OBJECTIVE

The aims of this study were to investigate the association of renal cell carcinoma (RCC) displaying rhabdoid features and morphologically mesenchymal characteristics with epithelial to mesenchymal transition (EMT), and to clarify the expression of EMT markers.

METHODS

We investigated the expression of EMT markers (E-cadherin, vimentin, Snail, Slug, ZEB1, ZEB2 and Twist1) using immunohistochemistry, Western blotting and real-time polymerase chain reaction in 18 cases of clear cell RCC (ccRCC) with rhabdoid features and 74 ccRCC cases with Fuhrman grade 1-3 (G1 to G3).

RESULTS

In ccRCCs with rhabdoid features, low E-cadherin and high vimentin expression were found. In G1 to G3 ccRCCs, low E-cadherin expression and high expression of vimentin, ZEB1 and ZEB2 were found. There was no significant difference in the immunoexpression of E-cadherin and vimentin between the two ccRCC groups.

CONCLUSIONS

The rhabdoid features may histologically and biologically be associated with EMT in ccRCC. There is a possibility that in G1 to G3 ccRCCs showing epithelial structures, other cell-cell adhesion mechanisms apart from E-cadherin adhesion may continue to work, and that ccRCC with rhabdoid features may be caused by an inactivation or loss of these mechanisms.

MicroRNAs have been found ectopically expressed in many cancers and play essential roles in tumor EMT progress. Recent studies identified decreased miR-940 expression in glioma cells and may serve as a tumor-suppressor. However, whether miR-940 involve in glioma EMT remain poorly understood. Here we confirmed that miR-940 was significantly reduced in glioma cells and tissues. Introduction of miR-940 dramatically suppressed invasion and migration of glioma cells. Gain-of-function experiments showed ZEB2 as a direct target of miR-940, knockdown of ZEB2 evidently repressed invasive capacity of glioma cells through EMT. Moreover, reintroduction of ZEB2 effectively reversed the tumor suppressive effect of miR-940 treatment. In vivo study showed reduced tumor cell motion in miR-940-injected groups. Spearman's correlation analysis indicated inversely correlated expression of ZEB2 and miR-940 in gliomas and NBTs. Altogether, miR-940-ZEB2 cascade may play important roles in glioma cells invasion and EMT progression, and might provide new therapeutic approaches for better outcomes of GBM patients.

Accumulating data have shown the involvement of microRNAs (miRNAs) in endometriosis pathogenesis. In this study, we used a novel approach to determine the endometriotic lesion-specific miRNAs by high-throughput small RNA sequencing of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissues together with eutopic endometria of the same patients. We found five miRNAs specific to epithelial cells--miR-34c, miR-449a, miR-200a, miR-200b and miR-141 showing significantly higher expression in peritoneal endometriotic lesions compared to healthy peritoneal tissues. We also determined the expression levels of miR-200 family target genes E-cadherin, ZEB1 and ZEB2 and found that the expression level of E-cadherin was significantly higher in endometriotic lesions compared to healthy tissues. Further evaluation verified that studied miRNAs could be used as diagnostic markers for confirming the presence of endometrial cells in endometriotic lesion biopsy samples. Furthermore, we demonstrated that the miRNA profile of peritoneal endometriotic lesion biopsies is largely masked by the surrounding peritoneal tissue, challenging the discovery of an accurate lesion-specific miRNA profile. Taken together, our findings indicate that only particular miRNAs with a significantly higher expression in endometriotic cells can be detected from lesion biopsies, and can serve as diagnostic markers for endometriosis.

Gallbladder cancer (GBC) is one of the most unfavorable prognostic tumor, and immediate growth and distant metastasis are important factors associated with the poor prognosis of patients with this disease. Standard and variant isoforms of CD44 are associated with tumor growth, metastasis, and epithelial-mesenchymal transition (EMT), although their roles in GBC are unclear. We investigated the relationship between the CD44 isoforms with EMT, chemotaxis, and tumorigenicity. We analyzed CD44 expression in the GBC cell line NOZ and found that it comprises a major population that expressed CD44std+/CD44v9- (CD44s) and the minor population that expressed CD44std-/CD44v9+ (CD44v). CD44s cells exhibited increased chemotaxis and invasiveness compared with CD44v cells in in vitro cell migration and invasion assays. CD44s cells expressed higher and lower levels of mRNAs that encode vimentin and E-cadherin, respectively, compared with those of CD44v cells. CD44s cells expressed high levels of the transcription factors ZEB1 and ZEB2 that mediate EMT, and low levels of a splicing factor ESRP1 that controls the CD44 isoform switch. We performed in vivo mouse xenotransplantation analyses of CD44s and CD44v cells and found that CD44v cells exhibited relatively increased tumorigenicity. Immunohistochemical analysis of tissue microarrays revealed that high levels of CD44v9 and CD44std were associated with poorer prognosis. The expression of CD44std was also associated with poorly differentiated tumors and distant metastasis. In conclusion, CD44s was associated with a mesenchymal phenotype, increased chemotaxis and invasiveness, and decreased tumorigenicity. In contrast, CD44v cells exhibited an epithelial phenotype, decreased chemotaxis, decreased invasiveness, and increased tumorigenicity. These findings suggest that CD44v and CD44s cells play differently important roles in the progression and metastasis of GBC and the isoform switch triggers EMT.

Modern treatment strategies provide better overall survival in cancer patients, primarily by controlling tumor growth. However, off-target and systemic toxicity, tumor recurrence, and resistance to therapy are still inadvertent hurdles in current treatment regimens. Similarly, metastasis is another deadly threat to patients suffering from cancer. This has created an urgent demand to come up with new drugs having anti-metastatic potential and minimum side effects. Thus, this study was aimed at exploring the anti-proliferative and anti-metastatic potential of colocynth medicinal plant. Results from MTT assay, morphological visualization of cells and scratch assay indicated a role of ethanol and acetone extracts of fruit pulp of the colocynth plant in inhibiting cell viability, enhancing cell cytotoxicity and preventing cell migration in various cancer cell types, including breast cancer cell lines MCF-7 and MDA-MB-231, and cervical cancer cell line SiHa, subsequently having a low cytotoxic effect on mononuclear PBMC and macrophage J774A cells. Our study in metastatic MDA-MB-231 cells showed that both ethanol and acetone pulp extracts decreased transcript levels of the anti-apoptotic genes BCL2 and BCLXL, and a reverse effect was observed for the pro-apoptotic genes BAX and caspase 3. Additionally, enhanced caspase 3 activity and downregulated BCL2 protein were seen, indicating a role of these extracts in inducing apoptotic activity. Moreover, MDA-MB-231 cells treated with both these extracts demonstrated up-regulation of the epithelial gene keratin 19 and down-regulation of the mesenchymal genes, vimentin, N-cadherin, Zeb1 and Zeb2 compared to control, suggesting a suppressive impact of these extracts in epithelial to mesenchymal transition (EMT). In addition, these extracts inhibited colony and sphere formation with simultaneous reduction in the transcript level of the stemness associated genes, BMI-1 and CD44. It was also found that both the plant extracts exhibited synergistic potential with the chemotherapeutic drug doxorubicin to inhibit cancer viability. Furthermore, GC-MS/MS analysis revealed the presence of certain novel compounds in both the extracts that are responsible for the anti-cancer role of the extracts. Overall, the results of this report suggest, for the first time, that colocynth fruit pulp extracts may block the proliferative as well as metastatic activity of breast cancer cells.

Background: Osteosarcoma (OS) is a type of malignant bone tumor with a growing incidence. Increasing studies indicate circular RNA (circRNA) has a vital function in tumorigenesis. Yet, how circRNA regulates OS development is not clear. In the present work, we aimed to investigate the roles of hsa_circ_0136666 in OS progression.

Results: hsa_circ_0136666 was shown to be upregulated in OS and correlated with advanced stage and poor prognosis. Functional investigation using CCK8, colony formation assay and Transwell assay demonstrated that hsa_circ_0136666 promoted OS proliferation, migration and invasion, but inhibited cell death. Additionally, we identified hsa_circ_0136666 was a molecular sponge for miR-593-3p to facilitate ZEB2 expression. MiR-593-3p and ZEB2 were inversely expressed in OS tissues. And hsa_circ_0136666 exerts oncogenic roles in OS relying on miR-593-3p and ZEB2.

Conclusion: Our results demonstrate the involvement of hsa_circ_0136666 in regulating OS tumorigenesis and it may be a therapeutic target.

Methods: The expression of hsa_circ_0136666 was analyzed by qRT-PCR in OS tissues and cell lines. Proliferation was measured via CCK8 and colony formation assays. Migration and invasion were determined through Transwell assay. Luciferase reporter assay was utilized to determine the interaction between hsa_circ_0136666 and miR-593-3p or between miR-593-3p and ZEB2. Animal experiment was performed to investigate the role of hsa_circ_0136666 in vivo.

Keywords: ZEB2; hsa_circ_0136666; miR-593-3p; osteosarcoma.

Aims: Hepatic capillaries are lined with specialised liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialisation is co-determined by transcription factors. The Zinc-Finger E-Box-binding Homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis.

Methods and results: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarisation'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarisation, HSC activation and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis.

Conclusion: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

Keywords: Capillarisation; Intussusceptive angiogenesis; Liver fibrosis; Liver sinusoidal endothelial cells; Zeb2.

Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 co-expression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS.

Factor analyses of attention-deficit/hyperactivity (ADHD) symptoms divide the behavioral symptoms of ADHD into two separate domains, one reflecting inattention and the other, a combination of hyperactivity and impulsivity. Identifying domain-specific genetic risk variants may aid in the discovery of specific biological risk factors for ADHD. In contrast with data available on genes involved in hyperactivity and impulsivity, there is limited information on the genetic influences of inattention. Transcriptional profiling analysis in animal models of disorders may provide an important tool to identify genetic involvement in behavioral phenotypes. To explore some of the potential genetic underpinnings of ADHD inattention, we examined common differentially expressed genes (DEGs) in the prefrontal cortex of SHR/NCrl, the most validated animal model of ADHD and WKY/NCrl, animal model of ADHD-inattentive type. In contrast with Wistar rats, strain representing the "normal" heterogeneous population, SHR/NCrl and WKY/NCrl showed inattention behavior in the Y-maze task. The common DEGs in the PFC of SHR/NCrl and WKY/NCrl vs. Wistar rats are those involved in transcription (e.g. Creg1, Thrsp, Zeb2), synaptic transmission (e.g. Atp2b2, Syt12, Chrna5), neurological system process (e.g. Atg7, Cacnb4, Grin3a), and immune response (e.g. Atg7, Ip6k2, Mx2). qRT-PCR analyses validated expression patterns of genes representing the major functional gene families among the DEGs (Grin3a, Thrsp, Vof-16 and Zeb2). Although further studies are warranted, the present findings indicate novel genes associated with known functional pathways of relevance to ADHD which are assumed to play important roles in the etiology of ADHD-inattentive subtype.

OBJECTIVE

The aim of the present work was to investigate the mechanism of transforming growth factor (TGF)-β1 and Sloan-Kettering Institute (Ski) in the pathogenesis of hypertrophic scars (HS).

BACKGROUND

Wound healing is an inherent process, but the aberrant wound healing of skin injury may lead to HS. There has been growing evidence suggesting a role for TGF-β1 and Ski in the pathogenesis of fibrosis.

METHODS

The MTT assay was used to detect the cell proliferation induced by TGF-β1. The Ski gene was transduced into cells with an adenovirus, and then the function of Ski in cell proliferation and differentiation was observed. Ski mRNA levels were measured by RT-PCR. Western blotting was used to detect the protein expression of α-SMA, E-cadherin, Meox1, Meox2, Zeb1 and Zeb2.

RESULTS

TGF-β1 can promote human skin fibroblast (HSF) cell proliferation in a time-dependent manner, but the promoting effect could be suppressed by Ski. TGF-β1 also induces the formation of the myofibroblast phenotype and the effect of TGF-β1 could be diminished by Ski. Also, Ski modulates the cardiac myofibroblast phenotype and function through suppression of Zeb2 by up-regulating the expression of Meox2.

CONCLUSIONS

Ski diminishes the myofibroblast phenotype induced by TGF-β1 through the suppression of Zeb2 by up-regulating the expression of Meox2.

BACKGROUND Epithelial-mesenchymal transition (EMT) is an essential progress for tumor cell invasion to both epithelial and non-epithelial cancers, and zinc finger E-box-binding homeobox 1/2 (ZEB1/2) is a well-known promoter of EMT. In glioma cell lines, both ZEB1 and ZEB2 have been demonstrated to facilitate cancer cell proliferation and invasion with experiments in vitro. However, the clinical significance of ZEB1 and ZEB2 in glioblastoma (GBM) is still controversial. MATERIAL AND METHODS We detected the expression of ZEB1 and ZEB2 in 91 cases of GBM with immunohistochemistry and investigated the correlation between clinicopathological factors and ZEB family expression with Fisher test. By univariate analysis with Kaplan-Meier test, we explored the prognostic significance of ZEB1/2 expression and the clinicopathological factors in GBM. By multivariate analysis with the Cox regression model, we identified the independent prognostic factors in GBM. RESULTS The percentages of ZEB1 high expression and ZEB2 high expression were 31.9% (29/91) and 41.9% (36/91), respectively. High expression of ZEB2 was significantly associated with lower survival rate of GBM patients (P=0.001). ZEB2, lower KPS score (P=0.004), gross total resection (P<0.001) and higher Ki67 percentage (P=0.001) were notably correlated to worse prognosis of GBM. With multivariate analysis, high expression of ZEB2 was demonstrated to be an independent prognostic factor indicating unfavorable prognosis of GBM (P=0.001, HR=3.86, and 95%CI=1.61-9.23). CONCLUSIONS High expression of ZEB2 is an independent prognostic factor predicting unfavorable prognosis of GBM, indicating that ZEB2 or its downstream proteins may be potential drug targets of GBM therapy.

Neurogenesis is the basis of stem cell tissue engineering and regenerative medicine for central nervous system (CNS) disorders. We have established differentiation protocols to direct human periodontal ligament-derived stem cells (PDLSCs) into neuronal lineage, and we recently isolated the neural crest sub-population from PDLSCs, which are pluripotent in nature. Here we report the neural differentiation potential of these periodontal ligament-derived neural crest stem cells (NCSCs) as well as its microRNA (miRNA) regulatory mechanism and function in NCSC neural differentiation. NCSCs, treated with basic fibroblast growth factor and epidermal growth factor-based differentiation medium for 24 days, expressed neuronal and glial markers (βIII-tubulin, neurofilament, NeuN, neuron-specific enolase, GFAP and S100) and exhibited glutamate-induced calcium responses. The global miRNA expression profiling identified 60 upregulated and 19 downregulated human miRNAs after neural differentiation, and the gene ontology analysis of the miRNA target genes confirmed the neuronal differentiation-related biological functions. In addition, overexpression of miR-132 in NCSCs promoted the expression of neuronal markers and downregulated ZEB2 protein expression. Our results suggested that the pluripotent NCSCs from human periodontal ligament can be directed into neural lineage, which demonstrate its potential in tissue engineering and regenerative medicine for CNS disorders.

UNASSIGNED

It is clearly necessary to discover prognostic biomarkers to identify stage I patients at risk of recurrence and give them timely postoperative treatment.

UNASSIGNED

Data of stage I lung adenocarcinoma were retrieved from four gene series in Gene Expression Omnibus (GEO) database (GSE50081, GSE30219, GSE37745, and GSE13213). Partek Genomics Suite software was used to identify survival-related genes for finding candidate indicators for early-stage patients at risk of recurrence. Differential expression of MTBP (MDM2 binding protein) in early-stage lung adenocarcinoma tissues was determined by immunohistochemical staining. The effects of MTBP interference expression and overexpression on viability, migration, and invasion capacity of lung cells were evaluated using Cell Counting Kit-8, wound healing, and Transwell assays. The tumor growth and lung metastasis in vivo were observed in chick embryo chorioallantoic membrane model. Human Exon 2.0 ST Array was used to analyze downstream regulation genes of MTBP in lung cancer cells. Involvement of ZEB2 and epithelial-mesenchymal transition (EMT) markers was investigated by Western blot.

UNASSIGNED

By mining GEO database, we identified MTBP as a poor prognostic indicator of stage I lung adenocarcinomas. In addition, increased expression of MTBP was also associated with poor survival in our early-stage lung adenocarcinoma cohort. Further experiment suggested that knockdown of MTBP suppressed the migration and invasion of A549 and H1975 cells in vitro and in vivo, whereas overexpression of MTBP in HCC827 and PC9 cells promoted the migration and invasion in vitro and in vivo. Furthermore, ZEB2 upregulation directly activated EMT to mediate the downstream effects of MTBP involved in lung cancer cells metastasis.

UNASSIGNED

MTBP is an independent indicator for poor prognosis in stage I lung adenocarcinomas and might promote the aggressive phenotype of non-small-cell lung cancer by inducing the EMT process through upregulating ZEB2 expression.

To investigate correlation relationship between serum miR-200b, miR-200c, miR-141 levels with liver metastases in colorectal cancer patients.

A total of 85 colorectal cancer patients with liver metastases and 54 colorectal cancer patients without liver metastases were divided into experimental group and control. Serum sample was collected before surgery and tested by Real-time PCR to evaluate miR-200b, miR-200c, miR-141 expressions.

The primary site of cancer of two groups was mostly derived from the colon cancer among the control and experimental group (there was no significant difference between the control group (61.1%) and experimental group (63.5%). Most of the invasive depth in the control group was T3 phase (50%), and most of the invasive depth in the experimental group was T4 phase (69.4%); therefore, the difference of the invasive depth between the control and experimental group is significant (p < 0.05). The majority of cases in experimental group were in stage IV, while they were in stage II in control. MiR-200b, miR-200c and miR-141 relate with tumor metastasis through epithelial mesenchymal transition (ETM) pathway, and target ZEB1 and ZEB2 genes. MiR-200b, miR-200c, and miR-141 have been confirmed to be related to tumor metastasis. miR-141 levels in serum from experimental group was higher significant compared to control group (p = 0.024). miR-200b levels in serum from experimental group was significantly increased compared to control group (p = 0.031). The miR-200c levels in serum from experimental group were significantly higher compared to control group (p = 0.015). Meanwhile, serum miR-141, miR-200b, miR-200c abnormal expressions in serum were related to tumor occurrence and development. Their levels were positively correlated with liver metastasis.

The probability of liver metastases in colorectal cancer patients was positively correlated with serum miR-141, miR-200b, and miR-200c expressions, which could be treated as new biomarker for early diagnosis of liver metastases in colorectal cancer.

MicroRNA-187 (miR-187) has been reported to be involved in the occurrence and development of several types of cancers; however, a role for miR-187 in osteosarcoma (OS) has not yet been reported. Here, miR-187 was found to be significantly downregulated in OS cell lines and tissue samples, and decreased miR-187 expression was shown to be correlated closely with the TNM stage and lymph node metastasis. miR-187 overexpression suppressed OS cell proliferation, colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT). Mechanically, zinc finger E-box binding homeobox 2 (ZEB2) was shown to serve as a direct target of miR-187 in OS cells and the overexpression of ZEB2 rescued the miR-187-induced suppression of proliferation, colony formation, migration, and invasion in OS cells. In clinical OS specimens, ZEB2 expression levels were elevated and were inversely correlated with miR-187 expression. These results suggest that miR-187 functions as a tumor suppressor in OS, partially by targeting ZEB2, and that miR-187 can serve as a promising candidate for OS.

MicroRNAs (miRNAs) play an essential role in the tumorigenesis of gastric carcinoma (GC). MiR-429 has been recently reported to inhibit GC growth; however, whether miR-429 may also regulate the invasion of GC cells is unknown. Here, we showed that miR-429 levels were significantly decreased and ZEB1 and ZEB2 levels were significantly increased in GC specimens, compared to the paired adjacent non-tumor gastric tissue. Moreover, the levels of miR-429 and ZEB1 or ZEB2 inversely correlated in GC specimens. Bioinformatics analyses showed that miR-429 targeted the 3'-untranslated region of both ZEB1 and ZEB2 mRNA to inhibit their translation, which was confirmed by luciferase reporter assay. Overexpression of miR-429 inhibited ZEB1/2-mediated cell invasiveness, while depletion of miR-429 augmented it. Together, our data suggest that miR-429 suppression in GC promotes ZEB1/2-mediated cancer cell invasion.

FAT10, an ubiquitin-like protein, functions as a potential tumor promoter in several caners. However, the function and clinical significance of FAT10 in breast cancer (BC) remains unclear. Here, we found that high FAT10 expression was detected frequently in primary BC tissues, and was closely associated with malignant phenotype and shorter survival among the BC patients. Multivariate analyses also revealed that FAT10 overexpression was independent prognostic factors for poor outcome of patients with BC. Function assay demonstrated that FAT10 knockdown significantly inhibited the metastasis abilities and the epithelial-mesenchymal transition (EMT) of breast cancer cell. Further investigation revealed that FAT10 directly bound ZEB2 and decreased its ubiquitination to enhance the protein stability of ZEB2 in BC cells. Moreover, our data shown that the pro-metastasis effect of FAT10 in BC is partially dependent on ZEB2 enhancement. Collectively, our data suggest that FAT10 plays a crucial oncogenic role in BC metastasis, and we provide a novel evidence that FAT10 may be serve as a prognostic and therapeutic target for BC patients.

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths. Early detection of tumor relapse is crucial for determining the most appropriate therapeutic management. In clinical practice, computed tomography (CT) is routinely used, but small tumor changes are difficult to visualize, and reliable blood-based prognostic and monitoring biomarkers are urgently needed. The aim of this study was to prospectively validate a gene expression panel (composed of GAPDH, VIL1, CLU, TIMP1, TLN1, LOXL3 and ZEB2) for detecting circulating tumor cells (CTCs) as prognostic and predictive tool in blood samples from 94 metastatic CRC (mCRC) patients. Patients with higher gene panel expression before treatment had a reduced progression-free survival (PFS) and overall-survival (OS) rates compared with patients with low expression (p = 0.003 and p ≤ 0.001, respectively). Patients with increased expression of CTCs markers during treatment presented PFS and OS times of 8.95 and 11.74 months, respectively, compared with 14.41 and 24.7 for patients presenting decreased expression (PFS; p = 0.020; OS; p ≤ 0.001). Patients classified as non-responders by CTCs with treatment, but classified as responders by CT scan, showed significantly shorter survival times (PFS: 8.53 vs. 11.70; OS: 10.37 vs. 24.13; months). In conclusion, our CTCs detection panel demonstrated efficacy for early treatment response assessment in mCRC patients, and with increased reliability compared to CT scan.

OBJECTIVE

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) insufficiency is commonly found in breast cancer patients with metastasis. We investigated the mechanisms by which PTEN affects breast cancer metastatic behavior.

METHODS

Migration and invasion assay, western blot, immunofluorescent staining and zebrafish animal model were applied.

RESULTS

We showed that PTEN insufficiency induced an increase in MCF-7 cell migration and invasion through induction of epithelial-mesenchymal transition (EMT), which was triggered by up-regulation of the EMT-inducing transcriptional factors Zeb1, Zeb2, Snail, Slug and Twist. Simultaneously, E-cadherin expression was inhibited and P-cadherin was up-regulated. Further, WNT1 inducible signaling pathway protein 1 (WISP1) and lipocalin-2 (LCN2) expressions were increased after PTEN knockdown in MCF-7 cells, which also exhibited increased filamentous actin (F-actin) synthesis and extracellular matrix metalloproteinase-2 (MMP-2) and MMP-9 expression. We further showed that PTEN knockdown in MCF-7 cells could increase cell migration in the xenograft zebrafish model.

CONCLUSIONS

Our findings reveal new therapeutic targets for breast cancer patients with PTEN insufficiency.

BACKGROUND

Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors.

METHODS

RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors.

RESULTS

Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFβ1 and TGFβ2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection.

CONCLUSIONS

The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

Mowat-Wilson syndrome (MWS) is a rare genetic syndrome characterized by a specific facial gestalt, intellectual deficiency, Hirschsprung disease and multiple congenital anomalies. Heterozygous mutations or deletions in the zinc finger E-box-binding homeobox2 gene (ZEB2) cause MWS. ZEB2 encodes for Smad-interacting protein 1, a transcriptional co-repressor involved in TGF-beta and BMP pathways and is strongly expressed in early stages of development in mice. Eye abnormalities have rarely been described in patients with this syndrome. Herein, we describe four patients (two males and two females; mean age 7 years) with MWS and eye malformations. Ocular anomalies included, iris/retinal colobomas, atrophy or absence of the optic nerve, hyphema, and deep refraction troubles, sometimes with severe visual consequences. All eye malformations were asymmetric and often unilateral and all eye segments were affected, similarly to the nine MWS cases with ophthalmological malformations previously reported (iris/chorioretinal/optic disc coloboma, optic nerve atrophy, retinal epithelium atrophy, cataract, and korectopia). In human embryo, ZEB2 is expressed in lens and neural retina. Using the present report and data from the literature, we set out to determine whether or not the presence of eye manifestations could be due to specific type or location of mutations. We concluded that the presence of eye malformations, although a rare feature in MWS, should be considered as a part of the clinical spectrum of the condition.

RNA-sequencing of a case of acute myeloid leukemia with the bone marrow karyotype 46,XY,t(2;14)(q22;q32)[5]/47,XY,idem,+?4,del(6)(q13q21)[cp6]/46,XY[4] showed that the t(2;14) generated a ZEB2-BCL11B chimera in which exon 2 of ZEB2 (nucleotide 595 in the sequence with accession number NM_014795.3) was fused to exon 2 of BCL11B (nucleotide 554 in the sequence with accession number NM_022898.2). RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript. All functional domains of BCL11B are retained in the chimeric protein. Abnormal expression of BCL11B coding regions subjected to control by the ZEB2 promoter seems to be the leukemogenic mechanism behind the translocation.

Recent studies have demonstrated that neural precursor cell expressed, developmentally downregulated 9 (NEDD9) is highly expressed in various tumor tissues and cell lines. However, research on the role of NEDD9 in gastric cancer (GC) is rare, and the potential mechanism in tumor progression has not yet been explored. In this study, we investigated the role and mechanism of NEDD9 in GC. The expression of NEDD9 in GC tissues and cell lines was measured by immunohistochemistry, qRT-PCR, and Western blot, respectively. Inhibiting NEDD9 expression was carried out by siRNA transfection, and upregulating of NEDD9 was via NEDD9 overexpression plasmid. The ability of proliferation, migration, and invasion was detected by MTT assay, scratch wound assay, and transwell assay, respectively. The expression of vimentin, E-cadherin, Zeb1, and Zeb2 was measured by Western blot and qRT-PCR. We found that NEDD9 expression was dramatically increased both in GC tissues and cell lines, and the expression was significantly related to GC development. Knockdown of NEDD9 in SGC-7901 strongly inhibited its malignant capacity in vitro. Meanwhile, upregulation of NEDD9 in GES-1 increased the malignant capacity. In addition, the expression of vimentin, Zeb1, and Zeb2 was positively correlated with NEDD9, while E-cadherin was opposite. Collectively, our findings suggest that NEDD9 acts as an oncogene and promotes GC metastasis via EMT.