Selective suppression of in vivo tumorigenicity by semaphorin SEMA3F in lung cancer cells

Abstract

Loss of the 3p21.3-encoded semaphorins, SEMA3B and SEMA3F, is implicated in lung cancer development. Although both antagonize VEGF binding/response to neuropilin (NRP) receptors, in lung cancer lines, SEMA3F is predominantly expressed and preferentially utilizes NRP2. In lung cancer patients, SEMA3F loss correlates with advanced disease and increased VEGF binding to tumor cells. In cell lines, VEGF enhances adhesion and migration in an integrin-dependent manner, and exogenous SEMA3F causes cells to round and lose extracellular contacts. Using retroviral infections, we established stable SEMA3F transfectants in two NSCLC cell lines, NCI-H157 and NCI-H460. When orthotopically injected into nude rats, both control lines caused lethal tumors in all recipients. In contrast, all animals receiving H157-SEMA3F cells, survived to 100 days, whereas all H157 controls succumbed. In H460 cells, which express NRP1 but not NRP2, SEMA3F did not prolong survival. This antitumor effect in H157 cells was associated with loss of activated alpha(v)beta(3) integrin and adhesion to extracellular matrix components. In addition, H157-SEMA3F cells, and parental H157 cells exposed to SEMA3F-conditioned medium, showed loss of p42/p44 MAPK phosphorylation. Thus, in this in vivo lung cancer model, SEMA3F has potent antitumor effects, which may impinge on activated integrin and MAPK signaling.

Figure 1

Establishment of H157 and H460 clones stably expressing SEMA3F. Southern blot (A) was performed with the SEMA3F cDNA probe and HpaI and EcoRI digested DNA from SEMA3F transformants and control (vector only) cells. DNA markers are indicated in kilobasepairs on the left. The arrow indicates the transfected SEMA3F DNA; the upper band is the endogenous SEMA3F. DNA from the Glc20 cell line was chosen as a negative control as these cells are homozygously deleted for SEMA3F [43]. SEMA3F protein was detected on the S1 and S2 subclones with an anti-Myc antibody by Western blot analysis (B) and immunofluorescence (C). The arrow indicates the SEMA3F band; protein markers are indicated in kilodaltons (B). (c) An enlargement of (b) Only background was detected by immunofluorescence in H157-CTL (a) Scale bar, 50 µm.

Figure 2

SEMA3F inhibits tumorigenicity of H157 cells but not H460. Survival curves are presented for rats injected into the lung with SEMA3F transfectants or control H157 (A) and H460 (B) cells.

Figure 3

Changes in cell morphology of SEMA3F-transfected H157 cells. Cell morphology was observed for H157 control and SEMA3F-transfected cells (A) several hours after plating. Cells were also observed at time 24 hours, at the periphery of an island of concentrated cells obtained with the CSM device (B). Scale bar, 100 µm.

Figure 4

Downregulation of activated α_vβ₃ integrin by SEMA3F. Immunocytochemistry was performed on H157 and H460 control and SEMA3F-transfected cells with WOW-1, an anti-activated α_vβ₃ integrin antibody (A), or an anti-α₆ integrin antibody (B). H157 cells were stained with DAPI (A). No change in staining was noticed with the anti-α₆ integrin, whereas loss of α_vβ₃ staining was observed for H157-S1 and H157-S2. Scale bar, 50 µm.

Figure 5

SEMA3F reduced the adhesion of H157 control cells to fibronectin and vitronectin. H157 control (CTL) cells were grown on different substrates: plastic (PL), fibronectin (F), vitronectin (V), and laminin (L) with a serum-free medium (+0) or control (+CTL), S1 (+S1), or S2 (+S2) conditioned media. The number of attached cells on each substrate is expressed as the mean number of cells. Assays were done in triplicate for two independent experiments. *P value < .05.

Figure 6

Downregulation of MAPK phosphorylation by SEMA3F. Western blot analysis of protein extracts of H157 and H460 control and SEMA3F-transfected cells with anti-p42/44 MAPK and anti-phosphorylated p42/44 MAPK antibodies (A). Parental cells were treated either with control or S1-conditioned media for 6 hours (B), and Western blot analysis was performed as in (A).