Promotion Of Epithelial-mesenchymal Transition Research Articles

MiR-19a and miR-424 target TGFBR3 to promote epithelial-to-mesenchymal transition and migration of tongue squamous cell carcinoma cells

ABSTRACTPrevious studies indicate that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), a novel suppressor of progression in certain cancers, is down-regulated in tongue squamous cell carcinoma (TSCC). However, the role of this factor as an upstream regulator in TSCC cells remains to be elucidated. The present study was designed to elucidate whether TGFBR3 gene expression is regulated by two microRNA molecules, miR-19a and miR-424. The study also aimed to determine if these microRNAs promote migration of CAL-27 human oral squamous cells. Immunohistochemistry (IHC) and western blot analyses demonstrated that TGFBR3 protein levels were dramatically down-regulated in clinical TSCC specimens. Conversely, bioinformatics analyses and qRT-PCR results confirmed that both miR-19a and miR-424 were markedly up-regulated in clinical TSCC specimens. In this study, we observed that transfection of a TGFBR3-containing plasmid dramatically inhibited epithelial-to-mesenchymal transition (EMT) and migration in CAL-27 cells. Co-immunoprecipitation analyses also revealed that TGFBR3 forms a complex with the β-arrestin 2 scaffolding protein and IκBα. Furthermore, overexpression of TGFBR3 decreased p-p65 expression and increased IκBα expression; these effects were subsequently abolished following knockdown of β-arrestin 2. Moreover, over-expression of miR-19a and miR-424 promoted migration and EMT in CAL-27 cells. We also observed that the promotion of EMT by miR-19a and miR-424 was mediated by the inhibition of TGFBR3. Our study provides evidence that miR-19a and miR-424 play important roles in the development of TSCC. These results expand our understanding of TGFBR3 gene expression and regulatory mechanisms pertaining to miRNAs.

TGFβ1 in fibroblasts-derived exosomes promotes epithelial-mesenchymal transition of ovarian cancer cells.

Cancer-associated fibroblasts (CAF), a major component of the tumor microenvironment, play an important role in interacting with neoplastic cells to promote ovarian cancer progression. Exosomes are nano-sized vesicles that mediate the cross-talk between different cell types. An increasing number of studies have focused on the fact that tumor cell-derived exosomes influence stromal cells. However, the mechanism by which CAF-derived exosomes modulate cancer cells in ovarian cancer remains obscure. To investigate the role of CAF exosomes in ovarian cancer, we examined the exosomal content of paired primary, metastatic and normal fibroblasts from seven stage IIIC ovarian cancer patients by ELISA. We found that in ovarian CAF-derived exosomes, TGFβ1 was upregulated compared to normal omentum fibroblasts (NOF). Exosomes derived from CAF were taken up by ovarian SKOV-3 and CAOV-3 cell lines during co-culture and induced malignant behaviors in cancer cells, including an enhanced migration and invasion ability and the promotion of epithelial-mesenchymal transition (EMT) by activating the SMAD signaling pathway. Our results indicate that the role of TGFβ1 in CAF exosomes triggers ovarian cancer cells into a more aggressive phenotype, suggesting that targeting CAF exosomes could be a potential treatment in ovarian cancer.

Periostin promotes epithelial-mesenchymal transition via the MAPK/miR-381 axis in lung cancer.

Periostin (POSTN, PN, or osteoblast-specific factor OSF-2) is a multifunctional cytokine that signals between the cell and the extracellular matrix. Periostin plays an important role in tumor development and is involved in carcinoma cell epithelial-mesenchymal transition (EMT), whereby mature epithelial cells undergo phenotypic morphological changes and become invasive, motile cells. Here, we discuss the molecular mechanisms involved in periostin-induced promotion of EMT in lung cancer cells. Online TCGA datasets demonstrate the prognostic relevance of periostin in lung cancer; a higher periostin level correlates with poor overall survival. Similarly, our IHC results show that high periostin expression is positively correlated with the EMT markers Snail and Twist, as well as stage of lung cancer. We found that recombinant periostin induces the EMT phenotype in lung cancer cells through the p38/ERK pathway, while pretreatment with chemical inhibitors prevented periostin-induced EMT induction. Moreover, we found that periostin regulates EMT by repressing microRNA-381 (miR-381) expression, which targets both Snail and Twist. Using the miR-381 mimic, we dramatically reversed periostin-induced Snail and Twist expression. Furthermore, periostin knockdown dramatically affected EMT markers and cell migration potential. The role of periostin in lung cancer progression is elucidated by the in vivo mouse model. Our findings indicate that changes in periostin expression in lung cancer may serve as a therapeutic target for the treatment of lung cancer metastasis.

Cellular proliferation and differentiation induced by single-layer molybdenum disulfide and mediation mechanisms of proteins via the Akt-mTOR-p70S6K signaling pathway

Single-layer molybdenum disulfide (SLMoS2) is a novel kind of 2D nanosheet that has attracted great attention regarding its use in biosensors, drug delivery, tissue engineering, and therapy. However, our results demonstrated that SLMoS2 accelerated proliferation and promoted myogenic differentiation and epithelial–mesenchymal transition (EMT) in human embryonic lung fibroblasts (HELFs). The abnormal proliferation and differentiation of HELFs contribute to idiopathic pulmonary fibrosis. Specifically, SLMoS2 significantly stimulated the expression of myofibroblast- and mesenchymal-associated genes and proteins. The Akt-mTOR-p70S6K signaling pathway plays a critical role in the acceleration of proliferation and promotion of myogenic differentiation and EMT in HELFs induced by SLMoS2. After cell uptake, SLMoS2 was primarily located in the cytoplasm and the perinuclear region and activated Akt-dependent signaling due to the generation of reactive oxygen species (ROS). Moreover, bovine serum albumin (BSA) binding markedly inhibited the cellular uptake of SLMoS2 and the production of intracellular ROS due to an increased thickness and reduced adhesion of HELFs. BSA binding also mitigated the SLMoS2-activated phosphorylation of Akt-dependent signaling pathways. This study is the first to illustrate the induction of cellular proliferation and differentiation by SLMoS2 and the related mediation by proteins through Akt-mTOR-p70S6K signaling pathway.

SOCS3 overexpression inhibits advanced glycation end product-induced EMT in proximal tubule epithelial cells.

Diabetic nephropathy (DN) is among the most severe complications of diabetes mellitus, and may lead to end-stage renal disease. Sustained exposure to advanced glycation end products (AGEs) typically causes renal tubular epithelial cells (TECs) to suffer from an epithelial-to-mesenchymal transition (EMT). However, there remains no consensus regarding the mechanism underlying the cause of EMT in TECs as induced by AGEs. In the present study, we investigated the promotion of EMT in TECs by AGEs, and the activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. In addition, we constructed a recombinant adenovirus (Ad) that overexpressed suppressor of cytokine signaling 3 (SOCS3), and examined the regulatory role of SOCS3 in the activation of JAK/STAT signaling and the promotion of EMT in TECs. The results demonstrated that AGE-bovine serum albumin (BSA) treatment significantly promoted the expression of EMT-associated proteins, while reducing the expression of the epithelial cell marker, E-cadherin. Furthermore, the Ad-mediated SOCS3 overexpression markedly inhibited the AGE-BSA-induced JAK2/STAT3 activation; phosphorylated JAK2 and phosphorylated STAT3 expression levels were reduced by the Ad-SOCS3 infection, compared with the control Ad (Ad-con) infection, in HK-2 cells subject to AGE-BSA. Moreover, the overexpression of SOCS3 markedly inhibited the AGE-BSA-promoted EMT in HK-2 cells. AGE-BSA-promoted EMT-associated proteins, such as α-smooth muscle actin and collagen I, were reduced by the Ad-SOCS3 virus infection, in contrast to the Ad-con virus infection. Furthermore, reduced E-cadherin expression was reversed by the Ad-SOCS3 virus infection, in contrast to the Ad-con virus infection, in epithelial HK-2 cells. In conclusion, the present study confirmed the inhibitory role of SOCS3 in the AGE-induced EMT in renal TECs, implying the protective role of SOCS3 in DN.

Targeting the Metabolic Reprogramming That Controls Epithelial-to-Mesenchymal Transition in Aggressive Tumors.

The epithelial-to-mesenchymal transition (EMT) process allows the trans-differentiation of a cell with epithelial features into a cell with mesenchymal characteristics. This process has been reported to be a key priming event for tumor development and therefore EMT activation is now considered an established trait of malignancy. The transcriptional and epigenetic reprogramming that governs EMT has been extensively characterized and reviewed in the last decade. However, increasing evidence demonstrates a correlation between metabolic reprogramming and EMT execution. The aim of the current review is to gather the recent findings that illustrate this correlation to help deciphering whether metabolic changes are causative or just a bystander effect of EMT activation. The review is divided accordingly to the catabolic and anabolic pathways that characterize carbohydrate, aminoacid, and lipid metabolism. Moreover, at the end of each part, we have discussed a series of potential metabolic targets involved in EMT promotion and execution for which drugs are either available or that could be further investigated for therapeutic intervention.

MTA1 modulated by miR-30e contributes to epithelial-to-mesenchymal transition in hepatocellular carcinoma through an ErbB2-dependent pathway.

MTA1 is a metastasis-associated protein, which is essential to epithelial-to-mesenchymal transition (EMT). However, information concerning its up- and downstream regulation is rare. We investigated its upstream regulation and downstream effector in human hepatocellular carcinoma (HCC). In total, 94 paired HCC and adjacent tissue samples were involved in the study, and the results indicated that decreased miR-30e levels were associated with increased MTA1 levels in human HCC. miR-30e exerted its regulation of MTA1 transcription by binding to its 3'-untranslated region, and was negatively associated with EMT. Furthermore, significantly higher expression of MTA1 was associated with overexpression of v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ErbB2) in human HCC, and MTA1 can promote transcription of ErbB2 by binding with histone deacetylase 2 (HDAC2) and acting as a promoter. The EMT promotion effect caused by MTA1 largely depended on ErbB2, and reducing the activity of ErbB2 can significantly attenuate EMT promotion by causing overexpression of MTA1 both in vitro and in vivo. In general, downregulation of miR-30e can increase levels of MTA1 in human HCC, and furthermore promote cell invasion and metastasis by promoting ErbB2.

Shp2 Plays a Critical Role in IL-6-Induced EMT in Breast Cancer Cells.

Accumulative evidence demonstrates that the protein tyrosine phosphatase Shp2 functions as a powerful tumor promoter in many types of cancers. Abnormal expression of Shp2 has been implicated in many human malignancies. Overexpression of Shp2 in cancer tissues is correlated with cancer metastasis, resistance to targeted therapy, and poor prognosis. The well-known function of Shp2 is its positive role in regulating cellular signaling initiated by growth factors and cytokines, including interleukin-6 (IL-6). Several recent studies have shown that Shp2 is required for epithelial-mesenchymal transition (EMT), triggered by growth factors. However, whether Shp2 is involved in IL-6-signaling-promoted breast cancer EMT and progression, remains undefined. In this study, we showed that exogenous and endogenous IL-6 can enhance breast cancer invasion and migration, through the promotion of EMT. IL-6 also induces the activation of Erk1/2 and the phosphorylation of Shp2. Knockdown of Shp2 attenuated the IL-6-induced downregulation of E-cadherin, as well as IL-6-promoted cell migration and invasion. Moreover, by using Shp2 phosphatase mutants, phosphor-tyrosine mimicking, and deficiency mutants, we provided evidence that the phosphatase activity of Shp2 and its tyrosine phosphorylation, are necessary for the IL-6-induced downregulation of E-cadherin and the phosphorylation of Erk1/2. Our findings uncover an important function that links Shp2 to IL-6-promoted breast cancer progression.

MicroRNA-222 Expression as a Predictive Marker for Tumor Progression in Hormone Receptor-Positive Breast Cancer.

PurposeThe microRNA-221/222 (miR-221/222) gene cluster has been reported to be associated with the promotion of epithelial-mesenchymal transition (EMT), downregulation of estrogen receptor-α, and tamoxifen resistance in breast cancer. We studied the expression of miR-222 in human breast cancer samples to analyze its relationship with clinicopathologic features of the tumor, including estrogen receptor status, expression of EMT markers, and clinical outcomes.MethodsQuantitative real-time polymerase chain reaction was performed to detect the expression of miR-222 in 197 invasive breast cancers. Expression of EMT markers (vimentin, smooth muscle actin, osteonectin, N-cadherin, and E-cadherin) was evaluated using immunohistochemistry.ResultsHigh miR-222 levels were associated with high T stage, high histologic grade, high Ki-67 proliferation index, and HER2 gene amplification. Its expression was significantly higher in the luminal B and human epidermal growth factor receptor 2-positive (HER2+) subtypes than in the luminal A and triple-negative subtypes. In the hormone receptor-positive subgroup, there was a significant negative correlation between miR-222 and estrogen receptor expression, and miR-222 expression was associated with EMT marker expression. In the group as a whole, high miR-222 expression was not associated with clinical outcome. However, subgroup analyses by hormone receptor status revealed that high miR-222 expression was a poor prognostic factor in the hormone receptor-positive subgroup, but not in the hormone receptor-negative subgroup.ConclusionThis study showed that miR-222 is associated with down-regulation of the estrogen receptor, EMT, and tumor progression in hormone receptor-positive breast cancer, indicating that miR-222 might be associated with endocrine therapy resistance and poor clinical outcome in hormone receptor-positive breast cancer.

Effects of upregulatioon of Forkhead box protein M1 on migration, invasion and epithelial-mesenchymal transition of human ovarian cancer cell

Objective To explore the effects and mechanism of over-expression of Forkhead box protein M1 (FoxM1) gene on the migration, and invasion and epithelial-mesenchymal transition (EMT) of human ovarian cancer cells. Methods FoxM1 over-expression of the virus vector was constructed. All HO-8910 cells were divided into three groups, the control group (Con-A), Lentiviral vector(LV) control group (Con-B) and over-expression group FoxM1. After three group HO-8910 cells was transfected with phosphate buffer (PBS), Lentiviral vector (LV), and LV-FoxM1, the protein levels of FoxM1 were determined by Western blotting, respectively. The migration and invasion ability was evaluated by wound healing assay and Transwell assay, respectively. The E-Cadherin and N-Cadherin of protein of cancer cells was examined by Western blotting assay. Results The results from Western blotting assay showed that the levels protein of FoxM1 were greatly increased in HO-8910 cancer cells transfected with LV-FoxM1. The results of the wound healing test showed that the migration distance of Con-A, Con-B, and LV-FoxM1 group was(156.80±2.26), (161.60±1.81) and (278.60±2.62) μm, respectively (P<0.01); The results of the Transwell invasion assay showed that the membrane cell number of Con-A, Con-B, and LV-FoxM1 group was (68.56±1.69) and (67.27±1.36) and (126.38±1.56), respectively (P<0.01); Compared with the Con-A, there were E-cadherin protein expression decreased; however, N-cadherin protein increased in HO-8910 cancer cells transfected with LV-FoxM1. Conclusion FoxM1 overexpression can promote human ovarian cancer cell migration and invasion, and its mechanism may be related to the promotion of epithelial-mesenchymal transition. Key words: Ovarian carcinoma; Forkhead box protein M1; Migration; Invasion; Epithelial-mesenchymal transition

SIX1 coordinates with TGFβ signals to induce epithelial-mesenchymal transition in cervical cancer.

Epithelial-mesenchymal transition (EMT) plays a critical role in promoting tumor invasion and metastasis. However, the key cofactors that modulate the signal transduction to induce EMT have note been fully explored to date. The present study reports that sine oculis homeobox homolog 1 (SIX1) is able to promote EMT of cervical cancer by coordinating with transforming growth factor (TGF)β-SMAD signals. The expression of SIX1 was negatively correlated with the expression of the epithelial marker E-cadherin in two independent groups of cervical cancer specimens. SIX1 could promote the transition of mesenchymal phenotype in the presence of active TGFβ signals in vitro and in vivo. TGFβ-SMAD signals were required for the SIX1-mediated promotion of EMT and metastatic capacity of cervical cancer cells. Together, SIX1 and TGFβ cooperated to induce more remarkable changes in the transition of phenotype than each of them alone, and coordinated to promote cell motility and tumor metastasis in cervical cancer. These results suggest that the coordination of SIX1 and TGFβ signals may be crucial in the EMT program, and that SIX1/TGFβ may be considered a valuable marker for evaluating the metastatic potential of cervical cancer cells, or a therapeutic target in the treatment of cervical cancer.

Heterogeneous expression of zinc-finger E-box-binding homeobox 1 plays a pivotal role in metastasis via regulation of miR-200c in epithelial-mesenchymal transition.

Although epithelial-mesenchymal transition (EMT) has been implicated as the pivotal event in metastasis, there is insufficient evidence related to EMT in clinical settings. Intratumor heterogeneity may lead to underestimation of gene expression representing EMT. In the present study, we investigated the expression of EMT-associated genes and microRNAs in primary colorectal cancer while considering intratumor heterogeneity. One-hundred and thirty-three multiple spatially separated samples were obtained from 8 patients with metastatic colorectal cancers and 8 with non-metastatic colorectal cancers, from the tumor center (TC), invasive front (IF) and metastasis. Differences in gene and microRNA expression were investigated by microarray and quantitative reverse-transcription PCR. Gene expression microarray analysis detected 7920 sites showing differing levels of gene expression among the TC, IF and metastasis. Expression of the EMT-associated gene zinc-finger E-box-binding homeobox 1 (ZEB1) significantly increased in the IF (p<0.01). To exclude individual differences, the expression ratio between TC and IF in each tumor was applied to analysis. This approach enabled recognition of the activation of the VEGF and Wnt signaling pathways, which were involved in metastasis via promotion of EMT. While no activation of these pathways was seen at the TC, regardless of whether tumors were metastatic or non-metastatic, they were preferentially activated at the IF in metastatic tumors, where high ZEB1 expression was seen in connection with decreased miR-200c expression. Multiple sampling in a tumor revealed that heterogeneous ZEB1 expression induced by EMT-associated signaling pathways played a pivotal role in metastasis via regulation of miR-200c.

Promotion of epithelial-mesenchymal transition by Frizzled2 is involved in the metastasis of endometrial cancer

The Wnt signaling pathway is essential for embryonic development, and genetic alteration in this network is closely correlated with tumorigenesis and progression. Previous research has shown that Wnt receptor Frizzled2(Fzd2) is elevated in many metastatic cancer cell lines and high grade tumors. Yet, little is known about the Fzd2 expression and activity in human endometrial cancer(EC). In this study, we present evidence of a direct role of Fzd2 in human EC. We found that Fzd2 expression was higher in EC than that in adjacent normal tissues, and was correlated with epithelial‑mesenchymal transition markers. Next, it was determined that the stable overexpression of Fzd2 in HEC-1B and Ishikawa cells promoted cell migration and induced an EMT phenotype. Conversely, RNA interference-mediated depletion of Fzd2 inhibited EC cell migration. Additionally, mechanistic investigation revealed that elevated Fzd2 expression activated canonical Wnt signaling and was blocked by canonical Wnt signaling inhibitor XAV939. However, Fzd2 did not influence the proliferation of EC cells. Thus, Fzd2 may be a potential marker for EC metastasis and a target for future therapies for this disease.

Abstract B52: PTK6 inhibition as a novel strategy to reverse EMT and suppress metastasis of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) poses clinical challenges due to intrinsic biological aggressiveness and lack of targeted therapies. Hypothesis: Protein tyrosine kinase 6 (PTK6) regulates epithelial-mesenchymal transition (EMT) of TNBC, as well as their growth and metastatic potential. We evaluated PTK6 as a promising therapeutic target to prevent metastases of TNBC. Experimental Procedures: Levels of PTK6 have prognostic significance; higher transcript levels are associated with poor outcomes for patients. We modulated the level of PTK6 in breast epithelial and TNBC cell lines, and determined effects on growth, migration, epithelial-mesenchymal transition and metastasis formation in vivo. Results: PTK6 promotes EMT; overexpression of kinase active PTK6 (Y447F) in non-transformed MCF10A cells promotes migration, suppresses expression of epithelial markers (E-cadherin, claudin-1), and increases expression of N-cadherin and fibronectin, markers of mesenchymal cells. Promotion of EMT is dependent on PTK6 kinase activity as overexpression of catalytically inactive PTK6 (K219M) failed to induce EMT. Downregulation of PTK6 in mesenchymal TNBC cells (human MDA-MB231 or murine MMTV-myc) restores epithelial properties and suppresses migration and growth in 3-D MatrigelTM cultures. PTK6 inhibition-induced E-cadherin expression is due to decreased expression of Snail, a transcriptional repressor of E-cadherin. PTK6 downregulation promotes the proteasome-dependent degradation of Snail protein, via a mechanism that is independent of GSK3β activity. PTK6 inhibition promotes anoikis of TNBC cells in vitro and prevents metastases in vivo; decreased metastasis formation is directly linked to PTK6-dependent regulation of EMT and E-cadherin levels. Conclusions: Our studies support PTK6 inhibition as a novel strategy to impair metastasis of TNBC cells via reversal of mesenchymal properties. The kinase dependency of EMT regulation by PTK6 highlight the potential for clinical translation of recently developed PTK6 kinase inhibitors as a therapeutic strategy for patients with triple negative breast cancers. Citation Format: Koichi Ito, Sun Hee Park, Hanna Y. Irie. PTK6 inhibition as a novel strategy to reverse EMT and suppress metastasis of triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B52.

Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression.

The closely related T-box transcription factors TBX2 and TBX3 are frequently overexpressed in melanoma and various types of human cancers, in particular, breast cancer. The overexpression of TBX2 and TBX3 can have several cellular effects, among them suppression of senescence, promotion of epithelial–mesenchymal transition, and invasive cell motility. In contrast, loss of function of TBX3 and most other human T-box genes causes developmental haploinsufficiency syndromes. Stephens and colleagues (1), by exome sequencing of breast tumor samples, identified five different mutations in TBX3, all affecting the DNA-binding T-domain. One in-frame deletion of a single amino acid, p.N212delN, was observed twice. Due to the clustering of these mutations to the T-domain and for statistical reasons, TBX3 was inferred to be a driver gene in breast cancer. Since mutations in the T-domain generally cause loss of function and because the tumorigenic action of TBX3 has generally been attributed to overexpression, we determined whether the putative driver mutations had loss- or gain-of-function properties. We tested two in-frame deletions, one missense, and one frameshift mutant protein for DNA-binding in vitro, and for target gene repression in cell culture. In addition, we performed an in silico analysis of somatic TBX mutations in breast cancer, collected in The Cancer Genome Atlas (TCGA). Both the experimental and the in silico analysis indicate that the observed mutations predominantly cause loss of TBX3 function.

Abstract B75: CCL2 promotes epithelial-mesenchymal transition (EMT) in prostate cancer cells via downregulation of dual-specificity phosphatase 22

Abstract CCL2 is a member of the CC chemokine superfamily that plays a crucial role in mediating macrophage recruitment and prostate cancer (PCa) metastasis. We have discovered that inhibiting the function of androgen receptor (AR) in prostate cancer cells results in the upregulation CCL2 and promotes disease progression by recruiting macrophages, activating signal transducer and activator of transcription 3 (STAT3), and by favoring the epithelial-to-mesenchymal transition. However, the molecular switches controlling CCL2 downstream signaling in PCa cells are not well understood. Here, we established an in vitro cell migration screening system using transwell migration chambers, and identified a higher amount of CCL2 in migrating PCa cells, establishing a link between CCL2 and a subpopulation of PCa cells. Importantly, we found that CCL2 expression correlated with the EMT marker, N-cadherin in migrating PCa cells. Importantly, CCL2 stimulation in PCa cells enhanced the expression levels of several EMT markers and induced STAT3 activation. In addition, CCL2 stimulation in PCa cells induced the downregulation of dual-specificity phosphatase 22 (DUSP22), which has been shown to act as a negative regulator of the IL-6-induced STAT3 activation through a direct interaction with STAT3. This supports that CCL2-induced DUSP22 downregulation in PCa cells as a potential mechanism to promote EMT. Therefore, we believe CCL2 may promote PCa metastasis in an autocrine manner through promotion of EMT in PCa cells with more aggressive motility behaviors. Targeting of the CCL2-STAT3 axis may offer new approaches to suppress the multifaceted support (immunosuppression, macrophage recruitment, and angiogenesis) of CCL2 for PCa cells in complex tumor microenvironment. Citation Format: Wen-Jye Lin, Kouji Izumi, Showping Lin, Hui-Min Ho. CCL2 promotes epithelial-mesenchymal transition (EMT) in prostate cancer cells via downregulation of dual-specificity phosphatase 22. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B75.

Twist induces epithelial-mesenchymal transition in cervical carcinogenesis by regulating the TGF-β/Smad3 signaling pathway.

Epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P<0.05). Depletion of Twist by stable shRNA-mediated knockdown decreased the migratory ability of cancer cell lines in vitro. Suppression or overexpression of Twist also resulted in an altered expression of the molecular mediators of EMT. Furthermore, exogenous TGF-β promoted EMT by upregulating the expression of Twist through the TGF-β/Smad3 pathway, and this effect was eliminated by Twist depletion in cancer cells as demonstrated in the in vitro study. The use of in vivo models revealed a decreased tumor proliferation potential in Twist-depleted cancer cells. The results suggested a novel function for Twist in the promotion of EMT via TGF-β/Smad3 signaling pathway. Thus, Twist constitutes a potential therapeutic target in human cervical cancer.

ZEB2/SIP1 as novel prognostic indicator in eyelid sebaceous gland carcinoma.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and metastasis in various malignancies. ZEB2/SIP1 is an important EMT regulator and down-regulates E-cadherin expression. The present study was planned to explore status of EMT-associated markers ZEB2/SIP1 and E-cadherin in eyelid sebaceous gland carcinoma (SGC) and to correlate with clinicopathological high-risk features. Expressions of ZEB2 and E-cadherin were evaluated by immunohistochemistry in 65 cases of histopathologically proven eyelid SGC. The results were correlated with clinicopathological high-risk features and survival of the patients to determine the prognostic significance of ZEB2, E-cadherin, and various high-risk features. Cytoplasmic overexpression of ZEB2 and membranous loss of E-cadherin were seen in 68% and 66% of cases of eyelid SGC, respectively. ZEB2 overexpression was significantly associated with E-cadherin loss (P = .002). Overexpression of ZEB2 also showed significant association with lymph node metastasis (P = .046), orbital invasion (P = .049), large tumor size (P = .018), and advanced tumor stages (P = .036). Survival analysis revealed that patients with ZEB2 overexpression had poor survival. ZEB2 overexpression and orbital invasion were found to be independent prognostic indicators (univariate analysis). However, multivariate analysis showed that ZEB2 (hazard ratio, 0.094; 95% confidence interval, 00.012-0.709; P = .022) was the best poor prognostic indicator of eyelid SGC. Our study demonstrates the role of both ZEB2 and E-cadherin in the promotion of EMT in eyelid SGC. The outcome of this study also points toward ZEB2 as an independent prognostic marker as well as a potential therapeutic target in eyelid SGC.

Expression of BMI1 and ZEB1 in epithelial-mesenchymal transition of tongue squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) is a crucial event required for the invasion and progression of carcinogenesis, inducing stem-like properties in epithelial cells. In the present study, the expression of BMI1, which controls self-renewal in stem cells, as well as that of ZEB1, a transcription factor that regulates EMT, was evaluated for its role in EMT and the carcinogenic processes of tongue squamous cell carcinoma (TSCC). Collagen invasion assays using two TSCC cells and 64 tongue specimens (32 carcinomas and 32 dysplasias) were employed and analyzed in the present study. We assessed the protein and mRNA expression levels of BMI1, ZEB1, vimentin and E-cadherin in the two cell lines and tumor tissues. The protein and mRNA expression of BMI1 and ZEB1 occurred at the invasion of TSCC. The elevated levels of BMI1 and ZEB1 were accompanied by the downregulation of E-cadherin and upregulation of vimentin at the invasive front, indicative of EMT in vitro and in vivo. The results showed that BMI1 and ZEB1 are important factors in association with the promotion of EMT and invasion of TSCC.

Combined targeting of high‐mobility group box‐1 and interleukin‐8 to control micrometastasis potential in gastric cancer

Micrometastasis is the major cause of treatment failure in gastric cancer (GC). Because epithelial-to-mesenchymal transition (EMT) is considered to develop prior to macroscopic metastasis, EMT-promoting factors may affect micrometastasis. This study aimed to evaluate the role of extracellular high-mobility group box-1 (HMGB1) in EMT and the treatment effect of combined targeting of HMGB1 and interleukin-8 (IL-8) at early-stage GC progression through interrupting EMT promotion. Extracellular HMGB1 was induced by human recombinant HMGB1 and pCMV-SPORT6-HMGB1 plasmid transfection. EMT activation was evaluated by immunoblotting, immunofluorescence and immunohistochemistry. Increased migration/invasion activities were evaluated by in vitro transwell migration/invasion assay using all histological types of human GC cell lines (N87, MKN28 SNU-1 and KATOIII), N87-xenograft BALB/c nude mice and human paired serum-tissue GC samples. HMGB1-induced soluble factors were measured by chemiluminescent immunoassay. Inhibition effects of tumor growth and EMT activation by combined targeting of HMGB1 and IL-8 were evaluated in N87-xenograft nude mice. Serum HMGB1 increases along the GC carcinogenesis and reaches maximum before macroscopic metastasis. Overexpressed extracellular HMGB1 promoted EMT activation and increased cell motility/invasiveness through ligation to receptor for advanced glycation end products. HMGB1-induced IL-8 overexpression contributed the HMGB1-induced EMT in GC in vitro and in vivo. Blocking HMGB1 caused significant reduction of tumor growth, and addition of human recombinant IL-8 rescues this antitumor effects. Our results imply the role of HMGB1 in EMT through IL-8 mediation, and a potential mechanism of GC micrometastasis. Our observations suggest combination strategy of HMGB1 and IL-8 as a promising diagnostic and therapeutic target to control GC micrometastasis.