Anna Sanchez Calle

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.

Synthesis, Biological Evaluation, Docking and QSAR Studies of SomeNovel Naphthalimide Dithiocarbamate Analogs as Antitumor and Anti-Inflammatory Agents

A series of novel naphthalimide dithiocarbamate 4a-f, 5a-f were efficiently synthesized via introduce dithiocarbamate and dithioate side chain onto the naphthalic anhydride core. The structures of the synthesized analogs were elucidated by spectroscopic methods, including IR,1H and 13C NMR, and (ESIHRMS) techniques. The anti-cancer activities of the generated naphthalimide derivatives 4c, 4d, 4e, 4f, and 5d were evaluated against 21 tumour cell lines; inculding 10 tumor subpanels using MTT assay. Analogue 4c offer antitumor activity with an IC50 of 10.54 μM against SKBR3 breast cancer cells. Compound 4d showed varying degrees of antitumor activities towards several tumour cell lines ranging from 21.1 to 71.7 μM. In addition to the antitumor activities; the synthesized compounds were evaluated for their In vitro anti-inflammatory activity. Compounds 4c and 4d revealed potent anti-inflammatory properties in comparison with the reference drug celecoxib. Molecular docking studies provided complementary theoretical support for experimental biological data.

Abstract LB-278: Cancer stem cells as the novel origin of cancer-associated fibroblast-like cells

Cancer associated fibroblast (CAF) is an indispensible component of stromal microenvironment mediating cancer progression notably in breast, prostate and pancreatic carcinoma. CAF heterogeneity is attributed to its possible multiple origin majorly from tumor resident fibroblast, mesenchymal stem cells, epithelial cells. The present work analyses the possibility of cancer stem cells (CSC) as the source of CAF like cells. For this, we have used the model of CSC established in our laboratory derived from mouse induced pluripotent cells (miPSCs) by culturing with conditioned medium from cancer cell lines mimicking tumor microenvironment. In the present study, human breast cancer derived T47D and BT549 cells have been cultured to collect the conditioned medium. Since the green fluorescent protein (GFP) gene was integrated under the control of Nanog promoter in the miPSCs used in this study, the resulting CSCs was assessed by the expression of GFP for the undifferentiated state. The differentiation of CSCs was tracked by the attenuation of GFP. The CSC population was multiplied by serial subcutaneous and orthotropic transplantation in nude mice. This population was further enriched by sphere formation. Then these spheres were allowed to differentiate into adhesive cells. The CAF-like cells were separated from the differentiated population. The expression of prominent CAF markers fibroblast specific protein (FSP), fibroblast activation protein (FAP), vimentin, collagen type1 α1, and smooth muscle actin (SMA) were assessed by real time PCR. Immuno-cytochemistry of the cells for the expression of FSP, FAP and SMA was further confirmed for the evidence that cancer stem cells should be a possible source of CAF. This model should elaborate the functional heterogeneity amongst CAFs and help design therapeutic interventions to inhibit such conversions. Citation Format: Neha Nair, Arun Vaidyanath, Kenta Hoshikawa, Anna Sanchez Calle, Tomonari Kasai, Masaharu Seno. Cancer stem cells as the novel origin of cancer-associated fibroblast-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-278.

Abstract LB-144: Derivation of a model of cancer stem cell from human induced pluripotent stem cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The existence of cancer stem cell (CSC) has been considered as one of the important reason as to why patients have a poor prognosis. However, heterotopic transplantation of embryonic stem cells and induced pluripotent stem cells has been shown to form teratoma, but not malignant teratoma. Since the microenvironment niche is playing a significant role for the proper differentiation of stem cells, the cancerous niche should drive stem cells into malignant cells in vivo. According to this hypothesis, we tried to generate cancer cells from human induced pluripotent stem (hiPS) cells. For the conversion into CSC, the conditioned medium from different human cancer cell lines was collected from confluent dishes and filtered using 0.22 micrometer filter. Then, hiPS cells, without MEF feeder cells, were maintained in the conditioned medium (CM) in the ratio of 1:1. The medium was changed every day with CM for 4 weeks. hiPS cells with the complete medium were used as control. For transplantation studies, 10^4 cells were suspended in HBSS and were xenotransplantated into NOD-SCID mice. After 3 months, tumors were excised and fixed in 10% neutral formalin buffer solution, or subjected to primary culture. The converted cells and primary cultured cells formed spheroids in suspension culture, and had tumorigenicity in vivo. The stemness of living cells was checked under fluorescent microscopy observation with rBC2LCN-FITC staining. The RNAs were extracted from converted cells and microarray analysis was perfomed. The RNA expression patterns of cell lines were visualized by sphered self-organizing map (sSOM) analysis. The sSOM analysis perfomed based upon various parameters shows the converted CSCs can be characterized into various cell types. Utilizing this method, we successfully established two different hiPS-CSC lines using CM from A172 and RERF-LC-KJ. The comprehensive understanding of cancer could be realized as the heterogeneity of cancer tissues is clarified and their component cells are identified. This study will lead to the development of the true personalized therapy of cancer in the future. Citation Format: Tomonari Kasai, Kenta Hoshikawa, Shuto Takejiri, Masashi Ikeda, Kazuki Kumon, Anna Sanchez Calle, Arun Vaidyanath, Akifumi Mizutani, Chen Ling, Masaharu Seno. Derivation of a model of cancer stem cell from human induced pluripotent 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 LB-144. doi:10.1158/1538-7445.AM2015-LB-144