Metastatic Lung Nodules Research Articles

Estrogen receptor alpha transcriptionally activates casein kinase 2 alpha: A pivotal regulator of promyelocytic leukaemia protein (PML) and AKT in oncogenesis

Protein kinase CK2α is frequently upregulated in different cancers. Alteration of CK2α expression and its activity is sufficient to induce dramatic changes in cell fate. It has been established that CK2α induces oncogenesis through modulation of both AKT and PML. CK2α has been found to be overexpressed in breast cancer. In contrary, statistical reports have shown low level of PML. However, the regulation of CK2α gene expression is not fully understood. In the current study, we found that CK2α and activated AKT positively correlate with ERα, whereas PML follows an inverse correlation in human breast cancer tissues. Modulation of ERα signalling leads to recruitment of activated ERα on the ERE sites of CK2α promoter, resulting in CK2α transactivation. Furthermore, the DMBA induced tumours in rat showed elevated level of active CK2α. Consequently it mediates enhancement of AKT activity and PML degradation, resulting in increased cellular proliferation, migration and metastasis. Syngeneic ERα overexpressing stable mouse 4T1 cells produce larger primary tumours and metastatic lung nodules in mice, corroborating our in vitro findings. Hence, our study provides a novel route of ERα dependent CK2α mediated oncogenesis that causes upregulation and consequent AKT activation along with degradation of tumour suppressor PML.

Magnetic resonance imaging and computed tomography features of alveolar soft-part sarcoma in the right deltoid muscle: A case report.

Alveolar soft-part sarcoma (ASPS) is a rare, highly vascular malignant soft-tissue tumor that predominantly affects young adults. The tumor can occur in any bodily region, but is most frequently observed in the lower deep soft tissues of the extremities, and is rarely observed in the upper extremities. The present study reported a case of ASPS of the right deltoid muscle. The patient presented with a 3-year history of a mass on the right shoulder that exhibited rapid growth in the month prior to diagnosis. Computed tomography (CT) and magnetic resonance imaging (MRI) were performed. The lesion mainly demonstrated isointense or mildly hyperintense signals compared with the muscle on the T1-weighted images of the MRI, and heterogeneous high signal intensity on the T2-weighted images. CT enhancement showed a homogeneous enhanced mass. The tumor was resected and submitted for histopathological examination. The diagnosis was verified as ASPS by microscopic examination and immunohistochemical analysis. No distant metastases were noted. No evidence of local tumor recurrence was seen at 6 weeks following the wide surgical excision. The CT scan revealed no metastatic nodules in either lung during the follow-up. ASPS should be considered as a possible diagnosis when a slow-growing, large mass is detected in young adults in the soft tissue of the extremities, with high signal intensity and numerous signal voids on T1-weighted images (T1WI) and T2WI, and strong contrast-enhancement.

Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells.

Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified.

Detection of metastatic tumors after γ-irradiation using longitudinal molecular imaging and gene expression profiling of metastatic tumor nodules.

A few recent reports have indicated that metastatic growth of several human cancer cells could be promoted by radiotherapy. C6-L cells expressing the firefly luciferase (fLuc) gene were implanted subcutaneously into the right thigh of BALB/c nu/nu mice. C6-L xenograft mice were treated locally with 50-Gy γ-irradiation (γ-IR) in five 10-Gy fractions. Metastatic tumors were evaluated after γ-IR by imaging techniques. Total RNA from non-irradiated primary tumor (NRPT), γ-irradiated primary tumor (RPT), and three metastatic lung nodule was isolated and analyzed by microarray. Metastatic lung nodules were detected by BLI and PET/CT after 6–9 weeks of γ-IR in 6 (17.1%) of the 35 mice. The images clearly demonstrated high [18F]FLT and [18F]FDG uptake into metastatic lung nodules. Whole mRNA expression patterns were analyzed by microarray to elucidate the changes among NRPT, RPT and metastatic lung nodules after γ-IR. In particular, expression changes in the cancer stem cell markers were highly significant in RPT. We observed the metastatic tumors after γ-IR in a tumor-bearing animal model using molecular imaging methods and analyzed the gene expression profile to elucidate genetic changes after γ-IR.

Timosaponin AIII inhibits melanoma cell migration by suppressing COX-2 and in vivo tumor metastasis.

Melanoma is the leading cause of death from skin disease, due in large part to its propensity to metastasize. We examined the effects of timosaponin AIII, a compound isolated from Anemarrhena asphodeloides Bunge, on melanoma cancer cell migration and the molecular mechanisms underlying these effects using B16-F10 and WM-115 melanoma cells lines. Overexpression of COX-2, its metabolite prostaglandin E2 (PGE2), and PGE2 receptors (EP2 and EP4) promoted cell migration in vitro. Exposure to timosaponin AIII resulted in concentration-dependent inhibition of cell migration, which was associated with reduced levels of COX-2, PGE2, and PGE2 receptors. Transient transfection of COX-2 siRNA also inhibited cell migration. Exposure to 12-O-tetradecanoylphorbal-13-acetate enhanced cell migration, whereas timosaponin AIII inhibited 12-O-tetradecanoylphorbal-13-acetate-induced cell migration and reduced basal levels of EP2 and EP4. Moreover, timosaponin AIII inhibited activation of nuclear factor-kappa B (NF-κB), an upstream regulator of COX-2 in B16-F10 cells. Consistent with our in vitro findings, in vivo studies showed that timosaponin AIII treatment significantly reduced the total number of metastatic nodules in the mouse lung and improved histological alterations in B16-F10-injected C57BL/6 mice. In addition, C57BL/6 mice treated with timosaponin AIII showed reduced expression of COX-2 and NF-κB in the lung. Together, these results indicate that timosaponin AIII has the capacity to inhibit melanoma cell migration, an essential step in the process of metastasis, by inhibiting expression of COX-2, NF-κB, PGE2, and PGE2 receptors.

A pH-Responsive Host-guest Nanosystem Loading Succinobucol Suppresses Lung Metastasis of Breast Cancer.

Cancer metastasis is the leading reason for the high mortality of breast cancer. Herein, we report on a pH-responsive host-guest nanosystem of succinobucol (PHN) with pH-stimuli controlled drug release behavior to improve the therapeutic efficacy on lung metastasis of breast cancer. PHN was composed of the host polymer of β-cyclodextrin linked with multiple arms of N,N-diisopropylethylenediamine (βCD-DPA), the guest polymer of adamantyl end-capped methoxy poly(ethylene glycol) (mPEG-Ad), and the active agent of succinobucol. PHN comprises nanometer-sized homogenous spherical particles, and exhibits specific and rapid drug release in response to the intracellular acidic pH-stimuli. Then, the anti-metastatic efficacy of PHN is measured in metastatic 4T1 breast cancer cells, which effectively confirms the superior inhibitory effects on cell migration and invasion activities, VCAM-1 expression and cell-cell binding of RAW 264.7 to 4T1 cells. Moreover, PHN can be specifically delivered to the sites of metastatic nodules in lungs, and result in an obviously improved therapeutic efficacy on lung metastasis of breast cancer. Thereby, the pH-responsive host-guest nanosystem can be a promising drug delivery platform for effective treatment of cancer metastasis.

Abstract A146: The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression

Abstract Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that multiple signaling pathways that affect motility, growth control, invasion, metabolism, cytokine release and the ability to escape the immune response, regulate melanoma development. Silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with shRNAi lentivirus (shR-SOCS-1) inhibits in vitro growth by cell cycle regulation with S phase arrest and increased size of cells and nuclei. It also inhibited tumor cell migration and invasion. Down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptors. Based on the results of SOCS-1 silencing, the present work aims at analyzing the expression of proteins relevant to tumor development, searching for signaling pathways related with SOCS-1. A protein microarray analysis of murine melanoma cells silenced for SOCS-1 by shRNAi-SOCS-1 lentivirus transduction was undertaken using as controls, cells transduced with the empty vector. Among 614 differentially expressed genes, c-kit, met and EphA3 cytokine/tyrosine-kinase (TK) receptors were downregulated. A significant decrease in the expression of TK receptors as observed by Western blotting of B16 shR-SOCS-1 extracts showed that SOCS-1 positively regulates the MAPK signaling pathways in these cells. A decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727) was observed. These results demonstrate that SOCS-1 plays an important role in tumor development and progression up-regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) and total AP-2 alpha were up-regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments using a syngeneic model and a prophylactic protocol showed that the subcutaneous immunization with shR-SOCS-1-transduced viable tumor cells, rendered protection against either subcutaneously grafted or metastatic murine melanoma cells, with significant reduction in tumor growth and lung metastatic nodules. Such immunization increased the expression of CD11c, CD80, CD86 and MHC-II in splenic dendritic cells, as compared to DCs from a group that received only B16F10-Nex2 cells. Of notice was the decreased expression of PD-L1 in SOCS-1-silenced B16F10-Nex2 cells (B16shR-SOCS-1). Such molecular phenotype may have a role in the immune response and protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells. By overexpressing PD-L1, WT-melanoma cells negatively regulates T-cell proliferation in the tumor environment. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma development and the potential of SOCS-1-silenced tumor cells in raising an effective anti-tumor immune response, inducing protection even against a metastatic melanoma challenge. Funded by FAPESP 2010/51423-0 and 2012/17473-6. Citation Format: Rodrigo Berzaghi, Esq., Vera SC Maia, II, Felipe V. Pereira, III, Filipe Manegatti De Melo, Luiz R. Travassos, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A146.

Cutaneous Metastasis of Giant Cell-Rich Osteosarcoma.

Dear Editor: Cutaneous metastasis from osteosarcoma is exceedingly rare, with only 11 cases reported in the literature1. Giant cell–rich osteosarcoma is a rare variant of osteosarcoma, accounting for 1%~3% of conventional osteosarcomas2. Herein, we present the rare case of a patient with cutaneous metastasis of giant cell–rich osteosarcoma, which closely resembled a giant cell tumor. Owing to the different prognoses and treatment strategies for these tumors, it is important to ensure that the correct differential diagnosis is made. A 43-year-old man presented with a 6-month history of nodules on his scalp. He had been diagnosed with giant cell-rich osteosarcoma in his right femur along with metastases to multiple organs, including the lungs and the mandible, 1 year ago. The metastatic lung nodules had increased in size despite 6 months of chemotherapy with ifosfamide, adriamycin, and high-dose methotrexate. Subsequently, oncologists changed the regimen to gemcitabine and docetaxel, but the tumor still progressed. Therefore, the regimen was changed once again, and he was admitted to receive a second cycle of cyclophosphamide and etoposide as a third-line regimen. Upon physical examination, several firm skin-colored to erythematous nodules were palpated on his scalp, each measuring approximately 5~12 mm (Fig. 1). Histopathological findings showed many uniformly distributed, multinucleated giant cells containing pleomorphic nuclei. Mononuclear tumor cells had hyperchromatic nuclei with a variety of morphologies, such as round, oval, polygonal, and spindle-shaped (Fig. 2). Although atypical mitoses or dominant osteoid formations were hardly observed, these histological findings were consistent with those of giant cell-rich osteosarcoma, which was previously confirmed by right femur biopsy (Fig. 2). Three months later, he died of sepsis following bone marrow failure caused by the continued chemotherapy. Fig. 1 Three 5 to 12-mm, flesh-toned to erythematous, firm nodules on the scalp (arrows). Fig. 2 (A) Hypercellular stroma composed of mononuclear tumor cells and many multinucleated giant cells (H&E, ×40). (B) Mononuclear tumor cells with a variety of morphologies and multinucleated giant cells containing large pleomorphic nuclei ... Several studies have reported that giant cell-rich osteosarcomas are sometimes misdiagnosed as giant cell tumors because of their similar clinicopathological features2,3,4. However, these tumors have extremely different clinical courses and prognoses. Giant cell tumors are usually benign with a metastatic rate of <2%, while the clinical course of osteosarcoma is extremely aggressive, with rapid hematogenous systemic dissemination and a tumor-related mortality of 75% within 5 years of diagnosis1,5. For these reasons, Huang et al.3 suggested the use of some key factors to distinguish these two tumors, including osteoid formation, nuclear pleomorphism, and atypical mitotic figures3. However, osteoid formation cannot be found in small biopsy samples or samples from early lesions4. Tumor cell pleomorphism is not obvious in some cases2. Unless malignant osteoid formation is found, as in this case, it is difficult to confirm an osteosarcoma diagnosis without knowing a patient's cancer history. Unfortunately, not all cases present with features as informative as those in the case reported here. Among 29 cases of giant cell-rich osteosarcoma, only 1 case of cutaneous metastasis had been reported in English literature2,3,4. With the histological similarity to giant cell tumors, this case emphasizes the value of careful examination of tumor cell pleomorphism and pathological mitoses when patients present with giant cell-rich tumors.

Clinical Value of a One-Stop-Shop Low-Dose Lung Screening Combined with (18)F-FDG PET/CT for the Detection of Metastatic Lung Nodules from Colorectal Cancer.

The aim of this study was to evaluate the clinical usefulness of additional low-dose high-resolution lung computed tomography (LD-HRCT) combined with (18)F-fluoro-2-deoxyglucose positron emission tomography with CT ((18)F-FDG PET/CT) compared with conventional lung setting image of (18)F-FDG PET/CT for the detection of metastatic lung nodules from colorectal cancer. From January 2011 to September 2011, 649 patients with colorectal cancer underwent additional LD-HRCT at maximum inspiration combined with (18)F-FDG PET/CT. Forty-five patients were finally diagnosed to have lung metastasis based on histopathologic study or clinical follow-up. Twenty-five of the 45 patients had ≤5 metastatic lung nodules and the other 20 patients had >5 metastatic nodules. One hundred and twenty nodules in the 25 patients with ≤5 nodules were evaluated by conventional lung setting image of (18)F-FDG PET/CT and by additional LD-HRCT respectively. Sensitivities, specificities, diagnostic accuracies, positive predictive values (PPVs), and negative predictive values (NPVs) of conventional lung setting image of (18)F-FDG PET/CT and additional LD-HRCT were calculated using standard formulae. The McNemar test and receiver-operating characteristic (ROC) analysis were performed. Of the 120 nodules in the 25 patients with ≤5 metastatic lung nodules, 66 nodules were diagnosed as metastatic. Eleven of the 66 nodules were confirmed histopathologically and the others were diagnosed by clinical follow-up. Conventional lung setting image of (18)F-FDG PET/CT detected 40 of the 66 nodules and additional LD-HRCT detected 55 nodules. All 15 nodules missed by conventional lung setting imaging but detected by additional LD-HRCT were <1cm in size. The sensitivity, specificity, and diagnostic accuracy of the modalities were 60.6%, 85.2%, and 71.1% for conventional lung setting image and 83.3%, 88.9%, and 85.8% for additional LD-HRCT. By ROC analysis, the area under the ROC curve (AUC) of conventional lung setting image and additional LD-HRCT were 0.712 and 0.827 respectively. Additional LD-HRCT with maximum inspiration was superior to conventional lung setting image of (18)F-FDG PET/CT for the detection of metastatic lung nodules from colorectal cancer (P < 0.05).

The decrease of cyclin B2 expression inhibits invasion and metastasis of bladder cancer

ObjectiveIt has been shown that cyclin B2 is commonly overexpressed in many malignant tumors. This study aimed to investigate the potential role of cyclin B2 in bladder cancer. Method and materialFixed tissues for immunohistochemistry and fresh tissues for western blotting and quantitative real-time polymerase chain reaction assay were randomly selected from Nanfang hospital. Normal bladder urothelial cell and bladder cancer cell lines was stored in our laboratory, the bladder cancer cells were transfected to develop bladder cancer cell clones expressing decreased cyclin B2 levels, the clones were used for cell growth and metastasis experiments in vitro and in vivo. ResultsWestern blot and immunohistochemical analysis both showed that the cyclin B2 protein expression was higher in bladder urothelial carcinoma than in normal bladder mucosa, especially in invasive cancer. Real-time polymerase chain reaction showed that the cyclin B2 messenger RNA expression exhibited the same trend. Results of cell lines experiments also showed higher cyclin B2 expression in cancer cells. In vitro tests the decrease of cyclin B2 expression that had little effect on cell growth and cell cycling according to the MTT assay and the Edu assay, whereas in the Boyden chamber transwell assay, the cyclin B2 low-expressing clones significantly inhibits the cells׳ invasion and metastatic abilities. This result was consistent with the scratch-wound assay result showing that the target clone needed more time for healing the wound. The in vivo experiment in nude mice produced similar results, the lung and liver target cell metastasis nodules were smaller and less than those of the negative control by the hepatic subcapsular injection assay, and the mice of the target clone group has longer survival time in no intervention observed test. ConclusionThese results indicate that the cyclin B2 was overexpressed in bladder cancer, and the down-regulation of cyclin B2 expression in bladder cancer greatly inhibits the cell׳s invasion and metastatic abilities, and it prolonged the survival time of nude mice in vivo.

Lung Metastatic Nodules as First Presentation of Synovial Cardiac Sarcoma

Synovial sarcomas are rare tumors accounting for approximately 1% from all cardiac neoplasms and present with a variety of symptoms depending on their location and size.

Inexplicable Outcome of Early Appearance of Hepatocellular Carcinoma in the Allograft After Deceased Donor Liver Transplantation: A Case Report

BackgroundThis case represents the earliest appearance of de novo HCC after liver transplantation (OLT) compared with cases of previously reported literatures. Case ReportA 45-year-old man underwent deceased donor OLT owing to decompensated liver cirrhosis. He had YMDD viral mutation and hepatitis B (HBV) and C (HCV) coinfection but no tumor was found in the liver on MRI before OLT. The donor was a healthy young female donor who was HCV and HBV negative. There was no tumor in the explant liver. After OLT, HCV RNA and hepatitis B surface antigen became undetectable with DNA-positive HBV. Nine months after OLT, a computed tomography (CT) scan was performed owing to abdominal pain, detecting a mass occupying the right lobe that depicted enhanced characteristics typical of HCC. The chest CT demonstrated metastatic lung nodules in the right basal lower lobe. Fluorescence in situ hybridization showed tumor cells from the recipient. ConclusionsTo the best of our knowledge, this is the first report of de novo hepatocellular carcinoma that has emerged within a short period of undergoing OLT.

Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in ERK-responsive lung cancer.

Metastatic spread of cancer cells portends a poor prognosis and mortality for lung cancer patients. Hypoxia-inducible factor-1α (HIF-1α) enhances tumor cell motility by activating the epithelial-to-mesenchymal transition (EMT), which is considered a prerequisite for metastasis. Recent studies of microRNA involvement in cancer invasion and metastasis have highlighted the role of such RNAs in tumor development. However, little work has been done to identify tumor suppressor microRNAs that target HIF-1α to down-modulate the EMT and thereby counteract the aggressiveness and metastasis of lung cancer cells. Here, we identified the 3'-untranslated region of HIF-1α mRNA as a target of miR-622 and established that miR-622-mediated down-modulation of HIF-1α correlates with decreased levels of mesenchymal proteins, including Snail, β-catenin, and vimentin. Functional analyses revealed that increased miR-622 expression inhibited lung cancer cell migration and invasion in vitro. miR-622 also inhibited the genesis of metastatic lung nodules as demonstrated in a lung cancer xenograft model in which nude mice were transplanted with A549 cells expressing miR-622. Mechanistic analyses showed that overexpression of EGF decreased the miR-622 level in A549 cells, and this reduction could be rescued by administrating U0126, an inhibitor of ERK. Moreover, miR-622 overexpression mediated by the transcription factor FOXO3a decreased the invasiveness of lung tumor cells by inhibiting HIF-1α via inactivation of ERK signaling in U0126-treated A549 cells. These findings highlight the pivotal role of the FOXO3a/miR-622 axis in inhibiting HIF-1α to interfere with tumor metastasis, and this information may contribute to development of novel therapeutic strategies for treating aggressive lung cancer.

Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model.

BackgroundMesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth.MethodsMurine mesenchymal stromal cells were loaded with Paclitaxel (Dr-MCsPTX) according to a standardized procedure and their ability to inhibit the growth of a murine B16 melanoma was verified by in vitro assays. The anti-metastatic activity of Dr-MCsPTX was then studied in mice injected i.v. with B16 melanoma cells that produced lung metastatic nodules. Lung nodules were counted under a dissecting stereomicroscope and metastasis investigated by histological analysis.ResultsWe found that three i.v. injections of Dr-MCsPTX on day 5, 10 and 15 after tumor injection almost completely abolished B16 lung metastasis. Dr-MCsPTX arrested into lung by interacting with endothelium and migrate toward cancer nodule through a complex mechanism involving primarily mouse lung stromal cells (mL-StCs) and SDF-1/CXCR4/CXCR7 axis.ConclusionsOur results show for the first time that Dr-MCsPTX are very effective to inhibit lung metastasis formation. Actually, a cure for lung metastasis in humans is mostly unlikely and we do not know whether a therapy combining engineered MSCs and Dr-MCs may work synergistically. However, we think that our approach using Dr-MCs loaded with PTX may represent a new valid and additive therapeutic tool to fight lung metastases and, perhaps, primary lung cancers in human.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0200-3) contains supplementary material, which is available to authorized users.

Suppression of motor protein KIF3C expression inhibits tumor growth and metastasis in breast cancer by inhibiting TGF-β signaling

Breast cancer is the most common cause of death among women. KIF3C, a member of kinesin superfamily, functions as a motor protein involved in axonal transport in neuronal cells. To explore the expression, regulation and mechanism of KIF3C in breast cancer, 4 breast cancer cell lines and 93 cases of primary breast cancer and paired adjacent tissues were examined. Immunohistochemistry, Real Time Polymerase Chain Reaction (RT-PCR), Western blot, flow cytometry, short hairpin RNA (shRNA) interference, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation techniques and xenograft mice model were used. We found that KIF3C was over-expressed in breast cancer tissues and such high KIF3C expression was also associated with tumor recurrence and lymph node metastasis. Silencing of KIF3C by shRNA inhibited epithelial-mesenchymal transition and metastasis by inhibiting TGF-β signaling and suppressed breast cancer cell proliferation through inducing G2/M phase arrest. The tumor size was smaller and the number of lung metastatic nodules was less in KIF3C depletion MDA-MB-231 cell xenograft mice than in negative control group. These results suggested that high expression of KIF3C in breast cancer may be associated with the tumor progression and metastasis.

Abstract 341: Breast cancer cell adhesion and degradome interact to drive metastasis

Abstract Although primary breast tumors are detected early in most cases, it is inevitable that many patients remain at risk for future recurrence and death due to micrometastases. We investigated the metastatic process with a focus on matrix metalloproteases (MMPs) within the degradome and on integrins and E-cadherin within the adhesome. We have performed comparative studies with a set of non-metastatic (BT-474, T47D, MCF7) versus metastatic (MDA-MB-231, SUM149, SUM159) human breast cancer cell lines and xenografts. Experiments were performed to measure growth rate, migration, invasion, and colony formation. Immunoblotting and immunohistochemistry revealed a higher expression of MMPs and heterophilic cell adhesion molecules integrin β1 and lower expression of homophilic cell adhesion molecule E-cadherin in the metastatic tumor cells and xenografts. Using zymography and activatable fluorescent probes for noninvasive imaging of MMPs, we demonstrated that metastatic breast cancer cells and tumors displayed an elevated degradome activity as compared to non-metastatic cells and tumors. Cell adhesion and cell aggregation assays showed an increased heterophilic and decreased homophilic adhesion in the metastatic models. Inhibiting MMPs reduced the expression of integrin β1, and stimulating integrin β1 resulted in higher MMP activities in metastatic cells. Re-expression of E-cadherin in metastatic cells led to a reduced expression of active integrin β1. Finally, circulating breast cancer cells and cells from lung metastatic nodules generated from metastatic tdTomato-fluorescing breast cancer cells displayed a reduced expression of active integrin β1 and less epithelial-mesenchymal-transition (EMT) marker protein S100A4. Our results indicate that proteolytic ECM remodeling by MMPs and cell adhesion by integrin β1 and E-cadherin are interdependent processes that control transformation from benign to invasive carcinoma and are critical for metastasis. Citation Format: Asif Rizwan, Menglin Cheng, Zaver M. Bhujwalla, Balaji Krishnamachary, Lu Jiang, Kristine Glunde. Breast cancer cell adhesion and degradome interact to drive metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 341. doi:10.1158/1538-7445.AM2015-341

Radiofrequency ablation inhibits lung metastasis ofbreast cancer in mice

To explore the effects of radiofrequency ablation(RFA) on immune system and lung metastasis in a mouse model of triple negative breast cancer 4T1. Mouse breast cancer 4T1 cells were injected into the right hind limb of female Bal B/c mice. When the tumor size was 6-8 mm in diameter, RFA was used to treat the transplanted breast cancer in mice. We examined the splenic lymphocyte subsets by flow cytometry at different time points after RFA. Fourteen days after treatment, we sacrificed the mice of both control and treatment groups, counted the number of lung metastatic nodules, and detected the changes of splenic lymphocyte subsets by flow cytometry. RFA basically eliminated the orthotopic carcinoma with a low local recurrence rate. After the RFA treatment, the amount of spleic CD4⁺ T cells, CD8⁺ T cells, B cells, NK and NKT cells was increased. Fourteen days after the RFA treatment, all mice were sacrificed, and the lung metastatic nodules were 24 ± 18 in the control group and 81 ± 35 in the RFA-treated group (P = 0.012). The mechanism of suppression of metastatic lung cancers was related to the increase of splenic CD4⁺ T cells, CD8⁺ T cells, B cells and NK cells, and the decrease of myeloid-derived suppressor cells. RFA can enhance the anti-tumor immunity and effectively inhibit lung metastasis of 4T1 cell-induced breast cancer, and has a good potential effect in the treatment of triple-negative breast cancer and the control of distant metastasis.

NANOG signaling promotes metastatic capability of immunoedited tumor cells.

Metastatic recurrence after cancer treatments with radiation, cancer drugs, or even immunotherapeutic agents (cytokine, antibody, lymphocyte etc.) is often intractable and fatal for cancer patients. Therefore, molecular understanding of metastatic recurrence is necessary. Recently, these recurrent and metastatic tumor cells with resistance to cancer drugs have been reported to possess stem-like attributes and epithelial-mesenchymal transition (EMT) phenotype. Previously, we also found that antigen-specific cytotoxic T lymphocyte (CTL)-mediated immunotherapy conferred tumor cells with immune-resistant and stem-like phenotypes by hyper-activating NANOG/TCL1/AKT signaling axis. In this study, we report that these immunoedited cells have high metastatic capability and phenotypes. These cells exhibit enhanced migration, infiltration, and invasiveness in vitro as well as formation of metastatic lung nodules in vivo. Moreover, they display EMT-like features characterized by increased expression of BMI1 and TWIST1. Importantly, these pleiotropic phenotypes of metastasis through the expression of the EMT-associated molecules were critically dependent on the NANOG/TCL1A/AKT signaling axis, which was also conserved across multiple types of human cancer. Thus, we provide proof of the principle that inhibition of the NANOG axis is an effective strategy to control metastasis of immunoedited cancer, particularly, after CTL-based immunotherapy.

Host protein C inhibitor inhibits tumor growth, but promotes tumor metastasis, which is closely correlated with hypercoagulability

IntroductionProtein C inhibitor (PCI), a member of the serine protease inhibitor family, is expressed in various human tissues, including liver and kidneys. In the plasma, PCI physiologically inhibits an anticoagulant serine protease, activated protein C (APC). PCI expressed by cancer cells suppresses tumor invasion by inhibiting urokinase-type plasminogen activator, and inhibits tumor growth and metastasis, which are independent of its protease-inhibitory activity. In the present study, we clarified the effects of host PCI on growth and metastasis of B16 melanoma (B16) cells by comparing between wild-type mice and mice transgenic for human PCI gene (hPCI-TG), which have a tissue distribution of PCI similar to that observed in humans. Materials and MethodsGrowth of intracutaneously-injected B16 cells was evaluated by measuring the tumor volume, and metastatic behavior of intravenously-injected B16 cells by counting the number of metastatic lung nodules. ResultsGrowth of intracutaneously injected B16 cells was significantly faster in wild-type mice than in hPCI-TG mice; however, hPCI-TG mice developed more metastatic nodules of B16 cells in the lungs. Immunohistochemical analysis using anti-mouse fibrinogen antibody revealed more fibrin deposition in the lung in hPCI-TG mice than in wild-type mice. Furthermore, the more invasive behavior observed in hPCI-TG mice was reduced by rabbit anti-human PCI IgG, APC, or soluble TM administration for 3 consecutive days including the day that B16 cells were injected. ConclusionsOur results suggest that like PCI expressed in tumor cells, host PCI also inhibits tumor growth, but host PCI promotes tumor metastasis via its procoagulant properties.

Comprehensive characterization of chemotherapeutic efficacy on metastases in the established gastric neuroendocrine cancer patient derived xenograft model.

The HuPrime® human gastric neuroendocrine carcinoma derived xenograft model GA0087 was established in this study. GA0087 PDX model showed high gene expression of vascular endothelial growth factors (VEGF)-A and B, and high potential of lung metastasis. Circulating tumor cells (CTCs) with either large or small size, circulating tumor microemboli (CTM) and lung metastatic lesions were detected in GA0087 PDX mice. The number of CTC correlated to the number of metastatic nodules in lung. Both primary tumor growth and metastasis in terms of the number of dynamically monitored CTCs and metastatic nodules were effectively suppressed by Cisplatin. Diverse subtypes of CTCs in the context of sensitivity to Cisplatin were specifically identified by subtraction enrichment (SE) integrated with in situ Phenotyping of cytokeratin 18 (CK18) and Karyotyping of chromosome 8 (in situ PK CTC by CK-iFISH). All the CK18-/diploid and majority of CK18+/diploid CTC subtypes were chemosensitive, whereas a higher percentage of CK18+/multiploid subtype of CTC were Cisplatin-insensitive. Combined histopathological examination of metastatic lesion and in situ PK CTC in a metastatic PDX (mPDX) tumor model are of particular significance, and may provide an unique and robust platform for cancer research as well as pre-clinical evaluation of therapeutic efficacy of new anti-cancer drugs.