Syk: a new player in the field of breast cancer

Breast tumor development and progression are thought to occur through a complex, multistep process, including oncogene activation (eg HER2/neu) and mutation or loss of tumor suppressor genes (eg p53). Determining the function of genetic alterations in breast carcinoma tumorigenesis and metastasis has been the focus of intensive research efforts for several decades. One group of proteins that play a critical role in breast cancer cell signaling pathways are tyrosine kinases. Overexpression of the tyrosine kinase HER2/neu is observed in many human breast cancers and is positively correlated with enhanced tumorigenesis [1]. Recently, another tyrosine kinase, Syk, has been implicated as an important inhibitor of breast cancer cell growth and metastasis [2]. This recent finding was unexpected, since Syk function has been predominantly linked to hematopoietic cell signaling, and is discussed further in this commentary.     

脾酪氨酸激酶与肿瘤

脾酪氨酸激酶(spleen tyosine kinase,Syk)在较长时间里被认为是造血细胞特有的信号分子,在淋巴细胞的成熟和免疫细胞的激活中起重要作用.近年来发现Syk在多种非造血细胞也有表达.最初观察到Syk在乳腺癌的演进中显著降低,后来发现Syk异常表达也见于其它许多肿瘤.研究表明,Syk有抑制肿瘤发生和转移的功能,但Syk抑癌的分子机制仍大部分未明.Syk基因启动子超甲基化是其沉默的机制之一.日益增多的临床研究显示,Syk的低表达与肿瘤转移形成呈正相关,表明Syk可能是一个潜在的肿瘤抑制因子.     

Biological and clinical significance of her2 overexpression in Breast Cancer

The product of the HER-2/neu proto-oncogene, HER2, is the second member of the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors and has been suggested to be a ligand orphan receptor. Ligand-dependent heterodimerization between HER2 and another HER family member, HER1, HER3 or HER4, activates the HER2 signaling pathway. The intracellular signaling pathway of HER2 is thought to involve ras-MAPK, MAPK-independent S6 kinase and phospholipase C-gamma signaling pathways. However, the biological consequences of the activation of these pathways are not yet completely known. Amplification of the HER2 gene and overexpression of the HER2 protein induces cell transformation and has been demonstrated in 10% to 40% of human breast cancer. HER2 overexpression has been suggested to associate with tumor aggressiveness, prognosis and responsiveness to hormonal and cytotoxic agents in breast cancer patients. These findings indicate that HER2 is an appropriate target for tumor-specific therapies. A number of approaches have been investigated: (1) a humanized monoclonal antibody against HER2, rhuMAbHER2 (trastuzumab), which is already approved for clinical use in the treatment of patients with metastatic breast cancer; (2) tyrosine kinase inhibitors, such as emodin, which block HER2 phosphorylation and its intracellullar signaling; (3) active immunotherapy, such as vaccination; and (4) heat shock protein (Hsp) 90-associated signal inhibitors, such as radicicol derivatives, which induce degradation of tyrosine kinase receptors, such as HER2.     

The nuclear receptor coactivator amplified in breast cancer-1 is required for Neu (ErbB2/HER2) activation, signaling, and mammary tumorigenesis in mice

Overexpression of the oncogene amplified in breast cancer 1 (AIB1)/steroid receptor coactivator-3 (SRC-3) induces mammary tumorigenesis in mice. In breast cancer, high levels of AIB1/SRC-3 and the growth factor receptor HER2/neu predict resistance to endocrine therapy and poor outcome. However, a mechanistic relationship between AIB1/SRC-3 and HER2/neu in the development of breast cancer has not been shown. Here, we show that deletion of one allele of SRC-3 significantly delays Neu-induced mammary tumor development in mice. Homozygous deletion of SRC-3 in mice completely prevents Neu-induced tumor formation. By ages 3 to 4 months, Neu/SRC-3(+/-) mice exhibit a noticeable reduction in lateral side-bud formation, accompanied by reduced cellular levels of phosphorylated Neu compared with Neu/SRC-3(wt) mice. In Neu-induced tumors, high levels of SRC-3, phosphorylated Neu, cyclin D1, cyclin E, and proliferating cell nuclear antigen expression are observed, accompanied by activation of the AKT and c-Jun NH(2) kinase (JNK) signaling pathways. In comparison, phosphorylated Neu, cyclin D1, and cyclin E are significantly decreased in Neu/SRC-3(+/-) tumors, proliferation is reduced, and AKT and JNK activation is barely detectable. Our data indicate that AIB1/SRC-3 is required for HER2/neu oncogenic activity and for the phosphorylation and activation of the HER2/neu receptor. We predict that reducing AIB1/SRC-3 levels or activity in the mammary epithelium could potentiate therapies aimed at inhibiting HER2/neu signaling in breast cancer.     

The Syk tyrosine kinase: a new negative regulator in tumor growth and progression

The spleen tyrosine kinase Syk was long thought to be a hematopoietic cell-specific signaling molecule. Recent evidence demonstrated that it is also expressed by many non-hematopoietic cell types and that it plays a negative role in cancer. A significant drop in its expression was first observed during breast cancer progression, but an anomalous Syk expression has now also been evidenced in many other tumor types. Mechanistic studies using Syk re-expression demonstrated its suppressive function in tumorigenesis and metastasis formation, which is surprising for a tyrosine kinase. Loss of Syk expression is regulated, albeit not exclusively, by its promoter hypermethylation. The molecular mechanism of its tumor-suppressive function remains largely unknown; the identification of its activators and effectors in non-hematopoietic cells will be a challenge for the years to come. An increasing number of clinical studies reveal a correlation between reduced Syk expression and an increased risk for metastasis formation, and assign Syk as a potential new prognostic marker in different tumor types.     

Transcriptome analysis of HER2 reveals a molecular connection to fatty acid synthesis

HER2 (erbB2/neu) is a member of the erbB family of receptor tyrosine kinases and is involved in regulating the growth of several types of human carcinomas. HER2 represents a successful therapeutic target of the biotechnology era as exemplified by the drug Herceptin (trastuzumab), which has clinical activity in a subset of breast cancer patients. Using DNA microarrays, we identified a cohort of genes that are differentially regulated by HER2 in breast epithelial cells. One of the HER2-regulated genes discovered was fatty acid synthase (FAS), which has been shown to be overexpressed in breast cancer as well as other cancers. FAS is implicated in tumorigenesis through its role in cell proliferation and membrane lipid incorporation of neoplastic cells. Here, we demonstrate that HER2-mediated induction of FAS is inhibitable by Herceptin and tyrosine kinase inhibitors of HER2. Through a phosphatidylinositol 3'-kinase-dependent pathway, HER2 stimulates the FAS promoter and ultimately mediates increased fatty acid synthesis. Interestingly, pharmacological inhibition of FAS preferentially induced apoptosis of HER2-overexpressing breast epithelial cells relative to matched vector control cells. These studies characterize a molecular connection between two genes individually implicated in tumorigenesis but never linked together.     

Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis

The receptor tyrosine kinase HER2 enhances tumor metastasis; however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the movement of malignant cancer cells to specific organs. Here, we show that HER2 enhances the expression of CXCR4, which is required for HER2-mediated invasion in vitro and lung metastasis in vivo. HER2 also inhibits ligand-induced CXCR4 degradation. Finally, a significant correlation between HER2 and CXCR4 expression was observed in human breast tumor tissues, and CXCR4 expression correlated with a poor overall survival rate in patients with breast cancer. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and establish a functional link between HER2 and CXCR4 signaling pathways.     

HER2/neu expression correlates with vascular endothelial growth factor-C and lymphangiogenesis in lymph node-positive breast cancer

Background: Vascular endothelial growth factor-C (VEGF-C) is the main inducer of lymphangiogenesis. VEGF-C overexpression is associated with lymphovascular tumor cell invasion, an increased rate of lymph node metastasis and adverse prognosis in various human cancers. However, little is known about the upstream inducers of VEGF-C expression. Recent studies have shown that human epidermal growth factor receptor 2 (HER2/neu) overexpression is associated with high VEGF-C levels in human breast cancer cells. In addition to blocking of HER2/neu, tyrosine kinase significantly decreased VEGF-C expression in vitro.Patients and methods: VEGF-C expression, lymphatic microvessel density (LMVD), lymphovascular invasion (LVI) and HER2/neu expression were evaluated with immunohistochemical/FISH methods in a collective of 150 lymph node-positive human breast cancers with long-term follow-up.Results: Cases with 3+ HER2/neu protein expression showed a significantly stronger VEGF-C expression than all others cases (P = 0.006). In addition, we found a significant correlation between VEGF-C expression and LMVD (P = 0.012) and a strong positive association between LMVD and LVI (P < 0.001).Conclusion: Our data provide evidence for a clinically relevant association between HER2/neu and VEGF-C expression in human breast cancer. Inhibiting HER2/neu may reduce tumor progression by blocking VEGF-C-mediated tumor cell proliferation and lymphogenic metastasis.     

HER2/neu over-expression induces endothelial cell retraction

Over-expression of the HER2/neu (HER2) proto-oncogene in breast carcinoma imparts an enhanced metastatic potential. Metastasis requires escape of the tumor cell from the vasculature into subjacent tissue, a transmigration event across an endothelial cell (EC) monolayer. EC retraction has been reported to precede transmigration in several tumor metastatic models. Using intact human iliac vein EC monolayers, we tested the abilities of MCF-7 breast cancer cells and HER cells, a transfected MCF-7 line over-expressing HER2, to induce EC retraction. We further analyzed whether HER2 signaling influenced cancer cell-induced EC retraction. MCF-7 or HER cells were co-cultured onto mature EC monolayers. More HER than MCF-7 cells induced EC retraction (76 +/- 19% vs. 17 +/- 12%, p < 0.001) with resultant exposure of subendothelial matrix (6.80 +/- 2.86% vs. 0.85 +/- 0.39%, p < 0.001). Blockade of HER2 signaling using Herceptin nearly eliminated EC retraction (p < 0.01), while stimulation of HER2 using heregulin-beta1-augmented EC retraction (p < 0.05). Further, there was no difference between cell lines in either the number of cells adhered or the strength of adherence to EC under shear stress. These data suggest that HER2 signaling enhances metastasis in breast cancer cells by inducing EC retraction, a process that appears to precede endothelial transmigration.     

HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion

The cancer stem cell hypothesis proposes that cancers arise in stem/progenitor cells through disregulation of self-renewal pathways generating tumors, which are driven by a component of 'tumor-initiating cells' retaining stem cell properties. The HER2 gene is amplified in 20-30% of human breast cancers and has been implicated in mammary tumorigenesis as well as in mediating aggressive tumor growth and metastasis. We demonstrate that HER2 overexpression drives mammary carcinogenesis, tumor growth and invasion through its effects on normal and malignant mammary stem cells. HER2 overexpression in normal mammary epithelial cells (NMEC) increases the proportion of stem/progenitor cells as demonstrated by in vitro mammosphere assays and the expression of stem cell marker aldehyde dehydrogenase (ALDH) as well as by generation of hyperplastic lesions in humanized fat pads of NOD (nucleotide-binding oligomerization domain)/SCID (severe combined immunodeficient) mice. Overexpression of HER2 in a series of breast carcinoma cell lines increases the ALDH-expressing 'cancer stem cell' population which displays increased expression of stem cell regulatory genes, increased invasion in vitro and increased tumorigenesis in NOD/SCID mice. The effects of HER2 overexpression on breast cancer stem cells are blocked by trastuzumab in sensitive, but not resistant, cell lines, an effect mediated by the PI3-kinase/Akt pathway. These studies provide support for the cancer stem cell hypothesis by suggesting that the effects of HER2 amplification on carcinogenesis, tumorigenesis and invasion may be due to its effects on normal and malignant mammary stem/progenitor cells. Furthermore, the clinical efficacy of trastuzumab may relate to its ability to target the cancer stem cell population in HER2-amplified tumors.     

Simultaneous detection of expression and gene mutations of HER2/neu in Chinese patients with gastric cancer

HER2/neu is one of the few identified oncogenes in tumorigenesis. Attention has been focused on the potential effect of HER2/neu mutations in the tyrosine kinase domain on carcinoma-targeted therapy. However, data concerning HER2/neu mutations in Chinese patients with gastric cancer (GC) are limited. This study aimed to detect the expression and somatic mutations of HER2/neu in Chinese patients with GC. Immunohistochemical staining for HER2/neu was performed on 72 formalin-fixed, paraffin-embedded specimens of GC (40 intestinal and 32 diffuse type). The correlation between the overexpression of HER2/neu and clinicopathological parameters was statistically analyzed. Somatic mutations in the tyrosine kinase domain of HER2/neu in the 72 patients were detected by direct sequencing. In the GC group, overexpression of HER2/neu was detected in 13 of the 72 GC patients and in 4 of the 72 adjacent tissues in the non-tumorous group (18.1 vs. 5.6%, P<0.05). Furthermore, the intestinal type of GC exhibited a higher rate of HER2/neu overexpression than the diffuse type (29.7 vs. 5.7%, P<0.05). The rate of HER2/neu overexpression in stage III-IV (TNM stage) GC cases was significantly higher than that in stage I-II (28.2 vs. 6.6%, P<0.05). HER2/neu overexpression correlated with a significantly less favorable patient survival (P=0.046). No somatic mutations in the tyrosine kinase domain of HER2/neu were detected in tumor tissues or the corresponding non-tumorous ones in the specimens obtained from the 72 Chinese GC patients. Results suggest that overexpression of HER2/neu is a frequent molecular event strongly associated with a poor patient prognosis, whereas the incidence of somatic mutations of the HER2/neu kinase domain is more likely a low-frequency event in Chinese GC patients.     

Breast tumor progression induced by loss of BTG2 expression is inhibited by targeted therapy with the ErbB/HER inhibitor lapatinib

The B-cell translocation gene-2 (BTG2), a p53-inducible gene, is suppressed in mammary epithelial cells during gestation and lactation. In human breast cancer, decreased BTG2 expression correlates with high tumor grade and size, p53 status, blood and lymph vessel invasion, local and metastatic recurrence and decrease in overall survival, suggesting that suppression of BTG2 has a critical role in disease progression. To analyze the role of BTG2 in breast cancer progression, BTG2 expression was knocked down in mammary epithelial cells. Suppression of BTG2 enhances the motility of cells in vitro and tumor growth and metastasis in vivo. The effects of BTG2 knockdown are mediated through stabilization of the human epidermal growth factor receptor (HER) ligands neuregulin and epiregulin and activation of the HER2 and HER3 receptors, leading to elevated AKT phosphorylation. Suppression of HER activation using the tyrosine kinase inhibitor lapatinib abrogates the effects of BTG2 knockdown, including the increased cell migration observed in vitro and the enhancement of tumorigenesis and metastasis in vivo. These results link BTG2-dependent effects on tumor progression to ErbB receptor signaling, and raise the possibility that targeted inhibition of this pathway may be relevant in the treatment of breast cancers that have reduced BTG2 expression.     

DNA vaccination against neu reduces breast cancer incidence and metastasis in mice

The gene for HER2/neu is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-gamma indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors.     

Preclinical studies of gemcitabine and trastuzumab in breast and lung cancer cell lines

Overexpression of the HER2/neu oncogene and receptor protein has been reported in 20%-30% of patients with breast cancer and is associated with a poor prognosis. HER2/neu expression in breast cancer patients assessed by fluorescence in situ hybridization or immunohistochemistry is a predictor for response to trastuzumab, a humanized monoclonal antibody against the HER2/neu cell-surface protein. Data regarding HER2/neu expression in lung cancer are more limited, and there is little information regarding HER2/neu expression and response to trastuzumab alone or in combination with chemotherapeutic agents. Gemcitabine is an active agent against non-small-cell lung cancer (NSCLC) and has demonstrated activity in breast cancer as well. In vitro modified tetrazolium salt growth assays were performed to determine whether the combination of trastuzumab/gemcitabine produced synergistic or additive effects on breast and lung cancer cell lines. The effects of trastuzumab alone, gemcitabine alone, and the trastuzumab/gemcitabine combination was evaluated on 4 NSCLC cell lines, 1 small-cell lung cancer (SCLC) cell line, and 2 breast cancer cell lines. HER2/neu surface protein expression was assessed by fluorescence flow cytometry and immunohistochemistry. Fluorescence in situ hybridization analysis was used to study gene expression. Trastuzumab treatment alone resulted in growth inhibition in all cell lines expressing HER2/neu and the inhibitive effect correlated with the level of cell surface HER2/neu protein expression. Treatment with gemcitabine alone resulted in growth inhibition in both breast and NSCLC cell lines. A synergistic growth inhibition effect was seen with the trastuzumab/ gemcitabine combination as indicated by combination index values < 1. The degree of synergy observed did not directly correlate with the level of surface protein expression, as synergy was seen even in cancer cell lines expressing low levels of HER2/neu. No treatment effect was seen in the SCLC cell line, which did not express HER2/neu. These preclinical studies indicate a need to study the clinical synergistic effects of the gemcitabine/trastuzumab combination in breast cancer and NSCLC patients whose tumors overexpress HER2/ neu.     

HER2/neu antisense targeting of human breast carcinoma

Overexpression of the HER2/neu oncogene is observed in approximately 30% of human breast carcinoma specimens. HER2/neu overexpression is a negative prognostic factor in breast cancer patients. Cancer cells that overexpress HER2/neu may also be less sensitive to chemotherapy. In order to further define mechanisms by which HER2/neu overexpression drives neoplastic cell growth and chemoresistance, antisense oligonucleotides (ODNs) have been utilized to selectively down-regulate HER2/neu expression in human breast cancer cells. Such antisense ODNs suppress HER2/neu mRNA and protein levels in a dose-dependent, sequence-specific manner. Down-regulation of HER2/neu expression in HER2/neu overexpressing breast cancer cells inhibits cell cycle progression in G0/G1 and results in apoptotic cell death. In tissue culture studies, combined treatment of HER2/ neu overexpressing breast cancer cells with HER2/neu antisense ODNs and conventional chemotherapeutic agents results in synergistic inhibition of cancer cell growth and activation of apoptotic cell death mechanisms. These studies have been extended to demonstrate synergistic antitumor effects following systemic treatment with antisense ODNs plus doxorubicin in nude mice bearing human breast carcinoma xenografts. Collectively these findings demonstrate that HER2/neu overexpression stimulates anti-apoptotic cell survival mechanisms and suggest that HER2/neu antisense ODNs may be of use in cancer therapeutics.     

Downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells

Receptor tyrosine kinase activity is essential for erbB2 (HER2/neu) promotion of breast carcinogenesis, metastasis and therapeutic resistance. erbB2 kinase can be activated by dimerization with another erbB receptor, most of which bind ligands. Of these, the erbB2/erbB3 heterodimer is the most potent oncogenic complex. erbB2 reportedly requires erbB3 to promote cellular proliferation, although this may occur without changes in erbB2 tyrosine kinase activity in some model systems. Our investigations focus on the role(s) of erbB3 in erbB2-associated kinase activity and tamoxifen resistance. Using tumor-derived cell lines from wild type rat c-neu transgenic mice and human breast cancers, we demonstrate that erbB3 plays a critical role in the activation of erbB2 tyrosine kinase activity and erbB2-associated tumorigenesis. Mechanistically, downregulation of erbB3 by specific siRNA reduces erbB2 tyrosine phosphorylation, decreases the PI-3K/Akt signaling, and inhibits mammary/breast cancer cell proliferation and colony formation. Specific erbB3 siRNA sensitizes erbB2 transfected MCF-7 cells (MCF-7/erbB2) to tamoxifen-associated inhibition of both cell growth and colony formation and enhances tamoxifen-induced apoptosis, in contrast to control siRNA transfected MCF-7/erbB2 cells which are tamoxifen-resistant. Our data indicates that erbB2/erbB3 heterodimerization is a prerequisite for erbB2 tyrosine kinase activation in mammary/breast cancer cells and that downregulation of erbB3 inhibits erbB2-associated procarcinogenic activity via inactivation of the PI-3K/Akt pathway. Furthermore, erbB3 also contributes to erbB2-mediated tamoxifen resistance and therefore may be a clinically relevant therapeutic target in addition to erbB2.