Lung Metastatic Tissues Research Articles

Novel oncogene COPS3 interacts with Beclin1 and Raf-1 to regulate metastasis of osteosarcoma through autophagy

BackgroundExpression of COP9 signalosome subunit 3 (COPS3), an oncogene overexpressed in osteosarcoma, has been demonstrated to be significantly correlated with tumor metastasis. However, the underlying mechanism by which COPS3 promotes metastasis of osteosarcoma and its role in autophagy remain unknown.MethodsThe expression of COPS3 was detected in primary osteosarcoma tissues and matching lung metastasis tissues by immunohistochemistry (IHC). The effect of COPS3 on the metastasis of osteosarcoma cells was investigated by transwell, wound healing assays and animal studies. Indicated proteins was analyzed by western blotting when COPS3 was knockdown or overexpressed. The COPS3 Interacting protein was determined by immunoprecipitation assay. The relationship between COPS3 and autophagy was detected by western blotting and immunofluorescence.ResultsWe found that knockdown of COPS3 significantly reduced the lung metastasis of osteosarcoma cells in a mouse model, coinciding with downregulation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling. The silencing of COPS3 also inhibited the epithelial–mesenchymal transition (EMT) through the 90 kDa ribosomal S6 kinases (RSK), a family of signal transduction proteins downstream of MEK/ERK. Reciprocal immunoprecipitation assays revealed that COPS3 directly interacts with Raf-1, an upstream regulator of MEK/ERK. Surprisingly, Beclin1, an important autophagic protein, appeared in the COPS3-immunoprecipitates, along with the autophagic markers LC3-I and LC3-II. Loss of COPS3 completely inhibited H2O2-induced autophagic flux and reduced Beclin1 expression. Additionally, autophagy inhibitor or silencing of Beclin1 both decreased cell metastasis.ConclusionsTaken together, these data reveal a novel function of COPS3 in the regulation of autophagy and highlight the relationship between autophagy and metastasis in osteosarcoma cells.

Abstract 2703: Proteomic identification of Galectin-1 in murine primary and metastatic triple-negative breast tumors

Abstract The purpose of this study was to identify proteins associated with Triple Negative Breast Cancer (TNBC) metastases using a proteomics approach, in order to gain insight into pathways and mechanisms that could help identify biomarkers or targets for intervention. For our investigations, we used a murine claudin-low tumor model that mirrors human claudin-low TNBC molecular profiles, T11. Cultured T11 cells were lysed, trypsin-digested, and analyzed by liquid chromatography mass spectrometry (LC/MS). A total of 104 proteins were identified with >95% confidence using Protein Pilot software. Among these proteins was Galectin-1 (Gal-1); seven distinct Gal-1 peptides were identified with protein coverage of 58%. Galectins are a family of ß-galactoside-binding proteins that share homology in the carbohydrate binding domain. Gal-1 was selected for further investigation due to its reported roles in cancer progression and immune evasion. Gal-1 expression was next examined in naïve murine lung tissue and in lung metastases from mice orthotopically implanted with T11. Lung tissue was homogenized, trypsin-digested and analyzed by LC/MS. Although Gal-1 was not identified in naïve lungs, three Gal-1 peptides (28% protein coverage) were identified with >95% confidence in lungs with macrometastases. LC/MS analysis did not identify any other galectin family members in primary tumor or lung metastases. Matrix-assisted laser desorption ionization (MALDI) imaging of trypsin-digested tissue sections revealed two distinct Gal-1 peptides localized to tumor regions within metastatic lung tissue. Immunohistochemistry (IHC) analysis showed higher Gal-1 expression in orthotopic T11 primary tumors compared to naïve mammary fat pad, and also higher Gal-1 expression in T11 lung metastases compared to naïve lung. Within tumors, Gal-1 was detected in extracellular matrix, in the cytoplasm of tumor cells and associated with tumor cell membranes. These findings are supported by our analysis of both murine and human genomic data sets, which showed Gal-1 overexpression in claudin-low TNBC compared to other breast cancer subtypes and normal breast tissue. There are several reports that exogenous Gal-1 induces an M2-like phenotype in myeloid cells, consistent with our prior findings that primary tumors, pre-metastatic and metastatic lungs exhibit significant M2-like myeloid cell infiltrates. Future investigations in our lab will focus on determining the role of tumor-produced Gal-1 in the regulation of myeloid cell function to promote cancer progression. Citation Format: Kassondra Balestrieri, Kim Kew, Mohamed Ramez, Keith Pittman, Nasreen Vohra, Kathryn Verbanac. Proteomic identification of Galectin-1 in murine primary and metastatic triple-negative breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2703.

Silencing of c-jun decreases cell migration, invasion, and EMT in radioresistant human nasopharyngeal carcinoma cell line CNE-2R.

BackgroundPreviously, we found that c-jun was highly expressed in radiation-resistant human nasopharyngeal carcinoma cells (CNE-2R) compared with human nasopharyngeal carcinoma cells (CNE-2).Materials and methodsIn this study, we first used the scratch assays and transwell assays to detect the migration and invasion of CNE-2R and CNE-2 cells and tested the epithelial mesenchymal transformation (EMT)-related proteins E-cadherin and N-cadherin by Western blot analysis. Subsequently, c-jun was knocked down to establish the effect of c-jun on EMT, migration, and invasion of CNE-2R cells both in vitro and in vivo.ResultsA high EMT level, CNE-2R cells were more capable of migration and invasion than CNE-2 cells. Moreover, silencing of c-jun has upregulated the expression of E-cadherin and downregulated N-cadherin in CNE-2R cells, and subsequently the migration and invasion capacity of the cells was decreased. Consistent with in vitro results, in vivo studies indicated that the c-jun silencing reduced pulmonary migration of CNE-2R cells. Immunohistochemistry of lung metastatic tumor tissue showed that E-cadherin was upregulated, and N-cadherin was downregulated.ConclusionThese findings suggest that silencing of c-jun in CNE-2R cells reduces cells migration, invasion, and EMT both in vitro and in vivo.

Abstract 39: LCN2, CD133 expressions in breast cancer spinal bone metastasis and CD151, TWIST1, EPHA2 in lung metastasis: Prognostic value and organ-specific relapse

Abstract Purpose: Breast cancer most often recurs and metastasizes to the distal organs that had their primary tumors surgically excised. Due to heterogeneous nature of the breast cancer, metastasis organotropism has poorly understood. In this study, we assessed the specific cancer-related gene expression changes occurring with metastatic breast cancer recurrence to distant organs comparing with non-metastatic breast cancer. Experimental design: We develop a clinically relevant metastatic syngeneic mouse model using two cell lines 4T1-luc2 that can only metastasize to lung and 4T1.2-luc2 metastasized to bone and lung. After the primary mammary tumor surgically excised from mice, we monitor mice using IVIS imaging for metastatic tumor lesion development in the distant organs followed by MRI scan to detect spinal bone metastases. Animals were sacrificed to collect spines, bones, and lung lesions for molecular analysis. We also validate the gene expression data using the RNA-seq cohort data from the Roswell Park Cancer Institute patient samples with advanced breast cancer bone metastases and matched non-metastatic breast cancer. Results: Our cell lines, RNA-seq data analysis suggested that there are at least 50 genes which are statistically highly expressed in 4T1.2-luc2 compared to 4T1-luc2 cells. Out of which only a few genes are well established for cancer metastasis biology, include ANGPTL77, SERPINE2, TSPAN11, ESM1, LCN2 which are expressed more than 8 fold in 4T1.2-luc2 compared to 4T1-luc2 cells. To our surprise, we found that most of these genes are differentially expressed when metastasized to distant organ. Our data revealed that LCN2 (7 fold) and CD133 (above 20 fold) are overexpressed in the spine and bone compared to the primary or lung met lesions formed by 4T1.2-luc2. Conversely, LCN2 and CD133 genes are downregulated in breast cancer lung metastasis tissues, whereas CD151, EPHA2, and TWIST1 genes are highly overexpressed in lung metastatic lesion compared to primary, bone or spine. Further, the RNA-seq data of patient samples explained that LCN2, CD133 expressions are significantly higher (8 out of 10 patients) in advanced breast cancer bone metastases compared with matching non-metastatic breast cancer. Conclusion: Our data suggested that LCN2, CD133 would be a prognostic marker for breast cancer spinal bone metastasis, whereas CD151, TWIST1, EPHA2 would be a prognostic marker for lung metastasis and organ-specific relapse. Citation Format: Aparna Maiti, Nitai C. Hait. LCN2, CD133 expressions in breast cancer spinal bone metastasis and CD151, TWIST1, EPHA2 in lung metastasis: Prognostic value and organ-specific relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 39.

Abstract 2904: Awakening metastatic breast cancer cells to chemotherapy

Abstract Breast cancer is the most common cancer in women with more than 316,000 new cases and expected 40,610 deaths in the US in 2017. Despite vast improvement in cancer management and treatment in the past years, not all subtypes of breast cancer have benefited equally. This includes the most aggressive form of invasive breast cancer, triple negative breast cancer (TNBC) subtype (ER-, PR- and HER2-), which accounts for about 20-25% of all breast cancer cases and presents a higher breast cancer-related mortality compared to other breast cancer subtypes. The poor survival rates attributed to TNBC is due to the lack of available biological targets and high rates of metastatic recurrence. Therefore, chemotherapy is the only therapeutic option for patients with metastatic TNBC. However, development of chemotherapy resistance is observed in the great majority of the cases and patients relapse within 5 years of a diagnosis. Given the unmet clinical needs, the discovery of novel therapeutic options that overcome chemoresistance of metastatic breast cancer is required. We have identified a group of genes which expression was enriched in chemotherapy resistant tumors cells, suggesting that these are potential therapeutic candidates to overcome chemotherapy resistance. Of these, CDKN1A (p21) was included. Furthermore, Immunostaining of metastatic lung tissue confirmed that surviving tumor cells under CTX treatment highly express p21. The enrichment of p21 in surviving tumor cells under chemotherapy, together with its well documented functions in cell-cycle arrest and regulation of apoptosis, provides a rationale to explore the underlying mechanisms that govern p21-induced chemoresistance and whether targeting p21 will re-sensitize the chemoresistant tumor cells in vivo. Citation Format: Ana Rita Lourenco, Jay Lopez, Michael Crowley, Vivek Mittal, Dingcheng Gao. Awakening metastatic breast cancer cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2904.

Somatic mutation landscape of a meningioma and its pulmonary metastasis

BackgroundExtracranial metastasis (ENM) of meningiomas is extremely rare, and typically occurs several years after a primary tumor is diagnosed. However, the genetic changes underlying ENM events have not yet been investigated.Case presentationA 58-year-old male patient was sent to the emergency room of our hospital because of a sudden fall. Magnetic resonance imaging detected a mass at the right frontal sagittal sinus. He underwent tumor resection and recovered well, but post-operative computed tomography revealed three lumps on the right side of his chest. Thoracic surgery was performed to remove two of the lumps. Pathological findings revealed that the brain and lung tumors were grade I meningiomas. The patient received no additional radiation or chemotherapy post-surgery, and there was no sign of tumor recurrence in the brain or progression of the remaining lump in the chest 1 year after surgery. We performed whole exome sequencing of the patient’s blood, primary brain tumor, and lung metastatic tumor tissues to identify somatic genetic alterations that had occurred during ENM. This revealed that a frameshift deletion of the neurofibromin 2 gene likely drove formation of the meningioma. Surprisingly, we found that the brain tumor was relatively homogeneous and contained only one dominant clone; both the pulmonary metastasis and the original brain tumor were derived from the same clone, and no obvious additional driver mutations were detected in the metastatic tumor.ConclusionAlthough ENM of meningiomas is very rare, brain tumor cells appear to be more adaptable to tissue microenvironments outside of the central nervous system than was commonly thought.

In Vivo Antitumor Effects of MK615 Led by PD-L1 Downregulation.

Background/Aim: MK615 extracted from Prunus mume was reported to have anti-inflammatory effects. In this article, we examined the in vivo antitumor effect of MK615 (an extract from Japanese apricot) using mouse tumor xenografts and focusing on the downregulation of PD-L1 (programmed death-ligand 1), a ligand of programmed cell death-1, a surface protein of activated T cells. Materials and Methods: B16/BL6 melanoma cells were injected into C57BL/6 or BALB/c-nu/nu mice to establish lung metastasis. BALB/c-nu/nu mice (nude mice) were used as a T cell–deficient model. The mice were given MK615 or saline orally every other day for approximately 8 weeks, and their survival was observed. NF-κB (nuclear factor-κB) and PD-L1 expressions of metastatic lung tissues were also examined. Results: The survival rate was improved only in the MK615-treated C57BL/6 mice (P < .05), not in the saline-given control mice or BALB/c-nu/nu mice. The downregulations of NF-κB and PD-L1 were observed in both MK615-treated C57BL/6 and BALB/c-nu/nu mice. These results suggest that the antitumor effects of MK615 are associated with T cell–mediated immunity activated by MK-615-induced PD-L1 downregulation in tumor cells. Conclusion: MK615 is beneficial for a prolonged host survival time in the B16/BL6 melanoma xenograft model associated with T cell–mediated antitumor immunity.

Overexpression of CD155 relates to metastasis and invasion in osteosarcoma.

The rapid development of metastatic lesions remains the leading cause of mortality for patients with osteosarcoma. CD155 serves a key role in cancer cell migration, invasion and metastasis. However, the function and mechanism of CD155 has not been explored in osteosarcoma metastasis. In the present study, we found that CD155 was significantly upregulated in lung metastatic tissue and the highly metastatic cell line K7M2-WT (K7M2) of osteosarcoma. Overexpression of CD155 in K7M2 cells enhanced lung metastasis, while inhibition of CD155 by an anti-CD155 monoclonal antibody reduced metastasis. Blocking of CD155 also decreased migration and invasion of K7M2 cells in vitro. A western blot analysis revealed that blocking of CD155 inhibits metastasis by downregulating focal adhesion kinase (FAK) and phosphorylated FAK (pFAK) in osteosarcoma. The results revealed that CD155 serves a crucial role in the metastasis of osteosarcoma by regulating FAK and may provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.

Abstract P5-03-07: Probing breast cancer cell activation in response to extracellular cues using well-defined synthetic microenvironments

Abstract Interactions between breast cancer cells and their microenvironments are essential in tumor growth, metastasis, and recurrence. The tumor stroma undergoes constant structural changes, including degradation, redeposition, and crosslinking of collagens with gradients in matrix stiffness and composition that drive invasion and metastasis. At metastatic sites, similar remodeling events that occur with injury and aging are hypothesized to promote reactivation of dormant tumor cells in recurrence. Approaches are needed for testing hypotheses about pivotal cell-matrix interactions in the progression of breast cancer for identifying key regulators and improving treatment strategies. In this work, we have designed well-defined materials to mimic key aspects of tumor microenvironments toward studying such complex phenomena in vitro. Specifically, we have created synthetic extracellular matrices with well-defined biophysical and biochemical properties that enable three-dimensional (3D) culture of breast cancer cells and niche cells over weeks. A bioinert polymer, multiarm poly(ethylene glycol) functionalized with thiols (Mn ˜ 20 kDa), was reacted with integrin-binding and cell-degradable peptides decorated with one and two allyloxycarbonyl protecting groups, respectively, by rapid light-triggered thiol-ene polymerization for independent control of matrix density and composition. The elasticity, or 'stiffness', of these matrices has been tuned to mimic a variety of soft tissues (Young's modulus E˜0.5-20 kPa), from healthy and cancerous mammary tissues to metastatic site bone marrow and lung tissues. Further, the biochemical content has been tuned with receptor-binding peptides derived from laminin (IKVAV, laminin receptor), collagen ((POG)3POGFOGER(POG)4, α2β1 and α1β1), and fibronectin/vitronectin (RGDS, αVβ3 and α5β1 amongst others) and a crosslinking peptide derived from collagen (GPQG↓IWGQ, degraded MMP-1, -2, and -9 amongst others). We hypothesized that a microenvironment rich in collagen and fibronectin/vitronectin, mimicking aspects of remodeled tissues, would activate breast cancer cells relative to a laminin-rich epthelium-like microenvironment, building upon seminal studies in naturally-derived matrices and in vivo. To test this, we cultured breast cancer cells of different metastatic potential (estrogen receptor positive [ER+, T47Ds] and triple negative [ER-, MDA-MB-231s]) within different matrix densities and compositions. Both cell types exhibited high viability (&amp;gt; 90%), and cell activation in response to different matrix compositions was assayed by examining proliferation (metabolic activity, Ki-67, cell/cluster number and volume) and phenotype (morphology; E-cadherin, vimentin). Increased matrix density decreased elongation of ER- cells and proliferation of both cell types. Increased collagen content increased the proliferation of the ER+ cells and proliferation and elongation of ER- with mass and stellate morphologies, respectively, like observed in naturally-derived matrices. These studies demonstrate a new tool for controlled 3D culture of breast cancer cells relevant for both fundamental and applied research, with on-going investigations incorporating niche cells and triggered matrix changes. Citation Format: Kloxin AM, Sawicki LA, Ovadia EM. Probing breast cancer cell activation in response to extracellular cues using well-defined synthetic microenvironments [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-07.

Cancer-associated fibroblasts induce epithelial-mesenchymal transition through secreted cytokines in endometrial cancer cells.

Endometrial cancer (EC) is the most common malignant gynecological disease. Cancer-associated fibroblasts (CAFs) serve an important role in the development and progression of EC through epithelial-mesenchymal transition (EMT). The aim of the present study was to examine the association between CAFs and EMT, and the possible mechanisms of action. Firstly, the CAFs and normal fibroblasts (NFs) were isolated and cultured, then an immunofluorescence assay was performed to analyze the purity and level of activation of CAFs and NFs, and then the conditional medium (CM) of CAFs and NFs was prepared. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting examined the expression levels of epithelial (E)-cadherin, neural (N)-cadherin and vimentin. A Matrigel® invasion assay and wound healing assay were used to analyze the effect of the CM on invasion and migration. An ELISA assay also measured the levels of various cytokines in the CM. In addition, EMT-associated proteins in metastatic lung tissues were detected by immunohistochemical assay. The results indicated that the CM of CAFs may decrease the level of E-cadherin, and increase the levels of N-cadherin and vimentin, while increasing the levels of invasion and metastasis in EC cells. The concentration of epidermal growth factor, transforming growth factor-β, hepatic growth factor and fibroblast growth factor in the CM of CAFs increased significantly, in comparison with the NFs group (P<0.05). The exogenous growth factors induced migration and invasion of EC cells. CAFs induced lung metastasis and the EMT process in vivo. These data suggested that cancer-associated fibroblasts may induce EMT through the secreted cytokines in endometrial cancer cells.

MiR-200b inhibits proliferation and metastasis of breast cancer by targeting fucosyltransferase IV and \u03b11,3-fucosylated glycans

Aberrant protein fucosylation is associated with cancer malignancy. Fucosyltransferase IV (FUT4) is the key enzyme catalyzing the biosynthesis of α1,3-linkage fucosylated glycans carried by glycoproteins on the cell surface, such as the tumor-associated sugar antigen Lewis Y (LeY). An abnormal increase in the levels of FUT4 and LeY is observed in many cancers and correlated with cell proliferation and metastasis. Some microRNAs (miRNAs) are known to negatively regulate gene expression. FUT4 is an oncogenic glycogene, and thus it is important to identify the specific miRNA targeting FUT4. In current study, we first identified miR-200b as a specific miRNA that inhibited FUT4 expression. We found that miR-200b level was decreased, whereas that of FUT4 was increased in tissues and serum of breast cancer compared with that in the control by real-time PCR, western blotting and enzyme-linked immunosorbent assay. The alterations of miR-200b and FUT4 level were recovered after chemotherapy. The results also showed that miR-200b suppressed FUT4 expression and inhibited tumor growth and metastasis in MCF-7 and MDA-MB-231 breast cancer cells, as well as in the xenografted tumor tissues and metastatic lung tissues. miR-200b decreased the α1,3-fucosylation and LeY biosynthesis on epidermal growth factor receptor (EGFR), as well as inactivation of EGFR and downstream phosphoinositide-3 kinase/Akt signaling pathway. In conclusion, the study highlights that FUT4 could apply as a novel target for miR-200b that suppress the proliferation and metastasis of breast cancer cells by reducing α1,3-fucosylation and LeY biosynthesis of glycoproteins. miR-200b and FUT4 are potential diagnostic and therapeutic targets for breast cancer.

Actin-like protein 6A is a novel prognostic indicator promoting invasion and metastasis in osteosarcoma

Osteosarcoma harbors highly metastatic properties, accounting for postoperative recurrence and metastasis. Actin-like protein 6A(ACTL6A) regulates cell proliferation, migration and differentiation. However, the biologic role of ACTL6A in osteosarcoma remains unknown. In this study, the results showed that, by analysis of frozen fresh primary tumor tissues, matched non-cancerous bone tissues(NCBTs) and biopsy lung metastatic nodule tissues from 30osteosarcoma patients after radical surgical resection, ACTL6A was overexpressed in osteosarcoma tissues compared with matched NCBTs, and its expression level was associated with osteosarcoma metastasis. Immunohistochemical analyses of osteosarcoma tissue samples from two independent cohorts of formaldehyde-fixed, paraffin-embedded osteosarcoma tissue samples from total of 186osteosarcoma patients showed that high ACTL6A expression correlated with malignant clinicopathological features such as larger tumor size, high Ennecking grade, high histologic grade, and advanced tumor node metastasis stage. High ACTL6A expression was associated with poor prognosis for patients with osteosarcoma, and an independent and significant risk factor for disease-free survival and overall survival after radical tumor resection. Both invitro and invivo assays showed that ACTL6A overexpression promoted osteosarcoma cell invasion and metastasis, whereas knockdown of ACTL6A expression reduced osteosarcoma cell malignant behavior such as invasion and metastasis. Furthermore, we proved that ACTL6A promoted osteosarcoma cells of metastasis through facilitating epithelial-mesenchymal transition(EMT). Inconclusion, data from the present study demonstrated that ACTL6A was associated with poor survival and promoted osteosarcoma cell metastasis through EMT, suggesting that ACTL6A may be a novel prognostic biomarker and therapeutic target for osteosarcoma.

Oxaliplatin Plus S1 for Treating Sorafenib-Refractory Recurrent Hepatocellular Carcinoma after Liver Transplantation

Recurrent hepatocellular carcinoma (HCC) following liver transplantation (LT) correlates with a poor prognosis due to the lack of effective therapeutic approaches after sorafenib (SORA) or everolimus (EVL) resistance. We assessed the efficacy of SOX (S-1 and oxaplatin) after failed SORA for HCC recurrence after LT based on the results of next generation sequences (NGS). A patient with HCC recurrence after LT was treated with SORA until severe side effect. And then NGS was performed on the primary and metastatic tumor tissues. Investigations on molecular aberrations in cellular pathways and SNPs of drug-related genes were applied. The result of the first NGS shows the benefit from platinum drugs and no abnormalities in PI3K/AKT/mTOR pathway. He received SOX regimen for 4 cycles with progression-free survival (PFS) as long as 4.3 months. NGS of lung and liver metastasis tissues after disease progression still did not match molecular target of everolimus, which he switched to for experimental therapy. He died of failure treatment with 17.3 months overall survival. SOX regimen may benefit recurrent HCC after LT. NGS may provide personalized clinical care for HCC patients.

Metabonomic analysis of metastatic lung tissue in breast cancer mice by an integrated NMR-based metabonomics approach

This study identified the common potential biomarkers for early lung metastasis of breast cancer in two models.

Polyphyllin I inhibits the growth of ovarian cancer cells in nude mice.

Polyphyllin I (PPI) is an active component in Rhizoma Paridis, which displays extensive pharmacological antitumor activities. In a previous study, we found that polyphyllin I exhibited inhibitory effects on cell growth in the human ovarian cancer HO-8910PM cell line, as well as promoting apoptosis and the inhibition of cell migration. Furthermore, gene expression was also profiled by microarray, which showed that numerous genes were altered by PPI; three genes were of particular note that were associated with tumor progression, namely, Caspase-9, C-jun and Wnt5a. In the present study, the effect of PPI on subcutaneous tumor growth (HO-8910PM cells) in nude mice was further evaluated, and immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to examine the expression of Caspase-9, C-jun and Wnt5a in subcutaneous and lung metastatic tumor tissues, in order to investigate the possible mechanisms involved. The results showed that PPI significantly inhibited the tumor growth in vivo without a marked impact on body weight, and through use of immunohistochemical staining and RT-PCR, it was found that the expression of Caspase-9 and Wnt5a was decreased, while the expression of C-jun was increased, in subcutaneous and lung metastatic tumor tissue; this was consistent with the in vitro results. In conclusion, the present study showed that PPI exerted antitumor activity on ovarian cancer cells in vivo, and indicated that the modulation of Caspase-9, C-jun and Wnt5a may be involved in the antitumor effect of PPI.

Downregulation of microRNA-497 is associated with upregulation of synuclein γ in patients with osteosarcoma.

The present study aimed to investigate the effects of microRNA (miRNA/miR)-497 expression levels on the expression levels of synuclein γ (SNCG) in serum samples, as well as osteosarcoma and lung-metastatic tissue samples, from patients with osteosarcoma. Between December 2010 and August 2013, fasting peripheral blood was collected from 36 patients with osteosarcoma for serum separation. In addition, osteosarcoma and lung metastatic tissues were resected from 15 osteosarcoma patients with lung metastasis by surgery. Bioinformatics was employed to predict the amount miRNA that binds to SNCG. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of SNCG and miR-497, and western blotting was performed to determine protein expression levels. It was observed that SNCG mRNA and protein expression levels were significantly upregulated in osteosarcoma tissues (P<0.01). Additionally, SNCG mRNA (P<0.01) and protein (P<0.05) expression levels were significantly upregulated in the blood of patients with osteosarcoma. SNCG mRNA and protein expression levels were also significantly upregulated in lung metastatic tissues (P<0.01). miR-497 was significantly downregulated in all three samples; therefore downregulation of miR-497 may lead to the occurrence, development and metastasis of osteosarcoma through the upregulation of SNCG mRNA. In summary, the upregulation of SNCG in blood, osteosarcoma tissue and lung metastatic tissue samples is associated with the dowregulation of miR-497, suggesting that miR-497 may be a potential marker and therapeutic target for the treatment of osteosarcoma.

Arsenic trioxide inhibits lung metastasis of mouse colon cancer via reducing the infiltration of regulatory T cells.

The purpose of this study was to investigate the effects of arsenic trioxide (As2O3) on the infiltration of regulatory T cells (Tregs) in the local lung metastasis of mouse colon cancer in vivo and the regulation of Tregs in cytokine-induced killer cells (CIKs) in vitro. A high Tregs infiltration mouse colon cancer lung metastasis model was established by intravenous injection of CT26 murine colon carcinoma cells. Tumor-bearing mice were randomly divided into three groups: control group, low-dose As2O3 group, and high-dose As2O3 group. For in vitro studies, CIKs were treated with vehicle control or 0.1, 1, or 5 μM As2O3. The level of Tregs was detected via flow cytometry, Foxp3 expression was assessed by immunohistochemistry and reverse transcription–polymerase chain reaction (RT-PCR), the level of interferon gamma (IFN-γ) was evaluated by enzyme-linked immunoassay (ELISA), and the cytotoxic activity of As2O3-treated CIKs was assessed through a lactate dehydrogenase (LDH) release assay. Obvious lung metastasis was observed 3 days after CT26 murine colon carcinoma cell injection. The numbers of Tregs in the lungs and spleens of tumor-bearing mice were significantly higher than those of the normal group (p < 0.01). As2O3 treatment increased the mouse weight as well as reduced the number of metastatic lung nodules and the lung/body weight ratio (p < 0.01). Moreover, As2O3 treatment significantly reduced the Tregs proportion and the Foxp3 messenger RNA (mRNA) levels in metastatic lung tissues (p < 0.01). In vitro, As2O3 significantly reduced the Tregs proportion and the Foxp3 mRNA levels (p < 0.01) and significantly increased the cytotoxic activity of CIKs and the IFN-γ levels in the supernatant of cultured CIKs (p < 0.01). As2O3 might inhibit lung metastasis of colon cancer by reducing the local infiltration of Tregs and increase the cytotoxic activity of CIKs by suppressing Tregs.

Abstract P3-06-07: Changes in the tumor microenvironment develop acquired resistance to pegylated liposomal doxorubicin in breast cancer mouse model

Abstract Pegylated liposomal doxorubicin (PLD) is one of the most widely used nanotherapeutics for the treatment of advanced/metastatic breast cancer. PLD accumulates in tumors utilizing so-called the enhanced permeation and retention (EPR) effect. Nevertheless, therapeutic efficacy and long term survival remain variable due to the development of acquired resistance. Elucidating the mechanisms of acquired resistance to PLD shall help developing new strategies to improve therapeutic outcome. It has been largely overlooked that the transport of therapeutics across biological barriers can significantly affect the efficacy of cancer therapies. Previously, we showed that the transport of PLD to tumors depends both on the tumor type and organ site. This effect is controlled by the extent to which endothelial cells (ECs) are covered by the collagen type IV (Col4) in the basement membrane, which in turn is influenced by the levels of MMP-9. Here, we have developed 4T1 tumor model which develops acquired resistance to PLD and spontaneous lung metastases. Our objective is to elucidate the resistant mechanism by evaluating the changes in the transport of PLD to the sensitive and resistant/metastatic tumors. BALB/c mice bearing 4T1 cancers were treated with PLD intravenously when tumor volumes reached a size of approximately 100-200 mm3 . Tumor volumes in all mice decreased after initial PLD injections (sensitive). However, tumors started to grow again after 20 days and didn't respond to the second/third injections (resistant). Furthermore, 13 out of 14 mice developed spontaneous lung metastases. To elucidate the mechanisms of the resistance, mice bearing sensitive or resistant tumors were sacrificed after 24 hours of PLD injection, respectively. PLD accumulation in tumors was evaluated by imaging fluorescence of doxorubicin. Immunofluorescence staining was performed to evaluate the expression of ECs, Col4, MMP-9, and efflux pump associated p-glycoprotein (P-gp) in the primary tumors, and the expression of ECs and Col4 were also evaluated in lung metastases. PLD accumulation was significantly decreased in the resistant tumors compared to the sensitive tumors, although P-gp expression was not increased in the resistant tumors. The amount of ECs and Col4 increased in the resistant tumors. Interestingly, ECs were covered more tightly by Col4 in the resistant tumors as compared with the sensitive tumors, which could decrease the EPR effect in the tumors. MMP-9 expression decreased in the resistant tumors, suggesting less degradation of Col4 in the basement membrane. Coverage of ECs by Col4 was similar between the metastatic lung tumors and uninvolved lung tissue as well as the resistant primary tumors, indicating the EPR effect is not increased in the metastatic tumors. In summary, ratio of ECs covered by Col4 is higher in the resistant/metastatic tumors as compared to that in the sensitive primary tumors. This structural change in the tumor microenvironment, impeding the sufficient PLD transport to the tumors after the initial PLD therapy, can be a cause of acquired resistance/development of lung metastasis. These changes should be taken into account to develop strategies for overcoming the acquired resistance and metastasis. Citation Format: Kai M, Liu YT, Saito Y, Ferrari M, Yokoi K. Changes in the tumor microenvironment develop acquired resistance to pegylated liposomal doxorubicin in breast cancer mouse model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-07.

Expression of Ceramide Galactosyltransferase (UGT8) in Primary and Metastatic Lung Tissues of Non-Small-Cell Lung Cancer.

Ceramide galactosyltransferase (UGT8) is an enzyme that regulates the synthesis of sphingolipids of the myelin sheath in nervous systems. The protein raises an increasing research interest as a potential marker of cancer progression in various organs. In the present study we seek to determine whether UGT8 could play a role of a therapeutic marker in non-small cell lung carcinoma (NSCLC). We addressed the issue by examining the intensity of UGT8 expression in tissue specimens of primary and corresponding metastatic lung tumors in 19 NSCLC patients undergoing surgery. The methodology was one of immunohistochemical tissue staining using light microscopy. The findings were that the majority of both lung primary and metastatic tumor tissues were positive in UGT8 signals. The cytoplasmic expression of UGT8 was found in 68.4 % of cases of primary tumors and 82.2 % of metastases, with a positive correlation between the UGT8 expression in both tumor tissues. The normal tissue adjacent to tumors showed no positive UGT8 staining. However, we failed to find any appreciable difference in UGT8 expression depending on the clinical stage of NSCLC or lymph node involvement. Nor was there any association between UGT8 expression in tumor tissues and patients' survival time. We conclude that it is unlikely that therapeutic targeting of UGT8 could inhibit cell proliferation and invasion of NSCLC. UGT8, although enhanced in NSCLC tissues, does not meet the criteria of a lung tumor marker. Thus, UGT8 cannot be considered as having diagnostic or therapeutic utility in NSCLC. The pathophysiological meaning of enhanced expression of UGT8 in lung cancer remains to be explored in further studies.

Abstract 3087: MicroRNA 141: A novel regulator of brain metastasis from breast cancer

Abstract Purpose: Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. The purpose of this study was to identify molecular mediators of brain metastasis from breast cancer using novel preclinical brain metastasis models we developed recently. Methods: GFP-labeled cells were injected via tail vein into SCID/beige mice and metastatic colonization to the brain and lung was evaluated by fluorescent stereomicroscope and histology 8-weeks after injection. miRNA microarray was performed with miRNA 3.0 Array. Stable knockdown and overexpression of miR-141 was achieved with lentiviral vectors. miR-141 serum levels in patients with metastatic breast cancer was measured using quantitative PCR and was associated with outcome data. Results: We developed novel brain metastasis models in which tail-vein injection of parental triple-negative and a HER2-overexpressing inflammatory breast cancer lines led to a high rate of brain metastases (67%) in SCID/Beige mice (SUM149, 6 of 9 mice; MDA-MB-IBC3, 10 of 15 mice). Sub-lines derived from brain metastasis (BrMS) or lung metastasis (LuMS) tissues were morphologically and molecularly distinct. The BrMS showed epithelial morphology and overexpressed epithelial markers and miR-141 while the LuMS showed mesenchymal morphology and overexpressed mesenchymal markers. Knockdown of miR-141 significantly inhibited metastatic colonization to the brain in both cell lines (miR-141 knockdown vs. control: SUM149, 0 of 8 mice vs. 6 of 9 mice, p = 0.009; MDA-MB-IBC3, 2 of 14 mice vs. 10 of 15 mice, p = 0.007). Ectopic expression of miR-141 in non-expressing MDA-MB-231 significantly enhanced brain metastatic colonization via tail-vein injection (5 of 9 mice vs. 0 of 10 mice, P = 0.02). On multivariate analyses high serum level of miR-141 was an independent predictor of progression free survival [HR 4.8 (95%CI, 2.6-8.7), P&amp;lt;0.001] and overall survival [HR 7.0 (95%CI 3.5-15.1), P&amp;lt;0.001], and was associated with shorter brain metastasis free survival (P = 0.03). Conclusion: We have generated novel brain metastasis models, demonstrated miR-141 as a key regulator of brain metastasis and provided clinical evidence supporting the prognostic relevance of miR-141 in patients with brain metastasis. We propose that miR-141 should be examined as a biomarker and potential target for the prevention and treatment of brain metastases from breast cancer. Citation Format: Bisrat G. Debeb, Lara Lacerda, Simone Anfossi, Parmesh Diagaradjane, Khoi Chu, Lei Huo, Caimiao Wei, Richard Larson, Adam Wolfe, Wei Xu, Li Li, Daniel Smith, Cristina Ivan, Pamela Allen, Savitri Krishnamurthy, George Calin, Xiang Zhang, Thomas Buchholz, Naoto Ueno, James Reuben, Wendy Woodward. MicroRNA 141: A novel regulator of brain metastasis from breast cancer. [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 3087. doi:10.1158/1538-7445.AM2015-3087