Comprehensive Analysis of miRNome Alterations in Response to Sorafenib Treatment in Colorectal Cancer Cells.

MicroRNAs (miRNAs) are master regulators of drug resistance and have been previously proposed as potential biomarkers for the prediction of therapeutic response in colorectal cancer (CRC). Sorafenib, a multi-kinase inhibitor which has been approved for the treatment of liver, renal and thyroid cancer, is currently being studied as a monotherapy in selected molecular subtypes or in combination with other drugs in metastatic CRC. In this study, we explored sorafenib-induced cellular effects in Kirsten rat sarcoma viral oncogene homolog olog (KRAS) wild-type and KRAS-mutated CRC cell lines (Caco-2 and HRT-18), and finally profiled expression changes of specific miRNAs within the miRNome (>1000 human miRNAs) after exposure to sorafenib. Overall, sorafenib induced a time- and dose-dependent growth-inhibitory effect through S-phase cell cycle arrest in KRAS wild-type and KRAS-mutated CRC cells. In HRT-18 cells, two human miRNAs (hsa-miR-597 and hsa-miR-720) and two small RNAs (SNORD 13 and hsa-miR-3182) were identified as specifically sorafenib-induced. In Caco-2 cells, nine human miRNAs (hsa-miR-3142, hsa-miR-20a, hsa-miR-4301, hsa-miR-1290, hsa-miR-4286, hsa-miR-3182, hsa-miR-3142, hsa-miR-1246 and hsa-miR-720) were identified to be differentially regulated post sorafenib treatment. In conclusion, we confirmed sorafenib as a potential anti-neoplastic treatment strategy for CRC cells by demonstrating a growth-inhibitory and cell cycle-arresting effect of this drug. Changes in the miRNome indicate that some specific miRNAs might be relevant as indicators for sorafenib response, drug resistance and potential targets for combinatorial miRNA-based drug strategies.

Relevance of MicroRNA200 Family and MicroRNA205 for Epithelial to Mesenchymal Transition and Clinical Outcome in Biliary Tract Cancer Patients.

Extensive stromal interaction is one reason for the dismal outcome of biliary tract cancer (BTC) patients. Epithelial to mesenchymal transition (EMT) is involved in tumor invasion and metastasis and is partly regulated by microRNAs (miRs). This study explores the expression of anti-EMT miR200 family (miR141, -200a/b/c, -429) and miR205 as well as the EMT-related proteins E-cadherin and vimentin in a panel of BTC cell lines and clinical specimens by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry, respectively. MicroRNA expression was correlated to (i) the expression patterns of E-cadherin and vimentin; (ii) clinicopathological characteristics; and (iii) survival data. MicroRNA-200 family and miR205 were expressed in all BTC cells and clinical specimens. E-cadherin and vimentin showed a mutually exclusive expression pattern in both, in vitro and in vivo. Expression of miR200 family members positively correlated with E-cadherin and negatively with vimentin expression in BTC cells and specimens. High expression of miR200 family members (but not miR205) and E-cadherin was associated with longer survival, while low miR200 family and high vimentin expression was a predictor of unfavorable survival. Overall, the current study demonstrates the relevance of the miR200 family in EMT of BTC tumors and suggests these miRs as predictors for positive outcome.

MiR-96-5p influences cellular growth and is associated with poor survival in colorectal cancer patients.

Expression of miR-96-5p is frequently altered in various types of cancer and the KRAS oncogene has been identified as one of its potential targets. However, the biological role of miR-96-5p expression in colorectal cancer (CRC) and its ability to predict the clinical course of patients have not been investigated yet. In this study, we explored miR-96-5p expression in 80 CRC patients and evaluated the impact on clinical outcome by Kaplan-Meier curves and multivariate Cox proportional models. In vitro miR-96-5p inhibition and overexpression were performed in CRC cells and the effects on cellular growth, anchorage-independent growth, apoptosis, and epithelial-mesenchymal transition (EMT)-related gene expression were explored. Low miR-96-5p expression levels in tumor tissue were associated with distant metastasis (P = 0.025) and multivariate Cox regression analysis identified low levels of miR-96-5p as an independent prognostic factor with respect to cancer-specific survival (hazard ratio = 1.78, 95%CI = 1.03-3.03, P < 0.038). In vitro overexpression of miR-96-5p led to a reduced cellular growth rate (P < 0.05), reduced colonies in soft agar (P < 0.05), corroborated by a decreased cyclin D1 and increased p27-CDKN1A expression (P < 0.05). Forced expression of miR-96-5p in CRC cells entailed no effects on apoptosis or EMT-related genes but decreased the expression levels of the KRAS oncogene (P < 0.05). Despite regulating KRAS expression, there was no significant association in miR-96-5p expression levels and response rates to EGFR-targeting agents. In conclusion, our data suggest that miR-96-5p influences cellular growth of CRC cells and low expression of miR-96-5p seems to be associated with poor clinical outcome in CRC patients.

microRNAs and Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common types of human cancer with high cancer-related morbidity and mortality rates. The development and clinical validation of novel therapeutic avenues have improved the clinical outcome, but metastatic CRC still remains an incurable disease in most cases. The interest in discovering novel pathophysiological drivers in CRC is intensively ongoing and the search for novel biomarkers for early diagnosis, for patient's stratification for prognostic purposes or for predicting treatment response are warranted. microRNAs are small RNA molecules that regulate the expression of larger messenger RNA species by different mechanisms with the final consequence to provide a fine tuning tool for global gene expression patterns. First discovered in worms, around 15 years ago it became clear that microRNAs are also existing in humans and that they are widely involved in human carcinogenesis. Within the last years, tremendous progress in the understanding of microRNAs and their role in CRC carcinogenesis has been developed. In this book chapter, several examples of previously identified microRNAs and how they influence colorectal carcinogenesis will be discussed. The information starting at the underlying molecular mechanisms towards clinical applications will be depicted and an overview what great potential these small molecules might carry in future colorectal cancer medicine, will be discussed.

SOX9 is a proliferation and stem cell factor in hepatocellular carcinoma and possess widespread prognostic significance in different cancer types.

SOX9 has been previously shown to be involved in hepatocellular carcinoma (HCC) and other types of cancer. However, prognostic studies so far involved rather small cohorts or lack external validation and experimental data. In this study, we firstly determined the histological expression pattern of SOX9 in human HCC by immunohistochemistry (n = 84) and evaluated its prognostic value. External cohorts of publicly available datasets were used to validate its prognostic relevance in HCC (n = 359) and other types of cancer including breast (n = 3951), ovarian (n = 1306), lung (n = 1926) and gastric cancer (n = 876). Functional SOX9 knock-down studies using siRNA and cancer stem cell models were generated in a panel of liver and breast cancer cell lines. High level of SOX9 was associated with poor survival even after adjustment for other prognostic factors in multivariate analysis (HR = 2.103, 95%CI = 1.064 to 4.156, p = 0.021). SOX9 prevailed a poor prognostic factor in all cancer validation cohorts (p<0.05). Reduced SOX9 expression by siRNA decreased the growth of liver cancer cells (p<0.05). SOX9 expression was associated with stem cell features in all tested cell lines (p<0.05). In conclusion, this study demonstrated in a large number of patients from multiple cohorts that high levels of SOX9 are a consistent negative prognostic factor.

Low spinophilin expression enhances aggressive biological behavior of breast cancer.

Spinophilin, a putative tumor suppressor gene, has been shown to be involved in the pathogenesis of certain types of cancer, but its role has never been systematically explored in breast cancer. In this study, we determined for the first time the expression pattern of spinophilin in human breast cancer molecular subtypes (n = 489) and correlated it with survival (n = 921). We stably reduced spinophilin expression in breast cancer cells and measured effects on cellular growth, apoptosis, anchorage-independent growth, migration, invasion and self-renewal capacity in vitro and metastases formation in vivo. Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells. Spinophilin expression was significantly lower in basal-like breast cancer (p<0.001) and an independent poor prognostic factor in breast cancer patients (hazard ratio = 1.93, 95% confidence interval: 1.24 -3.03; p = 0.004) A reduction of spinophilin levels increased cellular growth in breast cancer cells (p<0.05), without influencing activation of apoptosis. Anchorage-independent growth, migration and self-renewal capacity in vitro and metastatic potential in vivo were also significantly increased in spinophilin-silenced cells (p<0.05). Finally, we identified several differentially expressed genes in spinophilin-silenced cells. According to our data, low levels of spinophilin are associated with aggressive behavior of breast cancer.

Spinophilin expression determines cellular growth, cancer stemness and 5-flourouracil resistance in colorectal cancer.

The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC.

Loss of the putative tumor suppressor protein spinophilin is associated with poor prognosis in head and neck cancer.

The putative tumor suppressor protein spinophilin has been recently involved in the pathogenesis of lung, liver, and other types of cancer. Previous studies also indicate that a loss of spinophilin in combination with functional impairment of p53 drives tumor progression. To date, no data exist about the role of spinophilin in head and neck squamous cell carcinoma (HNSCC). In the present study, we evaluated spinophilin and p53 expression by immunohistochemistry in 85 patients with nonmetastatic HNSCC. Kaplan-Meier curves and multivariate Cox proportional models were used to define the prognostic relevance of spinophilin for patients with HNSCC. Overall, immunoreactivity for spinophilin was reduced in 40 tumors (47%). Nine cases (10.5%) showed complete loss of spinophilin. Kaplan-Meier curve analysis demonstrated that reduced spinophilin expression is associated with poor overall survival (P = .022). Concomitant analysis of spinophilin and p53 further showed that patients with reduced spinophilin expression and nuclear p53 staining have a significantly decreased overall survival (hazard ratio, 1.96; 95% confidence interval, 1.06-3.61; P = .030). In conclusion, the combination of reduced spinophilin expression and nuclear p53 staining indicates a poor prognosis in HNSCC patients. Based on our results, spinophilin might play a previously unrecognized role in the pathogenesis of HNSCC.

miR-181a is associated with poor clinical outcome in patients with colorectal cancer treated with EGFR inhibitor.

AIMS: miR-181a expression is frequently altered in different types of cancer. Members of the Wnt/ß-catenin signalling pathway, which is commonly altered in colorectal cancer (CRC), have been reported as molecular interaction partners of miR-181. However, the role of miR-181a expression in CRC and its ability to predict survival and response to agents targeting the epidermal growth factor receptor (EGFR) have not been explored yet. METHODS: In this study, we analysed 80 patients with wild type KRAS CRC undergoing treatment with the EGFR-targeting monoclonal antibodies cetuximab and panitumumab for metastatic CRC. The KRAS mutational status was determined by pyrosequencing and miR-181a expression was measured by quantitative RT-PCR in CRC tumour tissue and corresponding non-neoplastic colon tissue. The microRNA expression levels were correlated with clinicopathological characteristics. Cancer-specific survival was calculated by univariate and multivariate analyses, and progression-free survival (PFS) during treatment with EGFR-targeting agents was also evaluated. RESULTS: A low miR-181a expression level was associated with poor differentiation of CRC (p=0.04). A Kaplan-Meier curve showed a decreased survival time for patients with low miR-181a expression (p=0.019). Low miR-181a expression was furthermore associated with poor PFS (p=0.015). CONCLUSIONS: In conclusion, our data suggest that the miR-181a expression level is associated with poor survival in patients with CRC. Furthermore, miR-181a expression might predict PFS in EGFR-targeted therapy.

Preoperative neutrophil-to-lymphocyte ratio predicts clinical outcome in patients with stage II and III colon cancer.

UNLABELLED: AIM/ BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR), a combined indicator of inflammation and immunology, is as yet unidentified regarding the clinical outcome of stage II and III colon cancer patients. We evaluated the effect of NLR on time-to-recurrence (TTR) and overall survival (OS) in selected patients. PATIENTS AND METHODS: A total of 504 patients with stage II and III colon cancer were included in this retrospective study. Preoperative NLR with a cut-off level of 4 was associated with TTR and OS. RESULTS: In univariate analysis, elevated NLR was significantly associated with decreased TTR (p=0.001) and remained significant in multivariate analysis (p=0.006). Patients with NLR >4 showed a median TTR of 62.2 months. In contrast, patients with NLR ≤ 4 had a median TTR of 92.6 months. CONCLUSION: This study suggests that preoperative NLR may be an independent prognostic marker for TTR in stage II and III colon cancer patients.

Comprehensive analysis of alterations in the miRNome in response to photodynamic treatment.

Photodynamic therapy (PDT) is a local tumour treatment accepted for a number of indications. PDT operates via the cellular stress response through the production of reactive oxygen species and subsequent cellular damage, resulting in cell death. Although PDT-induced signalling and cytotoxicity mechanisms have been investigated, the effect of PDT on microRNA (miRNA) expression is largely unknown. Therefore, we conducted a comprehensive microarray-based analysis of the miRNome of human epidermoid carcinoma cells (A431) following in vitro photodynamic treatment using polyvinylpyrrolidone hypericin (PVPH) as a photosensitiser and nearly homogeneous apoptosis-inducing conditions. Using microarray analysis we found eight miRNAs to be significantly differentially expressed 5h post treatment compared with the baseline levels and three miRNAs with more than 2-fold differential expression that could be detected in 1 or 2 biological replicates. The verification of these results by quantitative RT-PCR including a detailed time-course revealed an up to 15-fold transient over-expression of miR-634, miR-1246, miR-1290 and miR-487b compared with the basal level. For these miRNAs, in silico mRNA target prediction yielded numerous target transcripts involved in the regulation of cell stress, apoptosis, cell adherence and proliferation. This study provides the first comprehensive miRNome analysis after PDT treatment and may help to develop novel miRNA-based therapeutic approaches to further increase the efficiency of PDT.

External validation of the derived neutrophil to lymphocyte ratio as a prognostic marker on a large cohort of pancreatic cancer patients.

BACKGROUND: With growing evidence on the role of inflammation in cancer biology, the presence of a systemic inflammatory response has been postulated as having prognostic significance in a wide range of cancer types. The derived neutrophil to lymphocyte ratio (dNLR), which represents an easily determinable potential prognostic marker in daily practise and clinical trials, has never been externally validated in pancreatic cancer (PC) patients. METHODS: Data from 474 consecutive PC patients, treated between 2004 and 2012 at a single centre, were evaluated retrospectively. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the prognostic relevance of dNLR, univariate and multivariate Cox regression models were applied. RESULTS: We calculated by ROC analysis a cut-off value of 2.3 for the dNLR to be ideal to discriminate between patients' survival in the whole cohort. Kaplan-Meier curve reveals a dNLR≥2.3 as a factor for decreased CSS in PC patients (p<0.001, log-rank test). An independent significant association between high dNLR≥2.3 and poor clinical outcome in multivariate analysis (HR = 1.24, CI95% = 1.01-1.51, p = 0.041) was identified. CONCLUSION: In the present study we confirmed elevated pre-treatment dNLR as an independent prognostic factor for clinical outcome in PC patients. Our data encourage independent replication in other series and settings of this easily available parameter as well as stratified analysis according to tumor resectability.

MicroRNAs in renal cell carcinoma: implications for pathogenesis, diagnosis, prognosis and therapy.

Renal cell carcinoma (RCC) is a potentially curable disease, especially if the tumor is limited to the kidneys and no systemic metastatic spread has occurred by the time of diagnosis. Despite the potential for successful surgical removal of the tumor-bearing organ in localized stages and the likelihood of treatment success, the complications and long-term morbidity and mortality of RCC are difficult to accurately predict. The currently used drugs were developed based on the understanding of the molecular details of pathogenesis at the time, which has improved over the past several decades. However, more efforts should be made to improve early diagnosis, the surveillance of patients who undergo resection and treatment for metastatic RCC. Recently, small non-coding RNAs (microRNAs) were found to play pivotal roles in the metastatic dissemination of tumor cells in different types of cancer. The aim of this review is to provide an overview of the role of microRNAs in the pathogenesis of RCC and to discuss their potential as diagnostic, prognostic and, ultimately, therapeutic biomolecules.