1. Smad4 (Dpc4) in Colon Cancer
Elizabeth Jarvis
March 22, 2007

2. Objectives: I. Cell Biological Role of Smad4
II. Biological Role of Protein (What does it do for the Organism?)
III. Connection to Cancer
Colon and Pancreatic

3. I. Cell Biological Role of Smad4

4. Smads Transcription Regulators Different Classes-
Smad2 and 3: Phosphorylated at carboxyl-terminal serines
Smad4: co-Smad
Smad2 and 3- “receptor-regulated smad”
Smad4- “common partner” smad; forms heteromeric complexes with recepor-activator Smads to enter nucleus

5. Cellular Machinery TGF-β = Serine/Threonine Kinase
Smad4 is NOT phosphorylated
Slide 4: TGF-B binds to its receptor(serine/threonine kinase) complex (1) leading to activation of the type I receptor (2). Smad2 and Smad3 are phosphorylated at the carboxyl-terminal serines by the activated type I receptor and form heteromeric complexes with Smad4 (3). Smad2/3/4 heteromeric complexes, consisting of an R-Smad and a Co-Smad, translocate into the nucleus (4) and bind to Smad responsive cis regulatory elements (SBE) on target genes and regulate transcription
Fazhi Li, M.D. et al. (2005). World J. Surg. 29,

6. How was this cell biological role determined?
Slide 5: Purpose of study was to determine whether smads are required for TGF- β signaling in colon cancer cells
MC-26 is a mouse colon cancer cell line with a functional pathway →found that cells express Smad2,3,4 by harvesting total RNA and doing reverse transcription PCR using cDNA
the cells were transfected with the p3tp-lux reporter plasmid; at 16 hours, cells were treated at dif. Dosage of TGF- β for 12 hours and harvested for analysis of luciferase activity-found that TGF- β induced luciferase activity in a dose-dependent fashion
also found that TGF- β inhibits cell growth and induces apoptosis (DNA fragmentation)- data not shown
Presence of Smad in MC-26 cell line→ Functioning TGF-β signaling pathway
Fazhi Li, M.D. et al. (2005). World J. Surg. 29,

7. How was this cell biological role determined?
Dominant Negative forms of Smads block TGF-β signaling
Slide 6: Cell line is still sensitive to tumor suppressor effects of TGF- β using dominant-negative Smad mutants
Used truncation mutation forms of Smads with deletions in last 41 to 43 carboxyl-terminal aa’s so they cannot be phosphorylated by TGF- β receptor
Luciferase Reporter Assay- to measure TGF- β signaling; p3TP-Lux reporter plasmid transfected into cells plus dominant negative mutant forms of Smad2,3,4; 16 hours later, cells treated with TGF- β for 12 hours and then cells were assayed for luciferase activity
Found that when cells were transfected with either smad mutant, the TGF- β induced reporter activity was significantly reduced and was even more reduced with a combination of 2 and 4 or 3 and 4
Fazhi Li, M.D. et al. (2005). World J. Surg. 29,

8. TGF/BMP Molecules Signal Through the SMADs
transforming growth factor- (TGF- ) ligands bind to a range of type II receptors to form complexes that interact with type I receptors. The receptors then form heterotetramers, which result in the phosphorylation and activation of receptor-regulated SMADs (R-SMADs) that subsequently form complexes with the common SMAD (Co-SMAD) SMAD4. This complex translocates to the nucleus, where it regulates gene transcription, directly or indirectly.
Jonas Larsson and Stefan Karlsson. (2005). Oncogene. 24, 5676−5692.

9. II. Biological Role of Protein What does it do for the Organism?

10. Disruption by Homologous Recombination
Embryonic lethal by day 7.5
Defective mesoderm and visceral endoderm development in Smad4 mutant embryos
(A) E6.5 Smad4 mutant embryos severely growth-retarded and unorganized as compared with wild-type littermates (left); (B) mutant embryos with a poorly defined extraembryonic region. The arrows point to the separation between the embryonic and extraembryonic regions. (C) E7.5 Smad4 mutant embryos have not developed considerably and start to be resorbed. (D) E8.5 wild-type embryos start organogenesis (left). While most of the Smad4 mutant embryos are in resorption, very few remnant embryos remain with no distinguishable structures (right on D and detail in E)
Christian Sirard et al. (1998). Genes and Development. 12,

11. Smad4 Gastrulation Defect
The gastrulation defect in Smad4 mutant embryos results from a defective visceral endoderm
NOT inability to respond to growth signals
  Wild-type visceral endoderm rescues the gastrulation defect of Smad4-deficient embryos. Whole mount in situ hybridization of E8.5 chimeric embryos generated from tetraploid aggregation experiments. (A,B) Embryos derived from Smad4 heterozygous ES cells were hybridized for T, or Krox-20 (K20) and Mox-1. (C,D) Dissected embryos derived from Smad4 homozygous mutant ES clone, C8-24. (C) Less affected embryo with malformation in headfold region (arrowhead). (D) Representative embryo with anterior truncation and a well developed posterior region with somites (arrow). (E,F) T expression (arrow) in embryos derived from Smad4 homozygous mutant ES clone F9-5 and C8-24, respectively. Embryo derived from clone F9-5 is less organized with abnormal head region (arrowhead). (G,H) Embryos derived from Smad4 homozygous mutant ES clone F9-5 and C8-24 respectively, probed with Krox-20 and Mox-1. Arrowhead points to head region. The arrows in B,G, and H point to Mox-1 expression.
Christian Sirard et al. (1998). Genes and Development. 12,

12. III. Connection to Cancer
Colon and Pancreatic Cancer

13. Smad4 mutation in the progression of colorectal carcinogenesis
176 tumors at varying stages [24]: being 0% (0/40) in adenomas, 10% (4/39) in intramucosal carcinomas, 7% (3/44) in invasive carcinomas without distant metastasis, 35% (6/17) in primary invasive carcinomas with distant metastasis, and 31% (11/36) in carcinomas metastasized to the liver and distant lymph nodes, or disseminated
Michiko Miyaki and Toshio Kuroki. (2003). Biochem and Biophysical Res. Comm. 306,

14. Somatic mutations of the Smad4 gene detected in human cancer
Majority of Smad4 gene mutations in human cancer are missense, nonsense, and frameshift mutations at MH2
majority of Smad4 gene mutations in human cancer are missense, nonsense, and frameshift mutations at the mad homology 2 region (MH2), which interfere with the homo-oligomer formation of Smad4 protein and the hetero-oligomer formation between Smad4 and Smad2 proteins, preventing the complex from entering the nucleus and resulting in disruption of TGFβ signaling.
Michiko Miyaki and Toshio Kuroki. (2003). Biochem and Biophysical Res. Comm. 306,