hcdc4 (archipelago)hcdc4 (archipelago)Endometrial CancerEndometrial Cancer

Andrew MarshallAndrew Marshall

April, 1, 2004April, 1, 2004

hcdc4hcdc4

• Highly conserved throughout evolutionHighly conserved throughout evolution– Ago (Drosophila), SEL-10 (nematode and mouse), and cdc4 Ago (Drosophila), SEL-10 (nematode and mouse), and cdc4

(yeast)(yeast)• Locus – 4q.32 (mutated in 30% human tumors)Locus – 4q.32 (mutated in 30% human tumors)• F-Box and WD40 Domains (FBXW7)F-Box and WD40 Domains (FBXW7)

– Repeated sequences of W (tryptophan) and D (aspartic acid)Repeated sequences of W (tryptophan) and D (aspartic acid)

(Strohmaier (2001))

hcdc4 and Cyclin E hcdc4 and Cyclin E

• Through various Through various screens, we know screens, we know the WD Domain the WD Domain serves as a dock for serves as a dock for Cyclin ECyclin E

• Together with other Together with other molecules forms a molecules forms a functional SCF-type functional SCF-type protein ubiquitination protein ubiquitination ligaseligase

What do we know about Cyclin What do we know about Cyclin E?E?

Cyclin E and Cell Cycle Cyclin E and Cell Cycle ControlControl

• The up and down The up and down cycles of Cyclin E cycles of Cyclin E are responsible for are responsible for the G1-S Transitionthe G1-S Transition

• But how are these But how are these levels controlled?levels controlled?

The Ubiquitination CascadeThe Ubiquitination Cascade

• Following the E1 to E2 Following the E1 to E2 and finally to E3 and finally to E3 Ubiquitin PathwayUbiquitin Pathway– Thus, marking the Thus, marking the

target (cyclin E) with a target (cyclin E) with a polyubiquitin chainpolyubiquitin chain

• Degradation by the Degradation by the ProteosomeProteosome

• Tumor Suppressor Tumor Suppressor (negative regulator of (negative regulator of cell proliferation)cell proliferation)

Putting It All TogetherPutting It All Together

What’s the Big Deal?What’s the Big Deal?

• We KnowWe Know– hcdc4 controls Cyclin E levels through the hcdc4 controls Cyclin E levels through the

ubiquitin cascadeubiquitin cascade– Cyclin E levels control the cell cycleCyclin E levels control the cell cycle

– The cell cycle is..... The cell cycle is..... ImportantImportant

Real Life Knockout ModelsReal Life Knockout Models

Sorry… not that kindSorry… not that kind

• The hcdc4 -/- (SEL-10) The hcdc4 -/- (SEL-10) mouse is inviable mouse is inviable – Halted development due Halted development due

to meiotic mistakes very to meiotic mistakes very early in development early in development

• Drosophila Model (Ago)Drosophila Model (Ago)– Over proliferation due to Over proliferation due to

failure to exit cell cyclefailure to exit cell cycle

– Photoreceptor problemsPhotoreceptor problems

(Moberg 2001)

Chromosomal InstabilityChromosomal Instability

• AneuploidyAneuploidy

• Gene AmplificationGene Amplification

• Chromosome Chromosome Rearrangements, Rearrangements, Inversions, Inversions, Translocations, and Translocations, and DeletionsDeletions

• Spindle Apparatus Spindle Apparatus failurefailure

• And in this Case, also And in this Case, also MicronucleiMicronuclei

(Rajagopalan (2004))

CheckpointsCheckpoints

• The Cell Cycle MustThe Cell Cycle Must– Introduce noIntroduce no

mistakesmistakes– Maintain diploidyMaintain diploidy

• Am I Big Enough?Am I Big Enough?

• Am I OK?Am I OK?

Prove It!Prove It!• A definitive association A definitive association

between hcdc4 between hcdc4 mutants and mutants and overexpression of overexpression of Cyclin ECyclin E– Without overexpression Without overexpression

of Cyclin E mRNAof Cyclin E mRNA

• LOHLOH

• Restoration of correct Restoration of correct cyclin E expression via cyclin E expression via retroviral transductionretroviral transduction

(Strohmaier (2001))

(Reed (2004))

Accumulation, Accumulation, Accumulation, Accumulation, AccumulationAccumulation

• Cancer takes more than one mutationCancer takes more than one mutation

• Increased chances withIncreased chances with– Increased CellsIncreased Cells

• Increased proliferation due to constitutively active Increased proliferation due to constitutively active Cyclin E pushing cells from G1 to S (proliferative Cyclin E pushing cells from G1 to S (proliferative advantage)advantage)

– DNA ProblemsDNA Problems• Chromosomal Instability due to that same push Chromosomal Instability due to that same push

towards the S Phasetowards the S Phase

• Accelerated lost of heterozygosityAccelerated lost of heterozygosity

What Do We Get?What Do We Get?

Cancer!Cancer!

• More specifically, endometrial cancerMore specifically, endometrial cancer– A cancer of the lining of the uterusA cancer of the lining of the uterus

Endometrial CancerEndometrial Cancer• How do we Really Know hcdc4/Cyclin E is How do we Really Know hcdc4/Cyclin E is

the cause?the cause?

(Spruck (2002))

Not So FastNot So Fast

• Not all endometrial cancers contain the hcdc4 Not all endometrial cancers contain the hcdc4 mutationmutation– 20% of cases with elevated Cyclin E levels20% of cases with elevated Cyclin E levels– Although, Those that do are associated with increased Although, Those that do are associated with increased

metastasis and severitymetastasis and severity

• Possible ReasonsPossible Reasons– Cyclin E regulated by different pathwaysCyclin E regulated by different pathways– hcdc4 mutations of WD40 region and amino terminus hcdc4 mutations of WD40 region and amino terminus

• Different variants of Different variants of hcdc4hcdc4

(Cassia (2003))

Still a MysteryStill a Mystery

• Although a great deal of research has Although a great deal of research has been done on hcdc4 of late, many of been done on hcdc4 of late, many of its functions remain to be understoodits functions remain to be understood

• Notch PathwayNotch Pathway

• Alzheimer'sAlzheimer's– Different Targets and Different RolesDifferent Targets and Different Roles

Works CitedWorks Cited• Cassia, Raul, Moreno-Bueno, Gema, Rodriguez-Perales, Sandra, Hardisson, David, Cigudosa, Juan C., Cassia, Raul, Moreno-Bueno, Gema, Rodriguez-Perales, Sandra, Hardisson, David, Cigudosa, Juan C.,

and and Palacios M.D., Jose. Cyclin E gene (CCNE) amplification and hCDC4 mutations in Palacios M.D., Jose. Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma. endometrial carcinoma. The Journal of PathologyThe Journal of Pathology 201 589-595 (2003). 201 589-595 (2003).

• Moberg, Kenneth H., Bell, Daphne W., Wahrer, Doke C.R., Haber, Daniel A., and Hariharan, Iswar K. Moberg, Kenneth H., Bell, Daphne W., Wahrer, Doke C.R., Haber, Daniel A., and Hariharan, Iswar K. Archipelago regulates Cyclin E levels in Archipelago regulates Cyclin E levels in DrosophilaDrosophila and is mutated in human cancer cell and is mutated in human cancer cell

lines. lines. NatureNature 413, 311-316 (2001).413, 311-316 (2001).

• Rajagopalan, Harith, Jallepalli, Prasad V., Rago, Carlo, Velculescu, Victor E., Kinzler, Kenneth W., Rajagopalan, Harith, Jallepalli, Prasad V., Rago, Carlo, Velculescu, Victor E., Kinzler, Kenneth W., Vogelstein, Bert, and Lengauer, Christoph. Inacivation of hCDC4 can cause chromosomal Vogelstein, Bert, and Lengauer, Christoph. Inacivation of hCDC4 can cause chromosomal

instability. instability. NatureNature 428, 77-81 (2004). 428, 77-81 (2004).

• Reed, Susanna Ekholm, Spruck, Charles H., Sangfelt, Olle, van Drogen, Frank, Mueller-Holzner, Reed, Susanna Ekholm, Spruck, Charles H., Sangfelt, Olle, van Drogen, Frank, Mueller-Holzner, Elisabeth, Elisabeth, Widschwendter, Marrtin, Zetterberg, Anders, and Reed, Steven I. Mutation of hCDC4 Widschwendter, Marrtin, Zetterberg, Anders, and Reed, Steven I. Mutation of hCDC4 Leads to Cell Leads to Cell Cycle Deregulation of Cyclin E in Cancer. Cancer Research 64, 795-Cycle Deregulation of Cyclin E in Cancer. Cancer Research 64, 795- 800 800 (2004).(2004).

• Spruck, Charles H., Strohmaier, Heimo, Sangfelt, Olle, Muller, Hannes M., Hubalek, Michael, Muller-Spruck, Charles H., Strohmaier, Heimo, Sangfelt, Olle, Muller, Hannes M., Hubalek, Michael, Muller-Holzner, Holzner, Elisabeth, Marth, Christian, Widscwendter, Martin, and Reed, Steven I. hCDC4 Gene Elisabeth, Marth, Christian, Widscwendter, Martin, and Reed, Steven I. hCDC4 Gene Mutations in Mutations in Endometrial Cancer. Cancer Research 62, 4535-4539 (2002).Endometrial Cancer. Cancer Research 62, 4535-4539 (2002).

• Strohmaier, Heimo, Spruck, Charles H., Kaiser, Peter, Won, Kwang-Al, Sangfelt, Olle, and Reed, Strohmaier, Heimo, Spruck, Charles H., Kaiser, Peter, Won, Kwang-Al, Sangfelt, Olle, and Reed, Steven I. Human Steven I. Human F-box protein hCdc4 targets Cyclin E for proteolysis and is mutated in a F-box protein hCdc4 targets Cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature breast cancer cell line. Nature 413, 316-322 (2001).413, 316-322 (2001).