MicroRNAs in tumor angiogenesis Weifeng Wang, Erdong Zhang, Changjun Lin PII: DOI: Reference:
S0024-3205(15)00349-5 doi: 10.1016/j.lfs.2015.06.025 LFS 14433
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
2 March 2015 3 June 2015 30 June 2015
Please cite this article as: Wang Weifeng, Zhang Erdong, Lin Changjun, MicroRNAs in tumor angiogenesis, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.025
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ACCEPTED MANUSCRIPT Review article
MicroRNAs in tumor angiogenesis
School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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a
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Weifeng Wang a, Erdong Zhang a, Changjun Lin a,*
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Corresponding author: Dr. Changjun Lin
P.R.China
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Lanzhou 730000
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Lanzhou University
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School of Life Sciences
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Phone: +86-931-8912563 Fax: +86-931-8912561 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract As it is necessary for tumor growth, angiogenesis has been an attractive target for
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drug therapy. Accumulating evidences indicate microRNAs (miRNAs), which are
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short non-coding RNAs, delicately regulate the angiogenic signals through targeting angiogenic factors and protein kinases. They can modulate pro-angiogenic signals induced by vascular endothelial growth factor (VEGF) and anti-angiogenic signals
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induced by thrombospondin-1 (TSP-1), and therefore promote or inhibit tumor
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angiogenesis. Receptor tyrosine kinases (RTKs) and hypoxia inducible factor (HIF) are also targeted by miRNAs. Moreover, miRNAs crosstalk with reactive oxygen
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species (ROS) influencing tumor angiogenesis. It is critical to understand the role of
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miRNAs in tumor angiogenesis due to their therapeutic potential to improve outcome
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for cancer patients. The following review discusses the current state of knowledge related to tumor angiogenesis-regulatory miRNAs and their targets.
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Key words: tumor angiogenesis; microRNAs; vascular endothelial growth factor (VEGF);
thrombospondin-1(TSP-1); receptor tyrosine kinases (RTKs); hypoxia
inducible factor (HIF);
reactive oxygen species (ROS)
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ACCEPTED MANUSCRIPT 1. Introduction In 1971, a milestone paper was published by Dr Judah Folkman, with the
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hypothesis that solid tumors caused new blood vessel growth (angiogenesis) in the
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tumor microenvironment through secreting pro-angiogenic factors (Folkman, 1971). Implicit in the paper was the notion that angiogenesis has potential value acting as therapeutic target for cancer treating. In 2010, Kristina M. Cook and William D. Figg
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gave a broad overview of the key mechanisms involved in tumor angiogenesis (Cook
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and Figg, 2010). They discussed the receptor tyrosine kinases (RTKs) signaling which controls angiogenesis and is activated by growth factors, as well as some other
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angiogenic factors. In recent years, microRNAs (miRNAs), the crucial regulators of
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gene expression at the post-transcriptional level, have been shown to regulate
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angiogenesis through directly targeting signaling protein or aniogneic factor mRNAs (Poliseno et al. , 2006, Urbich et al. , 2008, Wang et al. , 2008, Wang and
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Olson, 2009, Wu et al. , 2009). Because miRNA expression is tightly controlled and seems to be specific to different organs and cell types, developing angiogenesis inhibitors targeting miRNAs is a desirable anti-cancer therapy where fewer side-effects might be expected.
2. MiRNAs: New tumor angiogenic factors 2.1. Tumor angiogenesis Tumor requires sustenance in the form of nutrients and oxygen as well as an ability to evacuate metabolic wastes and carbon dioxide. Initially, the growth of a 3
ACCEPTED MANUSCRIPT tumor is fed by nearby blood vessels (Hanahan and Weinberg, 2011). Once a certain tumor size is reached, these blood vessels are no longer sufficient. In order to progress
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to a larger size, new blood vessels are required (Fig.1). The normal vasculature
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becomes largely quiescent in the adult. Pre-existing vessels develop new vessels by sprouting. Solid evidences have proved endothelial-to-mesenchymal transition (EndMT) plays an important role in angiogenic sprouting. EndMT enables the
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so-called tip cells, which lead an emerging vascular plexus, to migrate into adjacent
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tissue. During EndMT, resident endothelial cells delaminated from an organised cell layer, which lost endothelial markers, gained mesenchymal markers and acquired
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invasive and migratory properties, finally (Potenta et al. , 2008).
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The ability to induce and sustain angiogenesis seems to be acquired via an
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“angiogenic switch” from vascular quiescence (Folkman, 1995). Angiogenesis is turned on, as part of physiologic processes such as wound healing and female
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reproductive cycling, but only transiently. In contrast, the ‘‘angiogenic switch’’ is almost always long-lastingly activated during tumor development. A compelling body of evidence indicates that the “angiogenic switch” is the balance between pro-angiogenic and anti-angiogenic signals (Folkman, 1995). Vascular endothelial growth factor-A (VEGFA) and thrombospondin-1 (TSP-1) are the well-known prototypes of angiogenesis inducers and inhibitors, respectively. Additionally, VEGF gene expression can by upregulated both by hypoxia (Chen et al. , 2013) and by ischemia (Li et al. , 2014) in the microenvironment of tumor. TSP-1 evokes suppressive signals that can counteract pro-angiogenic stimuli through direct effects 4
ACCEPTED MANUSCRIPT on endothelial cell migration and survival, and through effects on VEGF bioavailability (Kazerounian et al. , 2008, Tolsma et al. , 1994). The “angiogenic
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switch” is turned on within tumors when pro-angiogenic signals are activated or
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anti-angiogenic signals are sequestered.
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Fig.1. Angiogenesis occurs during cancer development which is caused by activated
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pro-angiogenic signals or sequestered anti-angiogenic signals.
2.2. MiRNAs biogenesis and their targets
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MiRNAs are a class of highly conserved, single-stranded, non-coding small RNAs found in eukaryotes that regulate gene expression post-transcriptionally. The biogenesis of mature miRNAs is a complex and multistep process (Fig. 2). The primary miRNA transcripts (pri-miRNA) are generated by RNA polymerase II or III in the nucleus (Lee et al. , 2004). Pri-miRNA processing occurs in two steps. In the first step, pri-miRNAs are cleaved by the microprocessor complex to form the precursor miRNA (pre-miRNA) (Lee et al. , 2003). The microprocessor contains Drosha and the dsRNA-binding proteins (dsRBPs), such as DGCR8 protein. And the new formed pre-miRNA will be exported to the cytoplasm by Exportin 5 (Yi et al. , 5
ACCEPTED MANUSCRIPT 2003). Then pre-miRNAs are cleavage by Dicer (Kuehbacher et al. , 2007), assisted by transactivation-responsive (TAR) RNA-binding protein (TRBP) generating an
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~20-bp miRNA/miRNA* duplex (Maniataki and Mourelatos, 2005). Following
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processing, the guide strand of the miRNA duplex, which contains the seed sequence, associates with Argonaute 2 (Ago2) protein to form the miRNA-induced silencing complex (miRISC) (Donker et al. , 2007), whereas the other strand (passenger or
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miRNA*) is released and degraded. The miRISC then binds to the 3’-untranslated
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region (3’-UTR) of the target mRNA and mediates sequence-specific gene silencing through mRNA destabilization and translational repression (Fabian and Sonenberg,
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2012, Maniataki and Mourelatos, 2005). Mature miRNAs recognize their target
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mRNAs through base-pairing interactions between nucleotides numbering 2 to 8 of
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the miRNA (the seed region) and the complementary nucleotides in the 3’-UTR of the
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mRNAs (Fabian and Sonenberg, 2012) (Fig. 2).
Fig. 2. Biogenesis of mature miRNAs. Pri-miRNA is processed by Drosha and Dicer, operating in complexes with DGCR8 and TRBP, which generate a miRNA/miRNA* 6
ACCEPTED MANUSCRIPT duplex. The guide strand of the miRNA/miRNA* duplex is preferentially incorporated into a miRISC, whereas miRNA* strand is released and degraded. Following miRISC
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biogenesis, various target genes of miRNA are silenced at post-transcription level.
It is estimated that there are more than 1,000 miRNA genes in the human genome, and these could regulate more than one-third of the mRNAs produced (Lewis et al. ,
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2005). In recent years, some researches revealed that miRNAs modulate tumor
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angiogenesis through targeting pro-/anti-angiogenic factors including RTKs signaling proteins, hypoxia inducible factor (HIF), VEGF, TSP-1 and reactive oxygen species
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(ROS) causing tumor developed or arrested (Tab.1). It should be noticed that many
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miRNAs are expressed in cancer- and tissue-specific patterns, suggesting that
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miRNAs have cell type-specific functions. As miRNAs play an important role in tumor angiogenesis, we will discuss the latest examples that illustrate the role of
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miRNAs during tumor angiogenesis below.
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Tab. 1. MiRNAs target pro-/anti- angiogenesis factors in different cancer cells. cell type
up/down
pro-/anti-
target
regulated
cells
up
PTEN
Human prostate cancer
PTEN
cells
pro
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miR-21
human gastric cancer
Spred-1/PIK3R2
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miR-382
EC
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RTKs signaling-related miRNAs miR-126
references
angiogenesis
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miRNAs
(Wang et al., 2008) (Fish et al. , 2008)
pro
(Seok et al. , 2014)
pro
(Liu et al. , 2011)
HUVECs
up
RhoB
anti
(Sabatel et al. , 2011)
miR-26a
HCC
up
PIK3C2α
pro
(Chai et al. , 2013)
miR-218
mesenchymal GBM
down
anti
(Mathew et al. , 2014)
miR-18a
gastric cancer
up
mTOR
anti
(Zheng et al. , 2013)
p70S6K1
anti
(Xu et al. , 2012)
HIF-1α
anti
(Cha et al. , 2010)
HIF-1α
anti
(Yamakuchi et al. , 2011)
HIF-1β
anti
(Yamakuchi et al. , 2010)
cells
/PLCγ1/ARAF
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human colon cancer
PIK3C2α
down
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miR-145
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miR-21
HIF-targeted miRNAs
miR-107
up
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miR-22
HUVECs human colon cancer cells
down
human colon cancer cells
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miR-519c
VEGF-targeted miRNAs
miR-497
ovarian cancer cells
down
VEGFA
anti
(Wang et al. , 2014)
miR-503
HCC
down
VEGFA/FGF2
anti
(Zhou et al. , 2013)
down
VEGFA
anti
(Zhang et al. , 2013)
TSP-1
pro
(Sundaram et al. , 2011)
miR-126
human breast cancer cells
TSP-1-targeted miRNAs miR-194 miR-17-92 miR-467
HCT116 CRC cells ovarian endometrioma
down
TSP-1
pro
(Ramon et al. , 2011)
breast cancer cells
up
TSP-1
pro
(Bhattacharyya et al. , 2012)
pro
(Zhang et al., 2012)
ROS signaling-related miRNAs miR-21
human bronchial epithelial cells
TNFα/SOD3
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ACCEPTED MANUSCRIPT 3. Signals regulated by miRNAs in tumor angiogenesis Fig. 3 gives a frame of modulation of tumor angiogenesis by miRNAs. Fibroblast
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growth factor 2 (FGF2), VEGFA and EGF, three of the most potent pro-angiogenic
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factors, bind transmembrane receptors displayed by endothelial cells and thereby stimulate angiogenesis. FGF2 and VEGFA are respectively down-regulated by miR-503, miR-497 and miR-126, among which miR-503 binds 3’-UTRs of both
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FGF2 and VEGFA (Fig. 3). The combination of growth factors and their receptors
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activate RTKs signaling and related kinase proteins. Lots of miRNAs directly or indirectly repress kinase proteins involved in PI3K/mTOR or RAF/ERK pathways,
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including miR-382, miR-21, miR-126, miR-26a, miR-18a, miR-145 and miR-218
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(Fig. 3). Moreover, HIF complex is a major regulator of tumor angiogenic genes
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under low oxygen stress, down-regulated by miR-519c, miR-22 and miR-107 (Fig. 3). In addition, TSP-1, a key counterbalance in the “angiogenic switch”, could counteract
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pro-angiogenic signals induced by growth factors. And anti-angiogenic signals induced by TSP-1 are also influenced by miRNAs, such as miR-17-92, miR-194 and miR-467 (Fig. 4). ROS, as tumor angiogenesis promoters, also interact with RTKs signaling which is modulated by miR-21 (Fig. 3).
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Fig. 3. MiRNAs regulate pro-angiogenic signals and ROS. Pro-angiogenic factors,
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such as growth factors (VEGF, EGF and FGF2), bind to their receptors on the cell membrane which activate the downstream RTKs signaling pathway causing HIF and
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other angiogenic gene expression. MiRNAs modulate tumor angiogenesis by targeting the proteins related to RTKs signaling, HIF and growth factors. ROS regulated by
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miR-21also crosstalk with RTKs signaling.
3.1. RTKs signaling-related miRNAs AKT (protein kinase C) /extracellular signal-regulated kinase (ERK) signaling can be activated by the binding of a growth factor ligand to RTKs therefore to initiate processes like angiogenesis (Forough et al. , 2005, Jiang, 2008, Zhong et al. , 2000). The signaling cascades include the Phosphatidyl Inositol 3-kinase (PI3K)-AKTmammalian target of rapamycin (mTOR) and phosphor-lipase C-γ (PLCγ) -Protein Kinase C（PKC）-RAF kinase-EKR pathways. Some miRNAs control the expression 10
ACCEPTED MANUSCRIPT of kinase proteins in these pathways, leading to pro-/anti-angiogenesis, including miR-126, miRNA-382, miR-21, miR-26a and miRNA-218.
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MiR-126, an endothelial cell-restricted miRNA, was shown to enhance the
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pro-angiogenic actions of VEGF and FGF and promote blood vessel formation by repressing the expression of Spred-1 (Wang et al., 2008) and PIK3R2, intracellular inhibitors of AKT and RAF kinase (Fish et al. , 2008).
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MiR-382 and miR-21 both modulate angiogenesis by repressing phosphatase and
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tensin homolog (PTEN). MiR-382 induced by hypoxia in MKN1 human gastric cancer cells promoted angiogenesis. It acted as an angiogenic oncogene by
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down-regulating PTEN and its downstream target AKT/mTOR signaling pathway
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(Seok et al. , 2014). Similarly, miR-21 induced tumor angiogenesis through targeting
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PTEN, leading to activating AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression (Liu et al. , 2011). But another study
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revealed that miR-21 acted as a negative modulator of angiogenesis (Sabatel et al. , 2011). They found miR-21 overexpression in human umbilical vein endothelial cells （HUVECs）reduced endothelial cell proliferation, migration and the ability to form tubes through repression of RhoB, a gene that involved in angiogenesis regulation. In spite of the indirect impact on RTKs signaling, miR-26a and miR-218 have been confirmed to interact with PIK3C2α (class II PI3Ks) mRNA and inhibit its protein expression. miRNA-26a inhibited angiogenesis by down-regulating VEGFA through the PIK3C2α/ AKT/HIF-1α pathway in Hepatocellular Carcinoma（HCC） (Chai et al. , 2013). The expression of miR-218 was decreased significantly in highly 11
ACCEPTED MANUSCRIPT necrotic mesenchymal glioblastoma multiforme (GBM), and miR-218 targeted EGF receptor (EGFR) and multiple components of RTKs signaling pathways including
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PLCγ1, PIK3C2A, and v-raf murine sarcoma 3611 viral oncogene homolog (ARAF)
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(Mathew et al. , 2014). MiR-218 repression up-regulated the abundance and activity of multiple RTK effectors, which elevated RTKs signaling and promoted the
angiogenesis has not been determined.
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activation of HIF, most notably HIF2α. However, the direct effect of miR-218 on
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The miR-17-92 cluster has long been recognized as an oncogenic miRNA cluster and is amplified in multiple cancers (Hayashita et al. , 2005, Melegari et al. , 2006).
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Overexpression of miR-18a, a member of the miR-17-92 cluster, substantially
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reduced the phosphorylation of two mTOR substrates, S6K1 and 4E-BPl, indicating
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inactivation of the mTOR pathway in gastric cancer (Zheng et al. , 2013). Accompanying with the mTOR inactivation, the angiogenic factors, HIF-1α and
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VEGF, were significantly down-regulated. These data highlighted an important role for miR-18a in controlling gastric cancer growth and angiogenesis. MiR-145 is another miRNA directly repressing RTKs signaling, with down-regulated expression in colon and ovarian cancer tissues and cell lines. In human colon cancer cells, miR-145 inhibited post-transcriptional expression of p70S6K1, one of the downstream targets of mTOR, by binding to its 3’-UTR. The angiogenic factors HIF-1 and VEGF, which are downstream molecules of p70S6K1, were decreased by miR-145 overexpression (Xu et al. , 2012).
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ACCEPTED MANUSCRIPT 3.2. HIF-targeted miRNAs HIF is a heterodimeric complex that consists of a hypoxia-inducible, unstable
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α-subunit and a stable, constitutively expressed β-subunit. Three HIF-α isoforms have
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been identified. The HIF-1α subunit is continuously synthesized and degraded under normoxic conditions. Therefore, HIF-1 complex does not function during normal oxygen tension (Salceda and Caro, 1997). But hypoxia that occurs in the
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microenvironment of tumors can stabilize the HIF-1 complex. Once stabilized, the
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HIF-1 complex can bind to the promoter regions of its target genes and thereby induce target gene expression.
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HIF-dependent transcriptional changes regulate a broad spectrum of cellular
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functions, including angiogenesis (Liao and Johnson, 2007, Theodoropoulos et al. ,
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2004). Thus, miRNAs that target HIF are likely to have significant impacts on the angiogenesis pathways. Several miRNAs directly target HIF mRNA: miR-20a,
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miR-20b and miR-199a miR-519c, miR-22, miR-107, among which miR-20a, miR-20b and miR-199a have been discussed in another review (Madanecki et al. , 2013). The paper focuses on miR-519c, miR-22 and miR-107. Overexpression of miR-519c resulted in a significant decrease of HIF-1α protein levels and reduced the tube formation of HUVECs, suggesting an important role of miR-519c in HIF-1α–mediated angiogenesis (Cha et al. , 2010). In addition, hepatocyte growth factor (HGF), a known HIF-1α inducer, reduced the miR-519c levels through an AKT-dependent pathway. miR-22 was found to decrease its expression in human colon cancer compared with normal colon tissue, and acted as 13
ACCEPTED MANUSCRIPT anti-angiogenic factor through inhibiting HIF-1α expression, repressing VEGF production during hypoxia (Yamakuchi et al. , 2011). MiR-107 is a miRNA expressed
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in human colon cancer specimens directly regulated by p53 which can match 3’-UTR
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of HIF-1β. Furthermore, overexpression of miR-107 in tumor cells suppressed tumor angiogenesis, tumor growth, and tumor VEGF expression in mice. These data suggested that miR-107 could mediate p53 regulation of hypoxic signaling and tumor
3.3. VEGF-targeted miRNAs
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angiogenesis by targeting HIF-1β (Yamakuchi et al. , 2010).
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VEGF and its receptor tyrosine kinase (VEGFR) play key roles in angiogenesis
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and VEGF gene is a major transcriptional target of HIF (Ferrara et al. , 2003, Lee et
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al. , 2008). VEGF is actually a family of at least seven members, and the term VEGF typically refers to the VEGFA isoform, one of the most studied members and a major
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pro-angiogenic factor. VEGF binds to two tyrosine kinase receptors, VEGF receptor-1 (VEGFR1, and VEGFR2) in endothelial cells (ECs). The mitogenic and chemotactic effects of VEGF in ECs are mediated mainly through VEGFR2 which is activated through autophosphorylation of tyrosine residues in the cytoplasmic kinase domain. This event is followed by activation of downstream signaling pathways such as AKT, which is essential for EC migration and proliferation. Recent studies revealed that several miRNAs, such as miR-497, miR-503 and miR-126, were all able to match 3’-UTR of VEGFA mRNA. MiR-497 expression was down-regulated in human ovarian cancer tissues, which 14
ACCEPTED MANUSCRIPT was significantly associated with increased angiogenesis (Wang et al. , 2014). Further study disclosed that miR-497 exerted its function of anti-angiogenesis by suppressing
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VEGFA expression and, in turn, impairing the VEGFR2-mediated PI3K/AKT and
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MAPK/ERK pathways.
MiR-503 expression was down-regulated by hypoxia through HIF-1α in HCC cells and primary tumors. MiR-503 could simultaneously down-regulate VEGFA and
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FGF2 in cancers, demonstrating the anti-angiogenesis role in tumorgenesis (Zhou et
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al. , 2013).
MiR-126 was illustrated to antagonize tumor angiogenesis by inhibiting Spred-1
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and PIK3R2. In addition, miR-126 could directly target VEGFA, and its expression
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was decreased in human breast cancer, implying that miR-126 might play a role in
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tumorgenesis and growth by regulating the VEGF/PI3K/AKT signaling pathway
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(Zhang et al. , 2013).
3.4 TSP-1-targeted miRNAs Thrombospondin-1 (TSP-1) is a glycoprotein influencing angiogenesis by effects on ECs. In the extracellular matrix of ECs, TSP-1 inhibits metalloproteinases (MMPs) activation, and consequently the release of VEGF (Fig.4). Thence, there is a crosstalk between pro-angiogenic signals induced by growth factors and anti-angiogenic signals induced by TSP-1 (Fig. 4). Moreover, TSP-1 induces the activation of CD36 which leads to inhibition of migration and induction of apoptosis (Dawson et al. , 1997, Jimenez et al. , 2000) (Fig. 4). 15
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Fig. 4. Anti-angiogenic signals modulated by miRNAs. TSP-1 is the main
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anti-angiogenic factor influencing EC phenotype and their apoptosis, and it could be
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repressed by several miRNAs, such as miR-194, miR-17-92 and miR-467.
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TSP-1 is encoded by the THBS1 gene, whose promoter is activated by p53. However, it was reported that in HCT116 colorectal cancers (CRC) cells, p53 activated the THBS1 primary transcript, but failed to boost THBS1 mRNA or protein levels, implying potential post-transcriptional regulation by miRNAs. Further researches revealed that miR-194 negatively regulated THBS1 mRNA and protein levels.
Notably,
p53
can
upregulated
miR-194
expression
in
THBS1
retrovirus-transduced HCT116 cells, leading to decreased TSP-1 levels (Sundaram et al. , 2011). In addition, members of the miR-17-92 cluster, specifically miR-18a and miR-19, 16
ACCEPTED MANUSCRIPT negatively regulated TSP-1 levels during Myc-induced angiogenesis (Dews et al. , 2006). MiR-17-92 cluster localized on 13q31 was upregulated in colonocytes
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co-expressing K-Ras and c-Myc of human adenocarcinomas. The level of
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up-regulated miR-17-92 is directly proportional to the level of down-regulated TSP-1, reducing neovascularization of tumor (Dews et al. , 2006). Consistent with previous results, a research by Luis A. Ramon ´and his colleagues showed ovarian
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endometrioma had significantly higher expression of TSP-1 and lower expression of
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miR-17-5p than those of eutopic endometrium. Moreover, significant inverse correlations between miR-17-5p and TSP-1 protein levels, and between miR-222 and
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VEGF-A protein levels, were observed (Ramon et al. , 2011). Ramon et al. observed a
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significant increase in TSP-1 protein levels in endometrial cancer as well (Ramon et
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al. , 2012). Conversely, although TSP-1 is an anti-angiogenic factor, a significant positive correlation between TSP-1 protein levels and miR-210, which promoted
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angiogenesis in endometrial carcinoma, was obtained (Ramon et al. , 2012). MiR-467 is implicated in the control of angiogenesis in response to high glucose. It has also been identified as a translational suppressor of TSP-1 that is implicated in the pathogenesis of several diabetic complications (Bhattacharyya et al. , 2012). MiR-467 was upregulated by high glucose in microvascular endothelial cells and in breast cancer cells, where it suppressed the production of TSP-1 by sequestering its mRNA in the nonpolysomal fraction. In in vivo angiogenesis models, miR-467 promoted the growth of blood vessels, and TSP-1 was the main mediator of this effect (Bhattacharyya et al. , 2012). 17
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4. Crosstalk between ROS and miRNAs in tumor angiogenesis
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A lot of evidences suggest that ROS function as signaling molecules to mediate
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various growth-related responses including angiogenesis (Oshikawa et al. , 2010). Superoxide (O2.-) is the main initial ROS, which can be converted to other reactive species including hydrogen peroxide (H2O2). ROS can be generated by NADPH
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oxidase (NOX) or induced by extracellular stimuli such as ionizing radiation (IR). In
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turn, NOX is activated by numerous stimuli including VEGF, EGF, hypoxia and ischemia (Fig. 5). Interaction between ROS and miRNAs might be a critical factor for
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angiogenesis.
Fig. 5. Crosstalk between ROS and miRNAs in the regulation of tumor development. Certain concentrations of H2O2 up-regulates miR-200c expression level in HUVECs. In turn, miR-200c down-regulates VEGFA, leading to angiogenesis arrest. In various 18
ACCEPTED MANUSCRIPT types of tumor cells, miR-27a, miR-21 and miR-210 have close relationship with ROS
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generation and function as pro-/anti-tumor development.
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4.1. MiRNAs modulated by ROS
Oxidative stress has been demonstrated to play a crucial role in tumor angiogenesis and miRNAs have been shown to be modulated in cellular responses to
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redox imbalance. A miRNA profiling of HUVEC treated with H2O2, showed an
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up-regulation of miR-200 family members (Magenta et al. , 2011). This miRNA family consists of five members, miR-200c and miR-141 clustered on chromosome 12
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and miR-200a, miR-200b, and miR-429 clustered on chromosome 1. In particular,
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miR-200c and miR-141 are the most up-regulated miRNAs in HUVEC, exposed to
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H2O2 for different periods of time, whereas the other three clustered members are up-regulated to a lower extent (Magenta et al., 2011). And it has been reported that
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miR-200c targets VEGFA (Chuang et al. , 2012). Taken together, certain concentrations of H2O2-induced miR-200c leads to angiogenesis arrest through inhibiting VEGFA (Fig. 5).
GT-094, a nitric oxide chimera containing a nonsteroidal anti-inflammatory drug, down-regulated genes associated with angiogenesis, such as VEGF and its receptors (VEGFR1 and VEGFR2) (Pathi et al. , 2011). In colon cancer cells it was accompanied by decreased mitochondrial membrane potential (MMP) and increased ROS, which inhibited miR-27a expression. Down-regulation of miR-27a induced 19
ACCEPTED MANUSCRIPT ZBTB10, a repressor of specificity protein (Sp) transcription factors, and therefore caused repression of Sp and Sp-regulated gene products repression (Pathi et al., 2011).
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In addition, the miR-27a expression was significantly increased in VEGF-treated
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breast cancer stem like cells (BCSLCs). Increased miR-27a paralleling the reduced expression of ZBTB10 in BCSLCs promoted in vivo angiogenesis and tumor
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metastasis (Tang et al. , 2014) (Fig. 5).
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4.2. MiRNAs modulate ROS
Dicer is the key enzyme controlling miRNA biogenesis. Silencing Dicer in vitro
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impaired the angiogenic response of HUVECs and EA.hy.926 ECs (Shilo et al. ,
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2008). Global lowering of miRNA levels caused by Dicer knockdown withdrew the
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negative control of miRNA on HBP1 expression, resulting in higher levels of HBP1. Elevated HBP1, in turn, down-regulated p47phox expression and limited
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ROS-dependent angiogenic signaling (Shilo et al., 2008) . MiR-21 was reported to inhibit PTEN or RhoB, leading to pro-/anti-angiogenesis (Zhang et al. , 2012). In addition, miR-21 triggered the generation of ROS that promoted tumorigenesis (Zhang et al., 2012). MiR-21 inhibited the metabolism of O2.to H2O2, by directing attenuating SOD3 or by an indirect mechanism that limited TNFα production, thereby reducing SOD2 levels. Therefore, miR-21-induced tumorigenesis is partially due to the high O2.- level generated in the cells (Fig. 5). MiR-210, a main mitochondrial miRNA, has been demonstrated to play a key role in mitochondrial metabolism, therefore modulating ROS production (Qin et al. , 20
ACCEPTED MANUSCRIPT 2014) (Fig. 5). It was found that miR-210 increased the formation of ROS in MCF7 and HCT116 cell lines (Favaro et al. , 2010). In hypoxic HCC (Ying et al. , 2011) and
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CRC cells (Qin et al., 2014), vacuole membrane protein 1 (VMP1) was identified as
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the direct and functional downstream target of miR-210, which mediated cancer cell migration and invasion supported by angiogenesis. Meanwhile, miR-210 contained in exosomes released by cancer cells could be transported to endothelial cells to induce
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angiogenesis.
5. Tumor-specific miRNAs
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As limited information is known about tumor-type specificity of miRNAs, we
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here discuss some tumor-specific miRNAs which function differently.
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The expression level of miR-145 (Xu et al., 2012), miR-22 (Li et al. , 2011) and miR-126 (Zhang et al., 2013) decreased in colorectal cancer cells compared with the
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normal cells and these three miRNAs respectively targeted p70S6K1, HIF-1α and VEGF mRNA. HIF-1 and VEGF are downstream molecules of p70S6K1 pathway. Therefore, the expression of p70S6K1, HIF-1α and VEGF would get out of control, leading to promoted tumor angiogenesis. In addition, miR-126 acted as anti-angiogenic factor in non-small cell lung cancer cells by causing VEGFA arrest. In highly necrotic mesenchymal GBM cells, the expression of miR-218 was decreased significantly (Mathew et al., 2014). MiR-218 targets multiple components of RTKs signaling pathways, and miR-218 repression increases the abundance and activity of multiple RTK effectors, promoting the activation of HIF, most notably 21
ACCEPTED MANUSCRIPT HIF2α. The miR-218/RTK/HIF2α signaling promoted cell survival and tumor angiogenesis, particularly in necrotic mesenchymal tumors (Mathew et al., 2014).
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Similarly, in bladder cancer cells, miR-10b, 19a, 126, 145, 221, 296-5p and 378
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were significantly down-regulated, among which miR-145 is the most prominent one and the overall survival rate in patients with high expression of miR-21 is very low (Zaravinos et al. , 2012). Multivariate analysis revealed that miR-21, 210 and 378
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might serve as independent prognostic factors for overall patient survival. And
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miR-21 and 378 also could serve as independent prognostic factors for recurrence. Especially, miR-27a mediated hypoxia-induced down-regulation of a new angiogenic
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factor, AGGF1 in high-grade bladder urothelial carcinoma cells (Xu et al. , 2014).
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In contrast, in human gastric cancer cells, the expression of miR-382 (Seok et al.,
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2014) and miR-18a (Zheng et al., 2013) were up-regulated. MiR-382 could match to the 3’-UTR of PTEN leading to inhibition of tumor angiogenesis, while miR-18a
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functioned through inactivating the mTOR signaling pathway. Up-regulation of both miRNAs could impair tube formation of tumor. MiR-21 was reported to induce tumor angiogenesis through targeting PTEN in human prostatic cancer cells, leading to activating AKT and ERK1/2 signaling pathways, and thereby enhance HIF-1α and VEGF expression (Liu et al., 2011). HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.
6. Conclusion It is critical to understand the role of miRNAs in tumor angiogenesis due to their 22
ACCEPTED MANUSCRIPT therapeutic potential to improve outcome for cancer patients. Now abundance variation of many miRNAs in specific tumor have been tested. Wu and his colleagues
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produced a miRNA sensor, which not only enabled the quantitative and highly
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specific detection of multiplexed miRNAs in living cancer cells, but also allowed the precise and in situ monitoring of the spatiotemporal changes of miRNA expression (Wu et al. , 2015). Moreover, some miRNA expression profiles in cancer cells have
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been used in the diagnosis of cancer, such as miR-18a and miR-155 (Komatsu et al.,
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2014, Sochor et al. , 2014). Toward cancer therapy, various approaches have been used to regulate the aberrant expression of miRNAs. On one side, anti-miRNA
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molecules and miRNA scavengers have been used to attenuate the up-regulated
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oncogenic miRNAs (Tay et al. , 2015). The mice treated with antisense-miR-21-PS
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and antisense-miR-10b-PS delivered by polymer nanoparticles simultaneously antagonizing miR-21-induced anti-apoptosis and miR-10b-induced metastasis
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(Devulapally et al. , 2015). The treated mice showed substantial reduction in tumor growth compared to the control mice (Devulapally et al. , 2015). On the other side, some specific miRNAs are down-regulated in tumor cells, such as miR-26a in HCC cells. To counter such conditions, miRNA mimics have been used to restore the loss of function induced by the low expression of miRNAs (Kota et al. , 2009). Significant advances have been made for the discovery of the miRNAs, which play a crucial role in the angiogenesis of a specific tumor type by altering a whole network of target proteins. However, there have been far fewer reported successes in the accurate prevision of the combined effects of different angiogenesis-regulated 23
ACCEPTED MANUSCRIPT miRNAs on tumor developing. The number of discoveries, increasing so fast in the last few years, have paved the way for the use of miRNAs as the targeted therapy of
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the future, though there are some challenges.
Conflict of interest statement
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The authors declare that there are no conflicts of interest.
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Acknowledgments
The authors thank the members of the Free Radical Biology Laboratory (School
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of Life Sciences, Lanzhou University) for helpful discussion. This work was
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supported by the grant from the National Natural Science Foundation of China
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(31171121).
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Graphical Abstract
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S0024-3205(15)00349-5 doi: 10.1016/j.lfs.2015.06.025 LFS 14433
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
2 March 2015 3 June 2015 30 June 2015
Please cite this article as: Wang Weifeng, Zhang Erdong, Lin Changjun, MicroRNAs in tumor angiogenesis, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.025
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ACCEPTED MANUSCRIPT Review article
MicroRNAs in tumor angiogenesis
School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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a
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Weifeng Wang a, Erdong Zhang a, Changjun Lin a,*
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Corresponding author: Dr. Changjun Lin
P.R.China
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Lanzhou 730000
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Lanzhou University
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School of Life Sciences
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Phone: +86-931-8912563 Fax: +86-931-8912561 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract As it is necessary for tumor growth, angiogenesis has been an attractive target for
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drug therapy. Accumulating evidences indicate microRNAs (miRNAs), which are
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short non-coding RNAs, delicately regulate the angiogenic signals through targeting angiogenic factors and protein kinases. They can modulate pro-angiogenic signals induced by vascular endothelial growth factor (VEGF) and anti-angiogenic signals
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induced by thrombospondin-1 (TSP-1), and therefore promote or inhibit tumor
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angiogenesis. Receptor tyrosine kinases (RTKs) and hypoxia inducible factor (HIF) are also targeted by miRNAs. Moreover, miRNAs crosstalk with reactive oxygen
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species (ROS) influencing tumor angiogenesis. It is critical to understand the role of
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miRNAs in tumor angiogenesis due to their therapeutic potential to improve outcome
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for cancer patients. The following review discusses the current state of knowledge related to tumor angiogenesis-regulatory miRNAs and their targets.
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Key words: tumor angiogenesis; microRNAs; vascular endothelial growth factor (VEGF);
thrombospondin-1(TSP-1); receptor tyrosine kinases (RTKs); hypoxia
inducible factor (HIF);
reactive oxygen species (ROS)
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ACCEPTED MANUSCRIPT 1. Introduction In 1971, a milestone paper was published by Dr Judah Folkman, with the
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hypothesis that solid tumors caused new blood vessel growth (angiogenesis) in the
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tumor microenvironment through secreting pro-angiogenic factors (Folkman, 1971). Implicit in the paper was the notion that angiogenesis has potential value acting as therapeutic target for cancer treating. In 2010, Kristina M. Cook and William D. Figg
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gave a broad overview of the key mechanisms involved in tumor angiogenesis (Cook
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and Figg, 2010). They discussed the receptor tyrosine kinases (RTKs) signaling which controls angiogenesis and is activated by growth factors, as well as some other
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angiogenic factors. In recent years, microRNAs (miRNAs), the crucial regulators of
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gene expression at the post-transcriptional level, have been shown to regulate
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angiogenesis through directly targeting signaling protein or aniogneic factor mRNAs (Poliseno et al. , 2006, Urbich et al. , 2008, Wang et al. , 2008, Wang and
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Olson, 2009, Wu et al. , 2009). Because miRNA expression is tightly controlled and seems to be specific to different organs and cell types, developing angiogenesis inhibitors targeting miRNAs is a desirable anti-cancer therapy where fewer side-effects might be expected.
2. MiRNAs: New tumor angiogenic factors 2.1. Tumor angiogenesis Tumor requires sustenance in the form of nutrients and oxygen as well as an ability to evacuate metabolic wastes and carbon dioxide. Initially, the growth of a 3
ACCEPTED MANUSCRIPT tumor is fed by nearby blood vessels (Hanahan and Weinberg, 2011). Once a certain tumor size is reached, these blood vessels are no longer sufficient. In order to progress
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to a larger size, new blood vessels are required (Fig.1). The normal vasculature
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becomes largely quiescent in the adult. Pre-existing vessels develop new vessels by sprouting. Solid evidences have proved endothelial-to-mesenchymal transition (EndMT) plays an important role in angiogenic sprouting. EndMT enables the
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so-called tip cells, which lead an emerging vascular plexus, to migrate into adjacent
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tissue. During EndMT, resident endothelial cells delaminated from an organised cell layer, which lost endothelial markers, gained mesenchymal markers and acquired
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invasive and migratory properties, finally (Potenta et al. , 2008).
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The ability to induce and sustain angiogenesis seems to be acquired via an
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“angiogenic switch” from vascular quiescence (Folkman, 1995). Angiogenesis is turned on, as part of physiologic processes such as wound healing and female
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reproductive cycling, but only transiently. In contrast, the ‘‘angiogenic switch’’ is almost always long-lastingly activated during tumor development. A compelling body of evidence indicates that the “angiogenic switch” is the balance between pro-angiogenic and anti-angiogenic signals (Folkman, 1995). Vascular endothelial growth factor-A (VEGFA) and thrombospondin-1 (TSP-1) are the well-known prototypes of angiogenesis inducers and inhibitors, respectively. Additionally, VEGF gene expression can by upregulated both by hypoxia (Chen et al. , 2013) and by ischemia (Li et al. , 2014) in the microenvironment of tumor. TSP-1 evokes suppressive signals that can counteract pro-angiogenic stimuli through direct effects 4
ACCEPTED MANUSCRIPT on endothelial cell migration and survival, and through effects on VEGF bioavailability (Kazerounian et al. , 2008, Tolsma et al. , 1994). The “angiogenic
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switch” is turned on within tumors when pro-angiogenic signals are activated or
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anti-angiogenic signals are sequestered.
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Fig.1. Angiogenesis occurs during cancer development which is caused by activated
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pro-angiogenic signals or sequestered anti-angiogenic signals.
2.2. MiRNAs biogenesis and their targets
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MiRNAs are a class of highly conserved, single-stranded, non-coding small RNAs found in eukaryotes that regulate gene expression post-transcriptionally. The biogenesis of mature miRNAs is a complex and multistep process (Fig. 2). The primary miRNA transcripts (pri-miRNA) are generated by RNA polymerase II or III in the nucleus (Lee et al. , 2004). Pri-miRNA processing occurs in two steps. In the first step, pri-miRNAs are cleaved by the microprocessor complex to form the precursor miRNA (pre-miRNA) (Lee et al. , 2003). The microprocessor contains Drosha and the dsRNA-binding proteins (dsRBPs), such as DGCR8 protein. And the new formed pre-miRNA will be exported to the cytoplasm by Exportin 5 (Yi et al. , 5
ACCEPTED MANUSCRIPT 2003). Then pre-miRNAs are cleavage by Dicer (Kuehbacher et al. , 2007), assisted by transactivation-responsive (TAR) RNA-binding protein (TRBP) generating an
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~20-bp miRNA/miRNA* duplex (Maniataki and Mourelatos, 2005). Following
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processing, the guide strand of the miRNA duplex, which contains the seed sequence, associates with Argonaute 2 (Ago2) protein to form the miRNA-induced silencing complex (miRISC) (Donker et al. , 2007), whereas the other strand (passenger or
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miRNA*) is released and degraded. The miRISC then binds to the 3’-untranslated
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region (3’-UTR) of the target mRNA and mediates sequence-specific gene silencing through mRNA destabilization and translational repression (Fabian and Sonenberg,
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2012, Maniataki and Mourelatos, 2005). Mature miRNAs recognize their target
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mRNAs through base-pairing interactions between nucleotides numbering 2 to 8 of
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the miRNA (the seed region) and the complementary nucleotides in the 3’-UTR of the
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mRNAs (Fabian and Sonenberg, 2012) (Fig. 2).
Fig. 2. Biogenesis of mature miRNAs. Pri-miRNA is processed by Drosha and Dicer, operating in complexes with DGCR8 and TRBP, which generate a miRNA/miRNA* 6
ACCEPTED MANUSCRIPT duplex. The guide strand of the miRNA/miRNA* duplex is preferentially incorporated into a miRISC, whereas miRNA* strand is released and degraded. Following miRISC
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biogenesis, various target genes of miRNA are silenced at post-transcription level.
It is estimated that there are more than 1,000 miRNA genes in the human genome, and these could regulate more than one-third of the mRNAs produced (Lewis et al. ,
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2005). In recent years, some researches revealed that miRNAs modulate tumor
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angiogenesis through targeting pro-/anti-angiogenic factors including RTKs signaling proteins, hypoxia inducible factor (HIF), VEGF, TSP-1 and reactive oxygen species
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(ROS) causing tumor developed or arrested (Tab.1). It should be noticed that many
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miRNAs are expressed in cancer- and tissue-specific patterns, suggesting that
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miRNAs have cell type-specific functions. As miRNAs play an important role in tumor angiogenesis, we will discuss the latest examples that illustrate the role of
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miRNAs during tumor angiogenesis below.
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Tab. 1. MiRNAs target pro-/anti- angiogenesis factors in different cancer cells. cell type
up/down
pro-/anti-
target
regulated
cells
up
PTEN
Human prostate cancer
PTEN
cells
pro
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miR-21
human gastric cancer
Spred-1/PIK3R2
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miR-382
EC
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RTKs signaling-related miRNAs miR-126
references
angiogenesis
T
miRNAs
(Wang et al., 2008) (Fish et al. , 2008)
pro
(Seok et al. , 2014)
pro
(Liu et al. , 2011)
HUVECs
up
RhoB
anti
(Sabatel et al. , 2011)
miR-26a
HCC
up
PIK3C2α
pro
(Chai et al. , 2013)
miR-218
mesenchymal GBM
down
anti
(Mathew et al. , 2014)
miR-18a
gastric cancer
up
mTOR
anti
(Zheng et al. , 2013)
p70S6K1
anti
(Xu et al. , 2012)
HIF-1α
anti
(Cha et al. , 2010)
HIF-1α
anti
(Yamakuchi et al. , 2011)
HIF-1β
anti
(Yamakuchi et al. , 2010)
cells
/PLCγ1/ARAF
D
human colon cancer
PIK3C2α
down
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miR-145
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miR-21
HIF-targeted miRNAs
miR-107
up
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miR-22
HUVECs human colon cancer cells
down
human colon cancer cells
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miR-519c
VEGF-targeted miRNAs
miR-497
ovarian cancer cells
down
VEGFA
anti
(Wang et al. , 2014)
miR-503
HCC
down
VEGFA/FGF2
anti
(Zhou et al. , 2013)
down
VEGFA
anti
(Zhang et al. , 2013)
TSP-1
pro
(Sundaram et al. , 2011)
miR-126
human breast cancer cells
TSP-1-targeted miRNAs miR-194 miR-17-92 miR-467
HCT116 CRC cells ovarian endometrioma
down
TSP-1
pro
(Ramon et al. , 2011)
breast cancer cells
up
TSP-1
pro
(Bhattacharyya et al. , 2012)
pro
(Zhang et al., 2012)
ROS signaling-related miRNAs miR-21
human bronchial epithelial cells
TNFα/SOD3
8
ACCEPTED MANUSCRIPT 3. Signals regulated by miRNAs in tumor angiogenesis Fig. 3 gives a frame of modulation of tumor angiogenesis by miRNAs. Fibroblast
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growth factor 2 (FGF2), VEGFA and EGF, three of the most potent pro-angiogenic
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factors, bind transmembrane receptors displayed by endothelial cells and thereby stimulate angiogenesis. FGF2 and VEGFA are respectively down-regulated by miR-503, miR-497 and miR-126, among which miR-503 binds 3’-UTRs of both
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FGF2 and VEGFA (Fig. 3). The combination of growth factors and their receptors
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activate RTKs signaling and related kinase proteins. Lots of miRNAs directly or indirectly repress kinase proteins involved in PI3K/mTOR or RAF/ERK pathways,
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including miR-382, miR-21, miR-126, miR-26a, miR-18a, miR-145 and miR-218
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(Fig. 3). Moreover, HIF complex is a major regulator of tumor angiogenic genes
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under low oxygen stress, down-regulated by miR-519c, miR-22 and miR-107 (Fig. 3). In addition, TSP-1, a key counterbalance in the “angiogenic switch”, could counteract
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pro-angiogenic signals induced by growth factors. And anti-angiogenic signals induced by TSP-1 are also influenced by miRNAs, such as miR-17-92, miR-194 and miR-467 (Fig. 4). ROS, as tumor angiogenesis promoters, also interact with RTKs signaling which is modulated by miR-21 (Fig. 3).
9
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ACCEPTED MANUSCRIPT
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Fig. 3. MiRNAs regulate pro-angiogenic signals and ROS. Pro-angiogenic factors,
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such as growth factors (VEGF, EGF and FGF2), bind to their receptors on the cell membrane which activate the downstream RTKs signaling pathway causing HIF and
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other angiogenic gene expression. MiRNAs modulate tumor angiogenesis by targeting the proteins related to RTKs signaling, HIF and growth factors. ROS regulated by
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miR-21also crosstalk with RTKs signaling.
3.1. RTKs signaling-related miRNAs AKT (protein kinase C) /extracellular signal-regulated kinase (ERK) signaling can be activated by the binding of a growth factor ligand to RTKs therefore to initiate processes like angiogenesis (Forough et al. , 2005, Jiang, 2008, Zhong et al. , 2000). The signaling cascades include the Phosphatidyl Inositol 3-kinase (PI3K)-AKTmammalian target of rapamycin (mTOR) and phosphor-lipase C-γ (PLCγ) -Protein Kinase C（PKC）-RAF kinase-EKR pathways. Some miRNAs control the expression 10
ACCEPTED MANUSCRIPT of kinase proteins in these pathways, leading to pro-/anti-angiogenesis, including miR-126, miRNA-382, miR-21, miR-26a and miRNA-218.
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MiR-126, an endothelial cell-restricted miRNA, was shown to enhance the
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pro-angiogenic actions of VEGF and FGF and promote blood vessel formation by repressing the expression of Spred-1 (Wang et al., 2008) and PIK3R2, intracellular inhibitors of AKT and RAF kinase (Fish et al. , 2008).
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MiR-382 and miR-21 both modulate angiogenesis by repressing phosphatase and
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tensin homolog (PTEN). MiR-382 induced by hypoxia in MKN1 human gastric cancer cells promoted angiogenesis. It acted as an angiogenic oncogene by
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down-regulating PTEN and its downstream target AKT/mTOR signaling pathway
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(Seok et al. , 2014). Similarly, miR-21 induced tumor angiogenesis through targeting
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PTEN, leading to activating AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression (Liu et al. , 2011). But another study
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revealed that miR-21 acted as a negative modulator of angiogenesis (Sabatel et al. , 2011). They found miR-21 overexpression in human umbilical vein endothelial cells （HUVECs）reduced endothelial cell proliferation, migration and the ability to form tubes through repression of RhoB, a gene that involved in angiogenesis regulation. In spite of the indirect impact on RTKs signaling, miR-26a and miR-218 have been confirmed to interact with PIK3C2α (class II PI3Ks) mRNA and inhibit its protein expression. miRNA-26a inhibited angiogenesis by down-regulating VEGFA through the PIK3C2α/ AKT/HIF-1α pathway in Hepatocellular Carcinoma（HCC） (Chai et al. , 2013). The expression of miR-218 was decreased significantly in highly 11
ACCEPTED MANUSCRIPT necrotic mesenchymal glioblastoma multiforme (GBM), and miR-218 targeted EGF receptor (EGFR) and multiple components of RTKs signaling pathways including
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PLCγ1, PIK3C2A, and v-raf murine sarcoma 3611 viral oncogene homolog (ARAF)
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(Mathew et al. , 2014). MiR-218 repression up-regulated the abundance and activity of multiple RTK effectors, which elevated RTKs signaling and promoted the
angiogenesis has not been determined.
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activation of HIF, most notably HIF2α. However, the direct effect of miR-218 on
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The miR-17-92 cluster has long been recognized as an oncogenic miRNA cluster and is amplified in multiple cancers (Hayashita et al. , 2005, Melegari et al. , 2006).
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Overexpression of miR-18a, a member of the miR-17-92 cluster, substantially
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reduced the phosphorylation of two mTOR substrates, S6K1 and 4E-BPl, indicating
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inactivation of the mTOR pathway in gastric cancer (Zheng et al. , 2013). Accompanying with the mTOR inactivation, the angiogenic factors, HIF-1α and
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VEGF, were significantly down-regulated. These data highlighted an important role for miR-18a in controlling gastric cancer growth and angiogenesis. MiR-145 is another miRNA directly repressing RTKs signaling, with down-regulated expression in colon and ovarian cancer tissues and cell lines. In human colon cancer cells, miR-145 inhibited post-transcriptional expression of p70S6K1, one of the downstream targets of mTOR, by binding to its 3’-UTR. The angiogenic factors HIF-1 and VEGF, which are downstream molecules of p70S6K1, were decreased by miR-145 overexpression (Xu et al. , 2012).
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ACCEPTED MANUSCRIPT 3.2. HIF-targeted miRNAs HIF is a heterodimeric complex that consists of a hypoxia-inducible, unstable
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α-subunit and a stable, constitutively expressed β-subunit. Three HIF-α isoforms have
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been identified. The HIF-1α subunit is continuously synthesized and degraded under normoxic conditions. Therefore, HIF-1 complex does not function during normal oxygen tension (Salceda and Caro, 1997). But hypoxia that occurs in the
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microenvironment of tumors can stabilize the HIF-1 complex. Once stabilized, the
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HIF-1 complex can bind to the promoter regions of its target genes and thereby induce target gene expression.
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HIF-dependent transcriptional changes regulate a broad spectrum of cellular
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functions, including angiogenesis (Liao and Johnson, 2007, Theodoropoulos et al. ,
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2004). Thus, miRNAs that target HIF are likely to have significant impacts on the angiogenesis pathways. Several miRNAs directly target HIF mRNA: miR-20a,
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miR-20b and miR-199a miR-519c, miR-22, miR-107, among which miR-20a, miR-20b and miR-199a have been discussed in another review (Madanecki et al. , 2013). The paper focuses on miR-519c, miR-22 and miR-107. Overexpression of miR-519c resulted in a significant decrease of HIF-1α protein levels and reduced the tube formation of HUVECs, suggesting an important role of miR-519c in HIF-1α–mediated angiogenesis (Cha et al. , 2010). In addition, hepatocyte growth factor (HGF), a known HIF-1α inducer, reduced the miR-519c levels through an AKT-dependent pathway. miR-22 was found to decrease its expression in human colon cancer compared with normal colon tissue, and acted as 13
ACCEPTED MANUSCRIPT anti-angiogenic factor through inhibiting HIF-1α expression, repressing VEGF production during hypoxia (Yamakuchi et al. , 2011). MiR-107 is a miRNA expressed
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in human colon cancer specimens directly regulated by p53 which can match 3’-UTR
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of HIF-1β. Furthermore, overexpression of miR-107 in tumor cells suppressed tumor angiogenesis, tumor growth, and tumor VEGF expression in mice. These data suggested that miR-107 could mediate p53 regulation of hypoxic signaling and tumor
3.3. VEGF-targeted miRNAs
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angiogenesis by targeting HIF-1β (Yamakuchi et al. , 2010).
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VEGF and its receptor tyrosine kinase (VEGFR) play key roles in angiogenesis
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and VEGF gene is a major transcriptional target of HIF (Ferrara et al. , 2003, Lee et
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al. , 2008). VEGF is actually a family of at least seven members, and the term VEGF typically refers to the VEGFA isoform, one of the most studied members and a major
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pro-angiogenic factor. VEGF binds to two tyrosine kinase receptors, VEGF receptor-1 (VEGFR1, and VEGFR2) in endothelial cells (ECs). The mitogenic and chemotactic effects of VEGF in ECs are mediated mainly through VEGFR2 which is activated through autophosphorylation of tyrosine residues in the cytoplasmic kinase domain. This event is followed by activation of downstream signaling pathways such as AKT, which is essential for EC migration and proliferation. Recent studies revealed that several miRNAs, such as miR-497, miR-503 and miR-126, were all able to match 3’-UTR of VEGFA mRNA. MiR-497 expression was down-regulated in human ovarian cancer tissues, which 14
ACCEPTED MANUSCRIPT was significantly associated with increased angiogenesis (Wang et al. , 2014). Further study disclosed that miR-497 exerted its function of anti-angiogenesis by suppressing
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VEGFA expression and, in turn, impairing the VEGFR2-mediated PI3K/AKT and
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MAPK/ERK pathways.
MiR-503 expression was down-regulated by hypoxia through HIF-1α in HCC cells and primary tumors. MiR-503 could simultaneously down-regulate VEGFA and
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FGF2 in cancers, demonstrating the anti-angiogenesis role in tumorgenesis (Zhou et
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al. , 2013).
MiR-126 was illustrated to antagonize tumor angiogenesis by inhibiting Spred-1
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and PIK3R2. In addition, miR-126 could directly target VEGFA, and its expression
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was decreased in human breast cancer, implying that miR-126 might play a role in
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tumorgenesis and growth by regulating the VEGF/PI3K/AKT signaling pathway
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(Zhang et al. , 2013).
3.4 TSP-1-targeted miRNAs Thrombospondin-1 (TSP-1) is a glycoprotein influencing angiogenesis by effects on ECs. In the extracellular matrix of ECs, TSP-1 inhibits metalloproteinases (MMPs) activation, and consequently the release of VEGF (Fig.4). Thence, there is a crosstalk between pro-angiogenic signals induced by growth factors and anti-angiogenic signals induced by TSP-1 (Fig. 4). Moreover, TSP-1 induces the activation of CD36 which leads to inhibition of migration and induction of apoptosis (Dawson et al. , 1997, Jimenez et al. , 2000) (Fig. 4). 15
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Fig. 4. Anti-angiogenic signals modulated by miRNAs. TSP-1 is the main
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anti-angiogenic factor influencing EC phenotype and their apoptosis, and it could be
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repressed by several miRNAs, such as miR-194, miR-17-92 and miR-467.
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TSP-1 is encoded by the THBS1 gene, whose promoter is activated by p53. However, it was reported that in HCT116 colorectal cancers (CRC) cells, p53 activated the THBS1 primary transcript, but failed to boost THBS1 mRNA or protein levels, implying potential post-transcriptional regulation by miRNAs. Further researches revealed that miR-194 negatively regulated THBS1 mRNA and protein levels.
Notably,
p53
can
upregulated
miR-194
expression
in
THBS1
retrovirus-transduced HCT116 cells, leading to decreased TSP-1 levels (Sundaram et al. , 2011). In addition, members of the miR-17-92 cluster, specifically miR-18a and miR-19, 16
ACCEPTED MANUSCRIPT negatively regulated TSP-1 levels during Myc-induced angiogenesis (Dews et al. , 2006). MiR-17-92 cluster localized on 13q31 was upregulated in colonocytes
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co-expressing K-Ras and c-Myc of human adenocarcinomas. The level of
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up-regulated miR-17-92 is directly proportional to the level of down-regulated TSP-1, reducing neovascularization of tumor (Dews et al. , 2006). Consistent with previous results, a research by Luis A. Ramon ´and his colleagues showed ovarian
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endometrioma had significantly higher expression of TSP-1 and lower expression of
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miR-17-5p than those of eutopic endometrium. Moreover, significant inverse correlations between miR-17-5p and TSP-1 protein levels, and between miR-222 and
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VEGF-A protein levels, were observed (Ramon et al. , 2011). Ramon et al. observed a
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significant increase in TSP-1 protein levels in endometrial cancer as well (Ramon et
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al. , 2012). Conversely, although TSP-1 is an anti-angiogenic factor, a significant positive correlation between TSP-1 protein levels and miR-210, which promoted
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angiogenesis in endometrial carcinoma, was obtained (Ramon et al. , 2012). MiR-467 is implicated in the control of angiogenesis in response to high glucose. It has also been identified as a translational suppressor of TSP-1 that is implicated in the pathogenesis of several diabetic complications (Bhattacharyya et al. , 2012). MiR-467 was upregulated by high glucose in microvascular endothelial cells and in breast cancer cells, where it suppressed the production of TSP-1 by sequestering its mRNA in the nonpolysomal fraction. In in vivo angiogenesis models, miR-467 promoted the growth of blood vessels, and TSP-1 was the main mediator of this effect (Bhattacharyya et al. , 2012). 17
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4. Crosstalk between ROS and miRNAs in tumor angiogenesis
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A lot of evidences suggest that ROS function as signaling molecules to mediate
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various growth-related responses including angiogenesis (Oshikawa et al. , 2010). Superoxide (O2.-) is the main initial ROS, which can be converted to other reactive species including hydrogen peroxide (H2O2). ROS can be generated by NADPH
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oxidase (NOX) or induced by extracellular stimuli such as ionizing radiation (IR). In
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turn, NOX is activated by numerous stimuli including VEGF, EGF, hypoxia and ischemia (Fig. 5). Interaction between ROS and miRNAs might be a critical factor for
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angiogenesis.
Fig. 5. Crosstalk between ROS and miRNAs in the regulation of tumor development. Certain concentrations of H2O2 up-regulates miR-200c expression level in HUVECs. In turn, miR-200c down-regulates VEGFA, leading to angiogenesis arrest. In various 18
ACCEPTED MANUSCRIPT types of tumor cells, miR-27a, miR-21 and miR-210 have close relationship with ROS
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generation and function as pro-/anti-tumor development.
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4.1. MiRNAs modulated by ROS
Oxidative stress has been demonstrated to play a crucial role in tumor angiogenesis and miRNAs have been shown to be modulated in cellular responses to
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redox imbalance. A miRNA profiling of HUVEC treated with H2O2, showed an
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up-regulation of miR-200 family members (Magenta et al. , 2011). This miRNA family consists of five members, miR-200c and miR-141 clustered on chromosome 12
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and miR-200a, miR-200b, and miR-429 clustered on chromosome 1. In particular,
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miR-200c and miR-141 are the most up-regulated miRNAs in HUVEC, exposed to
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H2O2 for different periods of time, whereas the other three clustered members are up-regulated to a lower extent (Magenta et al., 2011). And it has been reported that
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miR-200c targets VEGFA (Chuang et al. , 2012). Taken together, certain concentrations of H2O2-induced miR-200c leads to angiogenesis arrest through inhibiting VEGFA (Fig. 5).
GT-094, a nitric oxide chimera containing a nonsteroidal anti-inflammatory drug, down-regulated genes associated with angiogenesis, such as VEGF and its receptors (VEGFR1 and VEGFR2) (Pathi et al. , 2011). In colon cancer cells it was accompanied by decreased mitochondrial membrane potential (MMP) and increased ROS, which inhibited miR-27a expression. Down-regulation of miR-27a induced 19
ACCEPTED MANUSCRIPT ZBTB10, a repressor of specificity protein (Sp) transcription factors, and therefore caused repression of Sp and Sp-regulated gene products repression (Pathi et al., 2011).
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In addition, the miR-27a expression was significantly increased in VEGF-treated
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breast cancer stem like cells (BCSLCs). Increased miR-27a paralleling the reduced expression of ZBTB10 in BCSLCs promoted in vivo angiogenesis and tumor
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metastasis (Tang et al. , 2014) (Fig. 5).
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4.2. MiRNAs modulate ROS
Dicer is the key enzyme controlling miRNA biogenesis. Silencing Dicer in vitro
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impaired the angiogenic response of HUVECs and EA.hy.926 ECs (Shilo et al. ,
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2008). Global lowering of miRNA levels caused by Dicer knockdown withdrew the
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negative control of miRNA on HBP1 expression, resulting in higher levels of HBP1. Elevated HBP1, in turn, down-regulated p47phox expression and limited
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ROS-dependent angiogenic signaling (Shilo et al., 2008) . MiR-21 was reported to inhibit PTEN or RhoB, leading to pro-/anti-angiogenesis (Zhang et al. , 2012). In addition, miR-21 triggered the generation of ROS that promoted tumorigenesis (Zhang et al., 2012). MiR-21 inhibited the metabolism of O2.to H2O2, by directing attenuating SOD3 or by an indirect mechanism that limited TNFα production, thereby reducing SOD2 levels. Therefore, miR-21-induced tumorigenesis is partially due to the high O2.- level generated in the cells (Fig. 5). MiR-210, a main mitochondrial miRNA, has been demonstrated to play a key role in mitochondrial metabolism, therefore modulating ROS production (Qin et al. , 20
ACCEPTED MANUSCRIPT 2014) (Fig. 5). It was found that miR-210 increased the formation of ROS in MCF7 and HCT116 cell lines (Favaro et al. , 2010). In hypoxic HCC (Ying et al. , 2011) and
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CRC cells (Qin et al., 2014), vacuole membrane protein 1 (VMP1) was identified as
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the direct and functional downstream target of miR-210, which mediated cancer cell migration and invasion supported by angiogenesis. Meanwhile, miR-210 contained in exosomes released by cancer cells could be transported to endothelial cells to induce
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angiogenesis.
5. Tumor-specific miRNAs
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As limited information is known about tumor-type specificity of miRNAs, we
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here discuss some tumor-specific miRNAs which function differently.
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The expression level of miR-145 (Xu et al., 2012), miR-22 (Li et al. , 2011) and miR-126 (Zhang et al., 2013) decreased in colorectal cancer cells compared with the
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normal cells and these three miRNAs respectively targeted p70S6K1, HIF-1α and VEGF mRNA. HIF-1 and VEGF are downstream molecules of p70S6K1 pathway. Therefore, the expression of p70S6K1, HIF-1α and VEGF would get out of control, leading to promoted tumor angiogenesis. In addition, miR-126 acted as anti-angiogenic factor in non-small cell lung cancer cells by causing VEGFA arrest. In highly necrotic mesenchymal GBM cells, the expression of miR-218 was decreased significantly (Mathew et al., 2014). MiR-218 targets multiple components of RTKs signaling pathways, and miR-218 repression increases the abundance and activity of multiple RTK effectors, promoting the activation of HIF, most notably 21
ACCEPTED MANUSCRIPT HIF2α. The miR-218/RTK/HIF2α signaling promoted cell survival and tumor angiogenesis, particularly in necrotic mesenchymal tumors (Mathew et al., 2014).
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Similarly, in bladder cancer cells, miR-10b, 19a, 126, 145, 221, 296-5p and 378
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were significantly down-regulated, among which miR-145 is the most prominent one and the overall survival rate in patients with high expression of miR-21 is very low (Zaravinos et al. , 2012). Multivariate analysis revealed that miR-21, 210 and 378
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might serve as independent prognostic factors for overall patient survival. And
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miR-21 and 378 also could serve as independent prognostic factors for recurrence. Especially, miR-27a mediated hypoxia-induced down-regulation of a new angiogenic
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factor, AGGF1 in high-grade bladder urothelial carcinoma cells (Xu et al. , 2014).
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In contrast, in human gastric cancer cells, the expression of miR-382 (Seok et al.,
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2014) and miR-18a (Zheng et al., 2013) were up-regulated. MiR-382 could match to the 3’-UTR of PTEN leading to inhibition of tumor angiogenesis, while miR-18a
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functioned through inactivating the mTOR signaling pathway. Up-regulation of both miRNAs could impair tube formation of tumor. MiR-21 was reported to induce tumor angiogenesis through targeting PTEN in human prostatic cancer cells, leading to activating AKT and ERK1/2 signaling pathways, and thereby enhance HIF-1α and VEGF expression (Liu et al., 2011). HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.
6. Conclusion It is critical to understand the role of miRNAs in tumor angiogenesis due to their 22
ACCEPTED MANUSCRIPT therapeutic potential to improve outcome for cancer patients. Now abundance variation of many miRNAs in specific tumor have been tested. Wu and his colleagues
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produced a miRNA sensor, which not only enabled the quantitative and highly
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specific detection of multiplexed miRNAs in living cancer cells, but also allowed the precise and in situ monitoring of the spatiotemporal changes of miRNA expression (Wu et al. , 2015). Moreover, some miRNA expression profiles in cancer cells have
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been used in the diagnosis of cancer, such as miR-18a and miR-155 (Komatsu et al.,
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2014, Sochor et al. , 2014). Toward cancer therapy, various approaches have been used to regulate the aberrant expression of miRNAs. On one side, anti-miRNA
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molecules and miRNA scavengers have been used to attenuate the up-regulated
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oncogenic miRNAs (Tay et al. , 2015). The mice treated with antisense-miR-21-PS
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and antisense-miR-10b-PS delivered by polymer nanoparticles simultaneously antagonizing miR-21-induced anti-apoptosis and miR-10b-induced metastasis
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(Devulapally et al. , 2015). The treated mice showed substantial reduction in tumor growth compared to the control mice (Devulapally et al. , 2015). On the other side, some specific miRNAs are down-regulated in tumor cells, such as miR-26a in HCC cells. To counter such conditions, miRNA mimics have been used to restore the loss of function induced by the low expression of miRNAs (Kota et al. , 2009). Significant advances have been made for the discovery of the miRNAs, which play a crucial role in the angiogenesis of a specific tumor type by altering a whole network of target proteins. However, there have been far fewer reported successes in the accurate prevision of the combined effects of different angiogenesis-regulated 23
ACCEPTED MANUSCRIPT miRNAs on tumor developing. The number of discoveries, increasing so fast in the last few years, have paved the way for the use of miRNAs as the targeted therapy of
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the future, though there are some challenges.
Conflict of interest statement
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The authors declare that there are no conflicts of interest.
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Acknowledgments
The authors thank the members of the Free Radical Biology Laboratory (School
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of Life Sciences, Lanzhou University) for helpful discussion. This work was
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supported by the grant from the National Natural Science Foundation of China
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(31171121).
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Graphical Abstract
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