proliferation, apoptosis, migration and invasion of squamous cell carcinoma.

Metastasis-associated colon cancer-1 (MACC1) plays an important role in cancer development, but the role and mechansim of MACC1 in squamous cell carcinoma (ESCC) remain unclear. In this study, we found that MACC1 expression was increased in ESCC, and correlated with lymph node metastasis. MACC1 knockdown suppresed ESCC cell proliferation, metastasis and enchanced cell apoptosis. Moreover, MACC1 knockdown inhibited ESCC cell autophagy, and 3-methyladenine was able to rescue MACC1-induced malignant phenotype of ESCC cells. Furthermore, MACC1 knockdown inactivated AMPK-ULK1 signaling pathway, and metformin could rescue MACC1-induced autophagy in ESCC cells. Collectively, this study found that upregulation of MACC1 in ESCC was associated with lymph node metastasis of patients, and MACC1 regulated ESCC cell proliferation, apoptosis, migration and invasion mainly through AMPK-ULK1 induced autophagy.

MicroRNA and target mRNA selection through invasion and cytotoxicity cell modeling and bioinformatics approaches in esophageal squamous cell carcinoma

This study analyzed microRNA(miRNA) and mRNA expression profiles and investigated the biological characteristics of ESCC by using invasion and cytotoxicity cell models. miRNA profiles were evaluated through miRNA microarray. Transwell chamber and nedaplatin(NDP) were used to construct invasion and cytotoxicity cell models. Invasion Transwell and cytotoxicity assays were performed to examine the invasiveness and proliferation in the cell models. Functional miRNAs were selected from dysregulated miRNAs through qRT-PCR. Biometric Research Program(BRB)-array tools, Cytoscape plugins, and DAVID were utilized to find potential mRNAs targeted by these two miRNAs between ESCC and paired normal adjacent tissues. Our microarray obtained 11 dysregulated miRNAs expressed in three paired ESCC samples from Kazakhs (ethnicity in Northwestern China). qRT-PCR demonstrated the miRNA expression in the invasion and cytotoxicity cell models. miR‑652-5p and miR‑21‑5p exhibited a consistent expression level in the microarray and cell models. Bioinformatics revealed that the potential targets of PLD1, MSH2, STC1, and DSG1 might be involved in ESCC invasion and proliferation. Cell models with bioinformatics approaches may help distinguish functional genes. miR‑652-5p, miR‑21‑5p, and their potential target genes may participate in ESCC development and metastasis.

MiR-424-5p participates in esophageal squamous cell carcinoma invasion and metastasis via SMAD7 pathway mediated EMT.

BackgroundsESCC is a life-threatening disease due to invasion and metastasis in the early stage. Great efforts had been made to detect the molecular mechanisms which led to the invasion and metastasis in ESCC. Recent evidence had suggested that deregulation of miR-424-5p took an important role in cancers. However, its role and functional mechanism in ESCC had seldom been elucidated.MethodsThe expression levels of miR-424-5p were detected in ESCC tissues and cell lines by real-time PCR methods. Then, the invasion, metastasis and proliferation ability of ESCC cell lines transfected with miR-424-5p mimics were analyzed separately by transwell invasion assay, wound healing assay and cell proliferation assay. Finally, the target gene of miR-424-5p was studied and verified by luciferase activity assay. And the role of miR-424-5p in EMT was also investigated by real-time PCR and western blot assay.ResultsWe showed that the expression levels of miR-424-5p were decreased both in ESCC tissues and cell lines. Furthermore, the expression levels of miR-424-5p were negatively linked to lymph node metastasis in ESCC tissues. Restoration of miR-424-5p in EC-1 cells by using miR-424-5p mimics could decrease the invasion, metastasis and proliferation of EC-1 cells, indicating its role in inhibition on the invasion and metastasis ability of ESCC cells and tissues. In addition, we demonstrated that SMAD7 was a specific target gene for miR-424-5p by luciferase activity assay and miR-424-5p could not only negatively regulate SMAD7 expression but also participate in EMT via SMAD7, because overexpression of SMAD7 could partly enhance the miR-424-5p anti-EMT function.ConclusionsOur results described that miR-424-5p -SMAD7 pathway contributed to ESCC invasion and metastasis and up-regulation of miR-424-5p perhaps provided a strategy for preventing tumor invasion, metastasis.