Enhanced expression of the stemness-related factors OCT4, SOX15 and TWIST1 in ectopic endometrium of endometriosis patients.

BACKGROUND: Current evidence suggests that endometrial-derived stem cells, spilled in the peritoneal cavity via retrograde menstruation, are key players in the establishment of endometriotic lesions. The aim of this study was to determine the presence and distribution of the stemness-related factors OCT4, SOX15, TWIST1 and DCAMLK1 in women with and without endometriosis. METHODS: Immunohistochemical analysis was used to determine stromal and epithelial expression of OCT4, SOX15, TWIST1 and DCAMLK1 in endometriosis patient (EP) endometrium (n = 69) and endometriotic tissue (n = 90) and in control endometrium (n = 50). Quantitative Real-Time PCR of OCT4, SOX15 TWIST1 and DCAMLK1 was performed in paired samples of EP endometrium and endometriotic tissue. Co-immunofluorescence staining was performed for OCT4 and SOX15. For statistical analyses we used unpaired t-test, Fisher combination test and Spearman test. For paired analyses, paired t-test and McNemar test were used. RESULTS: We detected a significant correlation between the expression of the established stem cell marker OCT4 and the stemness-related markers SOX15 (p < 0.001) and TWIST1 (p = 0.002) but not DCAMLK1. We showed a colocalization of SOX15 and OCT4 in epithelial and stromal cells of endometriotic tissue by coimmunofluorescence. A concordant expression of OCT4 and SOX15 in the same sample was observed in epithelial cells of the endometriotic tissue (71.7%). The expression of stemness-related factors was not associated with proliferative or secretory phase of the menstrual cycle in endometriosis patients but was found to be differentially expressed during the menstrual cycle in the control group. Increased expression of epithelial OCT4, SOX15 and TWIST1 was detected in endometriotic tissue compared to EP endometrium in paired (p = 0.021, p < 0.001 and p < 0.001) and unpaired analysis (p = 0.040, p < 0.001 and p = 0.001). CONCLUSION: Our findings support the hypothesis that upregulation of stem cell-related factors contribute to the establishment of endometriotic lesions. TRIAL REGISTRATION: The study was approved by the institutional review board (545/2010 on 6th of May 2014) of the Medical University of Vienna ( http://ethikkommission.meduniwien.ac.at/fileadmin/ethik/media/dokumente/register/alle_2010.pdf ).

Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose polarity and cell-to-cell contacts and acquire the migratory and invasive abilities of mesenchymal cells. These abilities are thought to be prerequisites for the establishment of endometriotic lesions. A hallmark of EMT is the functional loss of E-cadherin (CDH1) expression in epithelial cells. TWIST1, a transcription factor that represses E-cadherin transcription, is among the EMT inducers. SNAIL, a zinc-finger transcription factor, and its close relative SLUG have similar properties to TWIST1 and are thus also EMT inducers. MYC, which is upregulated by estrogens in the uterus by an estrogen response cis-acting element (ERE) in its promoter, is associated with proliferation in endometriosis. The role of EMT and proliferation in the pathogenesis of endometriosis was evaluated by analyzing TWIST1, CDH1 and MYC expression. METHODS: CDH1, TWIST1, SNAIL and SLUG mRNA expression was analyzed by qRT-PCR from 47 controls and 74 patients with endometriosis. Approximately 42 ectopic and 62 eutopic endometrial tissues, of which 30 were matched samples, were collected during the same surgical procedure. We evaluated TWIST1 and MYC protein expression by immunohistochemistry (IHC) in the epithelial and stromal tissue of 69 eutopic and 90 ectopic endometrium samples, of which 49 matched samples were analyzed from the same patient. Concordant expression of TWIST1/SNAIL/SLUG and CDH1 but also of TWIST1 and MYC was analyzed. RESULTS: We found that TWIST1, SNAIL and SLUG are overexpressed (p < 0.001, p = 0.016 and p < 0.001) in endometriosis, while CDH1 expression was concordantly reduced in these samples (p < 0.001). Similar to TWIST1, the epithelial expression of MYC was also significantly enhanced in ectopic endometrium compared to eutopic tissues (p = 0.008). We found exclusive expression of either TWIST1 or MYC in the same samples (p = 0.003). CONCLUSIONS: Epithelial TWIST1 is overexpressed in endometriosis and may contribute to the formation of endometriotic lesions by inducing epithelial to mesenchymal transition, as CDH1 was reduced in ectopic lesions. We found exclusive expression of either TWIST1 or MYC in the same samples, indicating that EMT and proliferation contribute independently of each other to the formation of endometriotic lesions.

FOLFOX4 Plus Cetuximab for Patients With Previously Untreated Metastatic Colorectal Cancer According to Tumor RAS and BRAF Mutation Status: Updated Analysis of the CECOG/CORE 1.2.002 Study.

BACKGROUND: This updated analysis of the CECOG/CORE 1.2.002 study investigated the association between clinical outcome and RAS and BRAF mutations in metastatic colorectal cancer (mCRC) patients treated with FOLFOX4 plus cetuximab. PATIENTS AND METHODS: Available DNA samples from CECOG/CORE 1.2.002 study patients with KRAS exon 2 wild type (wt) (at codons 12 and 13) tumors were screened for mutations at other loci in the KRAS and NRAS (RAS) coding regions by Sanger sequencing, and for BRAF codon 600 mutations by Sanger sequencing and pyrosequencing. Clinical outcome was compared among different mutation subgroups. RESULTS: Of 152 KRAS wt mCRC patients, 148 were evaluable for RAS and BRAF mutation status. Eleven RAS mutations were detected in 10 patients' tumors (7%). BRAF mutations were detected in 14 patients' tumors (9%). RAS and BRAF tumor mutations were mutually exclusive. Compared with patients with RAS wt/BRAF wt tumors (n = 124; median overall survival, 28.5 months), those with RAS mutations (n = 10; median, 16.3 months; hazard ratio, 0.43; 95% confidence interval, 0.20-0.89; P = .020) or BRAF mutations (n = 14; median, 11.7 months; hazard ratio, 0.23; 95% confidence interval, 0.12-0.41; P < .0001) had worse overall survival, which remained significant (P < .04) when adjusting for differences in baseline characteristics among the mutation subgroups. CONCLUSION: These findings support those from recent studies that RAS and BRAF mutations are associated with poor outcome in patients receiving an epidermal growth factor receptor-targeted monoclonal antibody in combination with oxaliplatin-based chemotherapy. Furthermore, mutation testing should not only include RAS codons 12 and 13 but should also be extended to the entire coding regions.

The L10P polymorphism and serum levels of transforming growth factor ß1 in human breast cancer.

The L10P single nucleotide polymorphism (SNP) is located in the signal sequence of the transforming growth factor ß1 (TGFß1) gene. The proline-encoding (Pro-) allele of this SNP has been associated with an increased breast cancer risk, which has been attributed to the elevated secretion of this TGFß1 variant observed in vitro and in male subjects. Here we investigated the association of the L10P SNP with serum levels of TGFß1 in female breast cancer patients and controls. We genotyped the L10P SNP in 276 breast cancer patients and 255 controls. Serum TGFß1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in a subset of the study population (n = 211). We found no evidence for an association of the L10P SNP with breast cancer risk (per-allele odds ratio: 0.91; 95% confidence interval: 0.71-1.16). However, patients with the Pro/Pro genotype exhibited a significantly younger age at breast cancer onset (55.2 ± 14.3 years) than Leu/Leu patients (60.6 ± 13.6 years; p = 0.04), which may reflect the ability of TGFß to promote tumor progression. Mean TGFß1 serum levels of Pro-allele carriers were 39.4 ± 7.4 ng/mL, whereas those of Leu/Leu subjects were 37.6 ± 6.0 ng/mL (p = 0.07). Thus, compared to a previous study of male subjects, we observed only a modest increase, if any, in TGFß1 levels of female Pro-allele carriers.

The Pro allele of the p53 codon 72 polymorphism is associated with decreased intratumoral expression of BAX and p21, and increased breast cancer risk.

BACKGROUND: The TP53 Arg72Pro polymorphism encodes two p53 variants with different biochemical properties. Here we investigated the impact of this polymorphism on the expression of key p53 target genes in a panel of human breast carcinomas, breast cancer risk, and age at onset. METHODOLOGY/PRINCIPAL FINDINGS: The Arg72Pro polymorphism was genotyped in 270 breast cancer patients and 221 control subjects. In addition, the Arg72Pro genotype of 116 breast tumors was determined, and correlated with intratumoral mRNA expression of TP53 and its key target genes MDM2, p21, BAX, and PERP, as quantified by qRT-PCR. We found a significantly increased breast cancer risk associated with the Pro-allele (per-allele odds ratio, 1.46; 95% confidence interval, 1.08-1.99), and a significantly later mean age at breast cancer onset for Pro/Pro patients (63.2±18 years) compared to Arg/Arg patients (58.2±15 years). The frequency of somatic TP53 inactivation was 25.4% in Arg/Arg, 20.9% in Arg/Pro, and 16.7% in Pro/Pro patients, which may reflect a higher selective pressure to mutate the Arg-allele. The median mRNA levels of p21 and BAX in the tumors of Pro-allele carriers were significantly reduced to 55.7% and 76.9% compared to Arg/Arg patients, whereas p53, MDM2 and PERP expression were hardly altered. CONCLUSIONS/SIGNIFICANCE: The p53(72Arg) variant appears to be a more potent in vivo transcription factor and tumor suppressor in human breast cancer than the p53(72Pro) variant. The Arg72Pro genotype has no significant effects in patients with TP53 mutated tumors, in which p53 is non-functional.

Activation of ectopic Oct-4 and Rex-1 promoters in human amniotic fluid cells.

Recently, amniotic fluid was suggested as a new source for stem-cell research and tissue engineering approaches. In order to enable isolation of stem cells and establishment of lines of such cells with an undifferentiated phenotype we have introduced green fluorescent protein regulated by the promoters of the stem cell-specific genes, Oct-4 or Rex-1, into human amniotic fluid cells. For the introduction of DNA into human amniotic fluid cells, we have optimized a specific transfection protocol. We found that human amniotic fluid contains cell populations which are able to activate these promoters. These undifferentiated cells expressing green fluorescent protein can be analysed on a flow cytometer. In addition, we have introduced a plasmid harboring a neomycin-resistance gene under the control of the Oct-4 promoter. G418 selection allowed the isolation of undifferentiated stem cells expressing Oct-4 protein out of human amniotic fluid samples. Our findings confirm the existence of stem cells within amniotic fluid. In addition, the ability to transfect human amniotic fluid cells and to isolate stem-cell marker-positive cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.

Neurogenic cells in human amniotic fluid.

OBJECTIVE: The purpose of this study was to determine whether human amniotic fluid contains cells that harbor the potential to differentiate into neurogenic cells. STUDY DESIGN: Amniotic fluid cells (uncultivated or cultivated in standard or in neurogenic differentiation medium) were analyzed for morphologic neurogenic differentiation and for expression of cluster of differentiation 133 (marker for neuronal stem cells), nestin (neuronal progenitor cells), neurofilament (neurons), the p75 common neurotrophin receptor, the brain-derived neurotrophic factor and neurotrophin-3 and cyclic nucleotide phosphodiesterase (oligodendrocytes). RESULTS: The appearance of neurogenic cells was not detected in uncultivated cells, was sporadic after cultivation in standard medium but strongly increased in neurogenic differentiation medium, and was accompanied by the induction of the expression of the analyzed marker genes. CONCLUSION: For the first time, this study provides evidence that human amniotic fluid contains cells that express markers for neuronal stem and progenitor cells, which harbor the potential to differentiate into neurogenic cells.

Insulin-like growth factor (IGF)-I and IGF-II serum concentrations in patients with benign and malignant breast lesions: free IGF-II is correlated with breast cancer size.

PURPOSE: Insulin-like growth factors (IGFs) are potent mitogens for breast cancer cells in vitro, and elevated IGF-I serum levels are a risk factor for breast malignancies. This study evaluated IGF-I and IGF-II serum levels in healthy women and in patients with benign and malignant breast lesions and correlated them with tumor size. EXPERIMENTAL DESIGN: Serum levels of the total and unbound fractions of IGF-I and IGF-II were analyzed in 65 patients with benign and malignant breast lesions and in 38 women without breast disease. ELISAs were used to detect serum IGF levels, with (total IGF) or without (free IGF) prior acid-ethanol extraction. RESULTS: Total IGF-I serum concentrations were lower in healthy women than in breast cancer patients (P < 0.001) or patients with benign breast lesions (P = 0.010), but no differences were observed in free IGF-I levels. Conversely, healthy women had higher serum levels of free IGF-II than women with breast lesions (P = 0.003), and the free/total IGF-II ratio was significantly reduced in patients with breast disease (P = 0.001). Although IGF-I or IGF-II serum concentrations of breast cancer patients were similar to those of patients with benign lesions, the size of a malignant tumor was correlated to the ratio free/total IGF-II (P = 0.002). CONCLUSIONS: Malignant breast tumors cannot be distinguished from benign breast lesions by systemic IGF serum levels. However, women with breast lesions have decreased IGF-II concentrations, and free IGF-II levels are clearly correlated to the size of a breast cancer, indicating an involvement in tumor growth.

Interleukin 1 system and sex steroid receptor expression in human breast cancer: interleukin 1alpha protein secretion is correlated with malignant phenotype.

PURPOSE: The interleukin 1 (IL-1) system plays an important role in human pathology and is involved in the local control of malignant disease. However, little is known about its expression in breast cancer and its correlation with prognostic parameters such as receptor status and grading. EXPERIMENTAL DESIGN: The expression of IL-1alpha and other IL-1 family members was analyzed by reverse transcription-PCR, ELISA, and immunohistochemistry in breast cancer cell lines, tumor-derived fibroblasts, and breast cancer tissue biopsies and compared with sex steroid receptor status and grading. RESULTS: In breast cancer cell lines, IL-1alpha and -beta gene expression was present in the phenotypically most malignant cell lines, whereas estrogen receptor (ER) alpha and progesterone receptor mRNA expression was confined to lines that exhibit a rather benign phenotype. Only the highly malignant receptor-negative tumor cell line MDA MB 231 expressed IL-1alpha protein, and none of the cell lines secreted IL-1beta. Biopsies from breast cancer tissue expressed various amounts of IL-1alpha, IL-1beta, and IL-1 receptor antagonist mRNA, but consistently high levels of IL-1tIR. IL-1alpha protein expression was detected in tumor cells and/or adjacent stroma in 88%, and epithelial protein expression was correlated with both poor differentiation (P = 0.002; r = 0.469) and decreasing epithelial ERalpha expression (P = 0.004; r = -0.387). Furthermore, stromal IL-1alpha was predominant in areas with low or absent ERalpha protein expression in neighboring tumor epithelium (P = 0.001; r = -0.457). CONCLUSION: We have demonstrated the presence of a functional IL-1 system in breast cancer and found that IL-1alpha is inversely correlated with local sex steroid receptor expression. We hypothesize that the unphysiological expression of IL-1alpha in less differentiated and ERalpha-negative tumors might contribute to their local invasiveness and malignant behavior.

Oct-4-expressing cells in human amniotic fluid: a new source for stem cell research?

BACKGROUND: It is the hope of investigators and patients alike that in future the isolation of pluripotent human stem cells will allow the establishment of therapeutic concepts for a wide variety of diseases. A major aim in this respect is the identification of new sources for pluripotent stem cells. Oct-4 is a marker for pluripotent human stem cells so far known to be expressed in embryonal carcinoma cells, embryonic stem cells and embryonic germ cells. METHODS: Cells from human amniotic fluid samples were analysed for mRNA expression of Oct-4, stem cell factor, vimentin and alkaline phosphatase via RT-PCR. Oct-4 protein expression was investigated by Western blot analysis and immunocytochemistry. Oct-4-positive cells were also analysed for the expression of cyclin A protein via double immunostaining. RESULTS: Performing RT-PCR, Western blot and immunocytochemical analyses revealed that in human amniotic fluid in the background of Oct-4-negative cells a distinct population of cells can be found, which express Oct-4 in the nucleus. Oct-4-positive amniotic fluid cell samples also express stem cell factor, vimentin and alkaline phosphatase mRNA. The Oct-4-positive amniotic fluid cells are actively dividing, proven by the detection of cyclin A expression. CONCLUSIONS: The results presented here suggest that human amniotic fluid may represent a new source for the isolation of human Oct-4-positive stem cells without raising the ethical concerns associated with human embryonic research.

The mouse mammary tumor virus-like env gene sequence is not detectable in breast cancer tissue of Austrian patients.

The mammary mouse tumor virus (MMTV) has been related to human breast cancer in previous studies, but these have yielded contradictory results. An MMTV env gene-like sequence was detectable in a relatively high proportion (38%) of human breast cancer tissues. The aim of this study was to determine the proportion of this 660 bp MMTV env gene-like sequence in a population of Austrian breast cancer patients. We performed PCR, repeat PCR, and nested PCR. We did not find any exogenous MMTV env gene sequences in the 50 DNA samples of human breast cancer tissue nor in 22 breast cancer cell lines including MCF-7, which has previously been described as a positive control.

Expression of sex steroid receptors and their co-factors in normal and malignant breast tissue: AIB1 is a carcinoma-specific co-activator.

The differential expression pattern of estrogen receptor alpha (ER-alpha), estrogen receptor beta (ER-beta) and their co-activator/co-repressor proteins is thought to modulate estrogenic action and to be present already during the early stages of tumorigenesis. It has therefore been postulated that certain co-activator and co-repressor proteins contribute to the development of breast cancer. There are some reports providing information on gene amplification and mRNA over-expression of certain co-factors in breast cancer, but to date there is only limited knowledge about their respective protein expressions. The aim of this study was to examine the expression of four steroid receptor co-activators (steroid receptor co-activator 1 (SRC-1), transcription intermediary factor 2 (TIF 2), protein 300 kDa/CREB binding protein (p300/CBP), amplified in breast cancer 1 (AIB1)), and of the co-repressor nuclear receptor co-repressor (NCoR), in malignant breast tissues and in matching normal breast biopsies of the same individuals. Protein expression was analyzed by immunohistochemistry and was compared to prognostic parameters such as lymph node involvement, tumor grading and receptor status. All members of the co-regulatory protein family were detected in both, benign and matching malignant tissue samples, except for AIB1, which was found to be expressed exclusively in malignant epithelium. AIB1 was preferentially present in carcinomas with high tumor grade (r = 0.48, p = 0.014), and was co-expressed with p300/CBP (r = 0.54, p = 0.006). TIF 2 correlated significantly to nodal status (r = 0.46, p = 0.025). Furthermore, protein levels of ER-beta p300/CBP and AIB1 were higher in invasive ductal carcinomas than in normal mammary tissue. The tumoral ER-alpha protein expression was significantly correlated with that of PgR (r = 0.61, p = 0.001) and NCoR (r = 0.4, p = 0.043), whereas ER-beta expression was associated with SRC-1 (r = 0.68, p < or = .001), TIF 2 (r = 0.64, p = 0.001) and NCoR (r = 0.48, p = 0.014) protein levels in malignant specimens. In our hands, 20% of ER-beta positive tumors did not express ER-alpha protein, thereby suggesting that a substantial fraction of ER-beta positive tumors is falsely considered to be 'estrogen receptor negative' if only ER-alpha specific antibodies are employed in the histological assessment of the ER status.

Brca1 and differentiation.

Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.

MMP-2 and MMP-9 expression in breast cancer-derived human fibroblasts is differentially regulated by stromal-epithelial interactions.

Tissue remodeling is a key element in the local invasion and metastasis of malignant breast tumors. The degradation of extracellular matrix that is associated with this process is thought to be mediated by a number of Zn2+-dependent matrix metalloproteinases (MMPs). In most cases these enzymes are not produced by the malignant epithelium itself but by adjacent breast stroma, suggesting an important role for cell-cell interactions. We have analyzed Gelatinase A (MMP-2) and Gelatinase B (MMP-9) gene expression in a panel of six breast cancer cell lines and six primary cultures of stromal cells deriving from breast cancer biopsies. With one exception we did not detect MMP-2 or MMP-9 gene expression in any of the established tumor cell lines. Conversely, tumor stroma-derived fibroblasts expressed MMP-2 mRNA. although no MMP-9 mRNA was seen in RNase protection assays. When fibroblasts were cultured in the presence of media conditioned by MCF-7 tumor cells, MMP-2 enzyme production increased but MMP-9 activity remained undetectable. However, when fibroblasts and MCF-7 tumor cells were co-cultured together, MMP-9 was induced. These observations were confirmed by immunocytochemical analysis of co-cultures of MCF-7 and tumor-derived fibroblasts in which MMP-2 and MMP-9 protein expression was confined to stromal cells adjacent to MCF-7 tumor cells. No MMP-2 or MMP-9 staining was detected in monocultures of the two respective cell types. We conclude that MMP-2 expression is present in the stroma of malignant tumors and is increased by paracrine stimulation mediated by soluble factors. In contrast, MMP-9 expression tumor-derived fibroblasts requires direct contact with malignant tumor epithelium.