MicroRNAs and Tumor Vasculature Normalization: Impact on Anti-Tumor Immune Response

REVIEW

MicroRNAs and Tumor Vasculature Normalization:Impact on Anti-Tumor Immune Response

Agata Matejuk • Guillaume Collet •

Mahdi Nadim • Catherine Grillon • Claudine Kieda

Received: 20 July 2012 / Accepted: 15 January 2013 / Published online: 11 April 2013

� L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2013

Abstract Inefficient immune response is a major glitch

during tumor growth and progression. Chaotic and leaky

blood vessels created in the process of angiogenesis allow

tumor cells to escape and extricate anti-cancer immunity.

Proangiogenic characteristics of hypoxic tumor microen-

vironment maintained by low oxygen tension attract

endothelial progenitor cells, drive expansion of cancer stem

cells, and deviantly differentiate monocyte descendants.

Such cellular milieu further boosts immune tolerance and

eventually appoint immunity for cancer advantage. Blood

vessel normalization strategies that equilibrate oxygen

levels within tumor and fix abnormal vasculature bring

exciting promises to future anticancer therapies especially

when combined with conventional chemotherapy.

Recently, a new group of microRNAs (miRs) engaged in

angiogenesis, called angiomiRs and hypoxamiRs, emerged

as new therapeutic targets in cancer. Some of those miRs

were found to efficiently regulate cancer immunity and

their dysregulation efficiently programs aberrant angio-

genesis and cancer metastasis. The present review

highlights new findings in the field of miRs proficiency to

normalize aberrant angiogenesis and to restore anti-tumor

immune responses.

Keywords MicroRNAs regulation � Hypoxia �Angiogenesis � Cancer � Vessels normalization �Tumor immune response

Introduction

Vasculogenesis and angiogenesis are highly regulated

processes. In physiological conditions they are abundant

during fetus development but restricted to wound healing

and menstrual cycles during adulthood. They become

prominent in pathological conditions such as cancer, dia-

betic retinopathy, macular degeneration, atherosclerosis,

and arthritis. On the other hand, inefficient angiogensis is a

concern during stroke and coronary arterial disease.

Hypoxia plays critical role in initiation of molecular events

directly leading to formation of new blood vessels.

Hypoxia-inducible factor (HIF) family by its pivotal

influence on a variety of genes including vascular endo-

thelial growth factor (VEGF) is a key regulator of tumor

angiogenesis (Gordan and Simon 2007; Gruber and Simon

2006). Hypoxia-dependent growth factors and pro-angio-

genic genes activate previously quiescent endothelial cells,

induce proinflamatory factors, degrade and remodel

extracellular matrix, and increase cancer cell mobility and

their dedifferentiation. All these events lead to progressive

tumor angiogenesis and result in immature, leaky, and

unevenly distributed vessels leading to inefficient engage-

ment of anti-cancer immune response and poor

accessibility of tumor cells to chemotherapeutic drugs.

Additionally, persisting low oxygen levels in tumor mass

render it unresponsive to radiotherapy (Jordan and Son-

veaux 2012).

Anti-angiogenic agents are now being approved and

introduced in clinics for the prevention and treatment of

A. Matejuk (&) � G. Collet � M. Nadim � C. Grillon � C. Kieda

Centre de Biophysique Moleculaire, CNRS UPR 4301, rue

Charles Sadron, 45071 Orleans, France

e-mail: [email protected]

A. Matejuk

Le Studium�, Loire Valley Institute for Advanced Studies, 3D,

avenue de la Recherche Scientifique, 45071 Orleans, France

M. Nadim

Libragen–Induchem Company, 3, rue des Satellites, Bat. Canal

Biotech, 31400 Toulouse, France

Arch. Immunol. Ther. Exp. (2013) 61:285–299

DOI 10.1007/s00005-013-0231-4

123

cancer. However, the benefits remain modest. As much as

these therapies bring exciting results majority of tumors

eventually become refractory. Monoclonal antibody-based

therapies are mostly directed towards the main key players

in angiogenesis: VEGF and its receptor VEGFR2. Indeed

they were found to efficiently target vasculature but the

effect was only transient (Carmeliet and Jain 2011a).

Especially anti-VEGF monotherapies failed (Carmeliet and

Jain 2011a; Cobleigh et al. 2003; Giantonio et al. 2007;

Jain et al. 2006) since anti-VEGF/VEGFR treatments boost

hypoxic state that further allow for selection of highly

resistant and aggressive stem-like cancer cells with pro-

gressively lower response to conventional therapies.

Accumulating data clearly show that cutting off the blood

flow to the tumor initiates atrocious compensatory mech-

anisms. In contrast, blood vessel normalization approaches

that fix the leaky, twisty blood vessels that feed tumors,

thereby improving the delivery of drugs are encouraging

and are under fast development (Carmeliet and Jain 2011b;

Jain 2009). Consequently, blood vessel normalization

concept covers and targets hypoxia, angiogenesis, cancer

stem cells differentiation, radio-, drug-response efficacy,

and reestablishment of anti-tumor immune response. Cur-

rent aims of anti-angiogenic therapy is to create a

normalization window where blood vessels regain their

typical structure with decreased permeability for better

drug penetration (Jain 2005; Tong et al. 2004; Wildiers

et al. 2003).

Recently, new opportunities for better understanding of

tumor biology came with discovery of microRNAs (miRs)

a new class of gene regulatory molecules. miRs are 19–25

single-stranded RNAs capable of targeting cleavage and/or

mRNA translational inhibition or inducing mRNA insta-

bility. Those non-coding RNAs control the expression level

of genes involved in development, proliferation, differen-

tiation, and apoptosis (He and Hannon 2004). Alterations in

miRs synthesis pathways and processing have important

consequences for both physiological and pathological

processes such as development and cancer (Winter et al.

2009). In fact, miRs have been shown to affect the hall-

marks of cancer, including angiogenesis and aberrant

immune response and their profile and level of expression

are changed in malignancies. Presently, more than 1400

human miRs are identified (Kozomara and Griffiths-Jones

2011) and the number is growing. A single miR can reg-

ulate a variable number of targets (Thomas et al. 2010).

Deeper understanding of the crosstalk between tumor and

immune cells in a language of angio-and/or hypoxa-miRs

should unravel miRs implications and potential new targets

for treatment and diagnostics. Recent review by Hartmann

and Thum (2011) describes in detail individual miRs

involved in vascular (dys)function. Caporali and Emanueli

(2011) present miRs that regulate angiogenesis. In this

review, we briefly discuss miRs that control tumor angi-

ogensis and have potential to target aberrant anti-cancer

immune responses.

Blood Vessel Normalization Paradigm

Vascular normalization hypothesis was first introduced by

Jain (2001). Tumor hypoxic environment boosts produc-

tion of VEGF and other proangiogenic factors and initiates

angiogenesis that results in abnormal blood vessel network.

Tumor vessels are highly irregular; the junctions among

endothelial cells and connective tissue cells such as peri-

vascular cells are loose, leading to leaky vascular structure

with multiple fenestrations (Fukumura et al. 2010; Nagy

et al. 2009). Pseudo-vessel formation is also linked to a

decrease in endothelial cell ability to adhere to each other.

Dysregulation of normal vessel maturation has catastrophic

consequences for any given anti-cancer therapy. Hyper-

permeable phenotype allows metastatic tumor cells to

escape and strongly prevents infiltration of anti-cancer

immunity and drug penetration to the tumor mass. It also

poorly delivers oxygen further enhancing hypoxic state

(Hockel and Vaupel 2001). Hypoxic condition induces

dedifferentiation of cells, epithelial-mesenchymal transi-

tion, increased cell migration and invasion, and high

resistance to chemo-, radio- (Teicher 1996), and immuno-

therapy (Noman et al. 2011). It also precludes cytotoxicity

of anti-cancer immunity (Ganss et al. 2004; Hamzah et al.

2008). In clinical applications, anti-VEGF monotherapy

that targets vascular recession by cutting off blood supply

was significantly unsuccessful. However, direct targeting

of VEGF as addition to standard chemotherapy showed

improvements in patient outcomes (Saltz et al. 2008;

Tebbutt et al. 2010). To explain this paradox Jain proposed

the ‘‘vascular normalization’’ hypothesis. It states that

cautious use of anti-angiogenic therapy creates a ‘‘nor-

malization window’’ that reverses aberrant angiogenesis,

leads to normalized vascular phenotype, and reduces vessel

permeability and hypoxia (Goel et al. 2011, 2012). Direct

or indirect anti-angiogenic therapy regulates equilibrium

between pro- and anti-angiogenic factors and controls

pathways crucial for maintenance of healthy angiogenesis.

Besides controlling VEGF levels, direct anti-angiogenic

therapies regulate angiopoietin-Tie2 pathway (Falcon et al.

2009), expression of placental growth factor (PlGF)

(Fischer et al. 2007; Van de Veire et al. 2010), endothelial

cell-specific integrins (Desgrosellier and Cheresh 2010;

Primo et al. 2010; Skuli et al. 2009), and the prolyl-4-

hydroxylase domain (PHD) tumor-oxygen sensor proteins

(Mazzone et al. 2009). The latter senses low oxygen ten-

sion and by predisposing to hypoxia generates abnormal

vessels. PHD2?/- mice displayed reduced metastasis due

286 Arch. Immunol. Ther. Exp. (2013) 61:285–299

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to decreased number of cancer cells from primary tumor in

blood stream (Mazzone et al. 2009). A recent study shows

that gene-targeting PHD2 improves tumor response to

chemotherapy and prevents side-toxicity (Leite de Oliveira

et al. 2012). Indirect inhibitors of angiogenesis target tumor

cell oncogenes such as HER-2, the PI3K-AKT-mTOR axis,

Ras and epidermal growth factor receptor, and include

endocrine therapies and metronomic chemotherapy (Goel

et al. 2012). The goal in ‘‘blood vessel normalization’’

strategy is to balance the action of pro-angiogenic factors

such as VEGF, basic fibroblast growth factor (bFGF), and

angiopoietin-2 (Ang-2) with action of anti-angiogenic

mediators such as thrombospondin-1 (TSP-1) and Ang-1

(Jain 2003; Relf et al. 1997). Ang-1 is an agonist of

endothelial Tie-2 receptor promoting blood vessel nor-

malization and Ang-2 is an antagonist of the same receptor

leading to increased angiogenesis. Ang-Tie-2 axis is crucial

in regulating both healthy and tumor-associated angio-

genesis (Huang et al. 2010). A recent approach that might

lead to blood vessel normalization is based on the regula-

tion of oxygen partial pressure within tumor mass.

Previously Kieda et al. (2006) and Sihn et al. (2007)

showed that myo-inositol trispyrophosphate (ITPP), a

ubiquitous carbohydrate and allosteric effector of hemo-

globin causing oxygen release, was able both in vitro and

in vivo to inhibit hypoxia-induced angiogenesis. Studies on

ITPP’s ability to normalize oxygen tension in tumor are

ongoing.

The Role of Blood Vessel Normalization Strategies

in Anti-Cancer Immune Responses

The ability to create metastasis and neovascularization

strongly depends on active interplay between tumor cells

and their microenvironment including extracellular matrix,

endothelial cells, and immune cells. Proper immune

responses achieved mainly by natural killer (NK) cells and

cytotoxic T cells are paramount in the eradication of can-

cer. As tumor growth progresses and hypoxic state

propagates, the development of immune tolerance and

activation of immune-related suppressor cells ascends.

Additionally, hypoxia surge resistance to cytotoxicity of

immune cells infiltrating tumor mass (Ganss et al. 2004).

Over the past few years there is a constant progression in

the development of new immunotherapies either with

active vaccinations or with adoptive cell transfers that

assure enhancement of cytotoxic anti-cancer immunity.

Vaccines targeting the neovasculature of tumors show

antitumor efficacy, inhibition of local suppression mecha-

nisms, and boosting anti-tumor immunity (Matejuk et al.

2011). However, the outcome of such therapies is still

modest. A few preclinical studies show increase of anti-

cancer immune responses when immunotherapy is com-

bined with anti-angiogenic approaches (Huang et al. 2002;

Li et al. 2006; Manning et al. 2007; Shrimali et al. 2010).

One of the possible mechanisms of such therapies is abo-

lition of immunosuppressive properties of VEGF.

Moreover, normalization of blood vessels can directly

improve infiltration of immune-competent cells. The role

of VEGF in immunosuppression is still not explained well.

It has been reported that VEGF upregulates adhesion

molecules on angiogenic vessels and eases adhesion and

rolling of NK cells (Melder et al. 1996). On the contrary,

Griffioen et al. (1996) showed that VEGF inhibits expres-

sion of leukocytes adhesion molecules and prevents

leukocyte tumor infiltration. In inducible nitric oxide syn-

thase (iNOS)-/- mice despite the increased level of

intratumoral VEGF, interleukin (IL)-12 induced a stronger

inhibition of blood vessel formation and recruitment of NK

cells (Bielawska-Pohl et al. 2010). Nitric oxide has been

shown to modulate endothelial interactions and expression

of junction molecules such as CD31 (Carreau et al. 2011).

In some studies VEGF has been shown to inhibit the

maturation and function of dendritic cells (Gabrilovich

et al. 1996; Osada et al. 2008). Additionally, VEGF was

proven to possess additive effect on immunosuppression in

tumor by attracting regulatory T cells (Tregs) and Gr1?

myeloid suppressor cells (Li et al. 2006; Shojaei et al.

2007). Besides tumor cells, myeloid cell that infiltrate

tumor site are main sources of VEGF. Stockmann et al.

(2008) by using mice lacking myeloid cell-derived VEGF

showed that myeloid cell-specific deletion of VEGF

resulted in vascular normalization, better oxygenation, and

better response to chemotherapeutic cytotoxicity. However,

unexpectedly, deletion of myeloid-cell VEGF-a resulted in

an accelerated tumor progression. Recently, Shrimali et al.

(2010) observed beneficial effect of anti-VEGF-a antibody

therapy combined with immunotherapy in melanoma

models by increased influx of antitumor cytotoxic T cells.

Vessel stability and normalization can also be obtained by

targeting Ang-Tie2 pathway. Ang-1 promotes maturation

of vessels and is produced by perivascular cells, whereas

Ang-2 produced by endothelial cells possesses proangio-

genic and vasculature destabilizing activity. Both growth

factors compete for Tie-2 receptor. The attempts to block

Ang-2 were successful in blood vessel normalization and

interfered with the proangiogenic, immunosupressive

activities of Tie-2 expressing monocytes (TEMs) (Coffelt

et al. 2010; Mazzieri et al. 2011). Abnormal vessel angi-

ogenesis is also mediated by PlGF. The histidine-rich

glycoprotein can downregulate PlGF and induce blood

vessel maturation, inhibition of hypoxia and switch from

pro-tumorogenic M2 macrophages to M1 with anti-tumor

activity (Huang et al. 2011b; Rolny et al. 2011). Thus,

regulation of abnormal tumor vessel phenotype has clear

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effect on the restoration of a proper immune response in

tumor environment (Fig. 1).

Angiomirs are Regulators of Angiogenesis

and Endothelial Cell Functions

miRs that tightly regulate the process of endothelial cell

function and regulate the vascular integrity, especially

angiogenesis, are referred as angiomiRs (Wang and Olson

2009). Pioneering work on miRs involved in the process of

angiogenesis came from studies on two key miRs biosyn-

thesis enzymes: Dicer and Drosha. The first report came

from the studies on mice homozygous for hypomorphic

allele of Dicer. Homozygous embryos displayed a retarded

phenotype, no angiogenesis, and died during gestation

period (Yang et al. 2005). Silencing of Dicer and Drosha in

endothelial cells inhibited endothelial cell migration and

tube formation; however, silencing Dicer only has a pro-

found effect on angiogenesis in vivo (Yang et al. 2005).

Later, several other in vitro studies on human endothelial

cells, using Dicer and Drosha knockdowns, demonstrated

inhibition of angiogenic events like proliferation, migra-

tion, and capillary sprouting (Kuehbacher et al. 2007; Shilo

et al. 2008; Suarez et al. 2007, 2008). Several key angio-

genic mediators such as VEGFs, their receptors: VEGFR1

and VEGFR2 as well as Ang-2 receptor Tie-1, endothelial

NOS (eNOS), and IL-8, were strongly affected by Dicer

deletion (Suarez et al. 2007; Yang et al. 2005). In addition,

TSP-1, a potent anti-angiogenic factor was significantly up-

regulated (Kuehbacher et al. 2007). Interestingly, recently a

novel Dicer-independent miRs processing pathway has

been described (Cifuentes et al. 2010). Rescue for endo-

thelial cell proliferation and morphogenesis in Dicer null

mice could be achieved by overexpression of miR-17-92

cluster members, especially miR-18a (Suarez et al. 2008).

Of note, the miR-17-92 cluster was the first oncogenic miR

described in mammals (He et al. 2005). MiR-17-92 toge-

ther with miR-155 are proven to cause oncogenesis

(Mendell 2008; O’Connell et al. 2008). Mounting evidence

indicates that miRs are emerging as important modulators

of angiogenesis in normal and pathological conditions such

as cancer.

First 15 miRs in human umbilical vein endothelial cells

(HUVEC) were identified by Poliseno et al. (2006). Pres-

ently 28 miRs are known as expressed by cultured

endothelial cells (Chen and Gorski 2008; Fasanaro et al.

2008; Fish et al. 2008; Kuehbacher et al. 2007; Suarez et al.

Fig. 1 Schematic representation of complex anti-cancer approaches.

The illustration shows the result of action of combinatory treatment of

blood vessel normalization stratagies that might include also miRs

characteristic for physioxia state (tissue-dependent proper oxygen

partial pressure) and conventional therapy (chemotherapy and/or

radiotherapy). It leads to reduction of hypoxia, downregulation of

hypoxiamiRs, and reduction of aberrant immune response mostly

derived from monocyte lineage. Restoration of physioxia and proper

anti-cancer immune response is achieved

288 Arch. Immunol. Ther. Exp. (2013) 61:285–299

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2007, 2008; Wurdinger et al. 2008). In vivo studies

revealed miR-126, -24 and -23a as specifically expressed

by microvascular endothelial cells, and predominant

expression of miR-145 by pericytes (Larsson et al. 2009).

In a recent study miR-145 was found to be expressed by

HUVEC and was able to control smooth muscle pheno-

types with potential to combat atherosclerosis

(Hergenreider et al. 2012). MiR-126 is the most habitually

expressed miR found in HUVEC and is characteristic of

endothelial cells from veins, arteries, and in brain and skin.

So far miR-126 seems to be a master regulator of vascular

integrity and angiogenesis that can inhibit tumor growth

and invasion. It positively regulates signaling downstream

of growth factors like VEGF, bFGF, and EGF (Fish et al.

2008; Kuhnert et al. 2008; Wang et al. 2008) thus inhib-

iting miR-126 might suppress cancer neovascularization.

Knockout of an endothelial cell-specific miR-126 in zeb-

rafish and mice caused leaky vessels, hemorrhaging, and

impaired neoangiogenesis (Fish et al. 2008; Kuhnert et al.

2008; Wang et al. 2008). Its importance in vascular

inflammation is evident by controlling leukocyte traffick-

ing to sites of inflammation and suppressing vascular cell

adhesion molecule-1, a molecule coordinating adhesion of

leukocytes to endothelium (Harris et al. 2008). Low levels

of miR-126 promoted adhesive interactions between leu-

kocytes and endothelium (Harris et al. 2008; van Solingen

et al. 2009) and its downregulation was observed in many

cancer cell lines (Crawford et al. 2008; Guo et al. 2008;

Tavazoie et al. 2008). Recently, miR-126 was reported to

increase endothelial VEGF signaling by pressure of blood

flow and the mechano-sensitive zinc finger transcription

factor klf2a (Nicoli et al. 2010). Besides miR-126, exam-

ples of other classic angio-miRs are miR-221/222, miR-23-

27, and miR-17-92 cluster (Wang and Olson 2009). miR-

221/222 modulate the angiogenic capacity of HUVEC.

They specifically target stem cell factor receptor and c-kit

and thus are able to inhibit angiogenesis by reducing

endothelial cell migration and proliferation (Li et al. 2009;

Matsui et al. 2004; Poliseno et al. 2006). A recent study in

zebrafish revealed the essential role for miR-221 in angi-

ogenesis with its critical role in tip cell formation and

migration (Nicoli et al. 2012). It has been shown that miR-

221/222 can regulate the level of eNOS involved in

endothelial cell migration, but the effect must be indirect

since they do not target 30-UTR of eNOS (Suarez et al.

2007). Minami et al. (2009) suggested that miR-221/222

are related to proliferative capacity of endothelial cells.

They showed that in patients with coronary artery disease

levels of miR-221/222 were higher which correlated with

lower number of endothelial progenitor cells (Minami et al.

2009). Of note, these miRs can also regulate p27(Kip1)

tumor suppressor promoting cancer cell proliferation (le

Sage et al. 2007). The crosstalk between miRs and

angiogenic factors is two-directional. The angiogenic fac-

tors produced by the tumor cells can change miR profile.

For example, miR-378 promotes angiogenesis and can-

cerogenesis in tumor models (Lee et al. 2007) by inhibiting

cell differentiation (Kahai et al. 2009; Lee et al. 2007) as

well as induction of VEGF expression (Hua et al. 2006).

MiR-378 competes with miR-125a for the same seed

region of VEGFs (Lee et al. 2007). Another angiomiR,

miR-296 expression increases during endothelial cells

co-stimulation with glioma cells and its inhibition reduces

vascularization of tumor xenografts (Wurdinger et al.

2008). miR-296 contributes to angiogenesis by targeting

the hepatocyte growth factor-regulated tyrosine kinase

substrate mRNA and reducing degradation of VEGFR2 and

PDGFRb (Hua et al. 2006; Wurdinger et al. 2008). The

secreted factor, TSP1 acting on angiogenesis inhibition is

targeted by the cluster of miRs-17 through -92 (Dews et al.

2006). miRs belonging to miR-17-92 family are known

oncogenes and are integrated components of the molecular

pathways that regulate tumor development and tumor

maintenance (Olive et al. 2010). All crucial events in

cancer development and progression such as uncontrolled

cell proliferation, inhibition of apoptosis, and tumor angi-

ogenesis are promoted by these miRs (Mendell 2008).

miR-17-92 cluster has potent tumor angiogenesis stimu-

lating activity. Inhibition of miR-17, -18a, and -20a

increased HUVEC sprouting in 3D spheroid model in vitro,

and in vivo increased number of blood vessels was

observed by blocking of miR-17 and miR-20a with a lesser

effect done by miR-18a and miR-19a (Doebele et al. 2010).

MiR-17/20 targets proangiogenic genes including Janus

kinase 1 (Doebele et al. 2010). Inhibition of miR-17/20

specifically augmented neovascularization of Matrigel

plugs but did not affect tumor angiogenesis indicating a

context-dependent regulation of angiogenesis by miR-17/

20 in vivo (Doebele et al. 2010). Moreover, miR-17-92

cluster belongs to hypoxamiRs that can affect HIF inde-

pendently of hypoxia (Loscalzo 2010). There is an intricate

and finely tuned circuit involving this family of miRs,

c-myc, and HIF-1a that may play a role in cancer cell

proliferation under normoxia (Taguchi et al. 2008). Inter-

estingly, recent findings depict one of miR-17-92 cluster

members, miR-92a, to be a negative regulator of new blood

vessel formation. AntagomiR that counteracted the effect

of miR-92a caused induction of blood vessel growth and

reduced damages caused by ischemia and myocardial

infarction in mouse model (Bonauer et al. 2009). MiR-21

and miR-31 possess pro-angiogenic function and are

upregulated in cancer promoting invasion and metastasis.

VEGF produced by tumor cells, crucial for angiogenesis, is

suppressed by several miRs including miR-15b, -16, -20a,

and -20b (Hua et al. 2006). Those miRs might be of special

interest in vessel normalization strategy since transfection

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to the cells can specifically target VEGF. miR-16 nega-

tively regulates VEGF translation by binding to VEGF 30-UTR (Karaa et al. 2009). Recently Chamorro-Jorganes

et al. (2011), reported that miR-16-like family members

(possessing the same seed sequence), including miR-15,

-16 and -424 critically regulate expression of VEGF-a,

their receptor VEGFR2, and fibroblast growth factor

receptor-1 (FGFR1). miR-15 and miR-16, by targeting

Acvr2a, a ligand belonging to transforming growth factor

(TGF)-b superfamily, interfere with TGF-b signaling. They

are also negatively regulated by Wnt/b-catenin pathway

(Martello et al. 2007). Members of another family miR-23-

27 cluster, miR-27a, and miR-27b are highly expressed in

endothelial cells and inhibition of miR-27b markedly

reduced endothelial sprouting in vitro (Kuehbacher et al.

2007). This family has been also found to regulate angio-

genesis (Zhou et al. 2011). Recently Urbich et al. (2012)

showed that miR-27a/b promote angiogenesis by targeting

the angiogenesis inhibitor Semaphorin 6A, which controls

repulsion of neighboring endothelial cells. Another potent

regulator of angiogenesis is miR-130a that induces novel

vessel formation by down-regulating anti-angiogenic

homeobox genes GAX and HOXA5 (Chen and Gorski

2008).

HypoxamiRs are Potential Targets for Blood Vessel

Normalization Strategies

Decrease in the partial pressure of oxygen to reach a low

level, called hypoxia, initiates genetic changes directly

leading to angiogenic response. Other cellular functions

controlled by hypoxia are related to proliferation, apopto-

sis, and metabolism. Hypoxic state is a hallmark of cancer

environment. Tumor cells under hypoxia tend to be resis-

tant to therapies and constitute a poor prognosis. Persistent

hypoxia correlates with metastasis and reduced survival in

patients (Hockel et al. 1999). Hypoxia triggers the stabil-

ization of oxygen-dependent transcription factor HIF that

further initiates the synthesis of the main pro-angiogenic

factors VEGF-A, -B, and -C (Rocha 2007). This leads to

initiation of neoangiogenesis and creation of new patho-

logic blood vessels within tumor environment. Thus VEGF

like many other factors is secondary to the hypoxia and as

much as current anti-VEGF approaches are helpful, even-

tually are over-compensated by progressive and persistent

hypoxia. Novel approaches to anti-cancer therapy by

modulating the oxygen tension in tumor environment and

thus directly targeting hypoxia are under development and

might bring new clinical applications. Aiming hypoxia in

cancer by specific hypoxamiRs can provide another layer

of controlling aberrant angiogenesis and blood vessel

normalization. Application of hypoxamiRs can also

directly lead to regulation of immune response in tumor

since HIF-1a has been demonstrated to regulate survival

and function of immune cells in the inflammatory micro-

environment such as cancer (Cramer and Johnson 2003;

Cramer et al. 2003; Palazon et al. 2012). Many cells

including macrophages express HIF-1a as an adaptation

mechanism in low oxygen environment. In T cells HIF-1anegatively regulates activation (Thiel et al. 2007). miRs

that target HIF-1a belong to miR-17-92 family and have

been shown to induce Th1 responses in lung cancer

(Taguchi et al. 2008). The term ‘‘hypoxamir’’ was initially

defined by Loscalzo et al. (Chan and Loscalzo 2010;

Loscalzo 2010). He classified hypoxamirs into three sub-

classes. The first group consists of HIF-dependent

hypoxamiRs, the second group involves miRs that are

induced by hypoxia and in turn affect HIF-1a expression,

and the last group comprises miRs that regulate HIF-1a in

hypoxia-independent manner. We added yet another group

called ‘‘miRs repressed by hypoxia’’ (Table 1) since sev-

eral new reports show that hypoxia not only induces

expression of diverse miRs but is also able to suppress their

expression. In rapidly growing field of miRs we realize that

our classification is only temporarily and many more newly

discovered molecules are on the horizon. Some of miRs

belong to two different categories since depending on the

conditions they might be induced or repressed by hypoxia.

The following are the examples of classical miRs whose

expression is related to hypoxia and/or HIF: cluster 17-92,

miR-20b, -31, -107, -199a, -210, -373, -424, and -519c.

miR-17-92 targets HIF-1a and is induced by oncogene

c-MYC (Taguchi et al. 2008). Similar to 17-92 cluster,

miR-519c suppresses expression of HIF-1a independently

on hypoxia (Cha et al. 2010). As found in MCF-7 breast

cancer cells, miR-20b reduces VEGF expression by tar-

geting HIF-1a and STAT3 (Cascio et al. 2010; Hua et al.

2006). miR-31 was identified as a pro-hypoxamiR inducing

expression of HIF-1a by affecting factor-inhibiting HIF

(Liu et al. 2010). miR-107 inhibits expression of HIF-1bsubunit of HIF-1 in response to p53 (Yamakuchi et al.

2010). miR-199a was found to decrease expression of HIF-

1a in cardiomyocytes (Rane et al. 2009). So far, the key

master of miRs that are steadily induced by hypoxia is

miR-210. Inhibition of miR-210 repressed the formation of

capillary-like structures in hypoxic environment and

reduced migration of cells in response to VEGF; endo-

thelial expression of miR-210 was induced by HIF-1a(Ivan et al. 2008). In hypoxia, miR-210 is a critical regu-

lator of endothelial cell survival, migration, and

differentiation (Fasanaro et al. 2008). Several target genes

have been identified for miR-210 such as ephrin A3,

RAD52, FGFRL2 and many others involved in angiogen-

esis, tumor propagation, cell cycle regulation, and stem cell

generation (Crosby et al. 2009b; Fasanaro et al. 2008;

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Pulkkinen et al. 2008; Tsuchiya et al. 2011). In vitro studies

show its strong up-regulation by hypoxia in cultured HU-

VEC, and in vivo in limb and brain ischemia (Jeyaseelan

et al. 2008; Pulkkinen et al. 2008). Another hypoxamiR,

miR-424, was shown to be specifically expressed by

endothelial cell in response to hypoxia (Ghosh et al. 2010).

VEGF-a, VEGFR2, and FGFR1 were identified as targets

of miR-424 in endothelial cells (Chamorro-Jorganes et al.

2011). Decreased miR-424 expression and increased levels

of MEK1 or cyclin E1 in senile hemangioma caused

abnormal cell proliferation in the tumor and abnormal

angiogenesis (Nakashima et al. 2010). With collaboration

of miR-155, -222 and -503, -424 regulates monocyte dif-

ferentiation (Forrest et al. 2010; Rosa et al. 2007).

Overexpression of miR-424 leads to maturation of the

monoblastic U937 cells and expression of monocyte/mac-

rophage characteristics. Another set of oncomiRs: miR-210

and miR-373 whose expression is dependent upon HIF has

been identified (Crosby et al. 2009a, b). miR-373 as onco-

gene was found to play an important role in colon cancer

induction via regulating expression of RAB22A gene

(Tanaka et al. 2011). In another study, miR-373 increased

the expression of metalloproteinase 9 by activation of the

Ras/Raf/MEK/Erk signaling pathway and nuclear factor

kappa-light-chain-enhancer of activated B cells (NF-jb)

(Liu and Wilson 2012). Opposite role for miR-373 as a

tumor suppressor was documented in estrogen receptor

negative breast cancer by targeting NF-jb and TGF-b sig-

naling pathways (Keklikoglou et al. 2012). From the breast

and colon cancer studies, a group of miRs regulated by low

oxygen level including miR-210, 26 and 181 has been

identified (Kulshreshtha et al. 2008). It becomes increas-

ingly clear that miRs are implicated in the regulation of

various aspects of hypoxia including angiogenesis and

appear as targets for novel therapeutic approaches in cancer.

The discovery of novel miRs (angiomiRs) that are able to

lead to physioxia state, a proper tissue-dependent oxygen

partial pressure, can be of special interest.

miRs that Regulate the Immune Responses

Pre-existing condition for tumor initiation is persisting

inflammation that eventually leads to tissue damage,

release of metalloproteinases, pro-inflammatory cytokines,

and chemokines. Unresolved inflammation activates

endothelial cells and angiogenesis, cell proliferation,

motility, and engagement of stem cells. Myeloid cells are

first to respond to local inflammation. Immune cells display

a relative plasticity and their developmental fate strongly

depends on microenvironmental stimuli. Myeloid cells

have potential to differentiate into monocytes and macro-

phages. The latter can further polarize into M1/M2

macrophages or DC1/DC2 dendritic cells depending on the

cellular and signaling milieu. Early immune response in

cancer is characterized by influx of these innate immune

effectors that also include neutrophils and NK cells.

Complex networks created by several immune-competent

cells such as dendritic cells, B cells, cytotoxic CD8? T,

CD4? T-helper and NK cells in combination with cyto-

kines, chemokines, and other immune mediators are

required for effective immune reactions against cancer.

Ineffective immune response to cancer and promotion of

M2 and DC2 phenotypes as well involvement of regulatory

T cells favor tumor development and expansion. Specific

regulation of immune function by miRs plays a particularly

important role in cancer immunology. In physiological

conditions, the immune system is cautiously regulated,

coordinated, and stabilized by multiple miRs (Baltimore

et al. 2008). There is a selective tissue expression of some

immune-related miRs. For example, miR-142 is found in

all hematopoietic tissues, miR-223 is almost solely

expressed in bone marrow, and miR-181 is typically pro-

duced in brain, lung, and thymus (Chen et al. 2004). miRs

constitute an important connection between the adaptive

and innate immune responses (O’Connell et al. 2010).

Immune cells can be recognized by the specific set of

miRs. For example, hematopoietic cells are characterized

by the following miRs: miR-142, -144, -150, -155, and -

223 (Landgraf et al. 2007). miR-150, -155, -223 as well as

miR-17-92 cluster and miR-181 actively participate in

maturation and differentiation of myeloid and lymphoid

lineages (Tsitsiou and Lindsay 2009). Different immune

cell types can express the same miRs; however, their

magnitude of expression can be altered, for example miR-

342 is 10-times more abundant in T cells than B cells

(Merkerova et al. 2008). miR-155 plays a crucial role in

development, maintenance, and function of immune sys-

tem (Rodriguez et al. 2007). It is a constituent of innate

response to a variety of inflammatory mediators (Sonkoly

et al. 2008; Tili et al. 2007) and is essential for proper T

cell responses. miR-155 as oncomiR directly links immu-

nity with oncogenesis and is dependent on MAPK

signaling (O’Connell et al. 2007). Disregulation of miR-

155 in hematopietic cells leads in mice to malignancy,

myeloproliferative disorder, and in human to acute myeloid

leukemia (Costinean et al. 2006; O’Connell et al. 2008).

miR-155 is induced by inflammatory mediators during

early macrophage responses (O’Connell et al. 2007). Dis-

ruption of gene encoding miR-155 in mice caused deficient

antigen presentation by dendritic cells, breakdown in

mounting memory, and T-cell-specific immune responses

(Rodriguez et al. 2007). It is a key regulator of adaptive

immunity and fate of Th1 vs Th2 cells (Thai et al. 2007).

Malfunction of miR-155 skews immune response towards

Th2 phenotype and production of IL-4, IL-5 and IL-10

Arch. Immunol. Ther. Exp. (2013) 61:285–299 291

123

cytokines. miR-155 inhibits interferon (IFN)-c signaling in

CD4 cells and its over-expression promotes Th1 responses

(Banerjee et al. 2010). It also promotes the production of B

cell memory responses, high-affinity B cell clones that

undergo isotype-switching and produce high-affinity anti-

bodies, and its malfunction leads to plasma cell

transformation (Calame 2007). In contrast to miR-155,

-146a is a negative regulator of inflammation. miR-146a

knockout mice display autoimmunity and immunoprolif-

erative disorders (Boldin et al. 2011). miR-146a has been

found to be a NF-jb-dependent gene and plays an impor-

tant role in control of Toll-like receptor (TLR) and pro-

inflammatory cytokine signaling (Taganov et al. 2006).

NF-jb dysregulation in miR-146a deficient mice drives the

development of myeloid malignancies (Zhao et al. 2011).

Bone marrow-derived macrophages in miR-146a deficient

mice show increased cytokine production such as IL-1b,

IL-6 and TNF-a upon lipopolysaccharide stimulation

(Boldin et al. 2011). miR-146a is a negative regulator of

the IFN pathway targeting TLR and STAT-5 (Tang et al.

2009). miRs that target T cells include miR-181a and

members of miR-17-92 family. miR-181a controls T cells

positive and negative selection in thymus and its overex-

pression causes augmentation of sensitivity to peptide

antigens in mature T cells (Li et al. 2007). miR-17-92

expression in activated T cells promotes Th1 responses

such as increase production of IFN-c (Xiao et al. 2008).

Overexpression of this family renders T cells more sensi-

tive to T cell receptor antigenic stimulation (Xiao et al.

2008). miR-223 and miR-150 are selectively expressed by

B and T cells (Merkerova et al. 2008; Monticelli et al.

2005). miR-223 promotes differentiation of myeloid blast

cells in bone marrow (Fazi et al. 2005) and regulates

activation of granulocytes (Johnnidis et al. 2008). miR-222

and miR-339 are oncomiRs that directly influence tumor

immune responses by suppression of intracellular cell

adhesion molecule-1, thus decreasing the receptiveness of

cancer cells to cytotoxic T lymphocytes (Ueda et al. 2009).

Deletion of Dicer and Dgcr8 had an impact on the

expression level of natural-killer group 2, member D

Table 1 Overview of hypoxamiRs

Hypoxamirs induced by HIF Hypoxamirs that

affect HIF

miRNAs that affect HIF

independent of hypoxia

miRNAs repressed by

hypoxia

miR-10b (Haque et al. 2011) miR-20b (Cascio et al.

2010; Lei et al.

2009)

miR-17-92 cluster(Taguchi et al.

2008)

miR-17-3p miR-15b (Hua et al. 2006)

miR-155 (Bruning et al. 2011) miR-130 (Saito et al.

2011)

miR-17-5p miR-16 (Dejean et al. 2011;

Hua et al. 2006)

miR-210 (Chan and Loscalzo 2010; Crosby et al.

2009a, b; Fasanaro et al. 2008; Giannakakis et al.

2008)

miR-145 (Bussolati

et al. 2012; Xu et al.

2012)

miR-18a miR-20a (Hua et al. 2006)

miR-373 (Crosby et al. 2009a, b) miR-155 (Bruning

et al. 2011)

miR-19a miR-20b (Hua et al. 2006)

miR-199a (Kang et al.

2012)

miR-19b miR-34a (Du et al. 2012)

miR-424 (Ghosh et al.

2010)

miR-20a

(Kang

et al. 2012)

miR-101 (Cao et al. 2010)

miR-92a miR-135a (Gonsalves and

Kalra 2010)

miR-21 (Liu et al. 2011) miR-199a (Gonsalves and

Kalra 2010; Rane et al.

2009)

miR-22 (Yamakuchi et al. 2011) miR200b (Chan et al. 2011)

miR-31 (Liu et al. 2010) miR-378 (Fang et al. 2012)

miR-107 (Yamakuchi et al. 2010) miR-449a/b (Muth et al.

2010)

miR-130 (Kulshreshtha et al. 2007;

Saito et al. 2011)

miR-138 (Song et al. 2011)

miR-199a (Mizuno et al. 2012)

miR-519c (Cha et al. 2010)

The miRs classifed by Loscalzo (2010) are written in bold

292 Arch. Immunol. Ther. Exp. (2013) 61:285–299

123

Table 2 Overview of miRs participating in immune response, their primary targets and functions

miRs Target(s) Effect(s) References

miR-9 NF-jB1/p50 Inhibition of NF-jB transcription Bazzoni et al. (2009)

miR-15 Acvr2a Interference with TGF-b signaling Martello et al. (2007)

miR-16 TNF-a, Acvr2a Suppression of pro-inflammatory cytokines Carballo et al. (1998), Lai et al.(1999)

Interference with TGF-b signaling Martello et al. (2007)

miR-17-92 E2F1, E2F2, E2F3, P21, TSP-1,Bim, CTGF, PTEN, HIF-1a,TGFBRII

Resistance to tumor-derived immunosuppressive factors andimprovement of Th1 reactivity

Sasaki et al. (2010), Xiao et al. (2008)

miR-21 PDCD4, IL-12p35 Derepression of IL-10 Sheedy et al. (2010)

miR-30e/miR-378

Granzyme B and perforin Negative regulation of human NK cell cytotoxicity Wang et al. (2012)

miR-31 Foxp3 Negative regulation of Foxp3 expression in natural Tregs Redouane et al. (2009)

miR-34a/c ULBP2 Decrease of tumor cell recognition by NK cells Heinemann et al. (2012)

miR-101 MKP-1 Regulation of the innate immune responses of macrophagesthrough targeting MKP-1

Zhu et al. (2010)

miR-105 TLR2 Modulation of TRL responses Benakanakere et al. (2009)

miR-106 IL-10 Suppression of IL-10 production Carballo et al. (1998)

miR-125b/miR-221,miR-579

TNF-a Regulation of TNF-a production El Gazzar and McCall (2010), Tiliet al. (2007)

miR-132/miR-200a/miR-212

STAT4 Modulation of IL-12 signaling pathway by reducing STAT4expression in primary human NK cells

Huang et al. 2011a

miR-145 TRAF6, IRAK1 Suppression of TLR signaling Starczynowski et al. (2010)

miR-146a/b IRAK1, IRAK2, TRAF6, FADD,STAT1, STAT5, TLR

Implication in TNF-a regulation in dendritic cells and viralinfection

Hou et al. (2009), Taganov et al.(2006)

Maintenance of human memory T cells Curtale et al. (2010)

Controlling of Treg cells suppressor function Lu et al. (2010)

Negative regulation of IFN pathway Tang et al. (2009)

miR-150 c-Myb Controlling B cell development Lin et al. (2008), Thomas et al.(2005), Xiao et al. (2007)

miR-155 SOCS1, SHIP1, c-Maf, Pro-inflammatory role in microglia and in the progression of theimmune response

Cardoso et al. (2012)

FADD, IKKe, Ripk1, c-Myb, Liu et al. (2012)

Regulation of human NK cell activation Okada et al. (2010)

Association in the response to TLR ligands, PAMPs and role indifferentiation of Th1 vs. Th2 T cells

Tili et al. (2007)

Regulation of the release of inflammatory mediators during theinnate immune response

Lu et al. (2009)

O’Connell et al. (2009)

Bezman et al. (2011)

Control of Tregs homeostasis

Controling of T and B cell responses

Control of NK and iNKT cells development

miR-181a/b Bim, AID, SHP-2, DUSP5,DUSP6, PTPN22

Activation of a miR-181a-dependent mechanism involvingdown-regulation of Bim protects B cell lymphoma cellsagainst apoptosis

Lwin et al. (2010)

Davidson-Moncada et al. (2010)

Regulation of B cell function by affecting class-switchrecombination

Okada et al. (2010)

Increasing of TCR response and CD4? T cell selection

miR-222/339 ICAM-1 Suppress ICAM-1 expression on tumor cells, and down-regulatethe susceptibility of tumor cells to CTL-mediated cytolysis

Ueda et al. (2009)

miR-223 NFI-A, Mef2c, TLR3, TLR4,IKKa

Regulation of human granulopoiesis and monocyte activation Fazi et al. (2005), Johnnidis et al.(2008), Li et al. (2010), Muzioet al. (2000)

Let-7 family TLR4, IL-6 Inhibition of inflammatory signaling Androulidaki et al. (2009),Asirvatham et al. (2009)

TCR T cell receptor, ICAM-1 intracellular cell adhesion molecule-1, iNKT invariant NKT, Th1 T helper type-1, TNF tumor necrosis factor, CTL

cytotoxic T lymphocyte, PAMP pathogen-associated molecular patterns, TLR Toll-like receptor

Arch. Immunol. Ther. Exp. (2013) 61:285–299 293

123

protein activating receptor on NK cells, and leads to

apoptosis of peripheral NK cells (Sullivan et al. 2012), key

players in direct lysis of cancer cells. Multiple miRs

coordinate the function of NK cells; however, miR-21

seems to be a key regulator of NK cell survival (Bezman

et al. 2010). Recently, the major role in NK development

and maturation has been attributed to miR-150, which was

found to differentially control the development of NK and

invariant NKT cell lineages by targeting c-Myb (Bezman

et al. 2011). Additionally, miR-378 and miR-30e were

proved to suppress human NK cell cytotoxicity (Wang

et al. 2012). To delineate the role of miRs in the biology of

Tregs that effectively inhibit antitumor responses and

interfere with antitumor therapies, mouse models with

depleted Dicer in Foxp3 Treg lineage were developed

(Liston et al. 2008; Zhou et al. 2008). Dicer deletion

resulted in lymphoproliferative autoimmune syndrome and

loss of Treg suppression activity in vivo (Cobb et al. 2006;

Zhou et al. 2008). There is a strong overlap in miR

expression between Tregs and activated T cells with the

exception of miR-223 and miR-146 (Cobb et al. 2006). A

few Treg-specific miRs are directly regulated by Foxp3;

one of them is miR-155 that is important in response of

Tregs to IL-2 by targeting SOCS1 and as an oncogene is

constitutively and abundantly expressed in Tregs (Lu and

Rudensky 2009; Lu et al. 2009; Marson et al. 2007; Zheng

et al. 2007). Table 2 provides the overview on miRs par-

ticipating in immune response that might relate to tumor-

suppressive or tumor-promoting activities for which the

specific targets are known.

Concluding Remarks

Continuous investigation of miRs as epigenetic agents can

reveal multiple mechanisms to avoid early events impor-

tant in aberrant angiogenesis and immunological

surveillance. Besides, as miRs are differentially expressed

in tumorogenesis, and are markedly tissue specific, they

can serve as new biomarkers. Finding similar miR profiles

regulating angiogenesis as well as developmental stages

and function of immune cells in tumor microenvironment

can offer another level of our understanding of the com-

plexity of tumor paradigm. It would be of particular

interest to recognize set of miRs that beneficially acts to

reverse aberrant angiogenesis and improve anti-cancer

immunity. One of such targets could be members of miR-

16-like family that specifically target VEGFs, as well as

their receptors and possess the ability to interfere with

TGF-b signaling. Moreover, one of them, miR-424, a

potent hypoxamiR that modulates HIF1-a, has been proven

to play a key role in immunity. Improving anti-tumor

immune responses by engineering miRs such as miR-17-

92, -155, -181a with properties for T cell-based immuno-

therapies represent another possible approaches. The vessel

normalization strategy based on application of miRs that

might have significant impact on the function of anti-tumor

immunity should efficiently eradicate aberrant immune

responses caused by tumor-associated macrophages,

TEMs, N2-effector neutrophils, and Tregs and increase

participation and cytotoxicity of NKs and CD8 T cells. As

much as this novel strategies bring hope we still need to be

aware of potential pitfalls such as probable distinct func-

tions of particular miRs in different cellular scenery or

interference and redundancy with physiological pathways.

Nevertheless, a lot of optimism exists with the future

clinical applications for miRs. Tissue-restricted expression

profile and abundance of circulating miRs in body fluids

such as blood, serum, plasma and saliva reflecting physi-

ological and pathological conditions make them good

candidates as non-invasive biomarkers. miR signature is

directly correlated to prediction of patients survival, out-

come for therapeutic responses, and can be used

successfully in molecular diagnostics for array of diseases

such as cancer and cardiovascular malfunctions. Such

approaches are being currently carried out. Moreover,

some miRs can act as oncogenes or tumor suppressors; thus

their activity modulation may regulate targeted cellular

behaviors in cancer treatments. Our continuous study on

miRs and their correlation with molecular and cellular

targets with disease phenotypes will illuminate new bio-

logical pathways and disease mechanisms. Gain- and loss-

of-function manipulations using oligonucleotide-based

inhibitors or miRs decoys/mimics open novel opportunities

for therapuetic interventions.

A new generation of anti-cancer drugs based on com-

binatory therapy built on set of specific miRs, blood vessel

normalization strategies, and conventional chemotherapy

that evidently will inhibit multiple cancer targets is eagerly

awaited.

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