Hsa-miR-375 is a predictor of local control in early stage breast cancer.

BACKGROUND: A long-term analysis by the Early Breast Cancer Trialist Group (EBCTG) revealed a strong correlation between local control and cancer-specific mortality. MicroRNAs (miRs), short (20-25 nucleotides) non-coding RNAs, have been described as prognosticators and predictors for breast cancer in recent years. The aim of the current study was to identify miRs that can predict local control after breast conserving therapy (BCT) in early stage breast cancer. RESULTS: Clinical data of 46 early stage breast cancer patients with local relapse after BCT were selected from the institutional database. These patients were matched to 101 control patients showing identical clinical features but without local relapse. The study was conducted in two steps. (1) In the pilot study, 32 patients (16 relapses versus 16 controls) were screened for the most de-regulated microRNAs (= candidate microRNAs) in a panel of 1250 miRs by microarray technology. Eight miRs were found to be significantly de-regulated. (2) In the validation study, the candidate microRNAs were analyzed in an independent cohort of 115 patients (30 relapses versus 85 controls) with reverse transcription quantitative polymerase chain reaction (RT-qPCR). From these eight candidates, hsa-miR-375 could be validated. Its median fold change was 2.28 (Mann-Whitney U test, corrected p value = 0.008). In the log-rank analysis, high expression levels of hsa-miR-375 correlated with a significantly higher risk of local relapse (p = 0.003). In a multivariate analysis (forward stepwise regression) including established predictors and prognosticators, hsa-miR-375 was the only variable that was able to distinguish the statistical significance between relapse and control groups (raw p value = 0.000195 HR = 0.76, 95 % CI 0.66-0.88; corrected p value = 0.005). CONCLUSIONS: Hsa-miR-375 predicts local control in patient with early stage breast cancer, especially in estrogen receptor α (ER-α)-positive patients. It can therefore serve as an additional molecular marker for treatment choice independently from known predictors and prognosticators. Validation in larger prospective studies is warranted.

Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues.

AIMS: Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation. METHODS AND RESULTS: In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays. CONCLUSION: This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.

MicroRNA expression profiling of specific cells in complex archival tissue stained by immunohistochemistry.

Global implementation of molecular diagnostics in modern pathology has been limited by the use of formalin-fixed, paraffin-embedded (FFPE) tissues in current routine diagnostic procedures because of modification and degradation of nucleic acids and protein molecules. In particular, molecular analysis of a specific cell type potentially important for biomarker identification is largely prevented in highly complex, solid tissues routinely used in histopathology. Accumulating data report on the substantial contribution of microRNA molecules (miRNA) to tumor development and malignant progression of most human malignancies. Our objective was to establish a sensitive and robust procedure to quantify miRNA expression in specific cells from complex archival tumor tissues identified by immunohistochemistry. Here, we show reliable detection of miRNA expression profiles determined from limited amounts of colorectal cancer FFPE tissues after routine staining procedure. The combination of routinely used FFPE specimens stained by immunohistochemistry with the molecular analysis of laser microdissected complex tumor tissue resulted in robust miRNA expression patterns exclusively obtained from epithelial tumor cells. This approach allows for a detailed molecular analysis of cancer cells and distinct stromal cell types and their in situ interaction in solid tumors. Hence, the methodology can offer new perspectives for basic research and, by comprehensive use of present archival tissue collections linked to clinical databases, facilitate miRNA biomarker identification in defined tissue cells for future diagnostic and therapeutic strategies.

Multicentre validation study of nucleic acids extraction from FFPE tissues.

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Invasion associated up-regulation of nuclear factor kappaB target genes in colorectal cancer.

BACKGROUND: : Colorectal cancer (CRC) displays intratumoral heterogeneity with less differentiated tumor cells at the invasive front (IF) than in the tumor center (TC). The authors previously observed that several genes were overexpressed at the IF of CRC with relations to inflammatory processes. Because nuclear factor kappaB (NF-kappaB), a dimeric transcription factor, is a major regulator of such processes, and because its target genes are involved in immune response, cell growth control, and cell survival, the expression of NF-kappaB target genes was investigated comparatively in CRC. METHODS: : By using gene array profiling, NF-kappaB target gene expression was assessed in CRCs that expressed human mutL homolog 1 (hMLH1), hMSH2, and nuclear beta-catenin by comparing expression at the IF, in the TC, and in normal mucosa. In addition, 5 NF-kappaB target genes with high differential expression were validated by using immunohistochemistry. RESULTS: : The expression of NF-kappaB target genes in the TC, at the IF, and in normal mucosa was distinct; whereas, specifically at the IF, most differentially expressed NF-kappaB targets were up-regulated. Moreover, the results indicated that the expression diverged between epithelial tumor cells and inflammatory stromal cells. CONCLUSIONS: : Because the results demonstrated that inflammation and the activation of NF-kappaB signaling promoted CRC invasiveness, the current study provided further evidence that downstream targets of NF-kappaB signaling may be specifically relevant in invasion and progression of CRC. Finally, as has been suggested for colitis-associated cancer, the authors of this report concluded that the inhibition of NF-kappaB signaling also may be an additional option for the treatment of sporadic CRC. Cancer 2009. (c) 2009 American Cancer Society.

The impact of microRNAs on colorectal cancer.

MicroRNAs are small RNAs that regulate gene expression at the post-transcriptional level. After their discovery 15 years ago, a new layer of gene regulation was introduced into every field of human biology and medicine. Considering the strong association between genetic alterations and neoplastic diseases, it is not surprising that there is a special focus on miRNAs and cancer. A multitude of experimental studies on colorectal cancer, the most common cancer site and furthermore the second most common cause of death due to cancer, deliver insight into miRNA-mediated, regulatory links to well-known oncogenic and tumour suppressor signalling pathways. Furthermore, several investigations have described the ability of microRNA expression patterns to predict prognosis in colon cancer and support diagnosis of poorly differentiated tumours. In this short review, we give a comprehensive overview focussed on miRNAs in colorectal cancer research.

p16INK4a is a beta-catenin target gene and indicates low survival in human colorectal tumors.

BACKGROUND & AIMS: Human colorectal carcinomas display an infiltrative front of invasion where tumor cells undergo an epithelomesenchymal transition associated with low survival. Epithelomesenchymal transition is regulated by a nuclear beta-catenin accumulation, and subsequently, activation of beta-catenin/TCF4 target genes similar to CYCLIN D(1). Unexpectedly, these tumor cells are characterized by low proliferation, which correlates with the expression of the cell cycle inhibitor p16(INK4A). Therefore, we investigated the molecular mechanism of the transcriptional regulation of p16(INK4A) in colorectal cancer and its correlation with survival. METHODS: Molecular biological techniques were used for investigating the transcriptional mechanisms of the p16(INK4A) gene regulation. Moreover, p16(INK4A) expression was correlated with the 10-year survival of patients with colorectal carcinomas. RESULTS: In colorectal carcinomas, expression of the p16(INK4A) gene is regulated by beta-catenin/TCF4 and correlates with low survival rates of patients with tumors displaying an infiltrative front of invasion. CONCLUSIONS: beta-catenin/TCF4 regulates cell cycle promoting (c-MYC, CYCLIN D(1)) and inhibiting genes (p16(INK4A)) at the same time in the mesenchymally differentiated tumor cells at the front of invasion. The function of p16(INK4A) seems to supersede in this context thus leading to low proliferation. Moreover, these tumor cells seem to govern the outcome of colorectal cancer independently of their proliferation.

The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer.

Invasion and metastasis are the hallmarks of malignant tumor progression and the main cause of death in cancer. The embryonic program "epithelial-mesenchymal transition" (EMT) is thought to trigger invasion by allowing tumor cell dissemination. Here, we describe that the EMT-inducing transcriptional repressor ZEB1 promotes colorectal cancer cell metastasis and loss of cell polarity. Thereby, ZEB1 suppresses the expression of cell polarity factors, in particular of Lgl2, which we found reduced in colorectal and breast cancers. We further show that retention of Lgl2 expression is critical for the epithelial phenotype and that its loss might be involved in metastasis. Thus, by linking EMT, loss of polarity, and metastasis, ZEB1 is a crucial promoter of malignant tumor progression.

Heterogeneous expression of Wnt/beta-catenin target genes within colorectal cancer.

Invasion of common colorectal adenocarcinomas is coupled with a transient loss of epithelial differentiation of tumor cells. Previously, we have shown that dedifferentiated tumor cells at the invasive front (IF) accumulate the transcriptional activator beta-catenin in the nucleus, in contrast to cells of the tumor center. To characterize the cells of these two morphogenic tumor areas, gene expression profiling was performed. Our study demonstrates that intratumorous heterogeneity in colorectal cancer correlates with differential expression of 510 genes between the central tumor region (TC) and the IF. Many genes differentially expressed at the IF are involved in cellular invasion processes like cell motility, cell adhesion and extracellular matrix interaction. This in vivo analysis shows overexpression of known Wnt/beta-catenin target genes either in the entire tumor tissue (compared to normal mucosa) or specifically at the IF. Thus, even though all tumor cells overexpress beta-catenin, the existence of at least 2 groups of Wnt/beta-catenin target genes selectively activated in different tumor regions is suggested. The concomitant high expression of inflammation- and tissue repair-related genes at the IF supports the hypothesis that an inflammation-activated microenvironment may trigger selective Wnt/beta-catenin target gene expression and contribute to the malignant progression of colorectal cancer.

Wnt/FZD signaling and colorectal cancer morphogenesis.

Malignant progression of colorectal carcinomas is characterized by an epithelial-mesenchymal transition (EMT)-like de-differentiation of the invading tumor cells. However a re-differentiation towards an epithelial phenotype, resembling a mesenchymal-epithelial transition (MET), is detectable in metastases. This indicates that malignant progression is based on dynamic processes, which can not be explained solely by irreversible genetic alterations, but must be additionally regulated by the tumor environment. The main oncoprotein in colorectal cancer is the Wnt-pathway effector beta-catenin, which in most cases is overexpressed due to mutations in the adenomatous polyposis coli (APC) tumor suppressor. EMT of tumor cells is associated with a nuclear accumulation of the transcriptional activator beta-catenin, which is reversed in metastases. Nuclear beta-catenin is involved in two fundamental processes in embryonic development: EMT and stem cell formation. Accumulating data demonstrate that aberrant nuclear expression of beta-catenin can also confer these two abilities to tumor cells, indicating the crucial role of aberrant Wnt-signaling for malignant tumor progression.

A transient, EMT-linked loss of basement membranes indicates metastasis and poor survival in colorectal cancer.

BACKGROUND & AIMS: Loss of the basement membrane (BM) is considered an important step toward tumor malignancy. However, the BM is still expressed in most typical colorectal adenocarcinomas; nevertheless, these tumors can invade and develop metastases. The aim of this study was to investigate the role, mechanisms, and clinical relevance of BM turnover in malignant colorectal cancer (CRC) progression. METHODS: Expression of BM components and their transcriptional regulation and clinical relevance were investigated in human CRCs and cell lines. RESULTS: Our data show new aspects in BM turnover in CRCs with impact on malignant tumor progression: (1) The BM is still expressed in the main tumor mass of most colorectal adenocarcinomas, but selectively lost at invasive regions of the tumor in many cases. (2) Selective loss of the BM at the invasive front has high clinical and tumor biologic relevance for distant metastasis and survival. (3) The BM is reexpressed in metastases, indicating that its loss is transient and regulated by environmental factors. (4) This transient loss is not only due to proteolytic breakdown but to a down-regulated synthesis and linked to an epithelial-mesenchymal transition (EMT) in tumor cells, and, thereby, zinc-finger-enhancer protein 1 (ZEB1) is the crucial transcriptional repressor of BM components in CRCs. CONCLUSIONS: A transient BM loss at the invasive front is correlated with increased distant metastasis and poor patient survival, indicating its tumor biologic relevance and usefulness as a prognostic marker. Targeting ZEB1 might be a promising therapeutic option to prevent metastasis.

Opinion: migrating cancer stem cells - an integrated concept of malignant tumour progression.

The dissemination of tumour cells is the prerequisite of metastases and is correlated with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumour progression. A stepwise, irreversible accumulation of genetic alterations is considered to be the responsible driving force. But strikingly, metastases of most carcinomas recapitulate the organization of their primary tumours. Although current models explain distinct and important aspects of carcinogenesis, each alone can not explain the sum of the cellular changes apparent in human cancer progression. We suggest an extended, integrated model that is consistent with all aspects of human tumour progression - the 'migrating cancer stem (MCS)-cell' concept.

beta-Catenin regulates the expression of tenascin-C in human colorectal tumors.

Tenascin-C (TN-C) is a component of the extracellular matrix (ECM). It is expressed during development and re-expressed in many types of cancers, where it is involved in the modulation of adhesion and proliferation. TN-C expression is especially high at sites of epithelial mesenchymal transition (EMT), which are found frequently at the invasion front of well-differentiated human colorectal adenocarcinomas. Tumor cells in this compartment are characterized by a strong nuclear expression of the oncogenic transcription factor beta-catenin. Here, we demonstrate that TN-C is a beta-catenin target gene in human colorectal tumors. Thus, by far the most common mutations in colorectal tumors, found in the Wnt-signaling pathway and leading to the stabilizing of beta-catenin, might influence invasion by altering adhesive properties and EMT of tumor cells.

Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin.

Invasion by colorectal carcinomas is characterized by an epithelial-mesenchymal transition (EMT)-like dedifferentiation of the tumor cells. However, a redifferentiation towards an epithelial phenotype, resembling a mesenchymal-epithelial transition, is detectable in metastases. This indicates that malignant progression is based on dynamic processes, which cannot be explained solely by irreversible genetic alterations, but must be additionally regulated by the tumor environment. The main oncoprotein in colorectal cancer is the Wnt pathway effector beta-catenin, which is overexpressed due to mutations in the APC tumor suppressor in most cases. EMT of the tumor cells is associated with a nuclear accumulation of the transcriptional activator beta-catenin, which is reversed in metastases. Nuclear beta-catenin is involved in two fundamental processes in embryonic development: EMT and stem cell formation. Accumulating data demonstrate that aberrant nuclear expression of beta-catenin can also confer these two abilities to tumor cells, thereby driving malignant tumor progression.

Down-regulation of the homeodomain factor Cdx2 in colorectal cancer by collagen type I: an active role for the tumor environment in malignant tumor progression.

The homeobox transcription factor Cdx2 specifies intestinal development and homeostasis and is considered a tumor suppressor in colorectal carcinogenesis. However, Cdx2 mutations are rarely found. Invasion of colorectal cancer is characterized by a transient loss of differentiation and nuclear accumulation of the oncoprotein beta-catenin in budding tumor cells. Strikingly, this is reversed in growing metastases, indicating that tumor progression is a dynamic process that is not only driven by genetic alterations but also regulated by the tumor environment. Here we describe a transient loss of Cdx2 in budding tumor cells at the tumor host interface, and reexpression of Cdx2 in metastases. Cell culture experiments show that collagen type I, through beta(1) integrin signaling, triggers a transient transcriptional down-regulation of Cdx2 and its intestine-specific target gene sucrase isomaltase, associated with a loss of differentiation. These data indicate an active role for the tumor environment in malignant tumor progression.

Evidence for acid-induced loss of Cdx2 expression in duodenal gastric metaplasia.

Gastric metaplasia in the duodenum (GMD) is characterized by transdifferentiation of intestinal epithelial cells into gastric foveolar cells within the duodenal mucosa. GMD is often associated with duodenal ulceration. Higher duodenal acidity due to increased gastric acid output into the duodenum has been implicated in the development of GMD. Intestinal development and homeostasis are controlled by the homeobox transcription factor Cdx2, which is considered to be the master regulator of intestinal differentiation. Using immunohistochemistry, the present study shows that GMD is associated with loss of expression of Cdx2 and its target gene product sucrase-isomaltase. Quantitative RT-PCR experiments using the intestinal cell line Caco2 revealed that Cdx2 and sucrase-isomaltase were down-regulated and gastric mucins MUC5AC and MUC6 were up-regulated under acidic culture conditions. Thus, it is suggested that increased acid exposure leads to GMD by impairing the transcription of Cdx2 and subsequently that of its intestine-specific target genes.

Tissue preparation for gene expression profiling of colorectal carcinoma: three alternatives to laser microdissection with preamplification.

Colorectal-carcinoma specimens are heterogeneous and include areas of nonmalignant mucosal and connective tissue. For those study designs in which laser microdissection and RNA preamplification are impracticable, the optimal yield of genuine cancer RNA is a key factor in gene-expression analysis. In this study we compared alternative methods of tissue purification. Three contiguous 0.5-cm(3) samples taken from an advanced primary adenocarcinoma of the sigmoid colon were processed immediately after surgery with the use of the following methods: (1) cryotomy after manual dissection (CMD), (2) microscopically assisted manual dissection (MAMD), and (3) tumor-cell isolation with the use of Ber-EP4 antibodies and Dynabeads (Dynal Biotech GmbH, Hamburg, Germany; technique abbreviated as DB). We generated gene-expression profiles with the use of GeneChip technology (Affymetrix, Santa Clara, Calif) and recorded preparation times, costs, and RNA quantity and quality. CMD took 60 minutes, MAMD 180 minutes, and DB 90 minutes to isolate 22, 8, and 23 microg of RNA, respectively. Expenses for materials amounted to 41, 23, and 91 US dollars for CMD, MAMD, and DB, respectively. The 3'/5' ratio, as determined with the GeneChips, for GAPDH/beta-actin was 1.01:1.03 for CMD, 1.13:1.28 for MAMD, 1.43:1.68 for DB, K-ras, APC, smad 2, transforming growth factor-beta, and p53 were marked as present in all cases, with the exception of APC, which was graded as marginal on DB. The correlation values of gene-expression profiles were 91% (CMD/DB), 93% (CMD/MAMD), and 97% (DB/MAMD). All 3 methods provided enough RNA, of sufficient quality, for gene-expression microarray analysis in colorectal carcinoma. Cross-methodologic analyses of array data should not be performed uncritically.

Beta-catenin up-regulates the expression of the urokinase plasminogen activator in human colorectal tumors.

Expression of the urokinase plasminogen activator (uPA) increases during the progression of colorectal tumors from adenomas to carcinomas. The highest amounts of uPA are found at the invasion front of carcinomas, which also displays a strong expression of nuclear beta-catenin and is therefore a region expressing beta-catenin target genes at high levels. Here we show that beta-catenin contributes to the transactivation of uPA. Therefore, beta-catenin might have an impact on the capacity of colorectal tumors for invasion and metastasis, as well as dormancy, which are hallmarks of cancer.

Beta-catenin activates a coordinated expression of the proinvasive factors laminin-5 gamma2 chain and MT1-MMP in colorectal carcinomas.

In colorectal carcinomas, loss-of-function mutations of the adenomatous polyposis coli (APC) tumor suppressor gene lead to a nuclear accumulation of the oncogenic transcriptional activator beta-catenin, predominantly at the invasive front within the tumor host interface. Various identified genes activated by beta-catenin are associated with tumor invasion. One prerequisite for malignant tumor invasion is the ability of tumor cells to migrate. We recently described the gamma2 chain of laminin as another beta-catenin target gene. Fragments of the laminin gamma2 chain, resulting from cleavage by the membrane type 1 matrix metalloproteinase (MT1-MMP), are strong inducers of epithelial cell migration. We here show a coordinated expression of nuclear beta-catenin, its target gene and MT1-MMP substrate laminin gamma2 chain, as well as MT1-MMP in tumor cells at invasive regions of colorectal carcinomas. We further demonstrate that MT1-MMP expression is regulated by beta-catenin/TCF through a TCF binding site in its promoter. These results suggest that nuclear beta-catenin activates the coordinated expression of the interacting proinvasive proteins laminin gamma2 chain and MT1-MMP, thereby leading to a promigratory activity at the invasive front of colorectal cancers. This further supports an important role of beta-catenin for invasion and metastasis of colorectal carcinomas.

Transcription of sonic hedgehog, a potential factor for gastric morphogenesis and gastric mucosa maintenance, is up-regulated in acidic conditions.

Gastric body mucosa atrophy predisposes one to gastric cancer. Disturbances in the gastric differentiation process might play a role in the evolution of gastric atrophy. Sonic hedgehog (Shh) has recently been implicated as a crucial factor in gastric organogenesis and gland differentiation. In this study we investigated the expression of key factors in the Shh pathway, namely Shh and its receptor Patched (Ptc), in normal and pathologic stomach mucosa. Furthermore, the potential role of pH for Shh dysregulation was analyzed. Ten gastric biopsy specimens each from normal gastric mucosa, chronic nonatrophic gastritis, atrophic gastritis, and gastric cancer were included. Expression of Shh and Ptc was analyzed by immunohistochemistry. In normal body mucosa and in nonatrophic body gastritis, Shh was strongly expressed in parietal cells. Ptc was also expressed in gastric chief cells. Shh expression was almost completely lost in atrophic gastritis and in gastric cancer and absent in intestinal metaplasia. Ptc was markedly reduced in atrophy and only weakly positive in intestinal metaplasia and gastric cancer. In in vitro experiments, gastric cancer cell line 23132 was found positive for Shh. In long-term culture as well as in culture conditions with low pH, transcription of Shh in 23132 was significantly increased in quantitative reverse transcription PCR analyses. We concluded that the decreased expression of the Shh pathway in atrophic gastritis and gastric cancer might reflect altered differentiation processes within the gastric unit and contributes to the development of gastric atrophy. The increase of gastric pH might play a role in the development of gastric mucosa atrophy via reduction of Shh transcription.