1. Smad4 in Colon & Pancreatic Cancers
Michael Cox

2. Smad4 gene alterations in various types of human cancer
M. Miyaki, T. Kuroki / Biochemical and Biophysical Research Communications 306 (2003) 799–804

3. Familial juvenile polyposis
Stem cells reside at crypt bottom.
Cells differentiate as they progress upward from crypt bottoms.
Mutations in Smad4 can lead to polyp formation in crypts.
Elena Sancho, Eduard Batlle, and Hans Clevers Annu. Rev. Cell Dev. Biol :695–723

4. Familial juvenile polyposis
Autosomal dominant disease.
Hamartomatous polyps-benign but can lead to malignancy.
Symptoms: Bleeding, diarrhea, abdominal cramps, anemia.
Usually occurs in first decade of life.
Smad4 mutations are only one of many causes.
No significant risk of cancer until multiple polyps form (>5).
Can be treated by colectomy.

5. Smad4 Gene
Member of Smad family (1-8), which mediates TGFβ signaling pathway.
Only known mammalian co-Smad
552 amino acids long
Contains 11 exons
Locus at 18q21.1
Homology to Drosophila Mad & C. elegans sma-2/3/4.

6. TGFβ Signaling Pathway
K. Pardali, A. Moustakas / Biochimica et Biophysica Acta 1775 (2007) 21–62

7. Smad4 Protein Acts as trimer, forms complexes with Smad2/3
M. Miyaki, T. Kuroki / Biochemical and Biophysical Research Communications 306 (2003) 799–804
Acts as trimer, forms complexes with Smad2/3
Smad2/3/4 proteins contain MH1 & MH2 regions at amino & carboxyl-terminals, respectively.
MH1: DNA binding
MH2: homo- and hetero-oligomerization
Smad4 binding element (SBE): 5’-GTCTAGAC-3’

8. Smad4 Knockout Mice Homozygous mutants embryonic lethal at day 7.5
Disorganized endoderm and extraembryonic regions.
Could rescue embryos with wt-visceral endoderm in tetraploid aggregation experiments.
Rescued embryos had defects in anterior/posterior patterning.
Could find no strong correlation between embryonic functions and tumor suppressor role as adult.
Sirard et al. GENES & DEVELOPMENT 12:107–119 © 1998

9. Smad4 Knockout Mice
Smad4 heterozygotes (+/-) were fertile and normal up to 1 year old.
50 weeks- gastric polyp development in 3 of 15 Smad4 (+/-) mice.
100 weeks- gastric polyp development in 15 of 15 Smad4 (+/-) mice.
Duodenal polyps in mice of 50+ weeks.

10. Role in Cancer Progression
APC-Adenomatosis polyposis coil
Taketo et al.- meiotic recombination to form APC/Smad4 cis-heterozygotes.
APC (+/-) Smad4 (+/-) polyps much larger than APC (+/-) Smad4 (+/+) mutants.
Development into adenocarcinomas in cis-compounds, not in simple APC mutants.
Inactivation of Smad4 results in malignant progression of intestinal polyps initiated by activation of APC gene.
M. Miyaki, T. Kuroki / Biochemical and Biophysical Research Communications 306 (2003) 799–804

11. Role in Cancer Progression
M. Miyaki, T. Kuroki / Biochemical and Biophysical Research Communications 306 (2003) 799–804

12. Overexpression of Smad4 in Pancreatic Cancer
Overexpression of Smad4 using a viral vector lead to suppressed tumor growth in certain pancreatic cancer cell lines
Peng et al. Clinical Cancer Research Vol. 8, , November 2002

13. Smad4 restoration in colon cancer cells leads to suppression of Wnt/β-catenin and migragtion.
Xiaoxiao Tian et al. Biochemical and Biophysical Research Communications Volume 380, Issue 3, 13 March 2009, Pages

14. Smad4 Inhibition 1) Inhibition by Smad6/7
M. Miyaki, T. Kuroki / Biochemical and Biophysical Research Communications 306 (2003) 799–804
1) Inhibition by Smad6/7
2) Removal from nucleus by monoubiquitination.
Ectodermin/TiF1γ-ubiquitinase
FAM/Usp9x-deubiquitinase
Jeffrey L. Wrana1,*Cell 136, January 9, 2009 ©2009 Elsevier Inc.

15. Smad4 Summary
Only mammalian co-Smad of the Smad family. An important molecule in the TGFβ signaling pathway.
Common in JPS, pancreatic, & colon cancers.
Loss of Smad4 function occurs at later stages of malignancy.
Overexpression can suppress tumor growth and migration in certain cancer cell lines.
Inhibited by Smad6/7 and monoubiquitination.