Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver.
BACKGROUND
Neuropilin-2 (NRP2) is a high-affinity kinase-deficient receptor for vascular endothelial growth factor (VEGF) and semaphorin 3F. We investigated its function in human colorectal cancers.


METHODS
Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA (shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for activation of the VEGFR1 pathway by immunoblotting. Soft agar assays, Annexin V staining, and Boyden chamber assays were used to examine anchorage-independent growth, apoptosis in response to hypoxia, and cell migration/invasion, respectively, in HCT-116 transfectants. Tumor growth and metastasis were analyzed in mice (groups of 10) injected with shRNA-expressing HCT-116 cells. The effect of in vivo targeting of NRP2 by small interfering RNA (siRNA) on the growth of hepatic colorectal tumors derived from luciferase-expressing HCT-116 cells was assessed by measuring changes in bioluminescence and final tumor volumes. All statistical tests were two-sided.


RESULTS
NRP2 expression was substantially higher in tumors than in adjacent mucosa. HCT-116 transfectants with reduced NRP2 levels had reduced VEGFR1 signaling, but proliferation was unchanged. Anchorage-independent growth, survival under hypoxic conditions, and motility/invasiveness were also reduced. In vivo, HCT-116 transfectants with reduced NRP2 demonstrated decreased tumor growth, fewer metastases, and increased apoptosis compared with control cells. Hepatic colorectal tumors in mice treated with NRP2 siRNAs were statistically significantly smaller than those in mice treated with control siRNAs (at 28 days after implantation, mean control siRNAs = 420 mm3, mean NRP2 siRNAs = 36 mm3, NRP2 vs control: difference = 385 mm3, 95% confidence interval = 174 mm3 to 595 mm3, P = .005).


CONCLUSIONS
NRP2 on colorectal carcinoma cells is important for tumor growth and is a potential therapeutic target in human cancers where it is expressed.
Gray
Michael J
MJ
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77230-1402, USA.
Van Buren
George
G
Dallas
Nikolaos A
NA
Xia
Ling
L
Wang
Xuemei
X
Yang
Anthony D
AD
Somcio
Ray J
RJ
Lin
Yvonne G
YG
Lim
Sherry
S
Fan
Fan
F
Mangala
Lingegowda S
LS
Arumugam
Thiruvengadam
T
Logsdon
Craig D
CD
Lopez-Berestein
Gabriel
G
Sood
Anil K
AK
Ellis
Lee M
LM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D018450:Disease Progression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008081:Liposomes; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism*; D051379:Mice; D009361:Neoplasm Invasiveness; D039943:Neuropilin-2 / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology* / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D039961:Semaphorins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism
D008081:Liposomes; D039943:Neuropilin-2; D034741:RNA, Small Interfering; D039961:Semaphorins; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D051057:Proto-Oncogene Proteins c-akt