In this study, we found that several breast cancer cell lines expressed VEGF receptors. Furthermore, we showed that reduced endogenous VEGF or VEGFR1 expression induced the apoptosis of MDA-MB-231 and MCF-7 breast cancer cells, whereas externally acting proteins (VEGF, VEGF antibody, soluble VEGFR1, and VEGFR1-blocking antibody) had no significant effects on breast cancer cell growth or survival. We also observed that internally expressed VEGFR1 induced the survival of breast cancer cells. These results indicate that VEGF plays a role as an internal autocrine survival factor in breast cancer cells via VEGFR1 and not through VEGFR2 or NRP1, and suggest that the role of the VEGF/VEGFR1 axis in breast cancer cells may not be governed by the classical paradigm of signal transduction involving interaction between ligands and cell surface receptors.
VEGF contributes to the growth of solid tumors, such as breast cancer, through angiogenesis rather than through a direct contribution to tumor cell survival. This effect seems to be derived partly from the poor response of tumor cells to externally acting molecules such as VEGF or antibodies to VEGF. However, this concept has now been challenged by recent reports showing that some cancer cells express VEGF receptors on their surfaces in vitro and in vivo, and that in several tumor cell types, including leukemia and melanoma cells, VEGF acts as an autocrine survival factor [22
]. Although breast cancer cell lines express both VEGF and the VEGF receptors VEGFR1, VEGFR2, and NRP1 [11
], the expression of these receptors in primary breast tumors is controversial [12
]. Dales et al [12
] reported that VEGFR1 and VEGFR2 were strongly expressed in endothelial cells within blood microvessels, and weakly expressed in tumor cells in a series of 918 patients. Univariate analysis showed that the expression of VEGFR1 in tumor cells was not correlated with survival, but was significantly correlated with high metastasis risk [12
]. In another study involving 905 cases of breast cancer tumors [29
], the expression of VEGFR1 was correlated with a high risk of local recurrence. On the other hand, VEGFR1 expression was rarely observed in 205 cases of ductal carcinoma in situ of the breast [13
]. Thus, there are conflicting reports regarding the expression and role of VEGFR1 in breast cancer. No correlation between survival and metastasis risk and relapse was found in breast cancer cells that expressed VEGFR2 [12
]. Therefore, the role of VEGF as an angiogenic factor and/or as a survival factor in tumor cells needs to be fully investigated.
Our siRNA studies on VEGF receptors revealed that VEGF can modulate the survival of breast cancer cells via VEGFR1, but not VEGFR2. Here, we show that VEGFR1 mediated AKT phosphorylation, a major survival pathway in breast cancer cells. It is known that VEGF mediates most angiogenic processes by interacting with VEGFR2 in endothelial cells. VEGF can also modulate VEGFR2 expression as well as its phosphorylation in endothelial cells [30
]. Thus, down-regulation of VEGF expression may induce apoptosis in the MDA-MB-231 clones AS-C1 and AS-C2 through the decreased VEGFR2 expression in these cells. However, our studies with siVEGFR2 demonstrated that VEGFR2 is not important for the survival of breast cancer cells. Although VEGFR2 has strong tyrosine kinase activity compared to VEGFR1 in endothelial cells, the level of VEGFR2 expression in MDA-MB-231 and MCF-7 breast cancer cells might not be sufficient to induce VEGF-mediated signaling in these cells. In fact, we almost could not detect VEGFR2 protein expression by immunoprecipitation, nor could we detect any expression of this protein by immunocytochemical analysis (A and B).
VEGFR2 tyrosine phosphorylation was reported to be enhanced by VEGF in breast cancer and to lead to increased ERK1/2 (extracellular signal-regulated kinase 1/2) and AKT phosphorylation, suggesting that VEGF stimulation is important in the regulation of cell growth, apoptosis, and differentiation [31
]. VEGFR2 was also shown to be expressed on the surface of breast cancer cells [31
]. However, these results differ from our data since the level of expression of VEGFR2 was very low in our cell system, and knockdown of VEGFR2 by siRNA treatment had no effect on the apoptosis of breast cancer cells. Additionally, we examined the effect of NRP1 on the survival of MDA-MB-231 cells, as it was previously reported that VEGF mediates the autocrine survival of these cells through NRP1 [14
]; we failed to show that NRP1 mediated VEGF-induced survival in MDA-MB-231 cells. NRP1 is known to interact with VEGFR1 but has a very short cytoplasmic domain [32
], suggesting that NRP1 may not directly transduce VEGF-mediated signaling in these cells. Although we can not fully explain these discrepancies, based on our results it is unlikely that VEGFR2 or NRP1 has a direct functional effect on VEGF-mediated survival in MDA-MB-231 cells. Knockdown of endogenous VEGF expression or addition of VEGFR synthetic inhibitors resulted in the apoptosis of hematopoietic stem cells [33
], whereas VEGF or soluble VEGFR1 did not have any effect on the survival of these cells. Therefore, the survival mechanism of VEGF in breast cancer cells might be different from that of VEGF in endothelial cells, and may mimic the VEGF-dependent survival system in hematopoietic stem cells [33
VEGFR1 was originally thought to be a receptor specifically expressed in vascular endothelial cells. However, some studies have shown that VEGFR1 is expressed in tumor cells and is involved in tumor growth and progression [5
]. VEGF has also been shown to increase the invasiveness of colorectal cancer cells or myeloma cells [15
], and VEGFR1-blocking antibody was reported to inhibit the VEGF-induced invasiveness of these cells [15
]. These results indicate that VEGF can act as an autocrine or paracrine factor in the invasiveness of both cell types via VEGFR1. These previously published studies are different from our present data, which show that neither exogenous VEGF nor VEGFR1-blocking antibody has any significant effects on the invasiveness or survival of breast cancer cells. We observed that VEGFR1 was expressed internally, but not on the surface of breast cancer cells. The localization of VEGFR1 expression in breast cancer cells is a novel finding that may support our observations that VEGF plays a role as an internal autocrine survival factor in these cells. It remains to be elucidated how VEGFR1 is expressed internally in breast cancer cells, and how it mediates VEGF-induced survival in these cells.
The results of our studies differ from those of studies by Vincent et al. [16
] showing that VEGF facilitated the growth and survival of human primary multiple myeloma cells via VEGFR1. In that study, VEGFR1 was shown to be present in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Vincent et al. [16
] reported that a monoclonal antibody against VEGFR1 inhibited the proliferation and migration of primary multiple myeloma cells via plasma membrane retention of VEGFR1 following the prevention of its nuclear translocation. In our study, however, externally acting growth factors, such as VEGF and VEGFR1-blocking antibody, had no effects on breast cancer cell growth or survival. Furthermore, VEGFR1 receptors were mostly expressed internally, and not on the surface of breast cancer cells, which may provide a mechanism for the lack of inhibition of breast cancer cell growth by antibody to VEGFR1. We also observed localization of VEGFR1 mainly in the nuclear envelope in breast cancer cell lines, primary breast cancer tumors, and normal mammary glands. These results suggest that the expression pattern, localization, and function of VEGFR1 may differ between breast epithelial cells and endothelial cells.
Although VEGF has a signal peptide that induces its direct secretion through the classical secretory pathway, VEGF can also translocate to the nucleus [35
]. Moreover, VEGF contains basic amino acids that could act as potential nuclear localization signals [36
]. Recent studies showed that VEGF protein colocalizes with the RNA-binding protein HuR in discrete nuclear compartments and that nuclear VEGF protein is increased in hypoxia [36
]. These results indicate that VEGF may have a nuclear function, especially during hypoxia. Furthermore, VEGF nuclear accumulation is correlated with phenotypic changes in endothelial cells [30
]. VEGF is internalized via the classical receptor-mediated endocytosis pathway and accumulates in the endosomal compartment, whereas in cells situated at the edges of a wound, VEGF is rapidly taken up and translocated to the nucleus [30
]. Therefore, it is possible that VEGF may be retained within the cell for its interaction with perinuclear VEGFR1. Future studies will address the function of VEGFR1 in live breast cancer cells and normal breast epithelial cells, and determine the kinetics, translocation, and binding of VEGFR1 to VEGF in these cells.
The limitation of our study is that the intracrine role of VEGF/VEGFR1 was examined in two breast cancer cell lines and in only a few samples of primary breast tumors. These results may not be fully applicable to all breast cancer samples, representing different stages of the disease. Thus, further studies of breast cancer cells from patients at different clinical stages of the disease will be necessary to define the pathophysiologic role of the VEGF/VEGFR1 axis in intracrine signaling. In addition, although we were able to examine the functional blocking of VEGFR1 using antibodies that were readily accessible to our laboratory, other VEGFR1 antibodies (that are currently unavailable to us) need to be examined for their effects on breast cancer cell growth and proliferation in vivo in this system, once these antibodies become available.
Recently it was reported that VEGFR1 expression was significantly increased in breast cancer patients with a poor prognosis [37
]. Consistent with that report, our data suggest that VEGFR1 might be an attractive target for therapeutic approaches in patients with malignant breast cancer. Furthermore, our data suggest that intracellularly acting inhibitors against VEGF and its receptor VEGFR1 might be a more effective therapeutic method for breast cancer patients as compared to the externally acting inhibitors. Specifically, the former approach will exert direct tumor-killing effects as well as antiangiogenic effects, while the latter approach will exert indirect tumor-killing effects through antiangiogenic activity alone. Hence, our study may provide an optimal strategy for tumor therapy based on the inhibition of angiogenesis.