hCDC4 & Endometrial Carcinoma Diana Rhyne

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

 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, (2001).

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, Substrate recognition Bridging protein Scaffolding Protein Ubiquitin conjugating enzyme CDC4

 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, (2001). Rajagopatan, H. et al. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, (2004).

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. Accessed March

hCDC4 & Cyclin E Pathway Cyclin E destruction pathway. Accessed march FBW7= hCDC4

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, (2004). Spindle dysfunction Micronucleus

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

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

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

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. ng_objectives.htm Accessed April ng_objectives.htm

…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

References: Article References:  Mao, J. et al. Fbxw/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene. Nature 432, (2004).  Moberg, K. et al. Archipelago regulates Cyclin E levels in Drosophilia and is mutated in human cancer cell lines. Nature 413, (2001).  Rajagopatan, H. et al. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, (2004).  Reed, S. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature Reviews 4, (2003).  Spruck, C. et al. hCDC4 gene mutation in endometrial cancer. Cancer Research 62, (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, (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, (2004). Website References:  Cyclin E destruction pathway. Accessed March  Endometrial Cancer. Accessed April  Endometrial Carcinoma. Accessed April  F-box and WD domain protein 7; Fbxw7. Accessed March  Gene Card for Fbwx7. Accessed March