Apoptosis

Programmed cell death or apoptosis is an important physiological process in multicellular organisms. The equilibrium between cell growth and division and the rate at which cells undergo cell death allows for dynamic adjustment of the cell number depending on internal or external parameters. For example, during the development of the vertebrate nervous system about half of the cells undergo apoptosis shortly after they have been formed. In an adult organism, this equilibrium is essential to maintain for example the size and function of organs and tissues. Dysregulation of this equilibrium oftentimes leads to cancer. Unlike necrosis which causes a potentially damaging inflammatory response after affected cells burst, apoptosis unfolds in a very organized way: the cell shrinks and condenses while the internal structures are disassembled and the DNA is fragmented. The dying cell is then rapidly phagocytosed by neighboring cells or macrophages.

At the core of the apoptotic process are caspases, a family of cysteine proteases. They are produced as pro-caspases which are rendered active subsequently to cleavage by other caspases. This caspase cascade is triggered when initiator procaspases (e.g. procaspases 8, 9, 10) are aggregated with the help of adaptor proteins, thus facilitating mutual activation due to low protease activity or conformational changes of the procaspases. The activated caspases are then free to activate effector caspases (e.g. caspases 3, 6, and 7) and promote apoptosis. Their effect is further regulated by Bcl-2 family proteins (e.g. Bcl-2, Bcl-xL) and IAPs (inhibitors of apoptosis, e.g. BIRC1, XIAP).

Apoptotic processes follow several pathways. Extrinsic death receptor pathways are induced through ligands that bind to a family of death receptor proteins (e.g. the FAS and TRAIL receptors) containing a cytoplasmic death domain. The intrinsic pathway is engaged in response to DNA damage or mitochondrial stress and is particularly relevant in cancer. Besides these canonical apoptotic pathways there are also caspase independent pathways, triggered e.g. by granzyme B and A. These caspase independent pathways are thought to have evolved in response to viruses that inhibit caspases.

Cell Division Cycle

All organisms (single and multi-cellular) rely upon reiteration of growth and division of existing cells under favorable conditions. During each round of this process, the cell cycles through an ordered series of events in which its genetic information is duplicated and then divided among two daughter cells. These events are tightly regulated and certain checkpoints must be passed in order for the cell cycle to be completed. The loss of control of these processes is a hallmark of cancer.

The cell cycle is split up into four major phases based on the events unfolding in the cell. During the first gap phase (G1) the proteins are produced that are essential for DNA replication. The genetic information is then being replicated during the synthesis (S) phase. In the second gap phase (G2) all the components that are necessary for the separation of the duplicated DNA during the subsequent mitosis (M phase). Cells that are not actively dividing are considered to be in a quiescent state in the resting phase G0.

Cell division is necessary for life, but unregulated division is dangerous and counterproductive. Therefore, the cell has an impetus for maintaining tight regulatory control over how and when division takes place. There are two major checkpoints in the cell cycle. The first choke-point the cell must pass is between the G1 and S phase. A second opportunity for arrest is provided between G2 and M phases. Once DNA replication has been initiated, it must be finished. Therefore, the major checkpoint in the cell cycle is the restriction (R) point between G1 and S phases, prior to DNA duplication, at which the cycle progresses depending on mitogenic or inhibitory factors such as DNA damage and signals from various signaling pathways.

Progression from one phase to another is controlled by cyclin dependent kinases (CDK) and their activators, cyclins. Latter proteins are unstable and their cellular concentration cycles throughout the cell cycle. Accordingly, specific cyclin-CDK complexes persist in an active form for a very short period of time after translation, and are then degraded or inactivated by the time that particular phase of the cell cycle completes. Activation of specific cyclin-CDK complexes are characteristic for the different cell cycle phases, and drive production of specific molecules associated with, and necessary for that phase of division. Additional levels of dynamic control are also provided by CDK inhibitors, which block CDK function even in the presence of their requisite cyclin.

Complement System

The complement system is part of the innate immune system and plays an important role in the host defense, inflammation, tissue regeneration and other physiological processes. Complement activation results in opsonization of pathogens and their removal by phagocytes. It also causes chemotactic attraction of phagocytes and macrophages. Furthermore, the complement system forms the terminal attack complex (MAC), a membrane channel causing osmotic lysis of the respective pathogen. While complement is not adaptable it does complement the adaptive immune system and it is also involved in B and T cell response regulation.

Activation of complement unfolds along three different complement activation pathways depending on the nature of the pathogen: the classical pathway, the lectin pathway, and the alternative pathway. All three converge into the common terminal pathway that leads to the formation of the MAC. In addition, anaphylatoxins C3a and C5a elicit a plethora of physiological responses that range from chemoattraction to apoptosis. The complement system consists of more than 30 proteins that are either present as soluble proteins in the blood or as membrane-associated proteins. Most exist as inactive zymogens that are then sequentially cleaved and activated. The central component in all three pathways is component C3, the most abundant complement protein found in the blood. Its activation induces the formation of the activation products C3a, C3b, and C5a and ultimately the MAC.

In addition to these three established pathways, it has been shown that factors such as kallikrein, plasmin, thrombin, and factor XIIa activate the complement system independently of the C3 protein.

DNA Damage Repair

DNA is the carrier of the genetic information that defines any living being. The genetic code fixed in DNA is crucial for processes on a subcellular scale up to the appearance and function of the organism as s whole. Nonetheless, DNA is constantly exposed to insults from endogenous sources such as hydrolysis, oxidation, alkylation, or replication errors. In addition, ionizing radiation, UV radiation, and a plethora of chemical reagents are external factors that threaten the integrity of DNA.

Unlike RNA and proteins, DNA is not being degraded and re-synthesized upon damage. Instead, various repair pathways are in existence to assure that the DNA remains intact. Francis Crick noted in 1974 that “we totally missed the possible role of enzymes in [DNA] repair. I later came to realize that DNA is so precious that probably many distinct mechanisms could exist.”

This presage holds true today: over a hundred genes have been characterized since that are involved in an intricate network of DNA repair pathways. DNA damage can be repaired via six different pathways depending on the nature of the lesion: chemical modifications, misincorporated nucleotides, and cross-links are reverted through direct reversal (DR), mismatch repair (MMR), and nucleotide excision repair mechanisms. DNA single strand breaks are being mended via base excision repair. Highly mutagenic DNA double strand breaks finally are repaired through a number of complex pathways that rely on homologous recombination (HR) with the sister chromatid (in the S or G2 phase of the cell cycle) or non-homologous end-joining (NHEJ) of both ends of the double strand break. In case a DNA lesion cannot be repaired in time, specialized DNA polymerases enable trans-lesion synthesis (TLS) in order to prevent the DNA replication fork from stalling. Mutations that render components of these repair pathways non-functional lead to diseases such as xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, and a predisposition for cancer.

Besides, these repair mechanisms are of high interest for current targeted genome editing approaches that typically take advantage of the cellular DNA repair machinery.

Hedgehog Signaling

Hedgehog (Hh) signaling is highly conserved across chordates of different taxonomical classification. It is essential for development of the embryo. The Hedehog pathway was first described and characterized in the fruit fly, Drosophila melanogaster. In mammals, Hh signaling regulates cell-fate, tissue polarity, and patterning during early embryogenesis and the morphogenesis of specific organs and tissues. It is subsequently silenced in most adult tissues but can be reactivated following injury to promote repair and regeneration.

Different Hh ligands, including homologs sonic hedgehog (SHH), indian hedgehog (IHH), and desert hedgehog (DHH) are produced as precursors that undergo autocatalytic cleavage, C-terminal cholesterol attachment, and N-terminal palmitoylation prior to secretion. Release and extracellular accumulation of the mature ligands is regulated by the homologs of the Drosophila dispatched (Disp) protein. Binding of secreted Hh ligands to the receptor homolog (PTCH1, PTCh3) triggers the stimulation of the signaling network: the repressive effect of PTCH on the transmembrane receptor SMO is relieved which leads to the activation of glioma-associated oncogene (GLI) transcription factors.

A key specialized structure in this process is the microtubule-based primary cilium. In the absence of the Hh signal unprocessed, non-activating GLI proteins as well as their regulator SUFU are concentrated in the distal tip of the primary cilium. Upon binding of the Hh ligand, PTCH relocates to the cell surface, thus rendering translocation of SMO to the primary cilium and the down-regulation of SUFU possible. Several structurally essential primary cilium proteins have also regulatory effects on the Hh signaling cascade.

Aberrant Hh/GLI regulation leads to major tissular disorders and the development of a wide variety of aggressive cancers. The Hh/GLI cascade has also been linked to the regulation of stemness genes and the survival of cancer stem cells.

JAK-STAT Signaling

JAK-STAT signaling in vertebrates relies on a network of protein kinases and transcription factors to integrate signals from various receptor systems. The multitude of stimuli include cytokines, growth factors, and hormones, binding of which ultimately effect processes such as immune response regulation and cell growth, survival, and differentiation.

The highly conserved pathway involves essentially three levels of processing of the incoming information depending on the function of the respective components:

• Binding of the ligand to the receptor triggers conformational changes of the receptor molecule(s).
• This steric change of the receptor brings two Janus Kinases (JAK) bound to the receptor or receptor subunits into close proximity, thus enabling trans-phosphorylation. The activated JAKs phosphorylate subsequently additional targets.
• The major phosphorylation targets are Signal transducer and activator of transcription (STAT). These transcription factors are inactive in the cytoplasm until phosphorylation by JAKs. Once the a conserved tyrosine toward the C-terminus of the STAT has been phosphorylated, it can acts as dimerization interface in conjunction with Sh3 domains of another STAT. These activated STAT dimers are then translocated to the nucleus and bind to specific DNA motifs to activate target gene transcription.

Besides, negative regulation of these processes takes place on multiple levels:

• Suppressors of cytokine signaling (SOCS) gene transcription is stimulated by activated STATs. SOCS inactivate signaling through binding to the phosphorylated JAKs or receptors or facilitate JAK ubiquitination.
• Protein inhibitor of activated STATs (PIAS) bind to activated STATs and prevent them from binding DNA.
• Protein tyrosine phosphatases (PTP) reverse the activity of JAKs.

The prototypical JAK-STAT signaling pathway is rather linear. There is however considerable crosstalk with other signaling cascades like MAPK pathways and JAK independent STAT phosphorylation through receptor tyrosine kinase (RTKs).

MAPK Signaling

Phosphorylation is the reversible process of attaching a phosphate group to a specific amino-acid residue on a protein. Functionally, phosphorylation acts as a simple molecular switch that can activate, deactivate, or modulate the function of a protein. Addition and removal of phosphate groups provide spatial and temporal control over protein activity. Phosphorylation is tightly controlled by a competing interdependent network of kinases - which donate phosphate groups to a substrate protein, and phosphorylases - which remove them from a substrate. Mitogen-activated protein kinases (MAPKs) are a highly conserved and ubiquitously expressed family of enzymatic kinases that phosphorylate many different target substrates. MAP-kinases are part of a larger, tiered phosphorylation cascade that includes MAP2Ks and MAP3Ks. This tiered organization affords flexibility, allowing a broad range of higher-order kinases to respond to stimuli and control cellular function through activation of a smaller subset of MAP-kinases that interact directly with other functional proteins. MAP-kinases play a major role in nearly every cellular process. MAPK dependent phosphorylation is implicated in signaling cascades that regulate cell-cycle progression, differentiation, development, and apoptosis. (Additional Reading: Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH (April 2001). "Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions". Endocr. Rev. 22 (2): 153–83.)

Microtubule Dynamics

Microtubules are highly-dynamic structural and functional intracellular highways. The term cytoskeleton conjures up an image of rigid, immutable, and permanent structure. In reality, the cytoskeleton is a highly dynamic interconnected network composed of three different principal components: microtubules (polymerized tubulin dimers), microfilaments (polymerized actin), and intermediate filaments. Microtubules are the largest cytoskeletal components. They play a role in nearly every cellular process. They give structural stability and form to an otherwise amorphous cell. They are the principal components in an interconnected intracellular “highway” by which all manner cellular cargo may be shuttled via a bustling network of molecular motor proteins. During mitosis, microtubule contraction, focused at one of two mitotic spindles, provides the force necessary to divide chromosomes.

The ever changing nature of the cellular microenvironment necessitates adaptability, particularly during development. Microtubules and other cytoskeletal components facilitate this adaptability by virtue of their polymeric structure. The fundamental unit of the microtubule is a tubulin dimer, composed of one alpha tubulin-subunit, and one corresponding beta-tubulin subunit. These dimers polymerize to form a hollow, tubular structure approximately 24nm wide. The natural state of unmodified tubulin is one of constant flux, with a nearly equal rate of polymerization and depolymerization. Several common GTPase families like Rac and Rho promote microtubule assembly indirectly by facilitating GDP/GTP exchange on regulatory members that effect assembly and disassembly of tubulin dimers. Other microtubule stabilizers take a more direct approach. Microtubule associated proteins like MAP1, MAP2, MAP4, or MAPtau bind polymerized microtubules stabilize the polymerized form, promoting assembly and microtubule growth. Most MAPs are activated by phosphorylation, and MAPK (map kinase) phosphorylation cascades add an additional layer of control to the process of microtubule assembly.

Conversely, cells have several different ways to regulate microtubule disassembly. Direct methods for promoting disassembly include components like Stathmin, which binds alpha/beta-tubulin dimers and prevents them from polymerizing. Microtubule severing enzymes like Katanin are able to break microtubules in the middle of their structure, while Kinesin I family members like KIF2 walk along microtubules and promote “fraying” and depolymerization.

NF-kappaB Signaling

NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) represent a group of homo- or heterodimeric transcription factors that are central in a network of various signal transduction pathways. A vast array of signals such as cytokines, growth factors and hormones, infections, oxidative stress, certain drugs, and chemical substances are transmitted to the NF-kB complexes and passed on to influence processes ranging from cell survival, apoptosis, and proliferation to immune response and inflammation. In addition, there is considerable crosstalk with other signaling pathways such as MAPK signaling and the p53 pathway. They are therefore highly pleiotropic, i.e. they influence a multitude of phenotypic traits².

NF-kB is a homo- or heterodimeric complex formed in mammals by the Rel homology domain (RHD)-containing proteins RelA/p65, RelB, c-Rel, NFKB1/p105/p50, and NFKB2/p100/p52. The heterodimeric RELA/p50 complex of the canonical pathway appears to be the most abundant one. All NF-kB proteins have an N-terminal RHD which plays an essential role in DNA binding, as dimerization interface and for the binding to IkB inhibitors. NF-kB proteins do however differ in their C-terminal: class I proteins RELA, RELB and c-Rel contain are characterized by a trans-activator domain whereas class II proteins NFKB1/p105/p50 and NFKB2/p100/p52 have ankyrin repeat transrepression domain³.

In the canonical pathway, NF-kappaB are kept in an inactive state in the cytoplasm through interaction with inhibitory IkB proteins. Upon stimulation of the NF-kB signaling by one of the abovementioned stimuli these regulators are phosphorylated by an IKK kinase complex⁴ composed of protein kinases IKK-alpha, IKK-beta, and NEMO. The phosphorylation marks the IkB inhibitors for proteasomal degradation, thus setting NF-kB free. Once freed, NF-kB are further activated by post-translational modification and translocated to the nucleus where it interacts with specific kB elements⁵. Depending on the cell type and nature of the received stimulus the non-canonical pathway⁶ can be engaged. The core regulator for this pathway is NF-kB inducing kinase (NIK) which activates IKK-alpha, thus leading to phosphorylation of p100. p100 is then processed to p52 leading to the activation and nuclear translocation of the p52/RelB NF-kB dimer.

The canonical pathway does not depend on protein synthesis and responds rapidly to numerous stimuli for a wide variety of downstream effects. In contrast, the non-canonical pathway is slow and persistent and responds only to a subset of signals for more specific effects.

Notch Signaling

The highly-conserved Notch signaling pathway is unique, as both the Notch receptor and most of its respective ligands (canonically the DSL or Delta/Serrate/lag-2 family members) are transmembrane proteins attached to the cell surface. Therefore, Notch signaling is limited to interaction between adjacent cells.

Communication between adjacent cells is paramount, particularly during early development, when cell fate and function are yet to be determined. Notch signaling provides a method for cells to specify their own identity, and to simultaneously influence the role and identity of neighboring cells through lateral inhibition.

The core of the Notch signaling pathway involves two adjacent cells, one expressing a DSL family ligand, and the other expressing the Notch (the receptor). When receptor and ligand interact, two separate protease enzymes cleave Notch into extracellular and cytosolic components. ADAM proteases cleave the extracellular portion of Notch, which remains bound to its respective ligand and is endocytosed by the signaling cell. γ-secretase cleaves the cytosolic portion of notch. This cytosolic region migrates to the nucleus where it binds to the transcription factor CSL, transforming it from a transcriptional repressor to an activator, and upregulating expression of Notch target genes.

p53 Signaling

PI3K-Akt Signaling

The PI3K/Akt pathway is pivotal for cellular homeostasis, neurological development, metabolism, and other processes. It regulates various aspects of cellular development such as apoptosis, cell cycle progression, and cell differentiation.

The key regulator of the PI3K/Akt pathway is Akt/PKB, a family of three closely related serine/threonine-protein kinases. Akt1/PKB alpha plays an important role in cell proliferation and cell metabolism. Akt2/PKB beta is of great significance for glucose metabolism. Akt3/PKB gamma is the least characterized member of the Akt family but it is mainly expressed in the brain where it is thought to regulate mitochondrial biogenesis.

Mutations in Akt contribute oftentimes to deregulation of these processes and the emergence of various forms of cancer. Akt hyperactivation also leads to increased glycolysis, thus supporting the metabolic transformation of cancer cells. Akt has been implicated in the development of cancer drug resistance and is therefore subject of preclinical and clinical research. However, Akt activation alone is usually not sufficient and other mutations are generally necessary to create the cancer phenotype.

Loss of function of the major Akt regulators PI3K and PTEN can lead to hyperactivation of Akt. PI3Ks are an enzyme family that phosphorylate PtdIns (Phosphatidylinositol) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). Upon activation by receptor tyrosine kinase PI3K phosphorylates PIP2 to PIP3 which then activates Akt signaling. PTEN acts as a negative regulator of the signal transduction by dephosphorylation of PIP3 to PIP2.

RTK Signaling

Receptor Tyrosine Kinases (RTKs) are membrane bound kinases that are activated upon binding of receptor specific ligands. They make up the largest class of membrane receptors that trigger signaling cascades through their inherent enzymatic activity. These structures, activation mechanisms and key components of the signaling pathways are highly conserved in metazoans. There are 58 known RTKs in humans, which are grouped into 20 classes depending on topology.

RTKs are fairly promiscuous receptors, and activating stimuli comprise a plethora of growth factors, hormones, and cytokines. Most RTKs form dimers and become active upon ligand binding. The active RTK phosphorylates activators of downstream signaling cascades such as NF-kB, MAPK, Ca2+ dependent signaling, and the JAK-STAT pathway.

RTKs affect a wide spectrum of processes ranging from cytoskeleton dynamics, cell growth and differentiation to inflammation, apoptosis, and tumor progression. In spite of the exceptionally high variety of receptors and outcomes, RTKs engage only a limited set of core processes. Therefore, quantitative analysis of factors like an RTK’s expression profile are crucial for the understanding of the signaling processes and predicting qualitative outcomes.

TLR Signaling

As part of the innate immune system the Toll-like receptor (TLR) signaling pathway contributes to the first line of defense against microbial pathogens. The innate immune system was historically considered nonspecific in response to different invading pathogens, targeting a wide array of pathogenic organisms, including viruses, bacteria, and fungi. This paradigm substantially shifted with the discovery of the Toll receptor in Drosophila.

To date, 10 members of the family have been identified in human and 13 in mouse. Homologs have also been discovered in plants, illustrating the high-degree of conservation in this receptor class.

Different TLRs recognize specific pathogen-associated molecular patterns (PAMPs). The chemical nature of these PAMPs is highly diverse; e.g. lipopolysaccharide (LPS) of gram-negative bacteria are recognized by TLR4 while TLR5 recognizes the bacterial protein flagellin. Ligands for TLR3, 7, 8, and 9 are nucleic acids, and TLR2 is specific for lipoproteins.

Binding of a TLR ligand to the N-terminal ectodomain of a TLR prompts the formation of TLR homo- or heterodimers. Following dimerization, TLR signals are transduced via a cytoplasmic C-terminal Toll IL-1 receptor (TIR) domain to a set of adapter proteins.

Downstream, TLR signaling engages two distinct pathways in which either TRIF (TICAM2) or MyD88 are the key component. Both pathways culminate in the induction of inflammatory cytokines (TNF, IL-6, IL-12), type I interferons (IFN-alpha, IFN-beta), or apoptosis. Furthermore, TLR signaling induces dendritic cell maturation and contributes consequently to the adaptive immune response.

WNT Signaling

Wnts are a class of evolutionarily-conserved, lipid-modified glycoproteins that play a pivotal role in development and homeostasis through a number of different paracrine and autocrine signal-transduction pathways. During early development, Wnt signaling plays a major role in axon guidance, cell polarity, and body axis specification.

Extracellular Wnts bind a variety of different receptors, and initiate signaling in several distinct pathways. Receptors include seven-pass transmembrane Frizzleds and receptor tyrosine kinases ROR and Ryk.

Wnt signaling pathways can result in changes to gene transcription. For example, in the canonical β-catenin signaling pathway Wnt signaling prevents destruction of the transcriptional regulator β-catenin. Wnt signaling can also prompt morphological changes to cellular structure e.g., the non-canonical planar cell polarity pathway induces a kinase cascade that results in reorganization of actin, a core component of the cytoskeleton.