Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Muscleblind-like 2 controls the hypoxia response of cancer cells SANDRA FISCHER,1 ANTONELLA DI LIDDO,2 KATARZYNA TAYLOR,3 JAMINA S. GERHARDUS,1 KRZYSZTOF SOBCZAK,3 KATHI ZARNACK,2 and JULIA E. WEIGAND1 1Department of Biology, Technical University of Darmstadt, Darmstadt, 64287, Germany 2Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany 3Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, 61-614, Poland ABSTRACT Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still lim- ited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abun- dance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA- binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A (VEGFA). MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes. Keywords: hypoxia; cancer; MBNL2; HIF target genes; alternative splicing INTRODUCTION Semenza 2016). Thus, tumor hypoxia signifies a more ag- gressive phenotype resulting from increased growth and Hypoxia is a common feature of solid tumors as the oxygen metastasis. Moreover, tumor hypoxia counteracts radio-, supply to the rapidly proliferating cancer cells is inade- chemo-, and immunotherapy, highlighting the importance quate. Two types of tumor hypoxia occur as a result of of developing effective hypoxia-targeted therapies structurally and functionally abnormal tumor vasculature. (Graham and Unger 2018). Well studied is the role of the “Chronic” hypoxia arises in cancer cells that are too far HIF (hypoxia inducible factor) transcription factors in the away from the blood vessels to be supplied with sufficient adaptation to hypoxia. HIFs are heterodimers consisting oxygen by diffusion. “Acute” hypoxia is the result of tem- of an oxygen-labile α subunit and a constitutively stable porarily limited perfusion by immature tumor vessels β subunit. Under hypoxia, HIFα subunits are stabilized, al- (Vaupel et al. 2004). Such hypoxic regions are spatially lowing for the transcriptional activation of several hundred and temporally dynamic due to tumor angiogenesis and target genes (Samanta and Semenza 2018). the reversibility of acute hypoxia, resulting in severe intra- Independent from transcriptional adaptation, hypoxia and intertumoral heterogeneity of cellular oxygen levels. triggers a widespread post-transcriptional response by The oxygen content in hypoxic areas of solid tumors is typ- altering alternative splicing (AS) decisions, translation effi- ically <1.4% and may reach complete anoxia (Vaupel et al. ciency, and mRNA stability. For example, at low glucose 2007). conditions, several mRNAs of HIF target genes are stabi- Hypoxia promotes virtually every step in tumor pro- lized under hypoxia (Carraway et al. 2017; Fry et al. gression contributing to metabolic reprogramming, 2017). While canonical protein synthesis is reduced due angiogenesis, extracellular matrix remodeling, epithelial to mesenchymal transition, cancer stem cell maintenance, immune evasion, and cell death resistance (Schito and © 2020 Fischer et al. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 Corresponding author: [email protected] months, it is available under a Creative Commons License Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. (Attribution-NonCommercial 4.0 International), as described at http:// 073353.119. creativecommons.org/licenses/by-nc/4.0/. 648 RNA (2020) 26:648–663; Published by Cold Spring Harbor Laboratory Press for the RNA Society Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press MBNL2 drives hypoxia adaptation to the limited ATP supply during hypoxia, alternative trans- knockdown of MBNL2 in cancer cells leads to reduced pro- lation pathways are activated, to sustain translation of pro- liferation and migration, emphasizing MBNL2 as a driver of teins essential for hypoxia adaptation (for review, see Chee cancer cell function in hypoxia. et al. 2019). Remarkably, ribosomal profiling of hypoxic primary hepatocytes showed that changes in translation RESULTS even precede the transcriptional response (Hettiarachchi et al. 2019). In addition, hypoxia-driven AS events diversify Chronic hypoxia induces similar changes in transcript the cellular response to hypoxia, both in primary and can- abundance, but divergent alternative splicing cerous cells (Weigand et al. 2012; Hu et al. 2014; Brady programs et al. 2017; Han et al. 2017; Bowler et al. 2018). The current knowledge of the factors that control these post-transcrip- We chose A549 lung and MCF-7 breast cancer cells to tional pathways is still very limited, but crucial in under- compare transcriptomic changes in response to chronic standing the tumor-promoting effects of hypoxia. hypoxia, as both cell lines have no deletions or mutations In line with the central role of RNA-binding proteins within the HIF pathway, the two major families of splicing (RBPs) in post-transcriptional gene control, aberrant RBP factors, the SR proteins (serin/arginine rich) and hnRNPs activities have been identified as potent cancer drivers (heterogeneous nuclear ribonucleoproteins) or core spli- (for reviews, see Anczuków and Krainer 2016; Pereira ceosomal genes, such as mutations in SF3B1 (splicing fac- et al. 2017). Just recently, MBNL proteins were found to af- tor 3B1), which are common in cancer (Cerami et al. 2012; fect tumorigenesis (Perron et al. 2018; Tabaglio et al. Gao et al. 2013; Seiler et al. 2018). First, we identified the 2018). The MBNL family consists of the three closely relat- lowest oxygen concentration that would allow continued ed paralogs, MBNL1, MBNL2, and MBNL3 (for review, see survival under hypoxic stress. We incubated A549 and Konieczny et al. 2014). MBNL1 and MBNL2 are ubi- MCF-7 cells for 48 h at two different oxygen levels, 0.5% quitously expressed, whereas MBNL3 expression is more and 0.2% O2. Cell viability after hypoxia treatment was restricted, with highest levels found in the placenta (Far- compared to normoxic (21% O2) controls using crystal vio- daei et al. 2002). All three MBNL paralogs encode two ami- let staining. While incubation at 0.5% O2 showed no differ- no-terminal tandem zinc finger (ZnF) domains. These bind ence in cell viability between normoxic and hypoxic cells to RNAs containing two or more clustered 5′-YGCY-3′ mo- (Fig. 1A), lower oxygen levels of 0.2% significantly reduced tifs (Lambert et al. 2014). The ability of MBNLs to bind RNA the viability of both cell types (Supplemental Fig. 1). is further altered by the structural context of the RNA tar- Further, VEGFA mRNA levels were quantified to control gets (deLorimier et al. 2017; Taylor et al. 2018). Binding for the induction of hypoxia response genes after 48 h at of MBNLs activates or represses mRNA alternative splicing 0.5% O2. VEGFA is a widely expressed, primary target of depending on MBNL binding location (Charizanis et al. HIF transcription factors and thus, a commonly used mark- 2012; Wang et al. 2012), with MBNL1 being the most po- er for the hypoxia response of cells (Forsythe et al. 1996; tent splicing regulator among the three paralogs (Sznajder Blancher et al. 2000). RT-qPCR confirmed a 3.6-fold or et al. 2016). Additionally, MBNL proteins are proposed to 7.2-fold increase in VEGFA mRNA levels in A549 and regulate mRNA stability (Masuda et al. 2012) and localiza- MCF-7 cells, respectively (Fig. 1B). Thus, incubation at tion (Adereth et al. 2005; Wang et al. 2012), alternative 0.5% O2 for 48 h robustly induces a hypoxia response in polyadenylation (Batra et al. 2014), and miRNA biogenesis both cell types without causing cell death and was there- (Rau et al. 2011). All three MBNL proteins undergo auto- fore used for all further experiments. regulatory feedback loops and can functionally compen- For the transcriptome-wide comparison of hypoxia re- sate each other in some cases (Konieczny et al. 2018). sponses, total RNA was extracted from normoxic and hyp- In order to identify RBPs central to the post-tran- oxic A549 and MCF-7 cells, depleted from ribosomal RNA scriptional response of cancer cells during hypoxia, we and subjected to deep sequencing (n = 2). Approximately obtained a comparative set of hypoxia-driven changes in 100 million reads were obtained for each sample. We de- gene and isoform expression as well as changes in RBP lev- tected expression of 18,214 genes