1. hCDC4 & Endometrial Carcinoma Diana Rhyne

2. hCDC4 – Gene Information  Homologs in S. cervisiae, Drosophilia, and C. elegans  Tumor Suppressor  Expressed in most human tissues  Located on Chromosome 4q31.3  Located in cell nucleus  Also called Fbxw7, Ago, Fbw7 http://www.genecards.org/cgi-bin/carddisp.pl?gene=FBXW7

3.  3 classes of F-box proteins: Fbw, Fbl, Fbx  Fbw7 class has WD-40 repeats  WD repeats are tryptophan and aspartic acid sequences  Substrate Recognition for ubiquitin-mediated proteolysis CDC4 is an F-box protein F-box Domain WD Repeats Moberg, K. et al. Archipelago regulates Cyclin E levels in Drosophilia and is mutated in human cancer cell lines. Nature 413, 311-316 (2001).

4. F-box proteins are found in E3 ligases  F-box binds to adaptor protein  WD repeats bind to substrate  Forms SCF Complex (Skp, Cullin, and F-box protein) Reed, S. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature Reviews 4, 855-864. Substrate recognition Bridging protein Scaffolding Protein Ubiquitin conjugating enzyme CDC4

5.  Mutations generally found on one of WD repeats, or in F-box  Mutations usually missense mutations or premature Stop codons  Mutations hinder substrate recognition hCDC4 Mutations Moberg, K. et al. Archipelago regulates Cyclin E levels in Drosophilia and is mutated in human cancer cell lines. Nature 413, 311-316 (2001). Rajagopatan, H. et al. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004).

6. CDC4 targets Cyclin E  Accumulates at G1-S transition  Activates CDK2  Critical for initiation of DNA Synthesis  If Cyclin E is stable, get continued division, no quiescence The Huber Lab. http://web.unbc.ca/~huber/biol424blog/ Accessed March 2007. http://web.unbc.ca/~huber/biol424blog/

7. hCDC4 & Cyclin E Pathway Cyclin E destruction pathway. www.Biocarta,comwww.Biocarta,com Accessed march 2007. FBW7= hCDC4

8. CDC4 Mutants show Genetic Instability  hCDC4 -/- cell lines show genetic instability including spindle dysfunction, micronuclei, and aneuploidy Rajagopatan, H. et al. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004). Spindle dysfunction Micronucleus

9. CDC4 -/- Mouse Knockout  CDC4 identified in mice screen for p53-dependent genes  CDC4-/- embryos lethal at 10.5 days  Mutants had complications such as deficiencies in hematopoietic, vascular, and heart chamber development  Elevated levels of Cyclin E were found, as well as inappropriate DNA synthesis

10. CDC+/- Mouse  Haploinsufficient tumor suppressor gene  CDC4+/- mice show greater susceptibility to tumorigenesis, BUT, most tumors retain and express the wild type allele (no LOH)  Mutant mice get range of tumors in epithelial tissues, such as lung, liver, and ovary tumors

11. CDC4 & Human Cancer  hCDC4 is deleted in ~30% of all human cancers  hCDC4 mutations also seen in breast and ovarian cancers  Sporadic Cancer (appears to have no familial inheritance)  hCDC4 is mutated in ~16% of human endometrial cancer “+” means hCDC4 mutation was present

12. Endometrial Cancer  Peak incidence occurs at age 50-65, 40,000 American women diagnosed each year  Risk factors may include exposure to chronic, unopposed estrogen (as would occur with anovulation), or obesity  Treatment involves surgery (hysterectomy), which is >90% effective in removing all cancer in Stage I patients  Prevention includes hormone therapy in post-menopausal women Endometrial Carcinoma. http://www.obgyn.ufl.edu/obgyn101/Objectives/learni ng_objectives.htm Accessed April 2007. http://www.obgyn.ufl.edu/obgyn101/Objectives/learni ng_objectives.htm

13. …In Review  hCDC4 is an F-box protein involved in the E3 ligase complex  hCDC4 functions to target Cyclin E for ubiquitination (inhibits Cyclin E)  When hCDC4 is mutated, get increase in susceptibility to tumorigenesis, especially in endometrial tissues

14. References: Article References:  Mao, J. et al. Fbxw/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene. Nature 432, 775-779 (2004).  Moberg, K. et al. Archipelago regulates Cyclin E levels in Drosophilia and is mutated in human cancer cell lines. Nature 413, 311-316 (2001).  Rajagopatan, H. et al. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004).  Reed, S. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature Reviews 4, 855-864 (2003).  Spruck, C. et al. hCDC4 gene mutation in endometrial cancer. Cancer Research 62, 4535-4539 (2002).  Strohmaier,H. et al. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413, 316-322 (2001).  Tetzlaff, M. et al. Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein. PNAS 101, 3338-3345 (2004). Website References:  Cyclin E destruction pathway. www.Biocarta,com Accessed March 2007.www.Biocarta,com  Endometrial Cancer. www.mayoclinic.com Accessed April 2007.www.mayoclinic.com  Endometrial Carcinoma. www.obgyn.ufl.edu/obgyn101 Accessed April 2007.www.obgyn.ufl.edu/obgyn101  F-box and WD domain protein 7; Fbxw7. http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=606278 Accessed March 2007. http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=606278  Gene Card for Fbwx7. http://www.genecards.org/cgi-bin/carddisp.pl?gene=FBXW7 Accessed March 2007.http://www.genecards.org/cgi-bin/carddisp.pl?gene=FBXW7