Rudolf Nenutil

Altered Expression of miR-21, miR-31, miR-143 and miR-145 Is Related to Clinicopathologic Features of Colorectal Cancer

Objectives: Development and metastases of colorectal cancer (CRC) are characterized by multiple genetic alterations. MicroRNAs (miRNAs) are endogenously expressed regulatory noncoding RNAs. Previous, mainly preclinical studies showed altered expression levels of several miRNAs in CRC. Methods: In our study, the expression levels of miR-21, miR-31, miR-143 and miR-145 in 29 primary colorectal carcinomas and 6 non-tumor adjacent tissue specimens were examined by real-time polymerase chain reaction. miRNA expression levels were also correlated with commonly used clinicopath-ologic features of CRC. Results: Expression levels of analyzed miRNAs significantly differed among tumors and adjacent non-tumor tissues: miR-21 (p = 0.0001) and miR-31 (p = 0.0006) were upregulated, and miR-143 (p = 0.011) and miR-145 (p = 0.003) were downregulated in tumors. For the first time, a high expression of miR-21 was associated with lymph node positivity (p = 0.025) and the development of distant metastases (p = 0.009) in CRC patients. Thus, expression of miR-21 correlated with CRC clinical stage (p = 0.032). Furthermore, tumors >50 mm in maximal tumor diameter were characterized by lower expression of miR-143 (p = 0.006) and miR-145 (p = 0.003). We found no correlation between analyzed miRNAs and serum levels of carcinoembryonic antigen. Conclusion: Our results suggest possible roles of miR-21, miR-31, miR-143 and miR-145 in CRC.

Differential expression of p63 isoforms in normal tissues and neoplastic cells

The p63 gene encodes at least six different proteins with homology to the tumour suppressor protein p53 and the related p53 family member p73. So far, there have been limited data concerning the expression patterns of individual p63 proteins, due to a lack of reagents that distinguish between the different isoforms. Three antibodies have been produced specifically directed against the two N‐terminal isoforms (TAp63 and ΔNp63) and the C‐terminal region of the p63α proteins. TAp63 proteins are located suprabasally in stratified epithelia compared with the N‐terminal truncated forms, which are more abundantly expressed in the basal cell layer, indicating a switch in expression of p63 isoforms during normal cellular differentiation. Analysis of squamous cell carcinomas shows ΔNp63α to be the most widely expressed isoform, compatible with a role for this protein in promoting neoplastic cell growth in these tissues. ΔNp63 protein expression is also restricted to basal cells in breast and prostate, whilst TAp63 isoforms are more widely expressed in these tissues as well as in tumours at these sites. TAp63, but not ΔNp63 or p63α, is detected in normal colon and in colon carcinoma. TAp63 proteins are also expressed in the nuclei of a sub‐population of lymphoid cells and in most malignant lymphomas, whereas ΔNp63 proteins are not expressed. Taken together, a hitherto unrecognized regulation of p63 isoform expression in vivo has been uncovered, with different p63 proteins expressed during differentiation and in different cell types. The data indicate roles for specific p63 isoforms not only in maintaining epithelial stem cell populations, but also in cellular differentiation and neoplasia. Copyright

Discriminating functional and non-functional p53 in human tumours by p53 and MDM2 immunohistochemistry.

Mutation and/or loss of the TP53 tumour suppressor gene is the single most common genetic abnormality in human cancer. The majority of TP53 mutations lead to stabilization of the protein, so that immunohistochemical staining for p53 can suggest mutation status in many cases. However, various false‐positive and false‐negative situations mean that simple immunostaining for p53 is not informative in a substantial number of tumours. In the present study, a series of 119 human cancers were immunostained using a highly sensitive technique that detects the low levels of wild‐type protein expressed in normal cells, such that homozygous gene deletion or non‐sense TP53 mutation can be identified by an absence of staining. TP53 gene status was also assessed using FASAY as a genetic/functional screen and in selected cases by direct sequencing. A quantitative scoring system was employed to assess p53 levels, and p53 post‐translational modification was evaluated using antibodies that detect specific phosphorylation sites. Phosphorylated p53 correlated with total p53 levels and did not improve the prediction of TP53 mutation status. The transcriptional activity of TP53 was determined by staining for two downstream target genes, p21WAF1 and MDM2, and statistical correlations between MDM2/p21WAF1 and p53 were found in tumours with wild‐type, but not mutant TP53. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost‐effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data. Copyright

Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis

The present pilot study constitutes a proof-of-principle in the use of a quantitative LC-MS/MS based proteomic method for the comparative analysis of representative low-grade breast primary tumor tissues with and without metastases and metastasis in lymph node relative to the nonmetastatic tumor type. The study method incorporated iTRAQ stable isotope labeling, two-dimensional liquid chromatography, nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid QqTOF platform (iTRAQ-2DLC-MS/MS). The principal aims of this study were (1) to define the protein spectrum obtainable using this approach, and (2) to highlight potential candidates for verification and validation studies focused on biomarkers involved in metastatic processes in breast cancer. The study resulted in the reproducible identification of 605 nonredundant proteins (p < or = 0.05). A quantitative comparison revealed 3/3 proteins with significantly increased/decreased level in metastatic primary tumor and 13/6 proteins with increased/decreased level in lymph node metastasis compared to nonmetastatic primary tumor (p < 0.01). Changes in selected differentially expressed proteins were verified with qRT-PCR. Although our pilot scale study does not warrant general biological conclusions, the synergic regulation of some proteins with related function (e.g., heme binding proteins, proteins of energetic metabolism, interferon induced proteins, proteins with adhesive function) determined in our sample set reflects the ability of our method in providing biologically meaningful data. The main conclusion from this pilot study was that our quantitative proteomic method constitutes a novel way of analyzing cancerous breast tissue biopsy samples that can be extended as part of a larger scale biomarker discovery program.

The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers

Transcriptomic screens in breast cancer cell lines have identified a protein named anterior gradient-2 (AGR2) as a potentially novel oncogene overexpressed in estrogen receptor (ER) positive tumours. As targeting the ER is responsible for major improvements in cure rates and prevention of breast cancers, we have evaluated the pro-oncogenic function of AGR2 in anti-hormone therapeutic responses. We show that AGR2 expression promotes cancer cell survival in clonogenic assays and increases cell proliferation and viability in a range of cancer cell lines. Chromatin immunoprecipitation and reporter assays indicate that AGR2 is transcriptionally activated by estrogen through ERα. However, we also found that AGR2 expression is elevated rather than inhibited in response to tamoxifen, thus identifying a novel mechanism to account for an agonistic effect of the drug on a specific pro-oncogenic pathway. Consistent with these data, clinical analysis indicates that AGR2 expression is related to treatment failure in ERα-positive breast cancers treated with tamoxifen. In contrast, AGR2 is one of the most highly suppressed genes in cancers of responding patients treated with the anti-hormonal drug letrozole. These data indicate that the AGR2 pathway represents a novel pro-oncogenic pathway for evaluation as anti-cancer drug developments, especially therapies that by-pass the agonist effects of tamoxifen.

Lack of correlation of stem cell markers in breast cancer stem cells

Background:Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers, identify the same population of cells, or equate to therapeutic response is controversial.Methods:We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo, comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin, docetaxol and radiotherapy.Results:CD24, CD44, ALDH and SOX2 expression, the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo, cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers, although ER-negative cells accumulate.Conclusions:Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications, rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer.

Surface-enhanced laser desorption/ionization time-of-flight proteomic profiling of breast carcinomas identifies clinicopathologically relevant groups of patients similar to previously defined clusters from cDNA expression

IntroductionMicroarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools.MethodsWhole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers.ResultsUnsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas.ConclusionOur results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the techniques potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.

Prediction of human papillomavirus 16 e6 gene expression and cervical intraepithelial neoplasia progression by methylation status.

Introduction: Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical intraepithelial neoplasia (CIN) and cervical cancer. We aimed to analyze the consequences of methylation of the E6 gene promoter in distinct stages of HPV-16-induced cellular transformation to assess its importance for disease progression. Methods: Human papillomavirus 16 was detected by sensitive polymerase chain reaction (PCR). Determination of E6 gene promoter methylation was analyzed by digestion with specific restriction endonuclease McrBC followed by PCR amplification. Expression of the E6 gene was determined by quantitative real-time PCR. Results: Of 103 cervical smears from asymptomatic women with no cytological and colposcopic abnormalities, 20.4% were HPV-16-positive. Human papillomavirus 16 was present in 44.4% of 18 patients with CIN I, in 62.2% of 143 patients with CIN II/III, and in 74.2% of 31 cervix carcinoma specimens. The incidence of HPV-16 in all lesions compared with asymptomatic women was statistically significant (P < 0.001, Pearson χ2 test). Methylation was detected in 81% (n = 21) of HPV-16-positive asymptomatic smears compared with 62.5% in CIN I (n = 8), 31.5% (n = 89) in CIN II/III, and 43.4% (n = 23) in carcinomas; a statistical significance between lesions and healthy women was found (P < 0.001, Pearson χ2 test). Expression of E6 mRNA correlated with methylation status (P = 0.010, Mann-Whitney U test). Conclusions: We conclude that methylation of the E6 gene promoter in HPV-16 genome is a predictive biomarker for cervical cancer progression by regulating the expression of the E6 oncogene.

Alterations of the Hsp70/Hsp90 chaperone and the HOP/CHIP co-chaperone system in cancer.

Activation of the Hsp90 chaperone system is a characteristic of cancer cells. The regulation of chaperone activities involves their interaction with cochaperones; therefore we investigated the expression of Hsp70 and Hsp90 and their specific co-chaperones HOP and CHIP in cancer cell lines and primary cancers. Inhibition of Hsp90 by 17AAG increased the levels of Hsp70, Hsp90 and HOP but not CHIP mRNA in cancer cells. These changes are linked to activation of the HSF1 transcription factor and we show that the HOP promoter contains HSF1 binding sites, and that HSF1 binding to the HOP promoter is increased following 17AAG. The lack of alteration in the co-chaperone CHIP is explained by a lack of HSF response elements in the CHIP promoter. Non-proliferating cells expressed higher levels of CHIP and lower HOP, Hsp70 and Hsp90 levels compared to proliferating cells. Decreased expression of CHIP in proliferating cancer cells is in keeping with its proposed tumor suppressor properties, while over-expression of HOP in proliferating cells may contribute to excessive Hsp90 activity and stabilization of client proteins in tumors. In a panel of colorectal cancer samples, increased expression of Hsp70 and an increased ratio of HOP to CHIP were found, and were associated with decreased median survival. These data indicate that multiple changes occur in the chaperone/co-chaperone system in cancer that impact patient survival. It is likely that the ability to identify individual alterations to this system will be beneficial for treatment strategy decisions, particularly those that employ chaperone inhibitors.

Transgelins, cytoskeletal proteins implicated in different aspects of cancer development

Transgelin is an abundant protein of smooth muscle cells, where its role has been primarily studied. As a protein affecting dynamics of the actin cytoskeleton via stabilization of actin filaments, transgelin is both directly and indirectly involved in many cancer-related processes such as migration, proliferation, differentiation or apoptosis. Transgelin was previously reviewed as a tumor suppressor; however, recent data based on a number of proteomics studies indicate its pro-tumorigenic role, for example, in colorectal or hepatocellular cancer. We summarize these contradictory observations in both clinical and functional proteomics projects and analyze the role of transgelin in tumors in detail. Generally, the expression and biological role of transgelin seem to differ among various types of tumor cells and stroma, and possibly change during tumor progression. We also overview the recent data on transgelin-2, a sequence homolog of transgelin, whose role in the tumor development might be contradictory to the role of transgelin.