APN has been implicated in attenuating mammary tumor progression in studies using in vitro
or xenografts models, partially attributable to a postulated role as a negative regulator of angiogenesis (16
). However, because there are important differences between the mechanism of tumor growth and angio-genesis in transplanted and autochthonous tumors (19
), we have analyzed, for the first time, the role of APN in mammary tumor progression in vivo
using the spontaneously occurring mammary tumor model of MMTV-PyMT. We did this through the use of APN KO mice and by taking advantage of neutralizing monoclonal antibodies that effectively clear APN from circulation. Contrary to the published data (16
), we show that APN can accelerate mammary tumor growth in vivo
at early stages when tumor lesions start to critically depend on angiogenesis for continued oxygen and nutrient supply. Such a role was also implied in an elegant recent article that was published in the course of the preparation of this article (41
). These authors suggested the existence of a molecular cross talk between the putative APN receptor T-cadherin and mammary tumor growth.
Previous in vivo
work found that APN promotes angiogenesis in response to tissue ischemia through the activation of an AMP protein kinase AMPK–dependent signaling pathway (25
), and that APN promotes endothelial cell survival, migration, and differentiation into capillary-like structures (37
). We therefore sought to determine the contribution of APN to tumor-associated angiogenesis. Our experiments suggest that APN supports the release of angiogenic factors such as VEGF-A and VEGF-B and can promote blood vessel formation as judged by an increase in endothelial cell markers such as CD31 and von Willebrand factor in the PyMT mammary tumors relative to PyMT/ APN KO–derived tumors. Our experiments using Matrigel implants suggest potent effects of APN that can lead to extensive reprogramming and induction of blood vessel formation. Importantly, these experiments, together with the data obtained from our microarray analysis on WT animals injected with APN, suggest a direct role for APN in the angiogenic response by paracrine and/or endocrine effects on cell types such as endothelial cells and inflammatory cells, rather than mediating its effects exclusively through action on the cancer cells.
It is generally accepted that obesity is a risk factor for breast cancer in postmenopausal, but not premenopausal women (13
). Because APN levels are inversely correlated with obesity (11
), it has been suggested that the decreased levels of APN may explain the increased risk of breast cancer with obesity (14
). In fact, numerous retrospective case-controls studies and a recent prospective study have shown that APN is inversely associated with breast cancer risk, an association that might be stronger for postmenopausal women (14
). Several observations in the PyMT model are consistent with this inverse association: (a
) a robust decline in circulating APN levels that is observed as the tumor progresses and (b
) an acceleration of mammary tumorigenesis in PyMT/APN KO compared with PyMT in advanced malignant stages of the disease. However, similar to the differential effect of menopausal status in women, a thorough analysis of the PyMT tumor model indicates a complex role for APN, suggesting a possibly biphasic effect of APN on tumor progression or, alternatively, the development of an adaptive mechanism at late stages to bypass the dependence of APN-driven angiogenesis. The switch from a lagging to a more aggressive tumor growth rate may be a reflection of a Darwinian dynamic similar to what is observed under conditions of exposure to angiogenesis inhibitors, during which tumors acquire the ability to survive and proliferate within the suboptimal tumor microenvironment by activating alternative ways to sustain tumor growth and angiogenesis (26
). Similarly, our data imply that following a prolonged antiangiogenic stress due to the absence of APN, an adaptive resistant mechanism takes place. A detailed gene expression analysis of the tumors arising in the PyMT/APN KO mice at late stages reveals easily interpretable changes in critical genes involved in cancer growth, survival, and enhanced glycolysis. In line with data that VEGF-A and SDF-1 can induce recruitment of endothelial progenitor cells (4
), we show an increase in local VEGF-A in late-stage APN KO tumor–bearing mice that potentially play a role in the observed robust mobilization of CEPs. This subpopulation of CEPs has the capacity to enhance tumor growth and has been postulated to be crucial in situations such as relapse after tumor shrinkage brought about by surgery or antiangiogenic therapies (27
). These results raise the possibility that the recruitment of this subpopulation contributes functionally to tumor growth in the PyMT/APN KO animals at late stage, rescuing the cells from their original nutrient-deprived status.
APN critically modulates insulin resistance, atherogenesis, and cardiac remodeling (11
). Our data presented here indicate that it also functions as an angiogenic mediator in mammary tumors. Our work suggests that vessel development is impaired in mammary tumors lacking systemic APN, leading to a nutrient-deprived tumor at the early malignant stages. Yet, alternative pathways are activated at later stages of tumorigenesis to bypass the dependence on APN and allow for further fueling of tumor growth. Compounds that modulate its circulating levels such as the PPARγ agonists thiazolindinediones (34
) may have a profound effect on mammary tumorigenesis through the angiogenesis axis (34
). Interestingly, PPARγ agonists have been shown to induce VEGF-A in adipocytes (47
) and, importantly, may be involved in adipose tissue–associated angiogenesis (48
). PPARγ is most abundantly expressed in adipocytes, yet it is also expressed in endothelial and tumor cells, macrophages, and a number of epithelial tissues including epithelia in the mammary gland (34
). Thus, this class of drugs clearly induces pleiotropic affects in multiple tissues. Taken together, the effects of PPARγ agonists on the enhancement of mammary tumorigenesis, as judged by the analysis of these models, may be mediated in part by the cross talk between APN and the vasculature axis, stimulating tumor growth. In contrast, PPARγ may act as an antimitogenic factor in tumor cells and as an anti-inflammatory factor in macrophages (34
). The net result of PPARγ stimulation on the transformed ductal epithelium of the mammary gland raises the distinct possibility that these agonists may enhance neovascularization of emerging tumors and should be used with caution in a diabetic population with newly diagnosed malignancies of the mammary gland. On the other hand, tumors arising in an environment with low APN (such as in an obese and/or diabetic population) may assume a more aggressive phenotype as suggested by the increased tumor growth at later stages in our APN KO mice.
Given that elevated levels of CEPs were observed in APN KO mice independent of the presence or absence of a tumor (data not shown), the proangiogenic role of APN may not only be relevant under tumor-associated conditions but may also contribute to its potent antidiabetic effects in our previously described mouse models with moderately elevated levels of APN achieved through transgenic overexpression. APN enables adipose tissue pads to massively expand during times of excess caloric intake, allowing a redistribution of excess ectopic lipid deposits. Despite the massive hyperplasia of the fat pads in these mice overexpressing APN, the fat pads remain minimally inflamed and fully insulin sensitive and do not display the classic hallmarks of expansion under suboptimal conditions, such as hypoxia and macrophage infiltration (29
Collectively, our detailed analysis suggests several possible strategies to delay an acquired adaptation to antiangiogenic stress under conditions when APN abundance is low, such as in the context of obesity. This includes treatment regimens targeting CEPs, an approach that has previously been shown to enhance treatment efficacy of cytotoxic chemotherapy (49
). These patients may potentially also show higher sensitivity to chemotherapy given their normalized vasculature (46
). A molecular understanding of the APN-mediated proangiogenic activity combined with the identification of key stimulating factors that are turned on at advanced stages of the disease has the potential to reveal novel therapeutic approaches for breast cancer in blocking both the angiogenic switch and the specific adaptive pathways that are triggered as a result of chronic angiogenic stress and nutrient deprivation.