The effects of sonic hedgehog signaling pathway components on non-small-cell lung cancer progression and clinical outcome

Abstract

Background: Researchers in recent studies have reported that the sonic hedgehog (Shh) signaling pathway plays a crucial role during tumorigenesis, angiogenesis and cellular differentiation. We investigated the clinical and pathological significances of the Shh pathway and of its lymphangiogenic components in non-small-cell lung cancer (NSCLC), namely, Shh, glioma-associated oncogene homolog zinc finger protein 1 (Gli1), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and vascular endothelial growth factor D (VEGF-D).

Methods: The expression of Shh, Gli1, LYVE-1 and VEGF-D in primary NSCLC tissue from 40 patients was examined using immunohistochemical assays, and relationships between expression and clinicopathological data, such as age, gender, histology, tumor size, nodal stage, visceral pleural invasion, lymphatic thromboembolism, recurrence and overall survival were investigated.

Results: Of the 40 specimens examined, 25 (62.5%), 20 (50.0%), 11 (27.5%) and 20 (50.0%) were positive for Shh, Gli1, LYVE-1 or VEGF-D expression, respectively. The expression of Gli1 and LYVE-1 were significantly associated (P = 0.011), and Shh and LYVE-1 expression was related to visceral pleural invasion and lymphatic thromboembolism, respectively (P < 0.05). Shh expression levels compared on survival curves were statistically significant in univariate logrank analysis (P = 0.020). However, other clinicopathological factors did not reveal any statistical significance in univariate and multivariate analyses.

Conclusions: To our knowledge, this the first report of the relationship between components of the Shh signaling pathway and prognosis in NSCLC. The expression of Shh, Gli1 and LYVE-1 was found to be associated with clinicopathological factors and survival. Thus, the overexpression of the Shh signaling pathway could serve as a predictor of malignant behavior, including lymphangiogenesis, in NSCLC.

Figures

Figure 1

Activation of the Shh signaling pathway and lymphangiogenesis in non-small-cell lung cancer. Immunohistochemical staining was performed on tumor tissues obtained from 40 non-small-cell lung cancer (NSCLC) patients for the Sonic hedgehog (Shh) signaling pathway (Shh, glioma-associated oncogene homolog zinc finger protein 1 (Gli1)) and lymphangiogenic factors (vascular endothelial growth factor D (VEGF-D), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)) using appropriate antibodies. Immunohistochemistry scores (IHC scores) were calculated from positive staining area percentages and intensity scores. Staining intensity scores were graded as follows: 0 = negative (A), 1 = weak (B), 2 = moderate (C) and 3 = strong (D). Scale bar = 50 μm. Positive area percentages were graded as follows: grade 0 (≤5%), 1 (6% to 15%), 2 (16% to 25%), 3 (26% to 50%), 4 (51% to 75%) and 5 (>75%). Positivity was determined using the following formula: IHC score = percentage score × staining intensity score. Positive expression was defined as an IHC score >6.

Figure 2

Sonic hedgehog signaling and non-small-cell lung cancer cell proliferation. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assays were performed to measure cell proliferation. (A) Proliferation of H1299 and H2009 cells treated with cyclopamine-KAAD (10 μM). (B) and (C) Proliferation of H1299 and Calu-1 cells transfected with Gli1, Gli2 or Gli3 siRNA or nonspecific control (Con) siRNA. Bars represent the standard deviations of three independent experiments conducted in triplicate. (D) H1299 and Calu-1 cells were transfected with Gli1, Gli2 or Gli3 siRNA or nonspecific control siRNA and harvested 48 hours later. RT-PCR was performed for endogenous Gli1, Gli2 and Gli3 transcripts. β-actin was used as a loading control.

Figure 3

The sonic hedgehog signaling pathway was regulated by lymphangiogenic factors. (A) H1299 and Calu-1 cells were treated with 500 ng/ml N-Shh for the indicated times. The total RNA was harvested and subjected to RT-PCR using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), vascular endothelial growth factor D (VEGF-D) or β-actin (loading control) primers. (B) H1299 cells were treated with 500 ng of N-Shh or 10 μM cyclopamine-KAAD for 24 hours, and RNAs were isolated and subjected to RT-PCR for Glioma-associated oncogene homolog zinc finger protein 1 (Gli1), LYVE-1, VEGF-D or β-actin (loading control).

Figure 4

Overall survival curves for 40 non-small-cell lung cancer patients positive or negative for sonic hedgehog expression. Survival was analyzed using the Kaplan-Meier method, and survival differences between the positive and negative groups were compared using a logrank test. Patients with a tumor positive for Shh were found to have significantly poorer survival (P = 0.020).