**4. Metastasis**

Many wound healing cellular actors, molecular mechanisms and signaling pathways are also implicated in metastasis [2]. Therefore, elucidating the link between wound healing and metastatic cancer progression may allow the development of better therapeutic strategies against these two pathologies.

### *4.1. Metastasis Hallmarks*

Metastatic spread comprises a complex succession of cellular biological events leading to the dissemination of cancer cells from the tumor to the surrounding tissues and to distant organs, through blood and lymphatic vessels [90]. Furthermore, it also involves crosstalk between cancer cells and components of the tumor microenvironment [91].

The metastatic process begins with the hypoxia at the primary tumor site due to excessive cell proliferation [92]. Reduced oxygen level induces HIF-1 α stabilization and its nuclear translocation, which promotes the expression of various genes involved among others in angiogenesis, glucose metabolism, extracellular matrix remodeling, epithelialmesenchymal transition, metastasis, cancer stem cell maintenance and immune invasion [93]. In parallel, hypoxia-induced necrosis results in a continuous release of cellular debris, notably High Mobility Group Box protein-1 (HMGB1) by dying tumor cells [94]. HMGB1 has been shown to be up-regulated in tissue biopsies from cancer patients [95]. Interestingly, HMGB1 plays opposite roles depending on its redox state. Oxidized HMGB1 induces the production of pro-inflammatory cytokines whereas the reduced form interacts with TAM therefore regulating monocyte recruitment, angiogenesis and immune suppression [96]. In fine, altering the redox status of HMGB1 may be considered as a therapeutic approach to combat metastasis and favor wound healing.

Angiogenesis provides oxygen and nutrients supply essential for cancer cells to dissociate from the basal membrane delineating the epithelial compartment from the stroma. This requires the degradation of the extracellular matrix (ECM), through the activation of matrix metalloproteinases [97]. Under normal circumstances, cells detachment from the ECM leads to the induction of an apoptosis called anoikis, a form of programmed cell death that occurs in anchorage-dependent cells [98]. However, cancer cells develop a transdifferentiation program known as epithelial–mesenchymal transition (EMT), which render the cells resistant to anoikis [99]. Anoikis plays an important role in the prevention of metastasis and promoting its induction might be an interesting therapeutic strategy. Finally, cells acquire stemness properties. Stemness is the ability of a cell to perform self-renewal and is capable of pluripotency. This is an important feature for supplying material for wound closure and for the establishment of cancer cells at the metastatic sites [100].

### *4.2. ROS and Metastasis*

One of the principal mechanisms underlying metastasis in human cells is the disruption of the redox balance. This imbalance in redox homeostasis is induced by an increase in free radicals, mainly ROS [101]. Cancer cells have elevated expression levels of NOXs (NOX1, NOX2, NOX4, NOX5), leading to high levels of ROS [101,102]. Consequently, cancer cells have been shown to be more tolerant to oxidative stress via increased expression of catalase and superoxide dismutase. However, the lack of robust anti-oxidant defenses

may have detrimental consequences in the tumor microenvironment and in the adjacent normal cells [103].


Although several processes of metastasis are redox-sensitive, it is still controversial whether ROS have oncogenic/metastatic or tumor suppressive functions. The answer appears to depend on ROS levels and the cancer stage, leading many authors to consider ROS as a "double-edged sword" [101]. Low to moderate ROS levels can promote survival of cancer cells by inducing EMT and stem cell differentiation, enhancing angiogenesis and switching to glycolytic metabolism. Conversely, excessive production of ROS induced by chemotherapy and radiotherapy is detrimental to the survival of cancer cells and causes cellular damage [104,105]. Concerning the stage of the disease, it has been reported that in the early stages of cancer, ROS promote cancer initiation by inducing base pair substitution mutations in pro-oncogenes such as Ras and tumor suppressor genes such as p53 [106]. As cancer progresses, an intracellular excess of ROS triggers apoptosis of tumor cells. To escape this ROS-induced apoptosis, tumor cells produce high levels of anti-oxidants [106]. In the last stages of tumor development, ROS have a pro-metastatic role promoting the spread of cancer cells.


Additionally, ROS are involved in angiogenesis. Angiogenesis is mainly mediated by VEGF whose expression can be regulated by nutrient deprivation and hypoxia, both of which increase levels of ROS [107,108]. Activation of angiogenesis by ROS can involve different signaling pathways. Firstly, ROS have been shown to activate PI3K/Akt/mTOR signaling cascade in different cancer cell lines (MCF-7, HepG2, H-1299, PC-3), enhancing HIF-1 α and VEGF expression and ultimately angiogenesis [109,110]. The role of ROS has been confirmed by several studies showing that catalase and glutathione peroxidase overexpression or NOX4 knockdown lead to a decrease in VEGF and HIF-1 α levels and inhibit angiogenesis in human ovarian cancer cells [111,112]. Further, oxidative stress can induce angiogenesis in a VEGF-independent manner through the activation of the TLR/NF-κB pathway. West et al. demonstrated the proangiogenic effects of TLR1/2 stimulation by oxidative stress, represented by lipid oxidation products, in murine and human melanoma [113]. In addition, angiogenesis is also mediated by matrix metalloproteinases and upregulated by ROS [114].


Several studies have proven that ROS are a major cause of EMT. ROS-induced EMT has been reported to be NOX4-dependent in human metastatic breast epithelial cells [115] and in lung cancer cells [116]. NOX4 is an important source of ROS induced by TGF-β and under hypoxia, two important mediators in cancer metastasis [117,118]. Furthermore, NOX4 inhibition significantly attenuated the distant metastasis of breast cancer cells to lung and bone [119].

Resistance to anoikis seems to concern not only the field of cancer but also this phenomenon may be interesting in wound healing. Indeed, ROS are considered as one of the key players in anoikis sensitivity. In recent studies, ROS generation induced by NOX4 has been involved in anoikis resistance of gastric [120] and lung cancer cells [121]. ROS promote EMT by inducing the expression and activity of MMPs that mediate proteolytic degradation of ECM components [122,123]. TGF-β1, a well-established player of EMT induction, regulates MMP-9 to facilitate cell migration and invasion via the activation of NF-κB through a ROS-dependent mechanism [123]. Similarly, ROS production induced MMP-2 secretion and activation results in pancreatic cells invasion [122]. In colorectal cancer, the EMT process is highly regulated through some of the classic tumorigenic signaling pathways, such as the NF-κB, HIF-1, and TGF-β1 pathways [124]. Intriguingly, TGF-β1 induces EMT through Nrf2 activation as well as ROS production in lung adenocarcinoma cells [116]. Indeed, Nrf2 is a key transcriptional regulator that drives anti-oxidant gene

expression and protection from oxidative damages. Oxidative stress plays a critical regulatory role in these pathways by degrading inhibitors or inducing nuclear translocation and consequent transcription [124].


Cancer stem cells possess a particular redox status, since they have lower ROS levels and increased anti-oxidant capacity than differentiated cancer cells [125,126]. Increasing evidence shows that these low amounts of ROS are actually needed to maintain the quiescence and self-renewal potential of cancer stem cells (CSC). Previous studies have demonstrated that ROS contribute to reduce stemness and to enhance differentiation of CSC. For example, glioblastoma stem cells have potent anti-oxidant defense mechanisms and H2O2 has been shown to inhibit their self-renewal and induce their differentiation [127]. ROS have been reported to promote hematopoietic stem cell differentiation with a progressive increase in ROS levels with the advancing differentiation stages. Moreover, inhibition of ROS production has been found to attenuate the differentiation of hematopoietic stem cells [128]. In summary, hypoxia-associated increase in ROS in tumor cells promotes stemness. Although oxidative stress promotes the development of CSC, ROS level declines after this acquisition of stemness, allowing the maintenance of the sub-population.

### *4.3. Oxidative Stress and Metastasis: Cellular Actors Involved*

Macrophages, neutrophils and fibroblasts are major ROS producers in the tumor microenvironment [92]. Here, we will focus on macrophages and fibroblasts since neutrophils activation in wound healing and metastasis has been already extensively reviewed [129].
