Cyclin-Dependent Kinase Inhibitors, p53 and G1 arrest

Cyclin-Dependent Kinase Inhibitors, p53, and G1 Arrest

Lecture 17 Overview

  • This lecture focuses on how the cell cycle is stopped, the roles of Cyclin-Dependent Kinase Inhibitors (CKIs), and the tumor suppressor p53.

Key Concepts

  • Cyclin/CDK Regulation: Cyclins and CDKs are regulated via phosphorylation, ubiquitination, and CKIs.
  • CKIs and RB Pathway: CKIs are linked to the Retinoblastoma (RB) pathway.
  • Regulation of CKIs: CKIs (p15, p16, p21, p27) are activated/inactivated by signals such as TGFβ and Akt.
  • CKIs Upregulation via p53: CKIs are upregulated in response to DNA damage via p53, a transcription factor and tumor suppressor.
  • p53 Identification: Discusses the identification of p53 and its ambiguous initial classification as an oncogene versus a tumor suppressor.
  • Comparison of p53 and RB: Compares and contrasts the roles of p53 and RB.

Learning Objectives

  1. Cell Cycle Arrest: Understand how the cell cycle is stopped.
  2. Cancer Genetics: Apply cancer genetics to understand molecular pathways regulating the cell cycle.
  3. RB-Cyclin-CKI Pathway: Identify cancers with an altered RB-cyclin-CKI pathway.
  4. Differentiation vs. Proliferation: Explain the molecular basis of the inverse relationship between differentiation and proliferation.
  5. p53 Identification: Describe how p53, the most frequently mutated gene in human cancer, was identified.
  6. p53 Initial Misclassification: Explain why the tumor suppressor p53 was initially thought to be an oncogene.

Regulation of Cyclin-CDKs

  • Cyclin-CDKs are regulated by multiple mechanisms:
    • Phosphorylation (PO4):
    • Ubiquitination (Ub):
    • Cyclin-dependent Kinase Inhibitors (CKI): (p21, p27, p15, p16).
A) Phosphorylation – Activating and Inactivating
  • Specific Phosphorylation Sites:
    • Tyrosine 15 (Tyr 15): Inactivating phosphorylation.
    • Threonine 161 (Thr 161): Activating phosphorylation.
  • Cdc25 Phosphatase: Removes inactivating phosphate groups.
  • Example: CDK/Cyclin B Activation at G2/M Transition:
    • MPF (Maturation Promoting Factor) = CDK1 + Cyclin B
B) Ubiquitination
  • Cyclins are degraded via the ubiquitin/proteasome pathway.
  • M-Cdk and M-cyclin are involved in mitosis.
  • S-cyclin and S-Cdk are involved in DNA replication.
C) CKI: Cyclin-Dependent Kinase Inhibitors
  • CKIs are small proteins that stoichiometrically bind and inhibit cyclin-CDK complexes.
  • CKIs are regulated by phosphorylation and ubiquitination.

CKI Families and Their Targets

  • Two families of CKIs:

    • INK4 (Inhibitors of CDK4):
      • p15, p16, p18, p19
      • Block association with cyclin D.
    • CIP (Inhibitors of CDK2):
      • p21, p27, p57
      • Block association with cyclins A/E.

  • Specific CKI targets:

    • Cyclin D/CDK4 complexes are inhibited by p16 (Ink4a).
    • Cyclin E/CDK2 and Cyclin A/CDK2 complexes are inhibited by p21, p27, etc.

CKIs and the RB Pathway

  • Mitogens cause translocation to the cytoplasm and degradation of CKIs.
  • E2F activates transcription of genes required to push the cell cycle forward.

*Active p16 -->> Cyclin D1 + Cdk4
Cyclin-CDK phosphorylates RB

  • Active RB (+HDAC): Represses S-phase genes.

  • Inactive RB: Releases E2F, activating S-phase genes.

  • Pathway Steps:

    • Mitogen → Active p16 → Cyclin D1/Cdk4 → Phosphorylates RB → Inactive RB → Active E2F → Transcription of S-phase genes.

Cancer Genetics and the RB Pathway

  • Evidence from cancer genetics shows a functional link between p16 and RB.

    • p16 (tumor suppressor): Inactivated in familial cancer syndromes (e.g., melanoma).
    • Cyclin D (and E) (oncogene): Amplified in many cancers, especially breast cancer.
    • RB (tumor suppressor): Inactivated in retinoblastoma and many cancers.
    • E2F (oncogene): Overexpression can cause a cell to enter S phase.

  • In tumors, inactivation of either RB or p16 is necessary but typically not both, indicating they are on the same pathway.

RB-Cyclin-CKI Pathway Mutations in Tumors

  • Many human tumors have mutations in the RB-cyclin-CKI pathway.

    • p15, p16, p21:
      • Mutated in esophageal, squamous cell, lung, bladder, pancreatic carcinoma, glioblastoma, familial melanoma.
      • Amplified in glioblastoma melanoma.
      • Overexpressed in esophageal, breast & squamous cell carcinoma, B cell lymphoma.
    • p27:
      • Mutated in retinoblastoma, small cell lung carcinoma, sarcomas, bladder carcinoma.
      • Overexpressed in breast carcinoma, HPV E7.

  • HPV E7 protein binds to RB, releasing E2F, which activates cell cycle and proliferation genes, present in cervical carcinoma.

Checkpoints and CKI Regulation

  • CKIs block the cell cycle at checkpoints and are activated/inactivated by a variety of signals.

  • Signals that activate checkpoints:

    • Contact inhibition.
    • Factors that stimulate differentiation (e.g., TGFβ via SMADs increase the expression of CKI genes).

  • Signals that inactivate checkpoints:

    • Factors that stimulate proliferation (e.g., Akt).

TGFβ and CKI Expression

  • TGFβ plays a role in differentiation by activating the expression of p15.
  • Pathway:
    • TGFβ binds and activates a receptor serine/threonine kinase.
    • SMADs (cytosolic transcription factors) are phosphorylated in response to TGFβ.
    • P-SMADs dimerize and translocate to the nucleus.
    • They activate expression of CKI p15 (and to a lesser extent p21), which inhibits CDK/cyclins.
  • Inactivating mutations in any component of the TGFβ pathway can lead to cancer.

Akt/PKB and CKI Regulation

  • Akt/PKB (PI3 kinase pathway activated by mitogens) phosphorylates CKI p21, causing it to be localized in the cytoplasm.
  • Cytoplasmic localization of a CKI promotes cell cycle progression.

p53 and DNA Damage Response

  • CKIs are upregulated in response to DNA damage via p53, a transcription factor and tumor suppressor.
  • It is important to upregulate CKIs in response to DNA damage to halt the cell cycle and allow for DNA repair.

p53 Mutations in Cancer

  • p53 is mutated in most Li-Fraumeni cancer patients, a family with a very high rate of cancers of different types.
  • p53-/- mice also have a very high rate of cancer, even without exogenous mutagens.
  • p53 is the most frequently mutated gene in human cancer:
    • Mutated in >50% of all human tumors and >90% in certain cancers.
    • >45,000 mutated p53 genes sequenced, with mutations at >140 different sites.

p53: Tumor Suppressor or Oncogene?

  • Properties indicating p53 is a tumor suppressor:
    • Mutated in a familial cancer syndrome.
    • Deletion in mice leads to increased tumors.
    • Activates another tumor suppressor (p21).
    • Missense mutations found in human tumors.
  • Initial misclassification as an oncogene:
    • Early findings suggested p53 from transformed cell lines cooperated with activated Ras in transformation assays.
    • Injection of an anti-p53 monoclonal antibody into cells lead to growth arrest (incorrectly assumed it inhibited p53 function).
    • Cells induced p53 when stimulated by growth factors.
    • Expression of p53 peaks at G1/S, consistent with a role in promoting S phase entry.

Re-evaluation of p53 as a Tumor Suppressor

  • 1987: Studies indicated virally-induced tumors correlated with the virus's insertion into the p53 locus → inactivation of the p53 gene.
  • 1989: Re-analysis revealed that p53 cDNAs that promoted Ras transformation were mutant; wild-type p53 antagonized transformation.
  • 1990: p53 knockout mice were made and found to contain lots of tumors.
  • 1995: Antibodies used in microinjection experiments were found to stimulate (not inhibit) p53-mediated transcription.
  • Post 1995: Sequencing revealed a large number of p53 missense mutations in colorectal cancers.

Accolades and Lessons Learned

  • Accolades:
    • Science magazine’s “Molecule of the Year” for 1993.
    • Designated “Guardian of the Genome” and “Cancer Killer.”
  • Lessons:
    • It is risky to clone your gene from a transformed cell line, as it might be mutated.
    • Discrimination between gain and loss of function mutations requires knowing the normal function.
    • Understanding the null phenotype, i.e., knockout mice, is critical.

Similarities Between p53 and RB

  • Both are tumor suppressor proteins.
    • Inherited:
      • p53: Li-Fraumeni syndrome (many different cancers).
      • RB: Familial retinoblastoma (mainly eye tumors).
    • Ubiquitous nuclear protein.
    • Role in G1 checkpoint.
    • Binds viral proteins:
      • p53: SV40 large T antigen, HPV E6, Adenovirus E1B.
      • RB: SV40 large T antigen, HPV E7, Adenovirus E1A.
    • Inhibits basal transcription.
    • Phosphorylated during cell cycle.

Differences Between p53 and RB

  • p53:
    • Present in low amounts.
    • Protein levels increase in response to stress.
    • Binds DNA directly.
    • Does not interact with E2F; interacts with other transcription factors.
    • Activates transcription.
    • Not essential for embryonic development.
  • RB:
    • Present in high amounts.
    • Protein level remains constant.
    • Does not bind DNA directly; interacts with E2F and other TFs.
    • Represses transcription.
    • Essential for embryonic development.