BACKGROUND: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. METHODS: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-a (ERa)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1a (HIF-1a), HIF-2a, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERa or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. RESULTS: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2a and CAIX, but not to HIF-1a or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERa or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. CONCLUSION: We conclude that in breast cancer, Mb is co-expressed with ERa and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.

Disruption of the Wnt pathway is thought to be crucial in the development of human cancer. Pathway inhibitory members of the secreted frizzled-related protein (SFRP) family were found to be downregulated due to epigenetic inactivation in various malignancies. To date, only SFRP1 has been studied in human breast cancer and we questioned whether other SFRP genes may be implicated in the pathogenesis of this disease as well. An initial real-time polymerase chain reaction analysis of SFRP5 expression in normal human tissues (n = 9) revealed weak expression in most tissues, including breast. Malignant mammary cell lines showed further SFRP5 expression loss in five of six cases. Consistently, in matched pairs of primary breast tumor/normal breast tissue, this downregulation (>5-fold) could be confirmed (n = 8/13; 62%). We identified promoter methylation as the predominant mechanism of SFRP5 gene silencing since SFRP5 promoter methylation correlated significantly with loss of SFRP5 expression in cell lines (P = 0.040) and primary tumors (P = 0.003). Moreover, cancerous cell lines re-expressed SFRP5 messenger RNA following treatment with DNA-demethylating drugs. Of 168 primary breast carcinomas, 73% harbored a methylated SFRP5 promoter, whereas 27% were unaffected by epigenetic alteration. Most interestingly, SFRP5 methylation was associated with reduced overall survival (OS) (P = 0.045) and was an independent risk factor affecting OS in a multivariate Cox proportional hazard model (hazard ratio): 4.55; 95% confidence interval: 1.01-20.56; P = 0.049). In conclusion, SFRP5 is a target of epigenetic inactivation in human breast cancer, supporting the hypothesis of its role as tumor suppressor gene. SFRP5 methylation may be a novel DNA-based biomarker potentially useful in clinical breast cancer management.

This study was designed to identify and validate potential new biomarkers for prostate cancer and to distinguish patients with and without biochemical relapse. Prostate tissue samples analyzed by 2D-DIGE (two-dimensional difference in gel electrophoresis) and mass spectrometry (MS) revealed downregulation of secernin-1 (P < 0.044) in prostate cancer, while vinculin showed significant upregulation (P < 0.001). Secernin-1 overexpression in prostate tissue was validated using Western blot and immunohistochemistry while vinculin expression was validated using immunohistochemistry. These findings indicate that secernin-1 and vinculin are potential new tissue biomarkers for prostate cancer diagnosis and prognosis, respectively. For validation, protein levels in urine were also examined by Western blot analysis. Urinary vinculin levels in prostate cancer patients were significantly higher than in urine from nontumor patients (P = 0.006). Using multiple reaction monitoring-MS (MRM-MS) analysis, prostatic acid phosphatase (PAP) showed significant higher levels in the urine of prostate cancer patients compared to controls (P = 0.012), while galectin-3 showed significant lower levels in the urine of prostate cancer patients with biochemical relapse, compared to those without relapse (P = 0.017). Three proteins were successfully differentiated between patients with and without prostate cancer and patients with and without relapse by using MRM. Thus, this technique shows promise for implementation as a noninvasive clinical diagnostic technique.

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Dickkopf 3 (DKK3) has been associated with tumor suppression of various tumor entities including breast cancer. However, the functional impact of DKK3 on the tumorigenesis of distinct molecular breast cancer subtypes has not been considered so far. Therefore, we initiated a study analyzing the subtype-specific DKK3 expression pattern as well as its prognostic and functional impact with respect to breast cancer subtypes. Based on three independent tissue cohorts including one in silico dataset (n = 30, n = 463 and n = 791) we observed a clear down-regulation of DKK3 expression in breast cancer samples compared to healthy breast tissue controls on mRNA and protein level. Interestingly, most abundant reduction of DKK3 expression was detected in the highly aggressive basal breast cancer subtype. Analyzing a large in silico dataset comprising 3,554 cases showed that low DKK3 mRNA expression was significantly associated with reduced recurrence free survival (RFS) of luminal and basal-like breast cancer cases. Functionally, DKK3 re-expression in human breast cancer cell lines led to suppression of cell growth possibly mediated by up-regulation of apoptosis in basal-like but not in luminal-like breast cancer cell lines. Moreover, ectopic DKK3 expression in mesenchymal basal breast cancer cells resulted in partial restoration of epithelial cell morphology which was molecularly supported by higher expression of epithelial markers like E-Cadherin and down-regulation of mesenchymal markers such as Snail 1. Hence, we provide evidence that down-regulation of DKK3 especially promotes tumorigenesis of the aggressive basal breast cancer subtype. Further studies decoding the underlying molecular mechanisms of DKK3-mediated effects may help to identify novel targeted therapies for this clinically highly relevant breast cancer subtype.

Background Following an accidental observation, we aimed to analyse expression, regulation and function of myoglobin (Mb) in breast cancer. Methods Immunohistochemistry/Immunofluorescence, microdissection, transmission electron microscopy, Western blot, qPCR, cell culture (under normoxia and hypoxia), shRNA and siRNA knockdown, in silico data mining, high resolution respirometry. Functional assays: proliferation, migration and colony formation. Results Mb protein is de novo expressed in 71% of invasive breast carcinomas (n=917) in association with the hypoxia inducible factor 2α (HIF-2 α), carbonic anhydrase IX and a better prognosis. Expression of Mb in breast cancer was confirmed on mRNA level and occurred irrespective of rhabdomyoid differentiation and was inducible by prolonged hypoxia in breast cancer cell lines (MDA-MB231, MDA-MB468, MCF7) with a median change fold of 3.4 after 72 hours. Interestingly, hypoxically induced MB mRNA originated from a novel, alternative transcription start site 6 kb upstream of the genuine ATG codon. Functionally, stable myoglobin knockdown in MDA-MB468 cells was associated with a stimulated O2 uptake during mild hypoxia, yet did not impact the O2 diffusion gradient in these small cells during limiting conditions. Knockdown of Mb also conferred a significant retardation of the cells’ in vitro motility to close an inflicted wound within the normoxic culture. Conclusions Regarding cancer cells, unconventional functions of myoglobin, not directly related to the transport of oxygen, thus need to be envisioned. These unprecedented findings may have fundamental implications for our understanding of non-muscle Mb in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 440.