ARF and p16: One Locus, Two Tumor

Suppressors

Brandon Bunker

Biology 445

Discovering ARF

ARF was discovered by a postdoc (Dawn Quelle) in the lab of Dr. Charles Sherr. Dawn was trying to clone the mouse p16 gene. Quick review…

CyclinD

CDK4CDK4

p16

p16

ARF Discovery Cont.

Transfected mouse cDNA into cells. Used known segment of p16 as probe.

Result: TWO genes! One was p16. One was unknown.

Overexpression of the unknown gene: Cell cycle arrest: The new gene was TUMOR

SUPPRESSOR!

http://www.sciencewatch.com/march-april2001/sw_march-april2001_page3.htm Accessed on March 24, 2007

• ARF and p16 share NO sequence homology.

• This economy of space is seen frequently in viruses- extremely rare in mammals.

Sherr, C.J. Genes Dev. 1998, 12, 2984-2991.

The p16-ARF locus

p16 reading frame: THE RED FOX HAD ONE HAT

ARF reading frame: HUH ARE DFO XHA DON EHA T

ARF Knockouts

ARF -/- Mouse Epidermal Fibroblasts (MEFs): Bypass senescence Become oncogenic

with ras over-expression

Less responsive to contact inhibition

Kamijo, T; et al. Cell, 1997, 91, 649-659.

ARF knockout mouse

Non-essential geneNormal development, but…

Blindness Spontaneous tumors (33%) Rapidly inducible cancers Mice died of cancer in 10-20 weeks.

Kamijo, T; et al. Cell, 1997, 91, 649-659.

McKeller, R.N.; et al. PNAS, 2002, 99(6), 3848-3853.

Cancer Types in Arf null mice

Kamijo, T; et al. Cell, 1997, 91, 649-659.

Analysis of ARF mutants

Cancers in ARF -/- mice Sporadic lymphomas Fibrosarcomas

Death occurred in 10-20 wks.

Cancers in p53 -/- mice Sporadic lymphomas Osteosarcomas

Death occurred in 10-20 wks.

Kamijo, T; et al. Cell, 1997, 91, 649-659.

Donehower, L.A., et al. Nature, 1992, 356, 215-221.

• p53 -/- cells do not respond to ARF over-expression.

• Tumors null for both p53 and ARF are rare.

Role and Importance of p53

Tumor suppressor Activated by:

DNA damage Deregulated oncogene expression

Initiates cell cycle arrest Gives cell time to repair its DNA

Initiates apoptotic response Mutated in ~50% of all cancers

Donehower, L.A., et al. Nature, 1992, 356, 215-221.

How does ARF work?

• ARF interacts with Mdm2.

• Remember Mdm2??

• E3 ubiquitin ligase that targets p53 for destruction.

• ARF prevents Mdm2 shuttling ubiquitinated p53 into the cytoplasm. Pomerantz, J. et al. Cell, 1998, 92, 713-723.

Llanos, S.; et al, Nat. Cell Bio, 2001, 3, 445-451.

Tao, W.; Levine, A.J. PNAS, 1999, 96, 6937-6941.

ARF Pathway and the Players

• ARF- Negatively regulates Mdm2

• Mdm2- E3 Ubiquitin Ligase of p53

• p53- Triggers cell cycle arrest or apoptosis

Growth arrest/ apoptosis

Eischen, C.M. Genes Dev. 1999, 13, 2658-2669.

How is ARF regulated?

• E2F protein/mRNA ↑ ARF ↑.

• Kinetics of ARF transcription is slow.

Bates, S. Nature, 1998, 395, 124-125.

ARF Regulation: Normal

Sherr, C.; Lowe, S.W. Curr Opin, 2003, 13, 77-83.

ARF Regulation: Stressed

Sherr, C.; Lowe, S.W. Curr Opin, 2003, 13, 77-83.

What this means for us

The p16/ARF locus is mutated in ~40% of all cancers.

Mutation Types: Point mutations- affects p16. Deletions- affects both. Hypermethylation- affects ARF, p16, or both.

ARF-only knockout mutations are rare.

Kim, W.Y; Sharpless, N. E. Cell, 2006, 127(2), 265-275.

ARF mutations in Human Cancer

Germline Mutations Melanoma predisposition

Cancers with ARF/p16 locus deletions:

Other Problems: Chemotherapy resistant cancers Highly aggressive tumors

Laud, K.; et al, J. Med. Genet.2006, 43(6), 39-47.

Kasahara, T. Anticancer Res. 2006, 6, 4299-4305.

Kim, W.Y; Sharpless, N. E. Cell, 2006, 127(2), 265-275.

• Lung • Brain

• Pancreatic • Skin

• Kidney • Colon

Summary

ARF and p16 are tumor suppressors that use different reading frames of the same locus.

ARF regulates p53 by inhibiting Mdm2. ARF/p16 locus is deleted or mutated in many

different cancer types.

Questions?

Comments?

Concerns?