Mechanisms of tumor angiogenesis and metastasis

Tumor angiogenesis and lymphangiogenesis

A distinctive feature of tumor vasculature is increased blood vessel permeability caused by immature, morphologically abnormal pericytes and an impaired connection between the basement membrane (BM) and perivascular and endothelial cells (EC). Another main feature is inadequate blood perfusion with vessel compression leading to hypoperfused and hypoxic tissues and, on the other hand, unequally distributed blood flow with elevated interstitial fluid pressure.

Thus, tumor blood vessels are leakier, tortuous and disorganized, leading to a worse therapeutic effect of chemo-/immunotherapy and radiotherapy with drug leakage, poor radiation sensitivity and low drug concentrations in hypoperfused areas. Vascular normalization is considered the main mode of action and effect of antiangiogenic agents. (Viallard, Larrivée, 2017)

Besides sprouting angiogenesis, tumors exploit additional mechanisms for neoangiogenesis. Intussusceptive angiogenesis, meaning splitting of pre-existing vessels into two functional vessels by inserting interstitial pillars into the lumen of vessels, is thought to be faster and less demanding metabolically. In vasculogenic

49 mimicry, tumor cells expressing vascular markers form perfused channel

structures and tubes. They can also integrate into the walls of tumor blood vessels, forming mosaic vessels. This mechanism has been reported also in ovarian cancer.

The fourth type of tumor angiogenesis is so-called vessel co-option or hijacking, where cancer cells grow along existing vessels and integrate them during cancer growth. Endothelial precursor cells from bone marrow have also been thought to possess the ability to differentiate and incorporate into the growing vessels and contribute to fast neoangiogenesis in many studies. Furthermore, cancer stem cells can participate in vasculogenic mimicry as previously described. (Viallard, Larrivée, 2017)

The molecular mechanism of tumor lymphangiogenesis is less understood, although several lymphangiogenic factors have been described. Generally, the endothelium of lymph vessels is thought to secede from venous trunks forming thin-walled capillaries, thicker collecting lymph vessels and finally large trunks of lymph. Lymphatic vasculature have loose overlapping cell junctions without pericytes or an intact basement membrane leading to suitable entry of invasive tumor cells and possible earlier spread of cancer to regional lymph nodes (Karpanen, Alitalo, 2008, Saharinen et al., 2004). Tumors may interact with lymph vessels by vessel co-option, chemotactic migration, invasion into lymph vessels and induction of lymphangiogenesis via growth factors and cytokines produced by tumor or stromal cells, tumor-associated macrophages and platelets (Sleeman, Thiele, 2009, Alitalo et al., 2005). Lymphatics in the periphery of tumors are thought to be functional whereas intratumoral lymph vessels are probably non-functional due to high intratumoral pressure (Achen et al., 2005, Padera et al., 2002).

VEGF-C and -D acting through VEGFR2 and VEGFR3 are the most well-known mediators of lymphangiogenesis. Lymphatic vessel hyaluronan receptor (LYVE-1), T1α/podoplanin, transcription factor protein (PROX1), interleukin-7 (Il-7) and gene encoding podoplanin (PDPN) have also been expressed in lymph vessels, which are thought to be active regulators of the tumor microenvironment and metastasis (Hirakawa, 2009, Schoppmann, 2005).

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Angiogenesis in metastatic process

It is thought that malignant tumors can grow only a maximum 2mm³ without vascular supply (Gavalas et al., 2013). Cancer growth and metastasis is a corner-stone of cancer progression, a complex process involving the degradation of the extracellular matrix, the epithelial-mesenchymal transition (EMT), tumor

angiogenesis and lymphangiogenesis, migration, invasion, the development of inflammatory tumor microenvironment and defects of programmed cell death, such as apoptosis, autophagy and necroptosis (Su et al., 2015). Despite passive exposure of peritoneum to the malignant cells in ovarian cancer, there is also evidence of hematogenic metastasis as a mechanism of typical omental tumorigenesis (Pradeep et al., 2014).

The microenvironment of the malignant tumors has been of particular interest in recent studies, and its significance in cancer behavior has elucidated lately.

Tumor-associated stroma, cross-talk between the epithelial carcinoma cells, various types of stromal cells and the endothelium of stromal vasculature via paracrine signaling and in concordance with intracellular pathways, has been shown to play a significant role in cancer formation (Watnick, 2012). Fibroblasts, activated by transforming growth factor -β (TGF-β) and tumor cells, deposit extracellular matrix in tumors, which increase interstistial fluid pressure and generates hypoxia, leading to the expression of hypoxia-inducible factor 1α (HIF-1α) and induction of VEGF, basic fibroblast growth factor (bFGF) and angiogenesis.

Cancer-associated fibroblasts have also been shown to secrete matrix

metalloproteins (MMP) that help degrade the basement membrane and promote tumor invasion in rat colon carcinoma cells and promote the proliferation of tumor cells at the metastatic site (Dimanche-Boitrel et al., 1994, Watnick, 2012).

Tumor-associated immune and inflammatory cells like macrophages, mast cells, neutrophils and dendritic cells are known to attract angiogenic factors and stimulate tumor angiogenesis by releasing proangiogenic factors like VEGF, prokinecitin-2 (Bv8) and MMP9 (Murdoch et al., 2008). Neutrophils are rich source of proteolytic enzymes that can release bioactive forms of growth factors and proangiogenic molecules (Liang, Ferrara, 2016). Bone marrow-derived cells have also been shown to possess antitumor activity and mediate antiangiogenic influence in many mouse and human tumor models (Watnick, 2012).

51 2.2.3 Angiogenic pathways

Angiogenesis is a process regulated by a balance between pro- and antiangiogenic factors. Pro-angiogenic factors can be divided into classical and non-classical subgroups (Table 5) (Marech et al., 2016). Factors bind their specific

transmembrane receptors and induce several downstream signaling pathways like mitogen-activated protein kinases (MAPK/ERK) pathway, and phosphoinositide 3 kinase (PI3K) (Figure 4). Phosphorylation of various intracellular kinases mediates each signaling pathway and promotes gene expression via nuclear transcription factors followed by migration and proliferation of endothelial cells, angiogenesis and survival of cancer cells (Marech et al., 2016).

Figure 4. Downstream signalling of VEGF-A, VEGFRs and examples of inhibitors of VEGF-A pathway (Madu et al., 2020).

52 Table 5. Pro- and anti-angiogenic factors (Annese et al., 2019, Marech et al., 2016) Classical Non-classical Pro-angiogenic Vascular endothelial growth factor A (VEGF-A) Fibroblast growth factor-2 (FGF-2) Platelet-derived growth factor (PDGF) Hepatocyte growth factor (HGF) Insulin-like growth factors (IGFs) Angiopoietins (Ang) Tumor necrosis factor-α (TNF-α) Interleukin-6 (Il-6) Transforming growth factor-β (TGF-β) Epidermal growth factor (EGF) Hypoxia inducible factor-1-alpha (HIF-1α) Matrix metalloproteinases (MMPs) Prolactin (PRL) Cyclooxygenase-2 (COX-2)

Erythropoietin (EPO) Angiotensin II (ANG-II) Endothelins (ETs) Adrenomedullin (AM) Proadrenomedullin N-terminal 20 peptide (PAMP) Urotensin-2 (Uts-2) Leptin (Lep) Adiponectin (Adipoq) Resistin (RETN) Neuropeptidi-Y (NPY) Vasoactive intestinal peptide (VIP) Pituitary adenylate cyclase-activating polypeptide (ADCYAP) Substance P Tryptase (TPSAB1) Chymase (CMA1) Granylosyte colony-stimulating factor (G-CSF) Granylosyte-macrophage colony-stimulating factor (GM-CSF) Bone morphogenetic proteins (BMPs) Netrins (NTNs) Semaphorins (SEMAs) Ephrins (EPHs) Slits-Roundabout Receptors (Slits-RoboR) MicroRNAs (miRNAs) Somatostatin (STT) Natriuretic peptides (NPP) Ghrelin (GHRL) Anti-angiogenic Thrombospondin (TSP) Angiostatin Endostatin Interferon-α (INF-α) Interleukin-12 (Il-12) Angiopoietin-2 (Ang-2) Tissue inhibitors of metalloproteinase (TIMPs) Growth hormone Dopamine Vasostatin

53 Vascular endothelial growth factors and VEGF-receptors

Native vascular endothelial growth factor (VEGF-A, VPF) is a heparin-binding homodimeric glycoprotein with a molecular weight of 45 kDA. According to earlier studies VEGF-A is the key mediator of angiogenesis, secreted by tumors, expressed in endothelial cells and binds two tyrosine kinase receptors VEGFR1 and VEGFR2.

(Carmeliet, 2005).

Human VEGF-A gene has been mapped to chromosome 6p21.3. Alternative mRNA exon splicing has yielded at least six different isoforms of VEGF-A: VEGF165, VEGF121,VEGF189 ,VEGF206 and the less frequent variants VEGF145 and VEGF183 (Houck et al., 1991, Ferrara et al., 2003). Isoforms of VEGF have different properties

concerning heparin-binding ability, diffusibility, mitogenic activity, bioavailability and biological potency. The balance between angiogenic and anti-angiogenic properties of different isoforms can regulate vessel growth and patterning.

VEGF165 corresponds closely to native VEGF, is heparin-binding and can be secreted or bound to the cell surface and extracellular matrix (Ferrara et al., 2003).

VEGF mRNA expression is induced by several mechanisms including hypoxia, acidosis, mechanical stress, growth factors like epidermal growth factor (EGF), TGF-α and -β, keratinocyte growth factor, insulin-like growth factor-1 (IGF-1), FGF and PDGF. Inflammatory cytokines Il-1α and Il-6 and hormones like thyroid-stimulating hormone (TSH), adrenocorticotropin (ACTH) and sex hormones can induce

expression of VEGF. In addition expression of oncogenes and mutations of tumor suppressor genes, like the RAS cell signaling pathway, upregulate VEGF (Ferrara et al., 2003).

VEGF-A is a main regulator in sprouting angiogenesis guiding endothelial cells to the tip of sprouts by functioning as a chemoattractive signal and inducing proliferation of endothelial stalk cells. VEGF also recruits monocytic cells from the bone marrow via VEGFR1 signaling and regulates arterial differentiation (Adams, Alitalo, 2007). It has been shown that autocrine VEGF is required for endothelial cell survival, whereas mainly paracrine secretion is not sufficient (VEGF-A/ kinase domain region, KDR, autocrine loop) (Lee, S. et al., 2007, Sher et al., 2009).

Furthermore, malignant ascites formation is believed to be the consequence of a VEGF-A-dependent effect on vascular permeability.

Four other members of the human VEGF- family are VEGF-B, VEGF-C, VEGF-D and placental growth factor (PLGF). Proteolytically processed forms of VEGF-C and VEGF-D bind to VEGF -receptors 2 and 3. They are expressed in endothelial,

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lymphatic and tumor cells. VEGF-C and -D play a crucial role in lymphangiogenesis, but they also stimulate angiogenesis. They may also suppress the immune

response to cancer (Chen, J. C. et al., 2012, Davydova et al., 2016, Stacker et al., 2001).

The VEGF-C gene was discovered in chromosome 4q34 in humans. Mature VEGF-C can bind to VEGFR2. When processed, VEGF-C can activate VEGFR3 homodimers and VEGFR2/VEGFR3 heterodimers. VEGFR3 homodimers are active in organizing endothelial cells and lumen formation, whereas heterodimers contribute to angiogenic sprouting. VEGF-C may stimulate tumor growth both in a paracrine and autocrine manner (Chen, J. C. et al., 2012).

Inflammatory cytokines like Il-6 and -17 can increase VEGF-C expression

promoting lymphangiogenesis. The activation of proto-oncogenes, growth factors, transcription factors and micro-RNAs may also regulate VEGF-C expression (Chen, J. C. et al., 2012, Adams, Alitalo, 2007).

Figure 5. VEGFs, corresponding receptors and co-receptors (Shibuya, M., Claesson-Welsh, 2006)

55 The three tyrosine kinase receptors VEGFR1 (Flt-1), VEGFR2 (KDR) and VEGFR3 (Flt-4) have a similar structure: an extracellular domain composed of six or seven immunoglobulin loops for ligand binding, a transmembrane domain, cytoplasmic juxtamembrane domain, catalytic tyrosine kinase domain split by a kinase

insertion domain and a C-terminal tail. Besides endothelial cells VEGF receptors have been indentified on bone marrow-derived cells, monocytes and some tumor cells. VEGFR1 binds not only VEGF-A but also PLGF and VEGF-B. Soluble VEGFR1 is an inhibitor of VEGF activity. Earlier it has been proposed that VEGFR1 could be a

`decoy` receptor to regulate VEGF activity with only mild mitogenic activity (Ferrara et al., 2003, Ferrara, 2004, Chen, J. C. et al., 2012). It is a positive regulator of macrophages and monocyte chemotaxis and stimulates inflammation and cancer metastasis. VEGFR1 can stimulate angiogenesis indirectly via VEGFR2 through binding of PLGF (Shibuya, M., 2006b).

VEGFR2 is the main mediator of the effects of VEGFs. VEGFR2 activation induces endothelial cell growth by activating the RAF-MEK-ERK pathway, with the

requirement of protein kinase C. VEGFR2 is also expressed in lymphatics and modulates lymphangiogenesis by binding VEGF-C and -D. A soluble form of VEGFR2 has also been discovered (Ferrara et al., 2003, Ferrara, 2004, Chen, J. C. et al., 2012).

VEGFR3, stimulated by VEGF-C and -D, is the key inducer of lymphangiogenesis, but it has also angiogenic effects. It can form pro-angiogenic heterodimers with VEGFR2 under the influence of processed VEGF-C. Besides lymphatics, VEGFR3 was shown to be expressed in the tip cells of angiogenic sprouts (Chen, J. C. et al., 2012, Tammela et al., 2008). VEGFR3 expression has been related to lymphatic

metastases in cancer.

Neuropilins are transmembrane non-tyrosine kinase glycoproteins that can act as co-receptors of VEGFs and modulate the effects of VEGFRs. Two neuropilin homologues, neuropilin (NRP) -1 and -2, lack the catalytic domain in the

cytoplasmic tail. NRP-1 is mainly expressed in arteries and NRP-2 in the lymphatic endothelium and at low levels in veins. NRP-1 mainly enhances the effect of

VEGFR2-mediated signal transduction by binding to mitogenic VEGF165. The binding of VEGF-C to NRP-2 induces downstream signal-transduction, leading to

lymphangiogenesis (Chen, J. C. et al., 2012, Ferrara, 2004) (Figure 5).

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Angiopoietins and Tie-receptors

Angiopoietins, ad 70 kDA glycoproteins, act primarily on vascular maturation and stabilization during the development of the vessels and angiogenesis. Four distinct angiopoietins have been described: Ang-1, Ang-2, Ang-3 and Ang-4. Angiopoietins activate the tyrosine kinase receptors Tie-2 and Tie-1 in Tie-1/Tie-2 heterodimers (Fagiani, Christofori, 2013).

Ang-1 -mediated Tie-2 activation promotes endothelial cell survival and quiescent vasculature, while Ang-2 levels are upregulated in diseases associated with vascular dysfunction, such as inflammation-enhanced angiogenesis. Ang-2 is a weak context-dependent agonist of Tie-2 and is thought to antagonize Tie-2 activation by preventing Ang-1 ligation (Thomas, Augustin, 2009).

Ang-2, produced by endothelial cells and stored in their Weibel-Palade bodies, can be rapidly secreted at sites of vascular remodeling as in tumors and acts in an autocrine manner in early stages of the angiogenic switch. It promotes the

dissociation of pericytes, increases vascular permeability and infiltration of proteases, cytokines and angiogenic myeloid cells. The impact of Ang-2 on

endothelial cells, whether it stimulates proliferation or regression, depends on the presence of VEGF-A, hypoxia and whether the cells are located in tips or stalks of the angiogenic sprouts (Fagiani, Christofori, 2013) (Figure 6). Ang-2 has also been discovered to induce cancer metastasis by increasing EMT in breast and lung cancer (Dong et al., 2018, Imanishi et al., 2007).

Targeting Tie (tyrosine kinase with immunoglobulin and EGF homology domains) receptors 1 and 2 is essential for vessel remodeling, maturation and integrity by recruitment of supporting pericytes and smooth muscle cells. Tie-1 is thought to be an orphan receptor without any specific ligand. It regulates Tie-2 activity by forming Tie-1/Tie-2 heterodimers. Activated Tie-2 stimulates several intracellular signaling pathways and is essential for the proliferation and maintenance of endothelial cells.

Ang-1 is a more active ligand of Tie-2 than Ang-2. Thus, the ratio of Ang-1 to Ang-2, under the influence of transcriptional factors, is determinant for vessel homeostasis and the state of stabilization or degradation of the host vasculature of tumors and activation of neoangiogenesis (Fiedler et al., 2003, Fagiani,

Christofori, 2013, Thomas, Augustin, 2009). Ang1-Tie-2 ligation is also essential for lymphatic development (Wu, X., Liu, 2010).

57 Figure 6. Angiopoietin signaling in normal vs activated endothelium (Fagiani, Christofori, 2013)

Other mechanisms regulating angiogenesis

As knowledge of the features of complex process of tumor angiogenesis has increased, the significance of reprogrammed macromolecules of extracellular matrix (ECM) in regulating malignant angiogenesis have become clearer. Vascular basement membrane harboring ECM components like proteoglycans can alter by promoting or inhibiting angiogenic process. Furthermore, thrombospondins (TSP) 1 and 2 were found to be natural inhibitors of angiogenesis while TSP1 has been shown to have a protumorigenic influence in glioblastoma and TSP4 enhances vascularization (Mongiat et al., 2019). MMPs can degrade extracellular proteins and process several bioactive molecules, modifying angiogenic homeostasis.

Non-coding RNAs, including micro-RNAs (miR) (21-25 nucleotides), control gene expression in a posttranslational manner. Micro-RNAs have been shown to

modulate the angiogenic process by targeting important angiogenic factors and signaling molecules (Khorshidi et al., 2016). miR-497 have been shown to inhibit

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angiogenesis in ovarian cancer by targeting VEGF (Goradel et al., 2019) and miR-204-5p to induce angiogenesis through TSP 1 (Chen, X. et al., 2019).

Effect of angiogenic factors in epithelial carcinoma cells

Tumor cells have been shown to express and secrete multiple angiogenic factors into the tumor microenvironment. Paracrine and autocrine stimulus of factors is needed for the survival of malignant cells under hypoxic conditions and without contact with other cells or ECM (VEGF-A/KDR loop) (Sher et al., 2009). Furthermore, paracrine secretion contributes to tumor development and malignant

transformation of cells via angiogenesis, growth and differentiation of tumors.

Paracrine secretion can also be essential in promoting tumor dissemination and metastasis through increasing EMT (Ang-2) (Dong et al., 2018).

In document Epithelial ovarian cancer and angiogenesis: biomarker and gene therapy study (sivua 50-60)