Non-metastatic Cell Line Research Articles

Abstract 140: Deregulated genes by abnormal promoter methylation in early and late stages in a linear model of melanoma progression

Abstract Metastatic melanomas are extremely aggressive tumors, accounting for 70% of skin cancer deaths, which incidence is increasing globally. Despite the advances in their treatment, metastatic melanomas are still associated with a poor prognosis, with a median survival of 6 to 12 months. The study of the molecular alterations affecting key pathways during melanoma progression may contribute to a better understanding of the biology underlying the aggressiveness of the disease and lead to the identification of biomarkers for prognosis and novel targets for therapy. In addition to mutations, epigenetic alterations are important contributors to malignant transformation and tumor progression. Aberrant DNA methylation is an epigenetic hallmark of cancer, known to play important roles in melanoma development. Although different genes were described as presenting methylation alterations in their promoters in melanomas, the dynamics of these alterations along melanocyte malignant transformation and melanoma progression is still unclear. Our laboratory developed a linear model of melanoma progression in which different pre-malignant and melanoma cell lines were established after subjecting murine melanocytes to a sustained stressful condition (adhesion impediment). In this study, we used the spontaneously immortalized melanocyte lineage (melan-a); pre-malignant melanocytes (4C), obtained after subjecting melan-a cells to 4 cycles of adhesion impediment; a non-metastatic tumorigenic cell line (4C11-), obtained after a limiting dilution of spheroids formed by 4C adhesion impediment; and a metastatic cell line (4C11+), resulting from the spontaneously loss of p53 by 4C11- cells. The aim of this work was to identify genes that have their expression altered by promoter and gene body methylation that are related to the malignant transformation of melanocytes and metastasis. Through the integrative analysis of methylome (ERRBS) with transcriptome (RNAseq) data, it was possible to identify the dynamic changes in DNA methylation in promoters and gene bodies, and their relation with gene expression in both early and late stages of melanoma progression. Among those genes are Foxd1, Lrrk2, Adcy1 and Adcy3, which had their expression validated by qPCR, and their epigenetic regulation validated by the treatment of the cells with epigenetic drugs (5-aza-2'-deoxycytidine and Trichostatin A). The gene expression and promoter methylation profiles found in the murine model of melanoma progression were correlated with clinical data from melanoma patients available in cancers cohorts, revealing potential novel prognostic markers. Supported by FAPESP, CNPQ and CAPES Citation Format: Débora Papaiz, Flávia E. Rius, Diogo Pessoa, Eduardo M. Reis, Christopher E. Mason, Gangning Liang, Miriam G. Jasiulionis. Deregulated genes by abnormal promoter methylation in early and late stages in a linear model of melanoma progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 140.

Abstract 3971: The role of obesity-associated adipose tissue inflammation in breast cancer metastasis

Abstract Although obesity is a known risk factor for breast cancer incidence, metastasis, and mortality, the mechanisms underlying this link remain elusive. This study therefore aims to uncover the relationship between obesity-associated adipose tissue and breast cancer metastasis by identifying specific inflammatory mediators in the adipose secretome. It is well known that obesity induces hypoxic conditions in the adipose tissue, stimulating an inflammatory response that ultimately creates a favorable environment for cancer cell growth and survival. The link between obesity-associated adipose inflammation and cancer metastasis, however, remains poorly described. We have previously shown that injection of basal-like E0771's, and claudin-low metM-Wnt-Lung and metM-Wnt-Liver metastatic breast cancer cell lines in mice fed a diet-induced obesity (DIO, 60% kcal from fat) diet displayed larger tumors with increased metastatic potential compared to lean mice fed a control (10% kcal from fat) diet. When diets were supplemented with Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), the enhancing effects of DIO on tumor growth and metastasis were mitigated. Gene expression microarray analysis of non-tumor bearing mammary fat pads from these mice revealed increased mammary expression of inflammation-related genes in tissues from DIO relative to control mice. Moreover, gene expression analysis of the tumor microenvironment from these mice revealed a signature of DIO-induced immunosuppression that was normalized by Sulindac supplementation. To further investigate the relationship between obesity-associated inflammation and breast cancer metastasis, we cultured our metastatic metM-Wnt-Lung, and metM-Wnt-Liver, and claudin-low, non-metastatic M-Wnt breast cancer cell lines with conditioned media (FCM) from the mammary fat pads of mice fed control, DIO, or DIO+Sulindac diets. We then conducted phenotypic assays to characterize differences in cell growth, proliferation, and metastatic potential. After normalizing the FCM concentrations to fat pad mass, we saw increased cell viability and migration in cells treated with FCM from DIO relative to control mice. Luminex assays identified several pro-inflammatory factors associated with the pro-cancer effects of FCM from DIO versus control mice, and additional characterization studies utilizing high-throughput proteomic screening and immune-depletion approaches are on-going. Taken together, our findings suggest an important role for the adipose secretome, and specifically inflammatory mediators, in obesity driven breast cancer progression. Citation Format: Lydia K. Eisenbeis, Shannon B. McDonell, Laura A. Smith, Alyssa J. Cozzo, Michael F. Coleman, Stephen D. Hursting. The role of obesity-associated adipose tissue inflammation in breast cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3971.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Abstract A11: STAT1 mediates resistance to experimental oral cancer that is associated with enhanced antitumor T-cell responses

Abstract Cancers of the oral cavity and pharynx remain among the 10 most prevalent cancers in the United States. Signal Transducer and Activation of Transcription 1 (STAT1) is a transcription factor primarily involved in mediating responses to IFNs, as well as interleukin (IL) 21, 27, and 35. STAT1 has been shown to promote or suppress tumors in different cancer types, but its role in oral cavity cancers has not been examined. We examined the role of STAT1 in oral cancer using orthotopic models in wild-type (WT) and STAT1-deficient BALB/c mice. Metastatic (LY2) or nonmetastatic (B4B8) HNSCC cell lines were injected into the oral cavity and tumor development was monitored over a period of three or six weeks, respectively. At terminal sacrifice, tumors, spleens, draining lymph nodes, and bone marrow tissues were analyzed for tumor growth, metastasis, and immunologic responses. Tumors from STAT1-deficient mice injected with B4B8 cells were twice as large as tumors from WT mice. Interestingly, LY2-injected STAT1-deficient mice possessed tumors that were five times as large as tumors from LY2-injected WT mice. The increased tumor burdens in metastatic tumors were associated with a decrease in CD4+ and CD8+ T-cell infiltration into the draining lymph nodes of tumor-bearing STAT1-deficient mice compared to WT mice. Further, lymph node and splenic CD4+ and CD8+ T cells in tumor-bearing STAT1-deficient mice produced significantly lower TNF-alpha compared to WT mice. Finally, tumor-bearing STAT1-deficient mice displayed a significant accumulation of CD11b+ Ly6G+ myeloid cells in the spleens and bone marrow compared to WT counterparts in both metastatic and nonmetastatic HNSCC models. Our results demonstrate that STAT1 mediates resistance to oral tumor development that is associated with enhanced T-cell migration, TNF-alpha mediated antitumor T-cell responses, and diminished accumulation of tumor-promoting CD11b+ Ly6G+ myeloid cells. Citation Format: Nathan Ryan, Kelvin Anderson, Greta Volpedo, Omar Hamza, Sanjay Varikuti, Abhay Satoskar, Steve Oghumu. STAT1 mediates resistance to experimental oral cancer that is associated with enhanced antitumor T-cell responses [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A11.

Fluidic substrate as a tool to probe breast cancer cell adaptive behavior in response to fluidity level

Although the roles of elastic components in breast cancer progression have been widely studied, the importance of matrix dissipative elements in regulating breast cancer behavior is still poorly understood. In this study, we designed viscosity-tunable fluidic substrates to investigate the effects of matrix viscosity on the alteration of breast cancer cellular fate using a hydrophobic molten polymer of poly(e-caprolactone-co-D,L-lactide) [P(CL-co-DLLA)] with different levels of fluidity. The high- and low-fluidity substrates used in this study were shown to behave as viscoelastic liquids at physiological temperature. A nonmetastatic breast cancer cell line (MCF-7) was cultured at the interface of the fibronectin-coated substrate, and its behavior towards the substrate fluidity level was thoroughly characterized. Despite fibronectin-mediated cell-substrate interactions, MCF-7 cells show sensitivity to substrate fluidity levels by forming types aggregates of different sizes and structures over time. Accordingly, MCF-7 cells were undergoing senescence on fluidic substrates, as shown by high metabolic activity over time, suppressed proliferation ability, and positive expression of senescence markers. Moreover, senescence implies more resistance towards anticancer drug treatment. This indicates that a fluidic substrate, as a two-dimensional synthetic matrix system, could demonstrate the importance of mechanical cues in redefining cellular function and cellular fate by changing the viscosity of the pure substrate. A fluidic substrate behaving as a hydrophobic viscoelastic liquid was designed as a tool to investigate the role of matrix viscosity on the alteration of breast cancer cellular fate. The fluidity level of fluidic substrate was tuned by modulating the molecular weight of poly(e-caprolactone-co-D,L-lactide). MCF-7 cells responded to the change in fluidity level of fluidic substrates by forming weak attachment. On high-fluidity substrate, MCF-7 cells formed 3D aggregates, while coalesced on low-fluidity substrate. More importantly, the fluidic substrate mechanically promoted senescence of MCF-7 cells regardless the fluidity level of substrate.

METCAM/MUC18 is a new early diagnostic biomarker for the malignant potential of prostate cancer: Validation with Western blot method, enzyme-linked immunosorbent assay and lateral flow immunoassay.

METCAM/MUC18 expression was increased with the malignant progression of prostate cancer and also a bona fide metastatic gene, capable of initiating and driving the metastasis of a non-metastatic human prostate cancer cell line to multiple organs. We explored if METCAM/MUC18 was detectable in human serum and a novel biomarker to predict malignant propensity of prostate cancer. Two antibodies were identified by Western blot analysis having the highest sensitivity and specificity to establish calibration curves from the recombinant METCAM/MUC18 proteins. They were used in ELISA and LFIA to determine the METCAM/MUC18 concentrations in serum samples from 8 normal individuals, 4 BPH patients, 1 with PIN, 6 with high-grade prostate cancer, and 2 treated cancer patients. Serum METCAM/MUC18 concentrations were statistically significantly higher in the patients with PIN and prostate cancer than those with BPH, the treated patients and normal individuals. The LFIA results were statistically better than ELISA and Western blot methods. Serum METCAM/MUC18 concentrations were in direct proportional to most of serum PSA concentrations.

Ceramide Kinase Is Upregulated in Metastatic Breast Cancer Cells and Contributes to Migration and Invasion by Activation of PI 3-Kinase and Akt.

Ceramide kinase (CerK) is a lipid kinase that converts the proapoptotic ceramide to ceramide 1-phosphate, which has been proposed to have pro-malignant properties and regulate cell responses such as proliferation, migration, and inflammation. We used the parental human breast cancer cell line MDA-MB-231 and two single cell progenies derived from lung and bone metastasis upon injection of the parental cells into immuno-deficient mice. The lung and the bone metastatic cell lines showed a marked upregulation of CerK mRNA and activity when compared to the parental cell line. The metastatic cells also had increased migratory and invasive activity, which was dose-dependently reduced by the selective CerK inhibitor NVP-231. A similar reduction of migration was seen when CerK was stably downregulated with small hairpin RNA (shRNA). Conversely, overexpression of CerK in parental MDA-MB-231 cells enhanced migration, and this effect was also observed in the non-metastatic cell line MCF7 upon CerK overexpression. On the molecular level, CerK overexpression increased the activation of protein kinase Akt. The increased migration of CerK overexpressing cells was mitigated by the CerK inhibitor NVP-231, by inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway and the Rho kinase, but not by inhibition of the classical extracellular signal-regulated kinase (ERK) pathway. Altogether, our data demonstrate for the first time that CerK promotes migration and invasion of metastatic breast cancer cells and that targeting of CerK has potential to counteract metastasis in breast cancer.

Nuclear translocation of Atox1 potentiates activin A-induced cell migration and colony formation in colon cancer.

BackgroundColorectal cancer remains a deadly cancer due to metastatic disease. To understand the molecular mechanisms of metastasis in colon cancer, we investigated whether the copper chaperone antioxidant-1 (Atox1) protein plays a role in this process. Recent findings indicate that Atox1 protein has transcription factor activities and plays a vital role in cell proliferation in cancer cells. However, the role of Atox1 in metastasis has not been examined.MethodsAtox1 expression was determined by immunofluorescence in a tissue microarray generated from a spectrum of CRC patients. Subcellular fractionation of colon cancer cell lines SW480 and SW620 cells was used to examine the cellular location of Atox1 in the face of activin A, a cytokine that stimulates colon cancer metastasis. Atox1 expression was genetically manipulated and cellular migration measured through trans-well assay and proliferation measured by colony formation assays.ResultsHere we demonstrate that in patients with metastatic colon cancer, there is a significant increase in the expression of nuclear Atox1. Interestingly, the metastatic CRC cell line SW620 has increased nuclear localization of Atox1 compared to its related non-metastatic cell line SW480. Further, inhibition of endogenous Atox1 by siRNA in SW620 decreased colony formation and reactive oxygen species generation via decreased expression of Atox1 targets cyclin D1 and NADPH oxidase subunit p47 phox, respectively. Additionally, overexpression of nuclear-targeted but not copper binding domain-mutated Atox1 in SW480 cells increased colony formation and cell migration that was further augmented by activin A stimulation, a known enhancer of colon cancer metastasis.ConclusionsOur findings suggest that nuclear Atox1 might be a new therapeutic target as well as a new biomarker for metastatic colorectal cancer.

Investigation of the effects of rapamycin on the mTOR pathway and apoptosis in metastatic and non-metastatic human breast cancer cell lines.

The aim of this study was to analyze the effects of rapamycin treatment on apoptosis via mTOR pathway in metastatic and non-metastatic human breast cancer cell lines by immunohistochemical and TUNEL analysis. MCF-7 and MDA-MB 231 cell lines were incubated under standard conditions forming Rapamycin and control groups. In immunohistochemical evaluation; mTOR pathway was evaluated with anti-IGF1, anti-PI3K, anti-pAKT1/2/3, anti-mTORC1, anti-mTORC2 and anti-ERK1 antibodies. The effect of apoptosis was also confirmed by TUNEL method. In this study, activation of PI3K/AKT/mTOR and related molecular pathways in the MDA-MB 231 and MCF-7 breast cancer cell line was evaluated and it was observed that these pathways could play a key role in cancer development. Increased apoptotic cells were observed in mTORC1 inhibition by Rapamycin administration. Targeting the mTOR pathway in breast cancer treatment may be a treatment option. In addition, the demonstration and confirmation of increased apoptosis in Rapamycin treated groups suggested that Rapamycin, an inhibitor of mTOR, is promising in the treatment of breast cancer (Tab. 2, Fig. 3, Ref. 66).

Enhancement of PDT-cytotoxicity via ROS induced by indomethacin in metastatic breast cancer

Breast cancer is one of the most common types of cancers prevalent in women. Several types of breast cancers can easily metastasize to bone and cause disease complications such as hypercalcemia and pathologic fracture, thus compromising the quality of life of people affected by it. Bisphosphonate drugs are often used for the treatment of bone metastasis to suppress osteoclastic bone resorption. However, bisphosphonate has adverse effects on the gastrointestinal tract and kidneys and also induces osteonecrosis of the jaw. Photodynamic therapy (PDT) is an alternative cancer treatment approach with minimal invasiveness. It is a combination treatment that uses photosensitizers, which accumulate in tumor cells, followed by laser irradiation. We previously reported that the cellular incorporation of 5-aminolevulinic acid (5-ALA), which was a precursor of protoporphyrin IX (PpIX), was regulated by reactive oxygen species derived from mitochondria (mitROS). In this study, we investigated the incorporation of 5-ALA, accumulation of PpIX, and subsequent effects on cell viability after laser irradiation of two different breast cancer cell lines with different metastaticites. The highly metastatic breast cancer cell line 4T1E/M3 showed a significant increase in ROS production after treatment with indomethacin (IND). In addition, IND treatment enhanced the cellular uptake of 5-ALA via PEPT1 upregulation in 4T1E/M3, but not in the non-metastatic cell line. Overall, metastatic breast cancer is likely to be sensitive to ROS and activate signaling pathways associated with 5-ALA transportation, suggesting that ALA-PDT could be an effective treatment with low invasiveness for metastatic breast cancer.

GREM1 is associated with metastasis and predicts poor prognosis in ER-negative breast cancer patients

BackgroundIn breast cancer, activation of bone morphogenetic protein (BMP) signaling and elevated levels of BMP-antagonists have been linked to tumor progression and metastasis. However, the simultaneous upregulation of BMPs and their antagonist, and the fact that both promote tumor aggressiveness seems contradictory and is not fully understood.MethodsWe analyzed the transcriptomes of the metastatic 66cl4 and the non-metastatic 67NR cell lines of the 4T1 mouse mammary tumor model to search for factors that promote metastasis. CRISPR/Cas9 gene editing was used for mechanistic studies in the same cell lines. Furthermore, we analyzed gene expression patterns in human breast cancer biopsies obtained from public datasets to evaluate co-expression and possible relations to clinical outcome.ResultsWe found that mRNA levels of the BMP-antagonist Grem1, encoding gremlin1, and the ligand Bmp4 were both significantly upregulated in cells and primary tumors of 66cl4 compared to 67NR. Depletion of gremlin1 in 66cl4 could impair metastasis to the lungs in this model. Furthermore, we found that expression of Grem1 correlated with upregulation of several stem cell markers in 66cl4 cells compared to 67NR cells. Both in the mouse model and in patients, expression of GREM1 associated with extracellular matrix organization, and formation, biosynthesis and modification of collagen. Importantly, high expression of GREM1 predicted poor prognosis in estrogen receptor negative breast cancer patients. Analyses of large patient cohorts revealed that amplification of genes encoding BMP-antagonists and elevation of the corresponding transcripts is evident in biopsies from more than half of the patients and much more frequent for the secreted BMP-antagonists than the intracellular inhibitors of SMAD signaling.ConclusionIn conclusion, our results show that GREM1 is associated with metastasis and predicts poor prognosis in ER-negative breast cancer patients. Gremlin1 could represent a novel target for therapy.

Silencing TTK expression inhibits the proliferation and progression of prostate cancer

PurposeThe main objective of our study was to explore changes in the expression levels of differentially expressed genes associated with prostate cancer progression and to design a series of experiments to verify the function of differentially expressed genes. MethodThe transcriptome datas of 499 cases of prostate cancer patients was downloaded from TCGA database. Differential genes associated with Gleason score were selected and filtered out by p < 0.05 and spearman coefficient >0.3. KEGG signaling pathway was enriched by differentially expressed genes, and TTK was selected as the research object. The expression of TTK was tested in prostate cancer tissues and prostate cancer cell lines. The changes of biological behavior of prostate cancer cell lines were verified after TTK was knocked out by siRNA and tumorigenic effect of TTK was verified by shRNA in vivo experiments. ResultThe expression of TTK was positively correlated with Gleason score of prostate cancer, and the expression of protein and mRNA in metastatic prostate cancer cell lines was higher than that in non-metastatic prostate cancer cell lines. Vitro biological experiments showed that TTK gene knockout could inhibit the proliferation, invasion and migration of PC3 and DU145 cells, and promote cell apoptosis. In vivo experiments showed that TTK knockout inhibited tumorigenesis in mice. It was found that the expression of CDK2 and CCNE1 decreased after TTK was knocked out. ConclusionOur results suggest that TTK is a gene associated with malignancy of PCa and could be a novel therapeutic target for clinical application.

Effect of immunosuppression in miRNAs from extracellular vesicles of colorectal cancer and their influence on the pre-metastatic niche

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer.

Epigenetic modifiers 5-aza-2'-deoxycytidine and valproic acid differentially change viability, DNA damage and gene expression in metastatic and non-metastatic colon cancer cell lines.

DNA methylation and histone modifications are major components of cellular epigenetic pattern determining gene expression. Cancer cells have their own epigenetic array, which can be different in cells of primary and metastatic tumors. In the present work we investigated effects of 1 mM valproic acid (VPA), a histone deacetylase inhibitor and 0.2 μM 5-aza-2'-deoxycytidine (5-aza-dC), a DNA demethylation agent, singly or in combination in two colorectal cancer cell lines Caco-2 (non-metastatic) and LoVo (metastatic). Cell viability, DNA damage and the mRNA expression of the CDC25C (cell division cycle 25C), CDKN1A (cyclin dependent kinase inhibitor 1A) and CHEK1 (checkpoint kinase 1), SQSTM1 (sequestosome 1) and ULK1 (unc-51 like autophagy activating kinase 1), RELA (RELA proto-oncogene, NF-κB subunit) and TP53BP1 (tumor protein p53 binding protein 1) genes important in cell cycle regulation, autophagy and cancer progression were investigated. Both drugs induced a moderate decrease in cell viability and significant DNA damage in both cell lines. LoVo cells were more sensitive to VPA and combined treatment than Caco-2. LoVo cells also showed higher expression of genes that are often associated with more aggressive tumors than Caco-2 cells and treatment with the modifiers increased this difference. In conclusion, 5-aza-dC and VPA can induce different effects in metastatic and non-metastatic cancer cell lines and this may be important in determination of epigenetic profile responsible for metastatic properties of cancer cells.

Abstract 3766: Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition

Abstract INTRODUCTION One of the unsolved problems of cancer is its progression into systemic, metastatic disease. Malignant cells are released into circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. We investigated a unique feature of CSCs, formation of cell clusters (spheroids or tumorspheres) in vitro. We found that it also takes place in vivo, cancer tumorspheres circulate in blood and we showed that they carry cancer stem cells and strongly correlate with metastatic disease. In this project we developed a filtration method to collect live circulating cancer stem cell in tumorspheres and searched for a method to establish a prognostic assay to predict their metastatic potential. METHODS The conventional approaches use knockout mice to assay metastasis (NOD-SCID mice, etc.), but he high cost, the complex procedure and the lengthy assay (5 weeks) ruled them out as a routine clinical assay. Our goal was to establish an in vivo translational assay to detect metastatic predisposition early, with a chance to prevent systemic progression. We found that the rapidly progressing environment in the chicken embryo offers critical advantages to test the metastatic potential of a patient’s cancer. Easy access to chorioallantois circulation and to the embryo, the lack of immune response, the rapid turnaround time (5 days) and the technical simplicity rendered the new embryonal metastatic assay a promising and cost effective translational method. We used tumorspheres of both highly metastatic and non-metastatic cell lines. For image analysis a highly metastatic cell line (MDA-MB-436) was labeled by Green Fluorescent Protein. To model the technique of labeling real life patient spheroids (DiI membrane dye and protein tracing by CFDA-SA), we used the highly metastatic MDA-MB-231 and the non metastatic MCF7 and T47D cell lines. For quantitative analysis of embryonal metastasis we used methods applied in microchimerism detection. Embryonal DNA/RNA was extracted 5-9 days after cancer spheroid implantation and used qPCR to detect human sequences. RESULTS In the embryonal metastatic assays, fluorescent and confocal imaging detected metastatic cells in the embryo. DiI and CFDA-SA labeling of native tumorspheres indicated a level of toxicity and needed to be optimized. Molecular detection of human microchimerism in the embryos was extremely sensitive and generated quantitative metastatic data. CONCLUSIONS The results indicate that the embryonal metastasis assay using molecular microchimerism detection has the potential for a sensitive and quantitative method to assess the metastatic predisposition of a cancer patient. The method needs only an incubator, DNA extraction and a thermocycler, easily available in most hospitals and offers cost effective, relevant in vivo prognostic data within days. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Viktoria Denes. Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3766.

Abstract 4703: Single Cell CNV End to End workflow to uncover tumor heterogeneity in a murine breast cancer model

Abstract Background: Tumor heterogeneity is a hallmark of cancer and can have significant impact on identifying drivers, including those that may be therapeutically relevant. Although, the traditional sequencing of bulk tumor specimens provides invaluable information, sequencing of individual cells provides a true representation of the cellular heterogeneity that exists within a cancer. Novel technologies such as single cell copy number variation (scCNV) enable copy number profiling at a single cell resolution. Single Cell CNV generates genome wide sequencing libraries from single cells to reveal tumor heterogeneity and evolution of subclonal populations. Methods: In this study, we utilized a scCNV assay to assess heterogeneity within the 4T1 murine breast tumor model. To accomplish this we have developed an end-to-end workflow including tissue dissociation, single cell library preparation, sequencing and data analysis. We injected 4T1 cells into the mammary fat pad of 4-6 week old BALB/c mice. Mice were sacrificed at 21 days and tumors were removed from the injection site and dissociated using Milteny’s Biotec Octo Dissociator. We utilized the 10X Genomics Chromium system to partition single cells and prepare sequencing libraries in parallel such that all DNA fragments produced within a partition are barcoded. Following library construction, the Illumina Novaseq 6000 system was used to sequence single cell libraries. Additionally, single cell libraries from a fraction of the 4T1 cells that were injected into the mammary fat and non-metastatic isogeneic cell line variants, 67NR and 168FARN, were also sequenced. Results: We observed a pair of amplifications (&amp;gt;8 copies) on chromosome 9 and three segments of amplification on chromosome 15 that were conserved throughout the injection site tumor cells. Interestingly, the amplifications on chromosome 15 encompass one of the most commonly observed breast cancer oncogenes, Myc, and the cell migration regulator, Skp2. Further, we observed three unique cluster of tumors cells which suggests the possibility of detecting major and minor clonal populations using this technology. Conclusion: Our findings suggest scCNV will help us to improve characterizations of the entirety of a patient’s disease to uncover potential clones that are primed to induce metastasis, drug resistance, and relapse. Citation Format: Lee D. Gibbs, Jing Qian, Michelle Webb, Sharon Chang, Zarko Manojlovic, Enrique I. Velazquez, Troy McEachron, David W. Craig, John D. Carpten. Single Cell CNV End to End workflow to uncover tumor heterogeneity in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4703.

Differential Response to 1,25-dihydroxyvitamin D in Metastatic and Non-Metastatic Breast Cancer Cell Lines in Hypoxia (P05-006-19)

ObjectivesPrevention of metastasis is of utmost importance for increasing survival in breast cancer patients. Oxygen tension is variable throughout tumors, creating regions of hypoxia that have been linked with poor cancer prognosis. Hypoxia increases glycolytic flux via hypoxia-inducible factor-1α (HIF1α), and can therefore alter growth and survival of cancer cells. Our objectives are to (1) characterize changes in metabolism and survival that occur when metastatic and non-metastatic mammary cancer cell lines are cultured in hypoxia, and (2) determine whether 1,25-dihydroxyvitamin D (1,25(OH)2D) reduces overall survival in hypoxia. MethodsWe utilized Wnt oncogene-driven murine mammary cancer cells that are non-metastatic (M-Wnt) or that preferentially metastasize to the lung in vivo (metM-Wntlung). Viability of M-Wnt and metM-Wntlung cells treated with 10 nM 1,25(OH)2D and/or 20 mM 2-deoxyglucose (2DG, an inhibitor of glycolysis) was measured with MTT. Expression of HIF1α protein was determined by Western blotting. ResultsWe show that 1,25(OH)2D treatment significantly decreased viability of metastatic metM-Wntlung cells grown in hypoxia by 41%, whereas viability of M-Wnt cells was not significantly impacted by 1,25(OH)2D treatment. Furthermore, treating cells with 2DG significantly decreased viability of both cells lines in hypoxia, with metM-Wntlung cells being more sensitive to 2DG. Interestingly, 1,25(OH)2D treatment partially rescued M-Wnt cells by 22% and metM-Wntlung cells by 24% when treated with 2DG in hypoxia. Finally, we show that M-Wnt cells have 1.9-fold increased expression of HIF1α protein compared to metM-Wntlung cells when grown in hypoxia. ConclusionsOur results collectively suggest that non-metastatic M-Wnt cells are less sensitive to treatment with 1,25(OH)2D and 2DG in hypoxia than metastatic metM-Wntlungcells. These data may be explained, in part, by elevated expression of HIF1α in M-Wnt cells, which may contribute to their improved survival in hypoxia. Funding SourcesNational Institute of Health and USDA.

HAX-1 promotes the migration and invasion of hepatocellular carcinoma cells through the induction of epithelial–mesenchymal transition via the NF-κB pathway

The expression of HS-1-associated protein X-1 (HAX-1) plays a major role in the development of hepatocellular carcinoma (HCC). However, the function of HAX-1 in HCC metastasis is unclear. Quantitative real-time PCR and western blotting were used to examine HAX-1 expression in HCC cell lines with different metastatic potential, and in tumor tissues with or without intrahepatic metastasis. HCC tissue arrays (n = 144) were used to assess correlations between clinicopathological parameters and HAX-1 expression. We also examined the effect of HAX-1 on promoting HCC cell metastasis in vivo and in vitro. The results showed that the expression levels of HAX-1 were higher in metastatic HCC cell lines than in non-metastatic HCC cell lines. HAX-1 was also significantly upregulated in primary HCC tissues with intrahepatic metastasis compared with those without intrahepatic metastasis. HCC in patients with high HAX-1 expression is more likely to metastasize. HAX-1 expression was associated with malignant progression and poor prognosis, and HAX1 silencing inhibited HCC cell migration and invasion in vitro and decreased HCC cell lung metastasis in vivo, whereas HAX-1 overexpression had the inverse effect. Moreover, HAX-1 increased HCC cell metastasis by promoting the epithelial–mesenchymal transition (EMT) process. Finally, we revealed that HAX-1 modulated EMT in HCC cells by increasing NF-κB/p65 nuclear translocation. In conclusion, HAX-1 promotes HCC metastasis by EMT through activating the NF-κB pathway, suggesting that HAX-1 could be a potential therapeutic target for HCC treatment.

Analysis of the proteasome activity and the turnover of the serotonin receptor 2B (HTR2B) in human uveal melanoma

Uveal melanoma (UM), although a very rare disease, remains a particularly aggressive type of cancer as near 50% of the UM presenting patients will also develop liver metastases within 15 years from the initial diagnostic. One of the most reliable predictive markers of UM at risk of evolving toward the formation of liver lesions is an abnormally elevated level of expression of the transcript encoding the 5-Hydroxytryptamine (serotonin) receptor 2B (HTR2B). In our previous study, we demonstrated that transcription of the HTR2B gene was under the regulatory influences of two transcription factors (TFs), NFI and RUNX1. However, the action of these TFs was insufficient to explain the elevated level of the HTR2B protein in metastatic UM cells or the discrepancies we observed between its expression at the transcriptional and protein levels, therefore suggesting that additional post-translational modifications may also contribute to the altered expression of HTR2B in UM cells. In the present study, we investigated whether the turnover of HTR2B by the proteasome could account at least in part for its deregulated expression. Microarray analyses performed with UM cell lines derived from both non-metastatic and metastatic UM primary tumors revealed important alterations in the expression of some of the transcripts encoding both the E3 ubiquitin ligases and the various subunits of the proteasome, and these modifications were further exacerbated by cell passaging in culture. These alterations also correlated with significant changes in the enzymatic activity of the proteasome. However, the highest proteasome activity and amount of ubiquitinated HTR2B observed in the metastatic T142 cell line, as revealed by immunoprecipitation of ubiquitinated proteins and Western blotting using the HTR2B antibody, apparently had little impact on the total content of HTR2B protein. This contrasts with the near total disappearance of this receptor in the non-metastatic T108 cell line. Our study therefore suggests that the inability of the proteasome to degrade HTR2B in metastatic UM cells might rely on an increased stability of the ubiquitinated receptor in these cells.

Abstract PD9-10: Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Cancer associated fibroblasts (CAFs) are the majority of stroma in BC and critical players in BC malignancy. For example, CAFs are the main source of SDF-1, a prominent chemokine in the tumor microenvironment (TME) that also imparts stem cell-like characteristics to BC cells. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. Stem-like CTCs and clusters have a greater propensity to establish metastasis. We recently identified circulating CAFs (cCAFs) in blood from patients with BC and in spontaneous, syngeneic, and xenograft mouse models of BC. cCAFs not only circulate individually, but are also found in clusters with CTCs. In this study, we examine the role of CAFs in promoting egress of stem-like CTCs (cCSCs), determine the capacity of stem-like CTCs to cluster with CAFs, and evaluate the involvement of CTC/cCAF clustering in augmenting BC metastasis. Methods: Our model employs NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines: the non-metastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis, and we used BALB/c mice injected with syngeneic 4T1 or 67nR cells to evaluate cCAFs, CTCs, and cluster egress in preclinical models. Mice were sacrificed at specific time points, and cardiac blood was collected. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were stained for IF and cCAFs, CTCs, cCSCs, and clusters were enumerated. Tumors from CAF co-injected mice were evaluated for their stem cell-like phenotype and re-implanted in mice to evaluate tumorigenicity and metastasis. Results: In spontaneous, syngeneic, and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs co-clusters appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. Co-inoculation of CAFs with BC cells resulted in a significant increase in tumor progression, metastasis, and in a substantially higher number of both individual cells and clusters in circulation. Dissociated tumor cells from CAF co-injected tumors had a higher proportion of CD44+stem cell-like cells (CSCs), enhanced ALDH-1 expression, and enhanced mammosphere formation. CD44+ CSCs, individually and in clusters, are found early on in the circulation of mice injected with dissociated tumor cells from CAF co-injected tumors. Upon re-implantation of CAF co-injected dissociated tumor cells without CAFs, dissociated tumor cells showed enhanced tumorigenicity and malignancy. Conclusion: CAFs are highly motile and cCAFs precede CTCs into circulation and can do so independently of tumor cells. CAFs sustain egress of tumor cells by augmenting malignancy and stemness of BC cells. cCAF clusters with the highly metastatic stem cell-like subset of CTCs bolster metastatic colonization. Targeting primary CAF function and/or cCAF/cCSC co-clusters may provide novel avenues to abrogate BC metastasis. Citation Format: Sharma U, Miller P, Medina Saenz K, Picon-Ruiz M, Morata-Tarifa C, Spartz A, Troness B, Park DN, Seagroves TN, Slingerland JM, Lippman ME, El-Ashry D. Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis [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 PD9-10.