A major component of the complex metastatic process involves the generation of new blood vessels within the tumor through angiogenesis, where VEGF plays a key role (Ferrara, 1999
). We have demonstrated that the targeted overexpression of VEGF in the context of Myc overexpression drastically alters the biologic behavior of mammary tumors leading to a high rate of lung metastases. As predicted, Myc/VEGF tumors are highly angiogenic, with increased vascularity and expression of angiogenic markers (VEGF, Flk-1, Flt-1, CD-31 and VE-cadherin).
Earlier studies from our group and others have demonstrated that VEGF may also act as an autocrine growth factor for breast tumor cells (Huh et al., 2005
; Schoeffner et al., 2005
). In this regard, the observation that Myc/VEGF compound transgenic tumors had an increased latency and decreased incidence, compared to tumors developed solely by targeted overexpression of Myc, was unexpected. Although the mechanism for this paradoxical effect remains unclear, the reduced tumor incidence and increased latency appear to correlate with a reduction in Myc levels in the Myc/VEGF tumors compared to Myc tumors. As both the transgenes operate off the MMTV promoter, it is possible that the transgenes compete for the same transcription factors, thus reducing the level of Myc transcription. Alternatively, it may be possible that VEGF overexpression inhibits transgene transcription or alters steady-state levels of Myc, or indeed that VEGF overexpression renders the mammary epithelial cells to be more refractory to the tumorigenic effects of Myc overexpression.
Presumably many molecular alterations in the primary tumors identified through our gene expression profiling studies occur through both direct and indirect effects of VEGF and are potentially involved in the increased metastatic phenotype observed in Myc/VEGF tumors. The molecular characterization of this Myc/VEGF model of metastases further credentials it as a relevant system in which to understand metastatic progression. Many differences in gene expression of ECM-related genes, such as collagens, proteases and adhesion proteins (fibronectin, laminins, integrins and tenascin-C) were observed between Myc/VEGF and Myc. Deregulation of ECM-related genes has also been reported as a key feature of metastatic signatures in several other microarray studies (Ramaswamy et al., 2003
; Eckhardt et al., 2005
; Minn et al., 2005
; Gupta et al., 2007
). Interestingly, it has been shown that VEGF requires interactions with ECM components to exert a proliferative effect on endothelial cells (Miralem et al., 2001
The Myc/VEGF tumors exhibited a fivefold increase in type 1 collagen α-1
) which was also found to be one of the 17 signature genes in a microarray meta-analysis predictive of metastasis (Ramaswamy et al., 2003
was also identified as a gene associated with high metastatic potential in MMTV-PyMT mammary mouse tumors (Qiu et al., 2004
). Laminins are a family of ECM proteins that are involved in adhesion and migration of a variety of cells (Miyazaki, 2006
). Of the many laminin isoforms, laminin-5 (α3β3γ2), upregulated in the Myc/VEGF mammary tumors, is important for the acquisition of tumor cell invasive properties (Giannelli and Antonaci, 2000
; Yamamoto et al., 2001
In order to utilize the array expression data generated from the mouse metastasis model as a filter to identify genes whose deregulated expression is also altered in metastatic human BC, we compared the dataseis from this study with a lung metastasis gene signature for human BC (Minn et al., 2005
). Five genes were similarly deregulated in the Myc/VEGF mouse tumor metastatic signature and in the lung metastasis signature from primary human BC: TNC, MMP-2, collagen-6-A1, mannosidase-α-1A
. Previous reports demonstrated that TNC modulates tumor and endothelial cell migration (Zagzag et al., 2002
; Chiquet-Ehrismann and Chiquet, 2003
), and is expressed in metastatic BC (Chiquet-Ehrismann and Chiquet, 2003
). Moreover, TNC upregulates MMP-9 cooperatively with transforming growth factor-β (TGF-β), in mammary cancer cells (Kalembeyi et al., 2003
). Therefore, we performed functional assays to determine the role of TNC in tumor growth and metastasis. We demonstrate that blockade of TNC strongly impairs cell migration, anchorage-independent cell proliferation and tumor growth. In addition, down-regulation of TNC causes a significant reduction in the ability of cancer cells to disseminate and grow in the lungs.
Knock down of TNC does not modify cell proliferation rates of attached cells in culture, but does decrease the clonogenic potential of cells cultured under anchorage-independent conditions in soft agar, and inhibits tumor cell proliferation in vivo. This suggests that the effects of TNC on proliferation require cellular interaction with the ECM. Our data indicate that reduced angiogenesis is not responsible for the decreased tumor size in shTNC tumors, as CD-31 levels were similar between shTNC tumors and control tumors. In addition, tumor recurrence following surgical removal is significantly more rapid in mice whose original tumors expressed high levels of TNC compared to tumors with low TNC expression. This may be due to a higher proliferative rate of remaining tumor cells following resection, or that cells expressing TNC are more invasive and less amenable to resection.
Importantly, we observed that the number of lung metastases correlates to the level of TNC expression in MDA-MB-435 and derivative cells. A significant (37.6%) decrease in the number of metastatic nodules was found in the lungs of mice injected with shTNC expressing cells compared to mice injected with the control cells. This is in contrast to a previous study in which loss of TNC expression in MMTV-PyMT mice did not decrease either the primary tumor growth or the rate of lung metastasis. It is likely that PyMT activates other metastatic pathways not dependent upon TNC (Talts et al., 1999
). Indeed, our previous microarray analysis of MMTV-PyMT primary tumors failed to find TNC as one of the metastatic signature genes in this model (Qiu et al., 2004
), suggesting that mechanisms of metastatic progression may depend upon earlier oncogenic events and the concerted effects of multiple genes and epigenetic phenomena. Thus, TNC may significantly contribute to metastatic progression in certain contexts. Therefore, an approach targeting several genes involved in metastasis may be necessary to reduce metastatic BC.