Allelic loss of BECN1
occurs with high frequency in human breast, ovarian and prostate cancers.29–32
Enhanced tumorigenesis in Becn1+/−
mice has been attributed to genomic instability resulting from increased metabolic stress and impaired autophagy.33
The mechanism by which Becn1+/−
mice exhibit increased tumorigenesis is not known.29
mice and mLECs derived from these mice, we demonstrate that Beclin 1 plays a crucial role in regulating tumorigenesis by suppressing angiogenesis. We observed that hypoxia-induced tumor growth and neovascularization were upregulated in Becn1+/−
mice compared to Becn1+/+
mice. Beclin 1 may regulate angiogenic responses by several distinct mechanisms. Although our data demonstrate that Beclin 1 deficiency in mice results in the enhanced production of the circulating factor EPO in vivo, they also implicate a regulatory role for Beclin 1 in the angiogenic response of endothelial cells. Our results show that Becn1+/−
mLECs displayed increased angiogenic activity under hypoxia, including capillary formation ex vivo, proliferation and endothelial cell migration. Recent studies have reported that endogenous anti-angiogenic molecules such as endostatin and angiostatin, induce autophagy in endothelial cells by modulating Beclin 1 and β-catenin levels.34,35
These studies were suggestive of an association of Beclin 1 with the regulation of angiogenesis. Autophagy as a response to hypoxia has also been recently implicated in the regression of hyaloid vessels in early developing retina.36
Here, we conclude that Beclin 1 is associated with the suppression of angiogenesis. However, it remains unclear whether active autophagy is required for the anti-angiogenic effects of Beclin 1, or whether the results reflect a regulatory signaling function of Beclin 1 that is independent of autophagy. The Becn1+/−
mice displayed reduced autophagosome formation in the lung during hypoxia exposure relative to wild-type mice. Interestingly, the Becn1+/−
mice also showed reductions in the expression and activation of autophagic protein LC3B in both normoxic and hypoxic conditions. These observations, taken together, suggest that the Becn1+/−
mice are at least partially compromised in autophagic function. Additional investigation to examine the relationship between autophagic flux and the regulation of angiogenesis may shed further light on this question.
Previous studies have shown that autophagy defects may increase cellular generation of ROS. For example, embryonic fibroblasts derived from Atg5
-deleted mice have enhanced mitochondrial ROS generation in the context of viral infection.23
Recently we have shown that macrophages derived from Becn1+/−
mice exhibit an enhanced production of mitochondrial ROS under basal conditions.24
Furthermore, previous studies have shown that autophagy defects can promote activation of transcription factors (i.e., Nrf-2) and pro-inflammatory regulators such as NFκB.25,26
In agreement with these observations, we also find that Becn1+/−
are primed for pro-inflammatory and stress-related responses, as suggested by elevations in the expressions of Nrf-2 and phospho-p65 (NFκB). Nrf-2 regulated genes (e.g., heme oxygenase-1) as well as NFκB-regulated cytokines can potentially impact angiogenic processes.37–39
Thus, the stress-primed phenotype of Becn1+/−
mice may contribute considerably to the pro-angiogenic balance.
In our study, the differential angiogenic response in Becn1+/−
mLEC was also associated with a switch from a HIF-1α to HIF-2α dominant phenotype in response to hypoxia. The mechanism for this alteration of the balance of HIF isoforms expression remains unclear. The stabilization of HIF-1α is known to be regulated by reactive oxygen species (ROS) tone.40
In light of these observations, the changes in HIF isoform expression evident in Becn1+/−
mLEC during hypoxic stimulation may be related to differential ROS production and/or pro-inflammatory signaling in endothelial cells and this warrants further investigation.
Although HIF-1α and HIF-2α both heterodimerize with HIF-1β, and recognize the same core element NCGTG in the promoter regions of target genes, these isoforms may regulate distinct transcriptional targets. The factors that govern the specificity and transcriptional activity of these factors with respect to the regulation of overlapping and non-overlapping gene targets, and the tissue-specificity of these responses remains incompletely clear.27
HIF-2α is highly expressed in embryonic vascular endothelial cells and activates the expression of target genes whose products modulate vascular function and angiogenesis.41,42
HIF-2α expression has been implicated in the growth and neovascularization of human tumors, including breast and bladder tumors.43,44
Mice with targeted endothelial cell-specific deficiency in HIF-2α developed normally, but displayed increased vessel permeability and aberrant endothelial cell ultrastructure.45,46
Our results indicate specificity for HIF-2α in the systemic regulation of the pro-angiogenic factor EPO. The Becn1+/−
mice displayed increased levels of circulating EPO relative to wild-type mice after hypoxic exposure, whereas the levels of VEGF, another major angiogenic factor remained unchanged. In the liver, EPO production is known to be preferentially regulated by HIF-2α.47,48
We cannot exclude the possibility that other proangiogenic factors may contribute to the phenotypic observations. Further studies using gene expression or cytokine profiling techniques may uncover additional pro-angiogenic factors that are specifically upregulated in Becn1+/−
Interestingly, HIFs have also been shown to regulate the autophagic pathway. HIF-1α promotes hypoxia-inducible autophagy through activation of the BNIP3 pathway.15
In contrast, HIF-2α downregulates autophagy in human and murine cartilage.49
Our studies suggest that Beclin 1 status is associated with the regulation of angiogenesis, which is demonstrated here in the presence of hypoxia. However, it should be noted that the tumor growth phenotype of Becn1+/− mice also contains a significant normoxic component as shown in , which is not evident in the in vitro assays of angiogenesis and cell proliferation as described in this paper. This normoxic component of tumor growth in Becn1+/− mice may be attributed to regulatory effects of Beclin 1 on tumor cell growth that are independent of vascular effects. On the other hand, the regulatory effects of Beclin 1 on tumor growth during hypoxia stimulation appear to involve elevated stress responses and differential regulation of HIF isoforms in host tissues with consequences on the production of circulating angiogenic factors and the regulation of angiogenesis.
In conclusion, the results of our study are the first to show an association between the autophagic protein Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development. We show that Becn1+/− vascular cells have elevated indices of stress and pro-inflammatory responses, which could account for in part, the pro-angiogenic phenotype of the corresponding mice. Among these changes, Becn1+/− display alterations in HIF-dependent signaling involving differential expression of the α-subunits of HIF. The mechanism(s) by which the expression of HIF-2α is altered in Becn1+/− mice remain unclear, but may involve the regulation of protein stability. Further experiments are warranted to determine whether Beclin 1 protein can directly regulate the DNA binding activity of functional HIF-1α/HIF-1β or HIF-2α/HIF-1β heterodimers. The resolution of Beclin 1-dependent anti-angiogenic mechanisms may lead to new treatments for vascular diseases and cancer.