Suppression of tumorigenesis and induction of p15(ink4b) by Smad4/DPC4 in human pancreatic cancer cells.
OBJECTIVE
The tumor suppressor gene Smad4/DPC4, a key transcription factorin transforming growth factor beta (TGF-beta) signaling cascades,is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-beta-mediated expression of cell-cycle regulatory genes p15(ink4b) and p21(waf1).


METHODS
Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGF-beta downstream target gene p21(waf1), regulation of the p15(ink4b) promoter, anchorage-independent growth, and tumorigenesis were examined.


RESULTS
We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15(ink4b), p21(waf1), and TGF-beta-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between -356 and -329 bp of the p15(ink4b) promoter. The p15(ink4b) promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15(ink4b) is one of the downstream target genes regulated by Smad/DPC4.


CONCLUSIONS
These results explain the role of Smad4/DPC4 in TGF-beta-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-beta-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.
Peng
Bailu
B
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Fleming
Jason B
JB
Breslin
Tara
T
Grau
Ana M
AM
Fojioka
Shuichi
S
Abbruzzese
James L
JL
Evans
Douglas B
DB
Ayers
Dan
D
Wathen
Kyle
K
Wu
Tianai
T
Robertson
Kimberly D
KD
Chiao
Paul J
PJ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000494:pharmacology*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D017930:Genes, Reporter; D006720:Homozygote; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; C493957:Cdkn1a protein, mouse; C493968:Cdkn2b protein, mouse; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D008156:Luciferases