3D Matrigel Research Articles

Abstract B45: De novo generation of highly aggressive human breast tumors by rapid serial passaging of oncogene-transduced starting populations of purified normal cells

Abstract Human breast cancers are clonal outgrowths that have diversified genetically and biologically by the time they are clinically evident. However, early events that predicate malignant transformation of normal human mammary epithelial cells and the importance of the specific cell type to first be altered has remained elusive. We have developed an in vivo system that allows analysis of the initial steps of tumorigenesis from defined subsets of normal human mammary cells within 8 weeks using a single (KRASG12D) or multiple oncogenes. Using a protocol in which we first isolate biologically, transcriptionally and phenotypically different human mammary cell types by FACS using EpCAM and CD49f or CD10 as distinguishing markers, and then transduce the cells with lentiviral constructs, and immediately transplant them into highly immunodeficient NOD/RAG1-/-IL2rγ-/- (NRG) female mice, we have found that invasive ductal carcinomas expressing a variety of basal and luminal-associated markers (such as CK5, CK8/18, EGFR and MUC1) can be obtained at high frequency from purified starting populations of both basal cells and luminal progenitors within 8 weeks. Many tumours could be passaged both in vivo and under multiple conditions in vitro (on collagen, in suspension “mammopshere” culture or 3D matrigel cultures). After only 4passages in vivo, an aggressive tumor cell line that is tumorigenic within 6 weeks in vivo at unit efficiency has emerged. Intravenous injections shown that these cells can colonize multiple organs including the lung, kidney and spleen. Molecular characterization of these aggressive tumors has established that they are EpCAM+ and CD44+. Additional examples of de novo tumors able to be serially passaged are now being characterized. This new tumorigenesis model will allow us to compare the sequential changes that accompany and underlie the full process of malignant progression and should thus enable key molecular determinants that drive cancer initiation and progression to be identified. Citation Format: Sylvain Lefort, Davide Pellacani, Long Nguyen, Connie J. Eaves. De novo generation of highly aggressive human breast tumors by rapid serial passaging of oncogene-transduced starting populations of purified normal cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B45.

Effects of mechanical stress and vitreous samples in retinal pigment epithelial cells

In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid.We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples.In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis.

Abstract B41: Targeting androgen-independent prostate cancer through epigenetic reprogramming

Abstract Background and aim of the study: The mainstay of therapy for patients with metastatic prostate cancer, including castration resistant disease (CRPC), is hormonal therapy targeting the androgen receptor (AR). However, tumors ultimately develop treatment resistance, which can include epithelial plasticity associated with loss of AR expression, clinical aggressiveness, and pathologic features of small cell or neuroendocrine carcinoma (NEPC). We recently preformed integrative molecular analyses of metastatic tumors from a large cohort of CRPC and NEPC patients. Emerging data from this study points to a key role of the Polycomb gene EZH2 and the epigenome in driving this adaptive response mechanism. In this study we are now exploring the hypothesis that the epigenetic modifier, EZH2, can lead the adaptive response towards an androgen-independent phenotype and towards the appearance of neuroendocrine features. Results: Based on the driving role of EZH2 in other tumor types and significant overexpression in NEPC tumors (confirmed at a protein level on Tissue Microarray), we evaluated the effects of the EZH2 inhibitor, GSK343, in NEPC cells (NCI-H660) and prostate adenocarcinoma cells (LNCaP and DU145) in 3D Matrigel cultures. GSK343 effectively inhibited H3K27me3 and resulted in a significant reduction of NCI-H660 viability, measured with an ATP-based assay, whereas LNCaP and DU145 cells were minimally affected after 7 or 14 days of treatment. Using a nanosting assay we also demonstrated a significant increase in the expression of AR signaling genes (such as PSA, PSMA) and decrease in NEPC-associated genes (chromogranin A, AURKA, ENO2) when neuroendocrine cells are treated with EZH2 inhibitor. These data suggest a modulation of the neuroendocrine phenotype via EZH2. We therefore extended these drug studies including patient-derived organoid models including both CRPC and NEPC organoids, and we observed similar results with preferential sensitivity of the AR-negative NEPC organoids to the AR-positive CRPC organoid, with similar reversion of downstream AR/NEPC gene expression. Conclusions: There is no approved drug that can specifically target AR- independent NEPC tumors. The restricted set of therapeutic options against this subtype of prostate cancer and consequent dismal outcome stem in part from our incomplete understanding of the molecular events underlying its pathogenesis. The discovery that epigenetics can be a key process in distinguishing tumors that are AR-independent and at high risk for NEPC progression represents an important step to highlight the use of epigenetic modifiers as therapeutic agents for this subtype of prostate cancer. Citation Format: Loredana Puca, Dong Gao, Myriam Kossai, Joanna Cyrta, Clarisse Marotz, Juan Miguel Mosquera, Theresa Y. MacDonald, Andrea Sboner, Rema Rao, Yu Chen, Mark A. Rubin, Himisha Beltran. Targeting androgen-independent prostate cancer through epigenetic reprogramming. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B41.

Three-dimensional Mammary Epithelial Cell Morphogenesis Model for Analysis of TGFß Signaling.

Culturing mammary epithelial cells in laminin-rich extracellular matrices (three dimensional or 3D culture) offers significant advantages over that in the conventional two-dimensional (2D) tissue culture system in that it takes into considetation the impact of extracellular matrix (ECM) microenvironment on the proliferation, survival, and differentiation of mammary epithelial cells. When grown in the 3D culture, untransformed mammary epithelial cells undergo morphogenesis to form a multicellular and polarized acini-like structure that functionally mimics the differentiated alveoli in the pregnancy mammary gland. This process is subjected to regulation by many growth factors and cytokines. The transforming growth factor-ß (TGFß) is a multipotent cytokine that regulates multiple aspects of development and tumorigenesis. In addition to its effects on epithelial cell proliferation, survival, and differentiation, it is also a potent regulator of the cell-matrix interaction. Thus, the 3D culture model may recapitulate the complex in vivo epithelial cell microenvironment and allow us to fully evaluate the role of TGFß signaling in multiple aspects of normal and cancerous cell behavior. In this chapter we provide detailed protocols for growing mammary epithelial cells in the 3D Matrigel for analysis of signaling pathways.

Abstract A22: Identification of novel tumor derived factors that inhibit angiogenesis

Abstract Background: Cell-cell communication in the tumor micro-environment drive tumor growth and facilitate neovessel recruitment from the adjacent vasculature. The present study was designed to identify novel cancer derived factors that modulate the vascular endothelial response in a paracrine manner. Methods: An array of cancer cell lines from multiple tissue types (Panc1; HepG2; LnCap; Skmel; Hep3B; PL45; SCC25; CaCo2; PaCa2; SKBR3) were grown under normoxic or hypoxic (1% oxygen) conditions for a total of 20 different conditions. After 48 hours, conditioned media was collected and filtered prior to testing on endothelial cells. The effects of the conditioned media on endothelial apoptosis, proliferation and tube formation were investigated. In tandem with these endothelial cell fate assays, the proteomic profile of these 20 conditions was profiled and assessed using the Berg Interrogative Biology™ platform. Results & Discussion: Our results reveal that conditioned media from all cancer cells tested can alter endothelial cell apoptosis, proliferation rate and tube formation in 3D matrigel assays i.e. critical cell fate decisions that enable formation of nascent vessel formation. As predicted, the predominant effect of tumor-derived conditioned media on endothelial apoptosis was a protective anti-apoptotic effect, with the discernable exception of normoxic Panc1 derived media. An unexpected finding was the general anti-proliferative effects of tumor-derived conditioned media on endothelial cell growth, with the exception of Hep2G, LnCap and PaCa2 cell derived media. Using a media fractionation strategy and Interrogative Biology™, we identified several endothelial anti-apoptotic candidates from Panc1 cells within a unique molecular weight range. Upon application of ASO gene knockdown in Panc1 cells, these targets were verified for apoptotic and proliferation effects on vascular endothelial cells. Conclusion: Specific factors that determine paracrine cell-cell interaction in the tumor microenvironment have been identified and validated employing Berg Interrogative Biology™. The technology presented herein represents a viable platform for identifying clinically relevant biomarkers and factors that alter cancer angiogenesis. Citation Format: Tony E. Walshe, Justin Bourdelais, Arleide Lee, Rakibou Ouro-Djobo, Vivek Vishnudas, Rangaprasad Sarangarajan, Niven Narain. Identification of novel tumor derived factors that inhibit angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A22.

New ex-ovo colorectal-cancer models from different SdFFF-sorted tumor-initiating cells.

Despite effective treatments, relapse of colorectal cancer (CRC) is frequent, in part caused by the existence of tumor-initiating cells (TICs). Different subtypes of TICs, quiescent and activated, coexist in tumors, defining the tumor aggressiveness and therapeutic response. These subtypes have been sorted by hyperlayer sedimentation field-flow fractionation (SdFFF) from WiDr and HCT116 cell lines. On the basis of a new strategy, including TIC SdFFF sorting, 3D Matrigel amplification, and grafting of corresponding TIC colonies on the chick chorioallantoic membrane (CAM), specific tumor matrices could be obtained. If tumors had similar architectural structure with vascularization by the host system, they had different proliferative indices in agreement with their initial quiescent or activated state. Protein analysis also revealed that tumors obtained from a population enriched for "activated" TICs lost "stemness" properties and became invasive. In contrast, tumors obtained from a population enriched for "quiescent" TICs kept their stemness properties and seemed to be less proliferative and invasive. Then, it was possible to produce different kinds of tumor which could be used as selective supports to study carcinogenesis and therapy sensitivity.

The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity.

BackgroundDeleted in Liver Cancer 1 (Dlc1) is a tumor suppressor gene, which maps to human chromosome 8p21-22 and is found frequently deleted in many cancers including breast cancer. The promoter of the remaining allele is often found methylated. The Dlc1 gene encodes a RhoGAP protein that regulates cell proliferation, migration and inhibits cell growth and invasion when restored in Dlc1 deficient tumor cell lines. This study focuses on determining the role of Dlc1 in normal mammary gland development and epithelial cell polarity in a Dlc1 gene trapped (gt) mouse.MethodsMammary gland whole mount preparations from 10-week virgin heterozygous Dlc1gt/+ gene-trapped mice were compared with age-matched wild type (WT) controls. Hematoxylin-Eosin (H&E) and Masson’s Trichrome staining of histological sections were carried out. Mammary glands from Dlc1gt/+ mice and WT controls were enzymatically digested with collagenase and dispase and then cultured overnight to deplete hematopoietic and endothelial cells. The single cell suspensions were then cultured in Matrigel for 12 days. To knockdown Dlc1 expression, primary WT mammary epithelial cells were infected with short hairpin (sh) RNA expressing lentivirus or with a scrambled shRNA control.ResultsDlc1gt/+ mice showed anomalies in the mammary gland that included increased ductal branching and deformities in terminal end buds and branch points. Compared to the WT controls, Masson’s Trichrome staining showed a thickened stromal layer with increased collagen deposition in mammary glands from Dlc1gt/+ mice. Dlc1gt/+ primary mammary epithelial cells formed increased solid acinar spheres in contrast with WT and scrambled shRNA control cells, which mostly formed hollow acinar structures when plated in 3D Matrigel cultures. These solid acinar structures were similar to the acinar structures formed when Dlc1 gene expression was knocked down in WT mammary cells by shRNA lentiviral transduction. The solid acinar structures were not due to a defect in apoptosis as determined by a lack of detectible cleaved caspase 3 antibody staining. Primary mammary cells from Dlc1gt/+ mice showed increased RhoA activity compared with WT cells.ConclusionsThe results illustrate that decreased Dlc1 expression can disrupt the normal cell polarization and mammary ductal branching. Altogether this study suggests that Dlc1 plays a role in maintaining normal mammary epithelial cell polarity and that Dlc1 is haploinsufficient.

Changes in microRNAs associated with Twist-1 and Bcl-2 overexpression identify signaling pathways

Epithelial–mesenchymal transition (EMT) is regulated by multiple signal transduction pathways. Twist-1 is one of the most important transcription factors in these pathways. In a previous study, we found that Bcl-2 enhanced the role of Twist-1 in EMT. Coexpression of Twist-1 and Bcl-2 may play an import role in vasculogenic mimicry (VM) through regulation of EMT. Moreover, regulators of EMT and VM are known to be important targets for microRNAs (miRNAs). To better understand how these critical pathways are induced by coexpression of Twist-1 and Bcl-2, we performed a comprehensive comparative bioinformatics analysis using microarrays on HCCs that overexpressed Twist-1 and Bcl-2. Eleven miRNAs associated with coexpression of Twist-1 and Bcl-2 were selected from the comprehensive analysis of miRNA microarray and ChIP-seq analysis. Changes in miRNAs were associated with significant differences in the expression of genes involved in signal transduction pathways related to processes including tumor invasion, metastasis, angiogenesis, and tumor cell shape. We confirmed the role of Twist-1 and Bcl-2 coexpression in HCC cells using wound healing assays, invasion assays, and 3D Matrigel assays. Furthermore, the role of miR-27a as a crucial regulator of EMT and VM was confirmed in HCC cells by RT-PCR and western blot analysis. These findings provide evidence that Bcl-2 enhances the role of Twist-1 in VM and EMT through miRNAs.

Abstract 4941: Profilin-1 is a key determinant for tumorigenic potential of breast cancer cells

Abstract A hallmark of oncogenic transformation is disruption of the actin cytoskeleton, which is partly attributed to altered expression and/or activity of proteins that bind to and regulate the state of polymerization of actin in cells. Profilin-1 (Pfn1) is a key regulator of actin polymerization that is downregulated in human breast cancer, and reduced Pfn1 levels were shown to facilitate dissemination-promoting activities of certain types of breast cancer cells. The objective of the present study was to investigate whether Pfn1 modulation affects tumor initiating capacity of breast cancer cells. Using a MMTV-PyMT transgenic mouse model, we demonstrated that mice engineered for conditional Cre-mediated homozygous deletion of Pfn1 (Pfn1−/−) in the mammary gland develop fewer spontaneous mammary tumors and exhibit longer survival than either wild-type or heterozygous Pfn1-knockout littermates. Haplo-insufficiency of Pfn1 (Pfn1+/−) causes the expected ∼50% downregulation of Pfn1 expression in tumors; however, mammary tumors isolated from Pfn1−/− mice exhibit residual Pfn1 expression that is at least comparable to the level found in tumors derived from Pfn1+/− mice. This scenario is consistent with a competitive disadvantage of Pfn1-null tumor cells for autonomous growth and/or survival, allowing selective outgrowth of tumor cell populations that have incomplete Cre-mediated gene excision of Pfn1, likely resulting from mosaic activation of Cre-driving MMTV-promoter in mammary gland. Acute deletion of floxed-Pfn1 alleles in normal mouse mammary epithelial cells by Cre-adenovirus transduction dramatically impairs their ability to outgrow when seeded sparsely on 3D matrigel. Likewise, human breast cancer cells exhibit retarded outgrowth on 3D matrigel upon stable silencing of Pfn1 expression even though their proliferation on 2D tissue-culture substrate was not affected. Correlated with these phenotypic changes, Pfn1 knockdown suppresses stem-cell-like behavior of breast cancer cells as marked by a significant reduction in both aldehyde dehydrogenase activity and mammosphere forming ability. Collectively, these findings suggest that a minimum level of Pfn1 is essential for tumor initiation of breast cancer cells. Interestingly, the outgrowth-deficient phenotype of Pfn1-depleted breast cancer cells on 3D-matrigel can be fully rescued by addition of collagen-I which suggests that alteration in cell-matrix adhesion and downstream signaling underlies the growth-related phenotypic changes induced by loss of Pfn1. Finally, the outgrowth-deficient phenotype of breast cancer cells can also be elicited in vitro and in vivo by Pfn1 overexpression, further suggesting that an optimum range of Pfn1 expression is conducive for tumor-initiating capacity of breast cancer cells. Based on these results, we conclude that Pfn1 expression level is a major determinant for tumorigenic potential of breast cancer cells. Citation Format: Chang Jiang, Zhijie Ding, Marion Joy, Laura Vollmer, Dave Gau, Su Hyeong Kim, Shivendra Singh, Andreas Vogt, Partha Roy. Profilin-1 is a key determinant for tumorigenic potential of breast cancer cells. [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 4941. doi:10.1158/1538-7445.AM2015-4941

Abstract 1989: Targeting Sin3-Pf1 complex: Novel site-specific epigenetic therapy for triple negative breast cancer

Abstract Introduction: Dearth of clinically validated drug targets is a major challenge in Triple Negative Breast Cancer (TNBC) that limits the treatment options to chemotherapy with intense cytotoxic consequences. We previously identified PAH2 domain of Sin3 protein as potential therapeutic target in TNBC and shown that protein-protein interactions of Sin3 via its PAH-2 domain can be disrupted by decoys (Mad1-SID peptide/small molecule inhibitors) designed based on the Sin3 interaction domain (SID) of transcription factor Mad1. Chromatin regulator Pf1 shown to be overexpressed in breast cancer and interacting with PAH2 domain of Sin3 can also be dissociated from Sin3 complex by Mad1-SID. In this study we identify Pf1 to be a significant contributor to the oncogenic phenotype associated with TNBC. Methods: Pf1 knockdown lines were generated by stably transfecting MDA-MB-231 cells with Pf1-shRNA or scr-shRNA. Cells were cultured in 2D and assayed for cancer stem cell (CSC) markers and ALDH activity by flow cytometry. For colony morphogenesis cells were cultured in 3D Matrigel. qRT-PCR were performed to assay expression of Nanog, Sox2 and Oct4. Fluorescence anisotropy and co-immunoprecipitation assays were used to test Sin3-Mad1 and Sin3-Pf1 interaction in the presence or absence of Mad1-SID and/or Pf1-SID. Results: MDA-MB-231 cells transfected with Pf1-shRNA had over two fold reduced ability to form colonies in 3D Matrigel cultures, with ∼95% of colonies non-invasive in contrast to the invasive star-like colonies formed by cells transfected with Scr-shRNA. Additionally reduction in Pf1 level was accompanied by a ∼1.5-fold reduction (p<0.05) in the tumorsphere forming ability of MDA-MB-231 cells. Pf1 depletion also significantly reduced RNA and protein of Nanog, Oct4 and Sox2. Confocal imaging revealed reduced levels and nuclear accumulation of Nanog, Sox2 and Oct4 in cells transfected with Pf1-shRNA compared to Scr-shRNA. Pf1 knockdown resulted in a 2.5-fold decrease in ALDH1 positive cells (6.55% in Scr-shRNA vs 2.59% in Pf1-shRNA). The CD44low/CD24low/neg population was enriched 3 fold over cells transfected with Scr-shRNA. From a therapeutic perspective we have designed a linear peptide corresponding to the Sin3 interaction domain of Pf1 (Pf1-SID). By fluorescence anisotropy we show that in comparison to Mad1-SID (IC50 = 1.4±0.3 μM), several hundred times higher concentration of Pf1-peptide (IC50 = 826±162 μM) is required to dissociate Sin3-Mad1 interaction. Co-immunoprecipitation assays show that Pf1-SID can specifically dissociate Pf1 from Sin3 without affecting the binding of Mad1 with PAH2 domain of Sin3. Pf1-SID treated MDA-MB-231 cells also have reduced invasive capacity and CSC traits. Conclusion: PAH2 domain of Sin3 and its interaction with Pf1 is potential drug target and Pf-1SID-mediated disruption of Sin3-Pf1 complex as translation relevance for first site-specific epigenetic therapy for TNBC. Citation Format: Nidhi Bansal, Joanna Wexler, Yeon-jin Kwon, Elena C. Gil, Boris Leibovitch, Rajal Sharma, Arthur Zelent, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. Targeting Sin3-Pf1 complex: Novel site-specific epigenetic therapy for triple negative 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 1989. doi:10.1158/1538-7445.AM2015-1989

Abstract 1975: Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion

Abstract ErbB2 overexpression gives rise to a highly invasive, aggressive phenotype associated with an increased activity of lysosomal cysteine cathepsins B and L and with a pericellular accumulation of lysosomes at the close vicinity of the cell membrane, enabling lysosomal exocytosis and secretion of lysosomal contents to the extracellular space. We recently showed that ErbB2 expression level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. We found that the increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and that it is mediated by a novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, Myeloid Zinc Finger 1 (MZF1) and ETS1, which regulate the ErbB2-induced expression of cathepsins B and L. We currently work on understanding the mechanisms that increase the transcriptional activity of MZF1 in breast cancer. Thus far we have identified two basic mechanisms that can increase MZF1 target gene expression: cancer-induced, increased levels of MZF1 and ErbB2 signaling-induced, increased transcriptional activity of MZF1. Our results thus far show that the level of MZF1 can be directly downregulated by specific, non-coding RNAs. The cancer-induced increase in the MZF1 level is most likely due to changes in the expression of these non-coding RNAs. Preliminary data employing two-dimensional SDS-PAGE as well as the factual involvement of several kinases downstream of ErbB2 for its transcriptional activity, suggests that MZF1, a prominent phosphoprotein, is activated by phosphorylation in response to ErbB2 signaling. Towards this end we have set up various experiments to verify the phosphorylation of MZF1 in response to ErbB2 signaling. We now aim at the identification of the ErbB2-regulated phosphorylation sites. With this work we expect to collect more information about the molecular mechanisms underlying ErbB2-induced invasion. Increased knowledge of the mechanisms is expected to enable the development of novel therapies and biomarkers for aggressive, ErbB2-positive breast cancer. Citation Format: Tuula Kallunki, Ditte M. Brix, Knut Kristoffer B. Clemmensen, Siri A. Tvingsholm, Bo Rafn, José Moreira, Irina Gromova, Marja Jäättelä. Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion. [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 1975. doi:10.1158/1538-7445.AM2015-1975

Abstract 3844: Targeting EZH2 in neuroendocrine prostate cancer

Abstract Background and aim of the study EZH2 is the catalytic subunit of the Polycomb repressive complex 2 (PRC2) and it is involved in controlling gene expression through trimethylation of histone H3 lysine 27 (H3K27me3), a key mechanism that regulates chromatin structure and gene silencing. EZH2 is overexpressed in a wide range of cancers including prostate. Recent findings show that EZH2 can form a complex with the androgen receptor (AR), enhancing its transcriptional activity. However the role of EZH2 in AR low/negative prostate cancer has not been previously investigated. Long-term exposure to AR targeted therapies can cause an adaptive response mechanism in a subset of advanced prostate cancers. Often this adaptation is associated with loss of AR expression, clinical aggressiveness, and pathologic features of small cell or neuroendocrine carcinoma. This AR independent resistant phenotype is broadly termed neuroendocrine prostate cancer (NEPC). Initial studies from our lab have highlighted a landscape of genomic alterations that characterize NEPC. In this study we explore the hypothesis that the epigenetic modifier, EZH2, can lead the adaptive response towards an androgen-independent phenotype and towards NEPC. Results Using Next Generation RNA-sequencing, we profiled tumors from a cohort of patients including 20 NEPC, 68 Prostate Adenocarcinoma (PCa), and 19 castration resistant prostate cancer (CRPC) and we discovered that EZH2 is highly expressed in NEPC (characterized by low or absent AR and morphologic changes). These findings were also confirmed at protein level by immunohistochemistry. Based on the driving role of EZH2 in other tumor types and significant overexpression in NEPC tumors, we evaluated the effects of the EZH2 inhibitor, GSK343, in NEPC cells (NCI-H660) and PCa cells (LNCaP and DU145) in 3D Matrigel cultures. GSK343 effectively inhibited H3K27me3 and resulted in a significant reduction of NCI-H660 viability, measured with an ATP-based assay, whereas LNCaP and DU145 cells were minimally affected after 7 or 14 days of treatment. Using a custom designed Nanostring assay, we also demonstrated a significant increase in the expression of AR signaling genes (e.g. PSA, PSMA) and decrease in NEPC-associated genes (e.g. chromogranin A, AURKA, ENO2) when NEPC cells were treated with GSK343. These data suggest a modulation of the neuroendocrine phenotype via EZH2. We therefore extended these drug studies to patient-derived organoid models including both CRPC and NEPC organoids and we observed similar results with preferential sensitivity of the AR-negative NEPC organoids compared to AR-positive CRPC organoids and similar reversion of downstream AR/NEPC gene expression. Conclusions Altogether, these data suggest that EZH2-mediated epigenetic changes contribute to the AR-independent NEPC resistant phenotype, and this effect can be reverted or delayed by targeting EZH2 activity. EZH2 represents a promising drug target and a potential modulator of the NEPC phenotype. Citation Format: Loredana Puca, Dong Gao, Myriam Kossai, Clarisse Marotz, Juan Miguel Mosquera, Theresa Y. MacDonald, Kyung Park, Rema Rao, Andrea Sboner, Yu Chen, Mark A. Rubin, Himisha Beltran. Targeting EZH2 in neuroendocrine prostate 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 3844. doi:10.1158/1538-7445.AM2015-3844

Abstract 1526: HOXA5 inhibits cell plasticity and stemness in breast cancer cells by reinforcing epithelial traits

Abstract Members of the Retinoic acid receptor (RAR) and HOX gene families are known to be associated with cellular differentiation. Nonetheless, how HOX genes drive differentiation in mammalian cells is poorly understood. HOX genes are transcription factors that orchestrate body segmentation during embryogenesis. But the continued expression of several members in adulthood indicates the necessity of their biological functions in specifying cell fate in adulthood. The mammary gland is an organ that continues to go through multiple cycles of development during life. Here, loss of specific HOX genes could be detrimental to normal development. We have previously shown that expression of HOXA5, a downstream target of the retinoic acid signaling pathway, is often lost in breast cancer. Loss of HOXA5 expression correlates with increasing pathological grades of breast cancer. Expression array analysis of HOXA5-depleted MCF10A (immortalized line of human breast epithelium) revealed its potential role in regulating several traits of the epithelial lineage, including expression of E-cadherin and CD24. Depleting HOXA5 in MCF10A enhanced cell motility, invasiveness and increased stem-cell population (CD24lo/CD44hi). Using an inducible HOXA5-depletion system, differentiation of the stem cells in MCF10A was shown to be mediated by HOXA5. Conversely, ectopic expression of HOXA5 in SUM149 breast cancer cells significantly reduced plasticity and stem cell population, confirmed by in vivo limiting dilution assays in mice. MCF10A-KRAS-HOXA5 depleted cells formed invasive structures in 3D matrigel cultures, and formed larger tumors, implanted orthotopically in mice. MCF10A-KRAS-tumors were histologically dedifferentiated and lost expression of luminal cell markers such as E-cadherin and cytokeratin 18. Collectively, our findings demonstrate the role of HOXA5 in retinoid-induced cell differentiation by regulating cell fate specific surface adhesion molecules. Citation Format: Wei Wen Teo, Vanessa Merino, Sean Cho, Preethi Korangath, Neil M. Neumann, Andrew J. Ewald, Saraswati Sukumar. HOXA5 inhibits cell plasticity and stemness in breast cancer cells by reinforcing epithelial traits. [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 1526. doi:10.1158/1538-7445.AM2015-1526

Abstract 2143: A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation

Abstract The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell surface of polarized MDCK cells. Previously, we showed that EREG basolateral trafficking is regulated by a conserved tyrosine residue within a YXXΦ motif (Y156ERV) in its cytoplasmic domain. Interestingly, a Y156A substitution led to apical mistrafficking of EREG and transformation of polarized MDCK cells (PNAS 110: 8960-5, 2013). We have identified an EREG mutation (R147stop) in human tumors that we now report mistrafficks to the apical surface. To test the hypothesis that apical mistrafficking of EREG can be a driver event, we have generated MDCK cells stably expressing inducible (Tet-ON) wild-type and Y156A mutant EREG. In 3D Matrigel cultures, we show that uninduced clones form normal-appearing cysts, regardless of EREG status. Induction of WT or mutant EREG leads to increased cyst diameter. However, only (Y156A)EREG-expressing cells form abnormal cysts with ectopic lumens and inward growth, features of in vivo transformation. Furthermore, we show that ligand processing and EGFR activity is required for EREG mistrafficking-induced ectopic lumen formation as preincubation with a broad-spectrum metalloprotease inhibitor, BB-94, abrogates ectopic lumen formation. We have extended these studies to polarizing human colon cancer cells, HCA-7, which preserve the trafficking fidelity of WT and mutant EREG. Furthermore, using EREG cytoplasmic domain as a bait in a yeast-2-hybrid screen, we have identified APOBEC3C as an interacting protein. APOBECs are a family of cytidine deaminases that have been identified as a major contributor to human cancer mutations. We have subsequently confirmed EREG-APOBEC3C interaction by co-immunoprecipitation. Results from MDCK cells stably expressing differentially inducible EREG (Tet-ON) and APOBEC3C (Cumate-ON) will be presented. Note: This abstract was not presented at the meeting. Citation Format: Bhuminder Singh, Galina Bogatcheva, Robert J. Coffey. A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation. [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 2143. doi:10.1158/1538-7445.AM2015-2143

Abstract 472: Galectin-3 modulates glioblastomas (GBM) tumor microenvironment, and is linked to glioblastoma invasion

Abstract Malignant glioblastomas (GBM) are lethal form of brain tumors, and are difficult to treat due to their invasion into the brain parenchyma. Aggressive and invasive nature of GBM is at least partly attributable to GBM associated stem cells (GSC). Soluble factors released by GBM and GSC can recruit inflammatory cells, including brain resident microglia to tumor micro-environment (TME) that support angiogenesis and invasion. We have previously shown that exogenous Galectin-3 (Gal-3), a carbohydrate binding lectin increases the migratory potential of BV2 microglial cells, and promotes the formation of pro-angiogenic structures in an in vitro HUVEC culture and 3D matrigel model. Furthermore, Gal-3 supports the survival of microglial cells under hypoxic environment. In this study, we investigated the role of Gal-3 in GBM by utilizing the biochemical and molecular approaches. Using well characterized GBM tumor cell lines, and GSC lines that exhibit various degrees of invasive potential, we have identified that the expression of the Gal-3 is significantly up-regulated in GSCs that are highly invasive as compared to those that form only focal tumors in a mouse xenotransplantation model. Gal-3 is also highly expressed in microglia that accumulated in and around the GSC tumors in mouse brain, as indicated by co-localization of Gal-3 and Iba1, the maker for activated microglia. Furthermore, Gal-3 expression is significantly increased in GBM tumor cells when exposed to in vitro hypoxic conditions. Importantly, Gal-3 increased the migratory potential of GBM tumor cells in vitro, indicating its role as a chemo-attractant. Taken together, these results suggest that Gal-3 plays a critical role in promoting GBM invasion through modulating the interaction of microglia in TME and GBM. Thus Gal-3 may be a potential therapeutic target for GBM. Citation Format: Umadevi V. Wesley, Esat Resad, Paul Clark, Carolina Larrain, John Kuo, Robert J. Dempsey. Galectin-3 modulates glioblastomas (GBM) tumor microenvironment, and is linked to glioblastoma invasion. [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 472. doi:10.1158/1538-7445.AM2015-472

Abstract 2237: Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells

Abstract Retinoids exert different effects on cell differentiation and malignant phenotype reversion through the modulation of Retinoid Acid Receptors (RAR). In this work we have analyzed the involvement of retinoid system induction on proliferation, self-renewal and differentiation of cancer stem cells (CSC) using the triple negative mammary cell line LM38-LP. In order to obtain a CSC enriched culture, cells where cultured under mammosphere conditions. We performed mammosphere cultures in order to enrich in CSC. The treatment with All-trans retinoid acid (ATRA1μM for 96 h) reduced CSC growth rate evaluated as mammosphere diameter (Control: 176±8 μm vs. ATRA: 129±10 μm; p≤0.05). Surprisingly, ATRA pre-treatment for 96 h increased CSC self-renewal evaluated as the number of secondary mammospheres (Control: 313±19; ATRA: 495±21; p≤0.05) and as the clonogenic capacity of LM38-LP CSC (Control: 6±0.5; ATRA: 11±1.2; p≤0.05). Concomitant with these biological effects, the same treatment showed, by RT-PCR, an increase in RARβ and RARγ levels in LM38-LP- CSC. Through the use of specific RAR antagonists, we found that RARγ inhibition impaired ATRA effects on self-renewal but RARβ inhibition increased the number of secondary mammospheres (Control: 382±27; ATRA: 735±11; ATRA/RARγ antagonist: 352±17; ATRA/RARβ Antagonist: 1260±177; p≤0.05). A similar result was observed when clonogenic capacity in RARγ blocked CSC was analyzed. Finally, we performed a matrigel 3D culture using cells from LM38-LP mammospheres. Under these conditions, colonies presented large irregular structures while ATRA treatment led to the formation of differentiated colonies with evidence of lumen. Our findings suggest that RARγ down modulation and RARβ induction could lead to CSC self-renewal inhibition and differentiation in triple negative mammary cancers. Citation Format: Damian E. Berardi, Carolina Flumian, Maria I. Diaz Bessone, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells. [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 2237. doi:10.1158/1538-7445.AM2015-2237

Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell–cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

Small Molecule Inhibitors of Plasminogen Activator Inhibitor-1 Elicit Anti-Tumorigenic and Anti-Angiogenic Activity.

Numerous studies have shown a paradoxical positive correlation between elevated levels of plasminogen activator inhibitior-1 (PAI-1) in tumors and blood of cancer patients with poor clinical outcome, suggesting that PAI-1 could be a therapeutic target. Here we tested two orally bioavailable small molecule inhibitors of PAI-1 (TM5275 and TM5441) for their efficacy in pre-clinical models of cancer. We demonstrated that these inhibitors decreased cell viability in several human cancer cell lines with an IC50 in the 9.7 to 60.3 μM range and induced intrinsic apoptosis at concentrations of 50 μM. In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant. Pharmacokinetics studies indicated an average peak plasma concentration of 11.4 μM one hour after oral administration and undetectable levels 23 hours after administration. The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis. These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer.

Myoepithelial and luminal breast cancer cells exhibit different responses to all-trans retinoic acid.

Breast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model. The bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo. We found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA. Our results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts.

The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative roleon CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions. • Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB. • H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB. • CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB. • S1P1/3 were involved on CM-induced morphogenesis and migration of MAB. • Matrigel plug assay showed the role of S1P axis in MAB-endothelial cell interaction.