1. hcdc4 (archipelago) Endometrial Cancer Andrew Marshall April, 1, 2004

2. hcdc4 Highly conserved throughout evolution Highly conserved throughout evolution –Ago (Drosophila), SEL-10 (nematode and mouse), and cdc4 (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) (Strohmaier (2001))

3. hcdc4 and Cyclin E Through various screens, we know the WD Domain serves as a dock for Cyclin E Through various screens, we know the WD Domain serves as a dock for Cyclin E Together with other molecules forms a functional SCF-type protein ubiquitination ligase Together with other molecules forms a functional SCF-type protein ubiquitination ligase

4. What do we know about Cyclin E?

5. Cyclin E and Cell Cycle Control The up and down cycles of Cyclin E are responsible for the G1-S Transition The up and down cycles of Cyclin E are responsible for the G1-S Transition But how are these levels controlled? But how are these levels controlled?

6. The Ubiquitination Cascade Following the E1 to E2 and finally to E3 Ubiquitin Pathway Following the E1 to E2 and finally to E3 Ubiquitin Pathway –Thus, marking the target (cyclin E) with a polyubiquitin chain Degradation by the Proteosome Degradation by the Proteosome Tumor Suppressor (negative regulator of cell proliferation) Tumor Suppressor (negative regulator of cell proliferation)

7. Putting It All Together

8. What’s the Big Deal? We Know We Know –hcdc4 controls Cyclin E levels through the ubiquitin cascade –Cyclin E levels control the cell cycle –The cell cycle is..... Important

9. Real Life Knockout Models

10. Sorry… not that kind

11. The hcdc4 -/- (SEL-10) mouse is inviable The hcdc4 -/- (SEL-10) mouse is inviable –Halted development due to meiotic mistakes very early in development Drosophila Model (Ago) Drosophila Model (Ago) –Over proliferation due to failure to exit cell cycle –Photoreceptor problems (Moberg 2001)

12. Chromosomal Instability Aneuploidy Aneuploidy Gene Amplification Gene Amplification Chromosome Rearrangements, Inversions, Translocations, and Deletions Chromosome Rearrangements, Inversions, Translocations, and Deletions Spindle Apparatus failure Spindle Apparatus failure And in this Case, also Micronuclei And in this Case, also Micronuclei (Rajagopalan (2004))

13. Checkpoints The Cell Cycle Must The Cell Cycle Must –Introduce no mistakes mistakes –Maintain diploidy Am I Big Enough? Am I Big Enough? Am I OK? Am I OK?

14. Prove It! A definitive association between hcdc4 mutants and overexpression of Cyclin E A definitive association between hcdc4 mutants and overexpression of Cyclin E –Without overexpression of Cyclin E mRNA LOH LOH Restoration of correct cyclin E expression via retroviral transduction Restoration of correct cyclin E expression via retroviral transduction (Strohmaier (2001)) (Reed (2004))

15. Accumulation, Accumulation, Accumulation Cancer takes more than one mutation Cancer takes more than one mutation Increased chances with Increased chances with –Increased Cells Increased proliferation due to constitutively active Cyclin E pushing cells from G1 to S (proliferative advantage) Increased proliferation due to constitutively active Cyclin E pushing cells from G1 to S (proliferative advantage) –DNA Problems Chromosomal Instability due to that same push towards the S Phase Chromosomal Instability due to that same push towards the S Phase Accelerated lost of heterozygosity Accelerated lost of heterozygosity

16. What Do We Get? Cancer! More specifically, endometrial cancer More specifically, endometrial cancer –A cancer of the lining of the uterus

17. Endometrial Cancer How do we Really Know hcdc4/Cyclin E is the cause? How do we Really Know hcdc4/Cyclin E is the cause? (Spruck (2002))

18. Not So Fast Not all endometrial cancers contain the hcdc4 mutation Not all endometrial cancers contain the hcdc4 mutation –20% of cases with elevated Cyclin E levels –Although, Those that do are associated with increased metastasis and severity Possible Reasons Possible Reasons –Cyclin E regulated by different pathways –hcdc4 mutations of WD40 region and amino terminus Different variants of hcdc4 Different variants of hcdc4 (Cassia (2003))

19. Still a Mystery Although a great deal of research has been done on hcdc4 of late, many of its functions remain to be understood Although a great deal of research has been done on hcdc4 of late, many of its functions remain to be understood Notch Pathway Notch Pathway Alzheimer's Alzheimer's –Different Targets and Different Roles

20. Works Cited Cassia, Raul, Moreno-Bueno, Gema, Rodriguez-Perales, Sandra, Hardisson, David, Cigudosa, Juan C., and Palacios M.D., Jose. Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma. The Journal of Pathology 201 589-595 (2003). Cassia, Raul, Moreno-Bueno, Gema, Rodriguez-Perales, Sandra, Hardisson, David, Cigudosa, Juan C., and Palacios M.D., Jose. Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma. The Journal of Pathology 201 589-595 (2003). Moberg, Kenneth H., Bell, Daphne W., Wahrer, Doke C.R., Haber, Daniel A., and Hariharan, Iswar K. Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413, 311-316 (2001). Moberg, Kenneth H., Bell, Daphne W., Wahrer, Doke C.R., Haber, Daniel A., and Hariharan, Iswar K. Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413, 311-316 (2001). Rajagopalan, Harith, Jallepalli, Prasad V., Rago, Carlo, Velculescu, Victor E., Kinzler, Kenneth W., Vogelstein, Bert, and Lengauer, Christoph. Inacivation of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004). Rajagopalan, Harith, Jallepalli, Prasad V., Rago, Carlo, Velculescu, Victor E., Kinzler, Kenneth W., Vogelstein, Bert, and Lengauer, Christoph. Inacivation of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004). Reed, Susanna Ekholm, Spruck, Charles H., Sangfelt, Olle, van Drogen, Frank, Mueller-Holzner, Elisabeth, Widschwendter, Marrtin, Zetterberg, Anders, and Reed, Steven I. Mutation of hCDC4 Leads to Cell Cycle Deregulation of Cyclin E in Cancer. Cancer Research 64, 795-800 (2004). Reed, Susanna Ekholm, Spruck, Charles H., Sangfelt, Olle, van Drogen, Frank, Mueller-Holzner, Elisabeth, Widschwendter, Marrtin, Zetterberg, Anders, and Reed, Steven I. Mutation of hCDC4 Leads to Cell Cycle Deregulation of Cyclin E in Cancer. Cancer Research 64, 795-800 (2004). Spruck, Charles H., Strohmaier, Heimo, Sangfelt, Olle, Muller, Hannes M., Hubalek, Michael, Muller-Holzner, Elisabeth, Marth, Christian, Widscwendter, Martin, and Reed, Steven I. hCDC4 Gene Mutations in Endometrial Cancer. Cancer Research 62, 4535-4539 (2002). Spruck, Charles H., Strohmaier, Heimo, Sangfelt, Olle, Muller, Hannes M., Hubalek, Michael, Muller-Holzner, Elisabeth, Marth, Christian, Widscwendter, Martin, and Reed, Steven I. hCDC4 Gene Mutations in Endometrial Cancer. Cancer Research 62, 4535-4539 (2002). Strohmaier, Heimo, Spruck, Charles H., Kaiser, Peter, Won, Kwang-Al, Sangfelt, Olle, and Reed, Steven I. Human F-box protein hCdc4 targets Cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413, 316-322 (2001). Strohmaier, Heimo, Spruck, Charles H., Kaiser, Peter, Won, Kwang-Al, Sangfelt, Olle, and Reed, Steven I. Human F-box protein hCdc4 targets Cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413, 316-322 (2001).