Interference with netrin-1 and tumor cell death in non-small cell lung cancer.
BACKGROUND
Netrin-1 may promote colorectal and breast tumorigenesis, by inhibiting apoptosis induced by its dependence receptors, deleted in colorectal cancer (DCC) and uncoordinated-5-homolog (UNC5H). The status of netrin-1 and its receptors in non-small cell lung cancer (NSCLC) was unknown.


METHODS
The levels of netrin-1 and its receptors were analyzed in a panel of 92 NSCLC and 25 human lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. In lung cancer cell lines that express netrin-1, the expression of netrin-1 was inhibited by using small interfering RNA (siRNA), or interference with netrin-1 was performed by treatment with a decoy recombinant DCC ectodomain protein (DCC-5Fbn). Cell death was monitored with a trypan blue exclusion assay or by measuring caspase-3 activity. The effect of netrin-1 interference on tumor growth was analyzed by DCC-5Fbn intratumoral or netrin-1 siRNA intraperitoneal injection in mice engrafted with lung cancer cell lines. All statistical tests were two-sided.


RESULTS
High levels of netrin-1 were found in 43 of the 92 NSCLC tumor samples (47%). Interference with netrin-1 in human lung cancer cell lines was associated with UNC5H-mediated cell death in vitro (percentage of cell death in untreated and in DCC-5Fbn-treated cells = 8% and 26%, respectively, difference = 18%, 95% confidence interval [CI] = 10% to 26%; P = .049) and with lung tumor growth inhibition and/or regression in xenografted nude mice (12 mice in DCC-5Fbn-treated group and 13 mice in control group). Mean volume of control and DCC-5Fbn-treated tumors on day 46 was 489 and 84 mm(3), respectively (difference = 404 mm(3), 95% CI = 145 to 664 mm(3); P < .001).


CONCLUSIONS
Almost half of the NSCLC tissue samples examined expressed high levels of netrin-1. Extracellular targeting of the interaction between netrin-1 and UNC5H may be a promising therapeutic approach for NSCLCs that express netrin-1.
Delloye-Bourgeois
Céline
C
Apoptosis, Cancer and Development Laboratory-Equipe labellisée La Ligue, CNRS UMR5238, Université de Lyon, Centre Léon Bérard, Lyon, France.
Brambilla
Elisabeth
E
Coissieux
Marie-May
MM
Guenebeaud
Céline
C
Pedeux
Rémy
R
Firlej
Virginie
V
Cabon
Florence
F
Brambilla
Christian
C
Mehlen
Patrick
P
Bernet
Agnès
A
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004396:Coloring Agents; D000075389:DCC Receptor; D064550:Death-Associated Protein Kinases; D018450:Disease Progression; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008175:Lung Neoplasms / Q000737:chemistry*; D051379:Mice; D008819:Mice, Nude; D009414:Nerve Growth Factors / Q000032:analysis* / Q000235:genetics; D000075386:Netrin Receptors; D000075388:Netrin-1; D010957:Plasmids; D034741:RNA, Small Interfering / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014183:Transplantation, Heterologous; D014343:Trypan Blue; D025521:Tumor Suppressor Proteins / Q000032:analysis* / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D004396:Coloring Agents; D000075389:DCC Receptor; C501909:DCC protein, human; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D000075386:Netrin Receptors; C000617353:Ntn1 protein, mouse; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D053148:Caspase 3; D014343:Trypan Blue