Modulators of Tumor Angiogenesis

#### **Chapter 3**

## Role of Exosomes in Tumor Induced Neo-Angiogenesis

*Joni Yadav, Nikita Aggarwal, Apoorva Chaudhary, Tanya Tripathi, Dikkshita Baruah, Suhail Chhakara, Divya Janjua, Arun Chhokar, Kulbhushan Thakur, Anna Senrung and Alok Chandra Bharti*

#### **Abstract**

Exosomes are the nanovesicles, belonging to the type of extracellular vesicles (EVs), produced by normal as well as tumor cells and function as a mode in cell-to-cell communication. Tumor cells utilize various approach to communicate with neighboring cells for facilitating tumor invasion and progression, one of these approaches has been shown through the release of exosomes. Tumor-derived exosomes (TEX) have the ability to reprogram/modulate the activity of target cells due to their genetic and molecular cargo. Such exosomes target endothelial cells (among others) in the tumor microenvironment (TME) to promote angiogenesis which is an important element for solid tumor growth and metastasis. So, exosomes play a vital role in cancer invasiveness and progression by harboring various cargoes that could accelerate angiogenesis. Here first, we will present an overview of exosomes, their biology, and their role in different cancer models. Then, we will emphasis on exosomes derived from tumor cells as tumor angiogenesis mediators with a particular importance on the underlying mechanisms in various cancer origins. In the end, we will unveil the therapeutic potential of tumor derived exosomes as drug delivery vehicles against angiogenesis.

**Keywords:** extracellular vesicles, angiogenesis, exosomes, tumor, endothelial cells (ECs)

#### **1. Introduction**

Tumor microenvironment interacts with tumor cells, creating an environment to suppress or contribute towards tumor development and progression [1]. For the tumor development, inflammation and angiogenesis are the processes which play vital roles from initial to the advanced stages of cancer [2]. Extreme angiogenesis and neoangiogenesis play a fundamental role in tumor progression, which is driven by various pro-and anti-angiogenic factors [3]. There are different ways for tumor cells to communicate with adjacent cells/tissues for facilitating tumor progression; one of these is through exosomes [4, 5]. Exosomes can transport various biomolecules like DNA

fragments, mRNAs, noncoding RNAs, proteins, and lipids from a source cell to target/ recipient cells that can enhance angiogenesis, which play a significant role in cancer progression [6]. There are evidences that various noncoding RNAs, particularly microRNAs and long non-coding RNAs (lncRNAs) play significant role in the regulation of angiogenesis [7]. Thus, alteration of angiogenesis has become a striking approach for development of effective cancer therapy [1].

#### **2. Extracellular vesicles (EVs)**

Prior to the discovery of exosomes it was assumed that the transmission of information between mammalian cells occurs in an indirect manner. In 1983, two pioneer studies carried out on the differentiation of reticulocytes into mature erythrocytes, reported release of transferrin receptors into extracellular space in form of small vesicles, which were later termed as "exosomes" by R.M. Johnstone [6, 8–10]. EVs are vesicles enclosed with phospholipid bilayer secreted in the extracellular matrix. Initially, they were initially considered as "garbage dumpsters" but now they are popularly being referred as "signal boxes" [11]. The presence of extracellular vesicles in solid tissue, physiological fluid, and cell culture supernatants has been demonstrated by a number of studies [12]. EV's are broadly categorized into different subtypes like microsomes, microvesicles, retrovirus-like particles and apoptotic bodies, different from each other on the basis of size, surface markers and their mode of biogenesis [13]. Extracellular vesicle is a collective term for exosomes and microvesicles. Microvesicles originate from through outward budding and fusion of plasma membrane whereas, exosomes are released via endocytosis and fusion with plasma membrane [14]. Exosomes are the smallest (30–100 nm) subpopulation of EVs. CD9, CD63 and Alix are the specific surface markers for these exosomes [13]. Exosome serve as important cell communication regulators and have gained more attention among all the diverse types of extracellular vesicles because they represent a more homogenous set of vesicular population more closely representing the parent cell of origin [15].

#### **2.1 Exosome biogenesis**

Exosomes are endosome derived extracellular vesicles. Multivesicular endosomes (MVEs) or multivesicular bodies (MVBs) are secreted via intracellular secretion pathway, from the plasma membrane. Early endosomes develop into MVBs which fuse with the cell membrane and release the exosomes or else undergoes degradation in lysosomes and autophagosomes. They are cup-or disc-shaped when observed under electron microscopy having a diameter of 30–150 nm [11, 16]. Various proteins and molecules like (ALIX, VPS4, and TSG101) are some of the major proteins involved in exosome biogenesis, content assembly and their secretion via endosomal sorting complex [16]. Exosome biogenesis supposedly occurs via two major pathways: Endosomal sorting complexes required for transport (ESCRT) dependent and ESCRT independent. The ESCRT dependent process includes ESCRT complex (0, I, and II) which are involved in recognizing and sequestering the ubiquitinylated proteins on the endosomal membrane. Exosomes are formed by membrane remodeling, involving bud formation by invagination of this endosomal membrane [17]. ESCRT independent pathway involves tetraspanins such as CD63 and lipid metabolism enzymes like neutral sphingomyelinase (nSMase) and rab family protein consisting of more than 60 GTPases that regulate intracellular trafficking of exosomes [16]. Anchoring of MVBs

*Role of Exosomes in Tumor Induced Neo-Angiogenesis DOI: http://dx.doi.org/10.5772/intechopen.104400*

#### **Figure 1.**

*Schematic representation of exosome biogenesis and secretion from eukaryotic cells. Exosome's formation starts with endocytosis, which involves inward budding of plasma membrane, leading to the formation of early and late endosomes. Further, small vesicles are generated by inward budding of late endosomes and forming multivesicular bodies (MVBs). The ultimate fate of MVBs can be either fusion with lysosome for degradation or fusion with plasma membrane to release exosomes. The exosome formation from MVBs proceeds through ESCRT-dependent and ESCRT-independent pathways. ESCRT-dependent pathway involves various ESCRT proteins like (ESCRT 0, I, II, and III) and ESCRT-independent includes lipids (ceramide) and the tetraspanins.*

and transportation of different exosomes is carried out by different RAB subtypes proteins. Early endosome transportation involves RAB5 and RAB21 proteins to mediate endocytosis pathway from early to late endosome and then to lysosome for degradation involves RAB7 protein. Tumor-associated vesicle trafficking requires a vital protein that is RAB27 and it is highly expressed in several tumors. Other than this, various RAB proteins which include RAB 3,11,26,27, 35, 37 and RAB 38 are linked with the exocytic pathway of vesicle trafficking [11]. RAB27 helps in the release of exosomes from mature endosomes enriched in TSG101, ALIX and CD63 whereas RAB11 & RAB35 are associated with the release of early nuclear endosomes which are enriched with PLP, Wnt and TfR. Finally, MVBs fused with the plasma membrane and exosomes are excreted out in the extracellular environment [12]. Diagrammatic representation of exosome biogenesis and secretion has been shown in **Figure 1**.

#### **2.2 Exosomal content**

Exosomes are nanovesicles enriched with a repertoire of biomolecules like proteins, nucleic acids and lipids [16]. Exosomes are dynamic and heterogeneous in nature with respect to their content which majorly depends on their cellular origin, pathological and physiological state of the parent cells. Exosomes from different cell types are enriched specifically in proteins like Alix, Tsg101, integrins, Rab GTPases,

tetraspanins (CD9) and (CD63), MHC class II proteins and heat shock proteins (HSP90, HSP70), which alsoserve as exosome marker proteins [16, 18]. Besides these, exosomes are also enriched with double-stranded DNA's and RNA population of different classes such as microRNA (miRNA), long noncoding RNA (lncRNA) [19]. ExoCarta and Vesiclepedia (http://microvesicle.org/), databases have cataloged the RNA, protein and lipid content of exosomes derived from different sources.

### **3. Mechanisms involved in exosomes-induced angiogenesis**

Tumor derived exosomes (TEXs) have been shown to play a significant role in tumor progression by accelerating angiogenesis [20]. New blood vessel formation occurred when angiogenic signaling pathways are activated by tumor-derived exosomes, when they are up taken by normal ECs [21]. Exosomal cargo once internalized into recipient cells present in the tumor microenvironment, can regulate their fate, function, and phenotype [22, 23]. Tumor cell derived exosomal cargo can activate/inhibit the various signaling pathway in ECs via receptor-ligand interaction [24]. There are several studies represent multiple avenues in which cancer-derived exosomes exert pro-angiogenic effects on ECs. Till date, the different signaling pathways that are involved in exosomes-induced angiogenesis are poorly known. However, the exosomal cargo which is involved in tumor progression and angiogenesis have been documented. Role of TEXs cargoes which is involved in tumor angiogenesis is showed in **Figure 2**. Also, a list of all mRNAs, proteins, and noncoding RNAs which are found in TEXs for regulating tumor angiogenesis are listed in **Table 1**.

#### **Figure 2.**

*Tumor derived exosomes as carrier of pro-angiogenic cargo from different cancer models promote neo-angiogenesis. Tumor-derived exosomes are enriched in proangiogenic proteins, mRNAs, miRNAs, and long noncoding RNAs which are transferred to recipient endothelial cells and activate various angiogenic signaling pathways involved in different angiogenesis process via cell proliferation, migration, and invasion.*

#### *Role of Exosomes in Tumor Induced Neo-Angiogenesis DOI: http://dx.doi.org/10.5772/intechopen.104400*

#### *Tumor Angiogenesis and Modulators*

#### *Role of Exosomes in Tumor Induced Neo-Angiogenesis DOI: http://dx.doi.org/10.5772/intechopen.104400*

*growth factor receptor; VHL/HIF-1α: Von*

*Hippel-Lindau/hypoxia*

 *inducible factor-1α; YAP-VEGF: yes-associated protein-vascular*

 *endothelial growth factor; ZO-1: zona occludens 1.*

#### *Tumor Angiogenesis and Modulators*

#### **3.1 Glioblastoma**

Exosomes derived from glioblastoma cells are known to carry different mRNAs, miRNAs and angiogenic factors which interacts with ECs and thus stimulate angiogenesis. Kucharzewska et al. demonstrated export of pro-angiogenic factors IL-8 and PDGF through exosomes derived from the hypoxic glioma cells and thus induce endothelial proliferation and cell migration by activating the PI3K/AKT signaling pathway [30]. Exosomes from glioblastoma cells showed enrichment of different noncoding RNAs that include, microRNAs (miRNAs): miR-148a-3p, miR-182-5p; long non-coding RNAs (lncRNAs): POU3F3, HOTAIR, CCAT2 in the regulation of glioma cell angiogenesis [22, 28, 29, 32, 33]. Exosomes derived from glioma cells are also known to carry pro-angiogenic proteins such as EGFRvIII, VEGF-A and DII4 which are important for tumor growth, survival and angiogenesis through the activation of Akt and MAPK signaling pathways [25–27, 31].

#### **3.2 Breast cancer**

Breast cancer derived-exosomes transfer majorly pro-angiogenic microRNAs: miR-10b, miR-101, miR-105, miR-122, miR-145, miR-210 and miR-373 responsible for tumor invasion, metastasis and lead to angiogenesis [34–36, 39–41]. However, Wu et al. found that exosomes secreted from breast cancer cells loaded with miR-497 are responsible for anti-angiogenesis by downregulating the VEGF and HIF-1 [37]. Maji et al. have observed that Annexin A2 was transferred via breast cancer exosomes to ECs and induces the process of vascularization and angiogenesis through the tissue plasminogen activator (tPA)-dependent manner *in-vitro* and *in-vivo* [38].

#### **3.3 Multiple-myeloma**

Multiple myeloid cancer cells derived exosomes are known to carry miR-135b and responsible for tube formation in ECs by suppressing its target FIH-1 [42]. Wang et al. observed that various pro-angiogenic factors are released into the exosomes derived from multiple myeloma cells such as angiogenin, bFGF and VEGF that promote tumor growth [43].

#### **3.4 Melanoma**

In a study conducted by Zhuang et al. demonstrated that exogenous miR-9 can advance tumor angiogenesis by downregulating the SOCS-5 levels, which can discordantly regulate the JAK-STAT signaling pathway [44]. Hood et al. have observed exosomes released from melanoma cells stimulate the expression of HIF-1α, HIF-2α and GM-CSF, which leads to angiogenesis in endothelial cells [46]. Moreover, Ekstrom et al. showed that the WNT5A signaling promotes the exosomal secretion from melanoma cells containing immunomodulatory and pro-angiogenic factors such as IL-6, MMP-2 and VEGF [45].

#### **3.5 Pancreatic cancer**

Pancreatic adenocarcinoma produced exosomes having high levels of tetraspanin Tspan8 (D6.1A) that promote migration, proliferation and sprouting in ECs. Moreover, these exosomes also help in maturation of endothelial progenitor cells [47, 48]. Guo et al. showed that lncRNA UCA1 was exported through exosomes derived from the hypoxic pancreatic cancer cells are responsible for angiogenesis via miR-96-5p/ AMOTL2 signaling pathway [50].

#### **3.6 Colorectal cancer**

Studying the exosomes from the colorectal carcinoma demonstrated that these exosomes carry pro-angiogenic factors Wnt 4, which helps in angiogenesis of ECs through Wnt/β-catenin pathway [49]. Hong et al. found that the exosomes released from SW480 colorectal cancer cell lines are loaded with M-phase related transcripts such as RAD21, CDK8, and ERH and regulate M-phase of the cell cycle and promotes proliferation and in turn enhance angiogenesis [51].

#### **3.7 Lung cancer**

Exosomes derived from small cell lung cancer (SCLC) cells are found to be enriched with miR-21 and miR-23a, which is correlated with the pro-angiogenic activities in ECs [52, 53]. A study of Mao et al. demonstrated that exosomes from SCLC cells are responsible for pro-angiogenic effect via miR-141/KLF12 pathway in targeted ECs [54]. In another recent study, Profilin2 protein was transferred from the lung cancer cells via exosomes and leads to angiogenesis by activating the t-PFN2 dependent pERK pathway in endothelial cells [55].

#### **3.8 Hepatocellular carcinoma (HCC)**

Vasorin (VASN), a type I transmembrane protein has an effective role in tumor progression and angiogenesis, was secreted by exosomes of hepatocellular carcinoma cells (HCC) and promotes the migration of HUVEC cells [56]. In another study of Xie et al. showed that angiopoietin-2 protein is transferred to ECs from HCC cells via exosomes and responsible for pro-angiogenesis [57]. Recently, it was found that miR-1290 is also released from the HCC cells through exosomes and responsible for angiogenesis by inducing the miR-1290 induced pro-angiogenic phenotype in endothelial cells, by targeting the SMEK1 [58].

#### **3.9 Renal cell carcinoma (RCC)**

Zhang et al. demonstrated that exosomes derived from renal cancer cell enhances angiogenesis by upregulating the expression of VEGF and downregulating the hepaCAM expression in ECs [59]. Moreover, exosomes derived from renal cancer 786-0 cells promotes invasion and migration of the endothelial cells through upregulation of chemokine receptors CXCR4 and MMP-9 [60]. A recent study of Hou et al. observed that the exosomes derived from renal clear cell carcinoma (RCCC) are loaded with miR-27a and inhibits SFRP1 expression which leads to accelerated angiogenesis in HUVECs [63].

#### **3.10 Bladder cancer**

Beckham et al. observed that the exosomes derived from urine of patients with bladder cancer and high-grade bladder cancer cell lines contain an angiogenic factor. Epidermal growth factor (EGF)-like repeats and discoidin I-like domain-3 (EDIL-3)

that facilitate cell proliferation and migration which leads to angiogenesis in endothelial cells. EDIL-3 activated EGFR signaling overrule this EDIL-3 induced bladder cell migration [24].

#### **3.11 Papillary thyroid cancer (PTC)**

In a recent study by Wang et al. observed that miR-181a is delivered by hypoxic PTC-secreted exosomes inhibits DACT2 by downregulating MLL3, leading to YAP-VEGF-mediated angiogenesis by increasing proliferation and forming capillary-like network in HUVECs. Further, angiogenic potential of hypoxic PTC-secreted exosomes was confirmed in-vivo, which was reversed in presence of hypoxic miR-181 inhibitor [64].

#### **3.12 Head and neck cancer (HNC)**

Chan et al. showed that nasopharyngeal carcinoma (NPC) derived exosomes are supplemented with pro-angiogenic factors, ICAM-1 and CD44v5, which helps in angiogenesis of endothelial cells [66]. In another study by Gu et al. recognized a vital role of PFKFB-3 in NPC derived exosomes, which helps in migration, proliferation and angiogenesis of HUVECs [67]. Exosomes derived from FaDu cells are highly enriched with miR-21, captured by monocytes present in the TME and responsible for increasing the expression of M2 polarization of TAMs markers, which helps in tumor progression by regulating the tumor invasiveness and angiogenesis [65]. In a recent study, it was observed that a nuclear protein HMGB3 is transferred to endothelial cells via exosomes released from NPC cells and responsible for accelerated angiogenesis *invitro* and *in-vivo* [68].

#### **3.13 Esophageal squamous cell carcinoma (OSCC)**

Zhang et al. demonstrated that exosomes released from esophageal squamous cells are enriched with lncRNA FAM225A, which accelerates esophageal squamous cell carcinoma progression and angiogenesis by sponging miR-206. Further, they showed the upregulation of NETO2 and FOXP1 expression when FAM225A absorbed the miR-206 thereby activating PI3K/Akt/NF-κB/Snail axis [69].

#### **3.14 Gastric cancer**

Exosomes derived from gastric cancer cell are enriched with miR-130a and plays a central role in tumor angiogenesis. They showed that exosomal miR-130a is able to facilitate angiogenesis by downregulating the c-MYB, which is an important transcription factor in different biological processes [70]. In another study by Li et al. demonstrated that exosomes released from irradiated gastric cancer cells promote invasiveness and proliferation of endothelial cells [89].

#### **3.15 Chronic myeloid leukemia (CML)**

LAMA84 a human CML cell line releases exosomes and are able to trigger diverse signaling pathways in ECs, leading to enhanced expression of important angiogenic

factor IL-8 [72]. Umezu et al. observed that exosomes from leukemia cells can transport miR-92a into ECs and responsible for enhanced tube formation and migration by downregulation of integrin-α5 [73]. In another study, it was found that leukemia cell derived exosomes are able to induce tube formation in HUVECs by activating Src [71]. It has been observed that exosomes released from K562 leukemia cells are loaded with miR-210 downregulate the receptor tyrosine kinase ligand, Ephrin A3 (EFNA3) [74]. However, in contrast, Taverna et al. showed that curcumin treatment deeply changes the molecular properties of exosomes released by leukemia cells, in particular, deplete the exosomes of the pro-angiogenic proteins and leads to enrichment of proteins with anti-angiogenic activity and miR-21 [75].

#### **3.16 Prostate cancer**

Exosomes derived from prostate cancer cells are known to carry TGF-β1 protein, which can induce the differentiation of recipient fibroblasts to myofibroblasts [76]. In a study by DeRita et al., showed that prostate cancer cell exosomes were loaded with, IGF-IR, FAK and c-src, which could promote tumor angiogenesis [77].

#### **3.17 Ovarian cancer**

Taraboletti et al. demonstrated that exosomes from ovarian cancer cells are known to carry pro-angiogenic growth factor VEGF, which helps in interaction between tumor and endothelial cells and is very important for angiogenesis [78]. Ovarian cancer exosomes are enriched with pro-angiogenic protein CD147, ATF 2, MTA1, SARS and ROCK1/2. They observed that these proteins can enhance the expression of vital angiogenic factors like VEGF, HIF-1α and MMPs and resulting in the enhanced angiogenesis of HUVECs [79, 80]. Additionally, Masoumi-Dehghi et al. observed that exosomes from ovarian cancer cells are enriched in miR141-3p, which helps in angiogenesis by activating the JAK/STAT and NF-kB signaling pathways [82].

#### **3.18 Chondrosarcoma**

Cheng et al. demonstrated that microarray analysis revealed that exosomes released from chondrosarcoma cells carried lncRNA RAMP2-AS1, which promotes HUVECs migration, proliferation, and tube formation which leads to angiogenesis through miR-2355-5p/VEGFR2 axis, thereby regulating the angiogenic ability of endothelial cells. Successive experiments showed that RAMP2-AS1 knockdown could decrease the pro-angiogenic effect of exosomes released from chondrosarcoma cells [86].

#### **3.19 Retinoblastoma**

Recently a study conducted by Chen et al. demonstrated that exosomes released by human retinoblastoma cell line WERI-Rb1, were enriched inmiR-92a-3p. The study, predicted that Krüppel-like factor 2 (KLF2) might activate target of miR-92a-3p, using bioinformatics tools & analysis. Thus, exosomal miR-92a-3p was found to modulate tumor angiogenesis by targeting KLF2 [87].

#### **3.20 Burkitt's lymphoma**

A study performed by Yoon et al. observed that miR-155 is transported from EBVpositive Burkitt's lymphoma cells derived exosomes which could induces angiogenesis in retinal epithelial pigment (RPE) cells (ARPE-19) by upregulation of transcriptional and translational levels of VEGF A via VHL/HIF-1α pathway. Thus, study demonstrated that miR-155 accumulation through exosomes affect nearby recipient cells [88].

#### **3.21 Cervical cancer**

Zhang et al. observed that exosomes released from cervical cancer cells harboring miR-221-3p, which accelerate the MVEC migration, proliferation, invasion and angiogenesis in cervical cancer cells by regulating MAPK10 [81]. In another study performed by Bhat et al. showed that cervical cancer exosomes were highly enriched with upstream proteins of hedgehog-GLI signaling includes, PTCH1, SMO, SHH and Ihh [83]. Also, they observed that these cervical cancer exosomes facilitate pro-angiogenic endothelial reconditioning through transfer of Hedgehog-GLI signaling components [84].

### **4. Therapeutic potential of tumor-exosomes in angiogenesis**

The discovery of exosomes as natural carriers of different mRNAs, miRNAs and lncRNAs makes them a suitable candidate as therapeutic drug vehicles and drug carriers to target cancer cells and modulation of tumor microenvironment. Recent advance in the field reveals several success stories (**Table 2**). The manipulation of

#### **Table 2.**

*Engineered exosomes as anti-angiogenic drug carriers in different cancer models.*

exosomes as drug carriers provides significant advantage for example their nonimmunogenic nature [95]. Exosomes are also known to carry different cell surface molecules due to which they have a commendable ability to transgress numerous biological barriers, such as the BBB (blood-brain barrier). They are highly stable in blood, which permits them to perform long distance intercellular communication [96]. Clinical data from various studies revealed that progression of cancer can be delayed or prevented when tumor angiogenesis is blocked [97]. So, angiogenesis during tumor development has now become the major emphasis of study and angiogenesis inhibition is evolving as a new method to treat cancer [98]. Recent investigations reported that exosomes can decrease or increase angiogenesis based on their molecular content. Thus, there is a lot of promise in developing engineered exosomes to transport numerous biological and synthetic genetic materials that can modify the expression of various genes involved in tumor angiogenesis [99]. For example, Ohno et al. demonstrated that modified exosomes carrying EGF or GE11 on their surface can deliver miR let-7a (tumor suppressor miR) to EGFR expressing breast cancer cells in RAG2/ mice model. Their previous investigation showed that GE11-exosomes which delivered miR-let 7a, effectively downregulated HMGA2 expression in cancer cells [90]. This study verifies that exosomes can be used as drug delivery vehicle to transport their cargo efficiently to the target cells. Exosomes have capability to act as carriers for delivering different small interfering RNAs (siRNAs) for targeted cancer treatment. Exosomes having HGF siRNA packed inside them can be transported into gastric cancer cells, where they downregulate the HGF expression [91]. Liu et al. demonstrated that exosomes are able to transport antisense RNA targeted to miR-150, which induces the expression of VEGF. They established that the neutralization of miR-150 downregulates the VEGF levels in mice and blocked angiogenesis [92]. Gupta et al. have shown that the bone marrow stromal cells (BMSCs) are involved in the tumor progression by secreting different pro-angiogenic factors, bFGF and VEGF [100]. In another study, it was observed that the miR content of exosomes derived from old and young BMSCs was different from each other. Young BMSC exosomes were highly enriched with miR-340, which inhibited the angiogenesis through HGF/c-MET signaling pathway in ECs. The antiangiogenic effect of older BMSCs was remarkably enhanced, when miR-340 was transferred to older BMSC exosomes that was highly expressed in young BMSC exosomes. Therefore, this investigation indicates the exosome-based cancer therapy via replenishment of miRNAs of exosomes [94]. The Arg-Gly-Asp (RGD) sequence containing peptide specifically bounds to αVβ3 integrin and plays an important role in endothelial cell survival, migration and angiogenic growth. In a study performed by Wang et al. showed successful binding of the RGD sequence containing peptide to the exosomal membrane surface and thereby binding of the αVβ3 integrin on the surface of angiogenic blood vessel. Thus, engineered exosomes are emerging as a new probable therapeutic motor for angiogenesis therapy [99]. In another study, it has been observed that curcumin treated CML cells released the exosomes, which are highly enriched with miR-21, which is further transferred to ECs and downregulates the expression of RhoB [75]. Docosahexaenoic acid (DHA) is a polyunsaturated omega-3 fatty acid (PUFA) and popularly known for its anti-cancer and anti-angiogenesis properties. A group of researchers demonstrated that exosomes released from the DHA-treated breast cancer cell lines are highly enriched with miRs, including miR-21, miR-27a/b, miR-23b, miR-320b, let-7 and let-7a, which are well known for their anti-angiogenic properties. They observed the increased expression of these miRs when exosomes were

co-incubated with the endothelial cells. Collectively, the exosomes show a strong therapeutic potential as natural nano carrier [93].

#### **5. Conclusion**

Herein, we have emphasized the current advances in the roles of tumor derived exosomes in cancers of different origins in tumor angiogenesis. Exosomes could modulate the angiogenic programming in target cells by transferring the angiogenic cargoes that include different mRNAs, miRNAs, lncRNAs and proteins. Angiogenesis is a very complex process in which aberrant growth of tumor and its metastasis occurs. So, the inhibition of angiogenesis is a pivotal point to control the progression of cancer. In spite of increasing amount of information about tumor derived exosomal cargo and changes prompted by them on target cells, the complexity of exosomal cargoes remains to be fully elucidated. There are several limitations and road blockers in the significance of exosomes in cancer therapy. These specifically pertain to exosomal yield, exosomes efficacy and specificity of targeting for effective cancer therapy. This field is yet elusive to assess the effect of exosomes on tumor angiogenesis and use them as potential means for different cancer therapies. So, future investigations should focus on identifying the fundamental exosomal cargoes and the mechanisms behind differential loading of different bioactive molecules, whose role could be implemented for designing noninvasive procedures to detect exosomes for cancer diagnosis and prognosis as well as development of effective therapeutic approaches based on exosomes.

#### **Acknowledgements**

Not Applicable.

#### **Funding**

Financial support from Science and Engineering Research Board Department of Science and Technology, Government of India (DST-SERB (EMR/2017/004018/ BBM)) and Institution of Eminence University of Delhi (Ref. No./IoE/2021/12/FRP) to ACB and grant from CCRH to ACB:SC:KT (17-51/2016–2017/CCRH/Tech/ Coll./DU-Cervical Cancer.4850) and Indian Council of Medical Research (ICMR-ICRC (No.5/13/4/ACB/ICRC/2020/NCD-III), are thankfully acknowledged. Study was partly supported by Junior Research Fellowship to TT (764/(CSIR-UGC NET JUNE 2019) and Senior Research Fellowship to AC [573(CSIR-UGC NET JUNE 2017)] by University Grants Commission (UGC), Senior Research Fellowship to NA (09/045 (1622)/2019-EMR-I) and JY (09/045(1629)/2019-EMR-I) by Council of Scientific and Industrial Research (CSIR); Junior Research Fellowship to DJ (09/0045/(11635)/2021- EMR-1) and AC (09/0045(12901)/2022-EMR-1).

#### **Conflicts of interest**

The authors declare that there are no competing/conflicts of interest.

*Tumor Angiogenesis and Modulators*

### **Author details**

Joni Yadav, Nikita Aggarwal, Apoorva Chaudhary, Tanya Tripathi, Dikkshita Baruah, Suhail Chhakara, Divya Janjua, Arun Chhokar, Kulbhushan Thakur, Anna Senrung and Alok Chandra Bharti\*

Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), Delhi, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Role of Exosomes in Tumor Induced Neo-Angiogenesis DOI: http://dx.doi.org/10.5772/intechopen.104400*

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#### *Tumor Angiogenesis and Modulators*

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#### **Chapter 4**

## Role of the IL-6/Jak/Stat Pathway in Tumor Angiogenesis: Influence of Estrogen Status

*José Manuel García-Castellano, David García-Padrón, Nerea Martínez-Aragón, Margarita Ramírez-Sánchez, Vicente Vera-Gutiérrez and Leandro Fernández-Pérez*

#### **Abstract**

Solid tumors, despite being hypervascularized, are hypoxic. This is due to the imbalance that exists between the inputs of the blood vessels that supply nutrients and O2 and that remove metabolic waste products, on one side; and the demands of the tumor cells that are part of the neoplasm that is forming, on the other. From this perspective, we briefly review the sequence of morphological events that occur during neo-angiogenesis; what chemical mediators are involved in this process; and we emphasize how the IL-6/Jak/Stat signaling pathway is involved in the control of these mediators. At the same time, we review how estrogens intervene in this control procedure, and how it opens the door to understanding the mechanism of action of these mediators. This would make it possible to propose alternative treatments, which can be added to the conventional ones, and which would exploit the findings described here in the search for new antitumor therapies.

**Keywords:** hypoxia, solid tumor, HIF, VEG, neovascularization, Jak/Stat, estrogens

#### **1. Introduction**

Blood vessels formation is an essential activity for the proper development of the organism. The development of new blood vessels is a well-regulated process, but it is a double-edged sword. Hence, in a physiological situation, such as embryonic development, it leads to the formation of a correct vascular network directed to provide the necessary nutrients and O2, as well as to waste products removal. However, in a tumoral scenario, is a problem since it uncontrollably feeds the tumor and provides the ways for its spread.

Paradoxically, although solid tumors are invariably hypervascularized, they contain hypoxic regions [1], with low pO2 levels. This is because the high rate of tumor growth is greater than the rate of new vessel formation [2] and there is no balance between supply and demand. This causes neoplastic cells to be too far from a blood vessel [1], generating a nutrient and O2 deficient state [3]. The hypoxia generated is also due to a poor O2

diffusion and to the fact that the cells of the neo-vessels are structurally abnormal [4]. The consequence of this tumor hypoxia leads to therapeutic radio- and chemo-resistance, as well as an increased probability of generating metastatic disease [4, 5]. The cellular change towards a state of tumoral hypoxia provokes an adaptive response that facilitates cell proliferation or angiogenesis, coordinated by the activity of HIF-1α [6]. The adaptive response to hypoxia generated by HIF-1α through angiogenesis and enhanced glucose metabolism confers a survival and growth advantage to hypoxic tumor cells [7].

#### **2. Hypoxic tumor cells generate new capillaries**

Neoangiogenesis is the process by which new capillary vessels grow out of preexisting ones (sprouting angiogenesis). These blood vessels will provide oxygen and nutrients and will remove the metabolic waste [6], which is regulated by a variety of pro- and anti-angiogenic factors [1].

The process of sprouting angiogenesis involves several sequential steps [8] that starts with the activation of endothelial cells due to diverse angiogenic stimulus, like hypoxia or inflammation [8]. The activity of endothelial cells, normally joined by adhesion molecules such as cadherins, is mediated by growth factors released after degranulation of platelet alpha granules [9, 10]. Pericytes, surrounding endothelial cells, inhibit the proliferation of the endothelial cells, also releasing cell survival signals such as VEGF and Angiopoietin-1.

As a consequence of this activation, there is a rupture of the endothelial cells tight junctions; the pericytes detach from the wall and the basement membrane, which, together with the extracellular matrix, will be degraded by activated proteases (metalloproteinases). Loss of junctions between endothelial cells allows them to invade into the surrounding interstitial tissue and, subsequently, proliferate and migrate through the matrix. These endothelial cells afterward become motile tip cells, which are located at the growing ends of the new vessels [11, 12].

Angiogenic factors, such as VEGF, increase the vascular permeability of endothelial cells, causing extravasation of plasmatic proteins and generating an extracellular matrix (ECM). In response to integrin signaling, cells migrate within that ECM, following the tip cells.

Endothelial cells move forward following the angiogenic signal sent by the tip cell that will guide them in the specific direction [12]. Adjacent cells to the tip cell will follow them, dividing to elongate the stalk and establish the lumen. This structure thus formed is an immature vessel [13].

Endothelial cells then rapidly proliferate [8], form tight and adherens junctions with other endothelial cells [11, 12], and finally, the endothelial cell migration and proliferation are inhibited.

The stabilization of the immature vessels is established by the recruitment of pericytes, which will line the capillary walls and stabilize the new vessels [11, 14]. Finally, a new extracellular matrix will be generated [15].

#### **3. Neo-angiogenesis is a well-regulated process**

The process of sprouting angiogenesis is tightly controlled by positive and negative regulators whose purpose is to control in a balanced way the structured formation of new vessels through the action of growth factors and cytokines (**Table 1**).

*Role of the IL-6/Jak/Stat Pathway in Tumor Angiogenesis: Influence of Estrogen Status DOI: http://dx.doi.org/10.5772/intechopen.104102*

#### **Table 1.**

*Relationship were the most common growth factors during the neo-angiogenesis process are reported, their function, and the most common cells that produce them.*

We will distinguish those factors that will improve the forming action of new vessels (enhancing factors), from those that are designed to modulate and stop the appearance or development of these vessels (inhibitory factors).

#### **3.1 Enhancing factors**

#### *3.1.1 Hypoxia-inducible factor*

Hypoxia-inducible factor (HIF-1α) is a transcription factor that regulates and coordinates the cellular response to hypoxia [31, 32], by activating genes encoding pro-angiogenic factors, such as VEGF, angiopoietin or PDGF.

When tissue and cellular oxygen levels are in a normal range, HIF-1α is degraded, disrupting the signaling cascade aimed at improving vascularization by means of pro-angiogenic factors [33].

Under low pO2 status, HIF-1α is involved in hypoxia response by binding to canonical DNA sequences (hypoxia-responsive elements or HREs) in the promoters or enhancers of target genes [34–38]. Also, HIF-1α, through the union of hypoxiaresponsive elements or HREs with the promoters of target genes, coordinates a broad response to counteract the effects of hypoxia. Under hypoxic conditions, proteasomal degradation of HIF-1α ends, and it translocates to the nucleus to activate hypoxiainducible genes [36, 39], such as VEGF, angiopoietin, PlGF, or PDGF [40].

#### *3.1.2 VEGF*

VEGF is a glycoprotein that plays an essential role in the development of new vessels. It is produced by tumor cells, macrophages, platelets, and endothelial cells, binding to the VEGF-R1/R2 receptors present on endothelial cells. This growth factor stimulates the endothelial cells survival, proliferation, and motility, initiating the growth of new capillaries by activating the RAS/MEK/ERK pathways or the PI3K/ AKT/mTOR pathway. The final effect is the stimulation of endothelial cell survival, proliferation, and motility, initiating the growth of new capillaries.

#### *3.1.3 FGF*

FGFs are a family of proteins, mostly with angiogenic effects. The best known are FGF-1 and FGF-2. They are essentially secreted by macrophages and vascular endothelial cells. They are involved in numerous processes, including the induction of endothelial cell differentiation, proliferation, migration, morphogenesis, and survival of endothelial cells; and extracellular matrix degradation by stimulating the secretion of proteases [19, 41]. FGF-1 is necessary for the differentiation and proliferation of all the cell types necessary for creating the vessel wall; while FGF-2 signaling is related to the preservation of vascular endothelial cell junctions and vessel permeability [19].

#### *3.1.4 PDGF*

Platelet-derived growth factor is a dimeric glycoprotein synthesized, stored (in the alpha granules of platelets), and released by platelets upon activation, it is also produced by other cells including smooth muscle cells, activated macrophages, and endothelial cells. PDGF is a potent mitogen for cells of mesenchymal origin.

#### *3.1.5 Angiopoietins*

Family of proteins involved in vascular repair. Ang-1 and Ang-2 are the best known. Its function is carried out by coupling an angiopoietin to its corresponding receptor (Tie-1 and Tie-2). These receptors are expressed specifically on vascular endothelial cells and on a certain type of macrophages involved in angiogenesis.

#### *3.1.6 Hepatocyte growth factor*

HGF is a factor secreted by mesenchymal cells in a paracrine manner that exerts its function through its c-Met receptor. This receptor is expressed in several cell types, such as endothelial cells, smooth muscles cells, and bone brown-derived endothelial progenitor cells. HGF stimulates mitogenesis, cell motility, and matrix invasion.

#### **3.2 Inhibitory factors**

#### *3.2.1 Angiostatin*

Angiostatin is a protein produced by autoproteolytic cleavage of certain proteins, like plasminogen. Its function is to inhibit endothelial cell proliferation and migration, tube formation, and tumor cell invasion. In addition, it decreases VEGF expression and induces endothelial cell-mediated apoptosis by thrombospondin-1.

#### *3.2.2 Endostatin*

Endostatin is a C-terminal type XVIII collagen fragment, cleaved by the proteolytic activity of MMP-7. It has anti-angiogenic activity by inhibiting FGF-2 and VEGF [1]. It also has an anti-migratory effect by binding to the α5-αv-integrins. It has the ability to directly combine to VEGFR2, inhibiting the VEGF-induced phosphorylation and consequently down-regulating receiver, as well.

#### *3.2.3 Platelet Factor 4*

It is a small protein belonging to the CXC chemokine family, usually associated with complexes with proteoglycans and released from alpha-granules of activated platelets during platelets aggregation. It is a potent inhibitor of angiogenesis, especially when acting in conjunction with the receptors of FGF2 and VEGF, leading to downstream effects on endothelial cell migration and proliferation.

#### *3.2.4 Thrombospondin-1*

TSP-1 is a glycoprotein that mediates intercellular interactions or with the ECM. This protein can bind to elements of this ECM (to fibrinogen, fibronectin, laminin, collagen types V and VII, and integrins alpha -V/beta-1), and exerts an inhibitory effect on the migration, proliferation, and survival of endothelial cells and the formation of capillary tubes.

#### **4. Role of the IL-6/Jak/Stat pathway on the neoangiogenesis process**

After a tissue injury, a cascade of events is set in motion aimed at repairing the damage. The products generated by tissue destruction stimulate the cells of the immune system. In response to this damaging process, immune cells in the tumor environment secrete multiple cytokines, such as histamine, serotonin, prostaglandins, leukotrienes; and inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and various chemokines. Many of them belong to the IL-6 family [42]. These substances help to repair healthy tissues but nevertheless have deleterious effects on tumors.

#### **4.1 IL-6/Jak/Stat pathway**

Janus kinase (Jak), the signal activation transducer (Stat) pathway, is recognized as an evolutionarily conserved signaling pathway (**Figure 1**). After binding the

#### **Figure 1.**

*IL-6/Jak/Stat pathway. After binding the cytokine to the receptor, Jak is activated by the specific tyrosine residues phosphorylation. Phosphorylated Jak, in turn, induces the phosphorylation of Stat, which, after dimerization, translocate into the nucleus where it regulates the transcription of numerous genes.*

cytokine to the receptor, Jak is activated by the specific tyrosine residues phosphorylation. Phosphorylated Jak in turn induces the phosphorylation of Stat, which, after dimerization, translocate into the nucleus where it regulates the transcription of numerous genes [43].

The IL-6/Jak/Stat pathway is overexpressed in various tumors, causing continuous transcription of cell growth factors that promote tumor progression. However, this pathway not only regulates aspects such as tumor proliferation, survival, and invasion, but also contributes significantly to tumor neo-angiogenesis [44, 45], enhancing endothelial cells survival, infiltration of the ECM by immune cells followed by activation of mesenchymal cells, and finally the generation of metastases [46].

Jak/Stat is activated upon stimulation by IL-6, among several effectors, promoting endothelial cell migration and tumor angiogenesis. This function is suppressed when Jak inhibitors are administered, ending the observed endothelial cell migration *in vitro* [47].

Regulation of tumor angiogenesis is dependent on VEGF and HIF-1α transcription by endothelial cells [48–50]. This action is induced by tumor IL-6 and mediated by Stat3 [51]. These results are validated by the fact that the aberrant expression of Stat3 causes an increase in the expression level of HIF- 1α and VEGF, as well as of the metalloproteinases MMP-9 and MMP-7, enhancing tumor progression and aggressiveness [52]. This pathway is reciprocally enhanced by the action of IL-6 secreted by endothelial cells on tumor cells [53]. This boost signal is also produced by other pathways, such as that promoted by EGFR, HER2, Ras, and Rho, which lead to Stat3 activation [46].

#### *Role of the IL-6/Jak/Stat Pathway in Tumor Angiogenesis: Influence of Estrogen Status DOI: http://dx.doi.org/10.5772/intechopen.104102*

On the other hand, IL-6-induced activation of Stat3 in tumor and stromal cells protects neoplastic cells from the immune surveillance system. This pathway promotes immune evasion [54], by modulating the secretion of various inflammatory factors such as IL-6 and TNF-α [55] and reducing natural killer cell activity [56, 57]. This favors tumor expansion by avoiding immunological control.

Furthermore, the metastatic process is regulated by Stat3, by controlling the capacity for cell migration and invasion of tissues. On one side, Stat3 acts directly on the promoter of MMP genes [58, 59], increasing their expression and thus the ability of cancer cells to degrade the basement membrane/extracellular matrix. Tumor cells then invade the surrounding ECM by migrating due to the action of RhoA on the cytoskeleton [60] after activation of the Stat3/ROCK-myosin pathway. The cells then spread through the circulatory or lymphatic system, forming metastatic foci in lymph nodes and distant organs.

#### **5. Effect of the IL-6/Jak/Stat pathway on neo-angiogenesis mediators**

Several authors show that the Jak/Stat pathway plays an important role in neoangiogenesis through these growth factors.

#### **5.1 HIF-1α**

In a hypoxic environment, the HIF-1α protein is stabilized and its proteasomal degradation rate is reduced by slowing down the protein ubiquitination of HIF-1α and thereby achieving enhanced HIF-1α protein levels [61]. This increases its half-life and the cellular concentration of HIF-1α. The IL-6/Jak/Stat3 pathway mediates in the regulation of this process; in such a way that Stat3 interacts with HIF-1α and with VEGF in order to generate greater tumor vascularization (**Figure 2**).

Similar results are obtained after sustained administration of the constitutively active form of Stat3, which causes an increase in HIF-1α transcription, with the consequent increase in HIF-1α protein levels. Changes in HIF-1α levels are also due to the interaction between this molecule and PIAS [62], a negative regulator of the Jak/Stat pathway. Hypoxia causes the interaction between molecules, promoting the stabilization of HIF-1α and prolonging its half-life.

#### **5.2 VEGF**

It is well-known that HIF-1α stimulates vascularization and metastasis upon activation of VEGF expression [63]. But there are evidences that show that Stat3 plays a central role in this response. Thus, Xu et al. [64] shows that in various types of human cancer cell lines Stat3 activation induces HIF-1α and up-regulates VEGF expression, promoting tumor angiogenesis [64]. The inhibition of Stat-1/Stat-3 phosphorylation was accompanied by a decrease in VEGF transcription and secretion due to the direct transcriptional action of the VEGF gene by Stat3 (**Figure 2**). On the other hand, Stat3 cooperates with HIF-1α, binding both simultaneously to the promoter region of the VEGF gene, leading to its maximum transcriptional activation and angiogenesis [65].

This action of Stat3 on the VEGF pathway also affects its VEGF receptor. Thus, it has been seen that indirubin suppressed severely the VEGFR-mediated Jak/Stat3 signaling pathway in prostate tumor cells, affecting angiogenesis and tumor growth [66]. Similarly, in pancreatic cancer cell lines, suppression of VEGFR-2 phosphorylation and Stat3-dependent expression of HIF-1α reduced the expression of the Rho-GTPases RhoC, which is downstream of VEGF signaling. This effect plays a vital role in tumor angiogenesis and metastasis [67] because RhoC plays an essential role in transmitting the VEGF signals downstream to angiogenesis and invasiveness [51].

In addition, inhibition of Stat-1/Stat-3 down-regulates other pro-angiogenic factors, such as eNOS, iNOS, MMP-2, and FGF-2 in HUVEC, associated with reduced capillary sprouting and tumor angiogenesis [68, 69]. These molecular findings, taken to clinical practice, translate into a reduction in cell viability, proliferation, adhesion, migration, and tube formation.

Lymphangiogenesis is carried out in a similar way, observing activation of the IL-6-Jak-Stat3-VEGF-C signaling pathway in the growth and invasion process [70, 71].

#### **5.3 PDGF**

On the other hand, other aspects must be taken into account. Thus, in the angiogenesis process, it is necessary to increase the cell population, either proliferating new cells or the chemo-attraction of others (**Figure 2**). To do this, PDGF, a growth factor that stands out for being a potent mitogen and chemoattractant for VSMC, stimulates the phosphorylation of Jak-2 and Stat3 in VSMCs [10, 72, 73] and contributes to PDGF-BB-induced mitogenesis [73] and VSMC motility [72]. In addition, PDGF helps regulate the IL-6/Jak-2/Stat pathway through phosphorylation of SOCS, a natural regulator of Jak, by platelet-derived growth factor receptor tyrosine kinase [64].

#### **Figure 2.**

*Diagram that summarizes the neo-angiotizing action of certain cytokines and growth factors that influence neoangiogenesis and how the mediators of the Jak/Stat pathway act on them.*

*Role of the IL-6/Jak/Stat Pathway in Tumor Angiogenesis: Influence of Estrogen Status DOI: http://dx.doi.org/10.5772/intechopen.104102*

#### **5.4 FGF**

New vessel formation is also regulated by growth factors such as FGF, another downstream effector to IL-6 that induces angiogenic activity in basal cell carcinoma cell lines [74], dependent on the activation of Jak/Stat3. Thus, IL-6 overexpression increases FGF-2 levels (**Figure 2**), tube formation by HUVEC cells, and consequently neoangiogenesis [74].

#### **5.5 Angiopoietins**

These molecules are also involved in relevant functions during neo-angiogenesis, such as vascular repair after binding with the endothelial cell-surface receptor tyrosine kinase, Tie2. It also highlights the regulatory activity of Stat on the cell survival, migration, and proliferation [75, 76] by Ang1/Ang2-Tie2 receptor activated. Thus, after Stat5, VEGFR-1 the Tie-2 receptor co-expressed, an increased expression of the cell cycle inhibitor p21 is induced [76], which will arrest cell proliferation (**Figure 2**). On the other hand, angiopoietin-like 4 stimulates Stat 3-mediated iNOS expression and enhances angiogenesis [77].

#### **5.6 HGF**

The IL-6/Jak/Stat signaling pathway is regulated by HGF, mediated by SOCS1 [78]. In the case of SOCS3 [79], it counteracted Stat3-dependent keratinocyte migration after being stimulated by HGF (**Figure 2**). In the case of EGFR, SOCS3 is involved in the regulation of IL-6/Jak/Stat signaling, attenuating the EGF signal [78, 80].

#### **5.7 Endostatin**

Endostatin activation in the extra cellular environment is enhanced by means of MMP-2/MMP-9 activation, which is accompanied by decreased tumor vascularization [81]. The administration of IL-35 to fibroblast-like synoviocytes produces an inhibition of vascularization due to an increase in the expression of endostatin and a decrease in the expression of VEGF, FGF-2, TNF-α, and IL-6, by means of Stat1 [82]. Synergism between endostatin and Stat3 suppression by a Stat3-siRNA has been observed. In the hepatocarcinoma model, each of both treatments had a potent antitumor effect; but, the combination had a superior effect. It was observed a decreased VEGF expression, decreased cell proliferation, induced cell apoptosis, and inhibited angiogenesis [83] (**Figure 2**).

#### **5.8 PF4**

PF4 may contribute to suppress tumor growth in the melanoma murine model, decreasing IL-17, IL-6, and p-Stat3 pathway (**Figure 2**) via up-regulation of SOCS3 expression [84].

#### **5.9 Leptin**

Leptin secreted by adipose tissue has a well-known paracrine effect on endothelial, stromal, and tumor cells, enhancing the aggressive tumor behavior. On

adipose-derived stromal cells, VEGFA, MMP-2, MMP-9, IGF-1, and b-FGF genes expression are up-regulated and angiogenesis is stimulated by the Jak/Stat3 pathway [85]. In addition, leptin increases the migration and proliferation of VSMC [86, 87], by inducing the phosphorylation of the tyrosine residue of Jak and the activation of its effectors Stat3 and MAPK [88, 89] (**Figure 2**). Jak, on the other hand, produces leptin-dependent up-regulation of TSP-1 [90].

#### **6. Effect of the estrogens on neoangiogenesis mediators**

Sex steroids cooperate with the pro- and anti-angiogenic factors involved in the tumor neo-vascularization process. The connection between the inflammatory pathway represented by the IL-6/Jak-Stat pathway, and the tumor estrogenic pathway is very close and is involved in the pathogenic processes of these diseases [43].

Recently, evidence has emerged showing that cytokines generated during the inflammatory process interact with estrogen signaling pathways [43]. On one side, there is a very close relationship between ER α protein levels and Stat 1 activity (**Figure 3**). Thus, if Stat1 levels are insufficient or its function is blocked, a decrease in ERα levels and cell proliferation is observed. This occurs through the direct action of Stat1 on the promoter region of ERα, regulating the transcription of mRNA levels [91]**.**

On the other hand, estrogenic activity has been found in adipose tissue and tumoral stroma. Thus, immunohistochemical studies have found the expression of cytochrome P450 aromatase, responsible for the aromatization of adrenal and testicular androgens into estrogens (**Figure 3**). It is also known that the IL-6/Jak/Stat pathway stimulates the cytochrome P450 aromatase expression, transforming tissue androgens into estrogens that will act in a paracrine manner on the tumor, causing tumor growth and development [92].

This connection between IL-6/Jak/Stat and estrogens is regulated, in such a way that there is negative regulation of Jak2 with respect to ERα [43] because Jak2 induces

#### **Figure 3.**

*Diagram summarizing the action of how estrogens and mediators of the Jak/Stat pathway interact during the process of neo-angiogenesis.*

the ubiquitination of ERα for being degraded in the proteasome (**Figure 3**). On the other hand, sustained treatment with E2 induces Jak-2 expression, thus controlling the formation and destruction of these molecules.

These observations are integrated by the adipose tissue cytokine leptin function. It activates the phosphorylation of the tyrosine residue of the receptor and causes the activation of its effectors Stat3 and MAPK (**Figure 3**). In this way, the estrogenic pathway is enhanced at the tissue level, since Stat3 induces the generation of estrogens by aromatization of androgens, and MAPK stops the proteasomal degradation of ERα [93], enhancing the estrogenic status [43].

Estrogens, in addition to synergizing with the IL-6/Jak/Stat pathway, regulate the action of mediators involved in the neo-angiogenesis process (**Figure 3**). Thus, regarding HIF-1α, estrogens stabilize the protein in normoxia by regulating its expression through the Akt pathway [63, 94].

In addition, IL-6 induces the expression of VEGF in granulosa cells through FSH mediation, favoring the expression of HIF-1α and COX2, thanks to the activation of the Jak/Stat3 pathway (**Figure 3**). Other evidence indicates that ovarian steroids increase the production of HGF by peritoneal macrophages, promoting the proliferation of endothelial cells and the organization of capillaries.

The angiopoietins, promote the formation of endothelial cells through the mediation of estrogens. Thus, the up-regulation of brain Ang-1 mRNA caused an increase in the capillary density. Besides, E2 acting through ERβ up-regulates Ang-2, increased Tie-2 phosphorylation, and promoted angiogenesis [95].

In ER-positive breast cancer tumor cells, estrogens control the production of TSP-1, which is under the direct control of estrogens, performing regulatory functions favorable to tumor growth [96].

It is also the case that a growth factor is influenced by both pathways. In the case of FGF, while estrogens potentiate its release, it signaling pathway was mediated by activated Stat1 [97].

All these coordinated measures between both systems are aimed at enhancing vascular neo-formation and thus potential metastatic dissemination.

#### **Conflict of interest**

"The authors declare no conflict of interest."

#### **Authorship**

José Manuel García-Castellano (JMGC); David García-Padrón (DGP); Nerea Martínez-Aragón (NMA); Margarita Ramírez-Sánchez (MRS); Vicente Vera-Gutiérrez (VVG); Leandro Fernández-Pérez (LFP).

### **Author details**

José Manuel García-Castellano1,2,3,4\*, David García-Padrón2† , Nerea Martínez-Aragón2† , Margarita Ramírez-Sánchez5 , Vicente Vera-Gutiérrez6 and Leandro Fernández-Pérez7

1 Orthopedic Surgery and Traumatology, Maternal and Child University Hospital Complex of Gran Canaria, Las Palmas de Gran Canaria, Canary Islands, Spain

2 Molecular Oncology Laboratory, Research Unit, Maternal and Child University Hospital Complex of Gran Canaria, Las Palmas de Gran Canaria, Canary Islands, Spain

3 Department of Medical and Surgical Sciences, University Institute of Biomedical and Health Research (IUIBS), University of Las Palmas de Gran Canaria, Spain

4 Spanish Sarcoma Research Group (GEIS), Spain

5 Physical Medicine and Rehabilitation Service, University Hospital of Gran Canaria Doctor Negrín, Las Palmas de Gran Canaria, Spain

6 Orthopedic Surgery and Traumatology, University Hospital of Gran Canaria Doctor Negrín, Las Palmas de Gran Canaria, Spain

7 Faculty of Health Sciences, Department of Clinical Sciences, Laboratory of Pharmacology, University of Las Palmas de Gran Canaria, Spain

\*Address all correspondence to<EMAIL_ADDRESS>
† Both authors contributed equally to this work.

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Role of the IL-6/Jak/Stat Pathway in Tumor Angiogenesis: Influence of Estrogen Status DOI: http://dx.doi.org/10.5772/intechopen.104102*

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#### **Chapter 5**

## Modulators of Tumor Angiogenesis: Insights into the Role of Galectin-3 and IL-17 Signaling

*Gordana D. Radosavljevic, Jelena Pantic, Bojana Simovic Markovic and Nebojsa Arsenijevic*

#### **Abstract**

Angiogenesis is a pivotal point in tumor progression driven by firmly orchestrated process of forming the new blood vessels relying on the complex signaling network. Here, the pleiotropic functions of Galectin-3 and IL-17 in tumor progression have been overviewed through their impacts on angiogenesis. As a key player in tumor microenvironment, Galectin-3 orchestrates practically all critical events during angiogenic cascade through interaction with various ligands and their downstream signaling pathways. Galectin-3 shapes chronic inflammatory tumor microenvironment that is closely related to angiogenesis by sharing common signaling cascades and molecules. In chronic inflammatory makeup of tumor microenvironment, IL-17 contributes to tumorigenesis and progression *via* promoting critical events such as angiogenesis and creation of immunosuppressive milieu. VEGF, as the master regulator of tumor angiogenesis, is the main target of Galectin-3 and IL-17 action. The better understanding of Galectin-3 and IL-17 in tumor biology will undoubtedly contribute to controlling tumor progression. Therefore, as important modulators of tumor angiogenesis, Galectin-3 and IL-17 may be perceived as the potential therapeutic targets in tumor including anti-angiogenic therapy.

**Keywords:** galectin-3, IL-17, VEGF, tumor angiogenesis, tumor progression

#### **1. Introduction**

Tumor angiogenesis or aberrant vascularization is considered a critical hallmark of tumor progression that is inevitable for tumor growth and metastatic spread [1]. This complex multistep process of new vasculature formation from pre-existing blood vessels is triggered by numerous signals from tumor cells in a phase of rapid growth [1]. The expression and secretion of various activators and inhibitors of angiogenesis are regulated by gene mutation (e.g., oncogenes and tumor-suppressor genes), and microenvironmental factors such as hypoxia and accumulation of different metabolites [2, 3]. As the growing tumor requires more blood vessels for nutrition and oxygen supply, angiogenic pathways are induced by tilting the balance toward pro-angiogenic molecules (angiogenic switch) to drive new blood vessel growth [3].

#### *Tumor Angiogenesis and Modulators*

High expression levels of pro-angiogenic factors reflect the tumor aggressiveness [4]. Within the angiogenic cascade, a diverse group of mediators are shown in **Figure 1**. These molecules participate in the establishment of new tumor vessels in various ways. Among them, vascular endothelial growth factor (VEGF), also called VEGF-A, is key "molecular player" that modifies the endothelial barriers [3]. Moreover, VEGF as master regulator of angiogenesis in tumor tissues and its receptors, particularly VEGFR-2, have been implicated in tumor vascularization [3]. Namely, activation of VEGF/VEGFR-2 signaling pathways triggers an angiogenic program in the endothelial cells (ECs) [3]. Thus, VEGF binds to its cognate receptor that results in autophosphorylation of specific tyrosine residues of VEGFR-2, and consequential activation of multiple downstream signaling networks in the vascular endothelial cells through the recruiting of the MAP kinase (ERK1/2 and p38), PI3K, AKT, PLC-γ, and JAK-STAT [5–7]. The final result is the activation of full range of

#### **Figure 1.**

*Pro-angiogenic mediators implicated in the tumor angiogenesis. Plethora of mediators that promotes tumor angiogenesis can be categorized into several groups. VEGFs-vascular endothelial factors; FGFs-fibroblast growth factors; PDGFs-platelet-derived growth factor; EGFs-epidermal growth factor; TGFs-transforming growth factors; MMPs-matrix metalloproteinases; uPA-urokinase-type plasminogen activator; TNF-α-tumor necrosis factor-α; NO-nitric oxide; PGE2-prostaglandin E2; S1P-sphingosine-1-phosphate.*

*Modulators of Tumor Angiogenesis: Insights into the Role of Galectin-3 and IL-17 Signaling DOI: http://dx.doi.org/10.5772/intechopen.102893*

biological responses that modulate angiogenesis, including vascular permeability as well as endothelial cell proliferation, survival, adhesion, and migration.

It is well established that VEGF is multifunctional molecule. VEGF has been first identified as vascular permeability factor, which exerts potent ability to increase vascular permeability, resulting in leakage of plasma protein and other molecules out of blood vessels [8]. Furthermore, VEGF is a potent mitogen that is highly specific for ECs and stimulates cell proliferation through VEGFR-2-mediated activation of the RAS/RAF/ERK/MAPK pathway [9]. Acting as survival factor for ECs, VEGF increases expression of the anti-apoptotic proteins Bcl-2 and A1 in the ECs [10]. On the other hand, VEGF also participates in tumor angiogenesis through increased migration and invasion of ECs by enhancing of matrix metalloproteinases (MMPs) release [3], and further amplifying angiogenesis by enhanced recruitment and homing of bone marrow derived vascular precursor cells [11]. PI3K/AKT signaling promotes VEGFmediated invasion and metastasis of ECs [12].

VEGF expression is tightly regulated by plethora of transcriptional regulators, such as transcription factor called hypoxia-inducible factor (HIF). Beside them, VEGF signaling is also upregulated by multiple stimuli, including cytokines and galectins by tumor microenvironments. We discuss the role of IL-17 and Galectin-3 in mediating angiogenesis, either directly or indirectly *via* induction of proangiogenic factors such as VEGF. The better understanding of Galectin-3 and IL-17 in tumor biology will undoubtedly contribute to controlling tumor progression. Namely, we will review the role of these two molecules in tumor angiogenesis and highlight the other mechanisms involved in the acceleration of tumor growth and metastases.

#### **2. Galectin-3 and IL-17: an important piece in the puzzle of tumor microenvironment**

The tumor microenvironment represents a complex ecosystem involving interactions between tumor cells, ECs, epithelial cells, immune cells, fibroblasts, and the extracellular matrix, as well as secreted cytokines and growth factors. All of these factors provide essential support for the tumor progression. The dynamic cross-talk between angiogenesis and tumor microenvironment is important to further accelerate tumor growth and metastasis [13]. Thus, released angiogenic factors can promote tumor immunosuppression by inhibiting maturation of dendritic cells, increasing mobilization of immunosuppressive cells, and suppressing CD8 + T cell activity [14]. The tumor microenvironment, in turn, produces numerous soluble molecules and growth factors that stimulate angiogenesis, thus forming a vicious circle for tumor progression [15]. Increasing evidence suggests that Galectin-3 and IL-17 are the significant pieces of that puzzle that shape angiogenesis and tumor progression in many ways (**Figure 2**).

Galectin-3, a unique chimaera-type member of the lectin family with selectivity for β-galactosides, is a versatile galectin involved in fundamental biological processes as well as various pathological circumstances [16, 17]. This evolutionary conserved molecule is usually overexpressed in variety types of tumor [18]. The ECs, immune cells, mesenchymal stem cells (MSCs), cancer-associated fibroblasts (CAFs), and myofibroblasts also produce and secrete Galectin-3 [19–21]. Galectin-3 expression is higher in endothelial progenitor cells as compared with normal ECs [22]. However, the tumor microenvironment, for example, tumor cells, inflammatory cells, and/or

#### **Figure 2.**

*Pro-angiogenic effects of Galectin-3 and IL-17 as a part of tumor progression machinery. Many cells and soluble mediators create tumor microenvironment characterized by hypoxia, chronic inflammation, and immunosuppression. Galectin-3 participates in all steps of angiogenic cascade via activation of different signaling pathways and/or polarization of macrophages toward pro-tumorigenic TAM2 phenotype. Galectin-3 affects the production of pro-inflammatory cytokines implicated in tumor promotion. Within the complex cytokine network in tumor microenvironment, IL-17 is recognized as one of the critical stimulators of the production of pro-angiogenic mediators, including VEGF. IL-17 mediates the recruitment of TAN2 thus augmenting angiogenic factors release. IL-23 and IL-33 seem to be significant co-workers in triggering angiogenic cascade. Both IL-17 and IL-33 induce recruitment of pro-angiogenic MDSC, while IL-23 further promotes function, survival, and expansions of Th17 lymphocytes, and subsequent IL-17 production. The activation, proliferation, and migration of endothelial cells, as well as sprouting and tube formation, precede the formation of new blood vessels critical for tumor progression. CAF-cancer-associated fibroblast; TAM-tumor-associated macrophage; TAN-tumorassociated neutrophil; MDSC-myeloid-derived suppressor cell; ECM-extracellular matrix.*

specific glycan-ligands on galectin-binding proteins, alters endothelial Galectin-3 expression as it provide most of the signals to which the ECs respond [23, 24]. Accordingly, pro-inflammatory cytokine IL-1β increases Galectin-3 expression by ECs [25]. ECs not only have a pivotal role in angiogenesis, but also they facilitate tumor invasion by secreting growth factors and extracellular matrix proteinases [26]. Released molecules sequentially increase chances that tumor cells enter to the circulation and metastasis [26].

#### *Modulators of Tumor Angiogenesis: Insights into the Role of Galectin-3 and IL-17 Signaling DOI: http://dx.doi.org/10.5772/intechopen.102893*

Depending on cell types and cellular localization, Galectin-3 drives force in the diverse processes critical in tumor biology, including apoptosis, invasion, metastasis, immune surveillance, gene expression, and inflammation [27]. The cytoplasmic Galectin-3 blocks apoptotic machinery in tumor cells [16] through several mechanisms [28]. Galectin-3 secreted by tumor cells contributes to immunosuppression within the tumor microenvironment by polarizing to pro-tumor phenotype of tumor-associated macrophages 2 (TAM2), restricting T cell receptor clustering, and triggering apoptosis of CD8 + T lymphocytes, further facilitating tumor escape [29]. The upregulation of Galectin-3 by TAMs in the hypoxic regions of breast cancer promotes tumor cell migration and invasion and TAMs-mediated metastasis, as well as angiogenesis [30]. Expression of Galectin-3 in CAFs in breast cancer has been associated with distant metastasis [31]. Galectin-3 is also found in extracellular vesicles released by tumor cells, and it seems that this galectin is critical regulator in cell-cell and cell-extracellular matrix interactions [32]. Endothelial Galectin-3 expression in the lungs cooperates with poly-N-acetyl-lactosamine on N-glycans of B16-F1 murine melanoma cells, as a ligand for Galectin-3 [33]. Our data demonstrated that host-derived Galectin-3 facilitates B16-F1 cell adhesion to the metastatic target and interferes with efficiency of the antitumor immune response, thereby accelerating melanoma metastasis [34].

Tumor angiogenesis and chronic inflammation are closely related and often share common signaling pathways and molecules [35]. In addition to angiogenesis, Galectin-3 participates in shaping of tumor inflammatory microenvironment likely through the recruitment of inflammatory cells and modification of their polarization [36], as well as the production of pro-inflammatory cytokines that have been implicated in tumor promotion (**Figure 2**, [37]). Overexpressed pro-inflammatory IL-1, IL-6, and TNF-α contribute to various steps of tumor progression [38]. This cytokine network, required for the establishment of chronic inflammation in the tumor microenvironment, facilitates tumor growth and metastasis, enhances angiogenesis, and inhibits immune surveillance [39]. In particular, tumor-infiltrating Th17 lymphocytes orchestrate the maintenance of chronic inflammation. IL-6, TGF-β, and IL-1β are pivotal drivers of development of Th17 cells that secrete IL-17 and other cytokines. Although IL-23 is not required for triggering Th17 differentiation, it is essential for the function, survival, and expansion of Th17 lymphocytes in the inflamed tissue [40]. To increase inflammation, IL-17 induces mobilization, recruitment, and activation of different immune cells [40]. Interestingly, the finding of correlation between serum Galectin-3 levels and IL-17 production in patients with colorectal carcinoma has suggested that Galectin-3 may be one of the important modulators in the regulation of inflammatory conditions (**Figure 2**, [41]).

IL-17A (commonly referred to as IL-17) is the first discovered and best characterized member of the IL-17 family. Currently, six structurally related cytokines of IL-17 family have been identified (IL-17A to IL-17F) [42]. It is well documented that IL-17 plays protective role in infections, but here, we will review the multifunctional impacts of IL-17 on tumor biology.

IL-17 is mostly produced and secreted by Th17 lymphocytes, but it can be also produced by a broad spectrum of other cell populations [42]. Many studies describe the Th17-rich microenvironment in various types of tumor and that Th17 lymphocytes are endowed with a unique functional plasticity [40, 43]. Tumor cells, CAFs, and myeloid-derived suppressor cells (MDSCs) have been found to produce cytokine milieu that elicits recruitment and/or generation of Th17 lymphocytes [44, 45]. In addition, metabolic conditions present in the tumor milieu including indoleamine

2,3-dioxygenase (IDO) and hypoxia drive the differentiation of CD4 + T lymphocytes toward the Th17 lineage [46, 47]. Type 17 CD8 + T cytotoxic (Tc17) lymphocytes among tumor-infiltrating lymphocytes (TILs) were detected in nasopharyngeal [48] and gastric cancer [49]. Further, the main IL-17-producing cells in breast cancer are tumor-infiltrating γδT cells [50], and it seems that these TILs can promote the breast cancer progression [51]. NKT cells and group 3 innate lymphoid cells (ILC3s) represent other innate lymphocytes capable to produce IL-17 in the tumor microenvironment [52]. On the other hand, IL-17R is widely expressed in ECs, epithelial cells, fibroblasts, hematopoietic cells [53], and tumor cells [54], which implicates pleiotropic effects of IL-17 in the tumor microenvironment.

It seems that IL-17, as Roman god Janus, exerts two opposite faces in the tumor: "dark face" that drives tumor progression and "light face" responsible for the development of effective antitumor immunity. By *in vitro* and *in vivo* experiments, IL-17 signaling was shown to be "malevolent player" that promotes tumorigenesis and tumor progression, in many ways. In general, IL-17 exerts pro-tumor properties by direct influence on the tumor cells *via* triggering malignant transformation and tumor growth [55, 56] and/or indirectly by controlling chronic inflammatory and immunosuppressive tumor microenvironment, as well as angiogenesis [40, 57]. The IL-17/IL-17R axis upregulates phosphorylated ERK1/2 in breast cancer cells lines thereby promoting their proliferation, migration, and invasion [58]. Also, IL-17 can indirectly support the cell proliferation and tumor growth by shaping of tumor microenvironment through the production of chemokines and cytokines [59]. IL-17 was shown to be able to promote hepatocellular carcinoma invasion and migration by upregulation of matrix metalloproteases, MMP-2, and MMP-9, *via* NF-κB signaling [60]. IL-17 promotes STAT3 activity in both tumor and stromal cells, leading to upregulation of anti-apoptotic Bcl-2 and Bcl-XL in an IL-6-dependent manner [61]. This may reflect the fact that IL-17 present in the tumor microenvironment may be an important survival factor and reason for tumor chemoresistance. Accordingly, IL-17 promotes resistance of breast cancer cells to chemotherapeutic docetaxel *via* activation of ERK1/2 pathway [58]. Based on these findings, it can be speculated that IL-17 contributes to development of chemoresistance in variety tumor cells *via* activation of prosurvival and/or proliferative signaling. Recent evidence suggests that IL-17 links inflammation to tumor progression. Indeed, long-term IL-17 activity leads to pro-tumor microenvironment by inducing the secretion of inflammatory mediators and reshaping the phenotype of stromal cells [62]. Additionally, IL-17 stimulates the chemokine and VEGF expression that favor the recruitment of specific subsets of immune cells to the sites of inflammation and angiogenesis, respectively [63]. This IL-17-mediated maintenance of inflammatory environment results in the stimulation of tumor growth and metastasis *via* subsequent expression of anti-apoptotic molecules and increased tumor cell survival [64]. Ironically, Wang et al. [57] illustrated that IL-17, as pro-inflammatory cytokine, contributes to immune paralysis in the tumor microenvironment. Namely, IL-17 increases the expression of programmed death-ligand 1 (PD-L1) inhibitor on MSCs that shape the immunosuppressive environment and facilitate tumor progression. Further, chemokines (e.g., CXCL1 and CXCL5) stimulate the recruitment of MDSCs in IL-17-depandent manner to establish a proangiogenic and immunosuppressive tumor microenvironment [62]. Alongside its pro-tumorigenic functions, IL-17 may act as a tumor regressor. The protective role of IL-17 in tumor relies on its property to induce the vigorous immune responses to attain tumor regression. In fact, it has been demonstrated that effective antitumor immune response is mediated by Th17 lymphocytes and highly depends

#### *Modulators of Tumor Angiogenesis: Insights into the Role of Galectin-3 and IL-17 Signaling DOI: http://dx.doi.org/10.5772/intechopen.102893*

on IFN-γ [65]. Further, IL-17 enhances the CTLs-mediated immune response directed against hematopoietic tumors by induction of IL-6 and IL-12 production [40]. Therefore, IL-17 is multifunctional cytokine with divergent actions on tumor that are highly context-dependent. It seems that epigenetic and transcriptional modifications as well as certain cytokine milieu in the tumor microenvironment specific to each tumor type and stage may account for the functional plasticity of IL-17 making difficult to predict its role. Finally, IL-17 brings different net outcome in a complex disease such as tumor.

#### **3. Galectin-3 as a tumor angiogenesis virtuoso**

The critical events during angiogenic cascade such as activation, proliferation, and migration of ECs, as well as sprouting and tube formation, largely depend on Galectin-3 [66]. Initially, it has been observed that soluble Galectin-3 affects the migration of human umbilical vein endothelial cells (HUVECs) and capillary tube formation indicating its potential as chemoattractant for ECs [19]. This result has been confirmed by the increased tumor angiogenesis in the presence of Galectin-3 *in vivo*. The direct binding of Galectin-3 for endothelial cell surface appeared to be carbohydrate recognition-dependent event as it may be inhibited by disaccharide lactose and modified citrus pectin (MCP) [19, 67, 68].

Ever since, Galectin-3 has been widely recognized as powerful pro-angiogenic molecule acting through various receptors on the ECs, subsequently activating distinct signaling pathways involved in tumor angiogenesis (**Figure 2**). Interactions between Galectin-3 and different integrins expressed on ECs supposed to be critical in controlling endothelial cell migration and adhesion. Pericyte-derived neural/glial antigen 2 (NG2) proteoglycan, Galectin-3, and α3β1 integrin form the membrane complex that triggers intracellular signaling involved in endothelial cell motility [69]. The blocking antibodies specific for αVβ3, α5β1, and α2β1 integrins interfere with endothelial cell adhesion to Galectin-3-coated surface [70]. In addition to integrins, Galectin-3 on endothelial cell migration markedly depends on direct binding to the membrane highly glycosylated cell adhesion molecule CD146, also known as melanoma cell adhesion molecule [71]. CD146 has been recognized as VEGFR-2 co-receptor and a potential target for anti-angiogenic therapy in tumors [72]. The interaction between Galectin-3 and CD146 is also responsible for secretion of pro-metastatic cytokines by ECs indicating that this axis regulates distinct events during tumor progression [73]. Galectin-3 interacts with glycoprotein endoglin expressed predominantly by ECs as a component of TGF-β receptor complex [74]. Endoglin is abundantly expressed by proliferating ECs indicating an important role of TGF-β/endoglin signaling in tumor vasculature formation [75]. Therefore, thanks to its carbohydrate-binding capacity, Galectin-3 interacts with different molecules expressed by ECs in tumor microenvironment. Moreover, truncated Galectin-3, containing CRD domain, interacts more efficiently with ECs in comparison with full-length molecule [76, 77]. Apart from CRD domain, it seems that angiostimulatory effect of Galectin-3 also depends on its N-terminal tail [78]. Full-length Galectin-3, including its ability to oligomerize through N-terminal domain, appears to be necessary to affect migration of ECs and capillary tube formation [78]. Taken together, angiostimulatory effect of Galectin-3 on distinct events during angiogenesis has been mediated by different parts of the molecule in both carbohydrate dependent and independent manner [68].

Further investigation of the molecular mechanisms responsible for Galectin-3 proangiogenic actions in tumors documented its involvement in modulation of VEGF and basic fibroblast growth factor (bFGF) signaling pathways. Galectin-3 binds N-glycans of integrin αvβ3 *via* CRD thus promoting its clustering and subsequent activation of focal adhesion kinase (FAK) in ECs [78]. FAK is a principal regulator of endothelial cell migration, proliferation, and survival, which participates in signal transduction triggered by integrins and growth factor receptors such as VEGFRs [79]. The expression of VEGFR-2, a major mediator of VEGF effects on ECs, is tightly regulated by FAK activation, its translocation to the nucleus, and subsequent regulation of VEGFR-2 gene transcription [79]. Given its carbohydrate-binding properties, Galectin-3 engages different N-glycosylated tyrosine kinase receptors including VEGFR-2 or FGF receptor-1 (FGFR-1) [80, 81]. It has been documented that Galectin-3 induces VEGFR-2 signaling during angiogenesis through modulation of expression and clustering of receptor on the ECs thus enabling its higher availability to VEGF [81]. However, the recent study has revealed that Galectin-3 amplifies the activation of VEGFR-2 and its downstream signaling only in the presence of VEGF [82]. Moreover, Galectin-3 is not necessary for VEGF-induced activation of VEGFR-2, nor it can activate the receptor in the absence of VEGF [82].

Galectin-3 has been described as a regulator of Jagged-1 (JAG1)/NOTCH1 signaling axis involved in tumor vasculature formation, in particular sprouting angiogenesis [83]. Under hypoxic condition, secreted Galectin-3 directly binds Notch ligand JAG1 in ECs thus activating pro-angiogenic JAG1/NOTCH1 signaling pathway. Galectin-3 prolongs the half-life of JAG1 over the Delta-like-4 (DLL4) thus affecting the balance between these molecules with opposite functions during angiogenic cascade [83, 84]. Interestingly, the proposed mechanism seems to be independent of VEGF/VEGFR signaling thus revealing novel potential targets in anti-angiogenic therapy.

In addition, Galectin-3 promotes the progression of hepatocellular carcinoma, including angiogenesis, through upregulation of β-catenin signaling [85]. Given its presence in different cellular compartments including nucleus, as well as its pleiotropic functions, Galectin-3 interferes with β-catenin pathway known to be active in various types of tumor. Galectin-3 activates PI3K/AKT signaling thus enhancing the phosphorylation and inactivation of key molecule of β-catenin degradation complex known as glycogen synthase kinase-3β (GSK-3β) [85, 86]. Subsequently, β-catenin accumulates in the nucleus and regulates the expression of genes involved in Galectin-3-mediated angiogenesis and epithelial-mesenchymal transition (EMT) [85].

Exosomes are vesicles secreted by living cells that participate in intercellular communication during essential processes such as proliferation, apoptosis, migration, and angiogenesis [87]. A highly glycosylated protein named lectin galactoside-binding soluble 3 binding protein (LGALS3BP), as a ligand for Galectin-3, has been previously recognized as a modulator of breast cancer angiogenesis that elevates VEGF expression via PI3K/AKT signaling pathway [88]. It has been shown recently that exosomes highly containing LGALS3BP affect endometrial cancer growth and angiogenesis [89]. The exosomes delivering LGALS3BP induce tumor cell proliferation and migration and HUVEC angiogenesis by triggering PI3K/AKT/VEGF signaling pathway [89].

The complex interplay between immunosuppression and angiogenesis is the integral part of tumor progression [29]. TAMs are the critical participants in tumor progression involved in the creation of immunosuppressive microenvironment thus enhancing metastasis and angiogenesis [90]. TAMs produce various pro-angiogenic molecules including growth factors (e.g., VEGF), chemokines, cytokines, as well

*Modulators of Tumor Angiogenesis: Insights into the Role of Galectin-3 and IL-17 Signaling DOI: http://dx.doi.org/10.5772/intechopen.102893*

as MMPs [90]. Galectin-3 promotes alternative activation of TAMs toward their pro-tumorigenic M2 phenotype (**Figure 2**, [29]). Increased angiogenesis in tumor is strongly associated with macrophage influx driven by elevated Galectin-3 expression [36]. Furthermore, Galectin-3 deficiency in both tumor tissue and stroma impairs angiogenesis *via* interfering with the responses of macrophages to the complex twoway VEGF and TGFβ-1 signaling pathways [91].

Collectively, thanks to its distinctive structure, Galectin-3 engages plenty of ligands both intracellularly and extracellularly, further interfering with various signaling pathways that regulate tumor angiogenesis. As a potential orchestrator of angiogenic cascade, Galectin-3 may be successfully targeted for anti-angiogenic tumor therapy.

#### **4. Cytokine regulation of tumor angiogenesis: pro-angiogenic activity of IL-17**

Apart from galectins, certain cytokine network within the tumor microenvironment contributes to angiogenesis mainly through sophisticated interplay between different cells and extracellular matrix components as well as stimulation of key pro-angiogenic mediator productions.

The data from human subjects have indicated the strong association between increased angiogenicity and high frequency of tumor-infiltrating Th17 lymphocytes [92, 93]. IL-17 overexpression has been associated with higher microvascular density (MVD) in tumors [92]. In general, IL-17 indirectly amplifies angiogenesis mostly by inducing VEGF upregulation, as well as another angiogenic factors by tumor cells and CAFs [94–96]. Also, IL-17 induces the recruitment of inflammatory cells with angiogenic phenotype (e.g., macrophages and neutrophils) and immunosuppressive cells to the tumor microenvironment, which contributes to different points of angiogenesis in many ways (**Figure 2**, [59, 97]). Even though the IL-17 overexpression has been detected in tumors, mechanisms of IL-17 that contribute to angiogenesis are still unclear. IL-17/IL-17RA axis promotes the activation of JAK-STAT3 signaling pathway resulting in phosphorylation and nuclear translocation of STAT3 [98]. STAT3 is important regulator of VEGF expression [96]. Furthermore, IL-17-mediated tumor angiogenesis involves the activation of STAT3/GIV (Gα-interacting vesicle-associated protein, Girdin) signaling pathway and subsequent upregulation of its downstream target VEGF [99]. Wu et al. [96] determined that IL-17 induces VEGF upregulation and neovascularization through STAT3-mediated signaling pathway in tumor cells that could be blocked by JSI-124, an inhibitor of phosphorylated STAT3. In addition, other mediators such as granulocyte colony-stimulating factor (G-CSF), EGF, FGF, PDGF, and IL-6 exhibit their pro-angiogenic functions *via* STAT3 signaling [61, 100]. IL-17 exerts synergistic effects with TNF-α by enhancing the secretion of potent angiogenic factors by stromal fibroblasts [94], which in turn triggers the angiogenic program in ECs and stimulates the new blood vessel development [95]. The inhibition of IL-17 suppresses VEGF expression in tumor tissue and decreases intratumoral MVD, which confirms important role of IL-17 in angiogenesis [101].

IL-17 stimulates the production of IL-8 [102]. IL-8 acts directly on ECs by promoting their proliferation, survival, and migration, as well as indirectly by increasing the recruitment of neutrophils that are important source of angiogenic factors in tumor microenvironment [103]. IL-17 activates ECs to produce pro-inflammatory

chemokines and cytokines, including CXCL1, IL-8, and granulocyte macrophagecolony-stimulating factor (GM-CSF), thus promoting neutrophil recruitment and adhesion to ECs [98]. It is well known that neutrophils release plethora of molecules that promotes angiogenesis. In particular, neutrophil-derived MMP-9 is critical for catalyzing angiogenic switch in tumor cells and releasing of sequestered growth factors (e.g., VEGF), as well as remodeling of extracellular matrix (ECM) components during angiogenesis [104].