Epithelial-mesenchymal Transition In Cancer Cells Research Articles

Targeting Wnt/β-Catenin Signaling by TET1/FOXO4 Inhibits Metastatic Spreading and Self-Renewal of Cancer Stem Cells in Gastric Cancer.

Simple SummaryMetastasis is the main cause of death for patients suffering gastric cancer. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critical attributes of metastasis, both of which are regulated tightly by DNA methylation and Wnt/β-catenin signaling. In this study, we unveiled a novel TET1-FOXO4-β-catenin signaling cascade, in which TET1 inhibits β-catenin activity and its nuclear translocation through transactivating FOXO4 expression. TET1 expression can significantly inhibit EMT and stemness properties of gastric cancer cells, while knocking-down endogenous TET1 induces metastasis and enhances self-renewal of CSCs by activating canonical Wnt signaling, which could be fully rescued by modulating FOXO4 expression. Our data also showed that low expression of TET1 or FOXO4 predicts poor survival of gastric cancer patients, suggesting reactivation of TET1 or FOXO4 might be a novel therapeutic approach to prevent gastric cancer metastasis.Metastasis is the main cause of death for patients suffering gastric cancer. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critical attributes of metastasis, both of which are regulated tightly by DNA methylation and Wnt/β-catenin signaling. Here, we studied the functions of DNA dioxygenase TET1 in regulating Wnt signaling and in gastric cancer metastasis. Knocking-down and overexpressing TET1 in gastric cancer cells promoted and inhibited metastatic spreading to the liver in immune-deficient mice, respectively. TET1 showed inhibitory effects on metastasis-related features -EMT and CSC, which were reversed by interfering with Wnt/β-catenin signaling. RNA-sequencing identified FOXO4 as a direct transactivating target of TET1. FOXO4 directly interacted with β-catenin and recruited it in the cytoplasm, so as to inhibit β-catenin-mediated transcription of Wnt target genes, including CSC marker EpCAM. Moreover, modulation of FOXO4 could reverse the effects of TET1 manipulation on EMT and self-renewal of CSCs. The analysis with clinical samples confirmed the value of FOXO4 as an independent prognostic predictor of patients’ overall survival. Taken together, regulation of Wnt signaling by TET1/FOXO4 is essential for metastasis-associated cellular properties, and targeting TET1/FOXO4/β-catenin pathway may serve as promising therapeutics in the prevention and treatment of gastric cancer metastasis.

FSCN1 Promotes Glycolysis and Epithelial-Mesenchymal Transition in Prostate Cancer through a YAP/TAZ Signaling Pathway.

Objective The aim of the study is to investigate the role and possible mechanism of fascin-1 (FSCN1) in the invasion, migration, glycolysis, and epithelial-mesenchymal transition (EMT) of prostate cancer. Methods Real-time quantitative polymerase chain reaction (qRT-PCR) was utilized to determine the mRNA expression level of FSCN1 in prostate cancer tissues and prostate cancer cells PC-3 and DU145. The transwell and the scratch test were applied to detect the invasion and migration abilities of cells, respectively. A metabolic assay was used for measuring the glucose consumption, lactate production, and the extracellular acidification rate (ECAR) in cells; western blot was used for checking FSCN1, EMT, and yes-associated protein/transcriptional co-activators with the PDZ-binding motif (YAP/TAZ) signaling pathway-related protein expression level in cells or tissues. Results FSCN1 was significantly highly expressed in prostate cancer tissues and cells. On the one hand, interference with the expression of FSCN1 could inhibit the invasion, migration, EMT, and glycolysis of prostate cancer cells. On the other hand, overexpression of FSCN1 promoted the invasion, migration, EMT, and glycolysis of prostate cancer cells. Besides, further mechanistic studies revealed that FSCN1 could activate the YAP/TAZ signaling pathway in prostate cancer cells. Conclusion FSCN1 promotes invasion, migration, EMT, and glycolysis in prostate cancer cells by activating the YAP/TAZ signaling pathway. FSCN1 may be used as a biomarker for the diagnosis or treatment in prostate cancer.

A Traditional Chinese Remedy for Hepatocellular Carcinoma Functions by Suppressing Exosomal miR-23a-3p/Smad Signaling

A decoction of Jianpi Huayu (JHP) has been used traditionally in orthodox Chinese medicine to treat tumors such as hepatocellular carcinoma (HCC), however, the mechanism is still a mystery. Intercellular communication in the cancer cell microenvironment points toward a strong role of exosomes, but their contribution to HCC is still not well understood. Xie and colleagues investigated the anti-neoplastic effects of JHD and the possible function of exosomal signaling. Nude mice models with subcutaneous cancer were used to gauge the effects of JHD. Migration, invasion, proliferation, and epithelial-mesenchymal transition (EMT) of cultured HCC cells were mitigated by JHD. From the EMT-induced HCC cells, exosomes increased the invasion, migration, and EMT of other cultured HCC cells, whereas post-treatment of JHD, exosomes from EMT-induced HCC cells had little effect. Exosomal miR-23a-3p which is significantly upregulated in cancer cells as compared to normal cells was reduced in JHD-treated cultured HCC cells. Post hepatectomy, patients with high miR-23a-3p levels had low survival rates. Smad signaling protein expression as well HCC cell proliferation was upregulated by miR-23a-3p. More critically, miR-23a-3p overexpression countered the EMT and Smad signaling inhibition brought about by JHD treatment. In vivo examinations revealed that treatment with JHD additionally diminished the development of HCC-derived tumors in naked mice, and decreased the levels of miR-23a-3p in serum exosomes and the degree of EMT in cancer cells. The authors conclude that the anticancer properties of JHD partially if not completely are due to the downregulation of exosome-mediated intercellular miR-23a-3p transfer and subsequent blockade of Smad signaling. Disrupting this exosomal miR-23a-3p/Smad signaling pathway may be an effective treatment. J Ethnopharmacol. 2022 Aug 10;294:115360.

A feedback loop between GATA2-AS1 and GATA2 promotes colorectal cancer cell proliferation, invasion, epithelial-mesenchymal transition and stemness via recruiting DDX3X

BackgroundColorectal cancer (CRC) is a common malignant tumor with a high risk of metastasis. Long non-coding RNAs (lncRNAs) have been reported to be implicated in cancer progression via regulating its nearby gene. Herein, we investigated the function of GATA binding protein 2 (GATA2) and lncRNA GATA2 antisense RNA 1 (GATA2-AS1) in CRC and the mechanism underlying their interaction.MethodsColony formation assay, flow cytometry analysis and transwell assay were implemented to detect cell proliferation, apoptosis and invasion. Western blot analysis and sphere formation assay were conducted to assess epithelial-mesenchymal transition (EMT) and cancer stemness of CRC cells. RNA pull down, RNA-binding protein immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and luciferase reporter assays were implemented to investigate the regulatory mechanism between GATA2-AS1 and GATA2.ResultsGATA2-AS1 and GATA2 were highly expressed in CRC cells. Knockdown of GATA2-AS1 and GATA2 impeded CRC cell proliferation, invasion, EMT and cancer stemness, and induced cell apoptosis. GATA2-AS1 expression was positively correlated with GATA2. GATA2-AS1 recruited DEAD-box helicase 3 X-linked (DDX3X) to stabilize GATA2 mRNA. GATA2 combined with GATA2-AS1 promoter to enhance GATA2-AS1 expression.ConclusionOur study confirmed that a feedback loop between GATA2-AS1 and GATA2 promotes CRC progression, which might offer novel targets for CRC treatment.

MFAP2 contributes to the proliferation and motility of lung cancer cells via Wnt/β-catenin pathway

Purpose: To measure microfibrillar-associated protein 2 (MFAP2) expression levels in lung cancer and identify its role in lung cancer.Methods: Immunoblots and PCR were used to determine the expression of MFAP2 in lung cancer cell lines. The effects of MFAP2 on the viability and apoptosis of lung cancer cells were evaluated by CCK-8, colony formation, and flow cytometry (FCM) assays, while wound healing and Transwell assays were used to assess the effects of MFAP2 on the motility and epithelial-mesenchymal transition (EMT) of lung cancer cells. Immunoblot assays were also performed to determine the effect of MFAP2 on the Wnt/β-catenin axis in lung cancer cells.Results: MFAP2 was highly expressed in lung cancer cells. Depletion of MFAP2 suppressed the viability and stimulated apoptosis in these cells (p < 0.01). In addition, knockdown of MFAP2 suppressed the motility and EMT of lung cancer cells (p < 0.01). MFAP2 mediated the Wnt/β-catenin axis and affected the viability and motility of lung cancer cells.Conclusion: MFAP2 is a promising target for lung cancer treatment.

MiRNA-651-3p regulates EMT in ovarian cancer cells by targeting ZNF703 and via the MEK/ERK pathway

miRNAs are non-coding single-stranded RNA molecules with many functions. Several miRNAs have been found to be dysregulated in ovarian cancer; however, the role of miR-651-3p in ovarian cancer remains unknown. Here, the expression level of miR-651-3p in ovarian tissue samples was determined via qRT-PCR, and then miR-651-3p was overexpressed and downregulated to study the functional changes in ovarian cancer cells. Based on previous research and database predictions, we analyzed the binding and regulatory effects of miR-651-3p on zinc finger protein 703 (ZNF703). We additionally evaluated the effect of miR-651-3p on epithelial–mesenchymal transition (EMT) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways in ovarian cancer cells. We found that miR-651-3p was downregulated in ovarian cancer tissues. miR-651-3p expression was associated with inhibited proliferation, invasion, and migration of ovarian cancer cells and promoted cell cycle arrest. Additionally, miR-651-3p was found to target ZNF703 and affect EMT in ovarian cancer by activating the MEK/ERK signaling pathway. MiR-651-3p was downregulated in ovarian cancer, and suppressed the malignant biological behavior of ovarian cancer by inhibiting ZNF703 and the MEK/ERK pathway. Our findings on miR-651-3p provided new insights for the diagnosis and treatment of ovarian cancer.

Abstract 6379: Dipeptidyl peptidase-4 inhibitor impairs the outcomes of patients with type 2 diabetes mellitus after curative resection for colorectal cancer

Abstract Background: Type2 diabetes mellitus(T2DM) is a risk factor for cancer. Recent studies have shown that DPP-4 inhibitor(DPP-4i) can either promote or suppress cancer progression. However, the detailed mechanisms remain unknown. Here, in this study, we investigated the effect of DPP-4i on tumor microenvironment and its effect on the prognosis of cancer patients. Method: We retrospectively examined the outcome of colorectal cancer (CRC) patients with T2DM who received curative surgery in Jichi Medical University Hospital and asked the impact of DPP-4i intake on their outcome. In addition, we performed immunohistochemistry to examine the phenotypes of TIL, TAM and epithelial-mesenchymal transition (EMT) of cancer cells in surgically removed CRC tissues in 40 CRC patients who had taken DPP-4i and propensity score-matched 40 patients who had not. Results: A total of 1696 patients underwent curative colectomy from April 2010 to March 2020. Among them, 260 patients had T2DM at the time of surgery, and 135 patients had been treated with drugs including DPP-4i. The postoperative disease-free survival rate (DFS) was significantly worse in the DPP-4i intake group compared with non-intake group (5 year DFS 18.5% vs 5.4%, HR=2.0, p<0.05). The number of Zeb1-positive tumor cells significantly increased in DPP-4i intake group. (Median(M)=29.0 (0-189)/mm2 vs 9.0 (0-71)/mm2, p<0.01). The number of CD3 (+) TIL in DPP-4i intake group was Median(M)=277.4(min 121.6-max 523.2)/mm2 which was significantly lower than those in non-intake group (M= 319.6, min 493.0-max 823.6/mm2). CD8(+) TIL in DPP-4i intake was reduced more significantly than in non-intake group (M=187.6, min 67.8-max 347.4/mm2 vs M=336.8, min 200.2-max 588.4/mm2, p<0.05) with less CD8/CD3 ratio (61.2% vs 67.1%, p<0.05). On the other hand, the density of CD68(+)CD163(+) TAM was significantly higher(M=202.6, min 122.8-max 257.0/mm2 vs M=126.6, min 94.8-max 247.6/mm2, p<0.05) in DPP-4i intake group. Conclusion: DPP-4i increases the number of M2-type TAM while decreases the number of effector TIL and promote EMT of cancer cells in tumor microenvironment, which might lead to the poor outcome of the patients with CRC. Citation Format: Akira Saito, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Mineyuki Tojo, Rie Kawashima, Hideyo Miyato, Naohiro Sata, Joji Kitayama. Dipeptidyl peptidase-4 inhibitor impairs the outcomes of patients with type 2 diabetes mellitus after curative resection for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6379.

Sorafenib inhibits ovarian cancer cell proliferation and mobility and induces radiosensitivity by targeting the tumor cell epithelial-mesenchymal transition.

Sorafenib, a pan-protein kinase inhibitor, inhibits the activity of various kinases (like vascular endothelial growth factor, platelet-derived growth factor, and rapidly accelerated fibrosarcoma) and clinically has been used to treat different human cancers. This study investigated its antitumor activity in ovarian cancer and the underlying molecular events. To achieve that, ovarian cancer SKOV-3 cells were treated with or without sorafenib (10 µM), transforming growth factor (TGF)-β1 (10 ng/mL), sorafenib (10 µM) + TGF-β1 (10 ng/mL), and TGF-β1 (10 ng/mL) + Ly2157299 (5 µM), followed by 8-Gy radiation. The cells were then subjected to cell viability, wound healing, Transwell, caspase-3 activity, and western blot assays. TGF-β1 treatment enhanced ovarian cancer cell epithelial–mesenchymal transition (EMT), whereas sorafenib and a selective TGF-β1 inhibitor Ly2157299 reversed tumor cell EMT, invasion, and expression of EMT markers (E-cadherin and vimentin). Sorafenib and Ly2157299 treatment also significantly reduced the tumor cell viability. Furthermore, both sorafenib and Ly2157299 significantly enhanced ovarian cancer cell radiosensitivity, as assessed by a caspase-3 activity assay. In conclusion, sorafenib inhibited ovarian cancer cell proliferation and mobility and induced tumor cell radiosensitivity. Molecularly, sorafenib could inhibit the TGF-β1-mediated EMT. Future studies will assess sorafenib anti-ovarian cancer activity plus TGF-β1 inhibitors in ovarian cancer in vivo.

Abstract 962: A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance

Abstract Introduction: Epithelial to mesenchymal transition (EMT) is essential for cancer dissemination but its role in overt metastasis establishment remains an open issue. Multiple EMT states and great phenotypic plasticity have also been described, indicating the need for in-depth characterization of biological properties associated with different EMT phenotypes. The so-called hybrid phenotype, characterized by presence of both mesenchymal and epithelial markers, might in fact identify cancer cells with enhanced metastatic propensity. Materials and Methods: To assess the metastatic potential of different EMT phenotypes, we exploited two lung cancer cell lines with different properties (A549/epithelial and LT73/hybrid) and generated their respective sublines through four sequential rounds of in vitro migration assay (named M4). TGFβ treatment was also used to induce EMT. With a previously validated score based on the ratio between expression of epithelial (CDH1) or mesenchymal genes (SNAI2), cells were classified as: epithelial (A549)>hybrid (A549-M4, LT73)>mesenchymal (LT73-M4, A549-TGFβ). The disseminating/metastatic properties of the different lines were tested in vivo by subcutaneous and tail-vein injections in SCID mice. Interaction with NK cells (NKs), known to specifically target EMT cancer cells, was also assessed. Results and Discussion: Compared to parental cells, M4 cells showed up-regulation of EMT-associated genes, expansion of the subset of CD133+ lung cancer stem cells (CSC) and displayed in vivo higher ability to disseminate from primary tumors to murine lungs (2-fold change, p=0.004). However, hybrid cells (LT73 and A549-M4) showed an increased ability to generate overt lung metastases in experimental metastasis assays compared to either epithelial (A549) or mesenchymal cells (LT73-M4 and A549-TGFβ). The higher metastatic ability of hybrid cells was correlated with CSCs expansion and reduced NKs infiltration to the metastatic lungs. Expression of IL-8, a cytokine involved in NKs recruitment, was decreased in hybrid cells and they were less prone to chemoattract NKs in vitro compared to both epithelial and mesenchymal cells. Finally, in vivo prevention of NKs recruitment to the lungs using a neutralizing antibody, massively increased metastasis formation and expansion of CSCs in mice injected i.v. with mesenchymal A549-TGFβ (p=0.0006), but had limited effects in groups injected with both epithelial A549 and hybrid A549-M4. Conclusions: Our data indicate that the hybrid phenotype correlates with the highest metastatic potential related to efficient cancer cell dissemination, increased induction of stemness features and ability to avoid NKs recruitment. Conversely, fully mesenchymal cells show limited metastatic potential, possibly due the control exerted by NKs on their CSC subsets. Citation Format: Giulia Bertolini, Monica Parodi, Massimo Milione, Fabio Murianni, Giovanni Centonze, Giulia Taiè, Gabriella Sozzi, Massimo Vitale, Luca Roz. A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 962.

Comprehensive Analysis of hsa-miR-654-5p’s Tumor-Suppressing Functions

The pivotal roles of miRNAs in carcinogenesis, metastasis, and prognosis have been demonstrated recently in various cancers. This study intended to investigate the specific roles of hsa-miR-654-5p in lung cancer, which is, in general, rarely discussed. A series of closed-loop bioinformatic functional analyses were integrated with in vitro experimental validation to explore the overall biological functions and pan-cancer regulation pattern of miR-654-5p. We found that miR-654-5p abundance was significantly elevated in LUAD tissues and correlated with patients’ survival. A total of 275 potential targets of miR-654-5p were then identified and the miR-654-5p-RNF8 regulation axis was validated in vitro as a proof of concept. Furthermore, we revealed the tumor-suppressing roles of miR-654-5p and demonstrated that miR-654-5p inhibited the lung cancer cell epithelial-mesenchymal transition (EMT) process, cell proliferation, and migration using target-based, abundance-based, and ssGSEA-based bioinformatic methods and in vitro validation. Following the construction of a protein–protein interaction network, 11 highly interconnected hub genes were identified and a five-genes risk scoring model was developed to assess their potential prognostic ability. Our study does not only provide a basic miRNA-mRNA-phenotypes reference map for understanding the function of miR-654-5p in different cancers but also reveals the tumor-suppressing roles and prognostic values of miR-654-5p.

The Regulatory Mechanism of EpCAM N-Glycosylation-Mediated MAPK and PI3K/Akt Pathways on Epithelial-Mesenchymal Transition in Breast Cancer Cells.

It was to investigate the regulation of motigen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathways mediated by epithelial cell adhesion molecules (EpCAM) N-glycosylation on epithelial-mesenchymal transition (EMT) in breast cancer cells. Breast cancer cells MCF-7 and MDA-MB-231 were taken in the control group; the blank vector plasmid transfection (blank plasmid group) and EpCAM N-glycosylation mutant plasmid transfection (Mutant-EpCAM group) were analyzed. The EpCAM in breast cancer cells was localized by immunofluorescence technique, the proliferation activity of cells in each group was detected by methyl thiazolyl tetrazolium (MTT) assay, and the clonality of cells was detected by plate cloning. and apoptosis-related proteins (Caspase-3, Bcl-2, and Bax), EMT-related molecular markers (E-cadherin, N-cadherin, and Vimentin), as well as MAPK and PI3K/Akt pathway-related proteins (p38, PI3K, and Akt) in cells were detected by western blotting (WB). EpCAM N-glycosylation mutations did not alter the expression localization of EpCAM in breast cancer cells. Compared with the control group and the blank plasmid group, the cell proliferation activity and the number of colonies formed were decreased in the Mutant-EpCAM group (P<0.05). The protein expressions of Caspase-3, Bax, and E-cadherin were up-regulated significantly, and the expressions of Bcl-2, N-cadherin, Vimentin, p-p38, p-PI3K, and p-Akt were greatly down-regulated (P<0.05). EpCAM N-glycosylation could regulate the EMT in breast cancer cells through MAPK and PI3K/Akt pathways.

Fangchinoline targets epithelial–mesenchymal transition process by modulating activation of multiple cell‐signaling pathways

Epithelial-mesenchymal transition (EMT) is a key process, which can promote the transition of tumor cells into other organs by weakening the cell-cell junctions. Tumor cell invasion and metastasis arising because of EMT can determine the prognosis of cancer. EMT can be induced by several growth factors including transforming growth factor-β (TGF-β), which can exert their effects by affecting several cell-signaling pathways. Fangchinoline (FCN), a kind of bisbenzylisoquinoline, belongs to the family Menispermaceae. FCN can display substantial antitumor effects against various malignant cell lines but its possible impact on EMT has not been explored. We examined the potential impact of FCN in affecting the activation of EMT in human colon cancer cells. We evaluated the influence of FCN on EMT in colon cancer cells by using Western blot analysis and reverse transcription-polymerase chain reactionassays. The cellular invasion and migration were observed by Boyden chamber and wound healing assays. Thereafter, the effect of the drug on proliferation and invasion was also evaluated by real-time cell analysis. FCN suppressed the levels of TGF-β-induced mesenchymal markers, such as fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail. However, FCN markedly enhanced the expression of epithelial markers such as occludin and E-cadherin. These results imply that FCN can potentially inhibit tumor metastasis through abrogating EMT. In addition, FCN downregulated c-Met/PI3K/Akt/mTOR and Wnt/β-catenin cell signaling pathways and mitigated tumor migration as well as invasion. Overall, our study suggests a potential novel role of FCN as an antimetastatic agent against human colon cancer cells.

Hsa_circ_0006732 regulates colorectal cancer cell proliferation, invasion and EMT by miR-127-5p/RAB3D axis.

Colorectal cancer (CRC) remains a malignancy tumor with high metastasis and poor prognosis. We aimed to explore the effect of circular RNA (circRNA) hsa_circ_0006732 in the progression of CRC. Hsa_circ_0006732 expression in CRC tissues and cell lines were detected using qRT-PCR. The relationship between hsa_circ_0006732 expression and clinicopathologic characteristics of patients with CRC was analyzed. Loss-of-function assay was conducted to determine the regulatory effect of hsa_circ_0006732 on CRC cell proliferation, migration and invasion by using the CCK-8, wound-healing assay and transwell assays. Protein expression changes on epithelial mesenchymal transition (EMT)-related factors were detected by western blotting. The downstream signaling pathway was investigated by bioinformatics, dual-luciferase reporter assay. Rescue assay was further examined for prediction validation. It was found that hsa_circ_0006732 was highly expressed in CRC tissues and cell lines. Downregulation of hsa_circ_0006732 suppressed the proliferation, migration, invasion and EMT of CRC cells. Further mechanistic investigations proved that hsa_circ_0006732 functioned as a competitive endogenous RNA (ceRNA) by directly sponging of miR-127-3p, which further affected the expression of Ras-related protein Rab-3D (Rab3D). Taken together, these findings indicated that hsa_circ_0006732 might be an oncogene in CRC through the regulation of the miR-127-5p/RAB3D axis. Thus, hsa_circ_0006732 might serve as a potential therapeutic target for the treatment of CRC.

From Tissue Physoxia to Cancer Hypoxia, Cost-Effective Methods to Study Tissue-Specific O2 Levels in Cellular Biology.

The human body is endowed with an extraordinary ability to maintain different oxygen levels in various tissues and organs. The maintenance of physiological levels of oxygen is known as physoxia. The development of hypoxic conditions plays an important role in the biology of several pathologies, including cancer. In vitro studies using normal and neoplastic cells require that culture conditions be carried out under appropriate oxygen levels, either physoxic or hypoxic conditions. Such requirements are difficult to widely implement in laboratory practice, mainly due to the high costs of specialized equipment. In this work, we present and characterize a cost-effective method to culture cells under a range of oxygen levels using deoxidizing pouches. Our results show that physoxic and hypoxic levels using deoxidizing absorbers can be achieved either by implementing a gradual change in oxygen levels or by a regimen of acute depletion of oxygen. This approach triggers the activation of an epithelial-mesenchymal transition in cancer cells while stimulating the expression of HIF-1α. Culturing cancer cells with deoxidizing agent pouches revealed PI3K oncogenic pathway exacerbations compared to tumor cells growing under atmospheric levels of oxygen. Similar to the PI3K signaling disturbance, we also observed augmented oxidative stress and superoxide levels and increased cell cycle arrest. Most interestingly, the culture of cancer cells under hypoxia resulted in the accumulation of cancer stem cells in a time-dependent manner. Overall, we present an attractive, cost-effective method of culturing cells under appropriate physoxic or hypoxic conditions that is easily implementable in any wet laboratory equipped with cell culture tools.

ARHGAP9 inhibits colorectal cancer cell proliferation, invasion and EMT via targeting PI3K/AKT/mTOR signaling pathway

AimIn digestive system, colorectal cancer (CRC) is a common malignant tumor. The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of the rapamycin (PI3K/AKT/mTOR) signaling pathway plays a central role in CRC, and the aberrant activation of this pathway is associated with tumorigenesis. We aimed to explore the role of Rho GTPase activating protein 9 (ARHGAP9) in the progression of CRC as well as its regulatory effects on the PI3K/AKT/mTOR pathway. MethodsThe expression of ARHGAP9 in CRC tumor tissues and cell lines were detected using reverse transcription-quantitative PCR (qRT-PCR). 5-ethynyl-2′-deoxyuridine (EdU) assay was applied to test the cell proliferation. Cell migration and invasion were both assessed through transwell assay. Xenograft mouse models were constructed to explore the effects of ARHGAP9 on CRC in vivo. The expressions of PI3K/AKT/mTOR-activating factors and epithelial-mesenchymal transition (EMT)-related factors were all determined using western blot. LY294002 was employed to block PI3K/AKT/mTOR pathway in CRC cells. ResultsThe expression of ARHGAP9 was down-regulated in CRC tumor tissues and cell lines when compared to normal tissues and cells. The over-expression of ARHGAP9 inhibited cell proliferation, invasion, migration and EMT in CRC cell lines while the knockdown of ARHGAP9 promoted them. In addition, ARHGAP9 up-regulation inhibited the activation of PI3K/AKT/mTOR signaling pathway in CRC cell lines while ARHGAP9 down-regulation led to an opposite effect. The over-expression of ARHGAP9 suppressed CRC tumor growth in vivo. When the PI3K/AKT/mTOR pathway was blocked in CRC cells, the effects of ARHGAP9 knockdown on cell proliferation, migration, invasion and EMT were all overturned. ConclusionARHGAP9 inhibited the malignant phenotypes of CRC cells via interdicting PI3K/AKT/mTOR signaling pathway.

Upregulated RACK1 Attenuates Gastric Cancer Cell Growth and Epithelial-Mesenchymal Transition via Suppressing Wnt/β-Catenin Signaling [Retraction

[This retracts the article DOI: 10.2147/OTT.S205869.].

YTHDF2 interference suppresses the EMT of cervical cancer cells and enhances cisplatin chemosensitivity by regulating AXIN1.

M6A reader YTH structural domain family 2 (YTHDF2) has been recognized to play an oncogenic role in numerous tumors, but its role in cervical cancer has not been extensively discussed yet. This paper was designed to explore the role of YTHDF2 in cervical cancer and identify its underlying mechanism. The expression of YTHDF2 was first determined in cervical cancer cells by quantitative reverse-transcription polymerase chain reactionand western blot. Then, the migration, invasion, and epithelial-mesenchymal transition (EMT) process were observed in YTHDF2-knockdown Hela cells using wound healing, transwell and immunofluorescence assays. The cisplatin chemosensitivity of Hela cells was also investigated by assessing cell activity with cell counting kit-8 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). After MeRIP-Seq assay and actinomycin D treatment to confirm the binding relationship between YTHDF2 and AXIN1, the migration, invasion, EMT process, and cisplatin chemosensitivity were assessed again in Hela cells silenced by YTHDF2 and AXIN1 or treated with Wnt agonist. YTHDF2 was increased in cervical cancer cells, and depletion of YTHDF2 led to reduced migration, invasion and EMT process but enhanced chemosensitivity of cisplatin in Hela cells. Furthermore, YTHDF2 could bind to and stabilize the expression of AXIN1. When the YTHDF2-knockdown Hela cells were further transfected with AXIN1 knockdown or treated with Wnt agonist, the effects of YTHDF2 knockdown on the migration, invasion and EMT process were partially abolished, together with reduced cisplatin chemosensitivity. To sum up, we reported that YTHDF2 interference could suppress the EMT of cervical cancer cells and enhance cisplatin chemosensitivity by regulating AXIN1.

Pancreatic cancer cell-derived exosomes induce epithelial-mesenchymal transition in human pancreatic cancer cells themselves partially via transforming growth factor β1.

Distant metastasis is a dismal prognostic factor of pancreatic cancer. Metastasis is established in several steps, but the mechanism underlying the very early stages remains unclear. Epithelial-mesenchymal transition (EMT) is involved in these stages. Although signaling molecules have been reported to induce EMT, the mechanism underlying their origin is unclear. In this study, we hypothesized that pancreatic cancer cell-derived exosomes induce EMT in cancer cells themselves, a notion we entertained because we found EMT in in vitro three-dimensional colonies of cancer cells, with vimentin-positive cells observed in some of the budding pancreatic cancer cells and in single cells outside the colony as well. First, we clarified that pancreatic cancer cell-derived exosomes induce EMT in cancer cells themselves. Next, we examined the involvement of transforming growth factor-β1 (TGF-β1), and TGF-β1 knock-down in pancreatic cancer cells with TGF-β1 siRNA significantly suppressed TGF-β1 gene expression in cancer cells, and exosomal TGF-β1 was significantly reduced in the secretory exosomes. Exosomes from TGF-β1 knock-down cells suppressed EMT induction in cancer cells themselves and TGF-β1 protein expression in target cells. Taken together, these findings suggest that TGF-β1 is involved in EMT induction via exosomes, results that may support the production of effective metastasis inhibitors.

Extracellular Matrix Protein 1 Regulates Colorectal Cancer Cell Proliferative, Migratory, Invasive and Epithelial-Mesenchymal Transition Activities Through the PI3K/AKT/GSK3β/Snail Signaling Axis

In prior reports, extracellular matrix protein 1 (ECM1) upregulation has been reported in colorectal cancer (CRC) patient tumor tissues, and has been suggested to be related to the metastatic progression of CRC, although the underlying mechanisms have yet to be clarified. In this study, we found that ECM1 was overexpressed in both CRC tissues and cell lines. Upregulation of ECM1 was correlated with tumor size, lymph node status and TNM stage in CRC patients. Knocking down ECM1 suppressed CRC cell growth, migration and invasion, in addition to reducing the expression of Vimentin and increasing E-cadherin expression. The overexpression of ECM1, in contrast, yielded the opposite phenotypic outcomes while also promoting the expression of p-AKT, p-GSK3β, and Snail, which were downregulated when ECM1 was knocked down. Treatment with LY294002 and 740 Y-P reversed the impact upregulation and downregulation of ECM1 on CRC cell metastasis and associated EMT induction. In vivo analyses confirmed that ECM1 overexpression was able to enhance EMT induction and CRC tumor progression. In conclusion, ECM1 influences CRC development and progression in an oncogenic manner, and regulates CRC metastasis and EMT processes via the PI3K/AKT/GSK3β/Snail signaling axis.

TGFβ selects for pro-stemness over pro-invasive phenotypes during cancer cell epithelial-mesenchymal transition.

Transforming growth factor β (TGFβ) induces epithelial–mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal–epithelial transition (MET) is induced by TGFβ withdrawal and correlates with metastatic colonization. Whether TGFβ promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E‐cadherin promoter. In 2D cultures, TGFβ induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFβ withdrawal. RFPlow cells generated robust mammospheres, with epithelio‐mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFβ receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFβ suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat‐pad‐transplanted mammospheres, in the absence of exogenous TGFβ treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFβ‐treated mammospheres revealed high tumour‐initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFβ to differentiate between pro‐stemness and pro‐invasive phenotypes.