Normal Tissues Of Patients Research Articles

Photoacoustic Imaging for Measuring Tumors and Normal Tissue in Patients With Head and Neck Cancer

Photoacoustic Imaging for Measuring Tumors and Normal Tissue in Patients With Head and Neck Cancer

P1.03-06 Integrative Omics Analysis Reveals Important Roles of Adenosine Diphosphate in Haemostasis and Platelet Activation in NSCLC

Lung cancer is the leading cause of cancer-related deaths in the world. The most prevalent subtype, accounting for 85% of cases, is non-small cell lung cancer (NSCLC). Lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the most common subtypes. Despite recent advances in treatment, the low 5-year survival rate of NSCLC patients (approximately 13%) reflects the lack of early diagnostic biomarkers and incomplete understanding of the underlying disease mechanisms. We hypothesised that integration of metabolomic, transcriptomic and genetic profiles of tumours and matched normal tissues could help to identify important factors and potential therapeutic targets that contribute to tumorigenesis. We integrated omics profiles in tumours and matched adjacent normal tissues of patients with LUSC (N = 20) and LUAD (N = 17) using multiple system biology approaches. We confirmed the presence of previously described metabolic NSCLC pathways, particularly those mediating the Warburg effect. In addition, through our combined omics analyses we found that metabolites and genes that contribute to haemostasis, angiogenesis, platelet activation and cell proliferation were predominant in both subtypes of NSCLC. The important roles of adenosine diphosphate (ADP) in promoting cancer metastasis through platelet activation and angiogenesis suggests this metabolite could be a potential therapeutic target.

Metabolomic, transcriptomic and genetic integrative analysis reveals important roles of adenosine diphosphate in haemostasis and platelet activation in non‐small‐cell lung cancer

Lung cancer is the leading cause of cancer‐related deaths in the world. The most prevalent subtype, accounting for 85% of cases, is non‐small‐cell lung cancer (NSCLC). Lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the most common subtypes. Despite recent advances in treatment, the low 5‐year survival rate of NSCLC patients (approximately 13%) reflects the lack of early diagnostic biomarkers and incomplete understanding of the underlying disease mechanisms. We hypothesized that integration of metabolomic, transcriptomic and genetic profiles of tumours and matched normal tissues could help to identify important factors and potential therapeutic targets that contribute to tumorigenesis. We integrated omics profiles in tumours and matched adjacent normal tissues of patients with LUSC (N = 20) and LUAD (N = 17) using multiple system biology approaches. We confirmed the presence of previously described metabolic pathways in NSCLC, particularly those mediating the Warburg effect. In addition, through our combined omics analyses we found that metabolites and genes that contribute to haemostasis, angiogenesis, platelet activation and cell proliferation were predominant in both subtypes of NSCLC. The important roles of adenosine diphosphate in promoting cancer metastasis through platelet activation and angiogenesis suggest this metabolite could be a potential therapeutic target.

LncRNA CASC11 promotes the cervical cancer progression by activating Wnt/beta-catenin signaling pathway

BackgroundStudies have shown that cancer susceptibility candidate 11 (CASC11), a newly discovered long non-coding RNA (lncRNA), was aberrantly overexpressed in hepatic carcinoma, gastric cancer and colorectal cancer. However, its effects on cervical cancer has been kept unknown up to now. The present study was aimed to investigate the relationship between lncRNA CASC11 and cervical cancer and further explore the mechanism of CASC11 effect on cervical cancer progression.MaterialsQuantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of CASC11 in cancerous and adjacent normal tissues of patients with cervical cancer as well as in cell lines. The proliferation, migration, invasion and apoptosis were assayed after transfecting the cell with si-CASC11 or pcDNA3.1-CASC11. TOP/FOP-Flash luciferase reporter assay and western blot were used to analysis the activation of Wnt/β-catenin signaling pathway. Si-CASC11-transfected HeLa cells were subcutaneously inoculated into male athymic (nude) mice to investigate the effect of CASC11 on the tumor formation.ResultsWe discovered that CASC11, the expression of which was positively associated with the tumor size and the FIGO staging and negatively related to the patients’ survival rate, was up-regulated in the cervical cancer tissues and cell lines. Silencing CASC11 inhibited the proliferation, migration as well as invasion and promoted the cell apoptosis. Conversely, overexpression of CASC11 facilitated the cancer cell’s proliferation, migration and invasion ability and suppressed the apoptosis. Further study showed that CASC11 promoted the migration and invasion of cervical cancer cells by activating Wnt/β-catenin signaling pathway and silencing CASC11 inhibited the tumor growth in vivo.ConclusionOur study demonstrated that CASC11 promoted the cervical cancer progression by activating Wnt/β-catenin signaling pathway for the first time, which provides a new target or a potential diagnostic biomarker of the treatment for cervical cancer.

Evaluation of the Methylation of MIR129-2 Gene in Gastric Cancer.

Genetic and epigenetic changes have strong role in the development of gastric cancer. The mutation of the MIR129-2 gene is one of the major causes in many cancers, especially gastric cancer. The aim of this study was to investigate the methylation changes of the MIR129-2 gene in tumor and normal tissue of patients with gastric cancer. In this study, 50 gastric cancer patients with Iranian Azari ethnic origin without any familial relations were included. Genomic DNAs was extracted from the tumoral and normal tissues. Then the promotor regions of the MIR129-2 gene were analyzed by methylation-specific PCR (MSP) to evaluate the presence or absence of methylated CpG sites. There was a statistically significant difference in methylation level of MIR129-2 gene between tumoral and normal tissues. It was observed that 84 out of 100 CpG cites were methylated in tumoral tissues in compression to 13 out of 100 CpG cites in normal tissues. MIR129-2 gene was hypermethylated in tumoral tissues, suggesting that methylation is involved in the development of gastric cancer.

A novel LARCassigner3 classification predicts outcomes in patients with locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy: a retrospective training and validation analysis.

Purpose: To build and validate a predictive model of outcome for patients with locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiotherapy.Materials and methods: We developed a LARCassigner3 classifier based on tumor and paired normal tissues of patients treated with neoadjuvant chemoradiation and surgery from January 2007 to December 2012 in Fudan University Shanghai Cancer Center. Excluding 23 pairs of tissues failed in the RNA quality test, rested 197 patients were divided into discovery (n=98) and validation (n=99) cohorts randomly. Median follow-up time was 58 months. We used the Kaplan–Meier method to estimate disease-free survival (DFS), overall survival (OS), local recurrent, and distant metastatic rate We constructed a multivariate Cox model to identify the variables independently associated with progression-free and OS.Results: We identified three classifier genes related to relevant colorectal cancer features (CXCL9, SFRP2, and CD44) that formed the LARCassigner3 classifier assay. In the discovery set, the median DFS was 48.1 months (95% confidence interval (CI) 47.3–49.5) in the low-risk group and 23.4 months (95% CI 22.1–24.8) in the high-risk group (p=0.0134); the median OS was 39.2 months (95% CI 38.4–40.3) in the high-risk group and 19.1 months (95% CI 18.3–20.7) in the low-risk group (p=0.0134); 5-year distant metastasis was 13.9% (95% CI 9.0–21.3) in the low-risk group and 49.8% (95% CI 38.7–60.9) in the high-risk group (p=0.0072). Additionally, the different responses to neoadjuvant chemoradiotherapy and the LARCassigner3 low-risk and high-risk groups was statistically significant (p=0.004) in the discovery cohort. Similar results were obtained in the internal evaluation cohort.Conclusions: Patients with LARCassigner3 low-risk tumors were associated with a good prognosis. The clinical utility of using LARCassigner3 subtyping for the identification of patients for neoadjuvant chemoradiotherapy requires validation in dependent clinical trial cohorts.

Evaluation of MET T1010I and MET rs40239 single-nucleotide polymorphisms in triple-negative breast cancer: a case-control study.

Aim: The purpose of this study is to evaluate the role of MET T1010I and MET rs40239 as potential risk factor and/or prognostic markers in patients with triple-negative breast cancer (TNBC).Methods: 114 samples of DNA from paraffin-embedded breast normal tissues of patients with TNBC and 124 samples of healthy controls were collected and analyzed for MET T1010I and MET rs40239 polymorphisms.Results: MET T1010I CT genotype was associated with increased risk of TNBC in both univariate and multivariate analysis. The status of rs40239 was not associated with a higher risk for TNBC at either the univariate or the multivariate analysis. None of the examined polymorphisms was associated with overall survival at the univariate or multivariate Cox regression analysis (adjusted HR=1.35, 95% CI: 0.31–5.97 for MET T1010I CT/TT vs CC; adjusted HR=1.78, 95% CI: 0.73–4.35 for rs40239 AG/GG vs AA).Conclusion: Our case–control study suggests that MET T1010I seems to be a risk factor for TNBC in the Caucasian Greek population, in contrast with MET rs40239, where no correlation was found.

GENE-03. CASE REPORT: CHOROID PLEXUS CARCINOMA TREATED WITH STEREOTACTIC RADIOSURGERY IN 2-YEAR-OLD WITH LI FRAUMENI SYNDROME

CASE REPORT: The child was diagnosed with choroid plexus carcinoma (CPC) at age 8 months. The tumor was positive for TP53 genomic alteration. Germline genetic test sent at age 10 months returned positive for heterozygous TP53 pathogenic mutation. She underwent surgical resection, chemotherapy, and repeat resection at 13 months. At 31 months, she had three small nodules concerning for recurrent disease surrounding the large surgical cavity. Repeat surgical resection was declined by her family. She therefore underwent stereotactic radiosurgery (SRS) to each lesion to a dose of 24 Gy in 1 fraction. Anesthesia was required at the time of planning CT simulation and at the time of treatment. At one year since the procedure, she has demonstrated no further recurrent disease and no evidence of secondary malignancy at this time. DISCUSSION: Retrospective studies approximate rates of p53 mutations in children with CPC at 40%. Radiation is avoided—unless absolutely necessary—in children less than 3 years old because of the risks of neurocognitive delay, endocrine and growth toxicities, and the risk of secondary malignancies. Patients with Li Fraumeni syndrome have further dramatic increase in rates of developing secondary malignancies after exposure to ionizing radiation. SRS provides dose escalation while minimizing ionizing radiation dose to normal surrounding tissue. CONCLUSIONS: Any child diagnosed with CPC should undergo genetic testing for p53 germline mutation because of high pre-test probability. In selected cases, SRS provides an alternative to surgery as local control for limited CPC. SRS minimizes the dose of ionizing radiation delivered to normal tissues in patients with p53 mutation, or in young patients in whom less-focused radiation techniques may cause increased negative neurocognitive effects. Because of the short treatment course, SRS also minimizes the number of days under anesthesia required for young patients, thereby minimizing risk incurred by repeated anesthetic requirements.

Upregulation of lncRNA AB073614 functions as a predictor of epithelial ovarian cancer prognosis and promotes tumor growth in vitro and in vivo.

Upregulation of lncRNA AB073614 is found in some cancer types and involved in tumor development and progression including ovarian cancer. However, the clinical value and functional role of lncRNA AB073614 in epithelial ovarian cancer (EOC) still needed to be investigated. We examined lncRNA AB073614 expression using quantitative real time polymerase chain reaction (qRT-PCR) in 75 paired of EOC tissue samples and adjacent normal tissues. Association of lncRNA AB073614 expression with overall survival (OS) was evaluated using Kaplan-Meier analysis. Univariate and multivariate analysis of factors associated with OS were assessed in EOC patients. After lncRNA AB073614 knockdown using siRNAs, the cell viability and cell colony forming assays were performed. Western blot analysis was used to assess relative protein expression. In present study, we demonstrated that lncRNA AB073614 was significantly upregulated in ovarian cancer tissues compared to adjacent normal tissues in patients. Higher lncRNA AB073614 expression significantly associated with tumor size, lymph node invasion, FIGO stage, and shorter OS rate of EOC patients. Furthermore, multivariate Cox regression analysis results showed that higher lncRNA AB0736141 was identified as an independent risk factor of OS in EOC patients. Moreover, we demonstrated that lncRNA AB0736141 knockdown suppressed EOC cell proliferation ability and cell colony formation in vitro. In vivo, we showed that AB0736141 knockdown suppressed tumor growth. We also revealed that lncRNA AB0736141 knockdown inhibited the PTEN/PI3K/AKT signaling pathway in EOC. Thus, these results indicated that LncRNA AB073614 may serve as a prognostic biomarker and potential target of treatment for EOC.

Predictive value of the ratio of 8‑hydroxydeoxyguanosine levels between cancerous and normal tissues in patients with stage II/III colorectal cancer

Reactive oxygen species (ROS) accumulation is known to induce carcinogenesis and accelerate cancer progression. 8‑Hydroxydeoxyguanosine (8‑OHdG) is a specific marker of ROS‑mediated DNA damage. Therefore, we analysed 8‑OHdG levels in cancerous and normal tissue DNA via enzyme‑linked immunosorbent assay (ELISA) using 97 tissue specimens obtained from surgically‑treated patients with stage II/III colorectal cancer (CRC). Additionally, 8‑OHdG levels in these tissues were also assessed via quantitative immunohistochemistry (qIHC). To eliminate individual background variables, the ratio of 8‑OHdG levels between cancerous and normal tissues was calculated using both techniques. A comparative analysis demonstrated that the 8‑OHdG ratio in DNA was significantly correlated with both lymph node metastasis and lymphatic invasion. Multivariate analysis revealed that a high 8‑OHdG ratio in DNA was independently correlated with poor prognosis. These results suggest that the 8‑OHdG ratio in DNA reflects ROS‑induced cancer progression. Conversely, a low 8‑OHdG ratio as estimated via qIHC was an independent factor for poor prognosis. In Kaplan‑Meier analysis, the combination of a high 8‑OHdG ratio in DNA (ELISA) and a low 8‑OHdG ratio in cytoplasm (qIHC) was associated with markedly worse patient prognosis than other combinations. Combined evaluation of the 8‑OHdG ratio using ELISA and qIHC may be pivotal for predicting surgical outcomes for patients with stage II/III CRC.

Modulation of Mitochondrial ERβ Expression Inhibits Triple-Negative Breast Cancer Tumor Progression by Activating Mitochondrial Function.

Breast cancer is a clinically and molecularly heterogeneous disease. Patients with triple-negative breast cancer (TNBC) have poorer outcomes than those with other breast cancer subtypes due to lack of effective molecular targets for therapy. The present study aimed to the identification of estrogen receptor (ER)β as a novel mitochondrial target in TNBC cells, together with underlying mechanisms. Expression of ERβ in clinical breast samples were examined by qRT-PCR, immunohistochemistry and immunoblotting. Subcellular distribution and binding of ERβ-Grp75 was determined by confocal microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. The effect of mitocondrial ERβ(mitoERβ) overexpression on cell proliferation and cell cycle distribution were assessed CCK-8 assays and FACS. Mitochondrial ROS, membrane potential, and Ca²⁺ level were measured using the specific fluorescent probes Mito-Sox, TMRE, and Rhod-2AM. The tumorigenic effect of mitoERβ overexpression was assessed using an anchorage-independent growth assay, sphere formation and a mouse orthotopic xenograft model. ERβ expression was lower in tumor tissue than in adjacent normal tissue of patients with breast cancer, and low levels of mitochondrial ERβ (mitoERβ) also were associated with increased tumor recurrence after surgery. Overexpression of mitoERβ inhibited the proliferation of TNBC cells and tumor masses in an animal model. Moreover, overexpression of mitoERβ increased ATP production in TNBC cells and normal breast MCF10A cells, with the latter completely reversed by mitoERβ knockdown in MCF10A cells. Grp75 was found to positively regulate ERβ translocation into mitochondria via a direct interaction. Coimmunoprecipitation and subcellular fractionation experiments revealed that ERβ-Grp75 complex is stable in mitochondria. These results suggest that the up-regulation of mitoERβ in TNBC cells ensures proper mitochondrial transcription, activating the OXPHOS system to produce ATP. Studying the effects of mitoERβ on mitochondrial activity and specific mitochondrial gene expression in breast cancer might help predict tumor recurrence, inform clinical decision-making, and identify novel drug targets in the treatment of TNBC.

Long non-coding RNA NEF inhibits proliferation and promotes apoptosis of laryngeal squamous cell carcinoma cells by inhibiting Wnt/β-catenin signaling.

The present study aimed to investigate the involvement of the recently identified long non-coding RNA neighboring enhancer of FOXA2 (lncRNA NEF) in laryngeal squamous cell carcinoma (LSCC). In this study, the expression levels of lncRNA NEF in tumor tissues and paired adjacent normal tissues were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Serum levels of NEF in patients with LSCC and healthy controls were also measured using RT-qPCR. Clinical and follow-up data of patients with LSCC were retrospectively analyzed. Diagnostic and prognostic values of serum NEF were evaluated by receiver operating characteristic curve and survival curve analysis, respectively. In addition, a NEF expression vector was constructed and transfected into human LSCC cells. The effects of NEF overexpression on cell proliferation, apoptosis and β-catenin expression were explored by Cell Counting kit-8 cell proliferation assay, MTT assay and western blotting. NEF was significantly downregulated in tumor tissues compared with in paired adjacent normal tissues of patients with LSCC. Serum levels of NEF were significantly lower in patients with LSCC than in healthy controls. Low serum levels of NEF distinguished patients with LSCC from healthy controls, and also indicated shorter postoperative survival. NEF overexpression inhibited proliferation and promoted apoptosis of LSCC cells, and also downregulated β-catenin expression. No significant effects of Wnt agonist on NEF expression were identified; however, Wnt agonist reduced the effects of NEF overexpression on cancer cell proliferation and apoptosis. In conclusion, lncRNA NEF may inhibit proliferation and promote apoptosis of LSCC cells by inhibiting Wnt/β-catenin signaling.

Preliminary functional inquiry of lncRNA ENST00000433673 in embryo implantation using bioinformatics analysis

ABSTRACTLong non-coding RNAs (lncRNAs), a class of non-coding RNA, have been shown to be essential in many diseases, such as infertility. Here, we found three candidate lncRNAs, ENST00000414116, ENST00000433673, and ENST00000448179, that are highly expressed in the uterus endometrial tissues of normal patients compared to the tissues of patients with adenomyosis, endometriosis, and recurrent implantation failure. lncRNAs ENST00000414116 and ENST00000433673 showed high expression in endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs), respectively, and lncRNA ENST00000448179 was specifically expressed in ESCs. The bioinformatics analysis results indicated that the target mRNAs of lncRNA ENST00000433673 were related to biological adhesion. Interestingly, intercellular adhesion molecule 1 (ICAM1), an interacting mRNA of the target mRNA integrin subunit alpha L (ITGAL), has been reported be an important regulator of embryo implantation. Further studies found that the target mRNA ITGAL and the interacting mRNA ICAM1 were highly expressed in the uterus endometrial tissues and EECs of normal patients. Based on our results, our study indicates that lncRNA ENST00000433673 might mediate the high expression of the target mRNA ITGAL, thereby promoting the expression of the interacting mRNA ICAM1 and the adhesion of EECs, which facilitates adhesion and implantation between the embryo and the mater.Abbreviations: AMs: adenomyosis; EMs: endometriosis; RIF: recurrent implantation failure; miRNAs: microRNAs; lncRNAs: Long non-coding RNAs; RT-qPCR: real-time quantitative PCR; ESCs: endometrial stromal cells; EECs: endometrial epithelial cells; BFE: free binding energy; PCDHB9: protocadherin beta 9; PARVG: parvin gamma; MAPK6: mitogen-activated protein kinase 6; LAF1: lymphocyte function-associated antigen 1

MRNA expression of programmed cell death ligand 1 and components of the phosphatidylinositol 3-kinase/AKT/phosphatase and tensin homolog pathway in epidermal growth factor receptor mutation-positive lung adenocarcinoma.

The objective of this study is to investigate the relationship between programmed cell death ligand 1 (PD-L1) expression and the mutation status of epidermal growth factor receptor (EGFR) in lung adenocarcinoma, as well as the correlation between the clinical features of patients and mRNA levels of PD-L1 and components of the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT)/phosphatase and tensin homolog (PTEN) pathways. mRNA levels of PD-L1, PI3K, AKT, and PTEN in tumor and matched normal tissues of patients with EGFR mutation-positive lung adenocarcinoma were determined by real-time polymerase chain reaction. Twenty-three patients with EGFR mutation-positive lung adenocarcinoma were enrolled, and 46 samples (23 pairs of tumor and matched normal lung tissues) were collected. PD-L1 and AKT mRNA levels were higher in EGFR mutation-positive lung adenocarcinoma than in matched normal lung tissues (P = 0.047 and P = 0.046, respectively), whereas PI3K and PTEN mRNA levels were significantly lower in the cancerous tissues (P = 0.009 and P = 0.039, respectively). PD-L1 expression was positively correlated with PI3K/AKT signaling pathway activation. PD-L1 upregulation in lung adenocarcinoma was positively correlated with EGFR exon 19 mutations (P = 0.034). AKT mRNA upregulation was correlated with lymph node metastasis (P = 0.034). PTEN mRNA downregulation was correlated with high tumor staging and lymph node metastasis (P = 0.035 and P = 0.014, respectively). Elevated PD-L1 mRNA expression in lung adenocarcinoma is associated with EGFR mutation and may be mediated through the PI3K-AKT pathway. EGFR exon 19 mutations closely correlate with increased PD-L1 mRNA expression. Increased AKT mRNA expression correlates with lymph node metastasis, while decreased PTEN mRNA levels correlate with advanced tumor stage and lymph node metastasis.

High orchiectomy and ipsilateral testicular sperm extraction in a man with a testicular tumor and Klinefelter syndrome: A case report

IntroductionTesticular epidermal cysts in Klinefelter syndrome are very rare. We report a case of Klinefelter syndrome associated with a testicular epidermal cyst. To our knowledge, this is the first report showing successful spermatozoa retrieval from the affected testis.Case presentationA 25‐year‐old married man was referred to our hospital with right scrotal induration, which was in lower pole of the right testis. Testicular cancer tumor markers were normal; endocrinological findings indicated hypergonadotropic hypogonadism. Semen analyses revealed azoospermia. Preoperative chromosome test result: 47, XXY karyotype; ultrasonography report: 1.9‐cm internal heterogeneous echoic mass in the right testis (malignancy not discarded). Because the patient hoped for children, he underwent high orchiectomy with ipsilateral testicular sperm extraction (200 spermatozoa from normal testicular tissue) for future fertilization procedures. Tumor pathology was an epidermal cyst.ConclusionWhile performing orchiectomy for testicular tumors, sperm retrieval should be attempted from normal tissues in patients planning for children.

Evaluation of pre-mir-34a rs72631823 single nucleotide polymorphism in triple negative breast cancer: A case-control study.

AimThe purpose of this study is to evaluate the role of pre-miR34a rs72631823 as potential risk factor and/or prognostic marker in patients with triple negative breast cancer.Methods114 samples of DNA from paraffin embedded breast normal tissues of patients with triple negative breast cancer and 124 samples of healthy controls were collected and analyzed for pre-miR34a rs72631823 polymorphism.ResultsPre-miR34a rs72631823 A allele was associated with increased TNBC risk both in univariate and multivariate analysis. The number of pre-miR34a rs72631823 AA subjects was very small and the association did not reach significance (p = 0.176, Fisher’s exact test). The examined polymorphism was not associated with overall survival at the univariate or multivariate Cox regression analysis (adjusted HR = 1.60, 95%CI: 0.64–3.96 for miR34 rs72631823 GA/AA vs. GG).ConclusionOur case-control study suggests that pre-miR34a rs72631823 A allele is associated with increased triple negative breast cancer risk.

Kinome-Wide siRNA Screening Identifies Src-Enhanced Resistance of Chemotherapeutic Drugs in Triple-Negative Breast Cancer Cells.

Background: Chemotherapy is the main treatment for triple-negative breast cancer (TNBC), which lack molecular markers for diagnosis and therapy. Cancer cells activate chemoresistant pathways and lead to therapeutic failure for patients with TNBC. Several kinases have been identified as chemoresistant genes. However, the involvement of kinases in the chemoresistance in TNBC cells is not fully understood.Methods: We employed a kinome siRNA library to screen whether targeting any kinases could increase the chemosensitivity of TNBC cell lines. The effects of kinase on cell viability in various breast cancer cells were validated with ATP level and colony formation. Protein expression and phosphorylation were determined by immunoblotting. The Cancer Genome Atlas (TCGA) dataset was collected to analyze the correlation of Src expression with prognosis of TNBC patients.Results: Primary screening and validation for the initial hits showed that Src kinase was a potential doxorubicin-resistant kinase in the TNBC cell lines MDA-MB-231 and Hs578T. Both siRNA against Src and the Src inhibitor dasatinib enhanced the cytotoxic effects of doxorubicin in TNBC cells. Moreover, phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), downstream effectors of Src, were accordingly decreased in Src-silenced or -inhibited TNBC cells. Additionally, TCGA data analysis indicated that Src expression levels in tumor tissues were higher than those in tumor-adjacent normal tissues in patients with TNBC. High co-expression level of Src and STAT3 was also significantly correlated with poor prognosis in patients.Conclusion: Our results showed that Src-STAT3 axis might be involved in chemoresistance of TNBC cells.

S100B regulates inflammatory response during osteoarthritis via fibroblast growth factor receptor 1 signaling.

The present study aimed to investigate the role of S100B in the inflammation process during osteoarthritis (OA). OA cartilage samples were collected for S100B expression analysis. S100B expression levels were significantly increased in patients with OA compared with the Controls (1.28±0.66 vs. 0.42±0.31; P=0.01) and were determined to be correlated with TNF-α (r=0.42; P=0.04) and IL-1β (r=0.73; P=0.001) expression levels. Orthopedic casting tape was used to immobilize the right knee at 180° extension of adult female New Zealand white rabbits for 4 weeks to establish an OA model. Cartilage specimens from the medial femoral condyle of these rabbits were used for histological confirmation and immunohistochemical analyses, whereas synovial fluid was used in ELISA assays for tumor necrosis factor (TNF)-α and interleukin (IL)-1β expression levels. Human synovial fibroblasts from the knee synovial tissues of normal patients with traumatic injury were transfected with S100B overexpression and knockdown plasmids and subjected to lipopolysaccharide (LPS) stimulation; subsequently, TNF-α and IL-1β expression levels in conditioned medium were determined by ELISA; S100B overexpression and knockdown significantly increased and decreased the TNF-α and IL-1β expression levels, respectively. Increased TNF-α (573.3±15.4 vs. 102.6±8.7 pg) and IL-1β (378.6±7.2 vs. 170.1±5.8 pg) expression levels were detected in OA model rabbits compared with the Control rabbits. Additionally, S100B, fibroblast growth factor (FGF)-1 and FGF receptor (FGFR)-1 mRNA and protein expression levels were increased in OA model rabbits compared with the Control group. FGFR1 knockdown significantly decreased TNF-α and IL-1β expression levels in LPS-stimulated S100B-overexpressing human synovial fibroblasts. S100B is involved in FGFR1 signaling-mediated inflammatory response during OA, which may be considered as a potential therapeutic target.

MED12 mutations and fumarate hydratase inactivation in uterine adenomyomas.

STUDY QUESTIONDo the uterine leiomyoma driver events – mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) overexpression, and fumarate hydratase (FH) inactivation – also contribute to the development of uterine adenomyomas?SUMMARY ANSWER MED12 mutations and FH deficiency occur in a subset of uterine adenomyomas, but at lower frequencies than in leiomyomas.WHAT IS KNOWN ALREADYUterine adenomyomas are benign tumours with clinical features very similar to uterine leiomyomas. Mutations affecting MED12, HMGA2 and FH account for up to 80–90% of leiomyomas, but their contribution to adenomyomas is not known.STUDY DESIGN, SIZE, DURATIONFormalin-fixed paraffin-embedded adenomyoma samples from 21 patients operated on during 2012–2014 were collected at the pathology department’s archives and analysed for uterine leiomyoma driver events.PARTICIPANTS/MATERIALS, SETTING, METHODSAdenomyoma diagnoses were verified by a specialized pathologist and representative areas were marked on haematoxylin-eosin slides. DNA was extracted from the tissue samples and sequenced to detect mutations in MED12. Expression levels of HMGA2 and 2SC, a robust indirect method to detect FH inactivation, were analysed by immunohistochemistry (IHC). The coding region of FH was sequenced in one adenomyoma sample showing strong 2SC staining as well as in the same patient’s normal tissue sample. All patients’ medical histories were collected and reviewed.MAIN RESULTS AND THE ROLE OF CHANCE MED12 mutation c.131G > A, p.G44D, the most common mutation in uterine leiomyomas, was identified in two samples (2/21; 9.5%). One adenomyoma displayed strong 2SC positivity and subsequent sequencing revealed a frameshift FH mutation c.911delC, p.P304fs in the tumour. The mutation was also present in the patient’s normal tissue sample, indicating that she has a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. HMGA2 protein expression was normal in all adenomyomas.LIMITATIONS, REASONS FOR CAUTIONRestricted sample size limits the determination of exact mutation frequencies of the studied aberrations in adenomyomas.WIDER IMPLICATIONS OF THE FINDINGSUterine leiomyoma driver mutations do contribute to the development of some adenomyomas. We also report an adenomyoma in the context of hereditary HLRCC syndrome. Despite clinical similarities, the pathogenic mechanisms of adenomyomas and leiomyomas are likely different. Large-scale genomic analyses are warranted to elucidate the complete molecular background of adenomyomas.STUDY FUNDING/COMPETING INTERESTSThis study was supported by The Academy of Finland, the Sigrid Jusélius Foundation, and the Cancer Society of Finland. The authors declare no conflict of interest.

Overexpression of High-Mobility Motor Box 1 in the Blood and Tissues of Patients with Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world. Extra- and intra-cellular high-mobility motor box 1 (HMGB1) proteins are invovled in the pathogenesis and prognosis of cancer. Regarding this, the present study was conducted with the aim of investigating the expression of HMGB1 protein and mRNA levels in the blood, tumor tissue, and marginal normal tissue of patients with head and neck squamous cell carcinoma (HNSCC) using the quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). This study was performed on 88 patients with HNSCC, who referred to the otorhinolaryngology and oral pathology departments, affiliated to Mashhad University of Medical Sciences, Mashhad, Iran, and a group of healthy subjects (i.e., control group) matched in terms of age and gender. RNA was collected from fresh tumor tissues, marginal tissues, and blood, followed by the implementation of quantitative PCR on the specimens. Furthermore, the expression of HMGB1 in tumor and normal margins was evaluated by means of IHC. The data were analyzed in SPSS software. According to the results the expression levels of HMGB1 protein and mRNA were significantly higher in the tumor tissue than in the normal margin tissues (P<0.01). In addition, there was a significant correlation between histologic grading and the expression of HMGB1 protein and mRNA in tissues (P<0.05). Furthermore, the receiver operating characteristic curve of the HMGB1 mRNA in tissue was located closer to the theoretical 100% sensitivity. The findings revealed a higher increase in the levels of mRNA and HMGB1 protein in HNSCC, compared to those in the normal margin tissues. In addition, HMGB1 mRNA showed a significant expression in the tissue and blood of the patients with lymph node involvement.