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Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non-small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed-forward loop in NSCLC cells in which tumor-derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration, and secretion of other angiogenic factors in A549, H1975, and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin-2 (ANGPT2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM-1), and platelet-derived growth factor bb (PDGF-BB). PI3K/AKT, RAS/ERK, and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post-transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention.

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.

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.

A range of target-based agents for the treatment of solid tumors are in development. The epidermal growth factor receptor (EGFR) has been identified as a relevant target as it is involved in regulating several cellular functions important in the proliferation and survival of cancer cells, is commonly expressed at high levels in a range of tumors, and high expression is often related to poor prognosis. EGFR is a member of the ErbB family of receptors which also includes ErbB-2, ErbB-3, and ErbB-4. These receptors form dimers of the same type (homodimers) or with other family members (heterodimers), each combination resulting in different downstream effects. Some of the most advanced targeted agents in development are the EGFR tyrosine kinase inhibitors (EGFR-TKIs), of which ZD1839 ('Iressa') is an example. In Phase II monotherapy trials, oral ZD1839 was well tolerated and demonstrated clinically meaningful antitumor activity and symptom relief in pretreated patients with advanced NSCLC. Preclinical studies have suggested that the antitumor activity of ZD1839 does not depend on the level of EGFR expression. Furthermore, in addition to an effect on EGFR signaling, treatment with ZD1839 as well as with other quinazoline EGFR-TKIs, may also affect signaling of other ErbB family members. EGFR-TKIs have been shown in preclinical studies to increase the efficacy of cytotoxic drugs and Phase III trials of such combinations are ongoing. On the basis that different signal transduction pathways contribute to the control of tumor growth, future therapeutic approaches are likely to involve combination of different targeted agents.

Human Cripto-1 (CR-1), a member of the epidermal growth factor-CFC (EGF-CFC) family of peptides, is expressed in the developing mouse mammary gland and can modulate mammary epithelial cell migration, branching morphogenesis and milk protein expression in vitro. In order to screen for a CR-1 receptor and to identify potential CR-1 target tissues, we constructed a fusion protein comprising the EGF-like domain of CR-1 and the Fc domain of a human IgG1. The recombinant CR-1 fusion protein (CR-1-Fc) was biologically active as it was able to activate the ras/raf/mitogen activated protein kinase (MAPK) pathway and to inhibit transcription of the milk protein beta-casein in NMuMG and HC-11 mouse mammary epithelial cells. By using immunocytochemistry and by an in situ enzyme-linked immunosorbent assay (ELISA), CR-1-Fc was found to specifically bind to NMuMG and HC-11 cells. Finally, immunohistochemical analysis using CR-1-Fc showed a specific localization of CR-1 binding to tissue sections from mouse mammary gland. In particular, more than 60% of the epithelial cells were intensely stained with the CR-1-Fc fusion protein in the lactating mouse mammary gland, whereas approximately 25% of the mammary epithelial cells were stained in the gland from pregnant mouse. Since expression of mouse cripto-1 (Cr-1) in the pregnant and lactating mouse mammary gland as well as its presence in milk has been previously demonstrated, these data strongly suggest that an autocrine pathway involving Cr-1 and its putative receptor is operating in the mouse mammary gland during pregnancy and lactation.