Josefine Gerhardt

FOXA1 Promotes Tumor Progression in Prostate Cancer and Represents a Novel Hallmark of Castration-Resistant Prostate Cancer

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer.

Periostin is up-regulated in high grade and high stage prostate cancer

BackgroundExpression of periostin is an indicator of epithelial-mesenchymal transition in cancer but a detailed analysis of periostin expression in prostate cancer has not been conducted so far.MethodsHere, we evaluated periostin expression in prostate cancer cells and peritumoural stroma immunohistochemically in two independent prostate cancer cohorts, including a training cohort (n = 93) and a test cohort (n = 325). Metastatic prostate cancers (n = 20), hormone refractory prostate cancers (n = 19) and benign prostatic tissues (n = 38) were also analyzed.ResultsIn total, strong epithelial periostin expression was detectable in 142 of 418 (34.0%) of prostate carcinomas and in 11 of 38 benign prostate glands (28.9%). Increased periostin expression in carcinoma cells was significantly associated with high Gleason score (p < 0.01) and advanced tumour stage (p < 0.05) in the test cohort. Whereas periostin expression was weak or absent in the stroma around normal prostate glands, strong periostin expression in tumour stroma was found in most primary and metastatic prostate cancers. High stromal periostin expression was associated with higher Gleason scores (p < 0.001). There was a relationship between stromal periostin expression and shortened PSA relapse free survival times in the training cohort (p < 0.05).ConclusionsOur data indicate that periostin up-regulation is related to increased tumour aggressiveness in prostate cancer and might be a promising target for therapeutical interventions in primary and metastatic prostate cancer.

Profiling Gastrin-Releasing Peptide Receptor in Prostate Tissues: Clinical Implications and Molecular Correlates

The gastrin‐releasing peptide receptor (GRPR) has emerged as an attractive target for both therapeutic and diagnostic appliances, but has only insufficiently been characterized in the human prostate so far. The aim of this study is to profile GRPR in a large cohort and correlate it with clinicopathologic and molecular parameters.

Endogenous myoglobin in breast cancer is hypoxia-inducible by alternative transcription and functions to impair mitochondrial activity: a role in tumor suppression?

Background: The role of MB in tumors cells is yet unclear. Results: MB is induced by hypoxia in breast cancer cell lines, possibly by an alternative transcription start site. Knockdown of MB in breast cancer cells is functionally relevant and significantly alters cellular respiration. Conclusion: MB might impair mitochondria in hypoxic cancer cells. Significance: MB might have tumor-suppressive functions, not described so far. Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here, we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/2-dependent transactivation. The hypoxia-induced MB mRNA originated from a novel alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O2 uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O2 consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O2. Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.

Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype

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-α (ERα)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1α (HIF-1α), HIF-2α, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERα 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-2α and CAIX, but not to HIF-1α or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERα 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 ERα 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.

Insulin-like growth factor II mRNA binding protein 3 (IMP3) is overexpressed in prostate cancer and correlates with higher Gleason scores

BackgroundThe oncofetal protein insulin-like growth factor II mRNA binding protein 3 (IMP3) is an important factor for cell-migration and adhesion in malignancies. Recent studies have shown a remarkable overexpression of IMP3 in different human malignant neoplasms and also revealed it as an important prognostic marker in some tumor entities. To our knowledge, IMP3 expression has not been investigated in prostate carcinomas so far.MethodsImmunohistochemical stainings for IMP3 were performed on tissue microarray (TMA) organized samples from 507 patients: 31 normal prostate tissues, 425 primary carcinomas and 51 prostate cancer metastases or castration-resistant prostate cancers (CRPC). IMP3 immunoreactivity was semiquantitatively scored and correlated with clinical-pathologic parameters including survival.ResultsIMP3 is significantly stronger expressed in prostate carcinomas compared to normal prostate tissues (p < 0.0001), but did not show significant correlation with the pT-stage, the proliferation index (MIB1), preoperative serum PSA level and the margin status. Only a weak and slightly significant correlation was found with the Gleason score and IMP3 expression failed to show prognostic significance in clinico-pathological correlation-analyses.ConclusionsAlthough IMP3 is overexpressed in a significant proportion of prostate cancer cases, which might be of importance for novel therapeutic approaches, it does not appear to possess any immediate diagnostic or prognostic value, limiting its potential as a tissue biomarker for prostate cancer. These results might be corroborated by the fact, that two independent tumor cohorts were separately reviewed.

The Androgen-Regulated Calcium-Activated Nucleotidase 1 (CANT1) Is Commonly Overexpressed in Prostate Cancer and Is Tumor-Biologically Relevant in Vitro

Previously, we identified the calcium-activated nucleotidase 1 (CANT1) transcript as up-regulated in prostate cancer. Now, we studied CANT1 protein expression in a large cohort of nearly 1000 prostatic tissue samples including normal tissue, prostatic intraepithelial neoplasia (PIN), primary carcinomas, metastases, and castrate-resistant carcinomas, and further investigated its functional relevance. CANT1 displayed predominantly a Golgi-type immunoreactivity with additional and variable cytoplasmic staining. In comparison to normal tissues, the staining intensity was significantly increased in PIN lesions and cancer. In cancer, high CANT1 levels were associated with a better prognosis, and castrate-resistant carcinomas commonly showed lower CANT1 levels than primary carcinomas. The functional role of CANT1 was investigated using RNA interference in two prostate cancer cell lines with abundant endogenous CANT1 protein. On CANT1 knockdown, a significantly diminished cell number and DNA synthesis rate, a cell cycle arrest in G(1) phase, and a strong decrease of cell transmigration rate and wound healing capacity of CANT1 knockdown cells was found. However, on forced CANT1 overexpression, cell proliferation and migration remained unchanged. In summary, CANT1 is commonly overexpressed in the vast majority of primary prostate carcinomas and in the precursor lesion PIN and may represent a novel prognostic biomarker. Moreover, this is the first study to demonstrate a functional involvement of CANT1 in tumor biology.

Abstract 440: Myoglobin in breast cancer: Regulation, function and relevance

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.

Abstract 1772: Calcium activated nucleotidase 1 (CANT1) promotes progression of prostate carcinomas

Aims: Previously, we identified Calcium-activated nucleotidase 1 (CANT1) mRNA to be overexpressed in human prostate cancer, however, so far no involvement of CANT1 in the progression of human tumours was reported. Therefore the protein expression level of CANT1 in prostate tumours was determined, followed by the elucidation of a functional implication of CANT1 overexpression in prostate cancer progression. Methods: Two tissue microarrays including 1100 prostate samples from two different institutions were constructed to examine the protein expression level of CANT1. The role of CANT1 in tumour progression was assessed in two prostate cancer cell lines in an RNA interference based approach. Cell number and DNA synthesis rate were determined by in vitro assays to measure cell proliferation, further cell migration was studied in transwell chamber assays. Results: A recurrent overexpression of CANT1 protein in human prostate cancers was confirmed on the tissue microarrays. Functionally, CANT1 knockdown induced a reduction of LNCaP as well as PC-3 cell number, which was caused by a diminished DNA synthesis rate. Moreover, a considerable decrease of cell migration towards a fibronectin gradient was observed in both cell lines upon CANT1 knockdown. This decreased cell mobility was also accompanied by morphological cell changes. Conclusions: Two cellular processes that are directly linked to tumour progression are markedly impaired upon CANT1 knockdown in prostate cancer cell lines, demonstrating for the first time a tumourbiological relevance of CANT1 expression. In future experiments the mechanism how CANT1 exerts its effects on proliferation and migration will be elucidated as well as the pathways that lead to CANT1 overexpression in prostate cancer. This will contribute to a better understanding of prostate carcinogenesis and to the evaluation of the potential of CANT1 as a therapeutic target. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 1772.