Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press 1 LncRNA-mediated regulation of SOX9 expression in basal sub-type 2 breast cancer cells 3 4 Aamira Tariq1,2*, Qinyu Hao2, Qinyu Sun2, Deepak K. Singh2, Mahdieh Jadaliha2, Yang 5 Zhang3, Neha Chetlangia2, Jian Ma3, Sarah E. Holton4, Rohit Bhargava4, Ashish Lal5, Supriya 6 G. Prasanth2, Kannanganattu V. Prasanth2* 7 8 1Department of Biosciences, Comsats Institute of Information Technology, Islamabad, 9 Pakistan 10 11 2Department of Cell and Developmental Biology, Cancer Center at Illinois, University of 12 Illinois at Urbana-Champaign, Urbana, IL, USA. 13 14 3Computational Biology Department, School of Computer Science, Carnegie Mellon 15 University, Pittsburgh, PA, USA. 16 17 4Department of Bioengineering and Beckman Institute of Advanced Science and 18 Technology, Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, 19 IL, USA. 20 21 5Regulatory RNAs and Cancer Section, Genetics Branch, Center for Cancer Research, 22 National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. 23 24 Running title: LncRNA promotes breast cancer cell proliferation. 25 26 Key words: Nuclear lncRNA, Basal-like subtype, TNBC, Regulation, Enhancer 27 28 * Corresponding authors Tariq et al 1 Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press 29 Aamira Tariq 30 Department of Biosciences, Comsats Institute of Information Technology, Islamabad, 31 Pakistan; Department of Cell and Developmental Biology, Cancer Center at Illinois, 32 University of Illinois at Urbana-Champaign, Urbana, IL, USA 33 E.mail [email protected] 34 Kannanganattu V. Prasanth 35 Department of Cell and Developmental Biology, Cancer Center at Illinois, University of 36 Illinois at Urbana-Champaign, Urbana, IL, USA 37 E.mail [email protected] Tariq et al 2 Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press 38 Abstract 39 40 Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer (BC) 41 subtypes with a poor prognosis and high recurrence rate. Recent studies have identified 42 vital roles played by several lncRNAs (long noncoding RNAs) in BC pathobiology. Cell type- 43 specific expression of lncRNAs and their potential role in regulating the expression of 44 oncogenic and tumor suppressor genes have made them promising cancer drug targets. By 45 performing a transcriptome screen in an isogenic TNBC/basal sub-type BC progression cell 46 line model, we recently reported ~1800 lncRNAs that display aberrant expression during 47 breast cancer progression. Mechanistic studies on one such nuclear-retained lncRNA, 48 linc02095, reveal that it promotes breast cancer proliferation by facilitating the expression 49 of oncogenic transcription factor, SOX9. Both linc02095 and SOX9 display co-regulated 50 expression in BC patients as well in basal sub-type BC cell lines. Knockdown of linc02095 51 results in decreased BC cell proliferation, whereas its overexpression promotes cells 52 proliferation. Linc02095-depleted cells display reduced expression of SOX9 concomitant 53 with reduced RNA polymerase II occupancy at the SOX9 gene body as well as defective SOX9 54 mRNA export, implying that linc02095 positively regulates SOX9 transcription and mRNA 55 export. Finally, we identify a positive feedback loop in BC cells that controls the expression 56 of both linc02095 and SOX9. Thus, our results unearth tumor-promoting activities of a 57 nuclear lncRNA linc02095 by facilitating the expression of key oncogenic transcription 58 factor in BC. 59 Tariq et al 3 Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press 60 Introduction 61 62 Breast cancer (BC) is a frequently diagnosed malignancy and a leading cause of death 63 amongst women across the globe (Siegel, Miller, & Jemal, 2016). Breast cancer, like most 64 other epithelial tumors, is a heterogeneous disease with diverse subtypes (Nguyen & 65 Massagué, 2007). These subtypes differ from each other in clinical behavior, therapeutic 66 response profiles, and the presence or absence of receptors such as estrogen receptor (ER), 67 progesterone receptor (PR), and human epidermal growth factor 2 (HER2). Based on 68 expression of these receptors, breast cancer is classified into different categories: luminal A 69 (ER+, PR+, and HER2-), luminal B (ER+, PR+ and HER2+/-), HER2-positive (ER-, PR-and 70 HER2-) and triple negative breast cancer (ER-, PR- and HER2-) (Jadaliha et al., 71 2016;(Weigelt, Baehner, & Reis-Filho, 2010). 72 Triple negative breast cancer (TNBC) has been further classified into two distinct 73 molecular subtypes; basal-like and claudin-low, based on their unique gene expression 74 profiles (Jadaliha et al., 2016). There are no targeted therapies available for TNBC, and 75 patients are typically treated with chemotherapy. However, TNBC patients display poor 76 outcomes due to disease heterogeneity and chemotherapy resistance(Lv et al., 2016) . 77 Less than 2% of the human genome encodes proteins; ~75% of the human genome encodes 78 non-coding RNAs that are: transcripts with no apparent protein-coding potential, such as 79 microRNAs (miRNAs), piwi-interacting RNAs (piRNAs) and the least understood long non- 80 coding RNAs (lncRNAs) (Prensner & Chinnaiyan, 2011). In general, lncRNAs range in size 81 between ~200bp to 100kb (Derrien et al., 2012). Genome-wide transcriptome analysis 82 revealed that the human genome harbors >16000 lncRNA genes 83 (https://www.gencodegenes.org/human/stats.html) (Jalali, Gandhi, & Scaria, 2016). Tariq et al 4 Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press 84 Mechanistic studies on a handful of them revealed that lncRNAs along with their interacting 85 protein partners contribute towards the regulation of diverse biological processes, 86 including cell proliferation (Prensner & Chinnaiyan, 2011; Schmitt & Chang, 2016; Ulitsky & 87 Bartel, 2013). They achieve this by modulating gene expression via different molecular 88 mechanisms such as chromatin modification, transcription, protein activity and localization, 89 and post-transcriptional gene regulation (Xu, Kong, Chen, Ping, & Pang, 2017). In the 90 context of BC, recent studies have identified several hundreds of lncRNAs as plausible 91 prognostic markers of BC (Van Grembergen et al., 2016). The aberrant expression of 92 lncRNAs like CCAT2 (Cai, He, & Zhang, 2015), MALAT1 (Jin, Lu, Lin, & Ma, 2016), H19 93 (Matouk et al., 2014), HOTAIR (Gupta et al., 2010) and ZFAS1 (Hansji et al., 2016) has been 94 associated with BC metastasis. Moreover, tissue type- and cell type-specific expression of 95 lncRNAs have made them promising candidates to address BC cancer cell heterogeneity 96 (Cabili et al., 2011). 97 In the present study, we investigated the potential role of a TNBC up-regulated lncRNA, 98 linc02095, in BC cell proliferation. We observed elevated expression of both linc02095 and 99 its neighboring protein-coding gene SOX9 in TNBC patient samples. Gain- and loss-of- 100 function experiments revealed that linc02095 promotes cell proliferation and BC 101 progression in vitro. Furthermore, we found that both linc02095 and SOX9 regulated the 102 expression of each other, and this co-regulation is required for enhanced tumorigenic 103 activities of BC cells. Taken together, our results imply that linc02095 could function as an 104 oncogenic lncRNA in breast cancer via its role in promoting the expression of oncogenic 105 and pro-metastatic transcription factor SOX9. 106 Results Tariq et al 5 Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press 107 108 Linc02095 is up-regulated in TNBC BC cells and patient samples 109 Human breast carcinoma could progress via sequential genetic modifications of benign 110 hyperplasia of mammary duct epithelial cells into atypical ductal hyperplasia, to ductal 111 carcinoma in situ, to invasive tumor localized to the breast or lymph node, ultimately 112 metastasizing to distant organs (Santner et al., 2001). In order to understand the role of 113 lncRNAs during breast cancer progression, we utilized a well-established isogenic 114 mammary epithelial cell line-derived triple negative breast cancer (TNBC or basal-sub type) 115 progression model system. This model system consists of three isogenic cell lines (M1, M3 116 and M4), all of them originally derived from non-tumorigenic MCF10A mammary epithelial 117 cells. The cell line series consist of MCF10A (M1), tumorigenic but less metastatic 118 MCF10CA1h (M3) and highly tumorigenic and metastatic MCF10CA1a.c11 (M4) cells. M3 119 gives predominantly well-differentiated low-grade carcinomas in the xeno-graft models (Fu 120 et al., 2010; Imbalzano, Tatarkova, Imbalzano, & Nickerson, 2009; Kadota et al., 2009; 121 Kadota et al., 2010; McKeen Polizzotti et al., 2012; B. Tang et al., 2003). 122 We recently performed poly A+ deep RNA-seq (~160-250 million paired-end 123 reads/sample) of M1, M2, M3 & M4 cells that were grown as three-dimensional (3D) acinar 124 or organoid-like structures in Matrigel for 7-10 days (Jadaliha et al., 2018). By analyzing the 125 RNA-seq, >1800 lncRNAs were de-regulated at least 2-fold in tumorigenic M3 cells 126 compared to the non-tumorigenic M1 cells (Jadaliha et al., 2018). Several of lncRNAs that 127 showed altered