LKB1 inhibits lung cancer progression through lysyl oxidase and extracellular matrix remodeling

Abstract

LKB1 loss-of-function mutations, observed in ∼30% of human lung adenocarcinomas, contribute significantly to lung cancer malignancy progression. We show that lysyl oxidase (LOX), negatively regulated by LKB1 through mTOR-HIF-1α signaling axis, mediates lung cancer progression. Inhibition of LOX activity dramatically alleviates lung cancer malignancy progression. Up-regulated LOX expression triggers excess collagen deposition in Lkb1-deficient lung tumors, and thereafter results in enhanced cancer cell proliferation and invasiveness through activation of β1 integrin signaling. High LOX level and activity correlate with poor prognosis and metastasis. Our findings provide evidence of how LKB1 loss of function promotes lung cancer malignancy through remodeling of extracellular matrix microenvironment, and identify LOX as a potential target for disease treatment in lung cancer patients.

The high mortality of lung cancer (1) is largely attributable to failure of early diagnosis and metastasis frequently observed at the time of diagnosis. Tumor suppressor loss of function is widely adopted in tumor initiation and progression. The roles of LKB1 as a tumor suppressor have emerged from the observation of increased risk of malignancy in gastrointestinal tract in Peutz–Jeghers syndrome (PJS) patients harboring germ-line LKB1 mutations (2, 3). Although rare in most types of human cancers (4, 5), LKB1 loss-of-function somatic mutations are frequently observed in human non–small-cell lung cancer (NSCLC) (6–10). Mice with oncogenic Kras^G12D mutant develop lung tumors with long latency and low aggressiveness. Concomitant loss of Lkb1 significantly shortened the latency, increased tumor burden, and promoted lung cancer invasion and distant metastasis, comparable to that from p53 loss (6). Yet, the molecular mechanisms involved remain largely unknown.

Cancer progression is a reciprocal process involving intimate interaction between tumor cells and tumor stroma, including extracellular matrix (ECM). ECM alteration and remodeling is one of the most frequently observed and most important events during malignancy progression, which subsequently modulates cell-matrix and cell-cell interaction and results in altered cell behavior (11). Increasing interests and efforts have been paid to those enzymes involved in ECM remodeling, among which lysyl oxidase (LOX) is of particular interest. LOX oxidizes lysine residues in collagen and elastin, resulting in covalent cross-linking and stabilization of these ECM structural components (12). Aberrant LOX expression or enzymatic activity has been linked to a variety of pathological conditions, including breast cancer and lung cancer (13–16). LOX is associated with hypoxia in human breast cancer and head and neck tumors, and is responsible for hypoxia-induced tumor metastasis (13). Although studies have implicated deregulated LOX mRNA and protein levels in lung adenocarcinomas (17, 18), the roles and molecular mechanisms of LOX involved in lung cancer progression are poorly understood.

Here we identify LOX as a target negatively regulated by LKB1 in both de novo murine lung tumors and human NSCLC cell lines. We further provide evidence that LOX potentiates lung cancer progression elicited by LKB1 deficiency via ECM remodeling, and may serve as a potential therapeutic target for lung cancer therapy.

Results

LOX Expression Level Correlates with LKB1 Status in Lung Cancer.

We have previously shown that loss of function of Lkb1 promoted lung cancer progression and metastasis in mouse model driven by Kras^G12D mutant (6). Integrative bioinformatic analysis on microarray datasets derived from lung tumors in Kras and Kras/Lkb1^L/L mice and from human NSCLC cell lines with or without LKB1 (6) has identified a series of differentially expressed genes, among which LOX, previously shown to be involved in breast cancer metastasis (13), drew much of our attention. Significantly increased Lox expression (∼15-fold assessed by quantitative RT-PCR) was observed in murine lung tumors with Lkb1 deficiency (Fig. 1A), but not in those with p53 deficiency (SI Appendix, Fig. S1). This is further confirmed by immunofluorescence staining on lung tumor sections (Fig. 1B). Consistently, increased LOX activities were observed in sera from mice with Lkb1-deficient lung tumors (Fig. 1C). Study of a panel of human NSCLC cell lines revealed that high LOX expression levels were evident in all three lines with mutant LKB1 but not in majority lines with wild-type LKB1 (SI Appendix, Fig. S2). To examine the clinical relevance of LOX levels to lung cancer progression, LOX activities in sera from a cohort of 80 lung adenocarcinoma patients were measured. LOX activity correlated significantly with clinical stages and metastasis status, but not with sex or smoking status (Table 1). Reevaluation of published microarray dataset of a cohort of 111 lung cancer patients (19) revealed significant association of high LOX expression with shorter overall survival in lung adenocarcinoma patients (19 vs. 49 mo median survival, P = 0.0009; Fig. 1D).

Fig. 1.

LKB1 down-regulates LOX in lung cancer. (A) Real-time PCR quantification of Lox mRNA levels in Kras and Kras/Lkb1^−/− lung tumors (three in each group). (B) LOX immunofluorescent staining on Kras and Kras/Lkb1^−/− lung tumor sections. (Scale bars: 100 μm.) (C) LOX enzymatic activity assay on serum samples from mice with Kras (n = 6) or mice with Kras/Lkb1^−/− lung tumors (n = 7). (D) Kaplan–Meyer plots show that lung cancer patients with high LOX expression levels had statistically significant shorter survival (P = 0.0009) than patients with low LOX expression levels. Data were presented as means ± SEM. Statistical analyses were performed using Student's t test. ***P < 0.001.

Table 1.

LOX serum activity correlates with human lung adenocarcinoma progression and metastasis

Characteristic	No.	LOX activity (mean ± SD)	P value
Clinical stage
I/II	36	1365.18 ± 1022.10
III/IV	44	2322.54 ± 2441.68	0.022*
Metastasis
Nonmetastatic	33	1332.24 ± 1075.75
Metastatic	47	2284.55 ± 2361.16	0.018*
Gender
Male	40	1633.64 ± 1165.18
Female	40	2149.80 ± 2545.95	0.249
Smoking status
Nonsmoker	52	2038.49 ± 2282.30
Smoker	28	1619.17 ± 1250.39	0.371

Nonmetastatic, no metastasis; metastatic, lymph node metastasis or distant metastasis.

*Denotes significant difference.

LKB1 Negatively Regulates LOX Transcription Through mTOR-HIF-1α Signaling Axis.

We next sought to investigate whether up-regulation of LOX is the direct effect of LKB1 deficiency. Ectopic expression of LKB1 in human NSCLC cell lines A549 dramatically decreased LOX mRNA levels, protein levels, and enzymatic activities (Fig. 2A). This regulation was also seen in LKB1 mutant cell line CRL-5800 and wild-type LKB1 cell line CRL-5807 (SI Appendix, Fig. S3 B and D). LKB1 knockdown in HTB-182 cells, a human NSCLC cell line with wild-type LKB1, resulted in a significant increase of LOX mRNA, protein levels, and enzymatic activities (SI Appendix, Fig. S4 A and B).

Fig. 2.

HIF-1α mediates LOX transcription downstream of LKB1. (A) Down-regulated LOX mRNA, protein levels, and activities in A549 cells reconstituted with LKB1. Anti-Flag is used for detection of Flag-LKB1. (B) Ectopic LKB1 expression down-regulated HIF-1α protein level in A549 cells. (C) Expression of either HIF-1α or PA mutant, a stable form of HIF-1α, up-regulated LOX levels in A549 cells. (D and E) Reintroduction of HIF-1α rescued the inhibition of LOX promoter activity (D) and down-regulation of LOX protein levels (E) by LKB1 in A549 cells. (F) Knockdown of HIF-1α decreased LOX protein levels in A549 cells with 200 μM CoCl₂. (G and H) mTOR inhibitor PP242 treatment significantly decreased LOX promoter activity (G) and protein levels (H) in A549 cells. Data are presented as mean ± SEM. Statistical analyses were performed using Student's t test. **P < 0.01, ***P < 0.001.

The altered LOX mRNA levels strongly indicate transcriptional level regulation. Consistently, ectopic expression of LKB1 dramatically decreased LOX promoter activity in multiple NSCLC cell lines, including A549, CRL-5800, and CRL-5807 (SI Appendix, Figs. S3 A and C and S5A). Hypoxia-inducible factor 1α (HIF-1α) was reported to regulate LOX expression in breast cancer via the conserved hypoxia-responsive elements (HREs) in LOX promoter (13). Ectopic LKB1 expression in A549 cells significantly decreased HIF-1α protein level (Fig. 2B), as well as its transcriptional activity (SI Appendix, Fig. S5B). Conversely, knockdown of LKB1 increased HIF-1α protein level in HTB-182 cells (SI Appendix, Fig. S4A). Overexpression of HIF-1α or HIF-1α-PA mutant, a stable form of HIF-1α, increased LOX promoter activity, transcription (SI Appendix, Fig. S5 C and D), and LOX protein levels in A549 cells (Fig. 2C). More importantly, reintroduction of HIF-1α could rescue the inhibitory effect of LKB1 on LOX promoter activity (Fig. 2D), mRNA level (SI Appendix, Fig. S5E), protein level (Fig. 2E), and enzymatic activity (SI Appendix, Fig. S5F). Similar results were seen in other NSCLC cell lines, including CRL-5800 and CRL-5807 (SI Appendix, Fig. S3). HIF-1α knockdown significantly decreased LOX expression (Fig. 2F and SI Appendix, Fig. S5 G and H). As a multifunctional protein kinase, LKB1 is involved in multiple signaling pathways, among which mTOR pathway has been shown to regulate HIF-1α (20). Inhibition of mTOR activation using either rapamycin or PP242 significantly decreased LOX promoter activity and LOX mRNA and protein levels in A549 cells (Fig. 2 G and H and SI Appendix, Fig. S6 A–C). Consistent with a previous report (21), mTOR inhibition significantly decreased HIF-1α transcriptional activity through down-regulation of HIF-1α protein level (Fig. 2H and SI Appendix, Fig. S6 C and D). Ectopic expression of HIF-1α rescued the inhibition of LOX enzymatic activity by rapamycin treatment in A549 cells (SI Appendix, Fig. S6E), indicating that HIF-1α mediates the regulation of LOX transcription downstream of mTOR. Therefore, LKB1 regulates LOX transcription directly through mTOR-HIF-1α signaling axis.

LOX Mediates Lung Cancer Cell Anchorage-Independent Growth and Migration.

LKB1 is a multitask tumor suppressor involved in regulation of cell proliferation and cell migration. We then asked whether LOX contributes to altered cell migration and/or cell proliferation in LKB1-deficient lung cancer cells. Consistent with previous reports (6), ectopic LKB1 expression significantly reduced lung cancer cell proliferation, anchorage-independent growth, and cell migration (Fig. 3 A and B and SI Appendix, Fig. S7 A–E). LOX overexpression had no effect on cell proliferation, nor could it rescue the inhibition on cell growth by LKB1 in A549 and CRL-5807 cells (SI Appendix, Fig. S7 C and E). Consistently, neither LOX knockdown in A549 or CRL-5844 cells nor inhibition of LOX enzymatic activity via β-aminoproprionitrile (BAPN) treatment in A549 cells had significant effect on cell proliferation (SI Appendix, Fig. S7 F–K), suggesting molecules other than LOX may mediate the antiproliferation effect of LKB1. However, reintroduction of LOX into A549 and CRL-5807 cells ectopically expressing LKB1 could partially rescue the inhibitory effects of LKB1 on anchorage-independent cell growth and cell migration (Fig. 3 A and B and SI Appendix, Fig. S8 A and B). The oxidase activity of LOX is necessary as LOX mutated in Lys320 or Tyr355, residues critical for LOX enzymatic activity, failed to rescue the inhibition of anchorage-independent cell growth and cell migration by LKB1 (Fig. 3 A and B). Conversely, LOX knockdown in A549 and CRL-5844 cells resulted in a significant decrease of anchorage-independent cell growth and cell migration abilities (Fig. 3 C and D and SI Appendix, Fig. S8 C and D).

Fig. 3.

LOX mediates lung cancer cell anchorage-independent growth and migration. (A and B) Ectopic expression of wild-type, but not enzymatically inactive LOX in A549 cell rescued the inhibitory effect of LKB1 on anchorage-independent cell growth (A) and cell migration (B). (C and D) Knockdown of LOX in A549 cells significantly impaired anchorage-independent cell growth (C) and cell migration (D). Data are presented as mean ± SEM. Statistical analyses were performed using Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

LOX Inhibition Alleviates Lung Cancer Malignancy Triggered by LKB1 Deficiency.

To test if LOX is essential for lung carcinogenesis provoked by LKB1 deficiency in vivo, BAPN, a widely used LOX pharmacological inhibitor, was administrated to previously established lung cancer mouse model with or without Lkb1 deficiency (6). BAPN treatment resulted in efficient inhibition of LOX activity (SI Appendix, Fig. S9A). BAPN treatment for 4 wk significantly decreased both tumor number and tumor volume in Lkb1-deficient mice model revealed by histological inspection, whereas it had no obvious effect on the progression of tumors with wild-type Lkb1 (Fig. 4 A and B and SI Appendix, Fig. S10 A and B). Interestingly, BAPN treatment dramatically reduced the number of large LKB1-deficient tumors (>0.5 mm²) but had no much effect on the number of those small tumors (Fig. 4C), indicating that LOX inhibition mainly impairs tumor progression. LOX activity inhibition resulted in much less cell proliferation, indicated by Ki67 positive staining, and enhanced tumor cell apoptosis, indicated by cleaved Caspase-3 staining (Fig. 4D and SI Appendix, Fig. S9). The roles of LOX in lung cancer metastasis were further confirmed by i.v. injection of A549 cells into nude mice. Knockdown of LOX in A549 cells resulted in significantly less tumors and smaller tumors in the mouse lung (SI Appendix, Fig. S9 C and D).

Fig. 4.

LOX inhibitor BAPN significantly alleviates lung tumor progression in de novo murine lung cancer with Lkb1 deficiency. (A) BAPN treatment significantly decreased the size of murine lung tumors. (Scale bars: Left, 500 μm; Right, 100 μm.) (B and C) Quantification of tumor volume and numbers in H&E-stained lung sections from Kras/Lkb1^L/L mice treated with BAPN or saline (eight mice in each group). (D) Quantitative proliferative and apoptotic indices in lung tumors from control and BAPN treatment group determined from more than 200 high-power fields (HPF). Data are presented as mean ± SEM. Statistical analyses were performed using Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Excess Collagen Deposition Increases Lung Cancer Cell Proliferation and Invasiveness Through Activation of β1 Integrin Signaling.

LOX functions as a key enzyme in collagen cross-linking, and therefore facilitates collagen deposition (12). We hypothesized that increased LOX activity in LKB1-deficient lung tumors may result in altered composition and architecture of collagen matrix and thus contribute to cancer progression. As shown by Sirius red staining, active ECM remodeling featured with collagen-rich fibrotic loci is evident in Lkb1-deficient tumors but not in those with wild-type Lkb1 (SI Appendix, Fig. S11A). BAPN treatment greatly diminished fibrotic loci in Lkb1-deficient lung tumors (SI Appendix, Fig. S11B), indicating LOX activity is responsible for the dense collagen deposition in Lkb1-deficient lung tumors. We next asked if the altered extracellular matrix, featured with excessive collagen deposition in LKB1-deficient lung tumors, facilitates cancer cell malignant transformation. Previous studies suggested that increased stiffness of the extracellular matrix promoted breast epithelial cell transformation (22). Similar to that observed in breast cancer cells, increased collagen concentration resulted in changed cell morphology and enhanced invasion ability of A549 cells in 3D matrigel culture in a collagen concentration-dependent manner (SI Appendix, Fig. S11C), underscoring fundamental roles of ECM remodeling with tensional change in cancer cell transformation and invasion ability. Immunocytochemistry analyses revealed disrupted cell polarity and increased cell proliferation within collagen-rich environment (Fig. 5A). β1 integrin is the major cell-surface collagen receptor. β1 integrin blocking antibody significantly attenuated A549 cell invasiveness in collagen-rich matrigel and reversed the disruption of cell polarity (Fig. 5A). Percentage of proliferating cells in collagen-rich matrigel was also decreased after β1 integrin antibody treatment (Fig. 5A). Binding of β1 integrin to collagen ligand activates intracellular signaling, including phosphorylation and activation of focal adhesion kinase (FAK) and Src. Knockdown of FAK impaired the aggressive cell behavior in collagen-rich matrigel, in a manner similar to that in β1 integrin blocking antibody treatment (Fig. 5B). Taken together, these data support a fundamental role of excess collagen deposition in lung cancer progression in response to elevated LOX activity through activation of β1 integrin signaling.

Fig. 5.

 Excess collagen deposition increases lung cancer cell proliferation and invasiveness through activation of β1 integrin. (A) β1 integrin antibody (20 μg/mL) blocked collagen-dependent cell morphology change and cell proliferation in 3D culture. (B) Knockdown of FAK retains A549 cell morphology and proliferation in collagen-rich environment. (Scale bars: Top, 200 μm; Middle and Bottom, 25 μm.) (Right) Quantification of percentage of proliferating cells. Experiments were performed in duplicates and repeated three times.

Discussion

The high mortality of lung cancer is mainly attributable to the poor understanding of lung cancer progression and metastasis. LKB1 loss-of-function mutations, which contribute to ∼30% of NSCLCs, warrant studies to explore downstream targets for development of effective cancer treatment strategies.

Here we identify LOX as an essential mediator of LKB1-deficiency-elicited lung cancer progression through ECM alteration, especially collagen matrix remodeling. LOX activity inhibition significantly alleviates LKB1-deficient lung cancer malignancy and invasion, underscoring the essential roles of LOX in cancer progression in lung tumors with LKB1 loss-of-function mutation. LKB1 loss triggers up-regulation of LOX expression through mTOR-HIF-1α signaling axis. Hypoxia and/or up-regulation of hypoxia-inducible factors are frequently observed in many types of cancers. Gene transcription mediated by HIF-1α promotes characteristic tumor behavior, including angiogenesis, invasion, metastasis, dedifferentiation, and enhanced glycolytic metabolism (23). VEGF, the well-known downstream target of HIF-1α, was also proposed to be involved in LKB1-deficient tumor progression. Sunitinib inhibition of VEGFR kinase activity resulted in a prolonged survival in Kras/Lkb1^L/L lung cancer mice via suppression of primary tumor growth without much impact on the malignancy progression (24). Thus, LOX and VEGF, two downstream targets of HIF-1α, may function synergistically in LKB1-deficient tumor progression.

Several recent studies have highlighted LOX as an important promoter of tumor malignancy. Erler et al. (13, 25) have shown that LOX is essential for hypoxia-induced tumor metastasis in human breast cancer, and further provide evidence that LOX facilitates the metastatic process via type-IV collagen cross-linking in the basement membrane and formation of “premetastatic niche.” Our data in lung cancer mouse model clearly show that the main function of LOX is to promote advanced-stage lung cancer progression and metastasis without much impact on initial neoplastic transformation. It is noteworthy that LOX mediates cell migration provoked by LKB1 loss with little impact on cell proliferation in 2D cell culture. However, lung cancer cells are more proliferative in collagen-rich matrigel, compared with those cultured in the absence of exogenously added collagen. LOX activity inhibition also affected lung tumor growth and progression in de novo animal model. The discrepancy could be due to the difference between 2D and 3D cell culture systems, and LOX may alter cancer cell growth potential via ECM remodeling.

Despite that breakdown of surrounding matrix is believed to be prerequisite for tumor metastasis, histopathological analyses had clearly shown correlation of poor outcome in patients with fibrotic lesions in a variety of cancers, including lung cancer, underscoring the essential roles of ECM remodeling during tumor progression and metastasis. The matrix stiffness, as well as the ECM composition and architecture, play fundamental roles in cell fate determination. Normal breast epithelial cells in stiff 3D microenvironment share characteristics with transformed breast cancer cells in disrupted cell adherent junction, enhanced cell proliferation, failure in lumen formation, and aberrant activation of intracellular signaling pathways and cytoskeleton rearrangement (22). Our data show that lung cancer triggered by LKB1 loss is frequently accompanied with fibrotic foci and significant ECM remodeling. The dense collagen matrix microenvironment provokes the increase of cancer cell invasion ability through activation of Src and FAK downstream of β1 integrin signaling, whereas β1 integrin blocking antibody and depletion of FAK significantly decreased cancer cell proliferation and invasiveness in a collagen-rich environment. The fundamental role of ECM remodeling is pivotal for lung cancer associated with LOX cross-linking of collagen and is of great importance for novel therapeutic strategies. The recent work by Weaver and coworkers (26) has also highlighted the importance of ECM remodeling in a broad range of cancer progression.

LOX was reported to be deregulated in multiple cancers, including breast cancer, head and neck cancer, and lung cancer (16). Our data show that high LOX expression or serum activity significantly correlates with lung cancer poor prognosis, and cancer stages and metastasis, respectively. A previous study found no correlation between LKB1 loss-of-function mutations and clinical outcome in stage I and II NSCLC patients (9). Although a larger-scale analysis of LKB1 mutation and/or expression is necessary to reach a conclusive point, it is conceivable that signaling components, e.g., mTOR and/or HIF-1α, which regulate LOX gene expression downstream of LKB1, may be commonly hyperactivated at late stages of lung cancer in a LKB1-dependent or -independent manner. From this aspect, our finding of increased LOX serum activity in advanced stages of lung cancer patients may provide an even broader application for lung cancer prognosis.

In conclusion, we provide strong evidence that LOX is an important downstream mediator of lung cancer progression and metastasis provoked by LKB1 deficiency. The identification of LOX links molecular pathways that control the progression and metastasis in lung cancers with LKB1 mutations to stromal ECM remodeling through collagen cross-linking and β1 integrin signaling activation. LOX is a potentially important therapeutic target for lung cancer treatment and a promising biomarker for lung cancer prognosis.

Materials and Methods

Additional information is provided in SI Appendix.

Reporter Gene Assay.

Luciferase activities were measured 48 h after transfection using the Dual-Luciferase Assay Kit (Promega) on a GloMax 20/20 Luminometer. pRL-SV40 or pEGFP-C1 was cotransfected as internal control. Experiments were performed in triplicate and repeated at least three times.

Mouse Treatment.

Kras^G12D, Lkb1^L/L, and P53 ^L/L mice were originally generously provided by T. Jacks (Cambridge, MA) and R. Depinho (Boston), respectively. Lung cancer mice models with Kras, Kras/Lkb1^L/L, and Kras/P53^L/L mice were generated as described previously (6). All mice were housed in a pathogen-free environment at Shanghai Institute of Biochemistry and Cell Biology and treated in strict accordance with protocols approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Detailed information is provided in SI Appendix.

Human Serum Sample Analysis.

The study was approved by the local ethic committees in Fudan University Shanghai Cancer Center, and sera were collected with the written consent of patients from July 2007 to March 2009. Additional details are provided in SI Appendix.

Histopathological Analysis and Immunological Studies.

Histopahtological analysis and immunological studies were performed as described (6, 27–29). Additional details are provided in SI Appendix.

LOX Enzymatic Activity Assay.

Serum from mice, human lung cancer patients, and phenol-red free conditioned media (CM) from confluent cells were collected for LOX enzymatic activity assessment as described (30). See SI Appendix for detailed information.

Statistical Analysis.

Data were analyzed by Student's t test; P < 0.05 was considered significant. Survival analysis on previously published microarray data (19) was performed using the Kaplan–Meier method. A P value <0.05 was considered statistically significant.