Molecular Pharmacology course lecture No.5

Mutagenic Signal Transduction and apoptosis

Dr.Omer Yahia ElhusseinB.sc. Pharmacy.M.sc. Molecular Medicine.

Normal Cell Signal Transduction Associated with tumor

• Wnt-B catenin path way.• Cell Cycle Arrest.• Growth Factor Tyrosine Kinase.• DNA Damage and repair.• Apoptosis.• Telomere Maintenance.

1. Wnt-B catenin path way. (Drosophila mutants :Nobel Prize 1995)

(the term Wnt is an amalgam of Wg and Int1)

• In resting cell cytoplasmic beta catenin turns over rapidly , with little entering the nucleus.

• Degradation is controlled by acytoplasmic complex that include the product of apc gene (tumor suppressor) and GSK , a kinase protein that phosphorylates beta catenin.

Wnt-B catenin path way

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• Mutation in APC phosphorylation site or loss of APC result in excess free beta catenin that enter the nucleus .

• eg: defect in APC in patient with familial Adenomatous Polyposis Coli, giving rise to multible pre cancerous polyps in the large intestine.

The gene rearrangements resulting from chromosome translocations frequently lead to the generation of oncogenes.

The first characterized example of oncogene activation by chromosome translocation was the involvement of the c-myc oncogene in human Burkitt's lymphomas and mouse plasmacytomas, which are malignancies of antibody-producing B lymphocytes (immunoglobulins).

For example, virtually all Burkitt's lymphomas have translocations of a fragment of chromosome 8 to one of the immunoglobulin gene loci, which reside on chromosomes 2, (k light chain), 14 (heavy chain), and 22 (g light chain).

Chromosomal abnormalities and cancer

The c-myc proto-oncogene is translocated from chromosome 8 to the immunoglobulin heavy-chain locus (IgH) on chromosome 14 in Burkitt's lymphomas, resulting in abnormal c-myc expression.

Cancer therapeutics that target components of

the canonical Wnt pathway

APC, adenomatous polyposis coli ; TCF, T‑cell factorBCL9, B‑cell lymphoma 9. P, phosphorylationCBP, CREB-binding protein; PS1, presenilin 1 CK1α, casein kinase;1α; Pygo, PygopusDKK, Dickkopf; RTKs, receptor tyrosine kinasesDVL, Dishevelled; WIF,Wnt inhibitory factor 1 GSK3β, glycogen synthase kinase 3β LEF, lymphoid enhancer factor;

LRP5, LDL-receptor related protein 5MAPK,mitogen-activated protein kinase;

NRTKs, non-receptor tyrosine kinases; SFRP, secreted Frizzled-related protein

Cancer therapeutics that target components of

the canonical Wnt pathway

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• The non-steroidal anti-inflammatory drug (NSAID) celecoxib, which targets the Wnt target gene COX2 (cyclooxygenase 2), is already approved for the treatment of patients with familial adenomatous polyposis (FAP)

• Therapeutic proteins, antibodies and low-molecular-weight products that interfere with aberrant activity of the canonical Wnt pathway are currently being developed or might be future candidates.

• Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor and an approved therapeutic for chronic myeloid leukaemia (CML), shows promise for the treatment of Wnt-induced gastrointestinal tumours.

• Interference with members of other signalling pathways, which directly or indirectly control β‑catenin stability (such as γ‑secretase or ADAM, a disintegrin and metalloproteinase ) might also provide therapeutics against disease

Note :• ADAM and γ‑secretase are • These metalloproteinase activities result in the

enhancement of migratory and invasive properties of endothelial cells, which are required for tumour angiogenesis.

2-Cell cycle Arrest

• Transforming growth factor‑β (TGFβ) signalling is transduced through Smad and non‑Smad pathways.

• TGFβ ligand binds to TGFBR2 and TGFBR1.• TGFBR2 phosphorylates (P) TGFBR1, which subsequently

phosphorylates and activates SMAD2 and SMAD3.• Activated SMAD2 and SMAD3 form a Smad complex with

SMAD4 and translocate into the nucleus. • In the nucleus, the Smad complex interacts with other

DNA‑binding transcription factors, and co‑activators and co‑repressors, binds to the promoter regions of TGFβ target genes and regulates the transcription of target genes.

• TGFβ also regulates non‑Smad pathways.• TGFβ receptors activate p38, JNK, Ras–Erk, PI3K–Akt, and

small GTPases such as RHOA and CDC42.• TGFβ stimulation inhibits cancer cell proliferation in some

cellular contexts and promotes it in others. • Numerous factors are involved in TGFβ‑regulated cell proliferation and keep its signalling pathways balanced1. It inhibit the proliferation of most adult cells (eg: TGF-beta null

mutation in mice causing death of inflammation in multiple organs caused by excessive proliferation of lymphocytes )

2. TGF-beta stimulate production extra cellular matrix, including collagen,proteoglycans and adhesion glycoprotein

• In addition to perturbation of TGFβ signalling, disruption or mutation of regulators of TGFβ signalling can lead to a loss of balanced TGFβ signalling, resulting in the generation and progression of tumours.

• TGFβ signalling in cancer cells has dual roles in the regulation of cell death and proliferation.

• TGFβ signalling has crucial roles in the maintenance of self‑renewal and tumorigenic activity of glioma‑initiating cells and leukaemia‑initiating cells , whereas the function of TGFβ signalling in breast cancer‑initiating cells is controversial.

• TGFβ signalling is involved in several cell responses during cancer cell metastasis, and cell type‑dependent and context‑dependent factors contribute to the regulation of tumour metastasis.

• Mutation in gene of TGF-beta or SMADS appear in up to 50% of some types of eg: heridetary hemorrhagic telangiecta

• The TGFβ pathway has been targeted for cancer therapy using multiple strategies. Some of them are currently in clinical trials

• Reserverotral (a potent antitumor factor) inhibit invasiveness of human SCC cell at least in part , through Down regulation of TGF-beta2 (Kopelovich L et al 2011)

3- FGFr signalling network.

Continue…• Following ligand binding and receptor dimerization, the kinase domains

transphosphorylate each other, leading to the docking of adaptor proteins and the activation of four key downstream pathways:

o RAS–RAF–MAPK signal transducer .o PI3K(inositol triphosphate kinase)–AKT signal transducer. o activator of transcription (STAT).o phospholipase Cγ (PLCγ) . • FGFRs have also been shown to bind and directly phosphorylate ribosomal S6

kinase17 .• Signalling can be negatively regulated at several levels by receptor

internalization or the induction of negative regulators, including o FGFR-like 1 (FGFRL1),o SEF, Sprouty (SPRY),o CBL, o MAPK phosphatase 1 (MKP1) and MKP3 (brown).

Continue…• These regulators may modulate ligand binding

(FGFRL1 and SEF) or interfere with intracellular signalling, principally through modulation of the MAPK pathway.

o DAG, diacylglycerol; o FRS2α, FGFR substrate 2α; o GRB2, growth factor receptor-bound 2; o IP3, inositol triphosphate; o P, phosphorylation; o PIP2, phosphatidylinositol-4,5 biphosphate;o PKC, protein kinase C;o Sos, son of sevenless.

The epidermal growth factor receptor signalling network

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• On activation (by ligand binding), the epidermal growth factor receptor (EGFR, also known as ERBB1) autophosphorylates tyrosine residues in its cytoplasmic domain, which serve as docking sites for the assembly of protein complexes that transduce EGF signals to generate specific biological responses.

• Scaffold protein : A protein that can simultaneously bind two or more other proteins, and thereby facilitate physical and functional interactions between the client proteins that bind to it.

o Kinases are light blue, o scaffolds are dark blue, o adaptor proteins are yellow, o G proteins are green and o transcription factors are orange.

Factor Principal Source

Primary Activity Comments

PDGF platelets, endothelial cells, placenta

promotes proliferation of connective tissue, glial and smooth muscle cells

two different protein chains form 3 distinct dimer forms; AA, AB and BB

EGF submaxillary gland, Brunners gland

promotes proliferation of mesenchymal, glial and epithelial cells

 

TGF- common in transformed cells

may be important for normal wound healing

related to EGF

FGF wide range of cells; protein is associated with the ECM

promotes proliferation of many cells; inhibits some stem cells; induces mesoderm to form in early embryos

at least 19 family members, 4 distinct receptors

NGF   promotes neurite outgrowth and neural cell survival

several related proteins first identified as proto-oncogenes; trkA (trackA), trkB, trkC

Erythropoietin kidney promotes proliferation and differentiation of erythrocytes

 

TGF- activated TH1 cells (T-helper) and natural killer (NK) cells

anti-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, inhibits macrophage and lymphocyte proliferation

at least 100 different family members

IGF-I primarily liver promotes proliferation of many cell types

related to IGF-II and proinsulin, also called Somatomedin C

IGF-II variety of cells promotes proliferation of many cell types primarily of fetal origin

related to IGF-I and proinsulin

Growth Factors

4- DNA damage and repair (Retinoblastoma , P53 , Mdm, E2F , Waf-1 , Bax)

• pRB primary function is to block cell cycle progression and it is in activated in response to mitogenic stimulation .

• So,loss of Prb can lead to in appropriate cycle progression and cancer

• Rare individuals who inherit one inactivated rb gene are predisposed to develop Retinoblastoma as children and Osteosarcomas as adult

• Homozygous loss of p RB is lethal during embryogenesis, as the protein is essential for promoting differentiation in various organs and tissues.

• E2F is a tumor suppressor because it opposes the cell cycle progression prior to the restriction point when partnered with p RB ; However it can act also as an Oncogene as it promotes the cell cycle when p RB in activated

Retroviral Oncogenes

• Viral oncogenes were first   defined in Rous Sarcoma   Virus (RSV), which transforms   chicken embryo fibroblasts in    culture and induces large   sarcomas within 1-2 weeks   after inoculation into chickens.

• In contrast, the closely related   avian leukosis virus (ALV)   replicates in the same cells as   RSV without inducing   transformation.

Both RSV and ALV infect and replicate in chicken embryo fibroblasts, but only RSV induces cell transformation.

5- Apoptosis and cancer • Cell dies because of :Old and defective - incur some damage - surplus to the requirement of

tissues ( like finger formation )

• Cell dies in different ways:1. Necrosis. 2. Apoptosis. 3. Autophagy.

Apoptosis : controlled process leading to cell death that is triggered by intracellular

damage (e.g., DNA lesions) or by external signals from neighboring cells. Also called programmed cell death.

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Extrinsic path way

T cell induced death

Intrinsic path way

5-Telomer Maintenance

• Telomerase is the enzyme for maintaining the end of the chromosome

• While the Telomerase enzyme is not active in most normal human cells , the enzyme is active in all types of cancer , but enables cancer cells that have telomerase too short for chromosome replication to divide indefinitely by lengthening the telomeres and stabilizing the chromosomes

• During successive cell cycles the telomeres shorten because the primer needed by DNA Polymerase for new DNA synthesis is removed.

• This action is counteracted by the action of telomerase, which adds new telomere repeat sequences to the ends of chromosomes.

• Eventually ,the telomeres become so short that the complex between sequences and the proteins that bind to them disrupted , and DNA damage occur.

Thanks