Inhibits Lung Cancer Metastasis Research Articles

Docetaxel-loaded Bovine Serum Albumin Nanoparticles Conjugated Docosahexaenoic Acid for Inhibiting Lung Cancer Metastasis to Bone

Cancer is one of the severest diseases in the world, and lung cancer is one of the five common cancers causing thousands of deaths every year. Moreover, most of the lung cancer patients die because of bone metastasis. This research was conducted with the aim of developing novel nanoparticles (NPs) of docetaxel (DTX) loaded bovine serum albumin (BSA) conjugated docosahexaenoic acid (DHA) using an emulsion/solvent evaporation method for inhibiting lung cancer metastasis to bone. The in vitro drug release of the DTX-DHA-BSA-NPs showed that the nanoparticles released in a sustained and controlled manner contributed to continual fight against cancer cells. The study results revealed that the DTX-DHA-BSA-NPs had higher antitumor efficacy in comparison with the DTXBSA- NPs or DTX in vitro. The study results also showed that the DTX-DHA-BSA-NPs had higher inhibiting efficacy of lung cancer metastasis to bone in comparison with the DTX-BSA-NPs or DTX in vivo. Furthermore, the mean survival time was longer with DTX-DHA-BSA-NPs (24.40 d) than that of DTX (20.95 d). In consideration of these results, the DTXDHA- BSA-NPs may hold potential for bone metastasis of lung cancer treatment.

3,4-Dihydroxybenzalactone Suppresses Human Non-Small Cell Lung Carcinoma Cells Metastasis via Suppression of Epithelial to Mesenchymal Transition, ROS-Mediated PI3K/AKT/MAPK/MMP and NFκB Signaling Pathways.

3,4-Dihydroxybenzalactone (DBL) was isolated from Phellinus linteus (PL), which is a folk medicine possessing various physiological effects. In this study, we used highly metastatic A549 cells to investigate efficacy of DBL inhibition of cancer metastasis and possible mechanisms. The results revealed DBL inhibited migratory and invasive abilities of cancer cells at noncytotoxic concentrations. We found DBL suppressed enzymatic activities, protein expression, and RNA levels of matrix metalloproteinase (MMP)-2 and MMP-9. Western blot results showed DBL decreased phosphoinositide 3-kinase (PI3K)/AKT, phosphorylation status of mitogen-activated protein kinases (MAPKs), and focal adhesion kinase (FAK)/paxillin, which correlated with cell migratory ability. DBL also affected epithelial to mesenchymal transition (EMT)-related biomarkers. In addition, DBL enhanced cytoprotective effects through elevated antioxidant enzymes including heme oxygenase 1 (HO-1), catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD). Moreover, DBL influenced the nuclear translocation of nuclear factor κB (NFκB), nuclear factor erythroid 2-related factor 2 (Nrf2), Snail, and Slug in A549 cells. Taken together, these results suggested that treatment with DBL may act as a potential candidate to inhibit lung cancer metastasis by inhibiting MMP-2 and -9 via affecting PI3K/AKT, MAPKs, FAK/paxillin, EMT/Snail and Slug, Nrf2/antioxidant enzymes, and NFκB signaling pathways.

Traditional Chinese medicine, Fuzheng Kang‑Ai decoction, inhibits metastasis of lung cancer cells through the STAT3/MMP9 pathway.

Lung cancer is a leading cause of cancer-associated mortality worldwide, including in developing countries such as China. Traditional Chinese medicine may provide a novel insight for the treatment of patients with lung cancer. The present study aimed to uncover the mechanism by which the Chinese herbal medicine, Fuzheng Kang-Ai (FZKA), functions on lung cancer cell metastasis. The results demonstrated that treatment with FZKA markedly inhibited cell viability, migration and invasion of lung cancer cells, as determined by cell viability and Transwell assays. Notably, the activity and expression of matrix metalloproteinase 9 (MMP9) was significantly inhibited by FZKA treatment on lung cancer cells, as determined by an MMP9 activity assay and western blot analysis. Furthermore, FZKA markedly inhibited epithelial-mesenchymal transition (EMT) of lung cancer cells by inhibiting the expression of the mesenchymal markers N-cadherin and vimentin. In addition, activation of the oncoprotein signal transducer and activator of transcription 3 (STAT3) was suppressed following treatment with FZKA. Conversely, overexpression of STAT3 was able to rescue MMP9 activity following FZKA treatment. The present study indicated that FZKA may inhibit lung cancer metastasis via the STAT3/MMP9 pathway and EMT, suggesting that FZKA may serve as a novel promising therapeutic strategy for the treatment of patients with late stage lung cancer.

NecroX-5 prevents breast cancer metastasis by AKT inhibition via reducing intracellular calcium levels

A major goal of breast cancer research is to prevent the molecular events that lead to tumour metastasis. It is well-established that both cytoplasmic and mitochondrial reactive oxygen species (ROS) play important roles in cell migration and metastasis. Accordingly, this study examined the molecular mechanisms of the anti-metastatic effects of NecroX-5, a mitochondrial ROS scavenger. NecroX-5 inhibited lung cancer metastasis by ameliorating migration in a mouse model. In human cancer cells, the inhibition of migration by NecroX-5 is cell type-dependent. We observed that the effect of NecroX-5 correlated with a reduction in mitochondrial ROS, but mitochondrial ROS reduction by MitoQ did not inhibit cell migration. NecroX-5 decreased intracellular calcium concentration by blocking Ca2+ influx, which mediated the inhibition of cell migration, AKT downregulation and the reduction of mitochondrial ROS levels. However, the reduction of mitochondrial ROS was not associated with supressed migration and AKT downregulation. Our study demonstrates the potential of NecroX-5 as an inhibitor of breast cancer metastasis.

Abstract A08: A novel lncRNA RGMB-AS inhibit NSCLC metastasis via upregulating the expression of target gene RGMB

Abstract Background: The relationships between long noncoding RNA (lncRNAs) and tumors have currently become one of the focuses on cancer studies. Our previous studies found repulsive guidance molecule b (RGMB) was downregulated in lung cancer which is associated with poor prognosis and cancer metastasis.The present study investigated the regulation of RGMB expression by lncRNA RGMB-AS and its role in lung cancer progression. Methods: The expression of lncRNA RGMB-AS and RGMB were detected in 56 paired NSCLC tissues and adjacent normal tissues by qRT-PCR assay. The relations of lncRNA RGMB-AS and RGMB expression with clinicopathological factors of NSCLC patients were explored. The lncRNA RGMB-AS was downregulated and overexpressed in A549 and H1299 cells, and its role in lung cancer was further investigated in vitro and in vivo. Results: The results of qRT-PCR showed that lncRNA RGMB-AS expression was significantly downregulated in NSCLC tissues than in adjacent normal tissues (P < 0.05). Correlation analysis indicated that the expression of lncRNA RGMB-AS and RGMB mRNA were positively correlated (P < 0.05). The downregulation of lncRNA RGMB-AS in NSCLC tissues were associated with poor overall survival status(P < 0.05). Downregulation of lncRNA RGMB-AS in A549 and H1299 cells subsequently results in reduced RGMB mRNA and protein expression. The downregulation/overexpression of lncRNA RGMB-AS significantly promoted/inhibited NSCLC cells metastasis in vitro and in vivo. Conclusion: We identified lncRNA RGMB-AS which was downregulated in NSCLC patients and thereby to reduce RGMB expression. lncRNA RGMB-AS might inhibit lung cancer metastasis via the regulation on RGMB and other tumor suppressors. Citation Format: Jin Li, Shi Xiaoshun, He Jianxing. A novel lncRNA RGMB-AS inhibit NSCLC metastasis via upregulating the expression of target gene RGMB. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A08.

Abstract B10: Effects of vascular normalization when combining miR-200b with radiation

Abstract Introduction: Lung cancer is the leading cause of cancer-related deaths worldwide, and patients usually die from metastatic disease. Radiation is a mainstay treatment in lung cancer, but its efficacy is largely dependent on tumor perfusion. We recently found that miR-200b is a novel AngiomiR capable of inhibiting metastasis and tumor angiogenesis while inducing vascular normalization. We are investigating whether vascular normalization with miR-200b can enhance therapeutic efficacy of radiation in a metastatic model of lung cancer. Methods: Using lung cancer cell lines, we assessed direct effects of miR-200b on radio-sensitivity using clonogenic assays and gamma-H2AX indices. Using an orthotopic lung cancer model (344SQ) in athymic nude mice, we determined peri-vascular biodistribution of intravenously administered chitosan nanoparticles (CH) containing Cy3-labled miRNA. Using the same model, a randomized therapeutic experiment was performed as follows (n=10 mice/group): 1) NC miR-CH, 2) miR-200b-CH, 3) NC miR-CH + radiation, or 4) miR-200b-CH + radiation. Radiation groups received four fractions of 4 Gy (16 Gy total). Following two weeks of treatment, mice were necropsied, and tumor burden was annotated and subsequently analyzed for microvessel density (MVD), pericyte coverage (CD31/Desmin) and tumor hypoxia (pimonidazole staining). Results: Following a single delivery of CH nanoparticles containing Cy3-labled miRs, we found that 37% of tumor vessels had Cy3 signal within 12.5 uM, 60% within 25 uM, and 82% within 50 uM. Compared to NC miRNA, we found that miR-200b overexpression in conjunction with radiation did not diminish colony formation or increase gamma-H2AX indices, suggesting miR-200b likely has little role in directly sensitizing cancer cells to radiation. In an orthotopic model of lung cancer, compared to NC miR-CH, while delivery of miR-200b-CH alone or treatment with NC miR-CH plus radiation showed no differences in metastasis, the combination of miR-200b-CH and radiation led to a 58% reduction in aggregate metastatic disease (p<0.05). Compared with the NC miR-CH treated group, MVD was significantly reduced by miR-200b-CH (27%), NC miR-CH plus radiation (50%) and miR-200b-CH plus radiation (77%), all p<0.0001. Dual treatment with miR-200b-CH and radiation had an additive anti-angiogenic effect compared with miR-200b-CH or NC miR-CH plus radiation alone, p<0.0001. Consistent with these dramatic reductions in MVD was an associated increase in the area of tumor hypoxia (ATH). Compared to NC miR-CH (16% ATH), while miR-200b-CH treatment did not significantly affect hypoxia (20% ATH, p=0.26), both radiation treatment groups had significantly increased tumor hypoxia (NC miR-CH plus radiation, 25% ATH, p<0.01; miR-200b-CH plus radiation, 41% ATH, p<0.0001). Again, dual treatment had a significantly additive effect compared to either treatment alone (miR-200b-CH v. miR-200b-CH plus radiation, p<0.01; NC-miR-CH plus radiation v. miR-200b-CH plus radiation, p=0.027). Intriguingly, compared with NC miR-CH treatment, only the miR-200b-CH delivery groups led to significant increases (40-50%) in pericyte coverage (both p<0.05). Discussion: Our work demonstrates that CH nanoparticle delivery of miR-200b can effectively target tumor vasculature. The combination of miR-200b delivery with radiation effectively inhibited lung cancer metastasis and was significantly associated with a marked decrease in MVD and increase in pericyte coverage. These additive effects appear to be independent of miR-200b directly radio-sensitizing cancer cells. However, we found that the profound anti-angiogenic effects of combination treatment (miR-200b-CH plus radiation) led to a 2.5 fold increase in tumor hypoxia. Our findings suggest that vascular normalization induced by miR-200b can be exploited to create an optimal “treatment window” for combination with radiation therapy. Citation Format: Salma Azam, Seth Miller, Trent Waugh, Vivek Somasundaram, Mitchell Smith, Adrienne D. Cox, Chad V. Pecot. Effects of vascular normalization when combining miR-200b with radiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B10.

Paeoniflorin inhibits macrophage-mediated lung cancer metastasis

Alternatively activated macrophages are more frequently involved in tumor growth, angiogenesis, and immunosuppression. A previous study showed that paeoniflorin, the major active constituent of Paeonia lactiflora Pallas, can inhibit tumor growth and lung metastases of Lewis lung tumor-bearing mice. This study tried to investigate whether paeoniflorin inhibited lung cancer metastasis by inhibiting the alternative activation of macrophages (M2 macrophage). Using a viability assay, the cytotoxicity of paeoniflorin on Lewis lung cancer cells and peritoneal macrophages were investigated. In vitro scratch wound and in vivo lung metastasis experiments were used to test the ability to inhibit the migration of paeoniflorin and the function of M2 macrophages. Flow cytometry was performed to test the cell cycle of Lewis lung cancer cells, and to test the M2 macrophages in peritoneal macrophages and subcutaneous transplantable tumor. It was found that paeoniflorin showed no inhibitory effect on the growth of Lewis lung cancer cells and peritoneal macrophages of mouse in vitro. Paeoniflorin could attenuate the migration of LLC stimulated by alternatively activated macrophages (stimulated for 24 h and 48 h, paeoniflorin 1, 3, 10, 30, 100 μmol·L−1, P < 0.01 or P < 0.05 vs control group). Paeoniflorin could decrease the cell populations at S phases (paeoniflorin 10, 30, 100 μmol·L−1, P < 0.05 vs control group) and increase the cell populations at G0-G1 phases of Lewis lung cancer cells (paeoniflorin 100 μmol·L−1, P < 0.05 vs control group) and reduce the numbers of M2 macrophages in peritoneal macrophages induced by IL-4 (paeoniflorin 1, 3, 10, 30, 100 μmol·L−1, P < 0.01 vs Control group). Paeoniflorin could reduce lung metastasis of Lewis lung cancer cells xenograft and decrease the numbers of M2 macrophages in subcutaneous xenograft tumour in vivo (paeoniflorin 20, 40 mg·kg−1, P < 0.01 vs control group). These results suggest that paeoniflorin could reduce lung metastasis of Lewis lung cancer cells xenograft partly through inhibiting the alternative activation of macrophages.

HuR-targeted nanotherapy in combination with AMD3100 suppresses CXCR4 expression, cell growth, migration and invasion in lung cancer.

The CXCR4 chemokine receptor plays an important role in cancer cell metastasis. The CXCR4 antagonist, AMD3100, has limited efficacy in controlling metastasis. HuR, an RNA-binding protein, regulates CXCR4 in cancer cells. We therefore investigated whether targeting HuR using a siRNA-based nanoparticle plus AMD3100 would suppress CXCR4 and inhibit lung cancer metastasis. We treated human H1299 lung cancer cell with HuR-specific siRNA contained in a folate-targeted lipid nanoparticle (HuR-FNP) plus AMD3100, and compared this with AMD3100 alone, HuR-FNP alone and no treatment. HuR-FNP plus AMD3100 treatment produced a G1 phase cell-cycle arrest and reduced cell viability above and beyond the effects of AMD3100 alone. HuR and CXCR4 mRNA and protein expression levels were markedly reduced in all treatment groups. Phosphorylated (p) AKTS473 protein was also reduced. P27 protein expression increased with HuR-FNP and combination treatment. Promoter-based reporter studies showed that the combination inhibited CXCR4 promoter activity more than did either treatment alone. Cell migration and invasion was significantly reduced with all treatment; the combination provided the most inhibition. Reduced matrix metalloprotease (MMP) -2 and -9 expression was associated with reduced invasion in all treatment groups. Thus, we found that combined HuR and CXCR4 targeting effectively controlled lung cancer metastasis.

MircoRNA-33a inhibits epithelial-to-mesenchymal transition and metastasis and could be a prognostic marker in non-small cell lung cancer.

Understanding the molecular mechanism by which epithelial mesenchymal transition (EMT)-mediated cancer metastasis and how microRNA (miRNA) regulates lung cancer progression via Twist1-activated EMT may provide potential therapeutic targets for cancer therapy. Here we found that miR-33a, an intronic miRNA located within the sterol regulatory element-binding protein 2 (SREBP-2) gene, is expressed at low levels in metastatic non-small cell lung cancer (NSCLC) cells and is inversely correlated with Twist1 expression. Conversely, miR-33a knockdown induces EMT and miR-33a overexpression blocks EMT by regulating of Twist1 expression in NSCLC cells. Bioinformatical prediction and luciferase reporter assay confirm that Twist1 is a direct target of miR-33a. Additionally, Twist1 knockdown blocks EMT-related metastasis and forced expression of miR-33a inhibits lung cancer metastasis in a xenograft animal model. Clinically, miR-33a is found to be at low levels in NSCLC patients and down-regulation of miR-33a predicts a poor prognosis. These findings suggest that miR-33a targets Twist1 and inhibits invasion and metastasis in NSCLC. Thus, miR-33a might be a potential prognostic marker and of therapeutic relevance for NSCLC metastasis intervention.

Triptolide Inhibits Lung Cancer Cell Migration, Invasion, and Metastasis

Triptolide is an extract from Tripterygium wilfordii used in traditional Chinese medicine to treat autoimmune disorders. Triptolide has anticancer effects in vitro and is reported to impair cancer cell migration. We studied whether triptolide inhibits lung cancer cell migration and metastasis. We determined the microRNA expression profile of triptolide-treated cells. We tested the effects of triptolide treatment on migration and invasion of lung cancer cells by using Transwell filters coated with fibronectin and Matrigel, respectively. Western blot analyses were used to compare expression of proteins involved in cell migration before and after 10 nmol/L triptolide treatment. Tail vein injections with H358 cells were performed. The mice were treated with 1 mg/kg triptolide or vehicle by intraperitoneal injection three times per week. Lung and liver metastases were compared at 9 weeks. Means of groups were compared by using a t test. Triptolide altered the expression of microRNAs involved in cellular movement and significantly decreased migration and invasion of lung cancer cells from approximately 18 to 3 cells per field (p < 0.001). Triptolide decreases focal adhesion kinase expression, which leads to impairment of downstream signaling. Finally, triptolide-treated mice injected with lung cancer cells significantly decreased metastatic colony formation in the lungs (p < 0.01). Triptolide decreases lung cancer cell migration and invasion in vitro and inhibits metastatic tumor formation in mice. Triptolide suppresses focal adhesion kinase, which causes deregulation of the migration machinery. These results suggest that triptolide inhibits lung cancer metastasis and should be investigated as a new lung cancer therapy.

Aerosolized MANS and BIO-11006 Peptides Inhibit Lung Cancer Metastasis in SCID Mice: Metastatic Non-Small Cell Lung Cancer

surgical resections. All specimens underwent histopathologic review, molecular testing for KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, MEK1, and NRAS using standard multiplexed PCR assays, and fluorescence in situ hybridization (FISH) testing for ALK and ROS1 gene rearrangements and MET amplification. Results: One hundred eighty-one patients with lung adenocarcinoma were evaluated. The median age was 65 (range 38-91), 54% female, and 22% never smokers. Whites, African Americans (AA), and Asians made up 66%, 30%, and 4% of the population, respectively. All stages of lung adenocarcinoma were included. Stage I (33%) and stage IV (44%) disease represented the largest portion of our patients. The results of the molecular analysis from the tumor samples from the overall population indicated that 95 (52%) patients were wild type (WT), and 86 (48%) had an identifiable driver mutation. Of these patients, the most frequently mutated gene was KRAS with 37 (20%) occurrences, followed by 23 EGFR (12.7%), 4 HER2 (2.2%), 3 BRAF, 3 Met amplified, 3 ALK-positive, 3 NRAS, 2 RET, 1 ROS1, and 1 IDH. Six patients harbored dual driver mutations including 2 KRAS and NRAS, and 1 each of EGFR and PIK3CA, EGFR and KRAS, EGFR and IDH, HER2 and RET. Amongst our cases, AA patients had a higher frequency of dual mutations than in other patient populations: (2 KRAS and NRAS, 1 KRAS and EGFR, 1 EGFR and IDH). Where treatment data was available, 99 (60%) patients underwent surgery, 51 (31%) had radiation, and 75 (47%) had chemotherapy. Fifteen (9%) patients were given targeted therapies based on the results of their molecular testing, with EGFR-directed treatments being the most frequent. Fifty-one patients (28%) demonstrated progression of disease and patient mortality was evenly split between WT and patients with identifiable mutations, with a total of 33 (18%) deaths. Conclusions: Approximately 50% of lung adenocarcinoma of all stages harbored an identifiable driver mutation. The higher representation of AA patients with dual mutations bears further investigation. Author Disclosure: C. Steuer: None. M. Behera: None. M. Rossi: None. K. Fisher: None. G. Sica: None. A. Gal: None. S. Pakkala: None. R. Pillai: None. T. Owonikoko: None. F. Khuri: None. S. Ramalingam: None.

PML represses lung cancer metastasis by suppressing the nuclear EGFR-mediated transcriptional activation of MMP2

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-β (IFNβ) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNβ induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.

Role and association of CD11b and Gr1+ cells in promotion of spontaneous lung cancer metastasis (TUM4P.920)

Abstract Microenvironment plays an important role in tumor growth and metastasis. We established a unique metastasis model by in vivo passaging the Lewis lung carcinoma (3LL) cells with 7-11 days of culture in between passages. When injected subcutaneously the parental (3LL) cells have minimal metastasis whereas the in vivo passaged (p3LL) cells showed robust metastasis. The goal of this study is to identify the immune population that enhances the metastatic propensity of tumor cells. Purified p3LL (CD45-) cells also yielded similar metastasis excluding a direct role of residual immune cells in promotion of metastasis. The p3LL cells metastasized at similar frequency in WT and Rag2-/- mice indicating that adaptive immune cells are not required for metastasis. Analysis of tumors derived from 3LL vs p3LL revealed differential accumulation of CD11b+Gr1+ subpopulations. 3LL tumors contained higher CD11b+ Gr1high cells whereas p3LL tumors showed higher levels of CD11b+ Gr1low population. Interestingly, when CD11b+ Gr1low populations were isolated from spleen of p3LL tumor bearing mice and co-cultured with 3LL cells for 5 days, these cells acquired enhanced metastatic capacity as compared to control 3LL cells. These results indicate a distinct association and a critical role of CD11b+ Gr1low population in promotion of metastatic phenotype and suggest that abundance of CD11b+ Gr1low population may serve as a marker and a potential therapeutic target for inhibiting Lung cancer metastasis.

MiRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3′-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.

MiR-133a suppresses cell proliferation, migration and invasion in human lung cancer by targeting MMP-14

Lung cancer is the leading cause of cancer‑related mortality worldwide. Over half of lung cancer cases are diagnosed after metastasis, for which the median survival time is approximately 8months. microRNAs (miRNAs), which are a class of single‑stranded endogenous non‑coding RNAs, are likely to be involved in most biological processes. miR‑133 plays roles in cardiac development and disease, and recent studies showed that miR‑133 is downregulated in various human malignancies, such as bladder and lung cancer. However, its detailed role in the processes of cancer remains to be determined. In the present study, we found that in the lung cancer cell lines A549 and NCI‑H1299 overexpression of miR‑133a suppressed cell proliferation, migration and invasion. The miR‑133a‑induced suppression of cell migration and invasion can be reversed by miR‑133a‑specific inhibitor. According to the mRNA sequence, matrix metalloproteinase (MMP)‑14, which is an important regulator of metastasis, is a predicted target of miR‑133a. This was confirmed by dual luciferase reporter assay. Moreover, miR‑133a overexpression decreases the mRNA and protein levels of MMP‑14. Collectively, these results suggest that miR‑133a may inhibit lung cancer metastasis by targeting MMP‑14 and may be used as an anti‑metastatic therapy in lung cancer patients.

Abstract 395: Farnesyl diphosphate farnesyltransferase induces metastasis via upregulating secreted phosphoprotein 1 in non-small cell lung cancer.

Abstract Identifying cancer progression associated genes that predicting patient outcome might provide opportunities for developing better therapeutic strategies for lung cancer patients. In the present study, we investigated whether farnesyl diphosphate farnesyltransferase (FDFT1), an enzyme of mevalonate pathway, is a potential biomarker of lung cancer metastasis. We further investigated its prognostic value and functional role in lung cancer invasion and metastasis. Our results by immunohistochemistry (IHC) analysis showed the expression of FDFT1 was significantly higher in tumors compared to normal lung tissues. Overexpression of FDFT1 was significantly associated with tumor size (P &amp;lt; 0.001), advanced tumor stage (P &amp;lt;0.001), lymph node metastasis (P = 0.02), distal metastasis (P = 0.043), and poor prognosis (P &amp;lt;0.001). Loss of FDFT1 expression significantly suppressed invasion and migration potential of lung cancer cell lines, whereas FDFT1 overexpression promoted invasion and migration in vitro and in vivo. Microarray analysis further identified secreted phosphoprotein 1 (SPP1) as an important mediator for FDFT1-induced invasion and migration. Furthermore, patients with high FDFT1 and high SPP1 expression showed dismal outcomes as compared to patients with low FDFT1 and low SPP1 expression with good prognosis. In conclusion, we identified the FDFT1 in this study as a new lung cancer progression biomarker that is associated poor prognosis and its overexpression resulted in enhanced tumor invasion and metastasis through upregulation of SPP1. Our findings may implicate the possibility of targeting FDFT1 as a novel therapeutic strategy to inhibit lung cancer metastasis. Citation Format: Yi-Fang Yang, Yi-Hua Jan, Michael Hsiao. Farnesyl diphosphate farnesyltransferase induces metastasis via upregulating secreted phosphoprotein 1 in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 395. doi:10.1158/1538-7445.AM2013-395 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Identification of ALDOA as a new Lung adeonocarcinoma predict gene involve cancer metabolism and tumor metastasis

The metabolic alterations in cancer progression remain unknown. In this study, we identified Aldolase A (ALDOA), a glycolytic enzyme that regulates glycolysis process, as the most significant marker in lung cancer by comparative microarray and prognosis database analysis. We observed overexpression of ALDOA mRNA was significantly associated with poor overall survival rate with the hazard ratio of (HR=3.0, P&lt;0.01). Microarray datasets also showed high ALDOA expression in tumor compared with corresponding normal tissue in 27 lung cancer patient (p&lt;0.01). Strong expression of ALDOA protein by IHC was further detected in 94.3% cases (99/105) and the expression level was higher compared to non‐tumor lung parenchyma (p&lt;0.01). High ALDOA expression was found associated with poor survival (p&lt;0.01) of lung cancer patients. ALDOA overexpression promoted invasion and migration in vitro. These complementary function expression consequences were also validated in metastasis colony forming assay in vivo and in‐situ invasion assay. Furthermore, overexpression of ALDOA induces upregulation of GAP1, and MMP2. Knockdown of GAP or MMP2 inhibited ALDOA induced invasion and migration in vitro, suggesting that ALDOA may promote lung cancer cell matastasis by the activation of GAP and MMP2. Our finding may implicate the possibility of targeting ALDOA as a novel therapeutic strategy to inhibit lung cancer metastasis.

CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation

CCN family protein 2 (CCN2), also known as connective tissue growth factor, is a secreting protein that modulates multiple cellular events. We previously demonstrated the metastasis-suppressive effect of CCN2 in lung cancer cells. In this study, we investigate the role of CCN2 in anoikis, a form of programmed cell death that is critical in suppressing cancer metastasis. CCN2 binds to the epidermal growth factor receptor (EGFR) and triggers ubiquitination by inhibiting the formation of the β-pix/Cbl complex, resulting in the degradation of EGFR. Binding of CCN2 to EGFR suppresses the phosphorylation of c-Src and extracellular signal-regulated kinase but increases the expression of death-associated protein kinase, which leads to anoikis. Overall, our findings provide evidence validating the use of CCN2 as an anti-metastatic therapy in lung cancer patients, and prospect a potential therapeutic synergy between CCN2 and the anti-EGFR antibody for the treatment of lung cancer.

Identification of anti-lung cancer extract from Chlorella vulgaris C-C by antioxidant property using supercritical carbon dioxide extraction

In this study, supercritical carbon dioxide extraction (SC-CO 2) technology was developed to extract the active components (such as antioxidants) from a novel microalga, Chlorella vulgaris C-C, due to its superior advantages over conventional solvent or ultrasonic extraction methods. Using SC-CO 2 and ultrasonic extractions, the polyphenol contents of C. vulgaris C-C were 13.40 and 0.46 (mg gallic acid/g lyophilized extract), respectively. The flavonoid content obtained from SC-CO 2 (3.18 mg quercetin/g lyophilized extract) was also significantly higher than from ultrasonic extraction (0.86 mg quercetin/g lyophilized extract). Investigation of C. vulgaris C-C extract from SC-CO 2 indicates strong antioxidant activities in radical scavenging, ferric reducing power and metal chelating abilities. In cell proliferation assay, the extract of C. vulgaris C-C inhibits human lung cancer H1299, A549, and H1437 cells in a dose-dependent manner. In addition, the treatment with extracts of C. vulgaris C-C effectively reduced the migration of tumor cells, suggesting the potential of using the C. vulgaris C-C extract to inhibit lung cancer metastasis.

Peonidin 3-Glucoside Inhibits Lung Cancer Metastasis by Downregulation of Proteinases Activities and MAPK Pathway

Anthocyanins, present in various vegetables and fruits as a nature colorant, have broad activities including anticarcinogenesis and antimutagenesis, which are generally attributed to their antioxidant activities. However, limited studies have been available concerning the inhibitory effect of peonidin 3-glucoside (P3G) for cancer metastasis. Here, we demonstrated that P3G could significantly inhibit the invasion (P < 0.001), motility (P < 0.05), secretion of matrix metalloproteinase (MMP)-2, MMP-9, and urokinase-type plasminogen activator (u-PA) of lung cancer cells. Meanwhile, P3G attenuated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, a member of mitogen-activated protein kinase (MAPK) family involved in the upregulation of MMPs and u-PA, and also inhibited the activation of activating protein-1 (AP-1) as shown by Western blot and electrophoretic mobility shift assay. Thus, the inhibitory effects of P3G may be at least partly through inactivation of ERK 1/2 and AP-1 signaling pathways as confirmed by abolishment of P3G-inhibited H1299 cell invasion by overexpression of MAPK kinase 1 (MEK1). Finally, P3G was evidenced by its inhibition on the metastasis of Lewis lung carcinoma cells in vivo (P < 0.001). Taken together, these findings suggested that P3G could reduce the metastasis of lung cancer cells, thereby constituting an adjuvant treatment for metastasis control.