ARID1A deficiency associated with MMR deficiency and a high abundance of tumor-infiltrating lymphocytes predicts a good prognosis of endometrial carcinoma

BackgroundARID1A alterations have been detected in 40% of endometrial carcinomas (ECs) and are associated with loss of its expression. The role of ARID1A in tumorigenesis and development is complex, and the prognostic role in EC remains controversial. Hence, it is of great significance to confirm the role of ARID1A in EC.MethodsA total of 549 EC patients (cohort A) from TCGA were evaluated to explore the prognostic role of ARID1A. NGS was performed for 13 EC patients (cohort B), and expression of ARID1A, CD3, CD8 and mismatch repair (MMR) proteins in 52 patients (cohort C) from our center was determined by immunohistochemistry (IHC). The Kaplan–Meier method was used to perform survival analyses.ResultsARID1A alterations were detected in 32% of EC patients and correlated with good disease-free survival (DFS, P = 0.004) and overall survival (OS, P = 0.0353). ARID1A alterations were found to co-occur with MMR-related gene mutations and correlated with higher PD-L1 expression. Patients concomitantly harboring ARID1A alterations and MMR-related gene mutations had the best prognosis (DFS: P = 0.0488; OS: P = 0.0024). A cohort from our center showed that ARID1A deficiency was an independent prognostic factor and predicted longer recurrence-free survival (P = 0.0476). ARID1A loss was associated with a tendency toward MSI-H (P = 0.0060). ARID1A alterations and expression loss were associated with a higher abundance of CD3+ (P = 0.0406) and CD8+ (P = 0.0387) T cells.ConclusionARID1A alterations and expression loss are tightly associated with MMR deficiency and a high abundance of tumor-infiltrating lymphocytes, which might contribute to the good prognosis of EC.     

Evaluation of the prognostic value of lymphadenectomy for low-grade serous ovarian cancer: A case-control multicenter retrospective study

BackgroundThe prognostic value of lymphadenectomy in low-grade serous ovarian cancer (LGSOC) remains uncertain.Materials and methodsA retrospective analysis of 155 patients with LGSOC who underwent surgery over a ten-year period (2011–2020) was performed. The propensity score matching (PSM) algorithm was performed between the lymphadenectomy and no lymphadenectomy groups, and Kaplan-Meier analyses were conducted to evaluate clinical prognosis. Finally, univariate and multivariate Cox proportional hazards regression analyses were performed to analyze high-risk factors associated with clinical prognosis.ResultsIn the pre-PSM cohort, 110 (71.0%) patients underwent lymphadenectomy. Of these, 54 (34.8%) experienced recurrence, and 27 (17.4%) died. There were statistical differences in disease-free survival (DFS) (P = 0.018) and overall survival (OS) (P = 0.016) in the post-PSM cohort. In the subgroup analysis, there were no statistically significant differences in DFS (P = 0.449) or OS (P = 0.167) in the FIGO I/II cohort. However, in the FIGO III/IV cohort, DFS (P = 0.011) and OS (P = 0.046) were statistically different between the two groups. Age > 50 years, FIGO stage III/IV, and suboptimal cytoreductive surgery were risk factors associated with prognosis. In the lymphadenectomy group, the histological status of pelvic lymph nodes had no significant effect on DFS (P = 0.205) or OS (P = 0.114).ConclusionLymphadenectomy was associated with DFS and OS, particularly in patients with advanced LGSOC patients. Age > 50 years, advanced FIGO stage III/IV, and suboptimal cytoreductive surgery were high-risk factors associated with clinical prognosis in patients with LGSOC.     

Surgery versus radiotherapy: Long term outcomes of T1 glottic cancer

AimThe aim of this study was to compare the outcomes, patterns of failure and laryngeal preservation rates in patients with T1N0 glottic cancer treated with surgery or radiotherapy.Materials/methodsRetrospective study of T1N0 glottic cancer patients treated in our institution between January 2007 and December 2017. Histologically proven squamous cell carcinoma patients, treated with upfront cordectomy/partial laryngectomy (S group) or radiotherapy (RT group) were included. Elective treatment of the neck was not permitted. Local failure (LF), disease-free survival (DFS), ultimate disease-free survival (UDFS), laryngectomy-free survival (LFS), disease-specific mortality (DSM) and overall survival (OS) were evaluated.ResultsTwo hundred and one patients were eligible (172 S group, 29 RT group), with a median follow-up of 38.8 months. Overall, 33 (16%) patients had a recurrence, 30 (17%) in the S group and 3 (10%) in the RT group. Local failure was the predominant site of failure (28 S, 2 RT). Overall, of all those that were salvaged, 17 (8%) underwent total laryngectomy (15 S, 2 RT). There was no significant difference in the 5-year cumulative incidence of LF (20.8% S, 8.1% RT, p = 0.138), 5-y LFS (85.0% vs. 91.7%, p = 0.809), 5-y DFS (67.5% vs. 82.1%, p = 0.343), 5-y UDFS (82.5% vs. 90.3%, p = 0.647) and 5-y OS (84.5% vs. 90.3%, p = 0.892). Multivariate analysis showed no correlation between initial treatment and the analyzed outcomes.ConclusionPrimary surgery or radiotherapy were similar first line options, since they do not differ in all outcomes. Patients’ and physician's preferences must be considered when choosing first treatment.     

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44⁺/CD24^low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44⁺/CD24^low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.     

Higher serum sPD-L1 levels after radiotherapy indicate poor outcome in hepatocellular carcinoma patients

BackgroundOur preclinical research reveals that radiotherapy (RT) promoted PD-L1 upregulation in tumor tissues and that higher PD-L1 after RT worsened the prognosis through immunosuppression. We sought to validate our experimental results in clinical cohorts and promote clinical application.Patients and methodsIn cohort 1, formalin-fixed paraffin-embedded samples were obtained from 46 HCC patients, 23 of whom received preoperative RT and the other 23 received direct surgery. A prospectively collected database contained 122 HCC patients treated with liver RT were enrolled in cohort 2. Blood samples were taken a day before and two weeks after RT. Patients in cohort 2 were further divided into two groups, exploration (73 patients) and validation (49 patients) groups.ResultsIn cohort 1, RT increased the expression of PD-L1 in tumor tissues (p = 0.001), and PD-L1 levels were associated with decreased cytotoxic T-cell infiltration and a trend toward poor prognosis (p = 0.14). Moreover, PD-L1 expression in tumor tissue positively correlated with soluble (s) PD-L1 in serum (R = 0.421, p = 0.046). Then, in cohort 2, we revealed RT increased sPD-L1 in serum (p < 0.001), which was associated with the number of circulating CD8+ T cells (R = -0.24, p = 0.036), indicating poor survival. Furthermore, patients with higher rate of sPD-L1 increase after RT have better treatment response (p < 0.001), PFS (p = 0.032) and OS (p = 0.045).ConclusionHigher post-RT serum sPD-L1, which may potentiate immune suppression effects, indicates a poor prognosis for HCC patients treated with RT.     

Markers of Epithelial to Mesenchymal Transition in Association with Survival in Head and Neck Squamous Cell Carcinoma (HNSCC)

Background

Elucidating the molecular phenotype of cancers with high metastatic potential will facilitate the development of novel therapeutic approaches to the disease. Gene expression profiles link epithelial to mesenchymal transition (EMT) phenotype with high-risk HNSCC. We sought to determine the role of protein biomarkers of EMT in head and neck squamous carcinoma (HNSC) prognosis.

Methods

Protein expression analysis of EGFR, β-catenin and E-cadherin was performed on a cohort of 102 patients with HNSCC recruited between 1992 and 2005 using automated quantitative protein analysis (AQUA). We evaluated associations with clinicopathological parameters and prognosis.

Results

There were 67 patients with primary squamous cell carcinoma of the head and neck in this cohort who met inclusion criteria and for whom we had complete E-cadherin, beta-catenin and EGFR expression data. High E-cadherin expressers had longer 5-year progression-free survival (PFS) compared to those with low E-cadherin (59.7% versus 40.6%, p = 0.04) and overall survival (OS) (69.6% versus 44.3%, p  = 0.05). Kaplan-Meier analysis showed that patients with low beta-catenin-expressing tumors trended toward worse 5-year PFS (p = 0.057). High EGFR expressers had inferior OS compared to low EGFR expressers (27.7% vs. 54%, p = 0.029). In the multivariable analysis context, E-cadherin remained an independent predictor of improved OS (HR = 0.204, 95% CI 0.043 to 0.972, p = 0.046) while EGFR trended towards significance for OS.

Conclusions

The putative markers of EMT defined within a panel of HNSCC using AQUA are associated with tumors of poor prognosis.     

Comprehensive Multiple Molecular Profile of Epithelial Mesenchymal Transition in Intrahepatic Cholangiocarcinoma Patients

Background

The aim of this study is to investigate the expression profile of multiple epithelial mesenchymal transition (EMT)-related molecules in intrahepatic cholangiocarcinoma (ICC) and the related prognostic significance.

Methods

Immunohistochemistry was performed to determine the expression of E-cadherin, Vimentin, Snail, slug and β-catenin in a tissue microarray consisting of tumor tissues of 140 ICC patients undergoing curative resection. The correlation between the expression of these molecules and the clinicopathological characteristics of ICC patients was analyzed, and their prognostic implication was evaluated.

Results

Reduced E-cadherin and increased Vimentin expression, the characteristic changes of EMT, identified in 55.0% and 55.7% of primary ICCs, respectively, were correlated with lymphatic metastasis and poorer overall survival (OS) and disease-free survival (DFS) of ICCs. The overexpression of snail and nonmembranous β-catenin, which are the major regulators of the EMT, were identified in 49.2% and 45.7% of primary ICCs, while little slug expression was detected in ICCs. Cytoplasmic/nuclear β-catenin did not significantly predict worse DFS and was not related with E-cadherin loss. The overexpression of snail predicted worse OS and DFS. Snail overexpression correlated with the down-regulation of E-cadherin and the up-regulation of Vimentin. Inhibition of snail in an ICC cell line decreased the expression of E-cadherin, enhanced the expression of Vimentin and impaired the invasion and migration ability of ICC cells.

Conclusions

These data support the hypothesis that EMT plays vital roles in ICC progression and suggest that snail but not slug and β-catenin plays a crucial role in the EMT induction of ICC.     

Tumor Infiltrating Lymphocytes and NHERF1 Impact on Prognosis of Breast Cancer Patients

BACKGROUND: Breast cancer (BC) is a heterogeneous disease, and patients with apparently similar clinicopathological characteristics in clinical practice show different outcome. This study evaluated in primary BCs and in the subgroup of the triple-negative breast cancers (TNBCs) the level of tumor infiltrating lymphocytes (TILs), Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression, and their association respect to the clinical outcome of patients. MATERIAL AND METHODS: NHERF1 expression was assessed by immunohistochemistry in 338 BC samples; the analysis of TILs was examined using hematoxylin and eosin stained slides, according to International TILs Working Group 2014. RESULTS: Multivariate analysis identified TILs as an independent prognostic factor for DFS in the entire cohort and in the TNBC subgroup (HR, 0.32; 95% CI, 0.12–0.87; P = 0.026; and HR, 0.22; 95% CI, 0.06–0.80; P = 0.022, respectively). Univariate and survival analysis by Kaplan–Meier method revealed that patients with cytoplasmic (c) NHERF1^-/TILs⁺ expression had better DFS than other patients (P = 0.049), and this result was also found in the TNBC subgroup (P = 0.031). Moreover, TNBC patients with cNHERF1⁻/TILs⁻ expression had a worse DFS and OS than other patients (P = 0.057 and P = 0.002, respectively). CONCLUSIONS: In the complex scenario of BC and in the era of tumor immunogenicity and immunotherapy, we found an association of TIL levels and cNHERF1 expression that could be useful to identify BCs and particularly TNBC patients with different prognosis and clinical outcome.     

N-Cadherin Expression Is Associated with Acquisition of EMT Phenotype and with Enhanced Invasion in Erlotinib-Resistant Lung Cancer Cell Lines

Background

The epidermal growth-factor receptor tyrosine kinase inhibitors have been effective in non-small cell lung cancer patients. However, acquired resistance eventually develops in most patients despite an initial positive response. Emerging evidence suggests that there is a molecular connection between acquired resistance and the epithelial–mesenchymal transition (EMT). N-cadherin is involved in the EMT and in the metastasis of cancer cells. Here, we analyzed N-cadherin expression and function in erlotinib-resistant lung cancer cell lines.

Methods

H1650 cell lines were used to establish the subline resistant to erlotinib(H1650ER). Then, induction of the EMT was analyzed using immunostaining and western blots in H1650ER cells. N-cadherin expression in the resistant cells was examined using FACS and western blot. In addition, an invasion assay was performed to characterize the resistant cells. The effects of N-cadherin on cell proliferation and invasion were analyzed. The association of N-cadherin expression with the EMT phenotype was investigated using immunohistochemical analysis of 13 archived, lung adenocarcinoma tissues, before and after treatment with erlotinib.

Results

In H1650ER cells, N-cadherin expression was upregulated, paralleled by the reduced expression of E-cadherin. The marked histological change and the development of a spindle-like morphology suggest that H1650ER cells underwent an EMT, accompanied by a decrease in E-cadherin and an increase in vimentin. A change in the EMT status between pre-and post-treatment was observed in 11 out of 13 cases (79%). In biopsies of resistant cancers, N-cadherin expression was increased in 10 out of 13 cases. Induction of the EMT was consistent with aggressive characteristics. Inhibition of N-cadherin expression by siRNA was tested to reduce proliferation and invasion of H1650ER cells in vitro.

Conclusions

Our data provide evidence that induction of the EMT contributes to the acquired resistance to EGFR-TKIs in lung cancer. It suggests that N-cadherin is a potential molecular target in the treatment of NSCLC.     

Low baseline vitamin D levels increase the risk of bone metastases among females with breast cancer − Hospital based cohort study

BackgroundSerum vitamin D (Vit-D) has been linked to the development of breast cancer (BC); however, their effect on pathological features and outcomes is undetermined. The purpose of this study was to examine the prognostic significance of baseline Vit-D levels and their effect on clinical outcomes.MethodsWe evaluated baseline serum Vit-D levels and baseline clinic-pathological features of female patients with non-metastatic BC between October 2018 and December 2019. A low Vit-D level was described as less than 30 nanogram per liter (ng/l). Patients were observed for a median of 24 months. To evaluate relationships between qualitative variables, the chi-square test was used. The Kaplan-Meier technique was used for survival analysis, and the log-rank test was used to compare the two survival curves. Correlation analysis was also used to examine the link between Vit-D levels and clinical outcomes.ResultsThe eligibility criteria were fulfilled by 221 patients. The median age of onset was (50.7). The median Vit-D level was (23.1 ng/l) with a range of (4–46 ng/l). Approximately half of the patients (56.5%) had Vit-D levels < 30 ng/l, with HER2 positive and triple negative (TNBC) patients having a greater proportion of low Vit-D levels (p = <0.001). Patients with low baseline Vit-D levels had a larger tumor size, more positive lymph nodes, and were diagnosed at a later stage. Following follow-up, Vit-D deficiency was associated with a significantly increased risk of bone metastases (HR 3.37, 95% CI 1.32–8.59, p = 0.006), and Vit-D levels were significantly correlated with disease-free survival (DFS) and overall survival (OS) (r = 0.850, r = 0.573, p < 0.00, p < 0.001, respectively).ConclusionsLow serum Vit-D is associated with advanced stage and adverse characteristics. It is more prevalent in HER-2 positive and TNBC patients; it increases the chance of bone metastases, and has a significant correlation with DFS and OS.     

Role of serum concentration of VEGFR1 and TIMP2 on clinical outcome in primary cervical cancer: Results of a companion protocol of the randomized,NOGGO–AGO phase III adjuvant trial of simultaneous cisplatin-based radiochemotherapy vs. carboplatin and paclitaxel containing sequential radiotherapy

ObjectiveAim of the present study was to analyze the expression-profile of IGF1, IGFBP3, sICAM1, sVCAM1, MMP2, MMP9, TIMP2, VEGFA, VEGFD, VEGFC and VEGFR1 in patients with high-risk FIGO-stage Ib-IIb cervical cancer.MethodsSerum from 68 cervical cancer patients treated within a phase-III-trial with either simultaneous cisplatin radiochemotherapy or sequential systemic carboplatin and paclitaxel followed by percutaneous irradiation was analyzed by ELISA. Both target expression and correlation with important clinicopathological factors were analyzed following standard statistic procedures.ResultsAll 68 patients underwent a primary radical hysterectomy with pelvic and/or paraaortic lymphadenectomy. 85.3% of the extirpated tumors had clear surgical margins (R0). Increased levels of VEGFR1, TIMP2 and MMP2 were significantly associated with positive surgical margins (p = 0.004, p = 0.018 and p = 0.004, respectively). High concentration of MMP2 and TIMP2 correlated additionally with an advanced age at time of diagnosis (p = 0.001 and p = 0.007, respectively). For the cut-off value of 100 pg/ml, an increased VEGFR1 was significantly associated with poor overall (OS) and progression-free (PFS) survival (p = 0.017 and p = 0.015, respectively). A TIMP2 concentration of lower than 90 ng/ml was significantly associated with poorer OS and PFS (p = 0.009 and p = 0.043, respectively). In the multivariate analysis, TIMP2 expression in serum was the only independent prognostic factor for OS (p = 0.032, HR = 6.51, 95% CI = 1.17–36.01).ConclusionsExpression-profile of specific biomarkers associated with tumor invasion, cell migration and angiogenesis seems to be of prognostic value for both OS and PFS in patients undergoing surgery due to primary cervical cancer. Further analyses are warranted to allow an implementation of such markers into clinical practice.     

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.     

Intravitreal injection of triptolide attenuates subretinal fibrosis in laser-induced murine model

BackgroundChoroidal neovascularization (CNV) is a common cause of irreversible blindness in elderly patients in developed countries, and subretinal fibrosis is an advanced stage of CNV. Currently, there is no effective clinical treatment for subretinal fibrosis.PurposeTo investigate whether intravitreal injection of triptolide (TP) could attenuate subretinal fibrosis and determine its underlying mechanisms.MethodsCNV was induced by laser photocoagulation in C57BL/6J mice. Immediately after laser photocoagulation, 1 μl of free TP (10 μg), TP-nanolip-PEG (TP-loaded PEGylated nanoliposomes containing 10 μg TP), or the same volume of phosphate-buffered saline (PBS) was intravitreally administered to each respective group. Areas and ratios of subretinal fibrosis were calculated seven days after laser injury. Additionally, expression levels of M2 macrophage-related markers, molecules of the transforming growth factor (TGF)-β1/Smad signaling pathway, and markers for epithelial-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) were detected both in vitro and in vivo.ResultsThe areas of subretinal fibrosis were significantly reduced in both the free TP (10993.87 ± 2416.90 μm²) and TP-nanolip-PEG (7695.32 ± 2121.91 μm²) groups when compared with the PBS group (15971.97 ± 3203.10 μm²) (p < 0.05, n = 6). The ratio of subretinal fibrosis in the free TP monomer (20.8 ± 4.2%) and TP-nanolip-PEG (12.5 ± 4.0%) groups was lower than that in the PBS control group (41.7 ± 5.3%) (p < 0.01, n = 6). Moreover, both TP and TP-nanolip-PEG suppressed the polarization of M2 macrophages and downregulated gene expressions of TGF-β1, Smad 2, Smad 3, α-SMA, and collagen I (p < 0.05), but upregulated the gene expression of E-cadherin (p < 0.05), thus reversing TGF-β1 induced EMT/EndoMT and attenuating subretinal fibrosis.ConclusionsTP could attenuate subretinal fibrosis by suppressing the polarization of M2 macrophages and TGF-β1 induced EMT/EndoMT. TP-nanolip-PEG enhanced the inhibitory effects of TP on subretinal fibrosis, suggesting its therapeutic potential for CNV-related subretinal fibrosis.     

Androgen Receptor (AR), E-Cadherin,and Ki-67 as Emerging Targets and Novel Prognostic Markers in Triple-Negative Breast Cancer (TNBC) Patients

BackgroundTNBC is an aggressive subset of breast cancer (BC) without specific target therapy.MethodsThis observational, retrospective study included 45 cases of TNBC. The aim of this study was to evaluate the expression of the AR, E-cadherin and Ki-67 in relation to histological type, time to relapse and overall survival (OS). Immunohistochemistry (IHC) was carried out on formalin-fixed paraffin-embedded tumor samples obtained from patients defined TNBC.ResultsThe AR was positive (IHC >10%) in 26.6%. E-cadherin (CDH1) expression was considered positive if the score was ≥ 2. This expression was negative in 53.3% cases. The Ki-67 index was ≥ 20% in 37.7%. Univariate analyses showed that AR, CDH1 and Ki-67 are significantly associated with OS. Multivariate analysis showed that AR and Ki-67 expression are independent variables associated with OS. The statistical analysis showed that patients with AR negative and Ki-67 positive expression have a significant correlation with poor outcome.ConclusionsOur data suggest that the combination of AR and E-cadherin expression as well as Ki-67 status might be useful prognostic markers in TNBC. Hence, these molecular determinants could play an interesting role to classify subgroups of TNBC.     

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor,saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.     

Fractionated radiotherapy might induce epithelial-mesenchymal transition and radioresistance in a cellular context manner

Despite the fact that radiotherapy is a main therapeutic modality in cancer treatment, recent evidence suggests that fractionated radiotherapy (FR) might confer radioresistance through epithelial-mesenchymal transition (EMT). Nevertheless, the effects of FR on EMT phenotype and the potential link between EMT induction and radioresistance development yet to be clarified. The aim of this study was to assess whether FR could promote EMT, and to elucidate if induction of EMT contributes to the acquisition of radioresistance. To this end, two human cancer cell lines (A549 and HT-29) were irradiated (2 Gy/day) and analyzed using wound healing, transwell migration and invasion assays, real-time polymerase chain reaction (for E-cadherin, N-cadherin, Vimentin, CD44, CD133, Snail, and Twist), clonogenic assay, Annexin V/PI, and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Irradiation of A549 (for 5 or 10 consecutive days) resulted in morphological changes including elongation of cytoplasm and nuclei and pleomorphic nuclei. Also, irradiation-enhanced migratory and invasive potential of A549. These phenotypic changes were in agreement with decreased expression of the epithelial marker (E-cadherin), enhanced expression of mesenchymal markers (N-cadherin, Vimentin, Snail, and Twist) and increased stemness factors (CD44 and CD133). Moreover, induction of EMT phenotype was accompanied with enhanced radioresistance and proliferation of irradiated A549. However, FR (for 5 consecutive days) did not increase HT-29 motility. Furthermore, molecular alterations did not resemble EMT phenotype (downregulation of E-cadherin, Vimentin, ALDH, CD44, CD133, and Snail). Eventually, FR led to enhanced radiosensitivity and decreased proliferation of HT-29. Altogether, our findings suggest that FR might induce EMT and confer radioresistance in a cell context-dependent manner.     

Nuclear NHERF1 expression as a prognostic marker in breast cancer

Our purpose was to investigate whether Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression could be linked to prognosis in invasive breast carcinomas. NHERF1, an ezrin-radixin-moesin (ERM) binding phosphoprotein 50, is involved in the linkage of integral membrane proteins to the cytoskeleton. It is therefore believed to have an important role in cell signaling associated with changes in cell cytoarchitecture. NHERF1 expression is observed in various types of cancer and is related to tumor aggressiveness. To date the most extensive analyses of the influence of NHERF1 in cancer development have been performed on breast cancer. However, the underlying mechanism and its prognostic significance are still undefined. NHERF1 expression was studied by immunohistochemistry (IHC) in a cohort of 222 breast carcinoma patients. Association of cytoplasmic and nuclear NHERF1 expression with survival was analyzed. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan–Meier method. Cytoplasmic NHERF1 expression was associated with negative progesterone receptor (PgR) (P=0.017) and positive HER2 expression (P=0.023). NHERF1 also showed a nuclear localization and this correlated with small tumor size (P=0.026) and positive estrogen receptor (ER) expression (P=0.010). Multivariate analysis identified large tumor size (P=0.011) and nuclear NHERF1 expression (P=0.049) to be independent prognostic variables for DFS. Moreover, the nuclear NHERF1(−)/ER(−) immunophenotype (27%) was statistically associated with large tumor size (P=0.0276), high histological grade (P=0.0411), PgR-negative tumors (P<0.0001) and high proliferative activity (P=0.0027). These patients had worse DFS compared with patients with nuclear NHERF1(+)/ER(+) tumors (75.4% versus 92.6% P=0.010). These results show that the loss of nuclear NHERF1 expression is associated with reduced survival, and the link between nuclear NHERF1 and ER expression may serve as a prognostic marker for the routine clinical management of breast cancer patients.     

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

PurposeAngiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma.MethodsThe expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma.ResultsWe noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R_s = 0.50; p = 0.002, R_s = 0.69; p = 0.0001, R_s = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R_s = 0.70, p = 0.0001, R_s = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R_s = 0.54, p = 0.0001, R_s = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half).ConclusionsBasing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.