1. SMAD4/DPC4: A Tumor Suppressor James Brooks March 23 rd, 2006

2. Found on chromosome 18 in humans, encodes 11 exons

3. A 552 amino acid protein found in the cytoplasm

4. SMAD Structure: MH1 and MH2 Domains

5. SMAD proteins exist as homo-oligomers The quaternary structure of SMADs consists of three monomers attached at their MH2 domains (homo-oligomers) MH2 domains are also responsible for hetero- oligomerization

6. SMAD4 is expressed at all stages of development Above: Northern blot (RNA) of SMAD4 (3.8 kb) at various stages of development in mouse (L32 is a loading control)

7. SMAD4 mutant mouse embryos exhibit early embryonic lethality By day 6.5, SMAD4 mutant mouse embryos appear much smaller than wild type ones; the difference in size is attributed to reduced cell proliferation By day 7.5, SMAD4 mutant mouse embryos are lethal

8. SMAD4 is the convergence point of several TGF-β pathways

9. SMAD4 activity regulates cell proliferation in the TGF-β signaling pathway

10. Left: SMAD4 distribution in absence of TGF-β Right: SMAD4 after translocation into nucleus following stimulation by TGF-β

11. SMAD4 activity regulates cell proliferation in the TGF-β signaling pathway

12. The Smad4 gene is often mutated in the MH2 domain

13. Familial Juvenile Polyposis (FJP) Clinical manifestation of germline SMAD4 mutation, phenotypically similar to that of familial adenomatous polyposis (FAP) from loss of APC function Autosomal Dominant inheritance pattern SMAD4 is mutated in approximately 50% of FJP cases Agrees with Knudson’s two-hit model Above: A juvenile polyp

14. Smad4 mutation is often seen in late-stage malignancies that have metastasized

15. This makes sense!

16. To sum things up… SMAD4 is a tumor suppressor that regulates cell proliferation through interaction with transcription factors When inactive, it is located in the cytosol. It is translocated into the nucleus once activated Loss of SMAD4 is associated with familial juvenile polyposis, and is often absent in malignant pancreatic and colon carcinomas

17. References Karyotype: http://www.amnh.org/learn/musings/FA01/ia/HW_01.jpghttp://www.amnh.org/learn/musings/FA01/ia/HW_01.jpg SMAD4 Crystal Structure: Shi et. al. A structural basis for the mutational inactivation of the tumour suppressor Smad4. 3 Jul 1997. Nature. SMAD MH1 and MH2 Domain Structure: http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/roth/discussion.html http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/roth/discussion.html Smad Mediated TGF-Beta Signaling Pathway: Miyaki et. al. Role of Smad4 (DPC4) inactivation in human cancer. 2003. Biochemical and Biophysical Research Communications. Smad4 Northern Blot: Sirard et. al. The tumor suppressor gene Smad4/DPC4 is required for gastrulation and later for anterior development of the mouse embryo. 1998. Genes and Development. Wild-type and Smad Mutant embryos: Sirard et. al. The tumor suppressor gene Smad4/DPC4 is required for gastrulation and later for anterior development of the mouse embryo. 1998. Genes and Development. TGF-Beta Superfamily Signaling Pathway: Waite and Eng. From developmental disorder to heritable cancer: it’s all in the BMP/TGF-β family. 2003. Nature. TGF-Beta Signaling Pathway: Lodish et. al. Molecular Cell Biology p. 957 SMAD4 Localization Photos: http://biochemistry.utoronto.ca/research/signal_transduction_and_regulation.html http://biochemistry.utoronto.ca/research/signal_transduction_and_regulation.html

18. References, Continued Mutations in SMAD4: Miyaki et. al. Role of Smad4 (DPC4) inactivation in human cancer. 2003. Biochemical and Biophysical Research Communications. Juvenile Polyp: http://www.surgery.uiowa.edu/surgery/physpubs/familialjuvenilepolyposis.htmlhttp://www.surgery.uiowa.edu/surgery/physpubs/familialjuvenilepolyposis.html SMAD4 in Cancer: Miyaki et. al. Role of Smad4 (DPC4) inactivation in human cancer. 2003. Biochemical and Biophysical Research Communications.