Ilka B. Schiffer

ERBB2-Mediated Transcriptional Up-regulation of the α5β1 Integrin Fibronectin Receptor Promotes Tumor Cell Survival Under Adverse Conditions

Oncogenic activation of the receptor tyrosine kinase ERBB2 is a key event in the development of a number of epithelial malignancies. In these tumors, high levels of ERBB2 are strongly associated with metastatic disease and poor prognosis. Paradoxically, an inherent cellular response to hypermitogenic signaling by ERBB2 and other oncogenes seems to be growth arrest, rather than proliferation. Molecular characterization of this yet undefined antiproliferative state in independent cell lines overexpressing either wild-type ERBB2 or the mutationally activated receptor unveiled a dramatic induction of the α5β1 integrin fibronectin receptor. α5 Integrin up-regulation is mainly a transcriptional response mediated by the hypoxia-inducible transcription factors (HIF), leading to a massive increase in membrane-resident receptor molecules and enhanced fibronectin adhesiveness of the respective cells. Functionally, ERBB2-dependent ligation of fibronectin results in improved survival of mammary adenocarcinoma cells under adverse conditions, like serum withdrawal, hypoxia, and chemotherapy. HIF-1α is an independent predictor of poor overall survival in patients with breast cancer. In particular, HIF-1α overexpression correlates significantly with early local relapse and distant metastasis, a phenotype also highly characteristic of ERBB2-positive tumors. As HIF-1α is known to be stabilized by ERBB2 signaling under normoxic conditions, we propose that α5 integrin is a major effector in this regulatory circuit and may represent the molecular basis for the HIF-1α-dependent aggressiveness observed in ERBB2-overexpressing breast carcinomas. Hypermitogenic ERBB2 signaling and tumor hypoxia may act synergistically to favor the establishment of chemoresistant dormant micrometastatic cells frequently observed in patients with breast cancer. This new insight could be the basis for additional approaches complementing current cancer therapy. (Cancer Res 2006; 66(7): 3715-25)

Metallothionein expression in ovarian cancer in relation to histopathological parameters and molecular markers of prognosis

Metallothioneins (MTs) and glutathione constitute the major fractions of intracellular thiol factors. Abundant nucleophilic sulfhydryl groups can interact with many electrophilic substances, including several anti‐neoplastic agents, participate in controlling intracellular redox potential, and act as scavengers of reactive oxygen species. In the present study, we examined the relation of MTs (alone and in combination with glutathione) to histopathological parameters and survival time of ovarian cancer patients. Expression of the major MT isoforms (MT‐1 and MT‐2) was determined by immunohistochemistry on paraffin‐embedded tumor specimens from 189 patients, 151 suffering from primary epithelial ovarian cancer and 38 from recurrences. MT was negatively associated with survival time when all patients with primary carcinomas (n = 151) were analyzed (p = 0.049, log‐rank test). However, no significant association between MT expression and survival was obtained when subgroups of patients with histological grade 1, 2 or 3 carcinomas were analyzed. Similarly, no significant association of MT expression and survival was obtained with the proportional hazards model adjusted for histological grade. This scenario can be explained by a correlation between MT expression and histological grade: MT was detectable in 26%, 48% and 62% of grade 1, 2 and 3 carcinomas, respectively (p = 0.008, χ2 test). An interesting hypothesis is generated by combined analysis of MT and total glutathione content (GSH). The product of MT and GSH levels (MT × GSH) was negatively associated with survival of grade 1 carcinomas (p = 0.021, log‐rank test) but not with grade 2 and 3 carcinomas (p = 0.176 and 0.403, respectively). When MT × GSH was greater than the median, 25% of patients with grade 1 carcinomas died within 235 days. In contrast, all patients with grade 1 carcinomas survived when MT × GSH in tumor tissue was smaller than the median. This suggests that high expression of sulfhydryl factors might facilitate survival and progression of low‐grade ovarian cancer cells. A significant correlation was obtained between MT expression and mutant p53 (p = 0.037, χ2 test). However, this might be an indirect effect since both MT (p = 0.008) and mutant p53 (p = 0.000) were associated with histological grade. In conclusion, MT expression as well as the product of MT and GSH were associated with histological grade of primary ovarian carcinomas. High expression of both sulfhydryl factors may identify a subgroup of low‐grade carcinomas with an increased risk of progression.

Lack of mutagenic and co-mutagenic effects of magnetic fields during magnetic resonance imaging

Mutagenic and co‐mutagenic effects of static, pulsed bipolar gradient, and high‐frequency magnetic fields, as well as combinations of them, were examined using the Ames test. The Ames test using Salmonella typhimurium bacteria, wild‐type strain RTA, preincubation assay, without metabolic activation, was performed. All combinations of magnetic fields were tested with and without co‐exposure to N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and benzo[a]pyrene‐4,5‐oxide, ethylene oxide, carboplatin, or cisplatin. As expected, chemical mutagens caused a clear‐cut increase of the revertants in the Ames test. However, neither the static fields nor a combination of a static magnetic field with the time‐varying bipolar gradient field or a pulsed high‐frequency magnetic field caused an alteration in the number of revertants in the Ames test. No co‐mutagenic effect of any magnetic field combination was observed. In conclusion, magnetic fields used during clinical magnetic resonance imaging (MRI) were neither mutagenic nor co‐mutagenic. J. Magn. Reson. Imaging 2001;14:779–788.

Dephosphorylation of p-ERK1/2 in relation to tumor remission after HER-2 and Raf1 blocking therapy in a conditional mouse tumor model.

Several studies have shown that HER‐2/neu (erbB‐2) blocking therapy strategies can cause tumor remission. However, the responsible molecular mechanisms are not yet known. Both ERK1/2 and Akt/PKB are critical for HER‐2‐mediated signal transduction. Therefore, we used a mouse tumor model that allows downregulation of HER‐2 in tumor tissue by administration of anhydrotetracycline (ATc). Switching‐off HER‐2 caused a rapid tumor remission by more than 95% within 7 d of ATc administration compared to the volume before switching‐off HER‐2. Interestingly, HER‐2 downregulation caused a dephosphorylation of p‐ERK1/2 by more than 80% already before tumor remission occurred. Levels of total ERK protein were not influenced. In contrast, dephosphorylation of p‐Akt occurred later, when the tumor was already in remission. These data suggest that in our HER‐2 tumor model dephosphorylation of p‐ERK1/2 may be more critical for tumor remission than dephosphorylation of p‐Akt. To test this hypothesis we used a second mouse tumor model that allows ATc controlled expression of BXB‐Raf1 because the latter constitutively signals to ERK1/2, but cannot activate Akt/PKB. As expected, downregulation of BXB‐Raf1 in tumor tissue caused a strong dephosphorylation of p‐ERK1/2, but did not decrease levels of p‐Akt. Interestingly, tumor remission after switching‐off BXB‐Raf1 was similarly efficient as the effect of HER‐2 downregulation, despite the lack of p‐Akt dephosphorylation. In conclusion, two lines of evidence strongly suggest that dephosphorylation of p‐ERK1/2 and not that of p‐Akt is critical for the rapid tumor remission after downregulation of HER‐2 or BXB‐Raf1 in our tumor model: (i) dephosphorylation of p‐ERK1/2 but not that of p‐Akt precedes tumor remission after switching‐off HER‐2 and (ii) downregulation of BXB‐Raf1 leads to a similarly efficient tumor remission as downregulation of HER‐2, although no p‐Akt dephosphorylation was observed after switching‐off BXB‐Raf1.

Possible mutagenic effects of magnetic fields

AIM To assess the potential mutagenic effect of static magnetic fields of 1.5 T and 7 T, gradient fields and high frequency magnetic fields. METHOD We used the Salmonella mutagenicity test (Ames test), which detects mutations in a gene of a histidine-requiring (his-) strain to produce a histidine-independent (his+) strain, Exposure to a static magnetic field of 1.5 T and 7.2 T, in a bipolar magnetic gradient and additionally in a high frequency field took place with and without known genotoxic chemicals. RESULTS No differences in the number of revertants between the bacterial strains of exposed and control cells could be detected and the exposure with known genotoxic chemicals showed no significant difference in mutagenicity. CONCLUSION In conclusion our data do not provide evidence that exposure to a static magnetic field exerts effects on the mutagenicity in our standard tester strains and whether the exposure took place in a diagnostic 1.5 T MR scanner which is used in the clinical routine or at 7.2 T which is a much stronger field made no difference. Also an exposure in a gradient field or in a high frequency field did not show any alteration in the number of revertants.Aim: To assess the potential mutagenic effect of static magnetic fields of 1.5 T and 7T, gradient fields and high frequency magnetic fields. Method: We used the Salmonella mutagenicity test (Ames test), which detects mutations in a gene of a histidine-requiring (his-) strain to produce a histidine-independent (his + ) strain. Exposure to a static magnetic field of 1.5 T and 7.2 T, in a bipolar magnetic gradient and additionally in a high frequency field took place with and without known genotoxic chemicals. Results: No differences in the number of revertants between the bacterial strains of exposed and control cells could be detected and the exposure with known genotoxic chemicals showed no significant difference in mutagenicity. Conclusion: In conclusion our data do not provide evidence that exposure to a static magnetic field exerts effects on the mutagenicity in our standard tester strains and whether the exposure took place in a diagnostic 1.5T MR scanner which is used in the clinical routine or at 7.2 T which is a much stronger field made no difference. Also an exposure in a gradient field or in a high frequency field did not show any alteration in the number of revertants.