1

Signalling Mechanism In Cell Growth

Speaker : Dr. Bijita Dutta

PGT/Pathology

Moderator :

Dr. D. Datta

Professor & HOD

Department of Pathology

Silchar Medical College

3

Cell Growth

The term cell growth is used in the contexts of cell development

and cell division.

• When used in the context of cell development, the term refers to increase in cytoplasmic and organelle volume (G1 phase) as well as increase in genetic material before replication (G2 phase).

• When used in the context of cell division, it refers to growth of cell populations, where one cell (the "mother cell") grows and divides to produce two "daughter cells” (M phase).

4

Cell Cycle

5

The cell cycle is broken up into four stages:

• G1, S, G2 and M

• S : DNA replication occurs during this S(“synthesis”) phase. • M : DNA packaging, chromosome segregation and cell

division (cytokinesis) occur in M(mitosis). • S phase and M phase are separated by Gap phases.

1.G1 is the gap between M and S.

Cell growth is one of the important events of G1.The

transition from G1 to S is the critical control point in the cell

cycle.

2. G2 is the gap between S and M, and provides time for proofreading to ensure that DNA is properly replicated and packaged prior to the cell division.

6

• G0 or quiescence occurs when cells exit the cell cycle due to the absence of growth-promoting signals or presence of prodifferentiation signals.

• The G1, S and G2 phases comprise interphase, which accounts for most of the time in each cell cycle.

• The M phase, mitosis, is relatively short (approximately 1 hour of a 24 hour cell cycle).

• Mitosis is itself divided into several phases,i.e,prophase,metaphase,telophase and anaphase.

7

8

Cell Cycle Control

Activators and Brakes

9

The mechanisms of regulation can be broken down into two

parts: • First, how is the cell cycle regulated so that the different

phases occur in the correct order? • Second, how do extracellular signals activate or inhibit the cell

cycle?

10

The orderly progression of cells through the various phases of the cell cycle is orchestrated by cyclin-dependent kinases (CDKs), which are activated by binding to cyclins, so called because of the cyclic nature of their production and degradation. The CDK-cyclin complexes phosphorylate crucial target proteins that drive the cell through the cell cycle.

11

12

The four key cyclin-Cdks that drive the cell cycle

13

G1 Regulation

• The G1 phase of the cell cycle is unique in that it represents the only time where cells are sensitive to signals from their extracellular environment.

• Cells require growth factor-dependent signals up to a point in late G1, referred to as the “restriction point” or “start”, after which the transition is made into S phase.

• In order to move from early G1 to late G1, the cell must synthesize cyclin E.

• Transcription of the cyclin E gene requires a transcription factor called E2F.

14

How the G1-Cdk turns on the G1/S Cdk

15

Types of cell signalling

According to the source of the ligand and the location of its receptors (i.e., in the same, adjacent, or distant cells) there are three general modes of signalling-

1. Autocrine

2. Paracrine and

3. Endocrine.

16

Autocrine Signalling

Cells respond to the signalling molecules that they themselves

secrete, thus establishing an autocrine loop.

Example :

1. Physiological- • Liver regeneration • Proliferation of antigen-stimulated lymphocytes

2. Pathological –• Tumors frequently overproduce growth factors and their

receptors, thus stimulating their own proliferation through an autocrine loop.

17

Autocrine Signalling

18

Paracrine Signalling

• One cell type produces the ligand, which then acts on adjacent target cells that express the appropriate receptor.

• The responding cells are in close proximity to the ligand-producing cell and are generally of a different type.

• Example-

1.Connective tissue repair of healing wounds, in which a factor produced by one cell type (e.g., a macrophage) has a growth effect on adjacent cells (e.g., a fibroblast).

2.Hepatocyte replication during liver regeneration.

3.Notch effects in embryonic development, wound healing, and renewing tissues.

19

Paracrine Signalling

20

Endocrine Signalling

• Hormones synthesized by cells of endocrine organs act on target cells distant from their site of synthesis, being usually carried by the blood.

• Growth factors may also circulate and act at distant sites, as is the case for HGF.

• Several cytokines, such as those associated with the systemic aspects of inflammation, also act as endocrine agents.

21

Endocrine Signalling

22

Signal Transduction

• The binding of a ligand to its receptor triggers a series of events by which extracellular signals are transduced into the cell resulting in changes in gene expression.

• So the components of signal transduction pathway are-

1. Ligands

2. Receptors

3. 2nd messengers

4. Transcription factors

23

24

Ligands (Primary Messengers)

• Growth factors : The proliferation of many cell types is driven by polypeptides known as growth factors.

• These factors can have restricted or multiple cell targets. All growth factors function as ligands that bind to specific receptors, which deliver signals to the target cells. These signals stimulate the transcription of genes that may be silent in resting cells, including genes that control cell cycle entry and progression.

25

26

Epidermal Growth Factor (EGF) and Transforming Growth Factor α (TGF-α)

• These two factors belong to the EGF family and share a common receptor (EGFR).• EGF is mitogenic for a variety of epithelial cells, hepatocytes and fibroblasts,• Widely distributed in tissue secretions and fluids. • TGF-α has homology with EGF, binds to EGFR, and shares most of the biologic

activities of EGF. • The “EGF receptor” is actually a family of four membrane receptors with

intrinsic tyrosine kinase activity.

The best-characterized EGFR is referred to as EGFR1, ERB B1, or simply EGFR. It responds to EGF, TGF-α, and other ligands of the EGF family.

• EGFR1 mutations and amplification have been detected in cancers of the lung, head and neck, breast, glioblastomas and other cancers, leading to the development of new types of treatments for these conditions.

• The ERB B2 receptor (also known as HER-2 or HER2/Neu), whose main ligand has not been identified, has received great attention because it is overexpressed in a subset of breast cancers and is an important therapeutic target.

27

Hepatocyte Growth Factor (HGF).• HGF was originally isolated from platelets and serum.

Subsequent studies demonstrated that it is identical to a previously identified growth factor isolated from fibroblasts known as scatter factor. .

• HGF has mitogenic effects on hepatocytes and most epithelial cells, including cells of the biliary epithelium, and epithelial cells of the lungs, kidney, mammary gland, and skin.

• The receptor for HGF, c-MET, is often highly expressed or mutated in human tumors, especially in renal and thyroid papillary carcinomas.

• Several HGF and c-MET inhibitors are presently being evaluated in cancer therapy clinical trials.

28

Platelet-Derived Growth Factor (PDGF).

• PDGF is a family of several closely related proteins, each consisting of two chains. Three isoforms of PDGF (AA, AB, and BB) are secreted as biologically active molecules. The more recently identified isoforms PDGF-CC and PDGF-DD require extracellular proteolytic cleavage to release the active growth factor.All PDGF isoforms exert their effects by binding to two cell surface receptors, designated PDGFR α and β, which have different ligand specificities. PDGF is stored in platelet granules and is released on platelet activation. It is produced by a variety of cells, including activated macrophages, endothelial cells, smooth muscle cells, and many tumor cells. PDGF causes migration and proliferation of fibroblasts, smooth muscle cells, and monocytes to areas of inflammation and healing skin wounds, as demonstrated by defects in these functions in mice deficient in either the A or the B chain of PDGF. PDGF-B and C participate in the activation of hepatic stellate cells in the initial steps of liver fibrosis and stimulate wound contraction.

29

Transforming Growth Factor β (TGF-β) and Related Growth Factors

• Native TGF-β is synthesized as a precursor protein, which is secreted and then proteolytically cleaved to yield the biologically active growth factor and a second latent component.

• Active TGF-β binds to two cell surface receptors (types I and II) with serine/threonine kinase activity and triggers the phosphorylation of cytoplasmic transcription factors called Smads (of which there are several forms, e.g., Smad 1, 2, 3, 5, and 8). These phosphorylated Smads in turn form heterodimers with Smad 4, which enter the nucleus and associate with other DNA-binding proteins to activate or inhibit gene transcription. TGF-β has multiple and often opposing effects depending on the tissue and the type of injury. Agents that have multiple effects are called pleiotropic; because of the large diversity of TGF-β effects, it has been said that TGF-β is pleiotropic with a vengeance.

30

• TGF-β is a growth inhibitor for most epithelial cells. • It blocks the cell cycle by increasing the expression of cell

cycle inhibitors of the Cip/Kip and INK4/ARF families. • The effects of TGF-β on mesenchymal cells depend on the

tissue environment, but it can promote invasion and metastasis during tumor growth.

• Loss of TGF-β receptors frequently occurs in human tumors, providing a proliferative advantage to tumor cells.

• At the same time TGF-β expression may increase in the tumor microenvironment, creating stromal-epithelial interactions that enhance tumor growth and invasion.

31

Receptors

1. Receptors with intrinsic tyrosine kinase activity

2. Receptors lacking intrinsic tyrosine kinase activity that recruit kinases

3. G protein–coupled receptors

4. Steroid hormone receptors

32

Receptors with intrinsic tyrosine kinase activity

• Ligands for these receptors : Include most growth factors such as EGF, TGF-α, HGF, PDGF, VEGF, FGF, c-KIT ligand and insulin.

• Structure: Receptors belonging to this family have

a. an extracellular ligand-binding domain,

b. a transmembrane region, and

c. a cytoplasmic tail that has intrinsic tyrosine kinase activity.

33

34

Receptors lacking intrinsic tyrosine kinase activity that recruit kinases

Ligands for these receptors: • many cytokines, such as IL-2, IL-3, and other interleukins; • interferons α, β and γ; • erythropoietin; • granulocyte colony stimulating factor;• growth hormone and• prolactin .

35

36

G protein–coupled receptors

Ligands include • chemokines, • vasopressin, • serotonin, • histamine, • epinephrine and norepinephrine, • calcitonin,• glucagon, • parathyroid hormone,• corticotropin, and • rhodopsin. • an enormous number of common pharmaceutical drugs

37

• Structure of G protein-coupled receptors :

They contain seven transmembrane α-helices and

constitute the largest family of plasma membrane receptors.

38

39

Steroid hormone receptors

Ligands other than steroid hormones include • thyroid hormone, • vitamin D, and • retinoids.

These receptors are generally located in the nucleus and

function as ligand-dependent transcription factors.

40

41

Transcription Factors

• Signal transduction systems used by growth factors transfer information to the nucleus and modulate gene transcription through the activity of transcription factors.

• Among the transcription factors that regulate cell

proliferation are products of several

a) growth-promoting genes, such as c-MYC and c-JUN, and

b) cell cycle–inhibiting genes, such as p53.

42

Structure of transcription factors :Transcription factors are modular in structure and contain

the following domains :• DNA-binding domain (DBD): attaches to specific sequences

of DNA adjacent to regulated genes.

DNA sequences that bind transcription factors are often referred to as response elements.

• Trans-activating domain (TAD) : contains binding sites for other proteins such as transcription coregulators.

• An optional signal sensing domain (SSD) (e.g., a ligand binding domain), which senses external signals.

43

• Growth factors induce the synthesis or activity of transcription factors.

• Cellular events requiring rapid responses depend on post-translational modifications for activation.

• These modifications include

(a) Heterodimerization ,e.g, the dimerization of the products of the protooncogenes c-FOS and c-JUN to form the transcription factor activator protein-1 (AP-1), which is activated by MAP kinase signaling pathways,

(b) Phosphorylation , as for STATs in the JAK/STAT pathway,

(c) Release of inhibition to permit migration into the nucleus, as for NF-κB, and

(d) Release from membranes by proteolytic cleavage, as for Notch receptors

44

45

46

Role Of Extracellular Matrix In Cell Growth

The ECM regulates the growth, proliferation, movement, and differentiation of the cells living within it. It is constantly remodeling.

• Control of cell growth : ECM components can regulate cell proliferation by signaling through cellular receptors of the integrin family.

• Establishment of tissue microenvironments. • Storage and presentation of regulatory molecules: For

example, growth factors like FGF and HGF are secreted and stored in the ECM in some tissues. This allows the rapid deployment of growth factors in the time of need.

47

• Integrins and proteoglycans are the major ECM adhesion receptors which cooperate in signalling events, determining the signalling outcomes and thus the cell fate.

48

49

Integration of cell signalling

At any given point of time, every single cell of our body is receiving multiple signals. All these signals work in combinations to regulate the behaviour of the cell, with each of the hundreds of thousands of different cell types in our bodies responding to this babble of signals differently. So cells integrate the many signals that they receive in deciding whether to survive, grow and divide (proliferate), differentiate, or die (apoptosis).

50

51

Signal Transduction Pathways In Cancer Cells

52

In cancer cells, these signalling pathways are often altered via mutations, gene amplifications or deletions and results in a phenotype characterized by uncontrolled growth and increased capability to invade surrounding tissues. Therefore, these crucial transduction molecules represent attractive targets for cancer therapy.

The most advanced targeted agents currently under development interfere with function and expression of several signalling molecules.

53

Growth Factor Overexpression

• Growth factors are frequently found overexpressed in a variety of tumors.

The result is that the respective receptors are stimulated at

a higher rate.• Often tumors are found to secrete growth factors

such as EGF,IGF-I and PDGF.

• These factors bind to their receptors and initiate growth and proliferative signals establishing an autocrine loop that leads

to tumor growth.

54

Receptor Mutation

• Receptors can be mutated in a way that they transmit the signal without ligand binding.

• For instance, tyrosine kinase receptors dimerize or oligomerize following ligand binding and carry out the signal transduction cascade. In various tumors tyrosine kinase receptors can be constitutively activated by mutations that render them active independent of ligand binding. Such mutations were found on NEU/c-erbB-2.

55

Mutation Of Non-receptor Tyrosine Kinase

• There are several such tyrosine kinases that are activated in tumors via mutations. Most of these mutations result from chromosomal translocations that give rise to hybrid gene products. A major example is the BCR-ABL in CML.

• The pathways that this protein uses to cause transformation are not clearly defined. It is known that it binds and activates GRB-2 which in turn, activates the Ras pathway, a key pathway for triggering MAPK activation and cell proliferation.

Contd.

56

• Another example of fusion proteins is TEL-ABL, present in acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia (AML) and chronic myeloblastic leukemia(CML)

with a reciprocal t (9; 12) translocation.

57

Cytoplasmic Molecules

• The MAP Kinase and the PI3Kinase cascades play a central role during cell activation and proliferation. Several oncogenes are known to act on these pathways and several molecules that participate on these cascades when deregulated they become oncogenic.

• Ras, a well-studied family of oncogenes, structurally altered in about 25% of all human tumors, functions on activating the MAPK cascade .

• Raf1,a serine threonine kinase that is activated by Ras, is also activated in some myeloid leukemias

Contd.

58

• Serine threonine kinases are another important group of oncogenes. This family of oncogenes includes the Akt family (Akt1, Akt2, Akt3).

• Akt2 is activated in pancreatic adenocarcinomas, small cell lung cancer, and ovarian cancers.

Contd.

59

Raf and Akt are kinases that contribute to the oncogenic phenotype through divergent mechanisms,e.g, they can

I. induce transcription of genes that are normally not expressed in these cells .

II. directly interfere with cell cycle machinery and promote progression through the cell cycle.

III. inhibit programmed cell death and, therefore, allow the survival of a cell that carries other defects and would otherwise apoptose.

60

Transcription factors

• In tumor cells a transcription factor can be mutated and activated independent of extracellular or cytoplasmic signals.

• NFkB is a transcription factor that regulates expression of several genes and was activated in a series of tumors such as breast tumors, pancreatic adenocarcinomas, lung cancers and acute T cell leukemias.

• C-myc is a transcription factor implicated in a variety of human tumors. When overexpressed it dimerizes with Max, a complex that elicits growth signals, while the Mad-Max complex promotes differentiation signals

• Overexpression of c-myc has been involved in a series of human tumors including colon, stomach, cervix, breast and hematological neoplasm.

61

Cell Cycle Control Proteins

• Deregulation of the cell cycle control is crucial for the development of a cancer cell since it has to proliferate at a faster than the normal rate. This effect can be direct,involving mutations of the cell cycle control proteins or indirect when an oncogenic protein targets the cell cycle regulators.

• Oncogenic processes exert their greatest effect by targeting particular regulators of the G1 to S phase progression.

• Inactivation of the Rb gene is a primary event in retinoblastomas.

• Inherited loss of INK4a gene that encodes p16 confers susceptibility to melanoma.

Contd.

62

• Although cell cycle transition depends on the underlying CDK cycle, superimposed checkpoint controls help ensure that certain processes are completed before others begin. The role of such mechanisms is to act as a brake on the cell cycle in the face of stress and damage and allowing repair to take place.

• The best-studied checkpoint regulator is the p53 gene and is most frequently mutated in human cancer.

• The p53 protein acts as a transcription factor and cancer related mutations cluster in its binding domain.

63

Apoptosis Related Proteins

• In cancer cells an anti-apoptotic mechanism is often activated to rescue the transformed cell from programmed cell death.

• The most common mechanism is activation of the bcl-2 family of proteins (Bcl-2, Bcl-xL, Bcl-W) that are able to inhibit cytochrome c release from the mitochondria and rescue the cell from apoptosis.

• Inactivation of the pro-apoptotic molecules Bax, Bak, Bid or Bim also contributes to rescuing the cell from apoptosis.

• Activation of oncogenic kinases such as Akt-1 protects cells from apoptosis by inhibiting the pro-apoptotic molecule Bad.

64

Signal Transduction Modulation

• The elucidation of signal transduction pathways in cancer cells, has fueled the design of drug molecules intended to act at specific proteins of the signal transduction cascade, often referred to as signal transduction modulators (STMs).

• STMs may interfere with signal transduction processes by blocking cell surface receptors, inhibiting growth factor receptor tyrosine kinases, or inhibiting the effects of further downstream genes, such as the mitogen-activated protein kinases.

• Many drug molecules directed against a wide range of signal transduction elements are being evaluated worldwide as potential anticancer therapies. Several STMs are currently in clinical trials; others are still in preclinical research and development.

65

Signal Transduction Modulators For Cancer Therapy

66

68