Kjetil Boye

S100A4 and Metastasis: A Small Actor Playing Many Roles

The calcium-binding protein S100A4 promotes metastasis in several experimental animal models, and S100A4 protein expression is associated with patient outcome in a number of tumor types. S100A4 is localized in the nucleus, cytoplasm, and extracellular space and possesses a wide range of biological functions, such as regulation of angiogenesis, cell survival, motility, and invasion. In this review, we summarize the evidence connecting S100A4 and cancer metastasis and discuss the mechanisms by which S100A4 promotes tumor progression.

Clinical relevance of microRNA miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145 in colorectal cancer

BackgroundMicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. In this study, using qRT-PCR, we examined the expression of six miRNAs (miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145) in tumors from 193 prospectively recruited patients with colorectal cancer, and associations with clinicopathological parameters and patient outcome were analyzed. The miRNAs were chosen based on previous studies for their biomarker potential and suggested biological relevance in colorectal cancer.MethodsThe miRNA expression was examined by qRT-PCR. Associations between miRNA expression and clinicopathological variables were explored using Mann–Whitney U and Kruskal-Wallis test while survival was estimated using the Kaplan-Meier method and compared using the log-rank test.ResultsMiR-101 was hardly expressed in the tumor samples, while for the other miRNAs, variable expression levels and expression ranges were observed, with miR-21 being most abundantly expressed relative to the reference (RNU44). In our study cohort, major clinical significance was demonstrated only for miR-31, as high expression was associated with advanced tumor stage and poor differentiation. No significant associations were found between expression of the investigated miRNAs and metastasis-free or overall survival.ConclusionsInvestigating the expression of six miRNAs previously identified as candidate biomarkers in colorectal cancer, few clinically relevant associations were detected in our patient cohort. Our results emphasize the importance of validating potential tumor markers in independent patient cohorts, and indicate that the role of miRNAs as colorectal cancer biomarkers is still undetermined.

Activation of NF-κB by extracellular S100A4: Analysis of signal transduction mechanisms and identification of target genes

The metastasis‐promoting protein S100A4 stimulates metastatic progression through both intracellular and extracellular functions. Extracellular activities of S100A4 include stimulation of angiogenesis, regulation of cell death and increased cell motility and invasion, but the exact molecular mechanisms by which extracellular S100A4 exerts these effects are incompletely elucidated. The aim of the present study was to characterize S100A4‐induced signal transduction mechanisms and to identify S100A4 target genes. We demonstrate that extracellular S100A4 activates the transcription factor NF‐κB in a subset of human cancer cell lines through induction of phosphorylation and subsequent degradation of the NF‐κB inhibitor IκBα. Concomitantly, S100A4 induced a sustained activation of the MAP kinase JNK, whereas no increased activity of the MAP kinases p38 or ERK was observed. Microarray analyses identified 136 genes as being significantly regulated by S100A4 treatment, and potentially interesting S100A4‐induced gene products include IκBα, p53, ephrin‐A1 and optineurin. Increased expression of ephrin‐A1 and optineurin was validated using RT‐PCR, Western blotting and functional assays. Furthermore, S100A4‐stimulated transcription of these target genes was dependent on activation of the NF‐κB pathway. In conclusion, these findings contribute to the understanding of the complex molecular mechanisms responsible for the diverse biological functions of extracellular S100A4, and provide further evidence of how S100A4 may stimulate metastatic progression.

B7-H3 expression in colorectal cancer: Nuclear localization strongly predicts poor outcome in colon cancer

In colorectal cancer there is a need for molecular markers that can complement the histopathological staging in predicting the likelihood of disease recurrence following curatively intended surgery. B7‐H3 is an immunoregulatory protein shown to be overexpressed in several cancer forms, often associated with more advanced disease and poor prognosis. We wanted to examine whether B7‐H3 could be a potential prognostic marker in colorectal cancer. Paraffin‐embedded samples from 277 colorectal cancer patients were immunostained with anti‐B7‐H3 antibody. B7‐H3 was expressed in the tumor cell cytoplasm and cell membrane in 62% and 46% of the samples, respectively. Unexpectedly, B7‐H3 was expressed in the nucleus in 30% of the tumors. The nuclear localization was confirmed by Western immunoblotting of subcellular fractions. Importantly, in colon cancer, nuclear B7‐H3 expression was independently and significantly associated with reduced metastasis‐free, disease‐specific and overall survival. B7‐H3 expression in tumor‐associated vasculature and fibroblasts was observed in the majority of samples, and endothelial B7‐H3 expression was also significantly associated with poor outcome in colon cancer. In rectal cancer patients, the only significant association was between fibroblast B7‐H3 expression and shorter metastasis‐free survival. Few significant associations to clinicopathological parameters were seen. The results indicate that nuclear B7‐H3 might be involved in colon cancer progression and metastasis, and suggest that nuclear B7‐H3 could become a useful prognostic marker in colon cancer.

Osteopontin - An important downstream effector of S100A4-mediated invasion and metastasis

Substantial evidence has linked the small calcium‐binding protein S100A4 to metastatic progression. S100A4‐mediated effects include stimulation of angiogenesis, regulation of cell death and increased cell motility and invasion, but the exact molecular mechanisms by which the protein exerts these effects are incompletely elucidated. In the present study, we demonstrate that S100A4 induces NF‐κB‐dependent expression and secretion of osteopontin (OPN) in a selection of osteosarcoma cell lines. OPN is, as S100A4, known to result in a variety of cellular effects potentially leading to increased angiogenesis and metastasis, and several of the activated signaling pathways are common for the two proteins. In our study, extracellular S100A4 was found to upregulate enzymes of the plasminogen activator system and matrix metalloproteinase (MMP) family, especially urokinase plasminogen activator and MMP‐13. Furthermore, increased motility and invasion was observed in vitro as a result of S100A4 treatment. OPN expression was inhibited by the use of siRNA molecules, and a partial blocking of S100A4‐induced effects on protease expression and invasive capacity was detected. In conclusion, our results suggest regulation of OPN as a downstream molecular mechanism of S100A4 signaling. This novel finding adds more information to how S100A4 mediates tumor development and metastatic progression. The observation of a link between S100A4 and OPN, and also identification of common downstream effect molecules, highlights them, their receptors or downstream proteins, as targets for therapeutic approaches.

Disseminated tumour cells as a prognostic biomarker in colorectal cancer

Background:The study was performed to determine detection rate and prognostic relevance of disseminated tumour cells (DTC) in patients receiving curatively intended surgery for colorectal cancer (CRC).Methods:The study population consisted of 235 patients with CRC prospectively recruited from five hospitals in the Oslo region. Bone marrow (BM) aspirates were collected at the time of surgery and the presence of DTC was determined by two immunological methods; immunomagnetic selection (using an anti-EpCAM antibody) and immunocytochemistry (using a pan-cytokeratin antibody). Associations between the presence of DTC and metastasis-free, disease-specific and overall survival were analysed using univariate and multivariate methods.Results:Disseminated tumour cells were detected in 41 (17%) and 28 (12%) of the 235 examined BM samples by immunomagnetic selection and immunocytochemistry, respectively, with only five samples being positive with both methods. The presence of DTC was associated with adverse outcome (metastasis-free, disease-specific and overall survival) in univariate and multivariate analyses.Conclusion:The presence of DTC was associated with adverse prognosis in this cohort of patients curatively resected for CRC, suggesting that DTC detection still holds promise as a biomarker in CRC.

Nuclear S100A4 is a novel prognostic marker in colorectal cancer

Current staging classifications in colorectal cancer are not able to accurately predict patient outcome, and the need for novel prognostic markers is evident. S100A4 is a Ca(2+)-binding protein which promotes metastasis in several tumour types, and the aim of the present study was to investigate the prognostic impact of S100A4 expression in colorectal cancer. Two hundred and forty two patients with curatively resected adenocarcinoma of the colon or rectum were prospectively included in the study at the time of surgery. S100A4 expression was analysed by immunohistochemistry, and associations with clinicopathological variables and patient outcome were investigated. Nuclear expression of S100A4 was observed in 29% and cytoplasmic expression was observed in 64% of the tumours. In univariate analysis, nuclear S100A4 was a negative predictor of metastasis-free (P=0.006) and overall survival (P=0.01), whereas cytoplasmic S100A4 was not associated with patient outcome. In multivariate analysis, nuclear localisation was inversely associated with metastasis-free (P=0.03) and overall survival (P=0.02). Interestingly, the prognostic impact was largely confined to TNM stage II, and stage II patients with tumours expressing nuclear S100A4 had a similar prognosis as stage III patients. In conclusion, nuclear expression of S100A4 is a novel prognostic marker in colorectal cancer, and the prognostic value in TNM stage II suggests that nuclear S100A4 could be used in the stratification of stage II patients for adjuvant treatment.

Signal transduction mechanisms involved in S100A4-induced activation of the transcription factor NF-κB

BackgroundThe metastasis-promoting protein S100A4 activates the transcription factor NF-κB through the classical NF-κB activation pathway. The upstream signal transduction mechanisms leading to increased NF-κB activity are, however, incompletely characterized.MethodsThe human osteosarcoma cell line II-11b was stimulated with recombinant S100A4 in the presence or absence of inhibitors of common signal transduction pathways, and NF-κB activity was examined using a luciferase-based reporter assay and phosphorylation of IκBα. mRNA expression was analyzed by real-time RT-PCR, protein expression was examined by Western blotting and IKK activity was measured using an in vitro kinase assay. The role of upstream kinases and the cell surface receptor RAGE was investigated by overexpression of dominant negative proteins and by siRNA transfection.ResultsThe Ser/Thr kinase inhibitors H-7 and staurosporine inhibited S100A4-induced IκBα phosphorylation and subsequent NF-κB activation. The protein tyrosine kinase inhibitor genistein and the phospholipase C inhibitor compound 48/80 had a partial inhibitory effect on IκBα phosphorylation, whereas inhibitors of protein kinase C, G-protein coupled receptors and PI 3-kinases had no effect on the level of phosphorylation. Interestingly, S100A4 treatment induced activating phosphorylations of IKKα/β, but neither H-7 nor staurosporine was able to significantly inhibit IKK activation. Dominant negative MEKK1 or NIK did not inhibit S100A4-induced NF-κB activity, and S100A4 stimulation did not influence AKT phosphorylation. Furthermore, diminished expression of the putative S100 protein receptor RAGE did not affect the observed phosphorylation of IκBα.ConclusionsS100A4 activates NF-κB by inducing phosphorylation of IKKα/β, leading to increased IκBα phosphorylation. The Ser/Thr kinase inhibitors H-7 and staurosporine attenuated S100A4-induced NF-κB activation and inhibited IKK-mediated phosphorylation of IκBα. S100A4-induced NF-κB activation was independent of the putative S100 protein receptor RAGE and the Ser/Thr kinases MEKK1, NIK and AKT. These findings lead to increased understanding of S100A4 signaling, which may contribute to the identification of novel targets for anti-metastatic therapy.

Molecular modelling and simulations in cancer research

The complexity of cancer and the vast amount of experimental data available have made computer-aided approaches necessary. Biomolecular modelling techniques are becoming increasingly easier to use, whereas hardware and software are becoming better and cheaper. Cross-talk between theoretical and experimental scientists dealing with cancer-research from a molecular approach, however, is still uncommon. This is in contrast to other fields, such as amyloid-related diseases, where molecular modelling studies are widely acknowledged. The aim of this review paper is therefore to expose some of the more common approaches in molecular modelling to cancer scientists in simple terms, illustrating success stories while also revealing the limitations of computational studies at the molecular level.

B7-H3 expression in colorectal cancer: associations with clinicopathological parameters and patient outcome

BackgroundWe have previously reported overexpression of the immunoregulatory protein B7-H3 in colorectal cancer and that nuclear expression predicted poor outcome in colon cancer patients. The present study was performed to examine the prognostic role of B7-H3 in an independent colorectal cancer cohort.MethodsUsing tissue microarrays from 731 colorectal cancer patients, tumour B7-H3 expression was assessed by immunohistochemistry. Associations with clinicopathological parameters and patient outcome were investigated.ResultsNuclear expression of B7-H3 in cancer cells was present in 27% of the samples in the total study cohort, while cytoplasmic/membrane and stromal expression was seen in 86% and 77% of the samples, respectively. Nuclear B7-H3 had no prognostic relevance in the complete outcome cohort, neither in colon cancer patients. However, nuclear B7-H3 was significantly associated with reduced recurrence-free survival in TNM stage I colorectal cancer patients.ConclusionsOverexpression of B7-H3 in colorectal cancer was confirmed, but in contrast to previous results, nuclear B7-H3 was not a strong prognostic biomarker in this cohort. The discrepancy might be related to the use of single-core tissue microarrays for detection of the heterogeneously expressed B7-H3, and the role of B7-H3 in colorectal cancer still needs further examination.