Integrin alpha 11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells

Abstract

Integrin alpha11 (ITGA11/alpha11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal alpha11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human alpha11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WT(shIGF2)) fibroblasts resulted in a decreased growth rate of A549+WT(shIGF2), compared with A549+WT tumors. The results indicate that alpha11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma-stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth.

Fig. 1.

Expression of integrin α11 chain in NSCLC. (A) Western blot shows α11 overexpression in three of four primary NSCLC samples (T, tumor samples; N, corresponding nonneoplastic lung tissue; loading control, Ponceau staining of total protein; M, marker). (B) Relative expression levels of α11 mRNA assayed by q-RT-PCR in microdissected stromal (S) relative to that in tumor cells in two unselected primary NSCLC tissues. (C) The mRNA expression of α11 and IGF2 in CAFs and their counterpart fibroblasts (CP). Four of five CAFs (520, 305, 619, and 836) showed higher α11 and IGF2 expression. All expression levels were relative to that of counterpart fibroblasts for sample 520. (D and E) Immunofluorescence images of an adenocarcinoma (D) and normal lung tissue (E) that were double stained with antibodies to α11 (red) and epithelial cell marker cytokeratin (green). The α11 staining was negligible in nonneoplastic lung tissue and was mainly confined to the stroma in the tumor sample.

Fig. 2.

The effect of integrin α11 expression in immortalized MEFs on the tumorigenicity of A549 lung adenocarcinoma cells. (A) Growth rates of tumors formed by cells implanted into the s.c. tissue of SCID mice. A549, KO, WT, and KI were tumor formation by respective cell lines alone, whereas A549+WT/KO/KI represent tumor formation by A549 cells coimplanted with α11-expressing MEF (WT), α11-deficient MEF (KO), and KO MEF that reexpressed human α11 (KI). (B and C) H&E staining of xenografts formed by A549 coimplanted with WT (B) and KO (C) MEFs. No apparent difference in the cellularity of carcinoma cells, compared with spindle fibrosarcoma cells, was noted. (D) Mouse α11 (filled bar) and human α11 (open bar) mRNA expression in tumors formed by WT, KO, and KI cells alone or when coimplanted with A549 cells. When WT was coimplanted with A549, there was an ≈10-fold increase in mouse α11 expression level compared with WT tumors. KI tumors only expressed human α11. The low level of mouse α11 detected in KO tumors was putatively from the host stromal cells. Note the lack of detection of human α11 in WT and KO tumors and the negligible level of mouse α11 in KI and A549+KI tumors, confirming the specificity of primers used. All expression levels were arbitrarily referenced to the mean of A549+KO tumors. (E) Vimentin mRNA expression in tumors. No significant difference was noted between A549+WT versus A549+KO, H460+WT versus H460+KO, and H520+WT versus H520+KO. All expression levels were relative to that of A549+KO. (F) CD31 mRNA expression levels in xenograft tumors specified in E. Error bar represents SE. The number in brackets indicates the number of mice in each group. The numbers of samples used for q-RT-PCR analysis were: WT, 7; KO, 7; KI, 4; A549+WT, 7; A549+KO, 8; A549+KI, 4.

Fig. 3.

Coimplantation studies with NCI-H460 and NCI-H520 NSCLC cell lines. Tumors formed by H460 (A) and H520 (B) cells coimplanted with the α11 WT MEFs were significantly greater than those with α11-deficient (KO) fibroblasts.

Fig. 4.

Regulation of IGF2 expression by integrin α11 in immortalized MEF tumors formed by MEFs coimplanted with NSCLC cells. (A) Mouse (m)IGF2 mRNA expression levels in tumors formed by α11-expressing (WT), α11-deficient (KO), and human (h)α11 forced-expressing KO MEF (KI) MEFs and the corresponding tumors formed when these MEFs were coimplanted with A549 cells. Note the lack of IGF2 expression when α11 was not expressed and its reexpression when h-α11 was reexpressed. (B) Corresponding expressions of mIGF1 and hIGF1 mRNA in the tumors described in A. The mIGF1 was only elevated in tumors formed by KI or A549+KI cells, and the level of induction was significantly less than that observed with IGF2, whereas the hIGF1 expression level remained unchanged. (C) Expression of hIGF2 and IGF1 receptor (IGF1R). Note the lack of hIGF2 and IGF1R expression in tumors formed by MEFs alone (WT, KO, and KI) and the low-level differences in coimplantation tumors, indicating the insignificant role they play in modulating the tumorigenicity of WT and KO MEFs, compared with IGF2. (D) The C_T values of h- and mIGF2 in tumors after normalization with the averaged C_T of hRPS13 and mGAPDH (Delta C_T). The mIGF2 expression level was approximately five C_T lower than that of hIGF2 in A549+WT tumors, indicating a 32-fold higher expression level of mIGF2 compared with that of hIGF2 because higher C_T indicates lower expression levels. (E) All three NSCLC cell lines expressed similar levels of IGF2 and IGF1R, with a <2-fold change observed among A549, H460, and H520 cells, compared with N135, an immortalized normal bronchial epithelial cell line. (F) Although mIGF2 expression was significantly higher in H460+WT and H520+WT tumors, compared with their counterparts with KO MEFs, NSCLC cells derived from hIGF2 and IGF1R expression levels were not influenced by the α11 expression status of the coimplanted fibroblasts. The numbers of samples used for q-RT-PCR analysis were: H460+WT, 4; H460+KO, 4; H520+WT, 5; H520+KO, 5.

Fig. 5.

The effect of IGF2 expression by MEFs on A549 cells. (A) Stable expression of shRNA for IGF2 resulted in an ≈60–70% down-regulation of IGF2 transcripts, compared with cells expressing luciferase (Luc) gene as control. Note that the IGF2 level in KO cells was not changed because these cells already expressed low levels of IGF2. (B) IGF2 expression in tumors formed by A549+WT_shLuc and A549+WT_shIGF2 MEFs showing a 70% down-regulation of IGF2 in the latter. (C) Tumor growth rates of A549 cells coimplanted with WT_shIGF2 and WT_shLuc (control) MEFs.