Bone Metastases In Non-small Cell Lung Cancer Research Articles

FMNL1 down-regulation suppresses bone metastasis through reducing TGF-β1 expression in non-small cell lung cancer (NSCLC)

Approximately 40% of patients with non-small cell lung cancer (NSCLC) develop bone metastasis. The formin protein formin-like 1 (FMNL1) plays a key role in the pathogenic processes of hematopoietic malignancies, and has been reported to be associated with the progression of multiple types of cancer. In the study, we found that FMNL1 expression was markedly up-regulated in primary NSCLC samples, and stronger expression of FNML1 was detected in bone metastasis. Reducing FMNL1 expression significantly suppressed cell proliferation in NSCLC cells. We also investigated the functional effects of FMNL1 knockdown on the inhibition of migration and invasion by meditating the expression of epithelial to mesenchymal transition (EMT)-associated signals in NSCLC cells. The transforming growth factor-β1 (TGF-β1)/SMADs signaling pathway was repressed in FMNL1-knockdown NSCLC cells. Further studies indicated that additional treatment with TGF-β1 could markedly abrogate FMNL1 knockdown-induced suppression of migration and invasion in NSCLC cells. In addition, NSCLC cell-induced osteoclastogenesis was also inhibited by FMNL1 deletion, as evidenced by the down-regulated expression of tartrate-resistant acid phosphatase (TRAP) and NFATc1. In vivo studies confirmed the results that FMNL1 knockdown markedly limited tumor growth. Importantly, decreasing FMNL1 reduced bone metastasis ability in vivo. Therefore, our results demonstrated that suppressing FMNL1 expression could inhibit bone metastasis in NSCLC through blocking TGF-β1 signaling, and FMNL1 might be a novel target for developing effective therapeutic strategy to limit the bone metastasis of NSCLC.

Bone metastasis unique plasma exosomal microRNA signature in non-small cell lung cancer.

e14534 Background: 20-40% of lung cancer patients develop bone metastasis (BM) with significantly decreased overall survival. Currently, BM is mainly diagnosed by CT scan or MRI when symptom develop. Novel biomarkers with higher prediction value of BM are needed. Tumor secreted exosomes that are enriched with microRNAs have been recently implicated in site-specific tumor metastasis for establishing a distant pro-metastatic niche. Methods: Plasma exosomes from 39 stage IV non-small cell lung cancer (NSCLC) patients (n = 23 with BM; n = 16 without BM) and 16 healthy individuals were separated by ultracentrifugation. Total RNAs were extracted followed by small RNA-sequencing. miRNA identification was performed by miRDeep2. Weighted correlation network analysis (WGCNA) was used for identifying co-expression networks of highly correlated miRNA clusters. miRNA enrichment analysis and annotation (MIEAA) was applied for pathway analysis. Differential expression of individual miRNA was analyzed by edgeR. Results: Hierarchical clustering based on the total miRNA profile can clearly separate cancer patients and healthy individuals (HP), but not patients with (BM+) or without (BM-) BM. WGCNA identified three consensus clusters (A, B, C) of highly correlated miRNAs, among which cluster A (144 miRNAs) showed significantly differential expression in lung cancer patients, especially in BM+ group. Pathway analysis of cluster B miRNAs revealed enrichment in glycolysis and gluconeogenesis pathways that may involve in metabolic preconditioning of the metastatic niche. Three differentially expressed miRNAs between BM+ and BM- patients within cluster A were identified as miR-574-5p, a suppressor of Wnt/β-catenin pathway, was down-regulated, while miR-328-3p and miR-423-3p, two activators of the same pathway, were up-regulated in BM+ patients. Cluster B miRNAs (n = 49) also showed trend of upregulation in BM+ patients. Interestingly, pathway analysis indicated that 43 of them are associated with chr14, which has been suggested to promote EMT and bone metastasis. Conclusions: NSCLC patients with BM displayed unique plasma exosomal miRNA profiles, which might involve in promoting BM, and be potential biomarkers for predicting BM in NSCLC.

The PI3K inhibitor buparlisib suppresses osteoclast formation and tumour cell growth in bone metastasis of lung cancer, as evidenced by multimodality molecular imaging.

Non-small cell lung cancer (NSCLC) metastasis commonly occurs in bone, which often results in pathological fractures. Sustained phosphoinositide-3-kinase (PI3K) signalling promotes the growth of PI3K-dependent NSCLC and elevates osteoclastogenic potential. The present study investigated the effects of a PI3K inhibitor on NSCLC growth in bone and osteoclast formation, and aimed to determine whether it could control symptoms associated with bone metastasis. A bone metastasis xenograft model was established by implanting NCI-H460-luc2 lung cancer cells, which contain a phosphatidylinositol- 4,5-bisphosphate 3-kinase catalytic subunit α mutation, into the right tibiae of mice. After 1 week, the tumours were challenged with a PI3K inhibitor (buparlisib) or blank control for 3 weeks. Tumour growth and burden were longitudinally assessed in vivo via reporter gene bioluminescence imaging (BLI), small animal positron emission tomography/computed tomography (CT) [18F-fluorodeoxyglucose (18F-FDG)] and single-photon emission computed tomography/CT [99mTc-methylene diphosphonate (99mTc-MDP)] imaging. Tibia sections of intraosseous NCI-H460 tumours were analysed by immunohistochemistry (IHC), western blotting and flow cytometry. Dynamic weight bearing (DWB) tests were further performed to examine the improvement of symptoms associated with bone metastasis during the entire study. Administration of buparlisib significantly inhibited the progression of bone metastasis of NSCLC, as evidenced by significantly reduced uptake of 18F-FDG, 99mTc-MDP and BLI signals in the treated lesions. In addition, buparlisib appeared to inhibit the expression of tartrate-resistant acid phosphatase and receptor activator of nuclear factor-κB ligand, as determined by IHC. Buparlisib also resulted in increased cell apoptosis, as determined by a higher percentage of Annexin V staining and increased caspase 3 expression. Furthermore, buparlisib significantly increased weight-bearing capacity, as revealed by DWB tests. The PI3K inhibitor, buparlisib, suppressed osteoclast formation in vivo, and exhibited antitumour activity, thus leading to increased weight-bearing ability in mice with bone metastasis of lung cancer. Therefore, targeting the PI3K pathway may be a potential therapeutic strategy that prevents the structural skeletal damage associated with bone metastasis of lung cancer.

Long noncoding RNA PXN-AS1-L promotes non-small cell lung cancer progression via regulating PXN

BackgroundIncreasingly evidences suggest that long noncoding RNAs (lncRNAs) play important roles in various cancers. LncRNA PXN-AS1-L is recently revealed to act as on oncogene in liver cancer. However, the expression, functions, and mechanisms of action of PXN-AS-L in non-small cell lung cancer (NSCLC) remain unclear.MethodsThe expression of PXN-AS1-L in primary NSCLC tissues, NSCLC bone metastasis tissues, and cell lines was measured by quantitative real-time PCR. The correlations between PXN-AS1-L expression and clinicopathological characteristics of NSCLC patients were analyzed by Pearson Chi square test and log-rank test. The roles of PXN-AS1-L in cell viability, proliferation, apoptosis, and migration of NSCLC cells, and in vivo NSCLC tumor growth were investigated by a series of gain-of-function and loss-of-function assays. The regulatory roles of PXN-AS1-L on PXN were determined by quantitative real-time PCR and western blot.ResultsPXN-AS1-L was up-regulated in NSCLC tissues compared with noncancerous lung tissues, and PXN-AS1-L was further up-regulated in NSCLC bone metastasis tissues. Increased expression of PXN-AS1-L was positively associated with advanced TNM stages and poor prognosis. Gain-of-function and loss-of-function assays showed that PXN-AS1-L increased cell viability, promoted cell proliferation, inhibited cell apoptosis, and promoted cell migration of NSCLC cells. Xenograft assays showed that PXN-AS1-L also promoted NSCLC tumor growth in vivo. Mechanistically, we found that PXN-AS1-L, as an antisense transcript of PXN, up-regulated the expression of PXN. PXN was also up-regulated in NSCLC tissues. The expression of PXN and PXN-AS1-L was positively correlated in NSCLC tissues. Furthermore, PXN knockdown attenuated the roles of PXN-AS1-L in increasing cell viability, promoting cell proliferation, inhibiting cell apoptosis, and promoting cell migration of NSCLC cells.ConclusionsOur data revealed that PXN-AS1-L is up-regulated and acts as an oncogene in NSCLC via up-regulating PXN. Our data suggested that PXN-AS1-L might serve as a potential prognostic biomarker and therapeutic target for NSCLC.

Impact of the homogeneous and heterogeneous risk factors on the incidence and survival outcome of bone metastasis in NSCLC patients.

Bone metastases (BM) is reported as the most frequent distant metastasis in non-small cell lung cancer (NSCLC), but the risk factors for the incidence and prognosis of BM patients in NSCLC have not been extensively elucidated. This study aimed to find risk factors to predict BM patients' morbidity and survival outcome in NSCLC. 63,505 patients of NSCLC in the Surveillance, Epidemiology and End Results database diagnosed from 2010 to 2015 were used to analyze risk factors for developing BM by conducting multivariable logistic regression. Of these patients, 6152 and 5664 BM patients diagnosed between 2010 and 2014 were selected to investigate predictive factors for BM overall survival (OS) and cancer-specific survival (CSS) using the multivariable Cox proportional hazards regression. There were overall 7486 (11.79%) BM patients in NSCLC. The homogeneous risk factors for BM patients' morbidity and survival outcome included male, higher T stage, lymph node involvement, poor differentiation grade, brain metastases, and liver metastases. Married status, adenocarcinoma type and lung metastases were positively correlated with BM incidence, while older age, white race, unmarried status, and SCC and other NSCLC types could predict poor OS and CSS of BM in NSCLC. The homogeneous and heterogeneous risk factors for morbidity and survival outcome of BM patients could help physicians in more precise and individualized screening and therapies for BM patients in NSCLC.

SOSTDC1 inhibits bone metastasis in non-small cell lung cancer and may serve as a clinical therapeutic target.

Bone metastasis occurs in ~40% patients with non-small cell lung cancer (NSCLC), resulting in serious morbidity and mortality. Sclerostin domain-containing protein 1 (SOSTDC1) has been demonstrated to be associated with the development and progression of multiple types of cancer. However, the role of SOSTDC1 in NSCLC bone metastasis remains unclear. In the present study, it was identified that SOSTDC1 was downregulated in NSCLC bone metastatic lesions compared with that in primary tumors, and low SOSTDC1 expression predicted poor prognosis for patients with NSCLC. Functionally, SOSTDC1 overexpression suppressed NSCLC cell proliferation, migration, invasion and cancer cell-induced osteoclastogenesis, while SOSTDC1 knockdown produced the opposite effect. In addition, a number of potential downstream target genes of SOSTDC1, which were demonstrated to be associated with tumor progression and bone metastasis, were identified in NSCLC cells by RNA deep sequencing and RT-qPCR assays. The results from the present study may provide useful insight for an improved understanding of the pathogenesis of NSCLC bone metastasis, and suggest that SOSTDC1 may be a potential prognostic biomarker and therapeutic target for NSCLC bone metastasis.

Diagnostic value of serum tumor markers in bone metastasis of non-small cell lung cancer

Objective To analyze the diagnostic value of serum tumor markers in bone metastasis of non-small cell lung cancer (NSCLC). Methods The clinical data and follow-up data of 160 patients with NSCLC from March 2013 to September 2015 in Tangshan People′s Hospital were retrospectively analyzed. According to the status of bone metastasis, the patients were divided into the observation group with bone metastasis (n=114) and the control group without bone metastasis (n=46). The positive rates and concentrations of 4 tumor markers including carcinoembryonic antigen (CEA), carbohydrate antigen CA125, CA19-9 and Ferritin were analyzed and compared. And their sensitivities and specificities to the diagnosis of NSCLC bone metastasis were analyzed. The Youden index was calculated to capture the performance of the diagnostic test. Results The concentrations of CEA, CA125, CA19-9 and Ferritin in the observation group were (19.74±6.71) μg/L, (45.62±19.53) kU/L, (35.89±15.66) kU/L and (334.02±79.43) μg/L respectively, which were higher than those in the control group [(2.36±0.92) μg/L, (14.37±5.43) kU/L, (15.31±7.22) kU/L, (122.58±69.46) μg/L], and the differences were statistically significant (t=7.200, P<0.001; t=7.180, P<0.001; t=6.032, P<0.001; t=11.152, P<0.001). The positive rates of CEA, CA125, CA19-9 and Ferritin in the observation group were 78.95%, 68.42%, 52.63% and 73.68% respectively, which were higher than those in the control group (8.70%, 4.35%, 4.35% and 13.04%), and the differences were statistically significant (χ2=66.746, P<0.001; χ2=53.822, P<0.001; χ2=32.193, P<0.001; χ2=48.975, P<0.001). The sensitivities of CEA, CA125, CA19-9 and Ferritin were 78.94%, 68.42%, 52.63% and 73.68% respectively, and the specificities of them were 91.30%, 95.65%, 95.65% and 86.96% respectively. The Youden indexes were 70.24%, 64.07%, 48.28% and 60.64% respectively. The sensitivity and specificity of multi-factorial combination were 99.17% and 72.63%, and the Youden index was 71.80%. Conclusion Four tumor markers including CEA, CA125, CA19-9 and Ferritin were correlated with NSCLC, and combined detection has better performance of dignosis than single factor. Key words: Carnicoma, non-small-cell lung; Tumor markers, biological; Neoplasm metastasis; Diagnosis

High Serum HDGF Levels Are Predictive of Bone Metastasis and Unfavorable Prognosis in Non-Small Cell Lung Cancer.

Hepatoma-derived growth factor (HDGF) is a heparin-binding protein possessing mitogenic activity and could be secreted from necrotic cells passively or actively, thereby functioning as a damage-associated molecular pattern (DAMP). The high expression of HDGF in non-small cell lung cancer (NSCLC) tissues is associated with unfavorable prognosis. However, the clinical significance of serum HDGF has not been elucidated in NSCLC yet. In the present study, we compared the serum levels of HDGF in 235 patients with NSCLC (141 adenocarcinoma and 94 squamous cell carcinoma cases) with those in 40 healthy subjects. Moreover, we explored the correlation between serum HDGF levels and clinicopathologic factors or the overall survival rates. We thus found that the serum HDGF levels were significantly higher in NSCLC patients than those in healthy subjects (P < 0.001). Moreover, there was no significant difference in the serum HDGF levels between adenocarcinoma and squamous cell carcinoma. Importantly, the higher serum levels of HDGF were significantly associated with bone metastasis and with lower overall survival rates. Thus, serum HDGF was identified as an independent prognostic factor indicating poor prognosis of NSCLC. Using A549 human lung adenocarcinoma cell line, we demonstrated that an autophagy inhibitor, chloroquine, could inhibit the HDGF secretion, while quercetin, an autophagy inducer derived from a traditional Chinese drug, could facilitate HDGF secretion. In conclusion, high serum levels of HDGF were significantly correlated to bone metastasis and poorer prognosis of NSCLC. We suggest that anti-HDGF therapy is potential to protect NSCLC patients with advanced stages from bone metastasis.

Abstract 1565: Cthrc1 mediated by myeloid-specific TGF-β signaling stimulates NSCLC bone metastasis

Abstract One third of non-small-cell lung cancer (NSCLC) patients develop bone metastasis and die within a year. Tumor growth in the bone microenvironment results in excessive bone resorption−osteolytic bone lesions, which releases many cytokines and growth factors stored in the bone matrix, such as transforming growth factor-β (TGF-β), which further stimulates metastatic tumor growth in the bone. It is not known how TGF-β signaling in resident cells of the bone microenvironment effect NSCLC-induced bone lesions. We found that blocking TGF-β signaling in cells of the myeloid lineage, LysMCre/Tgfbr2 knockout (KO), but not in cells of the mesenchymal lineage, Col1αcre/Tgfbr2 KO, decreases osteolytic bone lesion development of H1993 or H1975 NSCLC cells in a tibial-injected mouse model. In determining the mechanism by which LysMCre/Tgfbr2 KO decreases NSCLC bone lesion development, we demonstrated that basic-fibroblast growth factor (bFGF) of the mouse origin was significantly decreased in NSCLC injected tibiae of LysMCre/Tgfbr2 KO mice relative to controls. Exogenous bFGF partially rescues the reduced NSCLC bone lesions in these LysMCre/Tgfbr2 KO mice. bFGF is expressed by cells of the mesenchymal lineage, such as osteoblasts. However, bFGF has no effect on H1993 NSCLC cell proliferation. These data suggest that the decreases in NSCLC-induced bone lesions by loss of TGF-β signaling in myeloid lineage cells are dependent on the effects of bFGF on osteoblasts and osteoclasts. We therefore hypothesize that the regulation of bFGF expression in osteoblasts is indirect through a secreted myeloid-specific TGF-β signaling downstream target, collagen triple-helix repeat-containing 1 (Cthrc1). Cthrc1 is a TGF-β target gene and clastokine, which are proteins secreted by osteoclasts that couple osteoblasts. Cthrc1 is also a known biomarker for NSCLC progression and promotes NSCLC cell proliferation and invasion. We found Cthrc1 was significantly decreased in H1993-injected tibiae of LysMCre/Tgfbr2 KO mice relative to controls, and stimulates H1993 proliferation in a dose-dependent manner in vitro. Additionally, Cthrc1 is known to stimulate bone formation. However, the effect of Cthrc1 on osteoblasts is not known in the context of NSCLC bone metastasis. From our in vitro studies, we found that Cthrc1 has no significant effect on osteoblast differentiation, but significantly increases bFGF gene expression in MC3T3 cells. bFGF inhibits osteoblast differentiation, but stimulates osteoclast differentiation. We therefore conclude that myeloid-specific TGF-β signaling stimulates osteoclast Cthrc1 secretion, which promotes NSCLC tumor cell proliferation. Meanwhile, Cthrc1 increases bFGF expression from osteoblasts to inhibit osteoblast, but stimulate osteoclast differentiation. Taken together, these data suggests Cthrc1 is a potential novel target for effectively inhibiting NSCLC bone metastasis. Citation Format: Paul G. Daft, Xiangqi Meng, Alexandra Vander Ark, Austin Meadows, Xiaohong Li. Cthrc1 mediated by myeloid-specific TGF-β signaling stimulates NSCLC bone metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1565.

The role of lncRNA MALAT1 in bone metastasis in patients with non-small cell lung cancer.

lncRNA metastasis-associated lung adencarcinoma transcript1 (MALAT1) plays an important role in the metastasis of lung cancer. Yet, its role in bone metastasis and the related mechanism remain unknown. The present study aimed to investigate the role of lncRNA MALAT1 in the bone metastasis of non-small cell lung cancer (NSCLC), including the expression pattern in tumor tissues, and the effect on the apoptosis, proliferation, migration and invasion of NSCLC cells. The expression level of MALAT1 in NSCLC tissues with/without bone metastasis and in NSCLC cell lines with (ACC-LC-319/bone2)/without (SPC‑A1) bone metastatic ability was determined with qRT-PCR and compared with t-test. si-MALAT1 was used to downregulate the expression of MALAT1 in ACC-LC-319/bone2 cells. The proliferation ability was assessed by MTT assay, and the apoptosis, migration, invasion and tumorigenesis invivo were also assessed to detect the effect of MALAT1 expression on NSCLC cells. In conclusion, the present study found that MALAT1 was significantly highly expressed in NSCLC tissues with bone metastasis and in NSCLC cell lines with high bone metastatic ability (P<0.0001). Downregulation of MALAT1 expression significantly inhibited proliferation and induced cell apoptosis in comparing with the negative controls. Our results also revealed that MALAT1 significantly increased the migration, invasion and tumorigenesis invivo, which suggests its important role in the bone metastasis of NSCLC.

Diagnostic value of bone metabolic markers for bone metastasis of non-small cell lung cancer analyzed by Logistic regression combined with ROC curve

Objective To investigate the diagnostic value of bone metabolic markers (BGP, β-CTX and PINP) for bone metastasis of non-small cell lung cancer (NSCLC) analyzed by Logistic regression combined with ROC curve. Methods A total of 65 patients with stage Ⅳ NSCLC were enrolled in this study. The patients were divided into two groups based on radiological imaging, includirg bone metastasis group (30 cases) and non-bone metastasis group (35 cases). The serum concentrations of BGP, β-CTX and PINP were measured by electrochemical method. Logistic regression and ROC curve were applied to analyze the data and evaluate the diagnostic values. Results The concentrations of β-CTX [(0.54±0.39) ng/ml] and PINP [(103.64±81.86) ng/ml] were significantly higher in bone metastasis group than those [(0.31±0.16) ng/ml and (48.37±27.76) ng/ml, respectively] in non-bone metastasis group (P 0.05). The area under the ROC curve (AUC) for β-CTX and PINP was 0.662 and 0.678, respectively. The AUC for the new predictive variables created by Logistic regression was 0.761. Conclusion Combined detection of β-CTX and PINP in the serum and application of Logistic regression combined with ROC curve analysis can increase diagnostic accuracy on bone metastasis of NSCLC. Key words: Carcinoma, non-small-cell lung; Neoplasms metastasis; β-isomerized C-terminal telopeptides of type Ⅰ collagen; N-terminal propeptide of type Ⅰ procollage; Logistic models; ROC curve

When Should ⁹⁹mTc Bone Scintigraphy Be Performed in cT1N0 Non-Small Cell Lung Cancer Patients?

The aim of this retrospective study was to investigate the risk factors for bone metastases (BM) in clinical T1N0 non-small cell lung cancer (NSCLC) patients.From January 2010 to June 2012, 739 patients with primary diagnosed cT1N0 NSCLC were eligible for this study. Clinical variables, including sex, smoking history, age at diagnosis, tumor size, pathologic subtype, preoperative serum Carcino embryonie antigen (CEA) level, lesion imaging performance, and skeletal system symptom, were collected.BM were found in 7 patients (0.95%), in whom 6 patients had skeletal system symptom and 1 had silent metastasis. The frequency of BM was significantly high in younger patients (P = 0.007) and in patients with higher preoperative serum CEA level (P = 0.05). In multivariate analysis, age less than 50 years old (OR = 2.23, 95% CI: 1.56–4.21, P = 0.02), presence of clinical symptom (OR = 3.15, 95% CI: 1.98–6.42, P = 0.008), and CEA level over 5 μg/mL (OR = 2.14, 95% CI: 1.37–3.53, P = 0.03) were independently associated with BM in cT1N0 NSCLC patients.Presence of skeletal system symptom is not the unique criteria for performing BS. Younger age at diagnosis and higher preoperative serum CEA level are also risk factors for BM in cT1N0 NSCLC patients. Therefore, the selection of early-stage NSCLC patients being performed BS should be more precise in the future.

Abstract 2147: The effects of artemisinin, dihydroartemisinin and artemisinin-glycolipid on non-small cell lung cancer induced bone destruction

Abstract Lung cancer is the major cause of cancer-related death in males worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of entire lung cancer and largely metastasizes to bone. Bone consistently maintains the balance between osteoclasts and osteoblasts, but NSCLC metastasized to bone interacts with normal bone cells and abnormally enhances osteoclasts-mediated bone resorption. As a result, enhanced bone resorption results in the release of transforming growth factor-beta (TGF-β) and insulin-like growth factor -1 (IGF-1) which promote cancer progression. Plant-derived compounds have been received attention as therapeutic agents for many human diseases. Artemisinin (ART) which is isolated from chinese medicinal herb, Artemisia annua and its derivative dihydroartemisinin (DHA) have been known as outstanding anti-malarial agents and have also potent anti-cancer activities. ART-glycolipid is ART's derivative that combined ART with the glycolipid daumone in order to enhance anti-cancer activity. We found that cell viability was decreased in A549 cells treated with ART, DHA and ART-glycolipid. ART, DHA and ART-glycolipid decreased the migration of A549 cells treated with or without TGF- β1 and IGF-1 which are highly discovered in bone metastasis of NSCLC. In addition, they inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation in bone marrow macrophages (BMMs). In particular, ART-glycolipid showed the most potent inhibitory effect. Therefore, we expect that ART, DHA and ART-glycolipid can inhibit metastatic ability of A549 cells and bone resorbing activity of osteoclasts by reducing secretion of matrix metalloproteinases- 9 (MMP-9) and cathepsin K activities in BMM. These results indicate that ART, DHA and ART-glycolipid may serve as useful agents for attenuating NSCLC induced bone destruction. Citation Format: Gwang-Taek Ma, Kwang-Kyun Park, Won-Yoon Chung. The effects of artemisinin, dihydroartemisinin and artemisinin-glycolipid on non-small cell lung cancer induced bone destruction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2147. doi:10.1158/1538-7445.AM2014-2147

Characteristics and treatment of bone metastases in 322 cases non-small cell lung cancer: a retrospective study

背景与目的骨是非小细胞肺癌最常见转移部位之一，可引起疼痛病理性骨折等，严重影响患者生活质量。本研究探讨非小细胞肺癌骨转移的临床特点及预后因素。方法回顾性分析我科近5年收治的600例非小细胞肺癌患者，单光子计算机断层扫描（emission computed tomography, ECT）作为筛查方法，计算机断层扫描（computed tomography, CT）/磁共振成像（magnetic resonance imaging, MRI）/X线或病理学诊断作为骨转移的确诊方法。结果肺腺癌发生骨转移的比率最高，脊柱、骨盆、股骨等为骨转移高发部位。ECT显示3个浓聚灶及以上者，行CT/MRI/X线可以证实骨转移，确诊骨转移的几率远远高于ECT显示1个-2个浓聚灶的患者[80.6%（203/252）vs 50.79%（32/63）, P < 0.001]。出现骨转移后未发生骨相关事件（skeletal related events, SRE）的患者生存期长于发生SRE者，1年生存率和中位生存期分别为44.75%、14.74个月 vs 36.17%、12.25个月（P=0.022）。经Cox多因素分析，病理组织学诊断为非腺癌、骨转移病灶数少于3个，单纯骨转移为有益于生存期的预后因素。结论ECT异常浓聚灶的数目与骨转移影像学的确诊有关。病理组织学非腺癌、骨转移病灶数少于3个、单纯骨转移是非小细胞肺癌骨转移的独立预后因素。

Everolimus and zoledronic acid--a potential synergistic treatment for lung adenocarcinoma bone metastasis.

Non-small-cell lung cancer (NSCLC) frequently metastasizes to bone. It is known that zoledronic acid is cytostatic to tumors, and everolimus, the inhibitor for mammalian target of the rapamycin, could inhibit many types of cancer. Herein, we evaluated the effect of zoledronic acid alone and in combination with everolimus on treating lung adenocarcinoma bone metastasis in vitro and in vivo. Mice treated with zoledronic acid in combination with everolimus had more apoptotic lung cancer cells and more cells were arrested in the G1/G0 phase. The phosphorylation of p70S6K was inhibited in the combination treatment group. Lung cancer cell invasion was also significantly inhibited in the group with combination treatment in vitro. Bone nuclear scans revealed more metastatic lesions in controls compared with those in the combination treatment group. Bone scans and radiographic images indicated that combination therapy significantly reduced bone metastasis. The moderate survival rate suggested that the drug combination was synergistic, which can delay NSCLC bone metastasis and prolong survival in vivo.

Notch3 is important for TGF-β-induced epithelial-mesenchymal transition in non-small cell lung cancer bone metastasis by regulating ZEB-1.

The Notch signaling pathway plays an important role in the bone metastasis microenvironment. Although recent evidence suggests that Notch signaling contributes to bone metastasis in breast and prostate cancer, its role and possible mechanisms in non-small cell lung cancer (NSCLC) bone metastasis are not yet clear. Here, we show that Notch3 is overexpressed in NSCLC bone metastases. The inhibition of Notch3 by small interfering RNA transfection decreased the invasion ability of NSCLC cells and transforming growth factor (TGF)-induced interleukin (IL)-6 and parathyroid hormone-related protein (pTHrP) expression in vitro. We also observed that Notch3 induced a strong morphological transformation, promoting the epithelial-mesenchymal transition (EMT). Western blotting and real-time polymerase chain reaction assays revealed that the forced overexpression of Notch3 induced the expression and activity of ZEB-1 and subsequent suppression of E-cadherin and upregulation of fibronectin, contributing to EMT and invasion. Western blotting and immunofluorescence assays showed that RNA interference-mediated ZEB-1 suppression blocked Notch-induced EMT-like transformation and subsequently reversed the observed effects on E-cadherin and downregulated fibronectin. A luciferase reporter system showed that Notch-induced ZEB-1 requires a functional binding site in the ZEB-1 promoter. In vitro invasion assays showed that the inhibition of ZEB-1 can decrease Notch3-promoted invasion and the expression of pTHrP and IL-6. Our results demonstrated that Notch upregulates ZEB-1, which contributes to TGF-β-induced EMT-like transformation and bone metastasis in NSCLC.

RANKL Inhibition Blocks Osteolytic Lesions and Reduces Skeletal Tumor Burden in Models of Non–Small-Cell Lung Cancer Bone Metastases

Bone metastasis is a serious complication in patients with lung cancer, occurring in up to 40% of patients. Tumor cell-mediated osteolysis occurs ultimately through induction of RANK ligand (RANKL) within the bone stroma although this hypothesis has not been tested extensively in the setting of non-small-cell lung cancer (NSCLC). By using two novel NSCLC bone metastasis mouse models, we examined the effects of RANKL inhibition on osteolysis and tumor progression. We treated mice bearing skeletal NSCLC tumors with osteoprotegerin-Fc (OPG-Fc) to assess whether osteoclast inhibition through RANKL inhibition would affect bone metastases at early or late stages of bone colonization. Progression of skeletal tumor was determined by radiography, longitudinal bioluminescent imaging, and histological analyses. OPG-Fc reduced development and progression of radiographically evident osteolytic lesions and also significantly reduced skeletal tumor progression in both NSCLC bone metastasis models. In the H1299 human NSCLC bone metastasis model, OPG-Fc plus docetaxel in combination resulted in significantly greater inhibition of skeletal tumor growth compared with either single agent alone. The observed ability of RANKL inhibition to reduce NSCLC osteolytic bone destruction or skeletal tumor burden was associated with decreases in tumor-associated osteoclasts. These results demonstrate that RANKL is required for the development of tumor-induced osteolytic bone destruction caused by NSCLC cells in vivo. RANKL inhibition also reduced skeletal tumor burden, presumably through the indirect mechanism of blocking tumor-induced osteoclastogenesis and resultant production of growth factors and calcium from the bone microenvironment. RANKL inhibition also provided an additive benefit to docetaxel treatment by augmenting the reduction of tumor burden.

Lung cancer-derived Dickkopf1 is associated with bone metastasis and the mechanism involves the inhibition of osteoblast differentiation

Wnt/β-catenin signaling and Dickkopf1 (DKK1) play important roles in the progression of lung cancer, which preferably metastasizes to skeleton. But the role of them in bone dissemination is poorly understood. This study aims to define the role of DKK1 in lung cancer bone metastases and investigate the underlying mechanism. Our results demonstrated that DKK1 over-expression was a frequent event in non-small-cell lung cancer (NSCLC) blood samples, and serous DKK1 level was much higher in bone metastatic NSCLC compared to non-bone metastatic NSCLC. We also found that conditioned medium from DKK1 over-expressing lung cancer cells inhibited the differentiation of osteoblast, determined by alkaline phosphatase activity and osteocalcin secretion, whereas the conditioned medium from DKK1 silencing lung cancer cells exhibited the opposite effects. Mechanistically, DKK1 reduced the level of β-catenin and RUNX2, as well as inhibiting the nuclear translocation of β-catenin. Taken together, these results suggested that lung cancer-produced DKK1 may be an important mechanistic link between NSCLC and bone metastases, and targeting DKK1 may be an effective method to treat bone metastase of NSCLC.

Differential Expression of the RANKL/RANK/OPG System Is Associated with Bone Metastasis in Human Non-Small Cell Lung Cancer

BackgroundHuman non-small cell lung cancer (NSCLC) patients exhibit a high propensity to develop skeletal metastasis, resulting in excessive osteolytic activity. The RANKL/RANK/OPG system, which plays a pivotal role in bone remodeling by regulating osteoclast formation and activity, is of potential interest in this context.Materials and MethodsReverse transcriptase polymerase chain reaction, western blotting, and immunohistochemical analysis were used to examine the expression of RANKL, RANK, and OPG in human NSCLC cell lines with different metastatic potentials, as well as in 52 primary NSCLC samples and 75 NSCLC bone metastasis samples. In primary NSCLC patients, the expression of these proteins was correlated with clinicopathological parameters. Recombinant human RANKL and transfected RANKL cDNA were added to the PAa cell line to evaluate the promoter action of RANKL during the process of metastasis in vitro and in vivo.ResultsUp-regulated RANKL, RANK, and OPG expression and increased RANKL:OPG ratio were detected in NSCLC cell lines and in tumor tissues with bone metastasis, and were correlated with higher metastatic potential. The metastatic potential of NSCLC in vitro and in vivo, including migration and invasion ability, was significantly enhanced by recombinant human RANKL and the transfection of RANKL cDNA, and was impaired after OPG was added. The increased expression of RANKL and OPG correlated with tumor stage, lymph node metastasis, and distant metastasis.ConclusionsDifferential expression of RANKL, RANK, and OPG is associated with the metastatic potential of human NSCLC to skeleton, raising the possibility that the RANKL/RANK/OPG system could be a therapeutic target for the treatment of metastatic NSCLC patients.

Targeting receptor activator of nuclear factor-kappa B as a new therapy for bone metastasis in non-small cell lung cancer

The review aims at comprehensively discussing our current knowledge on bone metastases incidence in non-small cell lung cancer (NSCLC), their related complications as well as clinical impact in patients suffering from advanced disease. After evoking the use of zoledronic acid as the established standard of care until recently, the new class of drugs available to prevent skeletal related events and targeting receptor activator of nuclear factor-kappa B (RANK) will be emphasized, reporting on denosumab clinical trials, a RANK-ligand (RANKL) targeting monoclonal antibody. Biological hypothesis regarding their mechanisms of action as well a potential direct impact on tumor cells are described according to the most recent laboratory as well as hypothesis-generating clinical data. Targeting the RANK pathway is an efficient way to prevent complications of bone metastases in NSCLC. Interesting additional direct effects on tumor biology and evolution are being analyzed and prospectively assessed in clinical trials.