Author Manuscript Published OnlineFirst on May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Analysis of the Transcriptome: Regulation of Cancer Stemness in Breast Ductal Carcinoma In Situ by Vitamin D Compounds Naing Lin Shan1, Audrey Minden1,5, Philip Furmanski1,5, Min Ji Bak1, Li Cai2,5, Roman Wernyj1, Davit Sargsyan3, David Cheng3, Renyi Wu3, Hsiao-Chen D. Kuo3, Shanyi N. Li3, Mingzhu Fang4, Hubert Maehr1, Ah-Ng Kong3,5, Nanjoo Suh1,5 1Department of Chemical Biology, Ernest Mario School of Pharmacy; 2Department of Biomedical Engineering, School of Engineering; 3Department of Pharmaceutics, Ernest Mario School of Pharmacy; 4Environmental and Occupational Health Sciences Institute and School of Public Health, 5Rutgers Cancer Institute of New Jersey, New Brunswick; Rutgers, The State University of New Jersey, NJ, USA Running title: Regulation of cancer stemness by vitamin D compounds Key words: Breast cancer, cancer stemness, gene expression, DCIS, vitamin D compounds Financial Support: This research was supported by the National Institutes of Health grant R01 AT007036, R01 AT009152, ES005022, Charles and Johanna Busch Memorial Fund at Rutgers University and the New Jersey Health Foundation. Corresponding author: Dr. Nanjoo Suh, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, New Jersey 08854. Tel: 848-445-8030, Fax: 732-445-0687; e-mail: [email protected] Disclosure of Conflict of Interest: “The authors declare no potential conflicts of interest” 1 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Article Type: Research Article Word count: 5,286 Total number of figures and tables: Figures-4 and Tables-2 2 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Ductal carcinoma in situ (DCIS), which accounts for one out of every five new breast cancer diagnoses, will progress to potentially lethal invasive ductal carcinoma (IDC) in about 50% of cases. Vitamin D compounds have been shown to inhibit progression to IDC in the MCF10DCIS model. This inhibition appears to involve a reduction in the cancer stem cell-like population in MCF10DCIS tumors. To identify genes that are involved in the vitamin D effects, a global transcriptomic analysis was undertaken of MCF10DCIS cells grown in mammosphere cultures, in which cancer stem-like cells grow preferentially and produce colonies by self-renewal and maturation, in the presence and absence of 1α25(OH)2D3 and a vitamin D analog, BXL0124. Using next-generation RNA sequencing, we found that vitamin D compounds down-regulated genes involved in maintenance of breast cancer stem-like cells (e.g. GDF15), epithelial-mesenchymal transition, invasion and metastasis (e.g. LCN2, S100A4), chemo- resistance (e.g. NGFR, PPP1R1B, AGR2), while up-regulating genes associated with a basal-like phenotype (e.g. KRT6A, KRT5) and negative regulators of breast tumorigenesis (e.g. EMP1). Gene methylation status was analyzed to determine whether the changes in expression induced by vitamin D compounds occurred via this mechanism. Ingenuity pathway analysis was performed to identify upstream regulators and downstream signaling pathway genes differentially regulated by vitamin D, including TP63 and vitamin D receptor (VDR) mediated canonical pathways in particular. This study provides a global profiling of changes in the gene signature of DCIS regulated by vitamin D compounds and possible targets for chemoprevention of DCIS progression to IDC in patients. 3 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Breast cancer is the most common cancer and the second leading cause of cancer related deaths in women worldwide [1]. Based on the presence or absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2), breast cancers are divided into subtypes: luminal A (ER+ and/or PR+; HER2–), luminal B (ER+ and/or PR+; HER2+), basal-like (ER–, PR–, and HER2–), and HER2-enriched (ER–, PR–, and HER2+) [2]. Breast cancer development is a multi-step process that involves epigenetic and genetic changes contributing to aberrant cell growth [3]. Histologically, breast cancer can be staged into invasive ductal carcinoma, ductal carcinoma in situ (DCIS) and invasive lobular carcinoma. About 20% of breast cancers newly diagnosed in 2019 among US women will be classified as DCIS, amounting to over 48,000 cases [4]. DCIS is an early stage, non- invasive type characterized by proliferation of malignant epithelial cells in the ducts [5]. It arises from atypical ductal hyperplasia and may progress to invasive ductal carcinoma (IDC) and metastatic cancer [5]. It is predicted that up to 50% of the DCIS cases will progress to IDC within 10 years of initial diagnosis [6]. Gene expression and microRNA analyses have been performed to elucidate the molecular characteristics of DCIS progression to IDC [7, 8]. However, the natural history of progression of DCIS to IDC is yet to be fully determined. Cancer stem cells (CSCs) were first identified in breast cancer using the cell surface markers CD44+/CD24- [9]. This population is characterized by a stem-cell gene expression signatures, drug- resistant phenotype and self-renewal capacity in vitro and in vivo [10]. Human DCIS lesions form spheroids and duct-like structures in ex vivo organoid culture and tumors in immunodeficient mice, suggestive of the presence of CSC-like cells in these early tumors [11]. MCF10DCIS.COM was derived from the non-tumorigenic MCF10A human breast cell line and exhibits basal-like subtype properties [12]. It is similar to human DCIS with bi-potentiality that can give rise to both myoepithelial and luminal cells and spontaneous progression to invasive breast cancer in vivo [13], and it is widely used as a model for IDC development from precursor lesions. MCF10DCIS.COM cultures and tumors contain high ALDH1+ 4 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. and CD44+/CD49f+/CD24− subpopulations with increased self-renewal and tumor development capabilities, similar to CSCs of fully invasive tumors [14]. Vitamin D signaling is known to be a potential target for breast cancer chemoprevention [15]. Our laboratory has shown that vitamin D compounds inhibit triple negative breast cancer tumorigenesis by reducing expression of cancer stem-cell associated genes, including OCT4 and CD44, and by inducing differentiation and up-regulating myoepithelial markers [16]. These compounds reduce in vitro mammosphere formation and in vivo tumorigenesis, although the molecular mechanisms of these effects are not known. BXL0124 is an analog of calcitriol (1α25(OH)2D3) modified with an additional side chain at C21-methyl group, endowing it with more biological activity at lower concentrations without causing hypercalcemia, a limiting side effect of vitamin D [17]. BXL0124 also inhibits MCF10DCIS xenograft tumorigenesis more potently than 1α25(OH)2D3, apparently through the similar mechanism of suppressing cancer stem cells [18]. The present study was undertaken to develop global profiles of changes in gene expression and CpG methylation in MCF10DCIS cells induced by vitamin D compounds, to gain an understanding of the pathways involved in their overall effects and the molecular basis of their activities, and to identify potential targets that could be exploited in the chemoprevention of breast cancer progression from DCIS to IDC. Materials and methods Reagents and cell culture 1α25(OH)2D3 and a Gemini vitamin D analog (BXL0124; 1α,25-dihydroxy-20R-21(3-hydroxy-3- deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol, >95% purity) were provided by BioXell, Inc. (Nutley, NJ) [17]. Vitamin D compounds were dissolved in DMSO. MCF10DCIS.com human breast cancer cells (MCF10DCIS, RRID: CVCL_5552) were provided by Dr. Fred Miller at the Barbara Ann Karmanos Cancer Institute (Detroit, MI). Cells were maintained in DMEM/F12 medium supplemented with 5% horse serum, 1% penicillin/streptomycin, and 1% HEPES 5 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Author manuscripts have been peer reviewed and accepted for publication but have not yet been