The role of the EGFR signaling in tumor microenvironment.

The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB-2, ErbB-3, and ErbB-4) that are activated following binding to epidermal growth factor (EGF)-like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non-malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB-2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti-EGFR or anti-ErbB-2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor-associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth.

AZD3409 inhibits the growth of breast cancer cells with intrinsic resistance to the EGFR tyrosine kinase inhibitor gefitinib.

AKT and MAPK signaling are involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. RAS proteins are upstream mediators that transfer messages from surface receptors to intracellular signal transducers including MAPK and AKT pathways. AZD3409 is a novel prenyl inhibitor that has shown activity against both farnesyl transferase and geranylgeranyl transferase in isolated enzyme studies. We explored the activity of AZD3409 on breast cancer cell lines with high (SK-Br-3), intermediate (MDA-MB-361) or low (MDA-MB-468) sensitivity to gefitinib. We found that AZD3409 inhibits the growth of breast cancer cells in a dose-dependent manner, with the MDA-MB-468 and MDA-MB-361 cell lines showing higher sensitivity as compared with SK-Br-3 cells. Treatment with AZD3409 produced a significant reduction in the levels of activation of AKT in the three cell lines. AZD3409 also induced an increase in the expression of p27kip-1 and of hypophosphorylated forms of pRb2 in MDA-MB-468 cells that was associated with accumulation of cells in G0/G1 and the appearance of a sub-G1 peak suggestive of apoptosis. In contrast, AZD3409 produced a G2 arrest associated with reduced expression of pRb2 in MDA-MB-361 cells. A synergistic anti-tumor effect was observed when MDA-MB-468 or MDA-MB-361 cells were treated with both AZD3409 and gefitinib, whereas this combination was only additive in SK-Br-3 cells. However, treatment of breast cancer cells with AZD3409 and gefitinib did not produce a more significant blockade of AKT signaling as compared with gefitinib alone. These data suggest that AZD3409 might be active in gefitinib-resistant breast carcinoma.

The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib.

We investigated the role of the MEK/MAPK pathway in the sensitivity/resistance of breast carcinoma cells to the EGFR tyrosine kinase inhibitor gefitinib (IRESSA). We assessed the effects of gefitinib on the growth of three breast cancer cell lines that showed high (SK-Br-3; IC50 4 microM), intermediate (MDA-MB-361; IC50 5.3 microM), and low (MDA-MB-468; IC50 6.8 microM) sensitivity to the drug. Although treatment with gefitinib inhibited EGFR activation in the three cell lines in a similar fashion, significant reduction of both p42/p44-MAPK and AKT phosphorylation was observed in SK-Br-3 and MDA-MB-361, but not in MDA-MB-468 cells. The growth of MDA-MB-468 cells was significantly inhibited by treatment with either the PI3K-inhibitor LY294002 or the MEK-inhibitor PD98059. In agreement with these findings, treatment of MDA-MB-468 cells with a combination of PD98059 and gefitinib produced a synergistic anti-tumor effect, whereas this combination was only additive in SK-Br-3 and MDA-MB-361 cells. The combination of gefitinib and PD98059 also produced a significant increase in the levels of apoptosis in MDA-MB-468 cells as compared with treatment with a single agent. This phenomenon was associated with a profound decrease in MAPK activation, reduction of BAD (ser112) phosphorylation and a paradoxical increase in the levels of AKT activation. Finally, overexpression of a constitutively activated form of p42-MAPK in MCF-10A non-transformed human mammary epithelial cells resulted in a two- to three-fold increase in the IC50 to gefitinib. Taken together, these data strongly support the role of the MEK/MAPK pathway in the resistance to gefitinib, and provide the rationale for novel therapeutic approaches based on combinations of signal transduction inhibitors.

Epidermal growth factor receptor (EGFR) signaling in cancer.

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.