**2. Structural divergence of the cannabinoid receptors from class A GPCRs**

The CB1 receptor is a class A (Rhodopsin-like) GPCR (**Figure 1**). Different phylogenetic studies and multidimensional scaling analysis of Class A GPCRs classify cannabinoid receptors (CB1/CB2) into one cluster along with the endothelial differentiation G-protein coupled receptors (EDGRs) (including Sphingosine 1-phosphate receptors (S1P) and Lysophosphatidic acid receptors (LPA)) [9–12]. Receptors from those families, except for the LPA4–6, share common sequence divergence from other Class A GPCRs. Specifically, the absence of helix kinking proline residues in TMH2 and TMH5, and the absence of a disulfide bridge between the EC-2 loop and C3.25 at the EC end of TMH3. Instead, they share an internal disulfide bridge in the EC-2 loop, a conserved PxxGW motif at the EC end of TMH4, in addition to a Y5.39 that forms an aromatic pi-pi stack with W4.64 in that motif resulting in a similar shape of the EC2 loop as seen in the crystal structures for the CB1, S1P1 , and LPA1 receptors [6, 7, 13, 14]. At the

binding site, they share a common basic residue (K/R 3.28) on TMH3 and an aromatic residue (F/Y 2.57) on TMH2. In addition, the S1P receptors are like CB1/CB2 in the presence of E1.49 at TMH1. E1.49 has been reported to be a key interaction site for pregnenolone (an endogenous negative allosteric modulator that protects the brain from *cannabis* intoxication) with CB1 [15], while the LPA1–3 receptors share a W5.43 with CB1/CB2 that has been shown to affect

**Figure 1.** Helix net representation of the hCB1 receptor. The most highly conserved residue in each helix is shown in bold. Residues are numbered using the BW#: Ballesteros-Weinstein residue numbering system in GPCRs which uses the X.YY format; X denotes the transmembrane helix number and (YY) denotes residue position relative to the most

receptors recognize lipid-derived ligands that have been shown to bind to the receptor by diffusing from bulk lipid towards the binding site via a transmembrane portal [6, 7, 14, 17, 18].

, and the cannabinoid

Structural Insights from Recent CB1 X-Ray Crystal Structures

http://dx.doi.org/10.5772/intechopen.80783

35

antagonist binding to the cannabinoid receptors [16]. In addition, S1P1

conserved residue in the helix (X.50). Loop regions are numbered using absolute sequence numbers.

GPCRs have a common architecture of seven transmembrane helices (TMHs) joined by extracellular (EC) and intracellular (IC) loops of varied lengths, in addition to an extracellularly extending N terminus, and an intracellular C terminus that begins with an amphipathic alpha helical segment (Helix 8) oriented parallel to the cell membrane. In Class A GPCRs, the binding site for the endogenous ligand is generally formed by the EC core within the TMH bundle, and may extend to EC loops, referred to as the orthosteric binding site. Ligands may also bind

Due to the various physiological functions mediated by GPCRs, they are considered major targets for drug discovery and design of novel therapeutics. However, understanding the structure-function relationship of these proteins and the design of high affinity, selective ligands that target these receptors requires a detailed knowledge of the three-dimensional structure of the receptor in general and of the ligand binding site in specific. However, structural characterization for membrane proteins in general has been a challenge due to their low expression in recombinant hosts and their inherent instability in surfactants. It was not until the year 2000 that the first high resolution GPCR structure was resolved by X-ray crystallography, Rhodopsin in its inactive state [1]. The following 10 years witnessed the release of other inactive state crystal structures of class A GPCRs (e.g. the Adenosine A2A, and the β1 and β2 adrenergic receptors [2–4]), in addition to the release of the active state crystal structure of Rhodopsin in complex with a synthetic peptide resembling the C-terminus of the G-alpha subunit of transducing [5]. Available structures during that time served as templates for homology modeling for other GPCRs including the CB1 receptor.  A breakthrough in GPCR structural characterization has been achieved in the last 8 years with more than 200 structures for different GPCRs being deposited in the Protein Data Bank, including the CB1 inactive and active state crystal structures which have been resolved in 2016, and in 2017 respectively [6–8]. Before that, structural characterization of CB1 orthosteric as well as allosteric binding domains have been extensively studied via mutations, site-directed labeling, mass spectrometry, SAR studies, and in-silico methods, and will be

to distinct (allosteric) binding sites in the receptor.

34 Recent Advances in Cannabinoid Research

discussed in detail throughout this chapter.

loop as seen in the crystal structures for the CB1, S1P1

**A GPCRs**

**2. Structural divergence of the cannabinoid receptors from class**

The CB1 receptor is a class A (Rhodopsin-like) GPCR (**Figure 1**). Different phylogenetic studies and multidimensional scaling analysis of Class A GPCRs classify cannabinoid receptors (CB1/CB2) into one cluster along with the endothelial differentiation G-protein coupled receptors (EDGRs) (including Sphingosine 1-phosphate receptors (S1P) and Lysophosphatidic acid receptors (LPA)) [9–12]. Receptors from those families, except for the LPA4–6, share common sequence divergence from other Class A GPCRs. Specifically, the absence of helix kinking proline residues in TMH2 and TMH5, and the absence of a disulfide bridge between the EC-2 loop and C3.25 at the EC end of TMH3. Instead, they share an internal disulfide bridge in the EC-2 loop, a conserved PxxGW motif at the EC end of TMH4, in addition to a Y5.39 that forms an aromatic pi-pi stack with W4.64 in that motif resulting in a similar shape of the EC2

, and LPA1

receptors [6, 7, 13, 14]. At the

**Figure 1.** Helix net representation of the hCB1 receptor. The most highly conserved residue in each helix is shown in bold. Residues are numbered using the BW#: Ballesteros-Weinstein residue numbering system in GPCRs which uses the X.YY format; X denotes the transmembrane helix number and (YY) denotes residue position relative to the most conserved residue in the helix (X.50). Loop regions are numbered using absolute sequence numbers.

binding site, they share a common basic residue (K/R 3.28) on TMH3 and an aromatic residue (F/Y 2.57) on TMH2. In addition, the S1P receptors are like CB1/CB2 in the presence of E1.49 at TMH1. E1.49 has been reported to be a key interaction site for pregnenolone (an endogenous negative allosteric modulator that protects the brain from *cannabis* intoxication) with CB1 [15], while the LPA1–3 receptors share a W5.43 with CB1/CB2 that has been shown to affect antagonist binding to the cannabinoid receptors [16]. In addition, S1P1 , and the cannabinoid receptors recognize lipid-derived ligands that have been shown to bind to the receptor by diffusing from bulk lipid towards the binding site via a transmembrane portal [6, 7, 14, 17, 18].


**3. Creation theology and eco-theology**

#### **3.1 Eco-theology is not creation theology though these overlap**

Creation theology is just a chapter of systematic theology that deals with creation. It goes back to the Greek fathers of the Church. It has actually been codified at the Vatican I Council ([3], pp. 43–44). Eco-theology is much more recent. Eco-theology is a "theological perspective framed by concern for the environment" ([10], p. 2). Ernst Conradie, a South-African theologian, suggests Eco-theology should be regarded not as a sub-discipline of theology but rather as a mode of theological reflection or a reform movement that emerged in the years following the 1961 paper presented by Joseph Sittler, an American Lutheran theologian, at the New-Delhi meeting of the World Council of Churches ([10], p. 3). Sittler argued that the unity of the Church, founded in the reconciliation of all things (Col 1.15–20), is inseparable from ecology's fate. The beginning of Eco-theology as a movement approximately coincided with the beginning of public environmental concern ([10], p. 3). Thus feminist theology (Mary Daly, Elizabeth Johnson, Sally McFague, Rosemary Radford Ruether) and liberation theology as applied to the environment (Leonardo Boff), and Lutheran contributions by theologians Joseph Sittler, Paul Santmire, John Cobb, and Jürgen Moltmann mark the beginnings of Eco-theology ([3], pp. 61–65). "…Like feminist theology eco-theology engages in a 'twofold critique', in that it offers a critique from the perspective of Christian theology on cultural and social institutions that underlie the ecological crisis and at the same time engages in the critique of Christian theology and praxis from an ecological perspective" ([10], p. 3). Eco-theology has American roots except for liberation theology and seems until recently to be mainly an Anglo-Saxon concern. While the discovery of biological evolution goes back principally to Charles Darwin, the evolutionary perspective on creation theology is very recent. It was born in the context of science-religion relations and owe its origin to Anglican theology, the British philosopher and theologian Alfred North Whitehead, the French Jesuit paleontologist Pierre Teilhard de Chardin, and the following theologians: the German Karl Rahner, the American John Haught, the Dane Niels Gregersen who introduced the term "deep incarnation" (see infra), the British Christopher Southgate and Richard Bauckham, the Australian Denis Edwards, the American Celia Deane-Drummond and Sr. Ilia Delio among others. This current was imported in France and developed mainly by theologians François Euvé, Jacques Arnould, Jean-Michel Maldamé, Jean-Marc Moschetta and Fabien Revol.

In response to Lynn White's critique, his so-called "ecological complaint", "Santmire argues that the theological tradition is 'neither ecologically bankrupt' nor equipped with a great store of eco-theological traditions." ([10], p. 14). "Eco-theology should contribute to the ecological reformation of Christianity" ([11], p. 35) but "eco-theology is currently characterized by a number of different discourses" ([11], p. 64; [23], pp. 1954–1956) and "… does not have a particular theological methodology or group of methodologies which have enduring significance…" ([11], p. 64). Eco-theology also seeks an ortho-praxis, i.e. ways of living ecologically for a believer. The quest is not only moral; it is also spiritual.

Is Laudato si' a contribution to Eco-theology? It is obvious that panentheism, i.e. "the view that the divine reality is inclusive of and also immanent within the world" ([24], p. 2) is an important building block for Eco-theology, re-introduced in creation theology by Jürgen Moltmann ([3], pp. 66–67]. It is present in Laudato si'. Laudato si' does not, however, consider the universe as the body of Christ as did S. McFague ([3], p. 77). The former does not go as far as claiming that God suffers with the world, i.e.,

*God is rather an active participant, experiencing and suffering with the environment and the various creatures (including human beings) which belong to it. This is what divine immanence demands. Just as human beings suffer with their bodies, so God suffers with the divine body of creation… for God too is counted among the victims of our ecological disregard… [Quoting Paul Fiddes], if God suffers then God too protests and a God who protests against suffering cannot be the cause of it, or God would be protesting against God…. God is not causing or willing our ecological crisis, but rather suffering its detriments…. ecological suffering can be viewed as the sacramental presence of the suffering God. To see the suffering of other creatures is to also see the suffering of the God immanent to them… ([25], pp. 12–13). [Quoting David Gray Griffin], "divine influence is understood as part and parcel of the world's normal causal relations and never an interruption thereof" ([25], p. 14)*

*[Quoting Ivone Gebara], "to speak of pan-en-theism is to consider the potentialities of the universe, the potentialities of life, and the potentialities of human life as always open-ended." ([25], p. 14).*

*Although immanent to God, the world retains its own creative freedom, a freedom which God necessarily works in and through to achieve the goodness of what can be. The naturalism of this "in and through" emerges as an invitation to act, a call to actualize in the world what God can only do through creatures ([25], p. 15).*

Eco-theology questions that humans have a special status within creation ([26], pp. 95–117). Eco-theologians attack the "dominion" and the "stewardship" doctrine, which are anthropocentric. Basic tenets of eco-theology are the idea of divine immanence in the whole cosmos; a relational, ecological rather than hierarchical understanding of God, humans, and the created world; a radically reinterpreted view of human dominion over nature in terms of partnership with nature; a commitment to justice for all creatures, not just humans, highlighting the needs of the impoverished masses and endangered species around the globe.

Clearly, creation theology has been strongly influenced by Whitehead's process philosophy and theology that John Cobb introduced in Eco-theology and "that sets forth a deeply incarnational God who is the fellow sufferer who understands". Teilhard de Chardin that the Catholic Church is slowly but partially rehabilitating

plaid an important role as well. As Eco-theology raises issues that are technical, any eco-theological story—including mine!—needs screening by professional theologians. My purpose is to disseminate a story as I understand it and not either contribute to it or criticize it [27].

Moltmann's theology, especially his re-introduction of panentheism, is oecumenical and relies on Jewish theology as well ([3], pp. 66–67). One can thus talk of a unified Christian and Jewish creation theology. As Christians and Jews make approximately 1/3 of mankind, the eco-theological story, therefore, matters.

#### **3.2 The creation story revisited**

Theology is always context-dependent. The context is cultural, sociological, and cosmological. It has changed tremendously in the 19th century under the influence of the mathematics of non-linear systems, quantum physics, the discovery of biological evolution and, generally, the progress of the sciences ([28], pp. 44–47). The cosmological context has evolved from the static one underlying the book of Genesis to the one of an expanding universe. In Genesis and some psalms, the earth was thought to be floating on water or supported by columns sunk in water under a sky to which the sun, moon, and stars are anchored. Part of the Christian cultural landscape until the end of the 19th century was the Aristotelian philosophy of the static essences rediscovered in the Middle Ages (Thomism) to be replaced by the dynamic visions of History by Hegel, Marx, and Whitehead among others.

Creation theology is in full transformation. Everything moves; everything is dynamic! The world of the Bible is static. There was a golden age, the earthly paradise. This paradise was lost because of original sin. God sent his son in order to restore earthly paradise. This static vision of things, which originated in the Mediterranean view of the world three thousand years ago, was dominant roughly until the Renaissance: the stationary earth and humans were the center of the universe.

#### **3.3 The Christian eco-theological story of creation in point form as I understand it**

Theologian Paull Tillich said of the story of creation in the book of Genesis that it is a myth. A myth is not a made-up story. It is an anthropological story that illustrates the fundamental relationship between God, mankind, and the balance of creation ([3], p. 33, 50). The doctrine of creation asserts that, at each instant, God is the creative foundation of all reality. "The creation stories in the book of Genesis contain, in their symbolic and narrative language, deep teachings about human existence and on its historical reality" says Pope Francis ([2], par 66). The story about this relationship is not limited to Genesis but permeates the entire Bible. Evolution means not only change but also continuity through diversification and complexification ([28], pp. 193–195, 266–268). Creation is not, therefore, simply a past event. The creation story implies that creation finds its origin in God. It does not provide a history of the beginnings of the universe.

During the Renaissance, with the developments of astronomy and of corresponding measuring instruments, thus science, the static vision of the book of Genesis eroded progressively. Revelation is not limited to the books of the Bible but is extended to the "book of nature" that Saint-Augustine and Saint-Bonaventure already spoke about and that Anglo—Saxon natural theology built upon. Sciences play an important role in deciphering this book of nature and allow thus a certain universality of knowledge that cultural conflicts are able to hide (e.g. the dominance of the Mediterranean culture in the Western world).

The creation story is at the same time utopian and eschatological. It is utopian to the extent that it aims at undoing our current predicament by human means. It is eschatological to the extent that it relies upon a divine promise of a better world and upon Providence ([3], pp. 33–38). This dynamic vision illuminated by science completes the biblical vision anticipating the promise. Creation is God's language (Bonaventure) through which God reveals Himself. Thus God has revealed Himself from the beginning of the universe. "God …can be known with certitude by the natural light of human reason from created things" ([29], c.2; [30], par 6). Rather than seeing creation as a state to be restored, the contemporary creation story sees creation as a promise of a future state based on the hope of resurrection. Creation degradation results in the deterioration of God's language.

Before speaking of Messiah, savior, incarnation, the theme of the perpetual alliance between God and His creation is foremost in the Bible. The world, presently in constant evolution, is imperfect. It currently leaves room for corruption, to sin, to death. It tends however toward the fulfillment of the Promise. The incarnation is a project reflecting God's love for His creation despite the latter's imperfection. Incarnation is not so much due to the sin of mankind as to God's intent from time immemorial to enter into partnership (Alliance) with mankind. This is called deep incarnation. Incarnation is, therefore, not a Plan B but a Plan A ([31], p. 16).

Creation is also a gift ([2], par 76) completed with a promise. Within the anticipative perspective, creation is more a promise than a gift as creation is still unfinished. It is also a sacrament. In the Eucharistic bread, "creation is taut towards divinisation" ([2], par 236). The gift is the visible expression of God's love for an object, resulting in its goodness [Gn 1]), its intrinsic value, its holiness [1 Tim 4.3–5]. The sacrament reinforces creation's agency (e.g. water quenches thirst and purifies; bread and wine nourish; fire illuminates and purifies; oil feeds, perfumes, and illuminates). Sacraments do not sanctify the soul and body of humans only; they also deify nature. Environmental degradation desecrates creation; it is sacrilegious.

The universe will be deified, "eucharisticised" (Teilhard de Chardin), transfigured as Jesus was transfigured before the eyes of a few apostles [Mat 17.1–8, Mark 9.2–8, Luke 9.28–36]. Creation will become the resting place for God, the Shabbat of God, in which the entire creation will participate in a state of bliss. Creation will glorify its creator. The end of the universe is, therefore, not its demise but its eschatological transfiguration into a new creation, a process in, by, and for Christ: "the new Jerusalem" [Rev 21.2]. The purpose of the Shabbat—of the seventh day, of the seventh year—and of the biblical Jubilee is the restoration of the relations among humans and among them, the balance of creation and God. The Shabbat is a reminder of the Alliance among God, mankind, and the balance of creation. Humans occupy a special place in creation but, in exchange, they hold a special responsibility for the latter, its evolution, and well-being. This is where Eco-theology comes in.

Christ was present at the origin of the universe. He is responsible for its evolution. He will be responsible for its eschatological transformation into a new creation. "Christ is the redeemer of the whole process of creation" ([32], p. 106). This is a deep incarnation.

Christ is contemporaneous to the creation and, therefore, precedes the man Jesus. Christ fulfills a function: he participated in the original creation, partakes in the continuous creation, and will participate in the eschatological one as well. "Christ has not been an already accomplished character from the beginning of the universe. He achieves his accomplishment in the accomplishment of the world and the world

reaches its accomplishment in Christ" ([31], p. 5). Being Christ is not a name only but a function that does not coincide with the one of Jesus. Christ's function is a cosmic one. Christ is "…recognized as the universal and trans-historical figure of God who unites with creation without being shut off in the singularity of this union" [33].

When God sends his son in order to save the universe from its imperfection, the son fills the entire universe through his resurrection. The function of Christ is to perfect and unify the universe, to synthesize creation and redemption. "In him, everything holds together" [Col 1.17]. Christ is the beginning of everything because, without him, nothing can exist. He is also the first of the universe, i.e., its most important character. He is the end of the universe as well because the whole universe, through its evolution identified by science, tends toward him because of mankind's co-creative action in unity with Christ (Teilhard de Chardin's omega). "I am the Alpha and the Omega, the first and the Last, the beginning and the end." [Rev 22.13–14].

It is customary to divide the creation story into the original, the continuous and the new one.

#### *3.3.1 The original creation*

"All things came into being through him [God], and without him, not one thing came into being" [Jn, 1.3]. Creation is such an eternal act of intra-Trinitarian love that it is impossible to identify creation's beginning ([28], p. 218). Creation ex nihilo does not mean bringing into existence (efficient cause) but rather everlastingly optimizing goodness in creation through attraction (final cause) ([28], pp. 217–218).

The universe is completely distinct from God. This means that the universe is not God (no pantheism) even though God is present everywhere (panentheism).

#### *3.3.2 The continuous creation*

Since the universe is in constant evolution, it is imperfect. Moral imperfection is only one facet of this universal imperfection. An imperfect universe needs a redeemer. Original sin is not a historical event due to mankind only, therefore, but also to a condition that affects matter and History ([28], p. 343).

After the Great Flood episode, God made with Noah and the whole creation a perpetual Alliance whose sign is the rainbow [Gn, 9.8–13]. The Alliance is foremost in the Bible and was reaffirmed many times: to Abraham and his descendants first with the circumcision and the promised land [Gn 17, Gn 28. 13] then to Jacob [Lev 26.42] and then to Moses with the ten commands [Ex 34.1]) and the promised land again. It was renewed in the person of Jesus (the Savior, descendant from David) who said He came not to abolish the Law but to accomplish it [Mat 5.17–18]. It is nowadays being renewed constantly through the Eucharist, the new Alliance manifestation [Luke 22.20].

Creation is called the external alliance by the reformed theologian Karl Barth because creation needed to occur before one is able to establish an alliance therewith. Barth calls the incarnation the internal alliance because incarnation leads to salvation, i.e. justification through grace. The latter as well as creation are a gift from God. Salvation is the new creation that encompasses the whole of nature. Creation assigns a cosmic dimension to salvation [Rev 21, 22].

 When Genesis says that Adam was created in the image of God and to His likeness [Gn 1.26] and that Adam had the right to name the animals [Gn, 2.19–20], God did not yield the mastery of creation to mankind.

A person is only "dust from the ground" [Gn 2.7] and not separate from the balance of creation.

God's image is given to mankind. His likeness must be acquired through a virtuous life ([3], p. 70). The Greek fathers of the Church will fine-tune this statement: the deep meaning of mankind created in the image of God and cooperating with Him is that mankind becomes like God through the influence of the Holy Spirit. Humans are creation's stewards only, kings of creation in the manner of Christ, i.e. creation's servants. They empathize with creation's suffering, protect creation and make it grow (parable of the talents [Mat 25.14–30]). If they do not do this, they disobey God. Christ, being in the image of God as well, is also responsible for creation stewardship. The Catholic Church relies on the prophets [Hos 4.2–3; Is 24.4–5] to tie ecological injustice to social injustice. This tie needs to be restored ([32], p. 85).

God inhabits his creatures. This is the foundation of the sacramental approach to creation. The latter underlines the continuity between humans and the balance of creation. There is a continuity between the social and the ecological. Communion is the fundamental structure of everything which exists.

The relation of a person with a personal God must allow this person to personify the world (Vladimir Lossky). It is the care ethic that Laudato si' emphasizes and which leads to this personification which allows transcending matter's limits. This is why creation awaits with impatience the revelation of God's children [Rom 8.19]. The sacramental approach reveals the insufficiency of the stewardship concept discovered by hermeneutics because humans belong more to nature than the balance of creation belongs to humans.

The person is the priest of creation, i.e., celebrates the latter. Creation is called to enter into communion with its creator through the human person and conversely. All components of creation are integrated into celebrations to be offered to God and transformed through His spirit. "The Eucharist is intrinsically an act of cosmic love." ([2], par 236) "…The Eucharist is the privileged locus where God is present now to our world as a part of this world" [34].

#### *3.3.3 The new creation*

The future of the universe is a promise based on the hope for resurrection. Creation is not a gift only. The universe has no end. It will not be destroyed but will not subsist in its current state. Deuteronomy speaks of a promised land to all creation while Exodus speaks of liberation from a land of oppression and Saint Paul speaks of future liberation, conditional upon human behavior or service. "The natural world is spirituality's true home rather than a far-removed prophetic aspiration to a heaven elusive state of perfection detached from the earth" ([32], p. 41). The current cosmology replaces Genesis' cosmology. Mankind's well-being depends on the planet's well-being.

The person is prophet to the extent the former understands the divine design for creation.

When God sent his son to save the universe from its imperfections, the son filled the entire universe through his resurrection. Christ is cosmic: his role is to perfect and unify the universe, to synthesize creation and redemption. Christ is the beginning of everything because, without him, nothing can exist. He is also the first in the universe because the whole universe, in its evolution noted by science, tends towards Him because of the shared co-creative action of humans united in Christ (Teilhard de Chardin's omega). The fathers of the Greek Church in the first centuries of our

common era were interested in creation within the framework of the cosmic Christ, i.e., Christ present everywhere in the real world. The eschatological purpose of History is to "…reunite the whole universe under one leader, Christ, what is in heaven and on earth" [Ep 1.10].

Through the visible Christ (under His human appearance), we know God but in an imperfect manner [1 Cor 13. 12].

Christ existed before all creatures and is more than the latter. Therefore, everything which has been created, be they angels, humans, or the balance of nature, has been created for Christ and thus for God and is maintained in its existence by Him and for Him.

All of mankind is called to become in Christ similar to Christ. The whole universe in its diversity will be united to Christ because Christ is present in all its components (panentheism). The Church is destined to be the gathering around Christ of all humans united in one body.

Christ is the first to resurrect. Christ's resurrection secures the resurrection of the whole universe. Christ by His death and resurrection obtained victory over the cosmic forces of evil. The latter is responsible for suffering and death which affect the whole of nature. Nature, including humans, must also suffer and die to reach its transfiguration.

Victory over the cosmic forces of evil is a decisive victory, but it requires still extension in space and in time in order to reach the whole of mankind. Despite Christ's victory through His resurrection, the demonic elements of death, sin and chaos operate in the universe. Before His resurrection, Christ necessarily had to suffer and die since He assumed our human condition through his incarnation ([3], p. 197; [35], pp. 36–43).

The balance of nature is the passive victim of the Fall. "…in hope that the creation itself will be liberated from its bondage…" [Rom 8.20–21], creation awaits its liberation because of the active human cooperation towards this goal.

Christ's resurrection insured the redemption not only of humans but of the whole universe. This redemption was needed since creation is imperfect. Humans and nature are interdependent as asserted by ecology and the Fall story.

"God became man so as a man could become God" (Irenaeus) or, more exactly, according to all the Greek fathers of the Church "the Son of God became man so as for men to become sons of God" by adoption ([3], p. 197). This statement is not limited to mankind. The whole universe is recapitulated in Christ. Thus, incarnation, as well as redemption, are cosmic events.

#### **3.4 The eco-theological utopia**

Through her prophetic role, the human being understands the divine design of creation that Revelation teaches her, but creation needs the human being to manifest its potentialities. This is where the Christian utopia joins eschatology.

The transfiguration of nature depends on us because "creation itself will be liberated from its bondage to decay" [Rom 8.20] because of evil that exists in the imperfect creation. According to Maxim the Confessor, one of the Greek Church's fathers, God planned the universe so that the latter could unite with His divinity, but mankind turned its back on God's plan.

One action which is required from us is, with the help of the Holy Spirit, to wait with perseverance for Christ's return. Another is to change the existing situation of domination over other human beings and over the balance of nature ([2], par 224) while collaborating on the transformation of the universe in view of Christ's return. Because of its incompleteness, the universe laments about its disharmony and is divided.

It is humanity that must first find its unity again and then be the mediator and prophet between the balance of the universe and its creator in order for the universe to be transfigured through grace and to find its unity. This mediator's role consists in seeing the reason for the created world through contemplation, beyond appearances, the relation between a given creature and its creator as the one of "the rising sun with the things it illuminates"(Maxim the Confessor), and live in the cosmic energy that is love, according to God's design ([3], p. 198).

The whole of human destiny, as the one of the universe, is one of growth and maturation "day after day and ascending towards perfection, i.e. approaching the One who was not created, will not be complete in this life, but will continue in the other" (Irenaeus) ([3], p. 198). It is the prophetic meaning of the Christian utopia. This meaning would not exist without the eschatological one of the resurrection, which needed itself because of the myth of the Fall. O felix culpa (culpa as flaw)!

## **4. Conclusion**

Pope Francis considers it to be among his prerogatives to intervene in the ecological arena. He is convinced mankind needs to change its culture, to make an "ecological conversion". The predominant culture since the advent of the sciences has been that the environment is an open-access resource, i.e. a public good with unrestricted access. Since the nineteen-fifties, large-scale pollution of the air and of the rivers has been an unmistakable early warning sign that the open-access mentality was destroying the environment and its public good features, i.e. non-excludability of any economic agent from the good and non-rivalry in the latter's consumption.

The open-access culture is not biblical. The Bible sees the environment as a commons, a divine gift that mankind is expected to revere, develop, and share as a common inheritance within and across generations according to its creator's intent. The pope proposes Francis of Assisi's spirituality—articulated in a philosophical system by Bonaventure—as the model for the new culture. The stewardship model proposed by the Greeks, the Stoicians, and the Christian churches do not suffice. It needs to be completed by an ethics of care which not only assigns intrinsic value—whose origin is God himself—to all creation but treats the latter as close kin. The aboriginal culture does this to some extent already since kinship with the earth is part of the aboriginal identity. This is one reason why the latter needs preservation. The ethics underlying the relatively new relational valuation current may bridge the various ethical reference frameworks, stewardship, care, and virtue ethics especially. Whether the intrinsic value is assigned to everything created or to relations with everything created matters little as long as relational values are recognized. These are three alternative ethical frameworks that may jointly assign intrinsic value to creation and lead to the needed cultural change.

Climate change took over from place-based pollution as being the global pollution problem which, with the ozone hole and threatened global biodiversity, is affecting the entire planet. Planetary problems and the Anthropocene that they characterize had to be discovered by science first and, especially, by multidisciplinary science organized in networks. As the latter's conclusions were incompatible with vested interests in the statu quo, the validity of the science was systematically questioned as well as the degree of certainty of its results. Science is no substitute for policy, however; both do not use the same discourse. As, since the Middle Ages, the planet organized politically along with nation-states and since nation-states are generally not ecosystem-based, a coordinated solution to planetary problems has eluded the

#### *Laudato si', Six Years Later DOI: http://dx.doi.org/10.5772/intechopen.104441*

200-odd nation-states which cover the earth. No effective international environmental institutional regime exists yet. Nation-states have great difficulties implementing any international coordinated action within their own borders as well. Science tells us there is little time left for effective action. As environmental policies need to be supplemented with culture and life-styles changes and as these are slow-moving, it is likely that coordinated action will be implemented too little too late. Environmental problems of the Anthropocene are "wicked problems" [5].

The Compendium of the social doctrine of the Catholic Church is poorly integrated with ecology and thus does not achieve what Pope Francis wishes it to achieve ([6], Section 4). However, the potential for a mobilizing "grand-narrative" lies there. In particular, the theological introduction to the chapter of the Compendium dealing with environment is sound but could be enriched with eco-theology whose fundamental message is that "the future of God and the future of the world are integrated" ([28], p. 311). The Christian "grand-narrative" sufficiently jibes with other religions' narratives to make a religious alliance possible ([36], pp. 70-80). In 2012, 1/3 of mankind was Christian and 84% of mankind allegedly belonged to some sort of religion [37]. As the Moslem religious membership is expected to be the religious group growing the fastest and reach nearly the same proportion as Christians in 2050, where the effort has to be placed is obvious. Moslem creation theology has many similarities with the Christian one [38]. Pope Francis seems to understand this [39]. Environmentally-motivated people might be able to join this religious coalition if skillfully assembled. Whether this grand narrative, while in competition with other narratives, will be sufficient to mobilize the remaining religious people towards the required ecological conversion and towards an outward-oriented "new evangelization" with an ecological dimension remains to be seen. The eco-theological grand narratives, while still in their infancy and in need of dissemination, are hopefully capable of mobilizing as were Teilhard de Chardin's writings.


*7.1. Performance Metrics*

Recall (*R*), Precision (*P*), F1-score (*F1*), specificity (*S*), and accuracy were used as the performance criteria to examine deep-learning performance.

• Precision: This metric represented the fraction of genuine positives among the expected positives. As a result, true-positive (*TP*) and false-positive (*FP*) values were important.

$$P = TP / (TP + FP) \tag{3}$$

• Recall: The ratio of true positives accurately categorized by the model was the recall. The recall was calculated using *TP* and *FN* values.

$$R = TP/(TP + FN) \tag{4}$$

• Specificity: This was defined as the proportion of true negatives (those not caused by illness) correctly classified by the model. The *TN* and *FP* values were used to calculate specificity.

$$S = TN/(TN + FP) \tag{5}$$

• F1-Score: The F1-score measured the model's accuracy by combining precision and recall. Doubling the ratio of the total accuracy and recall values defined the F1-scores.

$$F1 = 2 \times (P \times R) / (P + R) \tag{6}$$

• Performance (Speed): This was an important performance metric in image detection and data classification and clustering, particularly when dealing with large datasets and real-time applications. It measured the time required to process and analyze the data and produce the desired output. In image detection, speed is important for applications such as autonomous vehicles, surveillance systems, and medical imaging, where the detection and analysis of images must be performed in real-time. The speed metric is usually measured in frames-per-second (FPS), which represents the number of images that can be processed in one second. In data classification and clustering, speed is important for applications such as recommendation systems, fraud detection, and customer segmentation, where large amounts of data must be analyzed and classified in a timely manner. The speed metric is usually measured in terms of processing time or throughput, which represents the number of data points that can be processed per unit of time.


**2.3 Water budget reestablishment**

Type and configuration of GI can not only regulate the baseflow but also affect the rest of the water budget for a single site [71, 72]. Designing a GI unit, therefore, needs to be reviewed in a broader sense. The configuration of each GI unit, though possibly having already accomplished the local-scale objectives, can be further tweaked to target the optimum goal of a greater scale such as of an urban watershed or an urban ecosystem. Then, the baseflow regulation by GI implementations eventually turns into the redesign of the water budget, such as the proposals for restoring the near-natural water budget [24, 35, 73].

Targeting water budget, however, may not be so straightforward to develop due to considerations for the integrated ecosystem management for each specific climate. From the ecological perspective, aquifer recharge might be beneficial ecologically only when the recharge amount matches the predevelopment condition [60]. So, the excessive rainwater should be harvested near the rain source [24]. However, in dry environments, ET can be dominant component of the predevelopment water budget before urbanization occurred [35]. Recovering the predevelopment ET ratio will be prohibitive in such urban settings [24]. Therefore, reestablishing a new water budget somewhere between the predevelopment and postdevelopment conditions is most feasible and beneficial for human and ecosystem water demands together. Regional water budget should be determined by the weights assigned between human water demand and ecosystem water demand.

The new equilibrium will need to integrate multiobjectives from different perspectives. For example, for the interests in urban heat island relief, GI is designed to enhance ET process, which requires the ET-focused GI with adequate storage capacity [1, 74]. For the interests in stormwater management in wet and cold regions with excessive return flows, the ET-focused GI is recommended to maximize the runoff reduction. In semi-arid environments with intermittent but intense rain events, high ET rates also guarantee the rapid update of storage capacity between storms, though irrigation supplement may be needed [75]. For regions with low recharge rate and high groundwater exploitation rate, the percolation-focused GI with highly permeable mediums might be a better option [76, 77]. In any case when increasing irrigation demand is most concerned, GI with low ET potential or drought-resistant plant species would be preferred [78].


**2. UPS components in ovarian cancer**

Ovarian cancer is characterized by multiple genetic and epigenetic abnormalities and several major (about seven) activated signaling pathways, which are directly or indirectly implicated with UPS. Moreover, several UPS components, E1s, E2s, E3s, DUBs and proteasomes are known to be deregulated or mutated in cancer (**Table 1**), suggesting their role in cancer signaling and cancer progression. This section discusses each UPS component implicated in ovarian cancer and the role of key players of each component in regulating ovarian cancer signaling (**Figure 4**).

#### **2.1. E3 ligases**

E3 ligases (E3s) are the most heterogeneous class of enzymes in UPS as they facilitate ubiquitination with exquisite spatial, temporal, and substrate specificity. There are more than 600 E3s in a human genome, indicating the precise substrate specificity of E3s [15]. E3s can be classified into three main types, RING E3s, HECT E3s, and RBR E3s depending on the presence of type-specific domains and on the mechanism of ubiquitin transfer to the substrate protein. RING E3s are the most abundant type of ubiquitin ligases. They are characterized by the presence of zinc-binding domain called Really Interesting New Gene (RING) and U-box domain. RING E3s mediate a direct transfer of ubiquitin to substrate, functioning as a scaffold to orient the ubiquitin-charged E2, whereas E3s with homologous to the E6AP carboxyl

**Table 1.** Cancer-associated alterations in UPS.

enzymes (DUBs) are another class of enzymes, which removes or edits the ubiquitin chains attached to a protein, making this a highly reversible process and thus highlighting the dynamic regulation of ubiquitin signaling in the cell (**Figure 3**). These enzymes together with proteasomes, a cellular machinery involved in ubiquitin-mediated protein degradation, comprise the

**Figure 3.** Enzymatic cascade of ubiquitin proteasome system. Ubiquitin is activated and conjugated to target protein by a conserved action of E1-ubiquitin-activating enzyme, E2-ubiquitin-conjugating enzyme, and E3 ubiquitin ligase.

**Figure 2.** Linkage-specific ubiquitin chains of different topologies. Each circle represents one ubiquitin moiety. (A) Monoubiquitination, (B) multi-monoubiquitination, (C) K48-linked chain, (D) K63-linked chain, (E) branched chain, and

(F) mixed chain.

138 Ovarian Cancer - From Pathogenesis to Treatment

RING domain for a potent E3 ligase activity and interaction with E2 UbcH5 [14, 21, 22]. BRCA1 E3 ligase substrate specificity is believed to depend on its phosphorylation-dependent binding to proteins containing phospho-SXXF motif such as CtIP, BACH1, and ABRA1 through a phospho-peptide recognition domain (BRCT) [23, 24]. The strong relation between BRCA1 tumor suppressor properties and E3 ligase activity is evident from the clustering of missense mutations that predispose to cancer in the Zn2+-binding residues of BRCA1 RING finger domain crucial for its ubiquitin ligase activity [25]. The full range of function of BRCA1/ BARD1 complex is not completely understood [18]. One of the most important functions of BRCA1 is to repair DNA double-strand breaks (DSBs). Following DNA damage, chromatinassociated histone H2AX phosphorylation by ATM and ATR at DNA damage site recruits an E3 ubiquitin ligase RFN8 and a phospho-module-binding mediator MDC1 at the damage site [17, 26–28]. RFN8 together with ubiquitin conjugase Ubc13, ubiquitinate histone H2A and H2B at chromatin lesions, which in turn translocate BRCA1 complex containing RAP80, a protein with ubiquitin-interacting motif (UIM), ABRA1, protein that interacts with BRCA1 BRCT domain and deubiquitinating enzyme, BRCC36 to Lys6- and Lys63-linked polyubiquitin chains at DSBs [17, 26, 29]. BRCA1 has also been implicated with the transcriptional activation of genes in response to DNA damage. The C-terminus of BRCA1 complexes with RNA polymerase II through RNA helicase, while N-terminus BRCA1/BRAD1 heterodimer binds to RNA polymerase II holoenzyme [30]. Identifying genes regulated by BRCA1 would shed a significant light on the transcriptional role of BRCA1. However, BRCA1 overexpression studies have shown induction in p53-responsive E3 ligase, mdm2, cell-cycle inhibitor, p21 and stress-response factor, and GADD45 in breast and small-cell lung cancer cell lines [31, 32]. Besides, BRCA1 also regulates G1/S, S-phase, and G2/M cell-cycle checkpoints through

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Over the last 10 years, significant information has been gained about the structure, function, and unique features of BRCA gene products, BRCA1 and BRCA2, which collectively contributes to the biological response to DNA damage through homologous recombination of DNA repair and regulation of cell-cycle checkpoints. BRCA1/2-deficient cancers, including ovarian cancer, are now recognized as the target for a class of drugs known as PARP (poly ADPribose polymerase) inhibitors [34]. PARP detects and initiates an immediate cellular response to metabolic or radiation-induced single-strand DNA breaks (SSB). It binds to DNA and synthesizes polymeric adenosine diphosphate ribose (poly ADP-ribose or PAR), which acts as signal to other DNA-repairing enzymes. PARP inhibition directly blocks the PARP enzymatic activity and subsequently leads to PARP accumulation on DNA, a process called PARP trapping, which converts an SSB into a double-strand DNA break through the collapse of replication fork [34]. BRCA-deficient tumor cells with impaired homologous recombination repair of double-strand DNA breaks are directed toward the error-prone repair process of non-homologous end joining which leads to genetic instability and cell death. Thus, BRCA1/2-deficient ovarian cancer cells with PARP inhibition undergo synthetically lethal cell death [34]. PARP inhibitor, Olaparib manufactured by AstraZeneca, is in phase I/II clinical trials for BRCAdeficient high-grade serous ovarian cancer [34]. Olaparib-treated ovarian cancer patients with BRCA1/2 mutation had a progression-free survival of 11.2 months compared to 4.3 months of patients receiving placebo [35]. In summary, BRCA is an ideal example of E3 ubiquitin ligase

interactions with RAD3, ATM/ATR, and Chk1/Chk2 [26, 30, 33].

playing an essential role in ovarian cancer and its intervention.

**Figure 4.** Key players of each UPS component involved in regulating ovarian cancer signaling. (A and B) DUBs and E3 ligases as candidate genes in ovarian cancer, (C) proteasomal activity and inhibitors in ovarian cancer, and (D) regulation of ovarian cancer oncogenic signaling by UPS.

terminus (HECT) domain transfer ubiquitin to the substrate in a two-step process—ubiquitin is first transferred to a catalytic cysteine on E3 and then to the substrate. Based on their N terminus extensions, HECTs are further classified into three subfamilies: Nedd4 family, HERC family, and other HECT that contain various domains. The RBR E3s are characterized by the presence of three RING domains, RING1 and RING2, separated by an in-between-RING (IBR) domain. RING1 recruits the ubiquitin-charged E2, RING2 possess catalytic cysteine. The IBR is called benign-catalytic domain as it lacks catalytic cysteine residue [15]. Given their cellular specificity and complexity, E3s are implicated in a number of pathophysiological conditions, which makes them an attractive therapeutic target in human diseases, including cancer [16].

#### *2.1.1. BRCA1*

The breast and ovarian cancer susceptibility gene, BRCA1, is a tumor suppressor gene [17]. Heterozygous mutations in BRCA1 gene predispose women to both familial and sporadic breast and ovarian cancers [18, 19]. Nonetheless, BRCA1 mutations are also associated with other cancers like stomach, pancreas, prostate, and colon [20]. BRCA1 acts as a hub protein, which participates in several different protein complexes to coordinate a diverse range of cellular functions including DNA repair, cell-cycle regulation, apoptosis, transcriptional regulation, and centrosome duplication to maintain genomic stability [17]. The structural analysis of BRCA1 protein suggested that it has a RING finger domain that harbors E3 ubiquitin ligase activity [14]. In addition, BRCA1 forms a heterodimer complex with BARD1, a protein with a RING finger domain [14]. BARD1 interaction stabilizes the proper conformation of BRCA1 RING domain for a potent E3 ligase activity and interaction with E2 UbcH5 [14, 21, 22]. BRCA1 E3 ligase substrate specificity is believed to depend on its phosphorylation-dependent binding to proteins containing phospho-SXXF motif such as CtIP, BACH1, and ABRA1 through a phospho-peptide recognition domain (BRCT) [23, 24]. The strong relation between BRCA1 tumor suppressor properties and E3 ligase activity is evident from the clustering of missense mutations that predispose to cancer in the Zn2+-binding residues of BRCA1 RING finger domain crucial for its ubiquitin ligase activity [25]. The full range of function of BRCA1/ BARD1 complex is not completely understood [18]. One of the most important functions of BRCA1 is to repair DNA double-strand breaks (DSBs). Following DNA damage, chromatinassociated histone H2AX phosphorylation by ATM and ATR at DNA damage site recruits an E3 ubiquitin ligase RFN8 and a phospho-module-binding mediator MDC1 at the damage site [17, 26–28]. RFN8 together with ubiquitin conjugase Ubc13, ubiquitinate histone H2A and H2B at chromatin lesions, which in turn translocate BRCA1 complex containing RAP80, a protein with ubiquitin-interacting motif (UIM), ABRA1, protein that interacts with BRCA1 BRCT domain and deubiquitinating enzyme, BRCC36 to Lys6- and Lys63-linked polyubiquitin chains at DSBs [17, 26, 29]. BRCA1 has also been implicated with the transcriptional activation of genes in response to DNA damage. The C-terminus of BRCA1 complexes with RNA polymerase II through RNA helicase, while N-terminus BRCA1/BRAD1 heterodimer binds to RNA polymerase II holoenzyme [30]. Identifying genes regulated by BRCA1 would shed a significant light on the transcriptional role of BRCA1. However, BRCA1 overexpression studies have shown induction in p53-responsive E3 ligase, mdm2, cell-cycle inhibitor, p21 and stress-response factor, and GADD45 in breast and small-cell lung cancer cell lines [31, 32]. Besides, BRCA1 also regulates G1/S, S-phase, and G2/M cell-cycle checkpoints through interactions with RAD3, ATM/ATR, and Chk1/Chk2 [26, 30, 33].

Over the last 10 years, significant information has been gained about the structure, function, and unique features of BRCA gene products, BRCA1 and BRCA2, which collectively contributes to the biological response to DNA damage through homologous recombination of DNA repair and regulation of cell-cycle checkpoints. BRCA1/2-deficient cancers, including ovarian cancer, are now recognized as the target for a class of drugs known as PARP (poly ADPribose polymerase) inhibitors [34]. PARP detects and initiates an immediate cellular response to metabolic or radiation-induced single-strand DNA breaks (SSB). It binds to DNA and synthesizes polymeric adenosine diphosphate ribose (poly ADP-ribose or PAR), which acts as signal to other DNA-repairing enzymes. PARP inhibition directly blocks the PARP enzymatic activity and subsequently leads to PARP accumulation on DNA, a process called PARP trapping, which converts an SSB into a double-strand DNA break through the collapse of replication fork [34]. BRCA-deficient tumor cells with impaired homologous recombination repair of double-strand DNA breaks are directed toward the error-prone repair process of non-homologous end joining which leads to genetic instability and cell death. Thus, BRCA1/2-deficient ovarian cancer cells with PARP inhibition undergo synthetically lethal cell death [34]. PARP inhibitor, Olaparib manufactured by AstraZeneca, is in phase I/II clinical trials for BRCAdeficient high-grade serous ovarian cancer [34]. Olaparib-treated ovarian cancer patients with BRCA1/2 mutation had a progression-free survival of 11.2 months compared to 4.3 months of patients receiving placebo [35]. In summary, BRCA is an ideal example of E3 ubiquitin ligase playing an essential role in ovarian cancer and its intervention.

terminus (HECT) domain transfer ubiquitin to the substrate in a two-step process—ubiquitin is first transferred to a catalytic cysteine on E3 and then to the substrate. Based on their N terminus extensions, HECTs are further classified into three subfamilies: Nedd4 family, HERC family, and other HECT that contain various domains. The RBR E3s are characterized by the presence of three RING domains, RING1 and RING2, separated by an in-between-RING (IBR) domain. RING1 recruits the ubiquitin-charged E2, RING2 possess catalytic cysteine. The IBR is called benign-catalytic domain as it lacks catalytic cysteine residue [15]. Given their cellular specificity and complexity, E3s are implicated in a number of pathophysiological conditions, which makes them an attractive therapeutic target in human diseases, including cancer [16].

**Figure 4.** Key players of each UPS component involved in regulating ovarian cancer signaling. (A and B) DUBs and E3 ligases as candidate genes in ovarian cancer, (C) proteasomal activity and inhibitors in ovarian cancer, and (D) regulation

The breast and ovarian cancer susceptibility gene, BRCA1, is a tumor suppressor gene [17]. Heterozygous mutations in BRCA1 gene predispose women to both familial and sporadic breast and ovarian cancers [18, 19]. Nonetheless, BRCA1 mutations are also associated with other cancers like stomach, pancreas, prostate, and colon [20]. BRCA1 acts as a hub protein, which participates in several different protein complexes to coordinate a diverse range of cellular functions including DNA repair, cell-cycle regulation, apoptosis, transcriptional regulation, and centrosome duplication to maintain genomic stability [17]. The structural analysis of BRCA1 protein suggested that it has a RING finger domain that harbors E3 ubiquitin ligase activity [14]. In addition, BRCA1 forms a heterodimer complex with BARD1, a protein with a RING finger domain [14]. BARD1 interaction stabilizes the proper conformation of BRCA1

*2.1.1. BRCA1*

of ovarian cancer oncogenic signaling by UPS.

140 Ovarian Cancer - From Pathogenesis to Treatment

#### *2.1.2. Cullin-RING ligases: cullin 4*

Cullin-RING ubiquitin ligases (CRLs), composed of CUL1, 2, 3, 4A, 4B, 5, and 7, are the largest family of E3s that ubiquitinate a wide array of substrates involved in cell-cycle, DNAdamage response, chromatin remodeling, and gene expression. Cullin (CUL) neddylation, a process of adding ubiquitin-like protein—NEDD8 to the cullin [36], is crucial for their activation. Neddylation is catalyzed by NEDD8-activating enzyme E1 (NAE), NEDD8-conjugating enzyme E2 (UBC12), and NEDD8-E3 ligase. The genome-wide analysis of human cancers revealed *CUL4A* amplification in 20% of the basal-like breast cancer subtype, characterized as "triple negative," and CUL4A levels were associated with aggressive growth and poor prognosis. Dysregulation of CUL4A in multiple tumor types leads to the hypothesis that CUL4A plays a role in promoting oncogenesis [36]. High CUL4A expression and activity in ovarian cancer is implicated with cancer cell proliferation and survival. NEDD8-activating enzyme inhibitor, MLN4924, which blocks cullin neddylation activation, is reported to induce cellcycle arrest, apoptosis, and tumor cell growth in epithelial ovarian cancer cells.  They reported the co-amplification of USP13 gene with PIK3CA (phosphatidylinositol-3-kinase catalytic subunit, α-isoform) in 29.3% of high-grade serous ovarian cancer patients and its association with poor clinical outcome. USP13 stabilized the protein levels of two key metabolic enzymes, ATP citrate lyase and oxoglutarate dehydrogenase, which in turn regulate the mitochondrial respiration, glutaminolysis, and fatty acid synthesis in ovarian cancer cells. USP13 inhibition suppressed ovarian tumor progression and sensitized the tumor cells to PI3K/AKT inhibitor [41]. Similarly, USP7 (also known as HAUSP, herpes virus-associated ubiquitin protease) plays a crucial role in ovarian cancer [42]. USP7 is a DUB for MDM2, which prevents MDM2 autoubiquitination, leading to its stabilization and consequent induction of p53 degradation. Treating an ovarian cancer xenograft model with a novel inhibitor of USP7, CDDO-Me suppressed tumor growth. CDDO-Me directly binds to USP7, which leads to a decrease in its substrate Mdm2, Mdmx protein levels [42]. USP4 overexpression is reported in invasive breast carcinoma, enhancing TGFβ signaling by stabilizing SMAD2/SMAD4 complex but not much is known about its role in ovarian cancer [11]. USP36 expression is increased in ovarian cancer cells compared to normal ovarian surface epithelium; however, further studies are needed to understand its role in ovarian cancer [43]. DUB UCHL1 (ubiquitin-carboxyl terminal hydrolase 1) plays a contradicting role in different cancers [11]; it is reported as a methylated tumor suppressor gene in ovarian cancer [44, 45], while it is overexpressed in lymphoma, esophageal squamous cell carcinoma, renal, lung cancers, and acts as an oncogene [46–48]. Under hypoxic conditions, UCHL1 is shown to deubiquitinate and stabilize HIF-1α and promote tumor metastasis [49, 50]. We for the first time identified the oncogenic overexpression of UCHL1 in high-grade serous ovarian cancer and association with poor clinical outcome (unpublished data). These studies suggest the emerging role of DUBs in ovarian cancer and the potential of DUB inhibi-

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The efficient and selective degradation of cellular proteins is essential for protein quality control and maintenance of cellular homeostasis [51]. Impaired protein quality control and degradation is associated with many human diseases such as cancer, cardiovascular diseases, and aging-related pathophysiological conditions such as Alzheimer's and Parkinson's. UPS mediates targeted protein degradation under both normal and malignant conditions [52]. However, cancer cells are more dependent on UPS-mediated degradation to promote the degradation of tumor suppressors and various cell-cycle checkpoint proteins as well as to reduce proteotoxic stress accumulated due to genomic aberrations [53]. The 26S proteasome is a multi-subunit complex that contains one barrel-shaped 20S catalytic core particle (CP) and 19S regulatory particle (RP) that binds to one or both the ends of barrel-shaped CP. The active degradation of proteins is regulated by 20S CP harboring proteolytic active sites while 19S RP regulates substrate binding and target protein entry

The amazing efficacy and clinical use of proteasome inhibitor Bortezomib (PS-341, Velcade) for the treatment of multiple myeloma and mantle cell lymphoma has encouraged researchers to explore the possibility of targeting other components of the UPS for cancer treatment [54]. However, Bortezomib has not demonstrated a significant activity against other solid

tors in neo-adjuvant therapies for ovarian cancer.

**2.3. Proteasomes**

into 20S [52].

The role of Skp2 and FBXW7 in ovarian cancer signaling is discussed in the next section.

#### **2.2. Deubiquitinating enzymes**

Reversibility is an important aspect of ubiquitin system, which is mediated by deubiquitinating enzymes or deubiquitinases (DUBs). DUBs are essential components of UPS that possess ubiquitin-isopeptidase activity and catalyze the removal of ubiquitin from the target proteins. Thus, DUBs play a crucial role in the regulation of ubiquitin-mediated regulatory and proteolytic signaling [11, 38]. DUBs activity affect the activation, recycling, localization, and turnover of multiple proteins, which in turn regulate cellular homeostasis, protein stability, and a wide range of signaling pathways [39]. DUBs also maintain ubiquitin homeostasis in the cell by generating free ubiquitin monomers, which is essential for ubiquitin-mediated regulation of cell function [38]. Consistent with this, an altered DUB expression or activity has been implicated with several diseases including cancer. Numerous DUBs have been characterized as oncogenes mediating cancer initiation and progression [11, 40]. Therefore, pharmacological interventions targeting DUB activity using small molecule inhibitors are being used as a rationale to search for novel anticancer drugs [11].

**Box 2.** About 98 DUBs are reported in human genome, which are mainly divided into five families based on their sequence and structural homology: Ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado Joseph disease proteases (MJD), and JAB1/MPN/Mov34 (JAMM) metallopeptidases. Most DUBs are cysteine proteases except JAMMs, which belong to catalytic class of metalloproteases. The recent discovery of DUBs with the selectivity of cleaving extended Lys-48-linked polyubiquitin chains belongs to new family of DUBs named Mindy. The DUB-substrate specificity somewhat depends on ubiquitin chain linkage and topology; however, by large, given the complexity of ubiquitin system, it remains unknown [38].

DUBs role is evident in several cancers including Fanconi anemia (USP1), prostate cancer (USP2), adenocarcinoma (USP4), non-small-cell lung carcinoma (USP7), glioblastoma (USP15), myeloma, and leukemia (USP9x) [11, 39]. Han et al. identified the role of USP13 as the master regulator of ovarian cancer metabolism [41]. They reported the co-amplification of USP13 gene with PIK3CA (phosphatidylinositol-3-kinase catalytic subunit, α-isoform) in 29.3% of high-grade serous ovarian cancer patients and its association with poor clinical outcome. USP13 stabilized the protein levels of two key metabolic enzymes, ATP citrate lyase and oxoglutarate dehydrogenase, which in turn regulate the mitochondrial respiration, glutaminolysis, and fatty acid synthesis in ovarian cancer cells. USP13 inhibition suppressed ovarian tumor progression and sensitized the tumor cells to PI3K/AKT inhibitor [41]. Similarly, USP7 (also known as HAUSP, herpes virus-associated ubiquitin protease) plays a crucial role in ovarian cancer [42]. USP7 is a DUB for MDM2, which prevents MDM2 autoubiquitination, leading to its stabilization and consequent induction of p53 degradation. Treating an ovarian cancer xenograft model with a novel inhibitor of USP7, CDDO-Me suppressed tumor growth. CDDO-Me directly binds to USP7, which leads to a decrease in its substrate Mdm2, Mdmx protein levels [42]. USP4 overexpression is reported in invasive breast carcinoma, enhancing TGFβ signaling by stabilizing SMAD2/SMAD4 complex but not much is known about its role in ovarian cancer [11]. USP36 expression is increased in ovarian cancer cells compared to normal ovarian surface epithelium; however, further studies are needed to understand its role in ovarian cancer [43]. DUB UCHL1 (ubiquitin-carboxyl terminal hydrolase 1) plays a contradicting role in different cancers [11]; it is reported as a methylated tumor suppressor gene in ovarian cancer [44, 45], while it is overexpressed in lymphoma, esophageal squamous cell carcinoma, renal, lung cancers, and acts as an oncogene [46–48]. Under hypoxic conditions, UCHL1 is shown to deubiquitinate and stabilize HIF-1α and promote tumor metastasis [49, 50]. We for the first time identified the oncogenic overexpression of UCHL1 in high-grade serous ovarian cancer and association with poor clinical outcome (unpublished data). These studies suggest the emerging role of DUBs in ovarian cancer and the potential of DUB inhibitors in neo-adjuvant therapies for ovarian cancer.

#### **2.3. Proteasomes**

*2.1.2. Cullin-RING ligases: cullin 4*

142 Ovarian Cancer - From Pathogenesis to Treatment

**2.2. Deubiquitinating enzymes**

rationale to search for novel anticancer drugs [11].

Cullin-RING ubiquitin ligases (CRLs), composed of CUL1, 2, 3, 4A, 4B, 5, and 7, are the largest family of E3s that ubiquitinate a wide array of substrates involved in cell-cycle, DNAdamage response, chromatin remodeling, and gene expression. Cullin (CUL) neddylation, a process of adding ubiquitin-like protein—NEDD8 to the cullin [36], is crucial for their activation. Neddylation is catalyzed by NEDD8-activating enzyme E1 (NAE), NEDD8-conjugating enzyme E2 (UBC12), and NEDD8-E3 ligase. The genome-wide analysis of human cancers revealed *CUL4A* amplification in 20% of the basal-like breast cancer subtype, characterized as "triple negative," and CUL4A levels were associated with aggressive growth and poor prognosis. Dysregulation of CUL4A in multiple tumor types leads to the hypothesis that CUL4A plays a role in promoting oncogenesis [36]. High CUL4A expression and activity in ovarian cancer is implicated with cancer cell proliferation and survival. NEDD8-activating enzyme inhibitor, MLN4924, which blocks cullin neddylation activation, is reported to induce cellcycle arrest, apoptosis, and tumor cell growth in epithelial ovarian cancer cells. In addition,

MLN4924 sensitized ovarian cancer cells to chemotherapeutic drug treatments [37].

The role of Skp2 and FBXW7 in ovarian cancer signaling is discussed in the next section.

Reversibility is an important aspect of ubiquitin system, which is mediated by deubiquitinating enzymes or deubiquitinases (DUBs). DUBs are essential components of UPS that possess ubiquitin-isopeptidase activity and catalyze the removal of ubiquitin from the target proteins. Thus, DUBs play a crucial role in the regulation of ubiquitin-mediated regulatory and proteolytic signaling [11, 38]. DUBs activity affect the activation, recycling, localization, and turnover of multiple proteins, which in turn regulate cellular homeostasis, protein stability, and a wide range of signaling pathways [39]. DUBs also maintain ubiquitin homeostasis in the cell by generating free ubiquitin monomers, which is essential for ubiquitin-mediated regulation of cell function [38]. Consistent with this, an altered DUB expression or activity has been implicated with several diseases including cancer. Numerous DUBs have been characterized as oncogenes mediating cancer initiation and progression [11, 40]. Therefore, pharmacological interventions targeting DUB activity using small molecule inhibitors are being used as a

DUBs role is evident in several cancers including Fanconi anemia (USP1), prostate cancer (USP2), adenocarcinoma (USP4), non-small-cell lung carcinoma (USP7), glioblastoma (USP15), myeloma, and leukemia (USP9x) [11, 39]. Han et al. identified the role of USP13 as

**Box 2.** About 98 DUBs are reported in human genome, which are mainly divided into five families based on their sequence and structural homology: Ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado Joseph disease proteases (MJD), and JAB1/MPN/Mov34 (JAMM) metallopeptidases. Most DUBs are cysteine proteases except JAMMs, which belong to catalytic class of metalloproteases. The recent discovery of DUBs with the selectivity of cleaving extended Lys-48-linked polyubiquitin chains belongs to new family of DUBs named Mindy. The DUB-substrate specificity somewhat depends on ubiquitin chain linkage and topology; however, by large, given the complexity of ubiquitin system, it remains unknown [38].

The efficient and selective degradation of cellular proteins is essential for protein quality control and maintenance of cellular homeostasis [51]. Impaired protein quality control and degradation is associated with many human diseases such as cancer, cardiovascular diseases, and aging-related pathophysiological conditions such as Alzheimer's and Parkinson's. UPS mediates targeted protein degradation under both normal and malignant conditions [52]. However, cancer cells are more dependent on UPS-mediated degradation to promote the degradation of tumor suppressors and various cell-cycle checkpoint proteins as well as to reduce proteotoxic stress accumulated due to genomic aberrations [53]. The 26S proteasome is a multi-subunit complex that contains one barrel-shaped 20S catalytic core particle (CP) and 19S regulatory particle (RP) that binds to one or both the ends of barrel-shaped CP. The active degradation of proteins is regulated by 20S CP harboring proteolytic active sites while 19S RP regulates substrate binding and target protein entry into 20S [52].

The amazing efficacy and clinical use of proteasome inhibitor Bortezomib (PS-341, Velcade) for the treatment of multiple myeloma and mantle cell lymphoma has encouraged researchers to explore the possibility of targeting other components of the UPS for cancer treatment [54]. However, Bortezomib has not demonstrated a significant activity against other solid tumors [55]. This conundrum has spurred the development of next-generation proteasome inhibitors, including MLN9708 (Millennium Pharmaceuticals), Carfilzomib and ONX0912 (Onyx Pharmaceuticals, South San Francisco, CA), and CEP18770 (Cephalon, Frazer, PA) [56]. Although these compounds target the same 20S CP, they differ in targeted active site and enzyme kinetics, resulting in activity differences based on tumor type and tumor location. Bazzaro et al. reported elevated levels of ubiquitinated proteins and 19S and 20S proteasome subunits in both low-grade and high-grade ovarian carcinoma tissues and cell lines compared to benign ovarian tumors and immortalized normal ovarian surface epithelium controls. They reported an increased sensitivity to apoptosis in proteasome inhibitor, PS-341 treated cells, and a reduced growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice [57]. In a similar study, proteasome inhibitor, MG132—a peptide aldehyde—showed an enhanced sensitivity of ovarian cancer cells, SKOV3 to cisplatin both *in vitro* and *in vivo* [58]. The effect of Bortezomib on ovarian cancer cells is also supported by the increased sensitivity of Bortezomib-treated chemoresistant ovarian cancer cells to TRAIL-induced apoptosis [59]. Together, these results indicate the essential role of proteasomes in mediating prosurvival signaling in cancer, which may also be due to altered proteasome composition resulting in an enhanced proteasomal activity [52].

have been reported in many cancers [63]. In addition, the expression and activity of Usp7, a deubiquitinating enzyme for Mdm2, is increased in several cancers including breast and ovarian cancer, which prevents Mdm2 ubiquitination and promotes its stability. Reduced tumor growth was seen in an ovarian cancer xenograft model treated with Usp7 inhibitor [42]. On the other hand, when p53 acquires gain-of-function (GOF) mutations as in the case of nearly half of the cancers, it gains oncogenic functions and loses its wild-type tumor suppressor properties. Thus, in these cancer cells, several mechanisms stabilize mutant p53 through its activation or by inhibition of its degradation by disrupting Mdm2 and mutant p53 binding. Several splice variants of Mdm2 are reported in cancer, which lack a p53-binding domain and thus stabilizes mutant p53 expression [63]. In addition, GOF mutation-induced conformational changes in mutant p53 allow the binding of Hsp90 (heat shock protein 90) to mutant p53, which prevents Mdm2 binding and Mdm2-mediated degradation of mutant p53 [60]. It is now well established that elevated mutant p53 levels correlate with more aggressive tumors and poor prognosis. About 96% of high-grade serous ovarian cancer patients have GOF p53 mutations, which orchestrate a distinct pro-tumorigenic transcription and oncogenic programs. Knowledge of a UPS component responsible for mutant p53 stabilization, which could be chemically manipulated, will be useful in HGSOC. Nonetheless, Mdm2 is a great therapeutic target and prognostic factor for ovarian cancer with wild-type p53, such as

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Genomic alterations in cell-cycle regulatory genes have been reported in almost every human carcinoma. Cyclins are the crucial regulators of cell-cycle progression [65]. A periodic increase in cyclin levels and their timed interplay with cyclin-dependent kinases (CDKs) is essential for the proper progression of cell cycle [65]. Their levels are regulated by a combination of transcription and ubiquitin-mediated degradation [18, 66]. About 30% of high-grade serous ovarian cancer patients have amplification of the CCNE1 gene, which encodes for G1/Sspecific cyclin E. Cyclin E-CDK2 interactions commit the cell to S-phase genome duplication [3]. Aberrant accumulation and overabundance of cyclin E leads to premature entry of the cell into S-phase, resulting in chromosome instability and tumor formation [67]. Cyclin E amplification is likely to be an early event in the development of high-grade serous ovarian cancer [3]. This subclass of patients has no apparent defect in homologous recombination as seen in patients with BRCA1 and BRCA2 mutations with defect in DNA repair pathways [3]. The overexpression of cyclin E is an indicator of poor overall survival of ovarian cancer patients. Cyclin E protein levels are maintained by a multi-subunit SCF ubiquitin ligase, which mediates its ubiquitination and degradation [68]. Cyclin E auto-phosphorylation after its association with CDK2 is recognized by the SCF-associated F-box protein 7 (FBXW7), which binds to cyclin E and facilitates its ubiquitination and degradation [68, 69]. More than 30% of human cancers have a deleted FBXW7 gene located on chromosome 4q32. FBXW7 also regulates mTOR, Myc, and Notch1 degradation, depending upon the type of tumor [70, 71]. FBXW7 is known to be mutated in breast and ovarian cancer cell lines with high cyclin E levels [3]. The loss of cyclin E or CDK2 results in cell-cycle arrest or apoptosis in HGSOC cell lines [3], sug-

gesting cyclin E inhibition as a novel therapeutic approach in ovarian cancer patients.

clear-cell carcinomas [64].

**3.2. Cyclin E**


**2. Vascular endothelial growth factors**

Vascular endothelial growth factor (VEGF) plays a key role in ocular angiogenesis and vas‐ cular permeability. Several VEGF family members have been discovered (VEGF-A, B, C, D

and PIGF). These isoforms of VEGF have different effects in ocular pathologies and may dif‐ fer in their neuroprotective abilities [5, 6]. RPE and Müller cells are the major sources of VEGF and they exert their effects through multiple receptors that are mostly expressed on endothelial cells and are also found on monocytes and macrophages [7].

trolled trials comparing the two most commonly drugs available were published recently. A summary of the available drugs (table 1), their mechanism of action and results from large

Anti VEGF Agents for Age Related Macular Degeneration

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This intravitreal RNA aptamer drug was the first anti-VEGF drug approved by the FDA in 2004 for use in neovascular AMD (nvAMD). It targets VEGFA-165[11]Its efficacy and safety

Patients with different types of sub foveal CNV secondary to AMD were randomized into four groups. Three groups received an intravitreal injection of pegaptanib sodium at a dose of 0.3mg, 1.0mg, 3.0mg to one eye respectively. The injection was given every 6 weeks for a period of 48 weeks in total. The forth group was the control group and subjects in this group received a sham injection every 6 weeks. Primary outcome was mean change in visual acui‐

Results from a combined analysis showed that for all three doses of pegaptanib (P<0.001 for the comparison of 0.3 mg with sham injection; P<0.001 for the comparison of 1.0 mg with sham injection; and P=0.03 for the comparison of 3.0 mg with sham injection) there was a significant difference between the patients receiving treatment and those receiving a sham injection. In the group dosed with pegaptanib 0.3 mg, 70 percent of patients lost fewer than 15 letters of visual acuity (VA), as compared with 55 percent among the controls (P<0.001). The risk of severe loss of VA (loss of 30 letters or more) was reduced from 22 percent in the sham-injection group to 10 percent in the group receiving 0.3 mg of pegaptanib (P<0.001). More patients receiving pegaptanib (0.3 mg), as compared with sham injection, maintained their VA or gained acuity (33 % vs. 23%; P=0.003). As early as six weeks after beginning ther‐ apy with the study drug, and at all subsequent points, the mean visual acuity among pa‐ tients receiving 0.3 mg of pegaptanib was better than in those receiving sham injections (P<0.002). During the second year, patients initially assigned to pegaptanib were re-random‐ ized (1:1) to continue or discontinue therapy for 48 additional weeks (8 injections). Those ini‐ tially assigned to sham were re-randomized to continue sham, discontinue sham, or receive 1 of 3 pegaptanib doses. The proportion of patients who lost more than 15 letters or more in vision between week 52 to week 96 was double (14 *vs* 7%), if treatment was discontinued compared to those who continued to receive pegaptanib injections. This suggests that there is a more favorable outcome when continuing treatment for at least two years [18].The Pe‐ gaptanib was found safe and there was no significant difference in serious systemic adverse events or severe ocular inflammation, cataract or glaucoma between the pegaptanib treated

The VA results of the VISION study are clearly inferior to those of the MARINA and AN‐ CHOR studies evaluating the efficacy of intravitreal ranibizumab for nvAMD (detailed lat‐

multicenter trials evaluating their efficacy and safety is presented below.

**3. Anti VEGF drugs**

ty from baseline.

**3.1. Pegaptanib sodium (Macugen) – OSI/Eyetech**

were evaluated in the large VISION trial [16, 17].

groups and the sham treated groups [16, 17].

VEGF-A, has been most strongly associated with angiogenesis and thus consists the target of most anti-VEGF treatments [8, 9]. VEGF-A signals through two receptor tyrosine kinases, VEGFR1 and VEGFR2, and is induced by hypoxia, unlike other VEGF isoforms [7, 10].

Alternative exon splicing of the human VEGF-A gene results in at least four major biologi‐ cally active isoforms, containing 121, 165, 189, and 208 aminoacids (five more are VEG‐ FA-145, VEGFA-162, VEGFA-165b, VEGFA-183, and VEGFA-206) [11].

Different VEGF-A isoforms may have different functions in ocular diseases.

VEGF 121 appears to be essential for normal retinal vascular function [12-13], and VEG‐ FA-165 is the predominant isoform in the human eye. It isa heparin-binding, homodimeric, 45-kDa glycoprotein that is predominantly secreted, although a substantial fraction is bound to the cell surface and to the extracellular matrix[13-14].It appears to be the isoform respon‐ sible for pathological ocular neovascularization.

Both isoforms are found in CNV tissue excised from patients with AMD.

In autopsy studies, VEGF levels were found to be elevated in the retinal pigment epithelium (RPE) and choroidal blood vessels within the macular area of eyes with AMD [15].

In summary VEGF-A acts through various pathways which result in promoting pathologic neovascularization:

Several anti-VEGF drugs have been studied and have been shown to be effective. However, effective, long-term drug-delivery remains a challenge. Two multi-center, randomized con‐ trolled trials comparing the two most commonly drugs available were published recently. A summary of the available drugs (table 1), their mechanism of action and results from large multicenter trials evaluating their efficacy and safety is presented below.