In this study, we identify IRS-2 as a hypoxia-responsive gene that contributes to breast carcinoma cell survival and invasion in hypoxic environments. Exposure of breast carcinoma cells to hypoxia increases IRS-2 expression, but not IRS-1 expression, at the level of gene transcription. Either HIF-1 or HIF-2 is required for this hypoxia-dependent increase in IRS-2 expression. IRS-2 is phosphorylated on tyrosine residues and recruits PI3K in response to IGF-1 stimulation in hypoxia, indicating that IRS-2 is functionally active to mediate signaling in low oxygen conditions. In this regard, activation of Akt in response to hypoxia is dependent upon IRS-2 expression. Functionally, IRS-2 can protect cells from apoptosis and promote invasion in hypoxic environments. Collectively, our results provide a novel mechanism by which IRS-2 contributes to the aggressive behavior and metastasis of hypoxic tumor cells.
IRS-1 and IRS-2 expression are differentially regulated by hypoxia in breast carcinoma cells, a finding that adds to a growing body of evidence that these homologous adaptor proteins are not functionally redundant. IGF-1-dependent signaling through IRS-1 or IRS-2 in human breast carcinoma cells stimulates proliferation or migration/invasion, respectively (8
). IRS-2, but not IRS-1, has been implicated in metabolic regulation in tumor cells, through the regulation of glycolysis (9
). In vivo
, mammary tumors that lack Irs-2 expression are significantly impaired in their ability to metastasize, and Irs-1 cannot compensate for this function (5
). In fact, in the absence of Irs-1 expression, Irs-2 expression and signaling increase in cell lines in culture and in tumors, and metastasis is enhanced (4
). Taken together, these findings infer that the balance of IRS-1 and IRS-2 expression can significantly impact tumor cell function and progression. Shifting the IRS balance in favor of IRS-2 would promote metabolic independence, invasive ability and survival, factors that contribute to the metastatic potential of a tumor. The fact that hypoxia concurrently suppresses IRS-1 expression while upregulating IRS-2 expression reveals a novel endogenous mechanism by which this balance is altered to favor tumor progression.
The regulation of IRS-2 gene expression by hypoxia implicates IRS-2 in the adaptation of tumor cells to hypoxia and reveals a novel mechanism by which hypoxic cells acquire a more aggressive behavior after exposure to low oxygen conditions. Hypoxia occurs in areas of tumors that are poorly vascularized, which results in decreased oxygen delivery to the tumor cells (16
). Overall, gene expression is suppressed in hypoxia as a mechanism to conserve energy in this stresssful microenvironment, which is often lacking in nutrient availability as well (15
). In general, the genes that are expressed in hypoxic environments are essential for tumor cells to survive in, and ultimately adapt to, low oxygen conditions. For example, genes that regulate anaerobic glycolysis are coordinately expressed in hypoxia to facilitate energy production when oxidative phosphorylation is inhibited by insufficient oxygenation (15
). Genes such as VEGF are also upregulated to increase angiogenesis and restore normoxic conditions (29
). Chronic exposure to hypoxia creates a selection for cells with a tolerance for hypoxia and these cells become more invasive and metastatic (16
). The impact of this selective pressure is highlighted by recent studies revealing that anti-angiogenic therapy alone may provide only short term benefit for many cancer patients because the disruption of blood vessels leads to increased hypoxia, and patients will go on to develop metastatic disease (30
). These studies underscore the importance of understanding how tumor cells maintain their viability in hypoxia. Our current findings that IRS-2 contributes to breast carcinoma cell survival in hypoxia, along with our previous demonstrations that IRS-2 regulates aerobic glycolysis and positively contributes to mammary tumor metastasis, identify this adaptor protein as a key mediator of signals that influence tumor cell responses to hypoxia.
Our data reveal that one mechanism by which IRS-2 contributes to the hypoxic tumor response is by sustaining activation of Akt in hypoxia. In our previous in vivo
studies, Akt signaling was upregulated in PyV-MT:Irs-1-/-
tumors that have enhanced Irs-2 expression and association with PI3K, providing evidence that our in vitro
findings linking IRS-2 with Akt activation are recapitulated in tumors (4
). A number of studies have implicated Akt signaling in positively regulating tumor cell survival in hypoxia and several mechanisms for its action have been proposed (32
). Akt negatively regulates the function of pro-apoptotic downstream effectors including the FOXO transcription factors and the pro-apoptotic protein Bad (33
). IRS-2 regulates FOXO function through Akt in mouse embryo fibroblasts, and this regulatory pathway has been proposed to control nutrient homeostasis (34
). Viability and growth are also influenced by the Akt-dependent regulation of genes that control energy production through the switch from oxidative phosphorylation to anaerobic glycolysis for ATP generation (35
). Akt signaling can also enhance the expression of HIF-1α to amplify the expression of HIF target genes (36
). In this regard, SUM149 cells, which lack PTEN and have elevated Akt activity, exhibited the greatest induction of IRS-2 expression in hypoxia (38
). As mentioned previously, tumors that arise in PTEN+/- mice also have elevated IRS-2 expression (6
). These findings raise the possibility that in tumor cells with PI3K pathway mutations, hypoxia provides a second “positive hit” by upregulating IRS-2 expression to counterbalance negative feedback regulation of IRS-2, and by doing so enhancing downstream PI3K signaling to promote tumor progression.
Hypoxic regulation of IRS-2 expression requires the function of either HIF-1 or HIF-2. The HIFs are major regulators of hypoxia-responsive gene transcription and each factor consists of two subunits, HIF-α and HIF-β/ARNT (39
). HIF-1α and HIF-2α subunits are highly homologous, and both contain basic helix-loop-helix (bHLH), Per/ARNT/Sim (PAS), and oxygen-dependent degradation (ODD) domains (39
). In low oxygen conditions, the HIF-α subunit is stabilized and it interacts with HIF-β to form the active HIF transcription factor, which binds to HRE sequences in target genes (40
). HIF-1 and HIF-2 can regulate both unique and common target genes (41
). Suppression of both HIF-1α and HIF-2α was required to prevent hypoxia-dependent upregulation, indicating that IRS-2 is a common target gene for HIF-1 and HIF-2. However, given the lack of a canonical HRE in the hypoxia responsive region of the promoter that we identified and the delayed timing of the increase in IRS-2 expression in response to hypoxia, additional factors are likely to contribute to the regulation of IRS-2 expression. A number of transcription factors have been identified that stimulate gene expression in response to hypoxia, and these factors either act in cooperation with HIF, or are regulated by HIF, to alter hypoxic gene expression (43
). Importantly, some of these transcription factors have been previously implicated in the regulation of IRS-2 gene expression including AP-1, the forkhead transcription factors FOXO1 and FOXO3a, and CREB (21
In summary, we have established a novel mechanism by which hypoxia selects for aggressive tumor behavior and promotes metastatic disease. The identification of IRS-2 as a hypoxia-responsive gene that regulates signaling pathways important for tumor cell survival and invasion in hypoxic environments opens a new avenue for investigation into how this pathway could be manipulated for therapeutic benefit.