Cell cycle and molecular basis of cancer

Cell cycle and molecular basis of cancer

Presenter: Dr. Durga.RIMS, Imphal, Manipur.

In the process of Development of unicellular fertilized egg into complex multi cellular organism- cellular replication, growth and differentiation.

Cellular replication occurs mitosis n it produces two identical daughter cells. Some daughter cells-specialise and bcom

teminally d/f cells of mature tissues. Other daughter cells- retain property of stem

cells.

Cell Cycle

Cell cycle is the interval period between beginning of one mitosis to the beginning of other division.

2 phases(in the order)

Interphase G1 phase S phase G2 phase

M phaseMitosis cytokinesis

Post mitotic phase. Accumulate the energy and prepares themselves for the synthesis of DNA. Active synthesis of RNA and protein.

Duplication of DNA and centriole

Formation of macro molecules for spindle formation.

G1 phase : It is the post mitotic phase and takes place at the end of

cell division. The newly formed cells accumulate the energy and prepares themselves for the synthesis of DNA . During this , active synthesis of RNA and protein takes place .

S phase : It is the synthesis phase during this phase duplication of

DNA and centriole takes places. The duplication of DNA results in the duplication of chromosomes . Point of no return. Commited to divide.

G2 phase : It is the pre- mitotic gap phase (invisible phase) the

synthesis of RNA and protein continues in this phase. The formation of macro molecules for spindle formation takes place and the cell prepare it self to go into the mitotic phase .

M PhaseNucleolus disappears

Cell Cycle Checkpoints

G1/ S checkpoint:• Checks for DNA damage before

replication.• Regulated by proteins called

cyclins and associated enzymes (CDK )& inhibitors (CDK inhibitors.)

• Cyclins D,E,A and B appear sequentially during the cell cycle and bind to CDK.

G2/M checkpoint: • Monitors the completion of DNA replication and

checks whether the cell can safely enter mitosis.• They delay cell cycle progression, thus providing time

for DNA repair. If damage is not repairable then apoptotic pathways are activated.

• Hence defects in these checkpoints will cause loss of normal cell cycle control ultimately leading to malignant transformation.

The orderly progression of cells through the various phases of cell cycle is regulated by Cyclins, cyclin dependant kinases (CDKs) & their inhibitors.

CDKs - expressed constitutively during cell cycle , in inactive form.

Cyclins- synthesized during specific phases of cell cycle ; activates CDKs

Cyclin D – First cyclin to increase in cell cycle - appears in mid G1 - binds to & activates CDK 4

Cyclin D- CDK4 complex phosphorylates retinoblastoma susceptibility protein (RB)

Phosphorylation of RB is a molecular on-off switch for cell cycle.

G0 G1 S G2 M

The key enzymes that control the transitions between the different states of the cell cycle, and the entry of nondividing cells into the cell cycle, are the cyclin-dependent protein kinases, or CDKs

Cyclin DCDK 4,6

Cyclin ECDK 2

Cyclin ACDK 2

Cyclin BCDK 1

Active Rb+E2F Inactive Rb and E2F

• Hypophosphorylated Rb is active form; binds E2F and wont let cell cycle to progress.

• E2F when released from Rb will form cyclin E A B and DNA polymerase and helps in cell cycle progression.

Mitogenic stimulus

2 classes of CDK Inhibitors1.Cip/Kip family (P21,P27,P57) (cell cycle non specific)2.INK4/ARF family (P16,P15,P18,P19) (cell cycle specific)

Neoplasia

Definition given by British oncologists Willis---

“Neoplasia is an abnormal mass or tissue , the growth of which exceeds and is uncoordinated with that of normal tissue and persists in same excessive manner even after cessation of stimuli which evoked the change.”

Neoplasia(Greek-New growth)

“Cancer is a result of accumulated genetic mutations and it acquire selective growth advantages over their non transformed counterparts, acquires adaptability even to everchanging environment and they proliferates.”

Mutations in genes result in altered proteins

Most cancers result from mutations in somatic cells

Some cancers are caused by mutations in germline cells

Molecular basis of cancer

1.Non lethal genetic damage is the heart of carcinogenesis . Mutations acquired from environment- exogenous and endogenous.

2.Clonality Tumors arise as clones from a single cell that has incurred genetic damage.

[ Tumors are monoclonal] The development of the malignant clone is due to mutations in DNA due to:

Random replication errors Exposure to carcinogens Faulty DNA repair process

Characteristics of Neoplasia

3. 4 principle targets of genetic damage:

a)Growth promoting protooncogenes. b)Growth inhibiting tumor suppresor genes. (Both)c)Genes that regulate apoptosis.d)Genes involved in DNA repair.

4. Carcinogenesis is a multistep process resulting from accumulation of multiple mutations; that leads to tumor progression.

Tumor progression: Mutations accumulate in different cells, generating subclones with variable ability to grow, invade, metastasize.

1. Self sufficiency2. Insensitivity to growth

inhibitory signals3. Evasion of apoptosis.4. Limitless replicative

potential.5. Angiogenesis6. Invasion &

Metastasis**sure sign of malignancy

7. Defect in DNA repair.

7 fundamental changes in cell physiology determine Malignant phenotype

Definition of terms:

Oncogenes- promote autonomous cell growth in cancer cells in the absence of growth signals also.

Protooncogenes -Unmutated cellular counter part of oncogenes

Oncoproteins- are the products of oncogenes

1.Self sufficiency

Under physiological conditions cell proliferation resolved into

1

2

3

45

Class I: Growth Factors

Class II: Receptors for Growth Factors and Hormones

Class III: Intracellular Signal Transducers

Class IV: Nuclear Transcription Factors

Class V: Cell-Cycle Control Proteins

Five types of proteins encoded by proto-oncogenes participate in control of cell growth:

Category Protooncogenes

MOA Associated human tumors

1Growth factorsPDGF B

SIS Overexpression

Astrocytomaosteosarcoma

FGF HST1INT2

OverexpressionAmplification

Stomach carcinomaBladder, Breast

TGF-A TGFA Overexpression

Astrocytoma,HCC

Selected protoncogenes & MOA

Growth factor driven proliferation contributes to malignant phenotype by increased risk of spontaneous or induced muatation in proliferating cell population.



Growth factor receptorsEGF receptor family

ERB1,2 Overexpression SCC

FMS like tyrosine kinase 3

FLT3 Amplification Breast ovary

Receptor for neurotrophic factor

RET Point muat MEN 2A 2B, MTC

PDGFR PDGFR B Overexpression Leukemia

Receptor for stem cell factor

KIT Point muatation

GIST, Seminomas, leukemias.

Oncogenic variant of normal receptors associated with constitutive dimerization of receptors and activation even with out the growth factor; giving out mitogenic stimulus. overrides the GF



Proteins involved in signal transduction

GTP binding K RASH RASN RAS

Point mutation Colon pancreasBladder kidneyHematological

Non receptor tyrosine kinase

ABL Translocation CML

RAS Signal transduction

BRAF Point mutation Melanomas

WNT signal pathway

B catenin Point mutation HCC-plays imp role in signalling cascade downstreasm.- Point mutation of RAS family genes is the single most common abnormality of dominant oncogenes in human tumors.- Approx. 15-20% of all human tumors contain mutated version of RAS proteins.



Nuclear regulatory proteins

Transcriptional activators

C MYCN MYCL MYC

Translocation

} Amplification

BurkittsNeuroblastoma, small cell carcinoma lung

Cell cycle regulatorsCyclins

Cyclin D

Cyclin E

Translocation, amplification

Overexpression

Mantle cell lymphoma, breast & oesophagus

Breast ca

CDK CDK4 Amplification glioblastoma

Insensitivity to growth inhibitory signals TUMOR SUPPRESSOR GENES

Normal function - inhibit cell proliferation LOSS OF FUNCTION Both gene copies must be defective Absence/inactivation of growth inhibitory signals-->

cancer

30

Rb Protein Rb protein , product of Rb gene chromosome 13q14. Mechanism :Binds E2F regulatory transcription factors

controlling progression of cell cycle. Controls the cell cycle progression b/n G1 and S phases. Exists in active hypophosphorylated state and binds

E2F(quiscent) And gets inactived by phosphorylation.• Phosphorylated Rb cannot bind E2F --> S phase

Cancer progression-– Disruption/deletion of Rb gene – Inactivation of Rb protein

--> uncontrolled cell proliferation

1.In sporadic form: both mutation of RB locus acquired after birth2.Familial: Born with one allele mutation one hit; the second hit acquired after birth leading to LOH

Gene is c/a TP53 – 17p13 and its protein P53 Molecular policeman. Prevents propogation of genetically

damaged cells It is a Transcription factor that is at the centre of large

network of signals ,sensing stress like DNA damage, shortened telomeres and hypoxia.

>50% tumors in humans show mutation in this gene. Acuired Inactivating mutation both the alleles are known to

be acquired in somatic cells Inherited- one mutant allele, other “second hit” i.e. LOH

needed to produce Li Fraumeni syndrome.

P53

Mechanism of action:

Phosphyorylated p53 activates transcription of p21 gene

p21 CDK inhibitor (binds CDK-cyclin complex --> inhibits kinase activity)

Cell cycle arrested buys time to let DNA to be repaired If damage cannot be repaire --> cell death (apoptosis)

Cancer progression: --> uncorrected DNA damage --> uncontrolled cell proliferation --> cancer

34

APC - 5q21 Main fxn – down regulate growth promoting signals.

B- Catenin protein fxn - 1. Regulates cell to cell adhesion 2. Gene transcription.(growth

pmoting signal)

APC/ B catenin

Imp fxn of APC- downregulate B catenin In case of cancer, mutations in B catenin prevents its destruction by APC “Loss of contact inhibition” mutation of E cadherin or B catenin.

GSK3B

Germline mutations- individual are born with one mutant allele develop thousands of adenomatous polyp by the age of 20 yrs and later carcinoma of colon.

Both the alleles to be lost for a tumor to arise.

Other genes functioning as Tumour suppressor genes: INK4a/ARF Locus The TGF-β pathway NF-1 & 2 gene VHL(Von Hippel Lindau) PTEN (phosphatase and tensin) WT-1(Wilms tumour) Cadherins KLF6 Patched(PTCH)

EVASION OF APOPTOSIS:

Normally apoptosis will cause damaged cell to undergo apoptosis, Mutations in genes that regulate apoptosis leads to accumulation of cells -neoplastic cells.

Overexpression of BCL-2 (anti apoptotic gene) Prototype of this category Downregulation of BAX pro apoptotic gene

Eg. In B cell lymphoma of follicular type, translocation t(14,18) (q32;q21) , BCL2 gene from 18q21 is translocated to 14q32 (Ig heavy chain locus), leading to overexpression of BCL2 & accumulation of B lymphocyte.

Additionally Mutations in p53 gene results in decreased transcription of BAX gene thus reducing apoptotic activity .

1)Reduced CD95 levels. 2) Inactivation of DISC. 3)Reduced egress of Cytc C, bcos of BCL2.4)Reduced levels of Proapoptotic BAX frm loss of P53. 5) Loss of APAF 1. 6) Upregulation of inhibitors of IAP

Telomeres and telomerase. The ends of each chromosome contains , highly variable

number of repeats of sequence (TTAGGG) called as telomeres.

Molecular count downclock- regulate the number of times a cell can divide.

With each division the length of telomere decreases. Shortened telomeres recognized by DNA repair machinery

as ds DNA breaks- cell cycle arrest by P53 and RB.– Ageing.

This telomere shortening is prevented by the enzyme telomerase in germ cells but most of the somatic cells lack this enzyme.

Cancer cells prevent telomere shortening by reactivation of telomerase enzyme thus causing unlimited proliferation

Limitless replicative potential

Development of sustained angiogenesis :

Tumor cells cannot enlarge beyond 2 mm of size unless they are vascularized.

Required for normal metabolism- oxygen and nutrients. Dual effects: Perfusion supplies required nutrients &

oxygen. Newly formed endothelial cells secrtes GF

and contributes for growth of new tumor cells.

Angiogenic switch involves production of angiogenic factors& loss of anti angiogenic factors.

Tumor associated angiogenic factors ( VEGF & b FGF )are produced by tumor cells & inflammatory cells (macrophages) which infiltrate tumors.

Additionally Mutational inactivation of both p53 alleles, causes ↓ in antiangiogenic factors like thrombospondin – 1 & ↑ in VEGF & Hypoxia inducible factor – 1 ( HIF – 1)

INVASION AND METASTASIS : Biologic hallmarks of malignant tumors. Invasiveness is a reliable feature that differentiates

malignant from benign tumors.

Metastasis – tumor implants discontinuous with the primary tumor.

Dissemination occurs with one of the three pathways-

a. Hematogenous spread b. Lymphatic spread c. Direct seeding of body cavities or surfaces.

Metastatic cascade can be divided in 2 phases : i. Invasion of extracellular matrix ii. Vascular dissemination and homing of tumor cells

Invasion of ECM

Active process involving the following steps.

1. Loosening up of the tumour cells from each other.

Downregulation of E Cadherin.

2. Degradation of ECM

proteolytic enzymes

MMP, Cathepsin D, UPA.

Another mxn- Ameboid migration :cell squeezes thru the spaces in the matrix .

3. Attachment of tumor cells to novel ECM proteins

Loss of adhesion in normal cell induces apoptosis.(Anoikis) Tumor cells resistant to apoptosis & additionally matrix modified. MMP2, MMP 9 produces novel sites that bind to tumor cells and stimulates migration.

4. Migration:Final step of invasion, tumor

propells thru the BM. Complex multistep process . Cells attach at the leading edge, detach from the matrix at trailing edge and contract the actin to move forward.

In the circulation- tumor cells forms clumps by homotypic and hetrotypic adhesions.

Forms tumor emboli At distant site tumor

attach to endothelium, egress thru BM

Favoured by adhesion molecules integrins, laminin receptors

Vascular dissemination and homing of tumour cells:

Humans literally swim in the sea of environmental carcinogens causing DNA damges

Ionising radiation Sunlight Dietary carcinogens ROS geneerated by cell metabolism

Such damage is regularly repaired by DNA repair systems. If the repair system not working optimally leads to accumulated mutations and leads to neoplastic transformation.

In some inherited disorders genes encoding DNA repair are defective leading to increased risk of carcinogenesis.-”Genomic instability syndromes”

Defects in DNA repair

Microsatellite- Are short repeats of Base sequences in genome.

Microsatellite instability- During cell division ,DNA polymerase creates another copy of

DNA from original strand. DNA polymerase inserts wrong base pairs; which usually gets rectified by a set of system – Mismatch repair system.

If mismatch repair system mutated or not fxng properly will lead to a state what is called as Microsatellite instability.

Seen in Colorectal carcinoma(15-20%). 2 principal mismatch repair genes mutated ar MLH1 &

MSH2.

Microsatellite instability

Defects in 3 types of DNA repair systems Defect in mismatch repair gene- Hereditary Nonpolyposis Cancer Syndrome.

Defect in Nucleotide excision repair system- Xeroderma Pigmentosa

Defect in recombination repair system- Ataxia telangiectasia, Bloom syndrome and Fanconi anemia.

BRCA-1 and BRCA-2 are the two genes that participate in the process of homologous recombination of DNA repair. Mutations in these genes will cause increased risk of breast, ovary , colon and several other cancers.

1. Robbins Pathologic basis of disease, 8th edition.

2. Recent advances in histopathology 20.3. Wheaters Functional Histology, 6th

edition. 4. Harpers illustrated biochemistry; 29th

edition.5. Internet sources.

References

Health & Medicine

Cell cycle and molecular basis of cancer