**4. The different functions of CD146**

CD146 was reported to be involved in many physiological processes. It has been described to play a role during the vascular development but also during the angiogenic process. As others junction molecules, it was described to be an actor during inflammation by modulating the migration of leucocytes through vascular endothelium.

## **4.1. CD146 during the vascular development**

The role of CD146 was studied during vascular development. To this end, a model of CD146 inactivation by antisense morpholino-oligonucleotides was developed in zebrafish. Authors observed a decrease in intersomitic vessels followed by a decrease in blood flow and a reduction in vessel lumen observed by microangiography after CD146 inactivation [33]. It was also shown an inhibition of the VEGF-dependent angiogenesis [34].

## **4.2. Permeability and leucocytes migration**

CD146 has been shown to be involved in endothelial permeability [33]. Using both a monocyte cell line, THP-1, and freshly isolated monocytes, it was also showed that it modulates monocytes transmigration. Junctional CD146 was shown to bind monocytes through a heterophilic interaction to increase their transmigration. In addition, an increased transmigration was observed following the binding of soluble CD146 on monocytes [33]. Another study showed that neo-expression of CD146 on lymphocytes induced new cellular properties. Indeed, an increase in the adhesion of CD146+ T lymphocytes effectors was observed after stimulation with IL-1 beta. This effect was blocked by the addition of anti-CD146 blocking antibodies [34]. An increase in the adhesion of CD4+/CD146+ T lymphocytes on endothelium was also observed after an inflammatory stimulus. In this study, *in vitro* transfection of the long isoform of CD146 in NKL.1 cell line induced a reduction of rolling cells and an increased adhesion to the endothelial monolayer. Moreover, these phenomena were accompanied by an increase in microvilli in T lymphocyte cell membrane. Another study showed an increased permeability of HMVEC (human microvascular endothelial cells) following incubation with an anti-CD146 antibody (P1H12) [35]. Finally, CD146 is coexpressed with CCR6 on a population of TH17 lymphocytic cells [36].

## **4.3. Angiogenesis**

Angiogenesis is an important mechanism, both in fetal life and at adulthood. Endothelial cells with angiogenic capacities are able to proliferate, migrate, adhere, and generate new capillaries from a preexisting one.

The injection of an anti-CD146 antibody (AA98) led to a decrease of 70% in the number of vessels in a membrane model, chorioallantoic membrane model, in chicken. Furthermore, in mice, this antibody reduced the number of vessels in different models of xenografted tumors (hepatocellular carcinoma, pancreatic, and leiomyosarcoma) [37], demonstrating a role of CD146 in tumor angiogenesis.

The recent discovery of the existence of two isoforms of CD146 and the description of a soluble form of CD146 led to study their implications in the angiogenic process. Specific siRNA directed against these two isoforms has shown that the absence of the short CD146 decreased the proliferation, migration, and adhesion of endothelial cells, whereas its overexpression led to the reverse phenomena. These experiments showed that the long CD146 was also necessary to generate pseudocapillaries in Matrigel *in vitro* by stabilizing the junctions of neovessels. It thus appears that both the short and long isoforms of CD146 display complementary effects to generate neovessels. The effects of the short CD146 were confirmed *in vivo* by the transplantation of endothelial colony-forming cells (ECFC) overexpressing this isoform in a mouse model of hind limb ischemia. Indeed, it increased the incorporation of ECFC in ischemic muscle and favored the generation of neovessels [23]. A study of the mechanism showed that the short CD146 is associated with VEGF-R2 [38], but also angiomotin and VEGF-R1 at the endothelial cell surface [39, 40]. This association is essential for these different pathways. Indeed, the absence of the short CD146 isoform decreases the phosphorylation of VEGF-R2 in endothelial cells and prevents the proangiogenic effect of vascular endothelial growth factor (VEGF).

Soluble CD146 is also able to increase the formation of pseudocapillaries *in vitro* and to induce neovascularization in a rat model of hindlimb ischemia. In addition, subcutaneous injection of Matrigel containing soluble CD146 in mice increased the recruitment of both mature and immature endothelial cells, as well as smooth muscle cells, resulting in the formation of capillary-like structures [41]. Of interest, it was reported that soluble CD146 stimulates the short CD146 isoform through its binding on angiomotin [39] and that the angiogenic properties of soluble CD146 are additive to those of VEGF [41]. The roles of the different forms of CD146 are summarized in **Figure 2**.

## **4.4. Cancer cell growth and dissemination**

CD146, which is neo-expressed on cancer cells, modulates their growth and dissemination. In prostate cancer, CD146 expression was observed in different cell lines. CD146 overexpression increased their invasiveness and metastatic potential [42]. CD146 overexpression was also observed in biopsies of patients. Its expression was correlated with a poor prognosis. In ovarian carcinomas, CD146 expression was also correlated with the increase of the metastatic potential. In addition, inhibition of CD146 protein expression in ovarian cancer cell lines led to inhibition of invasiveness, tumor spread and induced cancer cell apoptosis. This was explained by the fact that a lack of CD146 induced a decreased activity of Rho GTPase [43] involved in the invasion, proliferation, and metastatic spread of cancer cells.

an anti-CD146 antibody (P1H12) [35]. Finally, CD146 is coexpressed with CCR6 on a popula-

436 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Angiogenesis is an important mechanism, both in fetal life and at adulthood. Endothelial cells with angiogenic capacities are able to proliferate, migrate, adhere, and generate new capillar-

The injection of an anti-CD146 antibody (AA98) led to a decrease of 70% in the number of vessels in a membrane model, chorioallantoic membrane model, in chicken. Furthermore, in mice, this antibody reduced the number of vessels in different models of xenografted tumors (hepatocellular carcinoma, pancreatic, and leiomyosarcoma) [37], demonstrating a role of

The recent discovery of the existence of two isoforms of CD146 and the description of a soluble form of CD146 led to study their implications in the angiogenic process. Specific siRNA directed against these two isoforms has shown that the absence of the short CD146 decreased the proliferation, migration, and adhesion of endothelial cells, whereas its overexpression led to the reverse phenomena. These experiments showed that the long CD146 was also necessary to generate pseudocapillaries in Matrigel *in vitro* by stabilizing the junctions of neovessels. It thus appears that both the short and long isoforms of CD146 display complementary effects to generate neovessels. The effects of the short CD146 were confirmed *in vivo* by the transplantation of endothelial colony-forming cells (ECFC) overexpressing this isoform in a mouse model of hind limb ischemia. Indeed, it increased the incorporation of ECFC in ischemic muscle and favored the generation of neovessels [23]. A study of the mechanism showed that the short CD146 is associated with VEGF-R2 [38], but also angiomotin and VEGF-R1 at the endothelial cell surface [39, 40]. This association is essential for these different pathways. Indeed, the absence of the short CD146 isoform decreases the phosphorylation of VEGF-R2 in endothelial cells and prevents the proangiogenic effect of vascular endothelial

Soluble CD146 is also able to increase the formation of pseudocapillaries *in vitro* and to induce neovascularization in a rat model of hindlimb ischemia. In addition, subcutaneous injection of Matrigel containing soluble CD146 in mice increased the recruitment of both mature and immature endothelial cells, as well as smooth muscle cells, resulting in the formation of capillary-like structures [41]. Of interest, it was reported that soluble CD146 stimulates the short CD146 isoform through its binding on angiomotin [39] and that the angiogenic properties of soluble CD146 are additive to those of VEGF [41]. The roles of the different forms of CD146

CD146, which is neo-expressed on cancer cells, modulates their growth and dissemination. In prostate cancer, CD146 expression was observed in different cell lines. CD146 overexpression increased their invasiveness and metastatic potential [42]. CD146 overexpression was also

tion of TH17 lymphocytic cells [36].

**4.3. Angiogenesis**

ies from a preexisting one.

CD146 in tumor angiogenesis.

growth factor (VEGF).

are summarized in **Figure 2**.

**4.4. Cancer cell growth and dissemination**

It was also demonstrated that CD146 expression is increased in osteosarcomas as compared to nonpathological osteoblasts [44]. Injection of antibodies against CD146 decreased the amount of lung metastases in an immunodeficient mouse model injected with cells derived from human osteosarcoma [45].

In breast cancer, it was reported that CD146 would act as a tumor suppressor [46] while other studies have described CD146 as a poor prognosis marker [47]. Indeed, CD146 overexpression in a breast cancer cell line induced an increased motility and tumorigenicity [48]. Recent studies have also shown that CD146 induces the epithelial-mesenchymal transition (EMT) in so far as its expression is correlated with markers of EMT in gastric cancer [49]. Moreover, in triple negative breast cancers, an increase of CD146 expression in epithelial cells correlates with a loss of epithelial markers in favor of mesenchymal markers, increasing their invasiveness, migration, and the number of mammospheres. In addition CD44 expression increases and CD24 expression decreases on the cell surface suggesting that cells acquire phenotypic characteristics of cancer stem cells [50].

At present, there is no data on the differential expression and roles of the two membrane isoforms of CD146 on cancer cells. However, recent studies have shown an important role of soluble CD146 in tumor development. First, an increase of soluble CD146 concentration was described in blood of cancer patients with nonsmall cell lung cancer as compared to patients with respiratory inflammatory disease and healthy subjects [51]. In this chapter, we showed that association between an increased soluble CD146 concentration and an increased number of circulating endothelial cells (CEC) constitute a poor prognostic factor [51].

Recently, a study showed that human cancer cells that express membrane CD146 on their surface have also the ability to secrete the soluble form of CD146 [20]. This was described in melanoma, colorectal and pancreatic cancer cell lines. The authors demonstrated that soluble CD146 secreted by cancer cells could display autocrine effects on cancer cells and paracrine effects on vascular endothelial cells. Indeed, *in vitro* stimulation of cancer cells with recombinant soluble CD146 increased their proliferation and the production of protumorigenic factor such as VEGF. They also demonstrated that this stimulation protected cancer cells from apoptosis induced by H2 O2 and decreased cancer cell senescence. In particular, the c-myc signaling pathway appeared to be upregulated by soluble CD146. Soluble CD146 secreted by cancer cells also increased the proliferation of surrounding endothelial cells, stimulating tumor angiogenesis. These effects were confirmed *in vivo* in different models of xenografted mice and an antisoluble CD146 antibody was able to block these effects. Thus, soluble CD146 was described as a proangiogenic factor and seems to have a major role in tumor development.
