Published OnlineFirst May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 CANCER PREVENTION RESEARCH | RESEARCH ARTICLE Analysis of the Transcriptome: Regulation of Cancer Stemness in Breast Ductal Carcinoma In Situ by Vitamin D Compounds Naing Lin Shan1, Audrey Minden1,2, Philip Furmanski1,2, Min Ji Bak1, Li Cai2,3, Roman Wernyj1, Davit Sargsyan4, David Cheng4, Renyi Wu4, Hsiao-Chen D. Kuo4, Shanyi N. Li4, Mingzhu Fang5, Hubert Maehr1, Ah-Ng Kong2,4, and Nanjoo Suh1,2 ABSTRACT ◥ Ductal carcinoma in situ (DCIS), which accounts for one mesenchymal transition, invasion, and metastasis (e.g., out of every five new breast cancer diagnoses, will progress to LCN2 and S100A4), and chemoresistance (e.g., NGFR, potentially lethal invasive ductal carcinoma (IDC) in about PPP1R1B, and AGR2), while upregulating genes associated 50% of cases. Vitamin D compounds have been shown to with a basal-like phenotype (e.g., KRT6A and KRT5) and inhibit progression to IDC in the MCF10DCIS model. This negative regulators of breast tumorigenesis (e.g., EMP1). inhibition appears to involve a reduction in the cancer stem Gene methylation status was analyzed to determine cell–like population in MCF10DCIS tumors. To identify whether the changes in expression induced by vitamin genes that are involved in the vitamin D effects, a global D compounds occurred via this mechanism. Ingenuity transcriptomic analysis was undertaken of MCF10DCIS cells pathway analysis was performed to identify upstream grown in mammosphere cultures, in which cancer stem–like regulators and downstream signaling pathway genes cells grow preferentially and produce colonies by self- differentially regulated by vitamin D, including TP63 renewal and maturation, in the presence and absence of and vitamin D receptor –mediated canonical pathways a fi 1 25(OH)2D3 and a vitamin D analog, BXL0124. Using in particular. This study provides a global pro ling of next-generation RNA-sequencing, we found that vitamin D changes in the gene signature of DCIS regulated by compounds downregulated genes involved in maintenance vitamin D compounds and possible targets for chemopre- of breast cancer stem–like cells (e.g., GDF15), epithelial– vention of DCIS progression to IDC in patients. À À À þ Introduction and HER2 ), and HER2-enriched (ER ,PR , and HER2 ; ref. 2). Breast cancer development is a multi-step process that Breast cancer is the most common cancer and the second involves epigenetic and genetic changes contributing to aber- leading cause of cancer-related deaths in women worldwide (1). rant cell growth (3). Histologically, breast cancer can be staged On the basis of the presence or absence of estrogen receptor into invasive ductal carcinoma (IDC), ductal carcinoma in situ (ER), progesterone receptor (PR), and HER2, breast cancers are þ þ À (DCIS), and invasive lobular carcinoma. About 20% of breast divided into subtypes: luminal A (ER and/or PR ; HER2 ), þ þ þ À À cancers newly diagnosed in 2019 among U.S. women will be luminal B (ER and/or PR ; HER2 ), basal-like (ER ,PR , classified as DCIS, amounting to over 48,000 cases (4). DCIS is an early stage, noninvasive type characterized by proliferation 1Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, of malignant epithelial cells in the ducts (5). It arises from The State University of New Jersey, New Brunswick, New Jersey. 2Rutgers atypical ductal hyperplasia and may progress to IDC and Cancer Institute of New Jersey, New Brunswick, New Jersey. 3Department of Biomedical Engineering, School of Engineering, Rutgers, The State University of metastatic cancer (5). It is predicted that up to 50% of the New Jersey, New Brunswick, New Jersey. 4Department of Pharmaceutics, Ernest DCIS cases will progress to IDC within 10 years of initial Mario School of Pharmacy, Rutgers, The State University of New Jersey, New diagnosis (6). Gene expression and miRNA analyses have been 5 Brunswick, New Jersey. Environmental and Occupational Health Sciences performed to elucidate the molecular characteristics of DCIS Institute and School of Public Health, Rutgers, The State University of New Jersey, New Brunswick, New Jersey. progression to IDC (7, 8). However, the natural history of progression of DCIS to IDC is yet to be fully determined. Note: Supplementary data for this article are available at Cancer Prevention fi fi Research Online (http://cancerprevres.aacrjournals.org/). Cancer stem cells (CSC) were rst identi ed in breast cancer þ À Corresponding Author: Nanjoo Suh, Rutgers University, 164 Frelinghuysen using the cell surface markers CD44 /CD24 (9). This pop- Road, Ernest Mario School Of Pharmacy, Rutgers University, Piscataway, NJ ulation is characterized by a stem cell gene expression signa- 08854. Phone: 848-445-8030; Fax: 732-445-0687; E-mail: tures, drug-resistant phenotype, and self-renewal capacity [email protected] in vitro and in vivo (10). Human DCIS lesions form spheroids Cancer Prev Res 2020;13:673–86 and duct-like structures in ex vivo organoid culture and tumors doi: 10.1158/1940-6207.CAPR-19-0566 in immunodeficient mice, suggestive of the presence of CSC- Ó2020 American Association for Cancer Research. like cells in these early tumors (11). MCF10DCIS.COM was AACRJournals.org | 673 Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst May 28, 2020; DOI: 10.1158/1940-6207.CAPR-19-0566 Shan et al. derived from the nontumorigenic MCF10A human breast cell cell detachment solution. Cells were then grown in 6-well ultra- line and exhibits basal-like subtype properties (12). It is similar low attachment plates at a density of 10,000 cells/mL and to human DCIS with bi-potentiality that can give rise to both maintained in MammoCult Human Medium added with myoepithelial and luminal cells and spontaneous progression MammoCult proliferation supplement, hydrocortisone solu- to invasive breast cancer in vivo (13), and it is widely used as a tion, and heparin solution (Stemcell Technologies). Spheres a model for IDC development from precursor lesions. were treated with DMSO (0.01%), 1 25(OH)2D3 (100 nmol/L), MCF10DCIS.COM cultures and tumors contain high and BXL0124 (10 nmol/L) for 5 days. After 5 days in culture, þ þ þ À ALDH1 and CD44 /CD49f /CD24 subpopulations with the culture plates were gently swirled to cluster the spheres in increased self-renewal and tumor development capabilities, the middle of each well and photographed. Mammosphere similar to CSCs of fully invasive tumors (14). forming efficiency (MFE) was calculated by dividing the num- Vitamin D signaling is known to be a potential target for ber of mammospheres (≥100 mm) formed by the number of breast cancer chemoprevention (15). Our laboratory has shown single cells seeded in individual wells. Three independent that vitamin D compounds inhibit triple-negative breast cancer experiments were repeated. tumorigenesis by reducing expression of CSC-associated genes, including OCT4 and CD44, and by inducing differentiation Nucleic acid isolation and next-generation sequencing and upregulating myoepithelial markers (16). These com- MCF10DCIS mammospheres were treated with DMSO a pounds reduce in vitro mammosphere formation and in vivo (0.01%), 1 25(OH)2D3 (100 nmol/L), and BXL0124 tumorigenesis, although the molecular mechanisms of (10 nmol/L) for 5 days followed by extraction of RNA and these effects are not known. BXL0124 is an analog of calcitriol DNA using AllPrep DNA/RNA Mini Kit (Qiagen). The con- a fi (1 25(OH)2D3) modi ed with an additional side chain at C21- centrations and quality of the RNA and DNA were determined methyl group, endowing it with more biological activity at using NanoDrop spectrophotometer and Agilent 2100 Bio- lower concentrations without causing hypercalcemia, a limit- analyzer separately. We prepared three independent sets of ing side effect of vitamin D (17). BXL0124 also inhibits cultures with MCF10DCIS mammospheres treated with a MCF10DCIS xenograft tumorigenesis more potently than DMSO, 1 25(OH)2D3, and BXL0124, and extracted each 1a25(OH)2D3, apparently through the similar mechanism of separately to obtain three RNA samples and three DNA suppressing CSCs (18). This study was undertaken to develop samples. We pooled those samples for the RNA-sequencing global profiles of changes in gene expression and CpG meth- (RNA-seq) and methyl-sequencing (methyl-seq) analyses, ylation in MCF10DCIS cells induced by vitamin D compounds, which was carried out at RUCDR Infinite Biologics, a Rutgers to gain an understanding of the pathways involved in their University affiliated institution which provides next- overall effects and the molecular basis of their activities, and to generation sequencing (NGS) services. RNA-seq libraries were identify potential targets that could be exploited in the che- prepared using Illumina RNA Library Prep Kit v2 according to moprevention of breast cancer progression from DCIS to IDC. the manufacturer's user guide with 400 ng of RNA as input. The libraries were then quantified using KAPA Library Quantifi- cation Kit according to the manufacturer's user guide and Materials and Methods pooled with barcodes. The pooled libraries were sequenced Reagents and cell culture on Illumina NextSeq 550 System, using NextSeq 500/550 Mid a 1 25(OH)2D3 and a Gemini vitamin D analog [BXL0124; Output v2 Kit. The sequencing parameters used were 150 bp, 1a,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4- paired-end with around 20 million reads generated per sample. trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol, >95% The DNA samples were further processed using an Agilent purity] were provided by BioXCell, Inc. (17). Vitamin D SureSelect Human Methyl-seq Target Enrichment System compounds were dissolved in DMSO. MCF10DCIS.com (Agilent Technologies) and sequenced on an Illumina NextSeq human breast cancer cells (MCF10DCIS, RRID: CVCL_5552) 500 instrument with 76 bp single-end reads, generating 34–47 were provided by Dr.