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.     

Why the stroma matters in breast cancer

Survival and recurrence rates in breast cancer are variable for common diagnoses, and therefore the biological underpinnings of the disease that determine those outcomes are yet to be fully understood. As a result, translational medicine is one of the fastest growing arenas of study in tumor biology. With advancements in genetic and imaging techniques, archived biopsies can be examined for purposes other than diagnosis. There is a great deal of evidence that points to the stroma as the major regulator of tumor progression following the initial stages of tumor formation, and the stroma may also contribute to risk factors determining tumor formation. Therefore, aspects of stromal biology are well-suited to be a focus for studies of patient outcome, where statistical differences in survival among patients provide evidence as to whether that stromal component is a signpost for tumor progression. In this review we summarize the latest research done where breast cancer patient survival was correlated with aspects of stromal biology, which have been put into four categories: reorganization of the extracellular matrix (ECM) to promote invasion, changes in the expression of stromal cell types, changes in stromal gene expression, and changes in cell biology signaling cascades to and from the stroma.     

Tumor microenvironment: the role of the tumor stroma in cancer

The tumor microenvironment, composed of non-cancer cells and their stroma, has become recognized as a major factor influencing the growth of cancer. The microenvironment has been implicated in the regulation of cell growth, determining metastatic potential and possibly determining location of metastatic disease, and impacting the outcome of therapy. While the stromal cells are not malignant per se, their role in supporting cancer growth is so vital to the survival of the tumor that they have become an attractive target for chemotherapeutic agents. In this review, we will discuss the various cellular and molecular components of the stromal environment, their effects on cancer cell dynamics, and the rationale and implications of targeting this environment for control of cancer. Additionally, we will emphasize the role of the bone marrow-derived cell in providing cells for the stroma.     

Integrated analysis of co‐expression and ceRNA network identifies five lncRNAs as prognostic markers for breast cancer

Long non‐coding RNAs (lncRNAs), which competitively bind miRNAs to regulate target mRNA expression in the competing endogenous RNAs (ceRNAs) network, have attracted increasing attention in breast cancer research. We aim to find more effective therapeutic targets and prognostic markers for breast cancer. LncRNA, mRNA and miRNA expression profiles of breast cancer were downloaded from TCGA database. We screened the top 5000 lncRNAs, top 5000 mRNAs and all miRNAs to perform weighted gene co‐expression network analysis. The correlation between modules and clinical information of breast cancer was identified by Pearson's correlation coefficient. Based on the most relevant modules, we constructed a ceRNA network of breast cancer. Additionally, the standard Kaplan‐Meier univariate curve analysis was adopted to identify the prognosis of lncRNAs. Ultimately, a total of 23 and 5 modules were generated in the lncRNAs/mRNAs and miRNAs co‐expression network, respectively. According to the Green module of lncRNAs/mRNAs and Blue module of miRNAs, our constructed ceRNA network consisted of 52 lncRNAs, 17miRNAs and 79 mRNAs. Through survival analysis, 5 lncRNAs (AL117190.1, COL4A2‐AS1, LINC00184, MEG3 and MIR22HG) were identified as crucial prognostic factors for patients with breast cancer. Taken together, we have identified five novel lncRNAs related to prognosis of breast cancer. Our study has contributed to the deeper understanding of the molecular mechanism of breast cancer and provided novel insights into the use of breast cancer drugs and prognosis.     

Why the stroma matters in breast cancer: Insights into breast cancer patient outcomes through the examination of stromal biomarkers

Survival and recurrence rates in breast cancer are variable for common diagnoses, and therefore the biological underpinnings of the disease that determine those outcomes are yet to be fully understood. As a result, translational medicine is one of the fastest growing arenas of study in tumor biology. With advancements in genetic and imaging techniques, archived biopsies can be examined for purposes other than diagnosis. There is a great deal of evidence that points to the stroma as the major regulator of tumor progression following the initial stages of tumor formation, and the stroma may also contribute to risk factors determining tumor formation. Therefore, aspects of stromal biology are well-suited to be a focus for studies of patient outcome, where statistical differences in survival among patients provide evidence as to whether that stromal component is a signpost for tumor progression. In this review we summarize the latest research done where breast cancer patient survival was correlated with aspects of stromal biology, which have been put into four categories: reorganization of the extracellular matrix (ECM) to promote invasion, changes in the expression of stromal cell types, changes in stromal gene expression, and changes in cell biology signaling cascades to and from the stroma.     

Tumour-suppressive microRNA-424-5p directly targets CCNE1 as potential prognostic markers in epithelial ovarian cancer

An accumulated evidence supports that MicroRNAs (miRNAs) have shown a prominent role in pathological processes and different tumor onset. However, to date, the potential functional roles and molecular mechanisms by how microRNA-424-5p(miR-424-5p) affects cancer cell proliferation are greatly unclear, especially in epithelial ovarian cancer(EOC).In this study, we demonstrated that miR-424-5p was significantly down-regulated in EOC tissues and cell lines. The level of miR-424-5p was negatively correlated with tumor size, TNM stage, pathological grade, lymphatic metastasis of EOC. Restoring miR-424-5p expression in EOC cells dramatically suppressed cell proliferation and caused an accumulation of cells in G1 phase, and thus contributed to better prognosis of EOC patients. Mechanistically, miR-424-5p inhibits CCNE1 expression through targeting CCNE1 3′UTR, and subsequent arrest cell cycle in G1/G0 phase by inhibiting E2F1-pRb pathway. This study revealed functional and mechanistic links between miR-424-5p and CCNE1 in the progression of EOC and provide an important insight into that miR-424-5p may serve as a therapeutic target in EOC.     

Integrative analysis of DNA methylation and gene expression through machine learning identifies stomach cancer diagnostic and prognostic biomarkers

DNA methylation is an early event in tumorigenesis. Here, by integrative analysis of DNA methylation and gene expression and utilizing machine learning approaches, we introduced potential diagnostic and prognostic methylation signatures for stomach cancer. Differentially-methylated positions (DMPs) and differentially-expressed genes (DEGs) were identified using The Cancer Genome Atlas (TCGA) stomach adenocarcinoma (STAD) data. A total of 256 DMPs consisting of 140 and 116 hyper- and hypomethylated positions were identified between 443 tumour and 27 nontumour STAD samples. Gene expression analysis revealed a total of 2821 DEGs with 1247 upregulated and 1574 downregulated genes. By analysing the impact of cis and trans regulation of methylation on gene expression, a dominant negative correlation between methylation and expression was observed, while for trans regulation, in hypermethylated and hypomethylated genes, there was mainly a negative and positive correlation with gene expression, respectively. To find diagnostic biomarkers, we used 28 hypermethylated probes locating in the promoter of 27 downregulated genes. By implementing a feature selection approach, eight probes were selected and then used to build a support vector machine diagnostic model, which had an area under the curve of 0.99 and 0.97 in the training and validation (GSE30601 with 203 tumour and 94 nontumour samples) cohorts, respectively. Using 412 TCGA-STAD samples with both methylation and clinical data, we also identified four prognostic probes by implementing univariate and multivariate Cox regression analysis. In summary, our study introduced potential diagnostic and prognostic biomarkers for STAD, which demands further validation.     

Up-regulation of the novel proinflammatory adipokines lipocalin-2, chitinase-3 like-1 and osteopontin as well as angiogenic-related factors in visceral adipose tissue of patients with colon cancer

Background

Obesity is widely recognised as an important risk factor for colorectal cancer (CC).

Aim

The study aimed to evaluate the effect of CC on circulating concentrations and gene expression levels of inflammatory and angiogenesis-related factors in human visceral adipose tissue (VAT).

Methods

VAT biopsies were obtained from 18 healthy individuals and 11 patients with CC. Real-time polymerase chain reactions were performed to quantify gene expression levels and zymographic analyses were used to determine the activity of matrix metalloproteinases (MMPs).

Results

Patients with CC exhibited increased mRNA expression levels of lipocalin-2 (P=.014), osteopontin (P=.027), tumor necrosis factor-α (TNF-α) (P=.016) and chitinase-3 like-1 (P=.006) compared to control subjects in VAT. Gene expression levels of hypoxia-inducible factor-1 α, vascular endothelial growth factor and MMP-2 were significantly higher (P<.05) in VAT of patients with CC. The expression of insulin-like growth factor I, insulin growth factor binding protein 3 and MMP-9 followed the same trend, although no significant differences were reached. The enzymatic activity of MMP-9 was increased (P<.001) in patients with CC. Furthermore, individuals with CC showed increased (P<.05) circulating concentrations of the inflammatory markers interleukin-6, tumour necrosis factor α and hepatocyte growth factor, whereas levels of the anti-inflammatory adipokine adiponectin were decreased (P<.01).

Conclusion

These findings represent the first observation that mRNA levels of the novel inflammatory factors lipocalin-2, chitinase-3 like-1 and osteopontin are increased in human VAT of subjects with CC. This observation together with the up-regulation of angiogenic factors suggests that adipokines secreted by VAT may be involved in the development of colon cancer.     

A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor’s stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam ⁻ , AroA ⁻ ) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-γ release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8⁺ T-cell response against Legumain⁺ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.     

Matrix metalloproteases and TIMPs as prognostic biomarkers in breast cancer patients treated with radiotherapy: A pilot study

Breast cancer (BC) is the most common tumour in women and one of the most important causes of cancer death worldwide. Radiation therapy (RT) is widely used for BC treatment. Some proteins have been identified as prognostic factors for BC (Ki67, p53, E‐cadherin, HER2). In the last years, it has been shown that variations in the expression of MMPs and TIMPs may contribute to the development of BC. The aim of this pilot work was to study the effects of RT on different MMPs (‐1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐10, ‐12 and ‐13) and TIMPs (‐1 to ‐4), as well as their relationship with other variables related to patient characteristics and tumour biology. A group of 20 BC patients treated with RT were recruited. MMP and TIMP serum levels were analysed by immunoassay before, during and after RT. Our pilot study showed a slight increase in the levels of most MMP and TIMP with RT. However, RT produced a significantly decrease in TIMP‐1 and TIMP‐3 levels. Significant correlations were found between MMP‐3 and TIMP‐4 levels, and some of the variables studied related to patient characteristics and tumour biology. Moreover, MMP‐9 and TIMP‐3 levels could be predictive of RT toxicity. For this reason, MMP‐3, MMP‐9, TIMP‐3 and TIMP‐4 could be used as potential prognostic and predictive biomarkers for BC patients treated with RT.     

Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma: Key events in the progression from an in situ to an invasive breast carcinoma

The progression from in situ to invasive breast carcinoma is still an event poorly understood. However, it has been suggested that interactions between the neoplastic cells and the tumor microenvironment may play an important role in this process. Thus, the determination of differential tumor-stromal metabolic interactions could be an important step in invasiveness.

The expression of stromal Caveolin-1 (Cav-1) has already been implicated in the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Additionally, stromal Cav-1 expression has been associated with the expression of stromal monocarboxylate transporter 4 (MCT4) in invasive breast cancer. However, the role of stromal MCT4 in invasiveness has never been explored, neither the association between Cav-1 and MCT4 in the transition from breast DCIS to IDC.

Therefore, our aim was to investigate in a series of breast cancer samples including matched in situ and invasive components, if there was a relationship between stromal Cav-1 and MCT4 in the progression from in situ to invasive carcinoma. We found loss of stromal Cav-1 in the progression to IDC in 75% of the cases. In contrast, MCT4 stromal expression was acquired in 87% of the IDCs. Interestingly, a concomitant loss of Cav-1 and gain of MCT4 was observed in the stroma of 75% of the cases, when matched in situ and invasive carcinomas were compared. These results suggest that alterations in Cav-1 and MCT4 may thus mark a critical point in the progression from in situ to invasive breast cancer.     

幽门螺杆菌感染与胃癌相关基因的筛选及预后分析

目的 通过分析幽门螺杆菌感染胃黏膜组织和胃癌细胞系后的差异基因变化,并在癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和肿瘤基因芯片(Oncomine)数据库进行验证,探究幽门螺杆菌导致胃癌发生、发展的分子机制。 方法 分析基因表达汇编(Gene Expression Omnibus,GEO)数据库幽门螺杆菌感染相关芯片集GSE5081与GSE70394,绘制维恩图查找幽门螺杆菌感染后共同上调的差异基因。对共同上调的差异基因进行功能富集分析。通过TCGA和Oncomine数据库验证差异基因在胃癌中的表达。利用Kaplan Meier Plotter数据库和GEPIA数据库分析差异基因表达高低与胃癌患者预后是否存在相关性。 结果 通过差异基因筛选和维恩分析,两个芯片集共有21个共同上调差异基因。GO分析发现共同上调差异基因主要富集在对细菌来源分子的反应、趋化因子CXCR受体结合、中性粒细胞趋化作用等相关的基因功能上;KEGG通路主要富集在癌症通路、TNF信号通路、趋化因子信号通路等。STRING以及PPI数据库分析发现21个基因中PRDM1、IL10、NRP1、BIRC3、GNG13、CXCL1、CXCL2、CXCL3、CXCL8基因存在有网络关系,属于关键枢纽基因。通过TCGA和Oncomine数据库筛选及验证,发现在胃癌组织中NRP1、CXCL1、CXCL8 基因明显上调,结果差异有统计学意义(TCGA数据库中,三者P值均小于0.05,Oncomine数据库中,NRP1:t=4.607,P结论 不同的数据库均显示NRP1、CXCL1、CXCL8三个基因与幽门螺杆菌感染相关,同时在胃癌中高表达,并且NRP1的高表达与胃癌的不良预后相关,这些结果为进一步探究幽门螺杆菌导致胃癌发生、发展的分子机制提供了重要基础。     

A fine balance between CCNL1 and TIMP1 contributes to the development of breast cancer cells

Cyclin L1 (CCNL1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) are candidate genes involved in several types of cancer. However, the expression of CCNL1 and the relationship between CCNL1 and TIMP1 in breast cancer cells is unknown. Using patients’ breast cancer tissues, the expression of CCNL1 and TIMP1 was measured by cDNA microarray and further confirmed by real-time RT-PCR and western blotting. Overexpression or repression of CCNL1 and TIMP1, individually or together, was performed in breast cancer MDA-MB-231 cells by transient transformation methods to investigate their role in breast cancer cell growth. Simultaneously, mRNA and protein expression levels of CCNL1 and TIMP1 were also measured. CCNL1 and TIMP1 expression was significantly elevated in breast cancer tissues compared with that in peri-breast cancer tissues of patients by cDNA microarray and these results were further confirmed by real-time RT-PCR and western blotting. Interestingly, in vitro experiments showed a stimulatory effect of TIMP1 and an inhibitory effect of CCNL1 on growth of MDA-MB-231 cells. Co-expression or co-repression of these two genes did not affect cell growth. Overexpression of CCNL1 and TIMP1 individually induced overexpression of each other. These data demonstrate that there is a fine balance between CCNL1 and TIMP1, which may contribute to breast cancer development.