Reduction of Akt2 expression inhibits chemotaxis signal transduction in human breast cancer cells

Protein kinase Cζ PKCζ mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the research for its upstream regulator, we investigated the role of Akt2 in chemotaxis and metastasis of human breast cancer cells. Reduction of Akt2 expression by siRNA inhibited chemotaxis of MDA-MB-231, T47D, and MCF7 cells, three representative human breast cancer cells. Expression of a wild type Akt2 in siRNA transfected cells rescued the phenotype. EGF-induced integrin β1 phosphorylation was dampened, consistent with defects in adhesion. Phosphorylation of LIMK and cofilin, a critical step of cofilin recycle and actin polymerization, was also impaired. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during chemotaxis. Depletion of Akt2 by siRNA impaired the activation of PKCζ while inhibition of PKCζ did not interfere with EGF induced phosphorylation of Akt. Furthermore, EGF induced co-immunoprecipitation between PKCζ and Akt2, but not Akt1, suggesting that a direct interaction between PKCζ and Akt2 in chemotaxis. Protein levels of integrin β1, LIMK, cofilin, and PKCζ didn't alter, suggesting that Akt2 does not regulate the expression of these signaling molecules. In a Severe Combine Immunodeficiency mouse model, Akt2 depleted MDA-MB-231 cells showed a marked reduction in metastasis to mouse lungs, demonstrating the biological relevancy of Akt2 in cancer metastasis in vivo. Taken together, our results suggest that Akt2 directly mediates EGF-induced chemotactic signaling pathways through PKCζ and its expression is critical during the extravasation of circulating cancer cells.     

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.     

Disulfide-linked and noncovalent dimers of p185HER-2 in human breast carcinoma cells.

Enhanced levels of disulfide-linked dimers of the neu oncogene product have been suggested to be associated with the transformed state [Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI: Nature 338:230-231, (1989)]. We, therefore, investigated the properties of the dimeric forms of p185HER-2/neu from the human breast carcinoma cell line, SK-BR-3. We found disulfide-linked dimers as well as noncovalently associated dimers that were detected by cross-linking with bis(sulfosuccinimidyl) suberate (BS3). However, the disulfide-linked dimers did not exist in intact cells, since they were eliminated when the cells were lysed in the presence of the alkylating agent, sodium iodoacetate. Moreover, the disulfide-linked dimeric molecules were not the activated form of p185HER-2 since they incorporated about the same level of phosphate in an in vitro kinase reaction as the monomeric molecules. In contrast, the noncovalent dimers appeared to be present on the surface of intact cells and were phosphorylated at levels at least tenfold higher than monomers in an in vitro kinase reaction.     

Expression of HER2 in breast cancer

In breast cancer the membrane expression of HER2 receptor protein encoded by the HER2 proto-oncogene seems to have an ever growing clinical significance. In tissue cultures and animal experiments it was shown that the HER2 gene amplification induces malignant transformation and intensifies the aggressiveness of the tumour cells. Correlating with the so called pheno-and genotypic prognostic markers, the overexpression of HER2 in breast cancer predicts also poor prognosis and indicates enhanced potential for metastatisation. In some of the so called precancerous proliferations and "in situ" carcinomas we demonstrated the enhanced membrane staining of the HER2 receptor protein. In these cases we frequently observed DNA aneuploidy,the presence of p53 mutational protein and CD44v6 glycoprotein. The immunohistochemical studies of HER2 protein in invasive carcinomas have revealed, an interrelationship between the grade of differentiation, histological type, aggressiveness and biological behaviour of the "in situ" and invasive carcinomas. In clinical studies trastuzumab, a humanized monoclonal antibody recognizing extracellular domain of HER2 receptor protein, has proved to be effective in HER2 overexpressing metastatic breast cancer either as monotherapy or in combination with chemotherapeutical agents. The DAKO "HercepTest" is a semiquantitative, standardised method for the determination of HER2 overexpression.     

Dynamic chromatin remodeling on the HER2 promoter in human breast cancer cells.

Deregulation of the HER2 oncogene occurs in 30% of human breast cancers and correlates with poor prognosis and increased propensity for metastasis. Since the molecular basis of HER2 overexpression in human cancers is not known, we sought to determine whether chromatin remodeling pathways are involved in the regulation of HER2 expression. We report that compared with breast cancer cells expressing a low level of HER2, HER2-overexpressing breast cancer cells contained significantly higher levels of acetylated and phosphorylated histone H3, and acetylated histone H4 associated with the HER2 promoter. Decreased recruitment of histone deacetylases in the promoter is also noted in the HER2-overexpressing cell. The association of acetylated histone H4 with HER2 gene chromatin and HER2 expression in breast cancer cells was upregulated by an inhibitor of histone deacetylases. Treatment with histone deacetylase inhibitor also reduced the association of histone deacetylase-1 and -2 with the HER2 promoter. In addition, the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid stimulated the association of phosphorylated histone H3 on serine 10 with the HER2 promoter and also stimulated HER2 expression. These findings identify histone acetylation and histone phosphorylation as novel regulatory modifications that target HER2 gene chromatin, and suggest that elevated levels of these chromatin-relaxing components in the vicinity of the HER2 gene promoter may constitute an important non-genomic mechanism of HER2 overexpression in human breast cancer.     

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.     

Insight into p95HER2 in breast cancer: molecular mechanisms and targeted therapies

Breast cancer afflicts more than 1.3 million people worldwide and is the main cause of cancer-related deaths among women. Many efforts are underway to develop new therapeutic and biomarker strategies for the management of this disease. Hormone receptors and human epidermal growth factor receptor 2 (HER2) are currently the most important molecular tools in this regard. Moreover, targeted therapies including trastuzumab in particular are the primary treatment in both the adjuvant and recurrent settings. However, many studies reported that selected patients may present with resistance to trastuzumab due to the presence of p95HER2 fragments. To address this challenge, drugs such as lapatinib and others described in recent patents promise alternative therapeutic options. We discuss the most recent patents related to HER2 and p95HER2 fragments for breast cancer treatment.     

Target therapy in HER2-overexpressing breast cancer patients

The development of new therapeutic strategies, such as monoclonal antibodies directed against human epidermal growth factor receptor-2 (HER2), has offered new hopes for women with early breast cancer whose tumors overexpress HER2. We retrospectively analyzed the population-based data of Breast Cancer Registry of Palermo in 2004-2006, and selected 1401 invasive breast cancer cases, nonmetastatic at diagnosis, having HER2/neu oncogene expression determined. We have correlated this information to age, tumor stage at diagnosis (TNM), nodal involvement, and receptor status (ER and PgR). Survival analysis was conducted dividing the patients in two different groups according to date of diagnosis: one group diagnosed in 2004 and a second group in 2005-2006. In the 460 cases of 2004, nodal involvement, receptor status, age at diagnosis and TNM maintained a strong predictive value (p?相似文献   

Effects of LHRH-analogues on mitogenic signal transduction in cancer cells

The expression of luteinizing hormone-releasing hormone (LHRH) and its receptors has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Recent studies in our laboratory have demonstrated that the function of LHRH produced by ovarian cancer cells is the inhibition of their proliferation. Dose-dependent antiproliferative effects of LHRH-agonists have been observed by several laboratories in cell lines derived from the above cancers. Interestingly, also LHRH-antagonists have marked antiproliferative activity in most of the ovarian, breast and endometrial cancer cell lines tested so far, indicating that the dichotomy of LHRH-agonists/LHRH-antagonists is not valid for the LHRH-system in cancer cells. In addition, our data suggest that the classical LHRH receptor signal transduction mechanisms known from the pituitary (phospholipase-C, protein kinase C, adenylyl cyclase) are not involved in the mediation of LHRH effects in cancer cells. Data obtained by several groups, including ours, rather suggest that LHRH analogs interfere with the signal transduction of growth-factor receptors and related oncogene products associated with tyrosine-kinase activity. The mechanism of action is probably an LHRH-induced activation of a phosphotyrosine phosphatase, counteracting the effects of receptor associated tyrosine kinase. In our hands, LHRH analogs virtually blocked the EGF-induced MAP-kinase activity of ovarian and endometrial cancer cells. The pharmacological exploitation of this mechanism might provide promising new therapies for these cancers.     

HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells

Background

The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.

Methodology and Principal Findings

Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs'' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment–induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.

Conclusions and Significance

These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.     

p38 MAPK信号传导通路

丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄｐｏｒｏｔｅｉｎｋｉｎａｓｅ,ＭＡＰＫ）介导了生长、发育,分裂,死亡,以及细胞间的功能同步等多种细胞生理功能,在哺乳动物细胞中已发现和克隆了ＥＲＫ、ＪＮＫ／ＳＡＰＫ,ＥＲＫ５／ＢＭＫ１和ｐ３８／ＲＫ四个ＭＡＰＫ亚族,这些新的ＭＡＰＫ介导了物理,化学反激,细菌产物,炎性细胞因子等多种刺激引起的细胞反应,ｐ３８亚族至少包括ｐ３８（α）,ｐ３８β,ｐ     

Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells

Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells.     

细胞信号传导和肿瘤

信号传递系统的存在及其过程是近年细胞生物学,分子生物学和医学领域的研究热点之一。信号传递异常与肿瘤等多种疾病的发生,发展和预后直接相关。本所述是迄今认识比较清楚的与肿瘤密切相关的几条信号传导途径,阐明它们的作用机制对于洞悉肿瘤发病机制并最终攻克肿瘤均有重要意义。