Abstract
Rationale: Genetic alterations on 8p22 have been implicated in multiple cancers, including lung cancer. In this region, genetic variants of the class A scavenger receptor (SR-A) gene have been associated with prostate cancer risk and have been highlighted as a potential susceptibility gene of cancer.
Objectives: To determine whether common polymorphisms in the SR-A gene are associated with human lung cancer risk and to clarify the role of SR-A in lung carcinogenesis.
Methods: The relationship of three potentially functional polymorphisms (T-365C, T+25C, and Ala275Pro) in the SR-A gene with lung cancer risk was evaluated in 1287 lung cancer case subjects and 1261 control subjects from the Chinese population. At the same time, SR-A null mice were used to investigate its role in lung cancer development.
Measurements and Main Results: The T+25C polymorphism was independently associated with lung cancer risk and significantly correlated with decreased expression of SR-A. The decreased SR-A expression was also found in tumor tissues as compared with normal tissues. Depletion of SR-A boosted the growth and angiogenesis of implanted Lewis lung carcinoma in mice. The cancer-suppressing capability of SR-A was attributable to its expression in bone marrow–derived cells as evidenced by bone marrow transplantation. Further analysis revealed augmented expression of proangiogenic factors including matrix metalloproteinase-9 (MMP9) in SR-A-deficient mice, indicative of a more procarcinogenic microenvironment. Last, zoledronate, an MMP9 inhibitor, abrogated acceleration of tumor growth conferred by SR-A loss-of-function.
Conclusions: Evidence from the population study and mouse model strongly indicates that SR-A may function as a tumor modulator to inhibit lung cancer growth through affecting the tumor microenvironment.
The known susceptibility loci explain only a small proportion of lung cancer risk. Further studies on important genes are warranted to further understand the mechanism of lung cancer development. Class A scavenger receptor A (SR-A), encoded by MSR1 located at 8p22, which represents a hot spot and houses many cancer-related genes, is a pattern recognition receptor expressed primarily in macrophages. Its role in lung carcinogenesis is unknown.
The polymorphism T+25C in the SR-A promoter is independently associated with lung cancer risk and may increase human lung cancer risk through down-regulating SR-A expression. SR-A deficiency may promote lung cancer development via enhanced expression of matrix metalloproteinase-9 in mice.
Lung cancer is the leading cause of cancer-related deaths around the world. In China, the morbidity and mortality from lung cancer have increased rapidly in recent decades. Genome-wide association studies have identified six loci at 3q28 (TP63) (1), 5p15.33 (TERT-CLPTM1L) (2, 3), 6p21.33 (BAT3-MSH5) (3), 13q12.12 (MIPEP-TNFRSF19) (4), 15q25.1 (CHRNA5, CHRNA3, and CHRNA4) (5–7), and 22q12.2 (MTMR3-HORMAD2-LIF) (4) that are associated with altered susceptibility to lung cancer. However, in contrast to prostate cancer (8) and breast cancer (9), for which more than a dozen susceptibility loci have been discovered, the known susceptibility loci for lung cancer remain limited, which has impeded mechanistic investigations on lung cancer.
Genetic alterations on the short arm of chromosome 8, specifically 8p22, have been identified as being associated with multiple tumors, including lung cancer (10–12). The class A scavenger receptor (SR-A) gene is located within this region, indicative of its potential role in tumorigenesis. SR-A, also known as MSR1 and CD204, is a member of several structurally diverse receptors collectively termed scavenger receptors (13, 14). It is expressed primarily in macrophages, but can also be detected in endothelial, fibroblast, and vascular smooth muscle cells. A range of ligands, including modified low-density lipoproteins, fucoidan, advanced glycation end-products, denatured collagen, bacterial components (e.g., lipopolysaccharide), and apoptotic cells, have been reported to bind with SR-A and to engage SR-A in such important pathobiological processes as apoptosis and inflammation (15, 16). SR-A has multiple endocytic routes and its endocytosis is regulated by intracellular couplers (17, 18). Evidence has emerged that SR-A plays crucial roles in cancer development, with some rather controversial implications (19–22). For instance, Chen and colleagues portray SR-A as a potential tumor suppressor acting by regulating phosphatidylinositol-3-kinase (PI3K)–Akt and β-catenin in a mouse model of chronic myeloid leukemia (23). On the other hand, a study by Ohtaki and colleagues has correlated a high percentage of CD204-positive macrophages with a poor prognosis in lung adenocarcinoma, and proposed that CD204 could potentially serve as a marker of tumor-promoting macrophages that drive up tumor progression (24).
Genetic variations of SR-A have been connected to altered susceptibility in prostate cancer (25, 26), although the conclusiveness of these studies has been refuted (27). Interestingly, SR-A polymorphism has also been found to impact the outcome of chronic obstructive pulmonary disease, a risk factor for lung cancer (28). However, no study has thus far reported the role of common genetic variants of SR-A in lung cancer risk. Given the potential role that SR-A plays in cancer genetics, we hypothesized that common genetic variants of SR-A may be associated with lung carcinogenesis.
In the present study, we identified in a Chinese population a functional polymorphism in SR-A, the expression level of which is linked to lung cancer risk. More importantly, we conducted in vivo and in vitro studies to demonstrate that SR-A may negatively modulate lung cancer growth and angiogenesis by regulating matrix metalloproteinase-9 (MMP9) production. Therefore, targeting SR-A may provide viable strategies in combating lung cancer.
A total of 1,287 case subjects and 1,261 control subjects were included in this study, the details for which have been described previously (29). Briefly, patients with lung cancer were consecutively recruited from the Cancer Hospital of Jiangsu Province and the First Affiliated Hospital of Nanjing Medical University (Nanjing, China). Cancer-free control subjects were randomly selected from a pool of more than 30,000 participants in a community-based screening program for noninfectious diseases in Jiangsu Province. The control subjects were frequency-matched to the case subjects by age and sex. All study subjects were Han Chinese and signed informed consent forms. Individuals who smoked at least one cigarette per day for at least 1 year were defined as smokers; otherwise they were assumed to be nonsmokers. This study was approved by the Institutional Review Board of Nanjing Medical University.
For in vivo experiments, anesthetized 6- to 8-week-old C57BL/6J female mice were injected subcutaneously via the flank with, per mouse, 5 × 106 Lewis lung carcinoma (LLC) cells in phosphate-buffered saline. Tumor volume was calculated according to the following formula: volume = 0.5 × length × width2 (30). After 3 weeks, the mice were killed and tumors were dissected from the mice and weighed. Zoledronate (Zometa [ZA]; Novartis Pharma Stein AG, Stein, Switzerland) at a dose of 25 or 100 μg/kg was diluted in saline and delivered by subcutaneous injection every day for 3 days before LLC cells were injected (31). All aspects of the animal care and experimental protocols were approved by the Nanjing Medical University Committee on Animal Care.
Additional detail on the method is provided in the online supplement.
To determine whether SR-A genetic variants contribute to the development of lung cancer, we genotyped three potential functional single-nucleotide polymorphisms (T-365C, T+25C, and Ala275Pro) in a case–control study consisting of 1,287 case subjects and 1,261 control subjects. The characteristics of case subjects and control subjects are shown in Table E1 (in the online supplement). Frequency matching was performed with similar distributions of age (≤60 and >60 yr old) and sex between the case subjects and the control subjects (P = 0.949 and 0.942, respectively). As expected, the case subjects had a higher proportion of smokers and heavy smokers than control subjects (both P < 0.001). Of the 1,287 case subjects with lung cancer, there were 683 (53.1%) with adenocarcinoma, 357 (27.7%) with squamous cell carcinoma, 120 (9.3%) with small cell carcinoma, and 127 (9.9%) with large cell, mixed cell, or undifferentiated carcinomas.
The call rates were more than 97% for all three polymorphisms. The genotype distribution among control subjects was in Hardy-Weinberg equilibrium for each single-nucleotide polymorphism (P > 0.05). As shown in Table 1, T-365C and T+25C polymorphisms were significantly associated with lung cancer risk, with per-allele odds ratios of 1.19 (95% confidence interval, 1.06–1.34; P = 3.60 × 10−3) and 1.45 (95% confidence interval, 1.18–1.78; P = 3.84 × 10−4), respectively. When we tested the independence of the associations of these two polymorphisms with lung cancer risk in a conditional analysis, the effect of T+25C but not T-365C still reached the significance level (P = 4.91 × 10−3 and 0.057, respectively). However, there was no significantly different effect of the T+25C polymorphism on lung cancer risk between subgroups (Table E2).
| Case (n = 1,287) | Control (n = 1,261) | |||||||
| Location | Polymorphism | n | % | n | % | Crude OR (95% CI) | Adjusted OR (95% CI)* | P Value |
| 5′ flanking region (−365) | rs416748 | 1,244 | 1,244 | |||||
| TT | 224 | 18.0 | 277 | 22.3 | 1.00 | 1.00 | ||
| CT | 637 | 51.2 | 634 | 50.9 | 1.24 (1.01–1.53) | 1.24 (1.00–1.53) | 0.052 | |
| CC | 383 | 30.8 | 333 | 26.8 | 1.42 (1.13–1.79) | 1.42 (1.13–1.80) | 3.28 × 10−3 | |
| Per allele | 1.19 (1.06–1.33) | 1.19 (1.06–1.34) | 3.60 × 10−3 | |||||
| 5′ UTR (+25) | rs13306541 | 1,251 | 1,243 | |||||
| TT | 1,009 | 80.7 | 1069 | 86.0 | 1.00 | 1.00 | ||
| CT | 229 | 18.3 | 169 | 13.6 | 1.44 (1.16–1.78) | 1.42 (1.13–1.77) | 2.18 × 10−3 | |
| CC | 13 | 1.0 | 5 | 0.4 | 2.75 (0.98–7.75) | 2.83 (0.98–8.13) | 0.054 | |
| Per allele | 1.47 (1.20–1.79) | 1.45 (1.18–1.78) | 3.84 × 10−4 | |||||
| Exon 6 (Ala275Pro) | rs3747531 | 1,275 | 1,255 | |||||
| CC | 514 | 40.3 | 493 | 39.3 | 1.00 | 1.00 | ||
| CG | 593 | 46.5 | 593 | 47.2 | 0.96 (0.81–1.14) | 0.99 (0.83–1.18) | 0.902 | |
| GG | 168 | 13.2 | 169 | 13.5 | 0.95 (0.75–1.22) | 0.97 (0.75–1.25) | 0.807 | |
| Per allele | 0.97 (0.87–1.090) | 0.99 (0.88–1.11) | 0.810 | |||||
T+25C is located in the 5′ untranslated region of the SR-A gene, within the SR-A promoter. To evaluate whether the T+25C polymorphism that was independently associated with lung cancer risk was also correlated with the expression of SR-A, we examined SR-A mRNA expression in 51 lung cancer–adjacent normal tissues, using quantitative RT-PCR. The relative expression level of SR-A in tissues with the +25CT genotype (n = 7) was 0.59 ± 0.11, which was significantly lower than those with the +25TT genotype (n = 44) (mRNA level, 1.19 ± 0.11) (P = 0.043). These results showed that the +25C allele with low expression of SR-A could increase the risk of lung cancer, suggesting that SR-A may act as a tumor suppressor gene in the process of lung carcinogenesis.
We then compared expression levels of SR-A mRNA between lung cancer tumor tissues and matched adjacent normal tissues (n = 51). The relative levels of SR-A mRNA were 0.77 ± 0.09 and 1.10 ± 0.10 in tumor and normal tissues, respectively. The expression level of SR-A was significantly decreased in tumor compared with normal tissues. The average ratio of tumor-to-normal tissue expression level for SR-A mRNA was 0.75 ± 0.06 in lung cancer, indicating that SR-A may be down-regulated in lung carcinogenesis (Figure 1B). In addition, protein expression of SR-A was evaluated by immunohistochemistry. SR-A was expressed primarily in macrophages in normal lung tissue, and it was detected in macrophages and other stromal cells in lung tumor (Figure 1A). Indeed, significant differences in SR-A staining pattern were observed between human lung cancer tumor tissues (for which 11.6% showed strong staining for SR-A) and matched adjacent normal tissues (for which 46.5% showed strong staining for SR-A). Together, these data implicate SR-A as a potential negative modulator in lung tumorigenesis.
Figure 1. Class A scavenger receptor A (SR-A) expression was correlated with human lung cancer progression. (A) Representative images of SR-A staining of (b) human cancerous lung tissue (T) and (a) matched adjacent normal (N) lung tissue. Original magnification: left, ×100; right, ×400. Adjacent normal tissues showed strong SR-A staining and cancerous tissues showed weak staining. (c) Significant differences in SR-A staining pattern were observed between human lung cancerous and matched adjacent normal tissues (n = 43, P < 0.01, χ2 test). (B) Quantitative RT-PCR analysis of SR-A in human cancerous and matched adjacent normal lung tissues (n = 51, **P < 0.01).
[More] [Minimize]To further determine the exact role of SR-A in lung cancer development, 6- to 8-week old female mice deficient in SR-A (SR-A knockout [KO]) and their littermate controls (wild type [WT]) were injected subcutaneously with LLC cells via the flank. Tumor size was measured at 7, 14, and 21 days, respectively. LLC tumor size and weight were significantly greater in SR-A KO mice than in WT mice (Figure 2A), indicating that SR-A deficiency may nurture more robust growth of LLC tumor in mice. SR-A was expressed in the tumors dissected from WT, but not KO, mice (Figure E1A). There was no obvious SR-A expression in either LLC cells or SR-A KO macrophages (Figure E1B).
Figure 2. Class A scavenger receptor A knockout (SR-A KO) mice displayed enhanced tumor growth after Lewis lung carcinoma (LLC) cell inoculation. (A) LLC cells were inoculated subcutaneously into 6- to 8-week-old wild-type (WT) and SR-A KO mice. (a) Macrograph of tumors 3 weeks after LLC injection. (b and c) Quantification of tumor volume (WT, n = 6; SR-A KO, n = 7; **P < 0.01) and weight (WT, n = 14; SR-A KO, n = 14; **P < 0.01) 3 weeks after LLC injection. (B) (a) Immunohistochemical staining of tumor sections from WT and SR-A KO mice with an anti-PCNA (proliferating cell nuclear antigen) antibody. Original magnification, ×400. (b) Quantification of PCNA-positive areas (WT, n = 5; SR-A KO, n = 7; **P < 0.01).
[More] [Minimize]Next, we assessed the potential mechanisms underlying accelerated tumor growth as a result of SR-A ablation. To this end, size-matched tumors from SR-A KO and WT mice were bisected and processed for the analysis of tumor cell proliferation and angiogenesis. Immunohistochemical staining revealed an increase in proliferating cell nuclear antigen (PCNA) signal indicative of boosted proliferation of tumor cells in SR-A KO mice (Figure 2B). Importantly, stronger angiogenesis visualized by microvessel marker CD34 immunostaining was also detected in SR-A KO mice than in WT mice (Figure 3A). This was further confirmed by Matrigel plug assay. As shown in Figure 3B, SR-A KO mice exhibited more prominent angiogenesis than did WT mice, as evident by both gross observation and hematoxylin–eosin (H&E) staining. In further support of this assertion, SR-A KO mice showed a higher percentage of CD31-positive areas compared with WT mice (Figure 3C). Collectively, this set of evidence indicates that SR-A loss-of-function gives rise to a more deteriorated phenotype of lung cancer regarding both proliferation and angiogenesis.
Figure 3. Class A scavenger receptor A knockout (SR-A KO) mice exhibited enhanced angiogenesis in tumors and Matrigel plugs. (A) (a) Immunohistochemical staining of tumor sections from wild-type (WT) and SR-A KO mice with an anti-CD34 antibody. Original magnification, ×400. (b) Tumor specimens were scanned at low magnification to identify vascular hot spots. Areas of greatest vessel density were then examined at higher magnification (original magnification, ×400) and counted. Results are expressed as microvessel number per field (WT, n = 8; SR-A KO, n = 9; **P < 0.01). (B) Matrigel plugs supplemented with vascular endothelial growth factor and basic fibroblast growth factor were excised 10 days after implantation. Representative macroscopic appearance of plugs (a) and photographs of hematoxylin and eosin–stained sections (b; original magnification, ×100) from WT and SR-A KO mice are shown. (C) (a) Sections of Matrigel plugs obtained from WT and SR-A KO mice were immunostained with an anti-CD31 antibody. Representative images are shown (original magnification, ×400). (b) Quantification of CD31-positive areas (WT, n = 6; SR-A KO, n = 6; **P < 0.01).
[More] [Minimize]To determine whether aggravated LLC tumor growth was driven by SR-A insufficiency, we transplanted bone marrow–derived cells (BMDCs) from SR-A KO mice into WT mice. It was found that tumor growth and angiogenesis were significantly sped up compared with mice that received WT BMDCs (Figures 4A and 4B). The migration of transferred BMDCs into the tumor site in WT mice was identified by transplantation with BMDCs from enhanced green fluorescent protein (EGFP)-expressing mice. The EGFP-positive cells were readily detectable in the tumors of WT mice (Figure E2A). However, the expression level of SR-A in the tumor tissues of WT mice transplanted with SR-A KO BMDCs (SR-A KO to WT) was significantly lower than in those transplanted with WT BMDCs (WT to WT) (Figure E2B, part a). Moreover, the decreased expression level of SR-A originated mostly from macrophages (F4/80-positive cells) and endothelial cells (CD31-positive cells), not from fibroblasts (vimentin-positive cells), in the tumors of WT mice transplanted with SR-A KO BMDCs (SR-A KO to WT) (Figure E2B, part b). This line of evidence supports a tumor-suppressing role for SR-A in BMDCs, especially in macrophages and endothelial cells.
Figure 4. Class A scavenger receptor A (SR-A) in bone marrow–derived cells was sufficient to modulate tumor growth and angiogenesis. (A) Lewis lung carcinoma (LLC) cells were inoculated subcutaneously into mice 4 weeks after bone marrow transplantation. (a) Macroscopic appearances of tumors 3 weeks after LLC injection. Tumor volume (b) and weight (c) (wild-type [WT] to WT, n = 12; SR-A knockout [KO] to WT, n = 14; *P < 0.05) were measured. (B) (a) Immunohistochemical staining of tumor sections from WT to WT and from SR-A KO to WT mice, using an anti-CD34 antibody. Original magnification, ×400. (b) Quantification of microvessel formation (WT, n = 6; SR-A KO, n = 6; **P < 0.01).
[More] [Minimize]To further assess whether SR-A may target the migration/infiltration and polarization of macrophages, we measured mRNA levels of chemokine (C-C motif) ligand-2 (CCL2) and macrophage-colony stimulating factor (M-CSF), by which macrophages are recruited into tumors. There were slightly lower, but not statistically significant, levels of CCL2 and M-CSF messages in SR-A KO mice compared with WT mice (Figure E3A, parts a and b). Immunohistochemical staining of CD68, a marker of macrophages, exhibited similar expression in LLC tumors in WT and SR-A KO mice (Figure E3B). Flow cytometric analysis also showed that the proportion of F4/80+ macrophages in tumor was similar in WT and SR-A KO mice (Figures E3C, E3D, and E3E, part a). Furthermore, expression levels of the M1 macrophage markers (inducible nitric oxide synthase [iNOS] and CD32) and M2 macrophage markers (CD206 and Ym1) were not changed in SR-A KO mice (Figure E3A, parts c–f). This was corroborated by observation that a similar distribution in F4/80+CD11c+ M1 macrophages (Figures E3C and E3E, part b) and F4/80+CD206+ M2 macrophages was present in WT and SR-A KO mice, respectively (Figures E3D and E3E, part c). Taken together, these results revealed that accelerated tumor growth and enhanced angiogenesis observed in SR-A KO mice unlikely stemmed from altered macrophages homing or transdifferentiation.
Because SR-A loss-of-function did not affect macrophage infiltration or polarization in LLC tumors, we compared the expression of several genes that may influence the tumor microenvironment in mice in an attempt to unveil the possible mechanism whereby SR-A deficiency promoted tumor growth and angiogenesis. As expected, cyclooxygenase (COX)-2, stromal cell–derived factor-1 (SDF1) (Figure E4A, parts d and e), and vascular endothelial growth factor (VEGF) (Figure E4A, part f and Figure E4B, part b) expression, but not basic fibroblast growth factor (bFGF) (Figure E4B, part a) expression, were up-regulated in SR-A KO mice, which is consistent with enhanced neovascularization. Expression of proinflammatory mediators including IL-1β, IL-6, and tumor necrosis factor-α was not altered, in agreement with the observation that macrophage polarization remained unaffected in these mice (Figure E4A, parts a–c). Interestingly, MMP9, a linchpin in tumor progression implicated in remodeling the extracellular matrix and promoting sprouting and growth of new blood vessels, was significantly elevated in SR-A KO mice (Figures 5A and 5B). In accordance, circulating levels of pro-MMP9 in the plasma was also significantly increased in SR-A KO mice compared with WT mice (Figure 5D). Concomitantly, a gelatin zymography assay showed an increase in MMP9 activity in tumor tissues isolated from SR-A KO mice (Figure 5C).
Figure 5. Class A scavenger receptor A (SR-A) deficiency promotes the production of matrix metalloproteinase-9 (MMP9). (A) Quantitative RT-PCR analysis of MMP9 in Lewis lung carcinoma (LLC) tumors from wild-type (WT) and SR-A knockout (KO) mice (WT, n = 6; SR-A KO, n = 6; **P < 0.01). (B) (a) Western blot analysis of MMP9 in LLC tumors from WT and SR-A KO mice. (b) Quantification of MMP9 protein expression by densitometric analysis (WT, n = 6; SR-A KO, n = 6; *P < 0.05). (C) (a) Zymographic analysis of MMP9 activity in LLC tumors from WT and SR-A KO mice. (b) Quantification of MMP9 activity by densitometric analysis (WT, n = 6; SR-A KO, n = 6; *P < 0.05). (D) ELISA analysis of the plasma levels of pro-MMP9 in WT and SR-A KO mice (WT, n = 14; SR-A KO, n = 14; **P < 0.01). (E) (a) Quantitative RT-PCR analysis of MMP9 in WT and SR-A KO peritoneal macrophages cocultured with or without LLC. (b) ELISA analysis of the levels of pro-MMP9 in cell culture medium. (c) Quantitative RT-PCR analysis of MMP9 in LLC cells cocultured with WT or SR-A KO peritoneal macrophages. Experiments were performed in triplicate wells (*P < 0.05, **P < 0.01). GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
[More] [Minimize]MMP9 is believed to be expressed primarily by infiltrating macrophages in tumor tissue (32–34), raising the possibility that SR-A ablation may cultivate a tumor-friendly microenvironment by targeting MMP9. To test this hypothesis, primary peritoneal macrophages separated from WT and SR-A KO mice were cocultured with or without LLC cells. Basal MMP9 production was higher in SR-A KO macrophages, which was further up-regulated after LLC cell coculturing (Figure 5E, parts a and b), reflecting cross-talk between tumor tissue and myeloid cells. Reciprocally, LLC cells, under the influence of macrophages, scaled up their own synthesis of MMP9 (Figure 5E, part c), suggesting that both tumor cells and peripheral factors contribute to the formation of a protumorigenic niche. Collectively, these data indicate that SR-A deficiency may influence the tumor microenvironment by inducing macrophages and LLC cells to produce more MMP9.
To confirm the role of MMP9 in tumor growth promoted by SR-A deficiency in vivo, we tested the effects of pharmacological MMP9 blockade by systemic administration of the MMP9 antagonist ZA into mice at a dose of 25 μg/kg (31) or 100 μg/kg (31, 33, 35–37). We found that treatment with ZA at 100 μg/kg could significantly inhibit tumor growth in both WT and SR-A KO mice, whereas ZA at 25 μg/kg exhibited an inhibitive effect on tumor growth only in SR-A KO mice (Figure 6A). There was a dose-related response in inhibition of LLC tumor growth in SR-A KO mice. Last, we measured the expression of MMP9 in human lung cancer and matched adjacent normal tissues. In direct contrast to the SR-A staining pattern, MMP9 expression was significantly increased in tumorous tissues compared with matched adjacent normal tissues (Figure 6B), alluding to the importance of a potential SR-A–MMP9 axis in lung tumorigenesis.
Figure 6. Matrix metalloproteinase-9 (MMP9) is a modulator for accelerated tumor growth in class A scavenger receptor A knockout (SR-A KO) mice. (A) Wild-type (WT) and SR-A KO mice were injected subcutaneously daily with saline or zoledronate (ZA, 25 or 100 μg/kg) 3 days before Lewis lung carcinoma (LLC) cells were inoculated. (a) Macroscopic appearances of all the tumors. Tumor volume (b) and weight (c) were measured (WT, n = 16; WT/ZA 25 μg/kg, n = 12; WT/ZA 100 μg/kg, n = 14; SR-A KO, n = 16; SR-A KO/ZA 25 μg/kg, n = 14; SR-A KO/ZA 100 μg/kg, n = 16; *P < 0.05, **P < 0.01). (B) (a) Representative images of MMP9 staining in human lung cancerous (T) and matched adjacent normal tissues (N). Original magnification, ×400. (b) Significant differences for MMP9 staining pattern were observed between human lung cancers and matched adjacent normal tissues (n = 43, P < 0.01, χ2 test). (c) Quantitative RT-PCR analysis of MMP9 in human lung cancerous and matched adjacent normal tissues (n = 51, **P < 0.01).
[More] [Minimize]Roles of macrophages in carcinogenesis have drawn great attention (38). In this study, we identified a promoter polymorphism T+25C in the SR-A gene, encoding a pattern recognition receptor primarily expressed in macrophages, that was independently associated with lung cancer risk. The risk allele +25C resulted in decreased expression of SR-A in normal lung tissues. A mouse model revealed that SR-A knockout could promote the tumor growth and angiogenesis of lung cancer, possibly by cultivating a procarcinogenic microenvironment. Thus, the evidence from the population study and mouse tumor model indicates that SR-A acts as a suppressor gene for lung cancer, and that the T+25C variant is a candidate biomarker for lung cancer susceptibility.
Genetic alterations at 8p22 in multiple tumors, including lung cancer, have indicated the importance of the region in the development of malignancy (10–12). The SR-A gene, located at 8p22, has been proposed as one candidate at this region relevant to cancer. In an attempt to determine the role of SR-A in lung cancer, Yoshimura and colleagues performed mutation analysis of the coding sequence of the SR-A gene and identified a 6-bp deletion and a thymine-to-cytosine substitution in 1 of 30 primary lung cancer tissues, which showed a limited role of SR-A in lung cancer in terms of mutation (39). Genetic variants in SR-A have been linked to both hereditary and sporadic prostate cancer, and to lung cancer in the current study. Importantly, the genotype of T+25C was significantly correlated with the expression of SR-A in human beings. This discovery suggests that T+25C may contribute to lung cancer development by influencing SR-A expression.
Our population study also demonstrated that the expression of SR-A in lung cancer tumor tissues was down-regulated compared with adjacent normal tissues, implicating SR-A as a putative tumor-modulating gene. It has been reported that CD204-expressing stromal macrophage is associated with increased tumor aggressiveness in lung adenocarcinoma (24). In that study, the survival time of patients was significantly shorter in the high CD204-positive macrophage group, which seems to indicate that CD204 could potentially serve as a marker of tumor-promoting macrophages that drive up tumor progression. But the exact role of CD204 in tumor aggressiveness needs to be further validated by in vivo experiments. Our observation that deletion of SR-A resulted in more vigorous LLC tumor development than in WT mice seems contradictory to the report by Ohtaki and colleagues. The global SR-A deficiency in the present study may contribute to the discrepancy. The effect of gene knockout on the phenotype of lung cancer in mice is much stronger than attenuated SR-A expression attributable to the T+25C polymorphism in human beings, indicating that loss of SR-A function may increase lung cancer risk. This was confirmed by the observation that transplantation of SR-A KO BMDCs into WT mice aggravated LLC tumor growth and angiogenesis. BMDCs are composed of a heterogeneous myeloid, lymphoid, endothelial, and stromal cell population. Our results revealed that macrophages and endothelial cells may be responsible for the increased tumor growth in SR-A KO mice. SR-A is primarily expressed in macrophages but can also be detected in endothelial cells and fibroblasts (15, 16). The reason that transplantation of SR-A KO BMDCs did not change the expression level of SR-A in the fibroblasts of WT mouse tumors should be investigated. Future investigations with tissue-specific targeting of SR-A will likely give a more definitive answer to this question.
The occurrence and development of tumors are shaped by a complicated interplay between cancer cells, infiltrated immune cells, stromal cells, and numerous humoral factors (40). Infiltration of macrophages is linked to an unfavorable prognosis in patients with cancer (38, 41). Furthermore, in nonprogressing or regressing tumors, macrophages are biased to a classically activated M1 subset that is characterized by proinflammatory activity, antigen presentation, and tumor lysis. In malignant tumors, however, the phenotype of macrophages resembles that of alternatively activated macrophages (M2 type), accompanied by enhanced angiogenesis, tumor cell intra/extravasation, and growth (38, 41). However, we did not find a positive correlation between SR-A and macrophages transdifferentiation in our model. On the other hand, we found that MMP9, VEGF, SDF1, and COX-2 were markedly up-regulated in SR-A null mice, pointing to a putative role of SR-A in controlling tumor microenvironment in lung cancer. MMP9, a key orchestrator of the tumor microenvironment (34, 42), is an important cytokine generated mainly by macrophages in tumors (32, 33) as well as tumor cells (43, 44). MMP9 exerts its proangiogenic role by liberating VEGF from the extracellular matrix to elicit angiogenesis (32, 34). Intriguingly, MMP9 production was up-regulated in both cancer cells and macrophages after coculturing, reflecting a dynamic dialogue between tumor cells and surrounding tissues that collectively contribute to the formation of a proangiogenic and progrowth tumor microenvironment. Indeed, we showed that ZA could inhibit LLC tumor growth in a dose–response manner in both SR-A KO mice and WT mice. Its current application in cancer therapy is for the treatment of bone metastases and associated pain. In addition to these activities, ZA can inhibit MMP9 to impair VEGF availability, thus reducing tumor angiogenesis (33). However, the therapeutic effects of ZA may well go beyond MMP9 inhibition. Several studies have demonstrated that ZA inhibits factors involved in angiogenesis, such as VEGF and platelet-derived growth factor (45, 46). In addition, ZA can inhibit the chemotactic effect induced by SDF1 and reduces COX-2 expression (47). These effects of ZA may also be exerted in the present study because VEGF, SDF-1, and COX-2 in tumors were up-regulated in SR-A KO mice.
In summary, our data provide strong evidence that an SR-A gene promoter polymorphism (T+25C) leads to the down-regulation of SR-A expression and may increase lung cancer risk in humans. Moreover, loss of SR-A promotes lung cancer development by cultivating a procarcinogenic microenvironment in mice. As such, fine-tuning SR-A activity may yield promising therapeutic solutions against lung cancer in the future.
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*J. Ben and G. Jin contributed equally to this work.
Supported by grants from the National Basic Research Program (973) (nos. 2012CB517503, 2011CB503903, and 2012CB945003) and by key grants from the National Natural Science Foundation of China (nos. 81230070 and 30730044) and National Natural Science Foundation of China (no. 81070120) to Q.C.; the National Natural Science Foundation of China (no. 81000118) and the College Natural Science Foundation of Jiangsu (no. 10KJB310005) to J.B.; the National Key Basic Research Program Grant (no. 2013CB910304), National Natural Science Foundation of China (no. 81001276), and the Jiangsu Natural Science Foundation (no. BK2012841) to G.J.; a key grant from the National Natural Science Foundation of China (no. 30730080) to H.S.; the National Natural Science Foundation of China (no. 30972541) to Z.H.; and by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Author Contributions: Study concept and design: J.B., G.J., Z.H., H.S., Q.C.; data collection: J.B., G.J., Y.Z., B.M., H.B., J.C., Hanze Zhang, Q.G., Xiaodan Zhou, Hanwen Zhang, L.Q., Xudong Zhu, X.L., Q.Y.; data analysis: J.B., G.J., Y.Z., B.M.; manuscript writing and editing: J.B., G.J., Z.H., Y.X., H.S., Q.C.
This article has an online supplement, which is available from this issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201204-0592OC on August 9, 2012
Author disclosures
