|Year : 2022 | Volume
| Issue : 2 | Page : 159-163
Molecular facets and biochemical cross-talk of angiogenesis: Potential therapeutic targets
Jinnenahalli Raju Yodhaanjali, Raghu Ram Achar
Division of Biochemistry, School of Life Sciences, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
|Date of Submission||28-Sep-2021|
|Date of Acceptance||18-Oct-2021|
|Date of Web Publication||17-Jun-2022|
Raghu Ram Achar
Division of Biochemistry, School of Life Sciences, JSS Academy of Higher Education and Research, Mysore - 570 015, Karnataka
Source of Support: None, Conflict of Interest: None
Angiogenesis is a well conserved biological process for vascular growth and development. A canonical approach towards angiogenesis as provided insight in understanding the molecular and biochemical mechanism which differs in cancer angiogenesis. Vascular sprouting is a critical process in cancer metastasis and invasion, cancer cells release certain growth factors that can activate downstream signalling pathways to initiate VEGFR2 gene transcription further instigating angiogenesis via VEGFR2 receptors. Furthermore, paracrine signalling through these growth factor can directly bind to VEFGR2 causing its activation. There are several factors that has been procured by cancerous cells to sustain its survival. Over a period, studies have shown that there are various downstream signalling pathways taking part in cancer prognosis as most of the signalling pathways aim to inhibit endogenous VEGFR2 inhibitory molecules such as Thrombospondin. Cancer is a multifactorial disease and therefore hypoxia, changes in cellular pH, metabolic reprogramming, mutations in proto-oncogenes and tumour suppressor genes have been the contributory factors for cancer cell growth. Understanding the biochemical and molecular mechanism have paved its way in unsnarling the potential therapeutic targets. In addition, the role of adhesion molecules has also been studies they act as an adaptor molecule for an example αvβ6 in hippo pathway activates VEGFR for tip cell activity. Thereafter, focusing on these aspects of angiogenesis can provide several targets that would be used for developing and designing inhibitory antagonist, oncogene targeting drugs or anti-cancer drugs.
Keywords: Angiogenesis, molecular targets, receptors, vascular system
|How to cite this article:|
Yodhaanjali JR, Achar RR. Molecular facets and biochemical cross-talk of angiogenesis: Potential therapeutic targets. Biomed Biotechnol Res J 2022;6:159-63
|How to cite this URL:|
Yodhaanjali JR, Achar RR. Molecular facets and biochemical cross-talk of angiogenesis: Potential therapeutic targets. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Jun 26];6:159-63. Available from: https://www.bmbtrj.org/text.asp?2022/6/2/159/347708
| Introduction|| |
Angiogenesis is a process of forming new blood vessels from preexisting vessels by the growth and migration of endothelial cells (ECs). Blood vessels supply oxygen, nutrients, and immune cells to our body organs, as they are the part of transport or circulatory system. Vasculogenesis leads to the assembly of vascular architecture, in the process, the embryonal angioblasts are deposited next to each to form an initial capillary network; further, angiogenesis expands the vascular network within the growing organ. Various experiments were carried out to understand the role of angiogenesis in cancer, in 1920s, it was observed that different vascular patterns in different types of tumor, and in late 1980s, endothelial mitogen, vascular endothelial growth factor (VEGF) was purified and characterized, and the presence of VEGF enhanced angiogenesis, and its absence due to genetic or immunologic means inhibited angiogenesis.
To meet the energy requirements during carcinogenesis, cells would require more nutrients and also fasten the synthesis of macromolecules such as glucose, nucleic acids, proteins, and lipids. In continuation, hypoxia is another condition, especially for cancer to overcome, cancer cells require more oxygen supply than normally functioning cells would require, to overcome this situation, cancer cells have the capacity to initiate angiogenesis, which can fetch cancer cells with all the nutrients and supply oxygen. However, this also leads to metastasis, thus spreading cancerous cells to different parts of the body. Yet, these hallmarks can be potential target site for the design of new therapeutics.
Angiogenesis is a molecular process which is initiated by the cascade of multiple signaling pathways. VEGF and their receptors are the family of tyrosine kinase, receptor tyrosine kinases (RTKs) major type of cell surface receptors, all the RTKs comprise an extracellular domain containing a ligand-binding site, single hydrophobic transmembrane α-helix, and cytosolic domain that includes a region with protein-tyrosine kinase activity. The mode of action of RTKs is ligand binding leads to the activation of intrinsic protein-tyrosine kinase activity and autophosphorylation of tyrosine reduces in its cytosolic domain, there are many cellular pathways observed that are capable of initiating angiogenesis. Angiogenesis is one such process opted by cancer cells for their survival and pathology, as uncontrolled growth of cells fails without sufficient nutrients and supporting Extra-Cellular Matrix (ECM) environment. The vasculature differs from that of normal cells, it is leady and torturous and provides direct entry, allowing cells easy access to the circulation and they differ at the molecular level, for examples, the integrins αvβ3 and αvβ5 are upregulated in angiogenic vessels. Furthermore, regulation of angiogenesis depends on the angiogenic inducers and inhibitor any imbalance leads to abnormalities that is increase level of inducers and decrease level of inhibitors leads angiogenesis increasingly. One such inducers are VEGF endothelial-–specific, three families of VEGF and their receptors have been identified – VEGF and VEGF receptors, angiopoietins and tie receptors, ephrins, and ephrins receptors. The important component for angiogenesis is VEGF, and many receptor subtypes have been identified, each receptor plays the specified role required. There are many pathways involved in the initiation of angiogenesis; the major pathways are described below.
| Vascular Endothelial Growth Factor - Vascular Endothelial Growth Factor Receptor Induced Pathway|| |
VEGF binds to the specific receptor thus carrying out ligand-receptor interacting which leads to the dimerization and autophosphorylation of cytoplasmic polypeptide chains, thus, many pathways are activated, among all the pathways, phosphatidylinositol 3-kinase (PI3-Akt) pathways are also activated from the membrane phospholipids phosphatidylinositol 4,5-biphosphate. The activated Akt phosphorylates many target proteins, for example – BAD and Bim, these are the proapoptotic regulatory proteins, phosphorylation of these proteins creates 14-3-3 chaperone protein binding site which degrades BAD, thus inhibiting apoptosis, and resulting in cell survival. Similarly, Akt phosphorylates FOXO which is involved in the stimulation of transcription genes that inhibit cell proliferation or induce cell death, thus inhibiting the function of FOXO as shown in [Figure 1].
|Figure 1: Vascular endothelial growth factor pathway with the downstream signaling to induce angiogenesis|
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Considering other cell signaling pathways that play vital role in promoting angiogenesis, extending the focus on two pathways – hedgehog signaling pathway and Salvador-Warts-Hippo pathway are very important.
| Hippo-Yap/Taz Pathway|| |
The importance for this pathway was studied in Drosophila melanogaster; most of the signaling components are conserved in mammals. Hippo pathway is involved in control of organ size, cell proliferation and metabolism, and also involved in apoptosis. Many experiments have shown its role in cancer and are one of the target sites for cancer therapeutics. Consider the role of this pathway in humans, two downstream regulators play major role in the signaling, YAP/TAZ these are the transcriptional coactivators. The upstream regulators are mammalian ste20-like (MST1/2), YAP/TAZ are activated in the absence of hippo pathway signaling, as YAP/TAZ gets phosphorylated by LATS1/2 which phosphorylates the conserved sequence HXRKXXS motif, thus creating the binding site for chaperone 14-3-3 (similar to FOXO) resulting in cytoplasmic retention. LATS1/2 mediated phosphorylation triggers subsequent phosphorylation by casein kinase 1δ/ε, thus resulting in SCFβ-TrCP E3 ligase-induced degradation. When hippo signaling is not active the unphosphorylated YAP/TAZ translocates into the nucleus where it binds to TEAD1-4 (DNA binding region) as depicted in [Figure 2], this complex leads to the expression of hippo responsive gene, resulting the cell proliferation and migration.
| Yap/Taz Through Multiple Signaling Pathways|| |
The tight junctions among the cells are maintained by the adhesion molecules and the extracellular matrix (ECM). ECs are the basic building block of vascular network. Moreover, for angiogenesis to occur, the ECs have to loosen their junction with other cells, thus increasing the motility (which helps in cell migration). Considering the role of YAP/TAZ, they target connective tissue growth factor, cry61, and angiopoietins 2 for the initiation of angiogenesis. Angiomotin (AMOT) plays an important role in tube formation and migration of ECs. One of the isoforms of AMOT, p130-AMOT contains PPXY motif which mediates its interaction with WW domain of YAP/TAZ, can inhibit transactivating function of YAP/TAZ by either initiating MST1, LATS, and YAP near the junction and increasing the phosphorylation of YAP/TAZ or by integrating YAP/TAZ to F-actin. One of the signaling molecules of hippo pathway LATS can regulate AMOT, through direct phosphorylation on LATS substrate conserved-sequence, HXRXXS which is S175 of AMOT; s-175 is known to have an interaction with F-actin. Phosphorylation of s-175 disrupts AMOT interaction with F-actin thus reducing adhesion due to F-actin, resulting in inhibition of EC migration (in vitro studies). Studying the role and effect of LATS-AMOT interaction on YAP/TAZ function in angiogenesis is an intriguing field and can be a therapeutic target site for treating metastasis in cancer. Studies have found that VEGEFR activation triggers PI3/MAPK signaling pathway as shown in [Figure 3], which activates the hippo effectors YAP/TAZ. It is found that these transcription factors also interact with CRY61 which is a cysteine-rich angiogenic inducer, an extracellular matrix protein inducing cell migration.
|Figure 3: Role of YAP/TAZ in angiogenesis- activation of YAP/TAZ through multiple signaling pathways|
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| Integrin Pathway|| |
Cell-to-cell communication is a crucial function required by cells to interact with one other and also with the external environment. The cells are connected to the external environment and to the adjacent cell through adhesion molecules, these molecules are the surface receptors which mechanically aids in binding of cell cytoskeleton to the ECM and also they sense the external signals and transmit those signals to the cells. Cell-to-cell adhesion is a selective process; cells are very specific they bind to cells which are similar with respect to function, structure, and location. For example, cells from liver tissue adhere only to liver tissue but not to the brain tissue, this specificity is brought by the cellular adhesion molecules. Integrin is transmembrane glycoproteins which are a vital adhesion receptor for almost all the cells linking them to neighboring cells as well as to the ECM. These molecules bridge the communication between cell and Extra-Cellular Matrix (ECM), allowing cells to respond to their external environment but have also shown to promote invasion and metastasis in human cancer. Integrin α6β4 which is predominantly expressed in epithelial cells and governs the formation of hemidesmosomes, integrin α6β4 binds to the fibrous laminin of EMC and provides a stable adhesion through the formation of hemidesmosomes. Hemidesmosomes are dynamic structures, involved in maintaining cellular integrity. Experimental evidence suggests the importance of integrin β4, integrin β4 knockout mice are born with epidermal blisters and separation. These observations notified the importance of integrin β4 in maintaining CELL-EMC junction. Corelating the role of integrin α6β4 in wound healing and promoting invasion in cancer is similar.,
For the wound healing, hemidesmosomes must be disintegrated loosening the tight junction thus allowing the cells to migrate to the sight of wound. This is facilitated by phosphorylation of cytoplasmic tail of integrin β4 by protein kinase C that releases integrin α6β4 from hemidesmosomes, for this process to occur, external stimulation is required and the stimulation occurs through epidermal growth factor. Further, α6β4 relocalizes from keratin cytoskeleton to actin. Actin is globular protein involved cell motility and binding of integrin to the actin forms the motility structures. With the stimulation from the receptors and subsequent signaling, pathways promote the cell migration to the site of wound and covering of the wound. Therefore, the similar process of phosphorylation occurs for the invasion of cancerous cells. In normal cells, α6β4 can trigger apoptosis in response to DNA damage through the activation p53 but tumor cells with mutated or deficient in p53, α6β4 promotes cell survival by activation of AKT/protein kinase B signaling pathways and through translational regulation of VEGF expression. Moreover, it is also involved in promoting angiogenesis, experimental studies done using knockout mice in which the signaling domain was deleted, it showed reduced angiogenesis in a retinal neovascularization models and the same study deduced that integrin β4 subunit could promote fibroblast growth factor and VEGF- induced angiogenesis by signaling through ERK pathway, the signaling pathway as shown in [Figure 4].
|Figure 4: Role of integrins in the crosstalks leading to VEGF functioning|
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Integrin β4 subunit can only bind to the subunit α6, thus inhibiting the binding of β4 and inhibiting the phosphorylation of cytoplasmic tail of integrin β4 tumor invasion can be controlled thus can be the therapeutic target site.
| Role of Cry61 in Interconnecting Integrin Avb6 and Hippo Pathway in Cell Tip Activity- The Bridge|| |
Experimental evidence also shows that Cry61 (cellular communication network factor-1) bridge the connection between integrin αvβ6 and hippo pathway, thus autoregulating tip cell activity. Expression of Cry61 RNA in EC is low compared to mesenchymal or cancer cells, it was observed that Cry61 could activate VEGF receptor through the binding with integrin αvβ6 (upregulated during vascular damage or angiogenesis), subsequently, downstream signaling pathways – PI3K, ERK and mDia1 are activated. It was also noted that Cry61 activated mDia1 which is required for activation of YAP/TAZ and leads to the filopodia formation through increased cdc42 activity. mDia1 is classified as member of formins and effector of Rho family, involved actin dynamics. Cdc42 is a GTPase of Rho family which controls many cellular functions including cell migration and also involved in polymerization of actin filament. Actin filaments rapidly cross-links into bundles and networks in the projecting filopodial, they are the cytoplasmic extensions found at the protruding edge in migrating cells. Investigations were done to show if mDia1 is involved in Cry61-induced filopodial formation in ECs. DIAPHI gene encodes a protein which is crucial in regulating cell morphology and cytoskeleton, it was noticed that in Cry61 treated HUVECS; Cry61 increased DIAPHI mRNA expression, and overexpression of DIAPHI increased filopodial number.
Dominant-negative mutations of DIAPHI decreased Cry61-induced filopodial formation, thus emphasizing the necessity of mDia1 for Cry61-induced filopodial formation in ECs. It was further identified that activation of mDia1 induced by Cry61 is responsible for YAP/TAZ activation (involved in hippo pathway). Nuclear localization of YAP/TAZ was induced by overexpression of DIAPHI as well as Cry61 treatment, but the dominant-negative mutants of DIAPHI could not induce the nuclear localization, thus indicating the involvement of mDia1 in nuclear localization of YAP/TAZ in Cry61-induced tip cell formation.
Integrins are involved in various pathways, like activating vascular endothelial growth factor receptor 2 (VEGFR2). Therefore, integrins αvβ3 and αvβ1 are involved in the migration of Endothelial Cells induced by Cry61 as shown in [Figure 5], the analysis showed strong interactions between VEGFR2 and αvβ3. Thus suggesting that Cry61-mediated tip cell activation is regulated by YAP/TAZ through αvβ3/VEGFR2 pathway. Hence, αvβ3 serves as a main mediator of hippo pathway induced by Cry61.
Experiments suggest that overexpression of YAP/TAZ increase Cry61 expression and secretion from cancer cells into the tumor microenvironment, thus increasing the expression of YAP/TAZ which further activates tip cell activity. In ECs VEGFR2 and DLL4 (delta-like protein 4), a single transmembrane domains are involved in inducing vascular sprouting and tumor angiogenesis. Cry61 increases activation of VEGFR2, ERK1/2, p38 MAPK, and PI3K signaling pathway by binding with integrin αvβ3 and VEGFR2. In turn, these downstream pathways induce YAP/TAZ signaling through the MAPK and PI3K nexus, LATS1 (tumor suppressor, which phosphorylates YAP/TAZ leading to its degradation) which is repressed by phosphorylation, thus enhancing the nuclear localization of YAP and Cry61 expression to carry out tip cell activity.
| Conclusion|| |
Angiogenesis is a combination of various processes including cell signaling pathways and metabolic pathways. It is a blend of all these processes that contribute in various stages of angiogenesis such as release of pro-angiogenic factors from tumor cells, endothelial tip cell selection with extension of filipodia along with loosening of EC junction, stalk cells proliferation, lumen formation, and tip cell fusion. It is very important to focus on each and every aspect of angiogenesis. The process of angiogenesis may also vary from malignant tumor to benign tumor and also on the type of cancer. The ECs involved in the progression of angiogenesis undergo many variations including metabolic reprogramming. There are many therapeutic target sites like targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 which is involved in regulation of glycolysis. Another way is targeting the cellular organelles which can induce apoptosis in normal conditions. Thus, controlling angiogenesis can be one of the ways to control the rapid invasion of cancer cells. Allosteric changes in the metabolic enzymes can cause lack of energy in the form of ATP, due to which the ECs cannot sustain the angiogenesis process thus can be an approach to target the cancer cells. One such approach can be targeting the ligand-receptor interaction by secondary metabolites which can subsequently inhibit the pathways involved in angiogenesis. Changing the stereochemistry of the ligand binding to specific receptors can be another approach. Considering these signaling pathways with molecular and biochemical factors revolving around angiogenesis gives an opportunity to have different potential therapeutic approaches.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]