ERBB2-mediated transcriptional up-regulation of the alpha5beta1 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 alpha5beta1 integrin fibronectin receptor. alpha5 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-1alpha is an independent predictor of poor overall survival in patients with breast cancer. In particular, HIF-1alpha overexpression correlates significantly with early local relapse and distant metastasis, a phenotype also highly characteristic of ERBB2-positive tumors. As HIF-1alpha is known to be stabilized by ERBB2 signaling under normoxic conditions, we propose that alpha5 integrin is a major effector in this regulatory circuit and may represent the molecular basis for the HIF-1alpha-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.

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.

Premature senescence is a primary fail-safe mechanism of ERBB2-driven tumorigenesis in breast carcinoma cells.

The receptor tyrosine kinase ERBB2 plays a central role in the development of breast cancer and other epithelial malignancies. Elevated ERBB2 activity is believed to transform cells by transmitting mitogenic and antiapoptotic signals. Here we show that tightly regulated overexpression of oncogenic ERBB2 in human breast carcinoma cells does not stimulate proliferation but provokes premature senescence, accompanied by up-regulation of the cyclin-dependent kinase inhibitor P21(WAF1/CIP1). A similar effect was caused by retrovirus-mediated overexpression of oncogenic ERBB2 in low-passage murine embryonic fibroblasts. In contrast to previous observations based on constitutively overexpressing cell lines, P21 induced by tetracycline-regulated ERBB2 localizes to the nucleus in arrested cells. P21 up-regulation seems to be independent of the P53 tumor suppressor protein, and senescence-associated phenotypic alterations are reversed by specific inhibition of P38 mitogen-activated protein kinases. Functional inactivation of P21 by antisense oligonucleotides is sufficient to prevent cell cycle arrest as well as the senescent phenotype, thereby identifying the P21 protein as the key mediator of hypermitogenic cell cycle arrest and premature senescence in breast carcinoma cells. Our results may thus indicate that premature senescence represents an inherent anticarcinogenic program during ERBB2-driven mammary tumorigenesis. We propose a multistep model for the process of malignant transformation by ERBB2 wherein secondary lesions either target P21 or downstream effectors of senescence to bypass this primary fail-safe mechanism.

Switching off HER-2/neu in a tetracycline-controlled mouse tumor model leads to apoptosis and tumor-size-dependent remission.

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (> or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.