Xenograft Lung Research Articles

Abstract P3-09-03: RelB facilitates cell migration and invasion in breast cancer via MMP1 upregulation

Abstract Background: RelB is a subunit of the nuclear factor-κB (NF-κB) signaling famliy and appears aberrant expression in breast cancer. The aim of the present study was to further investigate the role of RelB in breast cancer and whether RelB might be a therapeutic target. Methods: 120 cases of breast cancer patients were collected from the Jiangsu Province Hospital during 2015-2017. Immunohistochemistry (IHC) was performed to detect RelB expression in breast cancer tissues and para-cancer tissues. The relationship between RelB expression and clinical pathologic parameters were investigated. Detection the expression of RelB in 4 kinds of human breast cancer cell lines (MCF-7, T47D, MDA-MB-231, BT-549) by Western blot and RT-qPCR.Knockout of RelB in TNBC cell lines (BT-549 and MDA-MB-231) was using the CRISPR/Cas9 genome editing system. CCK-8 and flow cytometry were performed to observe the proliferation, cycle and apoptosis of MDA-MB-231 and BT-549 after knockout of RelB. Transwell and wound healing experiments were performed to observe cell invasion and migration ability. RT-PCR and Western blotting were performed to detect the expression of mRNA and protein of EMT marker and MMP1 respectively. The luciferase reporter gene was performed to detect whether RelB binding with MMP1 promoter region. In vivo efficacy was tested using the xenograft model and lung metastasis model. Results: RelB was highly expressed in triple-negative breast cancer (TNBC) tissues compared with that in adjacent tissues. RelB levels were remarkably associated with the pTNM stage (p=0.01981) and ER expression (p=0.4689). RelB expressions in TNBC cell lines were higher than that of hormone receptor positive MCF-7 cells. In vitro studies showed that RelB deletion promoted apoptosis and suppressed breast cancer cell survival, migration and invasion by decreasing snail, vimentin and MMP1. RelB deletion inhibited cell cycle progression and G1/S transition by upregulating p21 and p27 expression. In vivo studies showed that 231-RelB-KO cells completely impaired tumor formation in xenograft models and lung metastasis model in nude mice. Conclusion: RelB is aberrant constitutive expressed in clinical specimens and cell lines of breast cancer, especially in TNBC. RelB regulates malignant phenotypes of breast cancer, including cell survival, migration and invasion, as well as apoptosis and the cell cycle. Moreover, RelB can induce MMP1, which facilitates the invasion of tumor. Collectively, our findings highlight that RelB might serve as a therapeutic target for triple negative breast cancer. Citation Format: Wang M, Tang J. RelB facilitates cell migration and invasion in breast cancer via MMP1 upregulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-03.

Abstract P2-08-37: The prognostic impact of synaptojanin 2 expression in estrogen receptor α-positive breast cancer patients

Abstract Background: Synaptojanin 2 (SYNJ2) was reported to be a druggable mediator of metastasis. It is overexpressed and amplified in breast cancer, particularly estrogen receptor α (ERα)-positive breast cancer. SYNJ2 was also shown to promote cell migration and invasion in breast cancer xenograft cultures and lung metastasis in mice. Here, we investigated SYNJ2 mRNA expression in breast cancer patients during long-term follow-up. Materials and methods: A total of 434 invasive breast cancer tissues were analyzed for SYNJ2mRNA expression using TaqMan PCR, and the correlation of this expression with patient clinicopathological factors was determined. We also examined the expression of markers associated with tumor-initiating capacity, such as SNAI1, SNAI2, and VIM. Survival curves were analyzed using the Kaplan–Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Results: The median follow-up period was 10.7 years. We found positive correlations between high expression of SYNJ2 mRNA and shorter disease-free survival in breast cancer patients (P=0.049), which was limited to ERα-positive patients (P=0.020) and not seen in ERα-negative patients (P=0.863). High SYNJ2 mRNA levels were positively correlated with high tumor grade, ERα negativity, and node positivity. Multivariate analysis indicated that high SYNJ2 mRNA expression was an independent factor for poor disease-free survival in breast cancer patients. Multivariate analysis of poor disease-free survival DFS Mutivariate All patientsp valueHR (95%CI)Tumor size2cm and fewer1540.43151 (Reference) more than 2cm280 1.19 (0.78-1.85)Lymph node metastasisNegative2190.0001 and fewer1 (Reference) Positive184 5.61 (3.59-9.05) Unknown31 2.40 (0.89-5.48)Grade1,22330.38471 (Reference) 3195 1.20 (0.79-1.81) Unknown6 0.17 (1.78-3.49)ER statusPositive3370.02621 (Reference) Negative97 2.06 (1.87-4.07)PgR statusPositive2930.39511 (Reference) Negative141 1.30 (0.70-2.28)SYNJ2 mRNA expressionlow2170.02251 (Reference) high217 1.57 (1.07-2.34) Conclusion:High SYNJ2 expression was shown to be an independent predictive factor of poor prognosis in ERα-positive breast cancer patients. SYNJ2 could therefore be used as a candidate biomarker and therapeutic target in breast cancer. Citation Format: Nishikawa S, Kondo N, Wanifuchi-Endo Y, Hisada T, Uemoto Y, Katagiri Y, Dong Y, Kato H, Takahashi S, Toyama T. The prognostic impact of synaptojanin 2 expression in estrogen receptor α-positive breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-37.

Abstract P6-03-04: Successful development of patient-derived orthotopic xenograft models of brain and lung metastases of human breast cancer

Abstract Background: Metastatic breast cancer is the reason that we continue to lose 40,000 women every year in the US. Without appropriate pre-clinical model, success rate of clinical trials continue to suffer. Because syngeneic mouse models utilize murine neoplasm that may not represent human cancer, patient-derived xenografts (PDX) have emerged as a pre-clinical model that maintains human cancer features such as intratumoral heterogeneity. However, there is no established orthotopic PDX model for metastatic breast cancer even though the main cause of death is brain and lung metastasis. Orthotopic brain or lung PDX is expected to reproduce the original tumor microenvironment. We describe our new patient-derived metastasis orthotopic xenograft (PDMOX) mouse models of human breast cancer. Methods: All work was performed in female NSG mice of age 8-12 m. Breast cancer metastatic tumors from brain and lung that had been passaged 3x in mammary fat pads were used. Tumors of 1 mm3 were implanted orthotopically in two forms: solid piece, or minced tissue with 3 μl Matrigel. Tumor growth was monitored by MRI. Results: Two methods for brain PDMOX were compared. “Manual push” method implanted minced tumor through a frontal bone burr hole into right caudate putamen at 4 mm depth using forceps. “Pipette tip” method utilized either a pipetter for minced tissue or Hamilton syringe for solid tissue to inoculate tumor. One hour post-surgical survival was 37.5% (3/8) after “manual push”, and 100% (30/30) after “Pipette tip” method. All tumors engrafted in surviving mice with either method. However, the tumors formed on brain surface and parenchyma invasion was rare after “manual push” method, whereas solid tumor invaded parenchyma by “pipette tip” method. Therefore, it was no surprise to find large variation in tumor growth after “Manual push” (detection time 17±5.0 d, range: 17-26; volume 5.6±21.0 mm3, range 2.8-48.7). One mouse developed ptosis, and 2 out of 3 mice that underwent “Manual push” had sudden death. On the other hand, all mice that underwent “Pipette tip” method lived until tumor grew to 125-200 mm3, without neurological symptoms. These brain tumors could be passaged with 100% success (9/9). For right lung PDMOX, “thoracotomy” and “non-thoracotomy” methods were compared. “Thoracotomy” method implanted a solid tissue using forceps or 8-0 nylon suture, or injected minced tissue 1 mm below pleura. “Non-thoracotomy” method injected minced tissue using 23G needle. One hour post-surgical survival was 30% (9/30, 8/30) after “thoracotomy” method using forceps or suture, resp. However, survival using suture method could be significantly improved to 97% (29/30) by reducing thoracotomy length (<10 vs. ≥10 mm: t test P = 0.003). Post-operative survival was not affected by age, weight, or operation or anesthesia time. On the other hand, all mice after “non-thoracotomy” method survived, but chest wall implantation occurred in 67% (4/6) when the method was performed using a cell line. Conclusion: By simple modifications of surgical techniques, we could establish orthotopic brain and lung xenografts of breast cancer tumors with almost zero mortality and 100% engraftment. Our novel PDMOX models can be powerful tools for preclinical studies. Citation Format: Oshi M, Okano M, Takabe K. Successful development of patient-derived orthotopic xenograft models of brain and lung metastases of human breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-03-04.

The effect of flow on blood oxygen level dependent (R*2 ) MRI of orthotopic lung tumors.

Blood oxygen level dependent (BOLD) MRI based on measurements can provide insights into tumor vascular oxygenation. However, measurements are susceptible to blood flow, which may vary accompanying a hyperoxic gas challenge. We investigated flow sensitivity by comparing measurements with and without flow suppression (fs) in 2 orthotopic lung xenograft tumor models. H460 (n = 20) and A549 (n = 20) human lung tumor xenografts were induced by surgical implantation of cancer cells in the right lung of nude rats. MRI was performed at 4.7T after tumors reached 5 to 8 mm in diameter. A multiecho gradient echo MRI sequence was acquired with and without spatial saturation bands on each side of the imaging plane to evaluate the effect of flow on . fs and non-fs MRI measurements were interleaved during an oxygen breathing challenge (from air to 100% O2 ). -weighted signal intensity changes (ΔSI(%)) and measurements were obtained for regions of interest and on a voxel-by-voxel basis and discrepancies quantified with Bland-Altman analysis. Flow suppression affected ΔSI(%) and measurements in each tumor model. Average discrepancy and limits of agreement from Bland-Altman analyses revealed greater flow-related bias in A549 than H460. The effect of flow on , and hence BOLD, was tumor model dependent with measurements being more sensitive in well-perfused A549 tumors.

Epstein-Barr virus-coded miR-BART13 promotes nasopharyngeal carcinoma cell growth and metastasis via targeting of the NKIRAS2/NF-κB pathway

Based on analysis of Epstein-Barr virus (EBV) BART microRNA expression profiles, we previously reported that EBV-encoded miR-BART13 is upregulated in nasopharyngeal carcinoma (NPC) plasma specimens. However, the effects and molecular mechanisms of miR-BART13 in NPC remain largely unknown. We found that miR-BART13 was significantly upregulated in NPC tissue specimens. Ectopic expression of miR-BART13 promoted NPC cell proliferation, epithelial mesenchymal transition, and metastasis in vitro, and facilitated xenograft tumor growth and lung metastasis in vivo. Molecularly, NF-κB inhibitor interacting Ras-like 2 (NKIRAS2), a negative regulator of the NF-κB signaling, was identified to be a direct target of miR-BART13 in NPC cells, and NKIRAS2 mRNA and protein expression was inversely correlated with miR-BART13 in NPC tissues, respecitvely. Furthermore, the NF-κB signaling pathway was activated by miR-BART13. By rescued experiments, reconstitution of NKIRAS2 expression abrogated all the phenotypes upregulated by miR-BART13, and attenuated activity of NF-κB signaling pathway activated by miR-BART13 in NPC cells. Our findings indicated the newly identified miR-BART13/NKIRAS2/NF-κB signaling axis may provide further insights into better understanding of NPC initiation and development, and targeting of this pathway could be further studied as a therapeutic strategy for NPC patients.

Aldehyde dehydrogenase 1A1 confers erlotinib resistance via facilitating the reactive oxygen species-reactive carbonyl species metabolic pathway in lung adenocarcinomas.

Background: Acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as erlotinib is a major challenge to achieve an overall clinical benefit of the targeted therapy. Recently, aldehyde dehydrogenase 1 (ALDH1) induction has been found to render lung adenocarcinomas resistant to EGFR-TKIs, and targeting ALDH1A1 becomes a novel strategy to overcome resistance. However, the molecular mechanism underlying such effect remains poorly understood. Methods: Comprehensive assays were performed in a panel of lung adenocarcinoma cell lines and xenografts that acquired resistance to erlotinib. Cancer phenotype was evaluated by cell viability, apoptosis, migration, and epithelial-mesenchymal transition analysis in vitro, tumorsphere formation analysis ex vivo, and tumor growth and dissemination analysis in vivo. Reactive oxygen species (ROS) and reactive carbonyl species (RCS) were detected based on fluorescent oxidation indicator and liquid chromatography coupled to mass spectrometry, respectively. Protein target was suppressed by RNA interference and pharmacological inhibition or ecto-overexpressed by lentivirus-based cloning. Gene promoter activity was measured by dual-luciferase reporting assay. Results: Knockdown or pharmacological inhibition of ALDH1A1 overcame erlotinib resistance in vitro and in vivo. ALDH1A1 overexpression was sufficient to induce erlotinib resistance. Metabolomic analysis demonstrated lower ROS-RCS levels in ALDH1A1-addicted, erlotinib-resistant cells; in line with this, key enzymes for metabolizing ROS and RCS, SOD2 and GPX4, respectively, were upregulated in these cells. Knockdown of SOD2 or GPX4 re-sensitized the resistant cells to erlotinib and the effect was abrogated by ROS-RCS scavenging and mimicked by ROS-RCS induction. The ALDH1A1 overexpressed cells, though resisted erlotinib, were more sensitive to SOD2 or GPX4 knockdown. The ALDH1A1 effect on erlotinib resistance was abrogated by ROS-RCS induction and mimicked by ROS-RCS scavenging. Detection of GPX4 and SOD2 expression and analysis of promoter activities of GPX4 and SOD2 under the condition of suppression or overexpression of ALDH1A1 demonstrated that the RCS-ROS-metabolic pathway was controlled by the ALDH1A1-GPX4-SOD2 axis. The ROS-RCS metabolic dependence mechanism in ALDH1A1-induced resistance was confirmed in vivo. Analysis of public databases showed that in patients undergoing chemotherapy, those with high co-expression of ALDH1A1, GPX4, and SOD2 had a lower probability of survival. Conclusions: ALDH1A1 confers erlotinib resistance by facilitating the ROS-RCS metabolic pathway. ALDH1A1-induced upregulation of SOD2 and GPX4, as well as ALDH1A1 itself, mitigated erlotinib-induced oxidative and carbonyl stress, and imparted the TKI resistance. The elucidation of previously unrecognized metabolic mechanism underlying erlotinib resistance provides new insight into the biology of molecular targeted therapies and help to design improved pharmacological strategies to overcome the drug resistance.

MiR-126-3p suppresses the growth, migration and invasion of NSCLC via targeting CCR1.

MicroRNA (miRNA) plays vital roles in the development of different cancers. In the current work, we explored the function of miR-126-3p in the growth and metastasis of non-small-cell lung cancer (NSCLC) cell in vitro and in vivo. The expressions of miR-126-3p in NSCLC cell lines were assessed using the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, wound healing and transwell invasion were applied to reveal the role of miR-126b-3p on NSCLC cell growth, migration and invasion. The expressions of epithelial-mesenchymal transition (EMT) associated markers (E-cadherin and N-cadherin) were assessed by immunofluorescence staining. The Xenograft model and lung metastasis model were applied to explore the impact of miR-126-3p on the growth and aggressiveness of NSCLC cell in vivo. MiR-126-3p was significantly down-regulated in NSCLC cell lines and tissues. The up-regulation of miR-126-3p inhibited the growth, colony formation, migration and invasion of NSCLC cell. Furthermore, the xenograft model indicated that miR-126-3p suppressed NSCLC cell growth and lung metastasis by targeting chemokine (C-C motif) receptor 1 (CCR1). In addition, we demonstrated that the over-expression of CCR1 rescued the inhibitory effects of miR-126-3p on NSCLC cells growth, migration and invasion. Finally, knocked-down of CCR1 was able to mimic the inhibitory effects of miR-126-3p on the progression of NSCLC cell. These findings indicate that miR-126-3p plays an important role in the growth, migration and invasion of NSCLC by targeting CCR1.

MiRNA-101-5p inhibits the growth and aggressiveness of NSCLC cells through targeting CXCL6.

BackgroundThe purpose of this study is to explore the potential biological roles of miR-101-5p in the progression of non-small-cell lung carcinoma (NSCLC).MethodsThe levels of miR-101-5p and chemokine (C-X-C motif) ligand 6 (CXCL6) in NSCLC tissues and cells were detected using the quantitative real-time PCR (qRT-PCR) assay. Proliferation, colony formation, migration and invasion assays were conducted using miR-101-5p-transfected NSCLC cells in vitro. The expression of CXCL6 was measured using immunofluorescence assay. Xenograft model and lung metastasis model were constructed to further reveal the precise roles of miR-101-5p in the lung metastasis and growth of NSCLC cells in vivo.ResultsmiR-101-5p was underregulated in NSCLC tissues when compared with that in the normal controls. The levels of miR-101-5p were lower in NSCLC cells (H1975, A549, HCC827 and H1650) than in non-tumorigenic human bronchial epithelial cells (BEAS-2B). Overregulation of miR-101-5p restrained the aggressiveness phenotypes of NSCLC cells in vitro. Furthermore, overregulation of miR-101-5p reduced the tumor growth and pulmonary metastasis of NSCLC cells in vivo. CXCL6 was the target gene of miR-101-5p in NSCLC. The mRNA levels of CXCL6 were negatively associated with the levels of miR-101-5p in NSCLC tissues. Finally, the rescue experiments suggested that the inhibitory role of miR-101-5p was mediated by regulating the expression of CXCL6 in NSCLC.ConclusionThese findings indicated that overregulation of miR-101-5p restrained the progression of NSCLC cells by targeting CXCL6 and might function as a potential therapeutic target for NSCLC.

MiR-10b suppresses the growth and metastasis of colorectal cancer cell by targeting FGF13.

MicroRNA (miRNA) plays important roles in the progression of different cancers. In this study, we investigated the precise role of miR-10b in the growth and metastasis of colorectal cancer (CRC) cell. The levels of miR-10b in CRC cell lines were detected using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. A series of functional assays, including cell proliferation, colony formation, wound healing and transwell invasion were conducted using miR-10b transfected cells. The expressions of E-cadherin and N-cadherin were assessed by immunofluorescence staining. Xenograft model and lung metastasis model were constructed to investigate the impact of miR-10b on the growth and metastasis of CRC cell in vivo. We revealed that miR-10b was markedly down-regulated in CRC. The up-regulation of miR-10b suppressed the growth, colony formation, migration and invasion of CRC cell. Furthermore, the xenograft model indicated that miR-10b inhibited CRC cell growth and lung metastasis in vivo by targeting fibroblast growth factor 13 (FGF13). In addition, we demonstrated that the overexpression of FGF13 rescued the suppressive effects of miR-10b on CRC cells growth, migration and invasion. Finally, the knockdown of FGF13 was able to mimic the inhibitory effects of miR-10b on the progression of CRC cells. These results demonstrate that miR-10b plays an important role in growth, migration and invasion of CRC by targeting FGF13.

SDH5 Depletion Enhances Radiosensitivity by Regulating p53: A New Method for Noninvasive Prediction of Radiotherapy Response.

Radiotherapy is an effective treatment for lung cancer but lacks a reliable prediction method. Cell-free nucleic acids in plasma have been reported as a novel tumor marker. Here, we evaluate circulating succinate dehydrogenase 5 (SDH5) mRNA in plasma and SDH5 protein in tumors, assess their predictive value in lung cancer patients undergoing radiotherapy, and explore the underlying mechanisms.Methods: SDH5 expression was measured in peripheral blood samples and fresh tumor specimens from 208 non-small cell lung cancer (NSCLC) patients and correlated with clinical outcomes. SDH5 knockout mice and human xenograft mice were used to evaluate radiosensitivity. Cell growth, apoptosis, and the DNA damage response were assessed. Relevant RNA and protein levels were analyzed by qRT-PCR and Western blotting. Immunoprecipitation and GST pulldown assays were performed to detect protein-protein interactions. Polyubiquitination of p53 was examined by an in vitro ubiquitination assay.Results: Plasma and tumor SDH5 mRNA levels were positively correlated (rho=0.894, P<0.001). Patients with relatively low SDH5 levels in plasma (0.47, 0.12-0.89) and tumors (3.85, 0.96-7.23) had a better prognosis after radiotherapy (median PFS: 30.0 versus 15.0 months, hazard ratio: 0.276, 95% CI: 0.201-0.379, P<0.001). In SDH5 knockout mice, the lung epithelial cells exhibited increased DNA damage after radiation. In human lung xenograft mice, SDH5-deficient tumors had a smaller volume after radiotherapy. Furthermore, SDH5 depletion inhibits p53 degradation via the ubiquitin/proteasome pathway, which promotes apoptosis and enhances radiosensitivity in NSCLC.Conclusion: Our findings provide a novel noninvasive method for prediction of response to radiotherapy and may have significant implications for cancer radiotherapy.

Progress and challenges in lung xenotransplantation: an update.

Recent progress in genetic engineering has facilitated development of transgenic donor animals designed to overcome the known barriers to discordant xenotransplantation, and greatly accelerated progress in the field of xenotransplantation. Here we review and summarize recent progress in lung xenotransplantation, and discuss possible additional genetic modifications and other interventions that may further advance the use of pulmonary xenografts towards clinical applications based on known mechanisms of xeno lung injury. Ex-vivo lung perfusion experiments have shown that the addition of human complement (hCD46, hCD55), coagulation (hEPCR, hVWF, hTBM, hTFPI, hCD39), or anti-inflammatory pathway regulatory genes (HO-1, HLA-E), and the knockout (KO) of major porcine carbohydrates (GalT, Neu5Gc, B4Gal) have each protective effects on lung survival and function. The use of these transgenes in multitransgenic donor organs, targeting several known xenogeneic rejection mechanisms, combined with drug treatments addressing remaining known rejection pathways, have led to prolonged recipient survival of up to 31 days with in some cases preserved live-supporting organ function of the transplanted graft for several days. Pulmonary vascular resistance elevation, which has been found to be associated with high thromboxane levels and has been the major failure reason of xenogeneic lung grafts in the past years, has been successfully attenuated by the addition of a thromboxane synthase inhibitor (1-Benzylimidazole). Currently, the predominant failure mechanism of xenogeneic lung grafts is an inflammatory process, leading to vascular barrier function injury with interstitial and trachea edema. Work with other pig organs in primate models show that regimens based on costimulatory pathway blocking antibodies prolong xenograft function for months to years, suggesting that once initial lung inflammation mechanisms are fully controlled, clinically useful application of pig lung xenografts may be feasible. The use of multitransgenic donor pigs coupled with drugs targeting complement activation, coagulation, and inflammation have significantly improved the survival of xenogeneic pig lungs both during ex vivo human blood perfusion and in life-supporting in vivo models, and for the first time allowed consistent life-supporting function of lung xenografts. Overcoming delayed loss of vascular barrier function injury appears to be within reach, and will be essential to make lung xenografts a clinically relevant treatment option.

Astrocyte Elevated Gene 1 (AEG-1) Acts as a Promoter Gene in Clear Cell Renal Cell Carcinoma Cell Growth and Metastasis.

BackgroundClear cell renal cell carcinoma (ccRCC) is usually incurable once it progresses to metastatic stage. Hence, in-depth investigations to reveal the precise molecular mechanisms behind the metastasis of ccRCC are required to improve the therapeutic outcome of ccRCC.Material/MethodsThe level of astrocyte elevated gene 1 (AEG-1) in ccRCC tissues and cell lines was determined by quantitative real-time PCR (qRT-PCR) assay. The MTS, colony formation, wound-healing, and Transwell invasion assays were used to assess the role of AEG-1 in ccRCC cells growth, migration, and invasion in vitro, respectively. Xenograft model and lung metastasis models were constructed to analyze the functions of AEG-1 in ccRCC cells growth and metastasis in vivo.ResultWe found that AEG-1 was overexpressed in ccRCC and was associated with the progression of ccRCC. Knocked-down AEG-1 impaired the migration and invasion of ccRCC cells in vitro. Furthermore, under-expression of AEG-1 caused complete inhibition of ccRCC cells growth and metastasis in vivo. In contrast, overexpression of AEG-1 significantly increased the migration and invasion ability of ccRCC cells in vitro. Finally, we revealed that AEG-1 boosted the metastatic ability of ccRCC cells via regulating Notch homolog 1 (Notch1).ConclusionsThe AEG-1/Notch1 signaling axis plays a vital role in ccRCC cell growth and metastasis.

The Precise Diagnosis of Cancer Invasion/Metastasis via 2D Laser Ablation Mass Mapping of Metalloproteinase in Primary Cancer Tissue.

Cancer invasion and metastasis remain the major causes of over 90% of patient deaths. Molecular imaging methods such as computed tomography (CT)/magnetic resonance imaging (MRI) can precisely assess primary regional lymph node invasion and distant organ metastasis via body scanning; however, such diagnostic methods are often utilized too late for cancer therapy. To date, pathologic methods mainly provide information on differentiation/proliferation and potential drug therapy biomarkers of primary tumors rather than precisely reveal tumor regional invasion and distant metastasis in the body. We hypothesized that quantification of membrane type-1 matrix metalloproteinase (MT1-MMP) levels in primary tumor tissue will provide a precise assessment of tumor regional lymph node invasion and remote organ metastasis. In this work, we developed peptide-coated Au clusters with intrinsic red fluorescence and a specific mass signal. When these clusters labeled MT1-MMP in tumor tissue sections derived from the xenograft lung carcinoma model, human lung carcinoma and human renal carcinoma, we could directly observe MT1-MMP via optical fluorescence microscopy and quantitatively detect the MT1-MMP expression level via laser ablation inductively coupled plasma mass spectrometry 2D mapping (2D-LA-Mass Mapping). By observing and quantifying the MT1-MMP expression level in primary human lung carcinoma and human renal carcinoma tissue sections, we precisely assessed the risk of primary tumor invasion/metastasis. Importantly, the accuracy of this pathologic method was verified by CT/MRI molecular imaging of cancer patients and traditional hematoxylin and eosin (H&E) staining/immunohistochemistry (IHC)/immunofluorescence (IF) pathologic studies of primary tumor tissues.

MicroRNA-886-3P functions as a tumor suppressor in small cell lung cancer

ABSTRACTSmall cell lung cancer (SCLC) is a highly aggressive disease and miRNAs may play an important role in modulating SCLC progression. We have previously screened 924 miRNAs and found that miR-886-3P was negatively associated with SCLC survival. In the current study, we further investigated the role of miR-886-3P mimic in regulating SCLC cell phenotypic alteration in vitro and xenograft tumor formation in vivo. We found that transfection of miR-886-3P mimic significantly inhibited SCLC cell proliferation, migration, and colony formation, and induced mesenchymal-epithelial transition (MET) by suppressing TGF-ß1 synthesis in vitro. Furthermore, intra-tumor injection of miR-886-3P mimic lead to necrosis and suppression of tumor invasion to the surrounding tissue in the subcutaneous xenograft tumor, and intra-vein injection of miR-886-3P mimic suppressed xenograft lung cancer growth in vivo. These findings suggested that miR-886-3P functions as a tumor suppressor in SCLC and thus, it might be a potential therapeutic molecule in the treatment of lung cancer.

TIPE3 hypermethylation correlates with worse prognosis and promotes tumor progression in nasopharyngeal carcinoma

BackgroundIncreasing evidence recognizes that DNA methylation abnormalities play critical roles in cancer development. Our previous genome-wide methylation profile showed that tumor necrosis factor-alpha-induced protein 8 like 3 (TIPE3) was hypermethylated in nasopharyngeal carcinoma (NPC). However, the relationship between TIPE3 methylation and its mRNA expression, as well as its biological roles in NPC are unknown.MethodsBisulfite pyrosequencing and quantitative RT-PCR were performed to quantify the TIPE3 methylation and expression levels. Kaplan-Meier curves and Cox regression analysis were used to estimate the correlation between TIPE3 methylation levels and survival in two patient cohorts collected from two hospitals (n = 441). The MTT, colony formation, Transwell migration and invasion assays, and xenograft tumor growth and lung metastatic colonization models were used to identify the functions of TIPE3 on NPC cells.ResultsWe found that TIPE3 CpG island (CGI) was hypermethylated and its mRNA levels were downregulated in many cancers, including NPC. TIPE3 downregulation was associated with its CGI hypermethylation. Furthermore, NPC patients with high TIPE3 CGI methylation levels had poorer clinical outcomes than those with low methylation levels. The TIPE3 CGI methylation level was an independent prognostic factor. Moreover, restoring TIPE3 expression significantly inhibited NPC cell proliferation, migration and invasion in vitro, and suppressed tumor growth and lung metastatic colonization in vivo, while silencing TIPE3 acted in an opposite way.ConclusionsTIPE3 downregulation correlates with its CGI hypermethylation in several solid cancers. TIPE3 acts as a tumor suppressor in NPC, providing a further insight into NPC progression and representing a potential prognostic biomarker for NPC.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood.

Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms. GalTKO.hCD46 single pig lungs were perfused ex vivo with fresh heparinized human blood: lungs were either treated with 1-Benzylimidazole (1-BIA) combined with histamine receptor blocker famotidine (n=4) or diphenhydramine (n=6), 1-BIA alone (n=6) or were left untreated (n=9). Six of the nine control experiments (GalTKO.hCD46 untreated), "survived" until elective termination at 4hours. Without treatment, initial PVR elevation within the first 30minutes resolved partially over the following hour, and increased progressively during the final 2hours of perfusion. In contrast, 1-BIA, alone or in addition to either antihistamine treatment, was associated with low stable PVR. Combined treatments significantly lowered the airway pressure when compared to untreated reference. Although platelet and neutrophil sequestration and coagulation cascade activation were not consistently altered by any intervention, increased terminal wet/dry weight ratio in untreated lungs was significantly blunted by combined treatments. Combined thromboxane and histamine pathway blockade prevents PVR elevation and significantly inhibits loss of vascular barrier function when GalTKO.hCD46 lungs are perfused with human blood. Platelet activation and platelet and neutrophil sequestration persist in all groups despite efficient complement regulation, and appear to occur independent of thromboxane and histamine antagonism. Our work identifies thromboxane and histamine as key mediators of xenolung injury and defines those pathways as therapeutic targets to achieve successful xenolung transplantation.

CPEB4 promotes growth and metastasis of gastric cancer cells via ZEB1-mediated epithelial- mesenchymal transition.

BackgroundCytoplasmic polyadenylation element-binding protein 4 (CPEB4) has previously been reported to be associated with biological malignancy in various cancers. However, its function in tumor growth and metastasis in gastric cancer (GC) remains obscure. Here, we explored the functional and molecular mechanisms by which CPEB4 influences GC.Materials and methodsThe expression of CPEB4 was assessed using Western blot and immunohistochemistry in GC specimens. The roles of CPEB4 in GC cell proliferation, migration, and invasion were investigated by cell-counting kit-8 (CCK-8), colony formation, and EdU assay; wound-healing assay; and transwell assay, respectively. Quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining were performed to detect the expressions of CPEB4 and epithelial–mesenchymal transition (EMT)-related markers. The function of CPEB4 on GC cell growth and metastasis was also determined in vivo through establishing subcutaneous xenograft tumor and lung metastatic mice model.ResultsThe results revealed that the expression of CPEB4 was increased in GC tissues compared with matched normal tissues. High expression level of CPEB4 was significantly associated with clinical metastasis and unfavorable prognosis in patients with GC. Furthermore, CPEB4 silencing remarkably inhibited GC cells’ proliferation, invasion, and metastasis in vitro and in vivo. Conversely, CPEB4 overexpression achieved the opposite effects. Mechanically, we proved that ZEB1-mediated EMT might be involved in CPEB4-facilitated GC cells’ proliferation, invasion, and metastasis.ConclusionOur findings implied that CPEB4 expression predicted a worse prognosis in patients with GC. Besides, CPEB4 contributed to GC cells’ proliferation, migration, and invasion via ZEB1-mediated EMT.

Release of c-FLIP brake selectively sensitizes human cancer cells to TLR3-mediated apoptosis

Toll-like receptor 3 (TLR3) mediates innate immune responses by sensing viral dsRNA, but also induces apoptosis selectively in cancer cells. Our analysis by immunohistochemistry revealed that TLR3 is frequently overexpressed in 130 non-small cell lung cancer (NSCLC) patients’ samples compared with normal bronchial epithelium (P < 0.0001, Mann–Whitney test), supporting the therapeutic potential of TLR3 ligand for this type of cancer. However, a proportion of TLR3-expressing cancer cell lines, including NSCLC, remain resistant to TLR3-mediated apoptosis, and the underlying mechanism of resistance remains unclear. We here investigated the molecular basis conferring resistance to non-transformed vs. transformed cells against TLR3-mediated cell death. In non-transformed epithelial cells cellular FLICE-like inhibitory protein (c-FLIP) and cellular Inhibitor of APoptosis (cIAPs) ubiquitin ligases exerted an efficient double brake on apoptosis signaling. In contrast, releasing only one of these two brakes was sufficient to overcome the resistance of 8/8 cancer cell lines tested. Remarkably, the release of the c-FLIP, but not cIAPs, brake only results in the sensitization of all human cancer cells to TLR3-mediated apoptosis. Taking advantage of the difference between transformed and non-transformed cells, we developed a rational strategy by combining the chemotherapeutic agent paclitaxel, which decreases c-FLIP expression, with TLR3 ligand. This combination was highly synergistic for triggering apoptosis in cancer cells but not in non-transformed cells. In vivo, the combination of paclitaxel with dsRNA delayed tumor growth and prolonged survival in a mouse xenograft lung tumor model. In conclusion, combining the release of the c-FLIP brake with TLR3 ligand synergizes to selectively kill cancer cells, and could represent an efficient and safe therapy against TLR3-expressing cancers such as NSCLC.

Epigenetic silencing of SFRP5 promotes the metastasis and invasion of chondrosarcoma by expression inhibition and Wnt signaling pathway activation

Backgroud/aimsAbnormal activation of the Wnt/β-catenin signaling, which may be antagonized by the members of secreted frizzled-related proteins family (SFRPs), is implicated in tumor occurrence and development. However, the function of SFRP5 relating to Wnt/β-catenin pathway in chondrosarcoma is not clear yet. This study was undertaken to investigate the potential role of SFRP5 promoter methylation in chondrosarcoma metastasis and invasion through activating canonical Wnt signaling pathway. Methods and resultsThe results demonstrated that SFRP5 promoter was hypermethylated and SFRP5 expression was significantly reduced in chondrosarcoma cell lines at the mRNA and protein levels. The canonical Wnt/β-catenin signaling was observably activated with β-catenin stabilization by dephosphorylation and translocation into the nuclear. 5-Aza-2′-deoxycytidine (5-Aza-dC), the DNA methyltransferase inhibitor, significantly inhibited the proliferation of chondrosarcoma cells by cell cycle arrest through repressing the methylation of SFRP5 and promoting its expression. Both 5-Aza-dC treatment and SFRP5 overexpression could significantly inhibited the metastasis and invasion of chondrosarcoma cells by inactivating Wnt/β-catenin signaling pathway and promoting chondrosarcoma cells mesenchymal-epithelial transition (MET). 5-Aza-dC also inhibited the xenograft growth and lung metastasis of chondrosarcoma cells in vivo via suppressing SFRP5 promotor methylation, inactivating Wnt/β-catenin pathway and inducing epithelial markers expression. ConclusionAll of our results revealed the epigenetic silencing of SFRP5 by promoter methylation plays pivotal roles in chondrosarcoma development and metastasis through SFRP5/Wnt/β-catenin signaling axis. Modulation of their levels may serve as potential targets and diagnostic tools for novel therapeutic strategies of chondrosarcoma.

Dicer1 Deficiency in the Idiopathic Pulmonary Fibrosis Fibroblastic Focus Promotes Fibrosis by Suppressing MicroRNA Biogenesis.

The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. To determine how IPF-ECM negatively regulates miR-29. We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.