G1/S Transition Regulation

Regulation of G1/S Transition

• Cyclin D and Cdk 4/6 form a complex in the G1 phase
• pRb is phosphorylated as a result of Cyclin D Cdk 4/6
• E2F gets released when pRb is phosphorylated
• Genes activating S phase entry include cyclin A and cyclin E
• Mitogens and growth factors are required for G1/S to occur
• pRb phosphorylation is the first step to S phase transition
• The G1/S transition is a highly regulated checkpoint that restricts proliferation
• Cells no longer require mitogens/growth factors after a certain point in the G1 phase, the restriction point (R point)
• Mitogenic signals promote the G1/S phase prior to the R point
• Increased E2F increases Cyclin E expression
• pRb is phosphorylated by CylinE-Cdk2
• A positive feedback loop is created, amplifying pRb phosphorylation
• Growth factors and mitogens activate transcription of cyclin D and inhibit CdkI INK4 with positive, growth-inducing signals
• Growth factor withdrawal and physiological/cellular stress are negative signals
• Growth factor withdrawal, anti-mitogenic factors, and cell adhesion inhibit cyclin D1
• Extracellular signals include growth factors and cytokines
• Signaling pathways and transcription factors promote cyclin D1

Cell Cycle Checkpoints

• There are cell cycle checkpoints at G1/S, S, intra S, and G2/M
• Checkpoints are activated to check for physiological stress and DNA damage

G1/S Checkpoint

• The ATM kinase phosphorylates and activates p53
• The P21CIP gene is transcribed and activated by P53
• The Cdk2/cyclin E complex is inhibited by P21CIP
• At the start of the S phase, replication is prevented by Cdk2/cyclin A

G2/M Checkpoint Controls G2/M Progression

• Cdc25 activation prevents the Cdk1/Cyclin B1 complex from inhibiting Cdk1/Cyclin B1
• The Cdk1/Cyclin B1 complex creates positive feedback
• ATM kinase activates CHK1/2 kinases, which in turn activate p53 and P21CIP

Cell Cycle in Cancer

• Uncontrolled proliferation occurs from changes in cell control
• S/G2/M proceeds past G1 once responsiveness to growth factor signals occurs in G1 phase
• Cancer cells can bypass growth factor stimuli and replicate independently since the G1/S control is often disrupted
• Hallmarks of cancer include sufficiency in growth signals and insensitivity to anti-growth signals
• Mechanisms of cancer include increased proliferation signals and decreased/inhibited regulatory pathways

pRb Pathway in Cancer Cells

• pRb inactivation occurs from pRb mutations, methylation of promoter, and viral oncoproteins
• Overexpression of cyclins like Cyclin D1 can be found in cancer cells involving gene amplification, increased transcriptional activation, and defects in degradation
• INK4 gene inactivation by deletion or methylation result in decreased protein expression
• Rare CDK4 gene amplification or mutations render cells unresponsive to INK4 inhibition.
• p53 mutations happen in 50+% of cancers
• p53 mutations can lead to the inability to activate checkpoints
• P21CIP activation is impaired from loss of p53 function, which then leads to failure to arrest in G1/S and G2/M
• Loss of cell cycle checkpoint activation happen as a result of mutations in regulators (ATM) of DNA damage signaling pathways

CDKs Inhibitors for Cancer Treatment

• CDKs are essential for cell proliferation
• Loss of cell control leads to unrestricted growth
• CDKs are overexpressed in cancer, and mutations are rare
• Targeting cyclins are not druggable
• 1st generation inhibitors targeted the ATP binding site of the CDKs which are toxic
• 2nd generation inhibitors targeted range of cyclins, also too toxic
• 3rd generation inhibitors are specific targeting of CDK4/6-cyclinD
• Palbociclib (Pfizer) is the 3rd generation inhibitor
• Third-generation drugs such as Palbociclib (Pfizer) bind to the ATP-binding pocket.
• Palbociclib is often combined with other drugs like mTOR inhibitors in advanced clinical trials

CDK2 Inhibitors

• Rationale for CDK2-specific inhibitors are that CDK2 activity is required for normal mammalian cell cycle progression
• Overexpression of CDK2 binding partners cyclin A and/or E is a key oncogenic process
• Cyclin-E deficient cells can develop a resistance to oncogenic transformation
• Some tumours can develop a resistance to CDK4/6 inhibitors
• Rb tumor suppressor may be lost in certain tumor types
• Most CDK2 inhibitors are not completely specific, can result in toxic side effects
• Many putative selective CDK2 inhibitors have entered early-phase clinical development

Case Study: Retinoblastoma

• A mother noticed a white glare in her daughter’s left eye
• Her pupil appeared white
• After examining the child’s eye, the doctor suspected retinoblastoma
• The doctor made a referral to an ophthalmologist, for more tests such as CT scan, MRI and/or ultrasound
• The doctor reassured the mother that retinoblastoma is treatable when in sporadic form

Retinoblastoma Details

• Occurs primarily in young children aged 1-3 years
• A malignant tumor of the retina
• Most common intraocular cancer of childhood, with an incidence of 1/20,000
• Leukocoria is a characteristic of retinoblastoma: White pupil or white glow
• Other signs of retinoblastoma includes Strabismus (crossing of the eyes)
• Sporadic retinoblastoma is somatic mutations in Rb that inactivates protein function
• One eye is affected (unilateral) in Sporadic retinoblastoma
• Treatment results in no further risk of cancer
• Familial retinoblastoma involves a genetic mutation inherited in an autosomal dominant pattern
• 20-40% of cases are familial
• A mutated gene predisposes for a second copy to be mutated
• Loss of heterozygocity during childhood
• Both eyes are affected (bilateral), leading to other cancers like bone cancer

Diagnosing Retinoblastoma

• Suspicions are confirmed, and further tests are recommended following a visit to an ophthalmologist
• Ultrasound is conducted first, followed by CT or MRI
• The sporadic form is determined from no family history and a unilateral case
• A small localized lesion is identified and thus does not spread to the optic nerve Lumbar puncture is not necessary since it has not spread into the optic nerve
• A bone marrow biopsy is not necessary since it has not spread into the bone marrow

Treatment for Retinoblastoma

• Laser therapy is recommended to treat smaller lesions.
• Laser therapy can preserve vision
• Radiation

Treating Tumours

• Surgery (Enucleation)
• Chemotherapy

Cell Cycle Summary

• The cell cycle is a highly regulated process resulting in cell duplication into two identical daughter cells
• Stages include G1, S, G2, M and are controlled by checkpoints
• Passage from each stage is controlled by cyclin-dependent kinases (Cdks, regulatory subunits and inhibitors)

G1/S Transition

• G1/S transition is an essential step in the cell cycle regulated by mitogenic stimuli
• pRb is an essential regulator of the G1/S transition

Additional Cell Cycle Information

• Checkpoints control progression through the cell cycle in response to DNA damage
• Cancer cells proliferate uncontrollably from deregulation of the cell cycle
• Cdks can be targeted for cancer therapy

Apoptosis

• Apoptosis is “programmed cell death”
• Apoptosis is an active process that activates cell destruction

Apoptosis Regulates Cell Number

• Essential during development: Frog tail regression of tadpole during metamorphosis, vertebrate mouse paw/human digits through elimination of tissue between developing digits
• Regulation of organ and body size: Intestinal epithelial cells eliminated by apoptosis, and regression of mammary gland from weaning of offspring through epithelial cells eliminates by apoptosis

Apoptosis is Activated by:

• Cellular stress from lack of nutrients, damaged DNA, or malfunction of organelles Including ER stress, excessive internal Ca+ concentrations, oxydative agents, and inhibition of protein synthesis
• Signals from the immune system

Cancer and Apoptosis

• Cancer cells inhibit apoptosis allowing the cells to survive internal stress signals

Characteristics of Apoptosis

• Apoptosis is a slow and active process
• Necrosis is the opposite and is fast and passive which leaks intracellular contents
• Apoptosis is when cells undergo collapse of cytoskeleton, degradation of nuclear envelope, chromatin compaction, and formation of membrane-bound apoptotic bodies
• Alterations in cells surface attract macrophages that engulf the apoptic cells
• Intracellular contents are not released during apoptosis preventing inflammatory responses

Caspases

• Proteases that cleave an Asp-XXX bond
• Caspases are essential in apoptosis, and caspase inhibitants abrogates/eliminates apoptotic stimuli
• Most proteolytic cleavages are a result of caspases
• There's two main types of caspases: initiators and executioners.

Caspases Activation

• Synthesized as procaspases
• Need to be cleaved into active form
• Two inactive procaspase molecules form a mature caspase
• Mature caspase is a tetramer with a large subunit and small subunit

Apoptosis through the Caspase Cascade

• Oligomerization of procaspases leads to their autoproteolytic activation
• Cleavage of effector procaspases leads to their activation resulting in death
• Chromosome and DNA fragment to disassemble cell structure and disable DNA repair

Activating Apoptosis

• Apoptosis requires an apoptotic signal

Pathways of Apoptosis

• Death receptor: Extracellular ligands bind to a transmembrane receptor, a “death receptor“ that activates apoptosis
• Mitochondrial pathway: Intracellular signals activate, and the central event is depolarization of mitochondria.

Intrinsic/Mitochondrial Pathway

• Intracellular signals induce DNA damage, damage/malfunction of organelles or alteration of cellular metabolic processes
• Pro-apoptotic and anti-apoptotic members make up the Bcl-2 family
• Pro-apoptotic consists of Bax/Bak which are pro-apoptotic vs anti-apoptotic Bcl-2/Bcl-XL/Mcl-1
• Cytochrome c is released in the cytoplasm from pro-apoptotic members promoting mitochondrial membrane depolarization
• Cytochrome c forms a complex with APAF-1 and procaspase-9 named "Apoptosome“
• Apoptosome converts procaspase-9 into active caspase-9.

Regulating Apoptosis

• The decision on whether to activate apoptosis is determined by the balance between pro- and anti- apoptotic factors of the Bcl-2 family
• When same or higher level of anti-apoptotic proteins and pro-apoptotic occurs, then nothing happens
• But when there is an excess of pro-apoptotic members compared to anti-apoptotic members, apoptosis is activated

Activating Apoptosis Summary

• BH3-only proteins bind and neutralize anti-apoptotic Bcl-2 members to free the pore-forming members for multimerization at the mitochondrial membrane.

Inhibiting Apoptosis

• Cancer cells inhibit apoptosis so they can survive

Extrinsic/Death Receptor Pathway for Apoptosis

• Extracellular signals induce ligand binding to a death receptor
• Ligand then induces a conformational change on the intracellular portion of the receptor
• The intracellular receptor domain then binds with FADD (Fas-Associated protein with Death Domain) to form DISC
• Procaspase-8 is recruited into DISC associating with and self-cleaving to yield activate caspase-8

Death Receptors

• About 30 different receptors in 5 families
• Bind to ligands that are members of the Tumour (TNF) necrosis factor family

Cancer Cells Inhibit Apoptosis

• Expression of decoy receptors, lack of appropriate intracellular domains, and reduced pro-apoptotic capacity
• p53 mutations result in the inhibition of apoptosis

Cancer Treatment and Sensitizing Cancers Cells

• Chemotherapy/radiotherapy create DNA damage, inducing apoptosis.
• Cancer drugs target the extrinsic pathway
• Apoptosis inhibition in cancer cells needs to be overturned

Inhibitors in Clinical Trials

• ABT-737 is a small molecule
• Mimetic engages the groove of a Bcl-2 protein, freeing Bax or Bak to trigger membrane permeabilization and caspase activation by targeting IAPs

Targeting Apoptosis in Cancer

• Some IAPs (cIAP1 and cIAP2) block caspases, preventing substrate binding and degrading some members, like SMAC/DIABLO

Follicular Lymphoma (FL

• Cancer of the lymphatic system
• One third of all lymphomas are non-Hodgkin's lymphoma, also known as Follicular lymphoma (FL)
• The thymus and the bone marrow is the primary lymphoid tissues
• Lymphocytes are formed in lymph nodes which are secondary

Follicular Lymphoma Symptoms

Most commonly presents as:

• Painless swelling of lymph nodes
• Fever, weight loss, fatigue and sweating Lymph node areas are examined as diagnosis to see for signs of lymphocytes

Diagnosing Lymphoma

Can diagnose with:

• Blood cell count,
• Radiograph and CT Scan is performed to determine the possibility of lymphocytes
• Node biopsy
• Bone marrow aspiration can also help establish this diagnosis

Follicular Lymphoma Tumor Stages

• Stage I - One involved lymph node or lymph node area
• Stage II - Two or more involved lymph nodes or lymph node areas on the same side of diaphragm
• Stage III - Involved lymph node or lymph node areas on both sides of diaphragm
• Stage IV - Disseminated disease, such as bone marrow, liver, or central nervous system involvement

Follicular Lymphoma Development

• 85% harbor t(14;18)(q32;q21), resulting in juxtaposition of B-cell-leukemia/lymphoma-2 (Bcl-2) gene with the immunoglobulin heavy chain (IgH) locus, causing a rearrangement of the Bcl-2 gene
• Translocation of the Bcl-2 protein results in the over expression blocking apoptosis

Treating Follicular Lymphoma

• Local radiation is performed for treatment
• Chemotherapy
• Has a survival rate of 72-77% over 5 years
• Median survival of 8-10 years
• Cancer can spread to organs as lymph tissue is in the bodies

Apoptosis Summary

• An active process that triggers important cell and tissue death
• Initiated by extra- and intracellular signals
• Involves "intrinsic" activation and "extrinsic" death receptors
• Cancer cells avoid apoptosis by modulating the expression of factors

Telomeres

• Barbara McClintock concluded that chromosomes have properties
• Muller gave them their names telomeres
• They are now known as non-coding

Telomere Structure

• They're at the ends of a chromosomes
• Tandem repeats of TTAGGG and CCCTAA over hundreds to thousands of base pairs
• Shelterin shields and protects the telomeres
• Leading strand ends with overhang

Telomere Research

• Cultured fibroblast cells have limited number of replicative cycles called Replicative senescence by Hayflick
• Cancer cells can acquire "immortality" by dividing

Telomere and Cell Division

• Telomeres shorten over time because they can't be fully processed due to the nature of the lagging strand synthesis
• Around 4 Kbp of telomere shortening results in "crisis" or cell senescence
• Cell division can result in shorter telomeres depending on number of telomeres
• If the cell is a fibroblast, then this activates senescence
• If the cell is anything else, then this can activate apoptosis

Shelterin Complex

• Essential to telomere function and hides them from cells
• Telomere shortening destabilizes the telomeric loop

When Telomeres Break

• Cellular levels of p53 must be lowered to survive
• Cancer needs TERT
• Telomere shortening may result in senescence
• De-protection leads to end-fusions mistaken as DNA damage

Telomerase

• Adds telmeric repeats to the 3' ends of the chromosome
• Germ/stem cells express telomerase
• Most cancerous cells contain 90% telomere and reactivate high levels of telomere
• Tumors initially have shorter telomeres because they are re-activating

Telomerase Inhibitors

• 1: TERT expressing immune cells product anti-tumor antigens
• 2: Bind to TERT and result in gradual telomere attrition

Premature Aging Syndromes

• Premature ageing syndromes are characterized by short telomeres, including Dykeratosis Congenita
• DC causes genetic mutations/instability

Types of Dyskeratosis Congenita

• Skin and nail abnormalities
• Leukoplakia
• Bone Marrow Failure
• Death
• Genetic testing identifies the majority of cases, DC is due to issues in telomere length maintanence