Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis.
BACKGROUND
Pancreatic cancer is one of the most aggressive malignancies, with high rates of invasion and metastasis and with generally poor prognosis. We previously found that metastasis was strongly associated with the expression of growth factor receptor-bound protein 7 (Grb7), which contains a Src homology 2 (SH2) domain. In this study, we evaluated Grb7 protein as a molecular target of therapy for metastatic pancreatic cancer.


METHODS
Grb7 protein expression was measured by immunohistochemistry in 36 human pancreatic cancer specimens and adjacent normal pancreatic tissue. We synthesized a nonphosphorylated peptide inhibitor that binds specifically to the SH2 domain of Grb7. Intracellular signaling was assessed by immunoprecipitation and immunoblot assays in cultured human pancreatic cancer cells. Cell migration was measured with a modified Boyden chamber method. Peritoneal metastasis of the pancreatic cancer cells was measured with a mouse model. All statistical tests were two-sided.


RESULTS
We found that 22 (61%) of 36 pancreatic cancer specimens had higher levels of Grb7 protein than their corresponding normal pancreatic tissue specimens. Grb7 expression was statistically significantly different between specimens from patients without lymph node metastasis (stage N0; two of the 10 patients) and patients with lymph node metastasis (stages N1 + N2; 20 of the 26 patients) (P = .006). The Grb7 peptide inhibitor selectively blocked the interaction between Grb7 and focal adhesion kinase and blocked the phosphorylation of Grb7 protein. In vivo Grb7 peptide inhibitor statistically significantly attenuated cell migration (for control peptide, 87.5 cells migrated, 95% confidence interval [CI] = 82.6 to 92.4 cells; for Grb7 peptide, 5.7 cells migrated, 95% CI = 2.3 to 9.0 cells; P < .001) and peritoneal metastasis of the pancreatic cancer cells in a mouse model, as assessed by the number of nodules (control = 72.6 nodules, 95% CI = 55.8 to 89.4 nodules; and for Grb7 peptide = 3.2 nodules, 95% CI = 1.6 to 4.8 nodules; P < .001, t test) and their weight (control = 4.13 g, 95% CI = 3.40 to 4.86 g; Grb7 peptide = 0.19 g, 95% CI = 0.06 to 0.32 g; P < .001, t test).


CONCLUSIONS
The Grb7 peptide inhibitor appears to be a promising molecularly targeted therapeutic agent against metastatic pancreatic cancer.
Tanaka
Shinji
S
Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Tokyo 113-8519, Japan. shinji.msrg@tmd.ac.jp
Pero
Stephanie C
SC
Taguchi
Kenichi
K
Shimada
Mitsuo
M
Mori
Masaki
M
Krag
David N
DN
Arii
Shigeki
S
D000230:Adenocarcinoma / Q000737:chemistry* / Q000188:drug therapy / Q000378:metabolism / Q000556:secondary; D000368:Aged; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002465:Cell Movement; D049109:Cell Proliferation; D004195:Disease Models, Animal; D005260:Female; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D051382:GRB7 Adaptor Protein / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008207:Lymphatic Metastasis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D010179:Pancreas / Q000737:chemistry; D010190:Pancreatic Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D010534:Peritoneal Neoplasms / Q000556:secondary*; D010766:Phosphorylation / Q000187:drug effects; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000032:analysis; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000235:genetics
D000970:Antineoplastic Agents; C509376:G7-18NATE; D010456:Peptides, Cyclic; D034741:RNA, Small Interfering; D051382:GRB7 Adaptor Protein; D051416:Focal Adhesion Protein-Tyrosine Kinases