PTGER3 knockdown inhibits the vulnerability of triple-negative breast cancer to ferroptosis.

Prostaglandin E receptor 3 (PTGER3) is involved in a variety of biological processes in the human body and is closely associated with the development and progression of a variety of cancer types. However, the role of PTGER3 in triple-negative breast cancer (TNBC) remains unclear. In the present study, low PTGER3 expression was found to be associated with poor prognosis in TNBC patients. PTGER3 plays a crucial role in regulating TNBC cell invasion, migration, and proliferation. Upregulation of PTGER3 weakens the epithelial-mesenchymal phenotype in TNBC and promotes ferroptosis both in vitro and in vivo by repressing glutathione peroxidase 4 (GPX4) expression. On the other hand, downregulation of PTGER3 inhibits ferroptosis by increasing GPX4 expression and activating the PI3K-AKT pathway. Upregulation of PTGER3 also enhances the sensitivity of TNBC cells to paclitaxel. Overall, this study has elucidated critical pathways in which low PTGER3 expression protects TNBC cells from undergoing ferroptosis, thereby promoting its progression. PTGER3 may thus serve as a novel and promising biomarker and therapeutic target for TNBC.

TAZ promotes vasculogenic mimicry in gastric cancer through the upregulation of TEAD4.

BACKGROUND AND AIM: Vasculogenic mimicry (VM) is a unique blood supply pattern in malignant tumors that is closely associated with metastasis and poor prognosis. The Hippo signaling effector TAZ is upregulated in several cancers, promoting cancer proliferation and metastasis. This study aimed to identify the function of TAZ and its regulatory mechanism in promoting VM in gastric cancer (GC). METHODS: The expression of TAZ and TEAD4 and their correlations with overall survival and VM-related markers were analyzed in 228 cases of GC. The regulatory mechanism of TAZ and its interaction with TEAD4 in epithelial-mesenchymal transition (EMT) and VM were investigated in vitro and in vivo. RESULTS: TAZ was highly expressed in GC samples and was associated with shorter patient survival time. TAZ expression was positively correlated with TEAD4 and VM in patients with GC. TAZ enhanced the migration and invasion capacity of GC cells through EMT in vitro and upregulated the expression of VM-associated proteins, including VE-cadherin, MMP2, and MMP9, thus promoting VM formation. Overexpression of TAZ accelerated the growth of subcutaneous xenograft and promoted VM formation in vivo. Co-immunoprecipitation assays showed that TAZ can directly bind to TEAD4, and in vitro experiments showed that this binding mediates the function of TAZ in regulating EMT and VM formation in GC. CONCLUSIONS: TAZ promotes GC metastasis and VM by upregulating TEAD4 expression. Our findings expand the role of TAZ in VM and provide new theoretical support for the use of antiangiogenic therapy in the treatment of advanced GC.

LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma.

Lysyl oxidase-like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid-Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.

Long noncoding RNA n339260 promotes vasculogenic mimicry and cancer stem cell development in hepatocellular carcinoma.

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self-renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high-resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)-cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.

The suppression of DUSP5 expression correlates with paclitaxel resistance and poor prognosis in basal-like breast cancer.

Basal-like breast cancer (BLBC) is resistant to endocrinotherapy and targeted therapy and new molecular therapies are needed for BLBC. In this study, we evaluated the role of DUSP1 and DUSP5, negative regulators of mitogen-activated protein kinase pathway, in the aggressiveness of BLBC. MDA-MB-231 cells were given paclitaxel (PTX) treatment and subsequently PTX resistant cell clones were established. Microarray analysis, real-time quantitative reverse transcription PCR (qRT-PCR), and online analysis of large cohorts of breast cancer patients were performed. The PTX resistant cells showed stronger cell proliferation ability by exhibiting the upregulation of CENPF, CDC6, MCM3, CLSPN and SMC1A expression. Furthermore, DUSP1 and DUSP5 expression was significantly downregulated in PTX resistant cells. In addition, in large breast cancer patients' database, both DUSP1 and DUSP5 correlated negatively with higher histological grade. DUSP1 low expression was obvious in HER2 positive and basal like while DUSP5 low expression was peculiar for basal like compared with other subtypes. Remarkably, low expression of DUSP5, but not DUSP1, was significantly correlated with poor survival of BLBC patients. In conclusion, our data suggest that loss of DUSP5 expression results in PTX resistance and tumor progression, providing a rationale for a therapeutic agent that restores DUSP5 in BLBC.

HnRNPM and CD44s expression affects tumor aggressiveness and predicts poor prognosis in breast cancer with axillary lymph node metastases.

HnRNPM is an essential splicing factor and its expression is closely correlated with invasion and metastasis of tumor cells. The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and CD44 splice variants have been implicated in specific oncogenic signaling pathways. To investigate the clinical significance and biological function of hnRNPM, immunohistochemistry, quantitative, and semiquantitative polymerase chain reaction, lentiviral transfection system and transwell invasion assays were performed. We found that hnRNPM expression was significantly upregulated in breast cancer tissues compared with benign breast lesions. Although there was no significant correlation between hnRNPM and total CD44 protein or mRNA level, there was a negative correlation between hnRNPM and CD44v6. HnRNPM and CD44s expression showed positive correlation and in particular, they were dually expressed in breast cancer tissues. Interestingly, cancer stem cells marker, ALDH1+ phenotype was positively associated with overexpression of CD44s or hnRNPM and negatively related to CD44v6. Patients with high hnRNPM tended to have higher levels of CD44s, shorter overall survival (OS) and higher rates of lymph node metastases (LNM). Remarkably, Kaplan-Meier and Cox regression analyses displayed that hnRNPM+ or CD44shigh was a poor prognostic factor for OS of patients with LNM. Upregulation of hnRNPM in MCF-7 cells caused a significant increase in cell invasion, and this effect may occur through the regulation of CD44s expression. In conclusion, overexpression of hnRNPM promotes breast cancer aggressiveness by regulating the level of CD44s, indicates a poor prognosis for patients with LNM, and has potential as therapeutic targets.

MMP-2 and MMP-13 affect vasculogenic mimicry formation in large cell lung cancer.

Matrix metalloproteinases (MMPs) have critical functions in tumour vasculogenic mimicry (VM). This study explored the mechanisms underlying MMP-13 and MMP-2 regulation of tumour VM formation in large cell lung cancer (LCLC). In our study, laminin5 (Ln-5) fragments cleaved by MMP-2 promoted tubular structure formation by the LCLC cell lines H460 and H661 in three-dimensional (3D) cultures. Transient up-regulation of MMP-13 or treatment with recombinant MMP-13 protein abrogated tubular structure formation of H460 cells in 3D culture. Treated cells with Ln-5 fragments cleaved by MMP-2 stimulated EGFR and F-actin expression. Ln-5 fragments cleaved by MMP-13 decreased EGFR/F-actin expression and disrupted VM formation. MMP-13 expression was negatively correlated with VM, Ln-5 and EGFR in LCLC tissues and xenograft. In vivo experiments revealed that VM was decreased when the number of endothelium-dependent vessels (EDVs) increased during xenograft tumour growth, whereas MMP-13 expression was progressively increased. In conclusion, MMP-2 promoted and MMP-13 disrupted VM formation in LCLC by cleaving Ln-5 to influence EGFR signal activation. MMP-13 may regulate VM and EDV formation.

OCT4 expression and vasculogenic mimicry formation positively correlate with poor prognosis in human breast cancer.

To evaluate the prognostic value of OCT4 expression and vasculogenic mimicry (VM) in human breast cancer, we examined OCT4 expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-schiff) double staining on 90 breast cancer specimens. All patients were followed up for five-149 months following surgery. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between OCT4 expression and VM formation (p<0.05). Both OCT4 expression and VM were also positively correlated with lymph node metastasis, higher histological grade, and Nottingham prognostic index (p<0.05). Patients with OCT4 expression or VM formation exhibited poorer overall survival (OS) and disease-free survival (DFS) than OCT4-negative or VM-negative patients (p<0.05). OCT4-positive/VM-positive patients also had the worst OS and DFS (p<0.05). In multivariate survival analysis, VM, Nottingham prognostic index (NPI), and Her2 were independent prognostic factors related to OS and OCT4-positive/VM-positive patients, whereas NPI and Her2 were independent predictors of DFS. These results suggest that a combined OCT4 expression/VM could improve the prognostic judgment for breast cancer patients.

UTP23 Functions in Breast Cancer Progression and Predicts Poor Prognosis of Luminal a Breast Cancer.

BACKGROUND: Luminal A breast cancer is the most common molecular subtype of breast cancer. Exploring biomarkers to identify luminal A breast cancer patients at high risk of recurrence and metastasis has important clinical significance. UTP23 is a component of ribosomal small-subunit processome, which is involved in ribosome synthesis and RNA maturation. The role of UTP23 in breast cancer has not been reported. METHODS: TCGA-BRCA data, LinkedOmics, STRING, Metascape and ssGSEA were used to analyze UTP23 expression in breast cancer and evaluate prognosis. Quantitative real-time PCR, western blot and in vitro cell experiment were used to demonstrate the role of UTP23 in breast cancer. RESULTS: UTP23 showed abnormally high expression in multiple cancers and was associated with poor prognosis. UTP23 was associated with T stage, lymph node metastasis, race, histological type, molecular subtypes and survival status in breast cancer. Importantly, UTP23 was significantly associated with poor OS in luminal A or early breast cancer, not in non-luminal A or advanced breast cancer. UTP23 expression was significantly correlated with immune cells infiltration. Enrichment analysis suggested that UTP23 might regulate cell cycle and cell division. Bioinformatics analysis showed DCAF13 might be downstream factor of UTP23. UTP23 expression promoted MCF-7 cells proliferation, migration and invasion possibly through regulating DCAF13 expression. CONCLUSIONS: UTP23 may function in breast cancer progression. The elevated UTP23 may be a potential prognostic biomarker for luminal A or early breast cancer.

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma.

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix-rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20-30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double-staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three-dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF-7 cells induced vessel-like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up-regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.

Slug promoted vasculogenic mimicry in hepatocellular carcinoma.

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial-mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.

LOXL2 serves as a prognostic biomarker for hepatocellular carcinoma by mediating immune infiltration and vasculogenic mimicry.

BACKGROUND: The development of human hepatocellular carcinoma (HCC) is a multistep process that is accompanied by progressive changes in the liver microenvironment, including immune evasion and angiogenesis. Lysyl oxidase-like 2 (LOXL2) has been suggested to contribute to tumour progression and metastasis; however, the underlying mechanism remains unclear. The purpose of the present study was to explore the relationship between LOXL2 and immune infiltration and vasculogenic mimicry (VM) and to identify the role of LOXL2 in HCC diagnosis prognosis evaluation. METHODS: The Cancer Genome Atlas (TCGA), UALCAN, GEPIA and Kaplan-Meier plotter databases were used to analyse LOXL2 expression and perform survival analysis. The Tumour Immune Estimation Resource (TIMER) was used to analyse immune cell infiltration, immune cell biomarkers and immune checkpoints. Immunohistochemistry (IHC) of 201 HCC samples was used to confirm the expression of LOXL2 and its relationship with VM. Coimmunoprecipitation (co-IP) and gain- and loss-of-function studies were performed to confirm the molecular mechanism of LOXL2 in VM. RESULTS: The expression of LOXL2 in HCC was higher than that in normal tissues at both the mRNA and protein levels. High expression of LOXL2 was associated with a poorer prognosis of HCC. The genetic alteration rate of LOXL2 was 5%. LOXL2 was positively related to immune cell infiltration and immune checkpoints (PD-1 and CTLA-4) in HCC. Co-IP showed that LOXL2 can interact directly with IQGAP1. Both gain- and loss-of-function studies showed that LOXL2 significantly induced cell migration, invasion and VM formation when IQGAP1 was upregulated. CONCLUSIONS: LOXL2 is involved in immune cell infiltration and promotes VM by upregulating IQGAP1. LOXL2 can be used as a novel biomarker for HCC diagnosis and prognosis prediction.

RSRC2 Expression Inhibits Malignant Progression of Triple-Negative Breast Cancer by Transcriptionally Regulating SCIN Expression.

Triple-negative breast cancer (TNBC) has a shorter survival time and higher mortality rate than other molecular subtypes. RSRC2 is a newly discovered tumor suppressor gene. However, the potential functional mechanism of RSRC2 in TNBC remains unknown so far. Multiple bioinformatics databases were used. A Human Transcriptome Array 2.0 analysis, ChIP-seq analysis, ChIP-qPCR, RT-qPCR, Western blot, cell function assays in vitro and a metastatic mouse model in vivo were performed to demonstrate the role of RSRC2 in TNBC. Through the analysis of various databases, RSRC2 expression was the lowest in TNBC tissues compared to other molecular subtypes. The low expression of RSRC2 was associated with a worse prognosis for patients with breast cancer. The transcriptome array, ChIP-seq and bioinformatics analysis identified that GRHL2 and SCIN might have a close relationship with RSRC2. The functional bioinformatics enrichment analysis and functional cell experiments showed that RSRC2 was involved in cell adhesion, cell proliferation, cell migration and invasion. Furthermore, RSRC2 expression suppressed SCIN expression but not GRHL2 expression. SCIN re-expression in the RSRC2 overexpression cells or SCIN knockdown in the RSRC2 knockdown cells reversed the cellular function caused by RSRC2. Mechanistically, RSRC2 transcriptionally inhibited SCIN expression. In summary, our study reveals that RSRC2 acts as a tumor suppressor in TNBC development and progression through negatively regulating SCIN-mediated cell function, thus providing a potential target for TNBC treatment.

GRHL2 Expression Functions in Breast Cancer Aggressiveness and Could Serve as Prognostic and Diagnostic Biomarker for Breast Cancer.

Background: Breast cancer (BC) is the most frequent malignancy in women worldwide and the leading cause of female cancer-associated death in the world. Grainyhead-like 2 (GRHL2) is an important gene involved in human cancer progression. However, the role of GRHL2 in BC is unknown. Methods: In this study, we used in vitro experiments to verify the role of GRHL2 expression in BC progression. We used 14 databases to analyse the expression level of GRHL2 in BC and its prognostic and diagnostic value. In addition, the correlation between GRHL2 expression and immune cell infiltration and DNA methylation was also analysed. Results: At the cellular level, overexpression of GRHL2 induced E-cadherin expression in BC cells with a mesenchymal phenotype and resulted in a hybrid epithelial/mesenchymal (E/M) phenotype, which is more strongly correlated with tumour aggressiveness than a pure mesenchymal phenotype. Through analysis of various databases, we found that tumour tissue had a higher expression level of GRHL2. High expression of GRHL2 was associated with worse prognosis of BC patients and indicated that GRHL2 had significant diagnostic value. Grainyhead-like 2 is also related to immune infiltration and regulated by DNA methylation. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses showed that GRHL2-related signalling pathways in BC were related to tumour cell proliferation, invasion, and angiogenesis. Conclusions: In summary, evidence indicates that GRHL2 can be used as a prognostic and diagnostic biomarker for BC.

LncRNA n339260 functions in hepatocellular carcinoma progression via regulation of miRNA30e-5p/TP53INP1 expression.

BACKGROUND: Currently, the molecular mechanism of the interaction between lncRNAs and microRNAs (miRNAs) and the target of miRNAs in tumor vasculogenic mimicry (VM) formation have not been clarified. Our aim is to study the interaction between lncRNA n339260 and miRNA30e-5p in the formation of VM. METHODS: Animal xenografts were established, 104 hepatocellular carcinoma (HCC) patients' frozen tissues were obtained and HCC cells in vitro were used to observe the role of n339260 in HCC progression. RESULTS: In vivo experiment showed lncRNA n339260 promoted tumor growth and VM formation. LncRNA n339260 and miRNA30e-5p were found to be associated with VM formation, metastasis and survival time in HCC patients. In vitro experiment showed that LncRNA n339260 could inhibit miRNA30e-5p expression and TP53INP1 was found to be the downstream targets of miRNA30e-5p. Snail, MMP2, MMP9, VE-cadherin, vimentin and N-cadherin overexpression and the downregulation of TP53INP1 and E-cadherin were observed in HCCLM3 and HepG2 cells overexpressing lncRNA n339260 or in cells with decreased expression of miRNA30e-5p. CONCLUSION: LncRNA n339260 promotes the development of VM, and lncRNA n339260 may enhance Snail expression by decreasing the expression of miRNA30e-5p, thereby reducing TP53INP1 expression. Therefore, a potential lncRNA n339260- miRNA30e-5p- TP53INP1 regulatory axis was associated with HCC progression.

Abnormal expression of Nek2 and ß-catenin in breast carcinoma: clinicopathological correlations.

AIMS: NIMA-related kinase 2 (Nek2) and ß-catenin are important centrosome regulatory factors. The aim of this study was to detect the possible disparity in their expression among normal breast tissue, invasive ductal carcinoma (IDC), concomitant ductal carcinoma in situ (DCIS), and pure DCIS, and to explore its correlation with clinicopathological factors. METHODS AND RESULTS: We used immunohistochemistry to detect protein expression of Nek2 and ß-catenin in breast cancer tissues from 60 cases of pure DCIS, 348 cases of IDC and 137 cases of concomitant DCIS with that in normal breast tissues from the same 137 concomitant DCIS patients as controls. As compared with normal tissue, expression of Nek2 and ß-catenin in the cytoplasm was significantly increased in IDC and DCIS (P < 0.05), and variation in expression was also observed in different grades of IDC (P < 0.01). Also, cytoplasmic expression of Nek2 and and of ß-catenin were correlated with each other in IDC and DCIS (P < 0.01). In addition, they were both related to Ki67 immunoreactivity (P < 0.05). Furthermore, our study also revealed a correlation between their expression and some clinicopathological factors. We found that Nek2 cytoplasmic expression was associated with grade and tumour size (P < 0.01) in IDC, whereas ß-catenin cytomembrane expression showed significant variation with grades, TNM stages, lymphoid node status, oestrogen receptor status, and molecular subtype (P < 0.05); a difference in expression was also observed between IDC and DCIS (P < 0.05). Also, ß-catenin cytoplasmic expression was associated with TNM stage (P < 0.05). Expression of Nek2 at the mRNA level was detected in 50 pairs of breast cancer specimens and matched normal tissues by reverse transcriptase polymerase chain reaction, and the result showed increased expression in IDC. CONCLUSIONS: This study suggests that abnormal expression of Nek2 and ß-catenin might be one of the mechanisms of tumorigenesis, especially of abnormal tumour proliferation. They may represent new potential targets for therapeutic intervention.

[Diagnostic and prognostic significance of FOXA1 expression in molecular subtypes of breast invasive ductal carcinomas].

OBJECTIVE: To investigate the expression of FOXA1 in breast invasive ductal carcinomas, observe its expression in all molecular subtypes of breast invasive ductal carcinomas and understand its diagnostic and prognostic significance. METHODS: Tissue specimens were obtained from 213 cases of breast invasive ductal carcinoma (IDC) and their expressions of FOXA1 and Ki67 detected by immunohistochemistry. And the expressions of CK5/6 and CK14 were detected to distinguish between normal breast-like subtype and basal-like subtype. RESULTS: The expression of FOXA1 was observed in 150 cases (70.4%). It correlated positively with ER and PR. But there was a negative correlation with histological grade, Nottingham prognostic index, p53 and Ki67. The expression of FOXA1 had difference between luminal and non-luminal subtypes. In luminal subtype, the expression of FOXA1 was associated with histological grade, PR, NPI, Ki67 and A subtype. In terms of prognosis, the expression of FOXA1 predicted a long disease-free survival and overall survival. CONCLUSION: The expression of FOXA1 is associated with a good prognosis. FOXA1 is a promising candidate for clinical identification of luminal A subtype. Prognostic analysis of FOXA1 in low-risk breast cancers may prove to be useful in clinical treatment decision-making.

The EMT transcription factor, Twist1, as a novel therapeutic target for pulmonary sarcomatoid carcinomas.

Pulmonary sarcomatoid carcinomas (PSCs) are a rare subtype of non­small­cell lung cancer and are typically biphasic neoplasms. No effective treatment for PSCs is currently available in clinical practice. The expression of the epithelial­mesenchymal transition (EMT) transcription factors, Twist1, Slug and Snail, as well as the EMT phenotype and vasculogenic mimicry (VM) were analysed in 41 PSC and 79 pulmonary squamous carcinoma (PSCC) samples. Compared with the PSCCs, the PSCs exhibited an EMT phenotype and VM, and they also exhibited an increased expression of the Twist1, Slug, Snail and VM markers. Twist1 expression was associated with metastasis and TNM stage. Twist1­positive patients exhibited a poorer prognosis for overall survival (OS) than those with Twist1­negative PSCs. Transforming growth factor ß1 (TGFß1) was used to induce an EMT transition in a PSCC cell line. SK­MES­1 cells treated with TGFß1 exhibited an increased expression of Twist1. The EMT phenotype, VM and increased migratory and invasive abilities were induced following TGFß1 treatment. Importantly, in cells treated with TGFß1, the EMT phenotype was reversed, VM marker expression was decreased, and the migratory and invasive ability of the PSCC cell line was decreased following Twist1 knockdown. Collectively, this study provides a new perspective of Twist1 in the aggressiveness of PSCs. The identification of Twist1 as an independent marker of poor prognoses may lead to the development of novel strategies for improving the treatment of patients with PSC.

Effect of EphA2 knockdown on melanoma metastasis depends on intrinsic ephrinA1 level.

PURPOSE: Upregulation of receptor tyrosine kinase EphA2 has been found to be associated with a poor prognosis in many types of cancer and is considered an attractive therapeutic target. As yet, few efforts have been focused on its tumor suppressive activity triggered by its ligand, ephrinA1. Here, we aimed to determine the potential of ephrinA1 as an important player in melanoma metastasis. METHODS: Data from the Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE) were analyzed to explore the expression and prognostic implications of EphA2 and ephrinA1 in melanoma. Western blotting, shRNA, colony formation and immunofluorescence assays, as well as two in vivo xenograft models (subcutaneous and metastatic) were used to evaluate the role of EphA2 in melanoma progression. Akt inhibition and ephrinA1-Fc were used to confirm the influence of Akt activation and ephrinA1 levels on the EphA2 effects. Immunohistochemistry (IHC) was performed on xenograft and patient melanoma tissues. RESULTS: We found that high levels of ephrinA1, but not EphA2, were negatively correlated with melanoma metastasis. The expression levels of EphA2 and ephrinA1 were not correlated. After EphA2 downregulation, colony forming abilities and lung metastatic growth were reduced in melanoma cell lines with a low ephrinA1 expression, but were increased in melanoma cell lines with a high ephrinA1 expression. EphA2-mediated colony formation in EphA2-high/ephrinA1-low cells was found to be Akt-dependent and to be inhibited by the addition of ephrinA1-Fc. IHC staining of primary melanoma specimens revealed that EphA2-high/ephrinA1-low patients exhibited poorer outcomes than EphA2-high/ephrinA1-high patients. CONCLUSIONS: From our data we conclude that evaluation of ephrinA1 levels may be helpful for the application of EphA2-targeted therapies and for prognostic predictions in melanoma patients.

Increased expression of centrosomal alpha, gamma-tubulin in atypical ductal hyperplasia and carcinoma of the breast.

Centrosomal abnormalities have been found in various cancer types. We sought to determine whether centrosomal dysfunctions occur in the atypical ductal hyperplasia (ADH)-carcinoma sequence of breast cancer. As alpha and gamma-tubulins are the structural components of centrosomes, we performed real time quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH) and immunnohistochemistry (IHC) to determine the DNA copy levels, messenger RNA (mRNA) expression, and protein expression of alpha and gamma-tubulins respectively. Gamma-tubulin staining was used for the localization and quantification of centrosomes. We found that alpha-tubulin or gamma-tubulin mRNA was increasingly expressed from normal breast tissue (NBT) to ADH, ductal carcinoma in situ (DCIS), and infiltrative ductal carcinoma (IDC), respectively, with the highest expressions being found in DCIS. The expression profiles of alpha, gamma-tubulin proteins were concordant with that of mRNA, except that the highest expression was found in IDC. Similarly, DNA copies of alpha, gamma-tubulins showed a rising tendency, with the highest level for gamma-tubulin attained in IDC and that for alpha-tubulin was found in DCIS. However, there was no significant difference of alpha, gamma-tubulin DNA copy levels, mRNA expression, and protein expression between DCIS and IDC. Our results demonstrate that centrosomal aberrations may play key roles in the early stage of breast tumorogenesis. The malignant transformation sequence is probably attributable to the amplification of centrosomal DNA leading to mRNA and protein over-expression of these centrosomal proteins. Furthermore, determination of alpha, gamma-tubulins using combined qPCR with ISH may be useful in assisting the diagnosis of premalignant lesions of the breast.