miR-210: A sensor for hypoxic stress during tumorigenesis

Abstract

In this issue of Molecular Cell, Huang et al. (2009) demonstrate that hypoxia inducible miR-210 acts as a rheostat for cellular adaptation and survival by inhibiting tumor initiation.

Hypoxia (low O₂) is a common feature of pathological conditions such as tissue ischemia and inflammation, as well as solid tumors (Keith and Simon, 2007). Multiple hypoxic responses impacting tumorigenesis are mediated through hypoxia inducible transcription factors (HIFs), composed of alpha (HIF-α) and beta (HIF-β; ARNT) subunits (Keith and Simon, 2007). In the presence of low O₂, HIF-α subunits are stabilized and heterodimerize with ARNT in the nucleus. This heterodimeric complex, along with transcriptional co-factors, binds to hypoxia response elements, modulating the expression of multiple target genes important for angiogenesis, cell survival and tumorigenesis. Two HIF-α proteins, HIF-1α and HIF-2α regulate the expression of overlapping and unique transcriptional target genes (Keith and Simon, 2007). In addition to the transcriptional activation of multiple genes, hypoxia is also involved in the regulation of miRNAs (Kulshreshtha et al., 2007). miRNAs are small non-coding RNAs ~ 22 nucleotides in length representing 1% to 2% of the eukaryotic transcriptome (Dalmay and Edwards, 2006). miRNAs are clearly involved in diverse biological processes including differentiation, cell proliferation, cell death and tumor progression (Dalmay and Edwards, 2006). However, very little is known about the role of miRNAs during hypoxia in tumorgenesis, and whether they are regulated by HIFs.

Recently, several studies have reported miR-210 as one of the highly upregulated miRNAs in hypoxic cells and have demonstrated its importance for cell survival (Fasanaro et al., 2008). In this issue, Huang et al. (2009) demonstrate that miR-210 is HIF-1α-dependent and also provide further insight into its functional role during tumor initiation. The authors first performed miRNA microarray profiling in hypoxic pancreatic cell lines and identified miR-210 as the most highly upregulated species. Furthermore, hypoxia induces miR-210 expression in pancreatic, breast, head and neck, lung, colon and renal cell lines, indicating a broad hypoxic response. Based on the analysis of various cell lines using short hairpin RNA (shRNA) and chromatin immunoprecipitation experiments, the authors concluded that HIF-1α regulates miR-210 expression. A recent study reported induction of miR-210 in 786-0/VHL cells under hypoxia, suggesting that HIF-2α could also regulate miR-210 expression (Zhang et al., 2009). These results imply that HIF-1α could be the predominant and sufficient regulator of miR-210; however in the absence of HIF-1α, HIF-2α could also mediate miR-210 expression.

Since each miRNA potentially regulates hundreds of genes, one of the biggest challenges in the miRNA field is conclusive identification of bona fide targets. The authors performed Argonaute 2 (AGO2) ribonucleoprotein immunoprecipitation (miRNP-IP) to enrich the pool of mRNAs associated with AGO2 (Huang et al., 2009). In order to specifically identify miR-210 targets, microarray experiments were performed, followed by comparison with potential miR-210 target genes predicted by various computational programs. A functional annotation of miR-210 target genes found that a majority are implicated in adaptation and cell survival under hypoxic stress. The authors further validated certain predicted miR-210 target genes by standard 3'UTR luciferase assay and confirmed HOXA1, HOXA9 and FGFRL1 as miR-210 target genes.

In addition to being one of the predominant hypoxia-inducible miRNAs, miR-210 is highly expressed in pancreatic tumors, breast cancers and glioblastomas. To delineate the functional role of miR-210 during tumor development, Huang et al. (2009) performed subcutaneous tumor xenografts with stable cell lines overexpressing miR-210. Given that hypoxia is a typical characteristic of many cancers, ectopic expression of miR-210 would be expected to exacerbate tumor growth. Intriguingly, xenografts from both pancreatic and head and neck cancer cell lines stably overexpressing miR-210 exhibited significant delay in tumor growth. However, this is not entirely unanticipated, as HIF-1α induces cell cycle arrest by antagonizing c-Myc transcriptional activity and inhibits canonical Wnt signaling to reduce tumor cell proliferation, arguably to facilitate tumor cell survival under hypoxic stress (Kaidi et al., 2007; Koshiji et al., 2004). Therefore, the role of HIF-1α-mediated miR-210 induction could be to promote cellular adaptation and survival in the stressful milieu of tumor hypoxia. On the other hand, miR-210 is also induced by HIF-2α in 786-0/VHL cells (Zhang et al., 2009), but previous studies have reported that HIF-2α promotes tumor cell proliferation (Gordan et al., 2007). Does this mean that HIF-2α induced miR-210 expression is not at the threshold required to cause an inhibitory effect, or c-Myc transcriptional activity is powerful enough to overcome the inhibitory effect of miR-210?

Ultimately, the inhibitory effect of ectopic miR-210 was overcome, suggesting that tumor cells attempted to override miR-210 inhibition. Huang et al. (2009) further demonstrated a partial rescue of miR-210 induced inhibitory phenotype by stable expression of miR-210 target genes, FGFRL1 or HOXA1. This partial rescue is consistent with the basic principle of miRNA regulation; specifically, multiple target genes are involved in the development of a cumulative phenotype. However, it is noteworthy that similar to miR-210 xenograft studies, rescue experiments with FGFRL1 or HOXA1 illustrate increased tumor growth during later stages. This raises an intriguing question: is the increase in tumor growth attributable to a more favorable tumor microenvironment where continuous repression of miR-210 target genes is not functionally critical?

Huang et al. propose that increased miR-210 expression inhibits tumor initiation which provides the tumor cells an opportunity to prevail in stressful conditions (Figure 1). If that is the case, will inhibition of miR-210 during tumor initiation facilitate aggressive tumor growth, or will the tumor cells fail to survive in the absence of miR-210, resulting in tumor suppression? It is impossible to speculate on the outcome of these studies due to the complexity involved in hypoxic regulation during tumorigenesis. Although the functional impact of miR-210 is not fully known, findings by Huang et al. extend our knowledge of the importance of miR-210 in tumorigenesis. Finally, recent advances in the field of miRNA and cancer, specifically the successful suppression of murine liver cancer by systemic delivery of miR-26a, demonstrate the potential of utilizing miRNAs as therapeutic agents (Kota et al., 2009). Altogether, a better understanding of the role of important miRNAs in tumorigenesis will open up new avenues for therapeutic opportunities.

Figure 1. Role of miR-210 in hypoxia mediated tumor progression.

Hypoxia mediated induction of miR-210 modulates the expression of genes promoting cell survival and adaptation during stress, followed by tumor progression under favorable conditions.