Accepted Manuscript

Title: The Role of Reactive Oxygen Species and Metabolismon Cancer cells and their Microenvironment

Author: Ana Costa Alix Scholer-Dahirel FatimaMechta-Grigoriou

PII: S1044-579X(14)00004-2DOI: http://dx.doi.org/doi:10.1016/j.semcancer.2013.12.007Reference: YSCBI 1097

To appear in: Seminars in Cancer Biology

Received date: 30-7-2013Revised date: 22-12-2013Accepted date: 30-12-2013

Please cite this article as: Costa A, Scholer-Dahirel A, Mechta-Grigoriou F, The Role ofReactive Oxygen Species and Metabolism on Cancer cells and their Microenvironment,Seminars in Cancer Biology (2014), http://dx.doi.org/10.1016/j.semcancer.2013.12.007

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The Role of Reactive Oxygen Species and Metabolism on Cancer cells and their Microenvironment

Ana Costa1,2*, Alix Scholer-Dahirel1,2* and Fatima Mechta-Grigoriou1,2#

* These authors contributed equally to this work

1 “Stress and Cancer” Laboratory, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05,

France 2 Inserm, U830, Paris, F-75248, France

# Correspondence should be addressed to: Dr. Fatima Mechta-Grigoriou, “Stress

and Cancer” Laboratory, Institut Curie, Inserm U830, 26 rue d’Ulm, 75248 Paris

Cedex 05, France. Email address: fatima.mechta-grigoriou@curie.fr; Phone number:

+33 (0)1 56 24 6653 ; Fax number : +33 (0)1 56 24 6650

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Keywords: ROS, oxidative stress, mitochondria, stroma, myofibroblast, CAF heterogeneity

ABSTRACT Compelling evidence show that Reactive Oxygen Species (ROS) levels are finely

regulated in the cell and can act as “second messengers” in response to diverse

stimuli. In tumor epithelial cells, ROS accumulate abnormally and induce signaling

cascades that mediate the oncogenic phenotype. In addition to their impact on tumor

epithelial cells, ROS also affect the surrounding cells that constitute the tumor

microenvironment. Indeed, ROS production increases tumor angiogenesis, drives the

onset of inflammation and promotes conversion of fibroblast into myofibroblasts.

These cells, initially identified upon wound healing, exhibit similar properties to those

observed in fibroblasts associated with aggressive adenocarcinomas. Indeed,

analyses of tumors with distinct severity revealed the existence of multiple distinct

co-existing subtypes of Carcinoma-Associated Fibroblasts (CAFs), with specific

marker protein profiling. Chronic oxidative stress deeply modifies the proportion of

these different fibroblast subtypes, further supporting tumor growth and metastatic

dissemination. At last, ROS have been implicated in the metabolic reprogramming of

both cancer cells and CAFs, allowing an adaptation to oxidative stress that ultimately

promotes tumorigenesis and chemoresistance. In this review, we discuss the role of

ROS in cancer cells and CAFs and their impact on tumor initiation, progression and

metastasis.

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INTRODUCTION ROS cellular levels are crucial for the cell fate. Accumulation of intracellular ROS in

normal cells contributes to the oxidation of various components, including nucleic

acids, proteins and lipids [1]. These various oxidative reactions cause multiple

damages, which usually promote apoptosis in case of overwhelming damages, but

can also drive to abnormal proliferation and lead to transformation. Anti-oxidant

mechanisms that can be either enzymatic (including catalases, dismutases and

peroxidases) or non-enzymatic (such as vitamin A, C or E) are critical to protect cells

against ROS-induced damages both at steady state and upon acute oxidative stress.

In addition, low levels of intracellular ROS are recognized to be signaling molecules.

Indeed both ROS concentration and sub-cellular compartmentalization are important

for effective signaling. Interestingly, ROS were found in redox-active endosomes

(redoxosomes) that may contribute to regulate their spatial and temporal regulation

[2]. ROS accumulation in tumors has a wide and severe impact on various biological

processes, eliciting proliferation, genomic instability, inflammation, resistance to

apoptosis and metabolic shift to glycolysis (Warburg effect). Ultimately, high levels of

ROS in tumors contribute to tumorigenesis and tumor progression. Considering that

the variation of ROS levels is a key early event in tumor initiation, understanding how

ROS are generated is relevant and will be addressed in this review as a starting

point. It is recognized that tumor microenvironment can drive the tumor

aggressiveness and metastasis. In this regard, the reactivity of stromal components,

such as CAFs, towards ROS plays a pivotal role in tumor initiation and sustains

tumor growth and dissemination. This review outlines the role of ROS as signaling

molecules, their role in myofibroblast conversion, metabolic reprogramming and

implications for tumor growth and progression.

1. ROS sources in cancer ROS comprise a group of oxygen derivatives resulting from distinct oxidation status

of O2, including radical forms (having free or unpaired electrons), such as superoxide

radical anion (O2•−), carbon dioxide free-radical (CO2•−) or hydroxyl free-radical (OH-)

and non-radical forms (such as hydrogen peroxide, H2O2) [3]. ROS are usually

generated through a cascade of reactions that starts with the production of

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superoxide anions (O2•−), which rapidly dismutate into H2O2 either spontaneously, at

low pH for instance, or through catalysis by superoxide dismutase (SOD). Most of the

damage caused by H2O2 and O2•− result from their conversion to even more reactive

and toxic species, such as OH-, generated from H2O2 through the Fenton reaction.

Yet, because of its long-life and high permeability across membranes, H2O2 still

remains the most reactive form of ROS for promoting signaling at long distance

inside one single cell or even between two different cells. ROS molecules have a

wide range of chemical reactivity and signaling properties, which will be addressed in

this review regarding their role on CAFs and on tumorigenesis.

Methods to measure ROS

ROS measurement in cells and tissues is of great interest. However, caution should

be taken as ROS can be compartmentalized at subcellular levels. Likewise, ROS

scavengers can also distort the assessment of ROS levels in the cells [4]. The

detection method to use depends on the form of ROS to be detected. One of the

most common methods to detect intracellular ROS levels relies on the use of cell-

permeant ROS-sensitive fluorescent dyes, such as 2’,7’-dichlorofluorescein (DCF).

The carboxy-derivative of DCF, 5,6-chloromethyl 2’,7’-dichlorodihydrofluorescein

diacetate (CM-H2DCFDA) carries additional negative charges that improve its

retention compared to noncarboxylated forms. After diffusion into the cell, DCFDA is

deacetylated by cellular esterases to a non-fluorescent compound, which is later

oxidized by ROS. DCF is a highly fluorescent compound, which can be detected by

fluorescence spectroscopy with maximum excitation and emission spectra of 495nm

and 529nm, respectively. DCF compounds being trapped into the cells by

deacetylation immediately after passive diffusion into the cytoplasm, their use

ensures specific labeling of intracellular ROS contents, and thus strictly excluding

staining of ROS produced by the cells in the extracellular medium. DCF compounds

are oxidized by various ROS, including HO. (Hydoxyl radical), superoxide anion (O2.-

), peroxyl radical (ROO.), peroxynitrite anion (ONOO-) and thus allow specific

quantification of these species. The increase in dye-fluorescence and thus

quantification of ROS production are the most often assessed using a flow

cytometer, as shown in [5, 6], but can also be evaluated, although with less

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precision, by fluorescence microscopy [7]. Another method commonly used for ROS

measurements is based on electron paramagnetic resonance spectroscopy (ESR).

ESR detects the absorption of microwave energy, which occurs on transition of

unpaired electrons in an applied magnetic field. ESR is thus the method of choice for

detecting compounds with unpaired electrons, such as O2.- or OH.. These species

are paramagnetic molecules and are highly reactive when submitted to a magnetic

field. Thus, they can be quantified by this methodology, as shown in [8]. Finally, in

contrast to the DCF-based method, which detects a variety of ROS, the Amplex Red

assay allows the specific detection of H2O2. This assay is based on the oxidation of

the Amplex Red molecule by horseradish peroxidase and H2O2 to resorufin, which is

fluorescent. One caveat of this technique is the instability of the Amplex Red dye.

Finally, O2•− levels can be detected by reduction of ferricytochrome C to

ferrocytochrome C by O2•−.. Ferricytochrome reduction results in increased

spectrophotometric absorbance in proportional to superoxide levels. However,

because other enzymes, such as xanthine oxidase, can also participate in the

cytochrome C reduction, this assay must be performed in the presence and absence

of SOD [9]. Overall, ROS are short-lived molecules. This property makes them

difficult to measure and the use of various methods to determine ROS levels is

advisable.

Main sources of ROS

Chronic oxidative stress can be the result of an imbalance between radical-

generating and radical-scavenging systems. Endogenous ROS are generally

produced as a byproduct of biological reactions involved in energy metabolism and

occurring mainly within mitochondria or peroxisomes. In addition, the NADPH-

oxidases (NOX) were the first enzymes identified as being able to generate ROS by

themselves. Initially isolated from phagocytes, this family of enzymes was rapidly

found in many other tissues. In this review, due to their importance regarding cancer

regulation, we will focus on two main sources of intracellular ROS: mitochondria and

NOX. Mitochondrial ROS are generated as a byproduct of the electron transport

chain. In the mitochondria two sites (site IQ- complex I and site IIIQo- complex III)

have been recognized as the predominant producers of ROS, such as superoxide

anions [10]. It is generally considered that two major conditions lead to significant

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O2•− production: when the Electron Transport Chain is defective in ATP production

and levels of NADH/NAD+ are high [11]. While other sites have been shown to

release superoxide anions to the mitochondrial matrix, mainly complex III produces

superoxide anions to both matrix and inter-membrane space. Superoxide anions

located in the inter-membrane space of the mitochondria have been proposed to

have implications to signaling in the cytoplasm [12] as they might access to

cytoplasm. Regardless the mitochondrial site of production, superoxide anions are

rapidly converted to H2O2 by superoxide dismutase 2 (SOD2), located in the

mitochondrial matrix. Other SOD enzymes, namely SOD1 and SOD3 (located

respectively in the cytoplasm and in the extracellular matrix) also play an important

role in regulating mitochondrial ROS. The second main source of intracellular ROS in

tumor cells is the family of NADPH-oxidases (NOX1-5 and DUOX1-2) [13]. All NOX

enzymes are transmembrane proteins (plasma membrane, endoplasmatic reticulum

and mitochondria) that transport electrons across biological membranes to reduce

oxygen into superoxide anions, which are further a target of SOD1 for the production

of H2O2. Due to place restriction, we will focus below on these two major sites of

ROS production in cancer cells, the mitochondria and the NOX enzymes.

Origins of oxidative stress in cancers

Tumor epithelial cells produce high levels of ROS resulting in constant exposition to

oxidative stress. Indeed, high levels of ROS have been detected in several cancers,

such as in breast, ovarian, liver or colon cancers. ROS in tumor epithelial cells can

derive from various causes including -among others- increased metabolism

associated with dysfunctional mitochondria, oncogene activity, abnormal expression

of NOX enzymes (especially NOX1 and NOX4), dysfunction of cycloxygenases

(COX), lipoxygenases (LOX) and thymidine phosphorylase or altered anti-oxidant

defenses (Fig. 1) [14]. One of the main sources of ROS in tumor epithelial cells

arises from mitochondrial dysfunction coupled with the metabolic readjustment to

generate ATP. The highly proliferative rates exhibited by tumor cells require high

levels of energy and building blocks for biomass production. Despite this high energy

requirement, cancer cells shift their oxidative metabolism to aerobic glycolysis, a

process referred to as the Warburg effect, which is observed regardless the

oxygenation levels. Indeed, aerobic glycolysis is favored compared to oxidative

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phosphorylation despite the low ATP amount being produced and independently of

the presence of O2. This process is referred to as the Warburg effect and is one of

the main hallmarks of cancer cells. In this context, the electron transport is

compromised, but the complex I and complex III of the respiratory chain are still

active. This condition is associated with a high membrane potential (ΔΨ), leading to

an increased leakage of electrons and an abnormal NADH/NAD+ ratio, which result

into significant O2•− production [15]. In addition, loss of pyruvate through reduction

into lactate decreases input into Krebs cycle thereby depriving cancer cells from the

antioxidant properties of Krebs cycle intermediates [16]. Thus the Warburg effect

contributes to oxidative stress by increasing ROS production and decreasing

antioxidant defenses. Although mitochondrial respiratory dysfunction was proposed

to be the major causes for such glycolytic switch / Warburg effect, this hypothesis

has been recently revisited. Indeed, while respiratory complex I is essential for

induction of the Warburg effect and adaptation to hypoxia, truncated mutation in

complex I subunit 1 enzyme confers anti-tumorigenic properties [141]. Moreover,

metabolic signatures identified several subsets of diffuse large B cell lymphoma, one

of them harboring an OxPhos-signature (genes involved in mitochondrial

metabolism). This subset of lymphoma cells display enhanced mitochondrial energy

transduction, greater incorporation into the tricarboxylic acid cycle and increased

glutathione levels [142]. Similarly, proteome profiling revealed an up-regulation in

enzymes of mitochondrial oxidative phosphorylation and ATP production in

Melanoma [143]. Thus, the Warburg effect has been recently revisited by various

studies showing metabolic heterogeneity, that affects drug resistance and may also

impact differently the stromal compartment.

Another feature of solid tumors responsible to generate and maintain the oxidative

stress is hypoxia. Indeed, hypoxia has been shown to be intimately associated to

mitochondrial ROS production, mainly by complex III [17]. Moreover, in the initial

phases of tumor growth, cancer cells suffer from the lack of O2, due to absence of

neo-vascularization. In this situation, the hypoxia inducible factor (HIF) is stabilized

and induces a series of signaling cascades that allow tumor cells to survive and

proliferate [18]. Among changes, HIF promotes the shift of oxidative phosphorylation

toward aerobic glycolysis. Indeed, HIF stimulates the transcription of genes involved

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in glycolytic metabolism, such as pyruvate dehydrogenase kinase 1 (PDK1), which

further inhibits pyruvate dehydrogenase (PDH), a key enzyme involved in pyruvate

entry into the Krebs cycle. Moreover, HIF-1 regulates several genes encoding

glucose transporters (GLUT1 and GLUT3), then enhancing the glucose uptake and

providing additional resources for ATP production. In a more advanced stage of the

tumor growth, neo-angiogenesis is induced to provide O2 to tumor cells. However,

the new blood vessels are often disorganized and leaky, which may cause periods of

hypoxia and reperfusion and perpetuates ROS release. These conditions in turn are

prone to stabilize the HIF transcription factor even under normoxic conditions [5, 6,

19]. An additional source of ROS in tumor cells is mediated by oncogenes

themselves, through the up-regulation of the NOX family members. Indeed, the RAS

family of oncogenes was observed to lead to the up-regulation of NOX1 and NOX4

expression [20-24]. In the same way, HER2-amplified breast tumors exhibit high

levels of NOX4 expression [25]. Furthermore, the expression of the NOX genes was

also found increased in prostate and colon cancers [26, 27]. Consistent with

increased ROS production, there is also evidence for enhanced activation of the

antioxidant program driven by the NRF-2 transcription factor in neoplasia, which has

differential impacts on tumorigenesis [28-30]. As discussed in [31], the impact of

NRF-2 on tumorigenesis could be context-dependent: when NRF-2 activity is

enhanced due to rises of ROS, NRF-2-dependent signatures could be associated

with a more favorable prognosis; in contrast, if increased NRF-2 activity results from

mutation in NRF-2/Keap1 genes, and thus independently of the redox state, NRF-2

activity could be linked to treatment resistance and poor prognosis [31]. Finally, the

increase of ROS in tumor cells can also be related to the decreased expression or

reduced activity of antioxidant enzymes such as catalase or manganese superoxide

dismutase (MnSOD), resulting from genetic or epigenetic regulation [32-34]. In this

line, it was recently observed that accumulation of ROS in the tumor

microenvironment can also be due to the inactivation of the extracellular SOD [35].

Lastly, it has been frequently observed that several cancers, including breast,

ovarian, colon and prostate tumors contain mutations in the mitochondrial DNA that

contribute to increase ROS levels in tumor cells [36]. Interestingly, Petros et al. [37]

found in prostate tumor cells that mutations in the mitochondrial DNA coding for

genes involved in oxidative phosphorylation lead to defective oxidative

phosphorylation and ultimately to ROS production. Mitochondrial dysfunction and

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RAS activation have been shown to cooperatively stimulate production of ROS, even

in neighboring cells with normal mitochondrial function, causing benign tumors to

exhibit metastatic behavior [38].

Taken together, there are several sources leading to an increase of ROS, and

possibly others remain to be investigated. Considering that ROS increase is a key

early event in signaling cascades and cancer growth, understanding how ROS are

generated and released may open new avenues for therapy. Although regarded as

unwanted products that lead to DNA damage, the relevance of ROS in the regulation

of diverse pathways is now evident. In fact, in cancer cells, the increase of ROS

induces signaling cascades that mediate the oncogenic phenotype. In the next

section, we discuss the impact of ROS in the cross-talk between epithelial tumor

cells and CAFs.

2. Role of ROS in CAF activation Although several studies have been initially focused on the cancer cell itself, the

critical role of the microenvironment in tumorigenesis, tumor progression and

metastatic spread of cancer cells has now been widely recognized [39-41]. In

particular, numerous studies have highlighted the key role of CAFs in tumor

progression. A large proportion of CAFs detected in aggressive adenocarcinomas

expresses smooth-muscle α-actin (α-SMA) and is therefore called myofibroblasts

[25]. Other markers such as Tenascin-C [42], integrin β1 [43], Platelet-Derived

Growth Factor Receptor-β (PDGFR-β) [44, 45], Caveolin-1 [46], Fibroblast Specific

Protein 1 (FSP1) also known as S100A4 [43, 47], Fibroblast Activation Protein (FAP)

[48], NG2 chondroitin sulfate proteoglycan (NG2) [49, 50] and Podoplanin [51, 52]

have also been used to identify fibroblast subtypes in carcinomas. To what extend

those markers identify distinct fibroblast populations remains unclear and defining

their specific role in tumorigenesis deserves detailed investigation. In this section, we

will focus on the contribution of ROS as a driver of CAFs activation.

ROS contribution to myofibroblast differentiation

The cellular origin of myofibroblast can be multiple. In this section, we describe the

underlying molecular ROS-controlled signaling mechanisms involved in myofibroblast

differentiation. In adenocarcinomas, it has been initially suggested that

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myofibroblasts can derive from epithelial cells through epithelial to mesenchymal

transition (EMT) [53-56]. Although some data indicate that the stromal compartment,

when microdissected from human breast tumors, exhibits genetic alterations, the

proportion of such alterations in fibroblasts remains rare suggesting that EMT cannot

be the main origin for myofibroblasts [57]. Recent data from human breast cancers

and animal models established that myofibroblasts can derive from bone marrow

derived cells, such as fibrocytes or mesenchymal stem cells [58-63]. Moreover,

various mesenchymal cell types, including endothelial cells, pericytes or pre-

adipocytes can also be converted into myofibroblasts in breast carcinomas [56, 64,

65]. Finally, local resident fibroblasts have also been considered as one of the major

sources of tumor-associated fibroblasts [25, 44, 66-68]. Myofibroblasts can be

induced by various signaling pathways, including transforming growth factor-β (TGF-

β), or the CXCL12(SDF-1)/CXCR4 axis, which also mediates fibroblast activation

during wound healing and organ fibrosis [25, 39, 43, 67, 69, 70]. Several studies

have highlighted the role of ROS in the transition of normal fibroblasts into

myofibroblasts. Indeed, mitochondrial ROS are required for TGF-β signaling.

Accordingly, pharmacological inhibition of mitochondrial ROS generation results in

reduced expression of NOX4, an enzyme that is required for TGF-β driven

conversion of fibroblasts into myofibroblasts [71]. Similarly, the CXCL12 chemokine,

which is up-regulated under stress conditions, promotes differentiation of fibroblasts

into myofibroblasts in a ROS-dependent manner [25, 67]. In addition, fibroblasts

suffering from chronic oxidative stress exhibit properties of myofibroblasts [25, 72].

Indeed in mouse models exhibiting chronic oxidative stress (such as JunD-/- or NRF-

2-/- mice, depleted for key anti-oxidant transcription factors [6, 73]) fibroblasts

inactivated for these factors are converted into myofibroblasts and long-term

antioxidant treatment reverses this phenomenon [25, 72]. Interestingly, HER2-

amplified human breast tumors exhibit enhanced CXCL12 signaling and a related

oxidative stress signature, which are correlated with high content of myofibroblasts.

Taken together those observations demonstrate that ROS can promote myofibroblast

differentiation in human tumors.

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In tumors, the oxidative stress that may account for fibroblast conversion into

myofibroblasts can arise from fibroblasts or from any other cell types present within

the tumor bed, including cancer cells themselves. Indeed, H2O2 can be mainly

produced by tumor epithelial cells as described above. Being a highly diffusible

species, H2O2 has been involved in intercellular communications [74], and could

induced fibroblast differentiation into myofibroblasts. In that matter, NOX enzymes,

located at the plasma membrane of tumor epithelial cells are overexpressed in

various carcinomas and might contribute to the production of H2O2 and the

conversion of surrounding fibroblasts into myofibroblasts (Fig. 2). Accordingly, NOX4

overexpression has been detected in HER2 breast cancers, in association with a

high myofibroblast content [25]. Alternatively, metalloproteases (MMPs) such as

MMP3 have been shown to modulate the activity of mitochondrial respiratory chain

thereby increasing cellular ROS contents [75]. At last, myeloid cells infiltrating the

tumor bed might also represent an important additive source of ROS.

Differential ROS effects on fibroblast subtypes

Despite the numerous publications illustrating the role of CAFs in cancer

progression, the molecular basis for fibroblast identification in the tumor tissues are

still unclear. Indeed, while α-SMA has long been recognized as a prominent

myofibroblast marker, various other markers have been used to detect stromal

fibroblasts in tumors. Such markers include: Tenascin-C [42], integrin β1 [43],

PDGFR-β [44, 45], Caveolin-1 [46], FSP1 (also known as S100A4) [43, 47], FAP

[48], NG2 [49, 50] and Podoplanin [51, 52]. To date, whether those markers identify

distinct fibroblast populations and their specific functions in tumorigenesis remain to

be determined. Yet, CAFs are a complex set of heterogeneous populations and one

might argue that ROS could impact differently the distinct fibroblast subtypes. In

addition to α-SMA+ fibroblasts (referred to as myofibroblasts above), another

fibroblast subtype that might be specifically affected by ROS is the PDGFR-β

fibroblasts. Indeed, PDGF is a potent stimulator of fibroblast growth and motility and

ROS have been shown to transduce PDGF signaling by inactivating phosphatases

[76-79,139,140]. The reversible inhibition of protein tyrosine phosphatases

(PTPases) by oxidation of their catalytic site is now established as a key regulatory

mechanism in growth factor signaling. The increase of ROS levels through binding of

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PDGF ligands on their receptors inhibits protein tyrosine phosphatase activity, further

enhancing PDGFR-signaling and ultimately increasing cell proliferation and migration

[76-79]. Reciprocally, PDGF-BB has been shown to induce ROS production in

normal human fibroblasts [80]. In a separate study, NOX4 and DUOX2, two enzymes

increasing ROS production, have been reported to regulate cell cycle entry upon

PDGF stimulation in normal human fibroblasts [81]. Finally, although studies on

PTPase oxidation have mainly be focused on pathways that regulate ROS levels,

recent data have highlighted the key role of the thioredoxin system in the reactivation

of PTPases [139]. Taken together, these results indicate that ROS can promote

PDGF signaling and might therefore specifically interfere with PDGFR-β+ CAFs

functions, in particular cell proliferation and migration. Caveolin-1 represents another

fibroblast marker that might be affected by ROS [82]. In vitro, caveolin-1 is degraded

in fibroblasts co-cultured with tumor epithelial cells in response to oxidative stress.

This degradation can be prevented by treatment with antioxidant and autophagy

inhibitors [83, 84]. Moreover, loss of caveolin-1 expression is also sufficient to induce

further oxidative stress suggesting the existence of a positive feedback loop.

Interestingly, different ROS have been described to affect differentially caveolin-1

expression in lung cancer cells: hydroxyl radical up-regulates caveolin-1 expression

whereas superoxide anion and hydrogen peroxide down-regulates caveolin-1

expression through a transcription-independent mechanism that involves protein

degradation via the ubiquitin-proteasome pathway [85]. In fibroblasts, caveolin-1

expression has been associated to metabolic switch, autophagy/mitophagy activity

and biochemical remodeling of the microenvironment. Those observations suggest

that ROS might have specific effects on the function of caveolin-1+ CAFs, which

might depend on the exact nature of ROS. Finally, NOX enzymes and derived ROS

are mandatory for the pro-survival signaling mediated by the extracellular matrix

receptor integrin β1, another CAF marker [86]. Taken together, these findings

suggest that CAFs consist of various distinct populations, whose differentiation state

and functions might be differentially affected by ROS.

3. ROS impact on CAF functions Modulation of CAF invasive properties by ROS

Previous studies have reproductively demonstrated that CAFs represent an

important determinant in the final outcome of cancer patients. Indeed, CAFs exhibit

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higher tumor-prone potential compared to their normal counterparts [25, 44, 67, 87-

92]. CAFs are sources of paracrine signals, which promote tumor cell proliferation,

survival and invasion, as well as neo-angiogenesis, inflammation and extracellular

matrix (ECM) remodeling, all processes being major hallmarks of cancer [93-95]. The

increase of ROS in the stromal fibroblasts results in the secretion of pro-invasive

signals (HGF, interleukin-6, VEGF, CXCL12, CXCL14- as discussed in section 2)

that trigger signaling pathways and promote tumor cell motility and neo-

angiogenesis, further increasing metastatic dissemination [5, 39, 45, 63, 90, 96, 97].

Moreover, CAFs can indirectly enhance migration of tumor cells (Fig. 2). Indeed,

CAFs, similarly as fibroblasts exposed to chronic oxidative stress, express genes that

encode for proteases involved in ECM remodeling, including collagens, cell adhesion

molecules and MMPs [25, 72]. Interestingly, imaging of invasive co-cultures of

squamous cell carcinoma cells and fibroblasts showed that the leading cell is always

a fibroblast and that cancer cells move behind the fibroblast in the extracellular tracks

[98]. Interestingly, this effect is mediated through the Rho-dependent pathway, which

generates contractile forces in stromal fibroblasts in a ROS-dependent manner to

remodel ECM and create tracks for collective migration of tumor cells [99]. CAFs

contribute to increase the invasion and metastasis by promoting also EMT of

carcinoma cells. Likewise, MMP3 overexpression, which has been observed in early

stages of human breast cancers, leads to the expression of Rac1b and to the

stimulation of mitochondrial production of ROS. This increase of ROS levels

orchestrates the induction of the EMT program [75]. The involvement of ROS and

CAFs in EMT was also shown in prostate cancer, where CAFs secrete MMPs that

lead to the production of ROS by COX-2 in tumor cells, which is required for EMT,

stemness properties and dissemination of tumor cells [100-102]. Hence, the oxidative

tumor microenvironment not only modulates the conversion of fibroblasts into

myofibroblasts but also the production of paracrine signals and matrix remodeling

enzymes that impact on tumor epithelial cells, leading to invasion and metastasis. In

that context, the role of the redox-regulation of protein tyrosine phosphatases

(PTPases) is emerging as a key mechanism regulating signaling from cell surface

receptors, i.e. tyrosine kinase receptors and integrins [139]. Indeed, PTPase

inactivation by reversible oxidation of their active site has been shown to be

important regulator of growth factor signaling, especially for PDGFR-β-mediated

signaling [140]. This reciprocal crosstalk between redox regulators and signaling

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pathways control tumor cell proliferation and migration. The molecular mechanisms

controlled by ROS that impact on CAF function and crosstalk with epithelial cells is

described below.

Metabolic cross-talk between CAFs and tumor cells

One main feature characterizing tumor cells is the metabolic reprogramming that

allows cell growth and division. Growing evidence highlights the role of CAFs

supporting the metabolic reprogramming of tumor cells. As discussed above, most

cancer cells predominantly produce energy through a high rate of glycolysis and

lactic acid fermentation, even in the presence of normal levels of O2, a process

referred to as the “Warburg effect” [103]. Recently, it has been proposed that tumor

epithelial cells also induce aerobic glycolysis in neighboring fibroblasts [104]. These

CAFs then undergo a myofibroblastic differentiation and secrete lactate and

pyruvate, energy-rich metabolites that could be taken up by tumor cells. In this

model, tumor cells use CAFs for providing them high energetic sources and

transform the normal stroma into a wound healing-like stroma. This scenario is called

the “reverse Warburg effect”. In that sense, it has been proposed that cancer and

stroma co-evolve to give advantage to tumor cell growth [105, 106]. The lactate

uptake by cancer cells increases the surrounding pH, thus also protecting cells from

the extremely acid microenvironment. Consistently, oxidative stress in CAFs leads to

the up-regulation of the mono-carboxylate transporter MCT4, which shuttles lactate

to the epithelial cells [107]. Indeed, high expression levels of the glucose transporter

MCT4 in tumor stroma correlate with poor prognosis in triple negative breast cancer

patients [108].

ROS, HIF and metabolism

It is recognized that metabolic reprogramming is under control of oxidative stress and

hypoxia. The transcription factors HIF-1 and HIF-2 play a crucial role in the metabolic

reprogramming of both CAFs and epithelial cells. Although their role remains

controversial under hypoxia, clear evidence now exist indicating that ROS stabilize

the HIF proteins under normoxic conditions through modulation of PHD enzyme

activity [5, 109-111]. Chiarugi and collaborators have shown that accumulation of

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ROS and HIF stabilization in CAFs result also from the down-regulation of SIRT3, a

mitochondrial NAD-dependent deacetylase [112]. HIF through SIRT3 can regulate

CAFs metabolism driving the Warburg phenotype. In addition, HIF-1 regulates

several genes involved in glucose metabolism, as glucose transporters (GLUT1 and

GLUT3) in order to increase the glucose uptake in the cell. Moreover, HIF targets

MCT4, responsible for the export of lactate [113]. Finally, HIF also activates pyruvate

kinase M2 (PKM2) transcription, contributing to reprogram the glucose metabolism of

epithelial cells [114]. In summary, oxidative stress induced by ROS production has

severe implications in CAFs metabolism, driving aerobic glycolysis that in turn has

profound effects on tumor microenvironment. In a simplistic view, CAFs feed the

cancers with high-rich compounds and induce anti-oxidant defense in cancer cells

allowing cancer cells to proliferate. Although the main focus of research has been

attributed until now to the interactions between CAFs and cancer cells, the effects of

oxidative stress on other components of the tumor microenvironment and the

interaction of “stressed”-CAFs with other cell types in the microenvironment remain

to be investigated, as well as further implications in tumor aggressiveness and

resistance to treatment.

ROS impact on CAF interaction with tumor epithelial cells

The high levels of ROS generated by mitochondrial dysfunction, NOX

overexpression or any other mechanisms, promote tumor cell proliferation and

motility in breast cancers [115]. In this regard, there are increasing reports in

literature demonstrating the role of ROS in several signaling pathways that control

properties of tumor cells. Here, we focus on two well-known examples of how ROS

modulate the cross-talk between cancer cells and CAFs that ultimately leads to

enhanced tumor cell motility and invasion. Cancer migration and metastasis are

mediated by chemokines receptors [116]. Chemokines can act as paracrine factors,

establishing the communication between tumor epithelial cells and the cellular

components of the tumor microenvironment to promote tumorigenesis, tumor growth

and metastasis. The two examples considered below, namely CXCL12(SDF-

1)/CXCR4 and HGF/c-met, are regulated by the HIF transcription factors under

normoxia in a ROS-dependent manner [25, 117-121].

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The activation of CXCL12/CXCR4 signaling pathway is one striking example of

cross-talk between tumor epithelial cells and CAFs that can be affected by ROS [90].

The cytokine CXCL12 is highly secreted by CAFs, while its receptor CXCR4 is found

mainly at the surface of tumor epithelial cells. CXCL12 signaling via its receptor

CXCR4 drives proliferation of tumor cells and promotes neo-angiogenesis by the

recruitment of endothelial progenitor cells into the tumor stroma [90].

CXCL12/CXCR4 signaling pathway has been found to be up-regulated in various

tumor subtypes, such as HER2 breast adenocarcinoma subtype where both CXCR4

and CXCL12 accumulate and play key roles in metastases. Indeed, up-regulation of

CXCR4 in HER2 cancer cells is essential to HER2-mediated tumor metastases [122].

In addition, accumulation of CXCL12 in the stroma of this breast cancer subtype

correlates with stromal alterations, including myofibroblast proportion and neo-

angiogenesis, NOX4 accumulation and enhanced rate of metastases [25, 123, 124].

Another example of a cross-talk between tumor epithelial cells and CAFs modulated

by ROS is the HGF/c-Met signaling pathway. Activation of HGF/c-Met pathway is

involved in the acquisition of an aggressive phenotype and resistance to therapy, as

shown in breast cancer. HGF is mainly expressed and secreted by tumor-associated

fibroblasts, while the proto-oncogene product c-Met is overexpressed at the surface

of tumor epithelial cells [125]. HGF binding to its cognate receptor c-Met increases

ROS levels in tumor cells, subsequently leading to c-Met phosphorylation and

activation of downstream signaling pathway. Activation of HGF/c-met signaling

promotes ultimately tumor cell motility and invasion [126]. Additionally, ROS levels in

the tumor bed can influence HGF bioavailability. Indeed, HGF can be sequestered in

the ECM by thrombospondin-1 [127] while high ROS levels inhibit thrombospondin-1

scavenger activity [128]. As a result, high ROS levels in the tumor bed lead to

increased HGF bioavailability and activation of the HGF/c-Met pathway.

Taken together, those observations indicate that oxidative stress in the tumor bed

can modulate the interactions between tumor epithelial cells and CAFs, contributing

to tumor progression and metastasis.

4. ROS and CAFs: similarities in cancer and wound healing

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The tumor microenvironment and the granulation tissue formed during wound healing

share remarkable histological resemblances [129]. Indeed, both tissues are

characterized by a reactive stroma composed of activated fibroblasts and specific

ECM components, immune cells and newly formed blood and lymph vessels.

However, in wounds, the stromal reaction is only a transient state while it persists in

tumors leading to Dvorak tumor definition as “wounds that do not heal”. Interestingly,

ROS have been involved in the initiation of the wound healing process, in particular

in cell attraction, migration and adhesion [130, 131]. Thus ROS-mediated signaling is

a key event share by both cancer and wound healing (Fig. 3).

ROS and inflammation: impact on CAFs

ROS, mainly H2O2, are generated immediately after injury and can act as

chemoattractant for immune cells [74, 132]. In zebrafish, tissue-scale gradient of

H2O2 allows leukocyte recruitment to the site of injury across distances of hundreds

of micrometers within minutes of wounding. Indeed, leukocyte recruitment coincides

with the presence of H2O2 in the blood vessels while H2O2 dissipation leads to a

decreased recruitment of immune cells [74]. Moreover, Feng et al. [133] showed,

using zebrafish as a model, that H2O2 is a key signal involved in early recruitment of

leukocytes to transformed cell burden. Interestingly, the authors provide evidence

that both transformed cells and normal surrounding cells contribute to generate H2O2.

To what extent H2O2 may play a similar role in higher organisms remains to be

established. In wound healing, a major consequence of leukocyte recruitment to the

wound is the secretion of inflammatory molecules. Thus, fibroblast activation and

inflammatory response are co-occurring in wound healing and cancer. Similarly,

chronic inflammation is also associated to fibroblast conversion into myofibroblasts

suggesting that inflammatory cells could induce CAF activation. Consistently,

inflammatory cytokines such as IL1β, TNF-α or TNF-β contribute to fibroblast

activation in chronic inflammatory disease. One might thus propose that H2O2-

recruited inflammatory cells could impact CAF heterogeneity and functions.

Ca2+ and ATP: primary signals inducing ROS in wound healing

Knowledge about the signals that mediate wound healing is emerging. The wound

healing response is a rapid process and the earliest molecular events identified are

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diffusible damage signals that include Ca2+ flashes, ATP release and H2O2 gradient.

These events induce short-term transcription-independent cellular effects while gene

expression is being adjusted [134]. Wounding triggers an instantaneous Ca2+ flash in

a variety of species including zebrafish, C.elegans and X.laevis [135-137]. Blocking

this Ca2+ flash inhibits H2O2 release at the wound site and leads to a reduction in the

number of immune cells migrating to the wound. The wound-induced Ca2+ flash was

shown to activate DUOX and thus to trigger the production of the attractant damage

cue H2O2 [132]. Consistently, in zebrafish and drosophila, inhibition of DUOX activity

abrogates the formation of H2O2 gradients [74, 132]. Accordingly, DUOX knockdown

results in decreased immune cell recruitment and reduces the number of transformed

cells, showing the clear impact of this signaling pathway in tumorigenesis [133]. ATP

release is also an early event triggered by wounding, and recent studies have shown

that ATP release can lead to an increase of Ca2+ and H2O2, serving as messenger

molecule to activate a cascade of signaling events in surrounding cells. This

signaling cascade is in part mediated by Ca2+ increase, subsequently leading to

activation of signaling pathways, such as EGF-dependent pathway, that ultimately

contributes to the healing of the wound [138]. As both Ca2+ and ATP have been

shown to modulate DUOX activity and subsequent H2O2 production, it is possible that

those diffusible signals might impact stromal components, such as CAFs. Consistent

with that hypothesis, H2O2, Ca2+ and ATP affect molecules, such as Rho GTPases,

that regulate the cytoskeleton and migratory properties similarly to phenotype of

tumor-associated myofibroblasts [25]. Further studies will be required to investigate

the role of Ca2+ flashes, ATP release, DUOX activity and H2O2 gradient in CAF

heterogeneity and functions and more generally in cancer biology in mammals. Being

the earliest events in wound signaling, and if homology is shared cancer, the control

of these signals could uncover new strategies in cancer therapy.

5. Conclusion Chronic oxidative stress has severe implications in tumor initiation, growth and

metastasis. ROS can be produced by tumor epithelial cells or various stromal cell

types. They act as crucial signaling molecules not only in cancer cells but also in

surrounding stromal components, through diffusion of the most stable or diffusible

ROS compounds, such as H2O2. ROS mediate cell motility and invasive properties of

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tumor cells, contribute to ECM remodeling, increase neo-angiogenesis and are

involved in the metabolic reprogramming of both tumor cells and CAFs. As described

in this review, one of the major effects of ROS is the conversion of fibroblasts into

myofibroblasts, thus shaping the tumor microenvironment. As CAFs have been newly

defined as heterogeneous populations, the effect of ROS in the genesis of these

different sub-populations and their biological functions needs to be investigated.

Moreover, ROS sustain the reactive stroma, which characterizes both wound healing

and cancers, not only by affecting CAFs but also by mediating the recruitment of

inflammatory cells. To what extent ROS-recruited inflammatory cells have an impact

on fibroblast heterogeneity is not yet known and deserves further investigation. In

summary, ROS are unarguably main drivers, which alter major stromal components

and affect the shape and the tension of the tumor microenvironment. Better

understanding of their functions in cancer prognosis and chemo-sensitivity could

therefore contribute to pave the way for new concepts of therapy.

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FIGURE LEGENDS Figure 1: Main sources of reactive oxygen species (ROS) in epithelial cancers

ROS generated from epithelial cells can result from increased metabolism associated

with dysfunctional mitochondria, oncogene activation or cytokine/chemokine

signalling that activate ROS-producing enzymes: NADPH oxidases (NOX),

clycloxygenases (COX, that mediates the production of prostanoids) and

lipoxygenases (LOX, that catalyze the dioxygenation of polyunsaturated fatty acids,

FA). In solid tumors, hypoxia could also contribute to maintain a certain rate of

oxidative stress. Additionally, levels of antioxidant defenses are often reduced. In the

tumor microenvironment, CAFs and inflammatory cells are also important producers

of ROS and thus contribute to persistent oxidative stress in tumors. Figure 2: Role of ROS in myofibroblast differentiation and crosstalk with tumor epithelial cells Chronic oxidative stress in tumor microenvironment, mainly represented by hydrogen

peroxide due to its highly diffusible properties, originate mostly from tumor epithelial

cells (e.g. by up-regulation of the NADPH oxidase (NOX) enzymes) and to a lower

extent from other cell types, such as myeloid cells or fibroblasts themselves. ROS

modulate several factors and signaling pathways (including HIF, CXCL12, PDGFR-β,

TGF-β, caveolin), which have been shown to play key roles in the conversion of

fibroblasts into myofibroblasts (α-smooth muscle actin-positive fibroblasts with high

migration propensity). In turn, ROS levels in activated fibroblasts stimulate paracrine

signals, which promote tumor epithelial cell motility and invasion, increase

extracellular matrix (ECM) remodeling, stimulate neo-angiogenesis and modulate

tumor cell metabolism, altogether contributing to tumor progression and metastasis.

Figure 3: Similarities between cancer and wound healing driven by ROS and CAFs

ROS, through hydrogen peroxide, modulate fibroblast conversion into myofibroblasts

and participate in the recruitment of inflammatory cells both in wound healing and

cancer. When healing process is achieved, ROS levels decrease and myofibroblasts

undergo nemesis. In contrast, in tumors, the chronic oxidative stress sustains a

persistent myofibroblastic and inflammatory environment. Moreover, in wound

healing, calcium waves and ATP release stimulate hydrogen peroxide release

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(through DUOX activation), while in tumors, these mechanisms are still poorly

characterized. Specifically, how ROS, calcium or ATP modulates fibroblast

heterogeneity and impact on their functions remains to be investigated.

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Figure3