bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 An EMT-primary -GLIS2 signaling axis regulates mammogenesis

2 and claudin-low breast tumorigenesis

3 4 5 6 7 Molly M. Wilson1, Céline Callens2, Matthieu Le Gallo3,4, Svetlana Mironov2, Qiong Ding5,

8 Amandine Salamagnon2, Tony E. Chavarria1, Abena D. Peasah6, Arjun Bhutkar1, Sophie

9 Martin3,4, Florence Godey3,4, Patrick Tas3,4, Anton M. Jetten8, Jane E. Visvader7, Robert A.

10 Weinberg9, Massimo Attanasio5, Claude Prigent2, Jacqueline A. Lees1, Vincent J Guen2* 11 12 13 14 1Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts 15 Institute of Technology, Cambridge, MA, USA. 16 2Institut de Génétique et Développement de Rennes - Centre National de la Recherche 17 Scientifique, Rennes, France. 18 3INSERM U1242, Rennes 1 University, Rennes, France. 19 4Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France. 20 5Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA, 21 USA. 22 6Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 23 USA. 24 7Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research and 25 Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia. 26 8Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health 27 Sciences, National Institutes of Health, Research Triangle Park, NC, USA. 28 9MIT Department of Biology and the Whitehead Institute, Cambridge, MA, USA. 29 30 *Corresponding author: [email protected] 31 32 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

33 Abstract

34

35 The Epithelial–Mesenchymal Transition (EMT) and primary ciliogenesis induce stem cell 36 properties in basal Mammary Stem Cells (MaSCs) to promote mammogenesis, but the underlying 37 mechanisms remain incompletely understood. Here, we show that EMT transcription factors 38 promote ciliogenesis at intermediate EMT transition states by activating ciliogenesis inducers, 39 including FGFR1. The resulting primary cilia promote BBS11-dependent ubiquitination and 40 inactivation of a central signaling node, GLIS2. We show that GLIS2 inactivation promotes MaSC 41 stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 42 inactivation is required to induce the proliferative and tumorigenic capacities of the Mammary- 43 Tumor-initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be 44 segregated from other breast tumor subtypes based on a GLIS2-dependent expression 45 signature. Collectively, our findings establish molecular mechanisms by which EMT programs 46 induce ciliogenesis to control MaSC and MaTIC biology, mammary gland development, and 47 claudin-low breast cancer formation. 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

65 Main text

66

67 Introduction

68

69 The mammary gland is a branched ductal tissue comprised of a stratified containing 70 luminal and basal cells surrounded by a basement membrane embedded in stroma(1). Mammary 71 gland development is fueled by basal multipotent and lineage-restricted unipotent mammary stem 72 cells (MaSCs) (1). The stem cell properties of basal MaSCs are induced by Epithelial- 73 Mesenchymal transitions (EMT), a cell-biological program that enables epithelial cells to acquire 74 an array of mesenchymal phenotypes (2-5). The EMT transcription factor (EMT-TF) Slug is 75 expressed in MaSC-enriched basal cells (2-4, 6, 7). The ability of these stem cells to form 76 organoids and reconstitute the mammary epithelium in transplantation experiments is enhanced 77 or impaired by Slug overexpression or loss, respectively (3, 4, 7). Consistent with these findings, 78 Slug-knockout (KO) mice demonstrate delayed mammary gland development (6).

79 Breast cancer comprises a variety of tumor types that are classified into therapeutic and molecular 80 subtypes (8). The Triple-Negative Breast Cancer (TNBC) therapeutic subtype, in particular, is 81 composed of breast tumors that do not express nuclear hormone receptors and HER2 and 82 includes the less differentiated claudin-low molecular subtype (8). Claudin-low breast tumors are 83 breast cancers characterized by low expression of epithelial cell- and high 84 expression of EMT and basal MaSC genes (8). In murine carcinoma models of claudin-low breast 85 cancer, the tumorigenic activity of Mammary Tumor-Initiating Cells (MaTICs) relies on EMT-TFs 86 (2, 3, 9-11).

87 Collectively, these studies revealed that EMT programs tightly regulate mammary gland 88 development and tumorigenesis by controlling both MaSCs and MaTICs. Recent work revealed 89 that EMT programs promote MaSC and MaTIC stemness through induction of primary ciliogenesis 90 (11). Primary ciliogenesis is a dynamic process in which a single -based structure, the 91 primary cilium, is assembled by a modified version of the mother centriole, called the , 92 at the plasma membrane and protrudes from the surface, where it acts as a cell signaling center 93 (12). Primary ciliogenesis requires the coordination of many cellular processes including a motor- 94 driven process that involves bidirectional movement of multiprotein complexes along the cilium, 95 called (IFT) (12). In the mammary gland, the molecular mechanisms linking 96 EMT, primary cilia, and stemness are largely unknown. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

97 Here, we show that EMT programs induce primary ciliogenesis at intermediate epithelial- 98 mesenchymal transition states in both human and mouse mammary glands. Additionally, we 99 establish that EMT-TFs induce the expression of direct ciliogenesis regulators and IFT inducers, 100 including FGFR1, to promote primary ciliogenesis. We show that primary cilia control BBS11- 101 dependent ubiquitination, and inactivation of a central signaling node, GLIS2. Furthermore, we 102 demonstrate that GLIS2 inactivation promotes the stemness of MaSCs and MaTICs, 103 mammogenesis and claudin-low breast tumorigenesis.

104 105 Results

106 EMT programs induce primary ciliogenesis at intermediate transition states in the 107 mammary gland. 108 We previously established that EMT programs are associated with primary ciliogenesis in the 109 mouse mammary gland (11), spurred by our discovery that primary cilia were present in a high 110 fraction of basal Slug-expressing cells present in ducts and terminal end buds in the murine 111 mammary epithelium (Fig. S1A) (11). To determine whether this was also true in the human gland, 112 we analyzed sections of human reduction mammoplasty tissue. Cells expressing the EMT-TF Slug 113 were detected in the basal layer of the human mammary epithelium and expressed significantly 114 lower levels of the epithelial marker E- than did the Slug-negative luminal cells (Fig. 1A). 115 Importantly, primary cilia (detected by the cilium marker Arl13b and the marker 116 gTubulin) marked the vast majority of Slug-expressing cells, in contrast to the Slug-negative cells 117 (Fig. 1A). Hence, basal mammary epithelial cells, which exist in an intermediate EMT transition 118 state, assemble primary cilia in both human and mouse mammary glands.

119 To determine whether primary ciliogenesis is a transient or a stable response to EMT activation, 120 we used HMLE cells, which are immortalized human mammary epithelial cells (harvested from a 121 mammoplasty and expressing hTERT and SV40 Large T ) (13). We previously generated 122 HMLE variants in which doxycycline induces ectopic expression of the Snail or Zeb1 EMT-TFs 123 (11). To eliminate pre-existing mesenchymal cells from the un-induced HMLE variant populations, 124 we used fluorescence-activated cell sorting (FACS) to isolate the most epithelial cells based on 125 their CD44Lo;CD24Hi phenotype (Fig. S1B) (2). We then tested our ability to induce EMT and detect 126 various EMT transition states in these epithelial-enriched populations by culturing them with 127 doxycycline for 12 days and examining their morphology and expression of epithelial (E-cadherin) 128 and mesenchymal (Vimentin) markers at various time points. At day 0, both HMLE variants 129 displayed the classic epithelial-like (E-like) cobblestone morphology (Fig. 1B), and lacked bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

130 detectable expression of either Snail or Zeb1 (Fig. 1C). Doxycycline treatment induced Snail and 131 Zeb1 expression with similar kinetics, both appearing by day 4 (Fig. 1C). Despite these similarities, 132 the two variant populations showed differences in their EMT transitions; the Snail-expressing cells 133 adopted an elongated mesenchymal-like (M-like) morphology progressively between days 8-12, 134 while the Zeb1-expressing cells gained an M-like appearance as early as day 4 and became more 135 distinctly mesenchymal at later time points (Fig. 1B). These changes were concurrent with E- 136 cadherin loss, which was only partial at day 4, and more pronounced at later timepoints in Snail- 137 expressing cells, but was already largely lost 4 days after Zeb1 induction (Fig. 1C). The 138 mesenchymal marker, Vimentin, increased gradually over 12 days in both HMLE variants (Fig. 139 1C).

140 Having defined the time windows in which the EMT transition states occurred, we next assessed 141 the representation of primary cilia across these states by staining cells for Arl13b alongside E- 142 cadherin. We observed a significant increase in primary ciliogenesis in response to Snail (day 8) 143 or Zeb1 (starting at day 2 and increasing dramatically on day 4) expression (Fig. 1D), which 144 coincided with the marked reduction of E-cadherin, intermediate expression levels of Vimentin, 145 and a partial M-like phenotype (Fig. 1B-D). Together, these data show that EMT programs induce 146 primary ciliogenesis at intermediate epithelial-mesenchymal transition states. 147 148 EMT-TFs activate the expression of positive regulators of cilium assembly to promote 149 primary ciliogenesis. 150 The mechanisms by which EMT programs induce primary ciliogenesis have been elusive. To 151 determine the underlying molecular mechanisms, we conducted comparative analyses of control 152 (CTL) and Snail-expressing HMLE cells under ciliogenesis-permissive conditions. To do so, CTL 153 and Snail cells were grown to high confluence and serum starved. Replicate samples were 154 subjected to immunofluorescent staining (Fig. S2A) or western blotting (Fig. S2B), which 155 confirmed that the Snail-expressing cells were highly ciliated relative to control cells (Fig. S2A), 156 and expressed lower levels of E-cadherin and higher levels of Fibronectin, N-cadherin, Vimentin, 157 and the Zeb1 and Twist1 EMT-TFs (Fig. S2B). Parallel replicate samples were subjected to RNA- 158 sequencing. This revealed substantial differences in in CTL versus Snail- 159 expressing cells (Fig. 2A). To identify ciliogenesis regulators whose expression was induced upon 160 EMT induction by Snail, we compared the list of upregulated genes in Snail-expressing versus 161 CTL cells to a cilium gene set, consisting of genes encoding centrosomal and/or ciliary proteins 162 (combined and curated MSigDB GO_Cilium, GO_Cilium Morphogenesis gene sets). We identified bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

163 47 genes that overlapped between the two gene signatures, which then represented candidate 164 ciliogenesis inducers and/or ciliary signaling regulators downstream of Snail (Fig. 2B, C).

165 We reasoned that this gene set likely included both direct and indirect Snail targets. To identify 166 potential direct Snail target genes, we reanalyzed the data from an existing Snail ChIP-seq study 167 in mouse mammary tumor cells (4), and compared the resulting gene list to our candidate 168 ciliogenesis inducers and cilia components. Remarkably, among these 47 genes, 6 of the 10 most 169 upregulated genes were identified as direct Snail targets: Fgfr1, Syne1, Rom1, Spata6, Hhip and 170 Map1b (Fig. S2C, Supplementary Table 1). Importantly, for all 6 of these genes, the peak scores 171 (which represent a normalized value of the read counts identified per gene) were similar to those 172 of known Snail targets (Fig. S2C). The Fgfr1 gene was particularly interesting. Multiple Snail 173 binding motifs associated with Snail-dependent transcriptional regulation were identified in close 174 proximity to or within the FGFR1 promoter (Fig. S2D), consistent with the notion that FGFR1 is 175 directly activated by Snail.

176 Over the last decade, FGFR1 has emerged as a key inducer of ciliogenesis in the embryonic 177 tissues of lower , including the zebrafish, xenopus, and chick (14, 15), and in human 178 lung carcinoma and rhabdomyosarcoma cells (16). FGFR1 directly controls intraflagellar transport 179 (IFT) by inducing expression of IFT system components and enabling their import into the cilium 180 (14, 15). FGFR1 has not been previously linked to ciliogenesis in the mammary gland, but it is 181 known to cooperate with FGFR2 to promote mammary gland development by regulating stemness 182 of MaSCs (17).

183 Given these observations, we hypothesized that EMT programs activate FGFR1 expression to 184 induce ciliogenesis. After validating the upregulation of FGFR1 in Snail-expressing versus CTL 185 HMLE cells by real-time qPCR analysis (Fig. S2E), we examined FGFR1 expression in 186 Snail- and Zeb1-expressing cells at various EMT transition states using our doxycycline induction 187 scheme. We found that FGFR1 expression was markedly induced by Snail and Zeb1 expression 188 at day 8 and day 4, respectively (Fig. 2D), coinciding with the induction of primary ciliogenesis 189 described earlier (Fig. 1D).

190 To directly test the role of FGFR1 in mammary ciliogenesis, we cultured our Snail-expressing 191 HMLE cells in ciliogenesis-permissive conditions in the presence of various concentrations of the 192 small-molecule FGFR tyrosine kinase inhibitor, SU5402, or DMSO vehicle control. Initially, we 193 examined the impact of drug treatment on FGFR1 activity by assessing the phosphorylation status 194 of two known FGFR1 downstream targets, AKT and MEK. We saw a significant decrease in the bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

195 phosphorylation of both targets when cells were treated at 10 µM of SU5402 (Fig. S2F). 196 Importantly, this drug treatment did not alter cell cycle withdrawal (as judged by FACS 197 quantification of G2/M cells; Fig. S2G) or the E-M status of the cells (as judged by western blotting 198 for E-cadherin and Vimentin; Fig. S2H), which could impact ciliogenesis indirectly. We then asked 199 whether it alters ciliogenesis by staining DMSO- and SU5402-treated cells for Arl13b and Cep170 200 (a centrosome marker). This revealed that FGFR1 inhibition is associated with a significant 201 decrease in the percentage of cells displaying primary cilia and in the length of the cilia that 202 succeeded in forming (Fig. 2E).

203 To evaluate the role of FGFR1 in ciliogenesis in a more physiological setting, we examined the 204 impact of SU5402 treatment on ciliogenesis in mammary organoids. For this, we isolated MaSC- 205 enriched basal cells from adult female mice by FACS based on their Lin-;CD24med;CD49fHi 206 phenotype (Fig. 2F). When plated in appropriate 3D culture conditions, these MaSCs form solid 207 organoids (Fig. 2F) that are mostly clonal, as judged by analyses of mixtures of MaSCs with or 208 without dTomato-expression (Fig. S2I). Importantly, these organoids form rudimentary branches 209 comprised of Krt8+;Krt14- cells surrounded by Krt14+;Krt8- cells, mimicking the complex cellular 210 architecture of the mammary gland (Fig. 2G). Moreover, the Krt14+ cells of the outer layer 211 expressed Slug and displayed primary cilia (Fig. 2G), similar to the human and mouse mammary 212 glands in vivo (Fig. 1A, S1A). 213 We tested the impact of SU5402 in this 3D assay at various concentrations. Consistent with 214 published data(18), we found that SU5402 suppresses organoid formation in a dose-dependent 215 manner (Fig. S2J), confirming the importance of FGF signaling for stemness. Additionally, we 216 found that SU5402 treatment significantly reduced the fraction of ciliated cells, as well as cilium 217 length (Fig. 2H). Together, these data showed that EMT-TFs activate the expression of 218 ciliogenesis inducers, including FGFR1, which positively regulate primary ciliogenesis in MaSC- 219 enriched basal cells of the mammary epithelium, and that FGF signaling is critical for MaSC 220 stemness. 221 222 Primary cilia control a BBS11-GLIS2 signaling axis. 223 Primary cilia act as cell signaling centers (19). To identify signaling pathways that are controlled 224 by primary cilia in mammary epithelial cells, we further expanded our gene expression analysis to 225 Snail-expressing cells grown to high confluence and serum starved in the presence of DMSO or 226 with Ciliobrevin A, a small-molecule ciliogenesis inhibitor (CilA) (20). As above, replicate samples 227 of cells were stained for Arl13b and gTubulin to assess the presence of primary cilia under the two bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

228 conditions (Fig. 3A), confirming that ciliogenesis was significantly inhibited by CilA-treatment 229 relative to DMSO treatment. Replicate samples were then subjected to RNA-sequencing (Fig. 3B). 230 This revealed substantial changes in the gene expression profiles of Snail-expressing cells treated 231 with CilA relative to DMSO-treated cells (Fig. 3B).

232 To identify signaling pathways induced by EMT and primary cilia, we performed gene set 233 enrichment analysis (GSEA). Here, we looked for significantly enriched gene sets that include 234 genes that are upregulated in Snail-expressing HMLE cells versus CTL cells (Fig. 2A) and are 235 downregulated in Snail-expressing CilA-treated cells versus DMSO-treated cells (Fig. 3B). This 236 revealed a gene set that is known to be regulated by GLIS2, a transcriptional repressor (Fig. 3C). 237 As we found, GLIS2 target genes are upregulated in the Snail-expressing cells and repressed 238 upon CilA treatment, suggesting that GLIS2 is inactivated by cilia.

239 GLIS2 is a transcriptional repressor that is essential for normal development of both the kidney 240 and incisor tooth epithelium (21-23). Recent studies showed that it acts as a central regulator of 241 stem and progenitor cell maintenance and differentiation(23, 24). Importantly, existing data 242 already suggested a link between GLIS2 and primary cilia. First, GLIS2 loss causes 243 nephronophthisis, a renal (21). Second, in the kidney and incisor tooth epithelial cells, 244 GLIS2 localizes to primary cilia (21, 23). However, the molecular basis of the interplay between 245 GLIS2 and primary cilia is unknown. Moreover, GLIS2 has not been previously implicated in 246 mammary gland biology. Our own data clearly suggest that primary cilia mediate GLIS2 inhibition 247 in the mammary epithelium.

248 To explore the underlying mechanism of this repression, we first analyzed GLIS2 RNA expression 249 in CTL, Snail, Snail+DMSO and Snail+CilA HMLE variants using our RNA-sequencing dataset, as 250 well as by real-time qPCR analysis. Neither the RNA-sequencing analysis nor the RT-qPCR 251 analysis indicated that GLIS2 expression is repressed upon EMT and ciliogenesis (Fig. S3A, B). 252 Notably, two of the few known GLIS2 target genes, GLI1 and CDH11, were indeed upregulated in 253 Snail versus CTL cells, and downregulated in Snail+CilA versus Snail+DMSO cells (Fig. S3B). 254 Thus, we concluded that GLIS2 is inhibited post-transcriptionally in a cilium-dependent manner.

255 GLIS2 can undergo ubiquitination-dependent inactivation, a process that is known to require 256 BBS11, an E3- ligase, and a member of the BBS family (25). This was particularly 257 intriguing, as BBS stands for Bardet Biedl Syndrome, a ciliopathy (26). We therefore asked 258 whether primary cilia inhibit GLIS2 by promoting its ubiquitination. To do so, an HA-tagged version 259 of GLIS2 was introduced in CTL or Snail-expressing HMLE cells, which were then treated with bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

260 DMSO or CilA. Total proteins were extracted, and ubiquitinated proteins were isolated by affinity 261 chromatography using Tandem Ubiquitin-Binding Entities (TUBE) and then analyzed by western 262 blot.

263 Comparable levels of total ubiquitinated proteins were recovered in all samples (Fig. S3C). 264 However, we detected a dramatic increase in the levels of ubiquitinated HA-GLIS2 in the 265 Snail+DMSO cells compared to the CTL + DMSO cells (Fig. 3D). Specifically, the lowest mobility 266 band, which we presume is the mono-ubiquitinated form, was present at higher levels in 267 Snail+DMSO versus CTL+DMSO cells (Fig. 3D, short exposure). We also detected multiple, high 268 molecular weight ubiquitinated HA-GLIS2 species that appeared to be unique to the Snail+DMSO 269 cells (Fig. 3D, long exposure). Remarkably, the levels and pattern of ubiquitinated HA-GLIS2 270 species in the Snail+CilA cells closely resembled that of the CTL+DMSO cells, not Snail+DMSO 271 (Fig. 3D), strongly suggesting that these are cilium-dependent events.

272 To more directly test whether primary cilia are indeed required for GLIS2 ubiquitination, we 273 examined the ubiquitination status of HA-GLIS2 in Snail-expressing cells in which KIF3A and 274 IFT20, two essential ciliogenesis regulators, were knocked-out (KO) using CRISPR. To ensure 275 complete gene KO, we selected 5 CRISPR single cell clones with total protein loss and then 276 pooled these knockout clones. Control pools of single cell clones were also generated. We 277 observed that ciliogenesis was abolished in KO cells relative to wild-type control cells (Fig. 3E, F). 278 Genetic inhibition of primary ciliogenesis decreased the levels and pattern of ubiquitinated HA- 279 GLIS2 species (Fig. 3G), in a comparable manner to the pharmacological inhibition of ciliogenesis 280 (Fig. 3D). Together, these data showed that primary cilia induce GLIS2 ubiquitination in mammary 281 epithelial cells.

282 We then asked if the E3 BBS11 contributes to the observed GLIS2 ubiquitination. 283 We found that BBS11 is significantly upregulated in Snail-expressing HMLE cells, relative to CTL 284 cells, at the level of protein (Fig. 3H) but not mRNA (Fig. S3D, E). We then knocked out the BBS11 285 gene (TRIM32) in Snail-expressing cells using CRISPR (Fig. 3I). Here, two out of the three 286 sgRNAs against TRIM32, #1 and #3, yielded cell populations exhibiting a marked reduction of 287 BBS11 protein levels (Fig. 3I). Weak BBS11 protein was still detectable in these populations due 288 to the cell-to-cell variability in the inactivation of TRIM32 in this polyclonal cell population. Next, 289 we grew the sgCTL and the two sgTRIM32 KO populations, #1 and #3, in ciliogenesis-permissive 290 conditions and found that BBS11 loss had no detectable impact on either the E-M status of the 291 cells (Fig. S3 F-G) or on primary ciliogenesis (Fig. 3J). We then examined ubiquitination of HA- bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

292 GLIS2 in the sgCTL and sgTRIM32#3 KO cells, as described above. In a comparable manner to 293 the pharmacological inhibition or genetic ablation of primary cilia, BBS11 loss reduced the levels 294 and pattern of HA-GLIS2 ubiquitination (Fig. 3K). Hence, we concluded that BBS11 is required for 295 primary cilium-dependent GLIS2 ubiquitination and acts downstream of primary cilium formation.

296 GLIS2 is a central regulator of basal MaSC and mammary gland development. 297 298 Our previously reported data established that EMT-induced primary ciliogenesis in MaSC- 299 enriched basal cells promotes their stemness (11). Given this finding, we undertook to test the 300 role of the primary cilium-GLIS2 signaling axis in this process. We initiated this study in our HMLE 301 model, in which the ability to form mammospheres, which are 3D floating structures comprised of 302 undifferentiated cells, serves as a measure of their SC-like properties (2).

303 To begin, we assessed primary cilia representation in mammospheres arising from HMLE cells 304 that have gained the ability to form these structures upon Snail expression, doing so by staining 305 for Arl13b and gTubulin (Fig. 4A, B). This showed that the undifferentiated cells in mammospheres 306 formed primary cilia (Fig. 4B). To investigate the role of GLIS2 in this process, we took advantage 307 of a previously described GLIS2 truncation mutant (GLIS2 c. 1_444del), termed GLIS2Cter, that 308 can act as a constitutive transcriptional repressor (27). Thus, we introduced GLIS2Cter or a control 309 vector into our Snail-expressing HMLE cells (Fig. S4A). We first confirmed the ability of GLIS2Cter 310 to mediate transcriptional repression using qRT-PCR to gauge the expression of the known GLIS2 311 target genes, GLI1 and CDH11, and found that both transcripts were downregulated, relative to 312 the control vector cells (Fig. S4B). We then showed that GLIS2Cter did not alter the M-like status 313 of these cells, as judged by morphology and expression of E-M markers (Fig. S4A, C), their ability 314 to form primary cilia (Fig. S4D), and their 2D proliferation rate (Fig. S4E). In subsequent 315 mammosphere assays, we found that GLIS2Cter expression significantly inhibited the 316 mammosphere-forming capacity of the Snail-expressing HMLE cells relative to control vector cells 317 (Fig. 4C).

318 Given these findings, we extended our analyses to primary basal MaSCs. Thus, we isolated 319 mouse basal MaSCs by FACS, as described above, and generated populations with ectopic 320 expression of either GLIS2Cter or a control vector. We then examined the ability of these cells to 321 generate organoids and observed a significant reduction in response to GLIS2Cter expression 322 (Fig. 4D). Taken together, these data showed that perturbation of the primary cilium-GLIS2 axis bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

323 results in inhibition of stem cell-like cells in both the HMLE population and the basal murine MaSCs 324 and thus impairs ex vivo organogenesis.

325 We reasoned that if Glis2 were important for regulation of MaSCs in vivo, its deletion should 326 perturb mammary gland development in mice suffering from germline KO of the Glis2 gene. We 327 further hypothesized that the resulting mammary defects would reflect inappropriate expansion of 328 MaSCs, at the expense of cell differentiation and proper lineage commitment. To test these 329 predictions, we examined mammary gland development in 4-week-old Glis2+/+ and Glis2-/- female 330 mice, through carmine-alum staining of whole-mount mammary glands. In agreement with our 331 hypothesis, ductal morphogenesis was profoundly impaired in Glis2-deficient mice, as evidenced 332 by the presence of only small mammary rudiments relative to the Glis2+/+ controls (Fig. 4E). 333 Additionally, H&E staining of paraffin sections showed that Glis2+/+ glands had the expected 334 bilayered epithelium, but this organization was disrupted in Glis2-/- mice (Fig. 4F). Most importantly, 335 Glis2-/- rudimentary ducts were enriched for TSPAN8-expressing cells relative to Glis2+/+ controls 336 (Fig. 4F). TSPAN8 is a plasma that is highly expressed in basal MaSCs and to 337 a lesser extent in luminal progenitor cells of the mammary epithelium (28). Collectively, our data 338 showed that GLIS2 regulates the expansion of basal MaSCs in a manner that is required for 339 normal mammary gland development. 340 341 Primary cilium-dependent regulation of GLIS2 controls claudin-low MaTIC stemness and 342 tumorigenicity. 343 344 We have previously shown that the role of primary cilia is not only important for normal basal 345 MaSCs but also promotes the tumor-initiating capacity of MaTICs in an orthotopic murine 346 carcinoma model (11). This analysis was conducted using HMLE cells transformed with H- 347 RASG12V (called HMLER), which, upon E-cadherin knockdown, become more M-like (Fig. S5A) 348 and acquire MaTIC properties (11). These cells can form tumorspheres comprised of non- 349 differentiated cells that display cilia in vitro (Fig. 5A-B) and generate ciliated tumors that display 350 the hallmarks of claudin-low breast cancers (Fig. S5B).

351 We therefore asked whether primary cilia control GLIS2 in these transformed HMLER cells. We 352 began by assessing the cilium-dependent gene expression events in HMLER variants. For this, 353 we grew HMLER shCTL and shE-cadherin (shEcad)-expressing cells in ciliogenesis-permissive 354 conditions, doing so with or without DMSO and CilA. For each condition, samples were stained 355 for Arl13b and E-cadherin to assess primary cilia representation. These analyses confirmed that bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

356 E-cadherin loss in HMLER cells led to a dramatic increase in the percentage of ciliated cells, 357 relative to shCTL cells (Fig. S5C), and that this ciliogenesis was reduced significantly by treatment 358 with CilA (Fig. S5C). Parallel samples were analyzed by RNA-sequencing, revealing substantial 359 changes in gene expression profiles between these HMLER populations (Fig. S5D). Importantly, 360 GSEA revealed that the GLIS2 target genes were upregulated in shEcad-expressing vs shCTL 361 cells and downregulated in shEcad-expressing CilA vs DMSO treated cells (Fig. S5E). Thus, 362 GLIS2 is inhibited in a cilium-dependent manner in transformed HMLER cells enriched for MaTICs 363 in a similar manner to that observed in their non-transformed HMLE counterparts.

364 We next asked whether GLIS2 activity influences the properties of these HMLER cells, doing so 365 by generating HMLER shEcad populations expressing GLIS2Cter. As with Snail-expressing 366 HMLE cells, this perturbation did not alter the E-M status, primary ciliogenesis, or proliferation rate 367 of the HMLER+shEcad cells in monolayer culture (Fig. S5F-I). However, GLIS2Cter did repress 368 GLI1 and CDH11 gene expression (Fig. S5J), validating its activity. Importantly, GLIS2Cter 369 expression decreased the tumorsphere-forming capacity of HMLER shEcad cells relative to the 370 control vector cells (Fig. 5C).

371 To further validate the key role of GLIS2 in tumors, we also compared the tumorigenic capacity of 372 GLIS2Cter-expressing and CTL HMLER shEcad cells in orthotopic tumor implantation 373 experiments. Consistent with our in vitro assays, the GLIS2Cter expression significantly reduced 374 the in vivo tumorigenic capacity of the HMLER shEcad CTL cells (Fig. 5D). These data show that 375 the primary cilium-dependent regulation of GLIS2 is required for the proliferative and tumorigenic 376 capacities of MaTICs, which form tumors displaying hallmarks of claudin-low breast cancers.

377 To further analyze primary ciliogenesis in claudin-low human breast cancers, we established a 378 tumor biobank by characterizing tumor samples in a previously assembled TNBC biobank. We 379 extracted total RNA from sections of frozen tumors (n = 62) and analyzed gene expression by 380 RNA-sequencing. Among the 62 TNBCs, 23 claudin-low tumors were then identified based on 381 their differential expression of a panel of genes (Fig. 6A), previously identified by Prat and 382 colleagues (29). We were able to stain paraffin sections for 20 of them, along with 3 luminal B 383 tumors (as controls) for H&E and for E-cadherin, Claudin 4, and Claudin 7.

384 We found that, in contrast to the luminal B tumors, which all demonstrated the typical appearance 385 of differentiated breast cancers, the claudin-low tumors displayed a poorly differentiated 386 phenotype (Fig. 6B). Luminal tumors expressed higher levels of epithelial markers than did 85% 387 of the claudin-low tumors (Fig. 6B, S6A). We also co-stained luminal and claudin-low tumor bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

388 sections for Vimentin, E-cadherin, Arl13b, and gTubulin in order to assess primary cilia 389 representation. This revealed ciliated cancer cells in claudin-low tumors that co-expressed E- 390 cadherin and Vimentin, but not in luminal tumors, indicating that they reside in an intermediate 391 EMT transition state (Fig. 6C, S6B). Moreover, these cells represented a small fraction of overall 392 tumor cells, consistent with the hypothesis that the ciliated claudin-low tumor cells are MaTICs.

393 To determine whether high expression of the GLIS2 signature is a hallmark of claudin-low tumors 394 across breast cancers in general, we compared the expression of GLIS2 target genes in 1082 395 breast cancers, using a public dataset from The Cancer Genome Atlas (TCGA) (30). Remarkably, 396 we found that high expression of GLIS2 target genes enables the segregation of claudin-low 397 tumors from other breast tumors in a manner similar to the Prat et al reference panel of genes 398 used to mark claudin-low cancers (Fig. 6D) (29). Moreover, GSEA revealed that the GLIS2 gene 399 set is significantly enriched in the signature that was initially used to define the claudin-low tumors 400 (Fig. S6C) (29). Collectively, our data showed that primary cilia are assembled in claudin-low 401 breast tumor cells that are enriched in MaTICs, in which they control GLIS2 inactivation to promote 402 the tumorigenic capacity of these cells. Furthermore, high expression of GLIS2 target genes 403 serves as a hallmark of claudin-low breast cancers.

404

405 Discussion

406

407 Our previously reported data established that EMT programs induce ciliogenesis in mammary 408 epithelial cells (11). However, we lacked a mechanistic understanding of the link between EMT 409 and primary ciliogenesis, including the degree to which ciliogenesis is a stable or a transient 410 response upon EMT activation, as well as the underlying molecular mechanisms. Our data 411 reinforce the connection between EMT programs and primary ciliogenesis both in the human and 412 mouse mammary glands. Specifically, we show that the propensity to form primary cilia closely 413 follows entrance into mixed epithelial-mesenchymal phenotypic states and that primary cilia are 414 assembled in mammary epithelial cells that display an array of M-like phenotypes. While we do 415 not see a decrease in the percentage of ciliated cells in the most mesenchymal phenotypic states 416 in our systems, we do not exclude the possibility that primary ciliogenesis is repressed at more 417 extreme mesenchymal EMT transition states. Indeed, this latter hypothesis is supported by an 418 elegant study of Epithelial-Myofibroblast Transition (EMyT), a more extreme type of EMT that can 419 occur in kidney epithelial cells (31). Here, primary cilia are induced during early transition states, bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

420 where they promote EMyT progression but are ultimately repressed when the cells acquire the 421 highly mesenchymal, myofibroblast phenotype (31). 422 In yet another context, the murine coronary vasculature, a link between EMT and primary 423 ciliogenesis was recently identified during development (32). Here, the authors found that 424 epicardial cells that undergo EMT assemble primary cilia, and that genetic disruption of 425 ciliogenesis in these cells perturbed EMT and migration, causing coronary artery developmental 426 defects (32). 427 These findings cement a strong connection between EMT and primary ciliogenesis in multiple 428 contexts and underscore the concept that EMT is not a simple binary switch between E and M 429 states, but instead generates a spectrum of transitional phenotypic states that can have a 430 distinctive relationship with the primary cilium biology. Our data established that EMT induces 431 primary ciliogenesis at intermediate and pronounced transition states in mammary epithelial cells 432 of the human and mouse mammary gland to support stemness. 433 434 The precise molecular mechanisms by which EMT programs induce primary ciliogenesis were 435 previously unknown. Our data show that EMT-TFs activate the transcription of ciliogenesis 436 inducers in mammary epithelial cells. Specifically, we found that FGFR1 expression is induced by 437 EMT-TFs at intermediate epithelial-mesenchymal transition states. We show that FGFR1 is 438 responsible, at least in part, for primary cilium assembly and elongation upon activation of EMT 439 programs. A recent study found that FGFR1 promotes ciliogenesis in the hair cells of the inner ear 440 of the chick embryo by phosphorylating the protocadherin Pcdh15 (15). Interestingly, we found 441 another member of the protocadherin family, PCDHB15, in our identified list of 47 putative 442 ciliogenesis inducers, together with FGFR1 (Fig. 2C), raising the intriguing possibility that FGFR1 443 cooperates with Protocadherin beta-15 in promoting primary ciliogenesis in mammary epithelial 444 cells. Of note, FGFR1 cooperates with FGFR2 to promote mammary gland development and 445 regeneration by enabling the stemness of MaSCs (17, 18). Our own observations show that FGFR 446 inhibition represses both ciliogenesis and organoid-forming capacity in MaSCs. Collectively, these 447 data suggest that FGFR1 controls ciliogenesis, thereby enabling basal MaSC stemness and 448 mammary gland development. 449 450 Our prior work revealed that primary cilia control Hedgehog signaling in mammary cells (11). In 451 the current study, we used gene expression analyses to gain a broader and unbiased insight into 452 the signaling pathways that depend on the primary cilium in mammary cells; these analyses 453 revealed that cilia control a GLIS2 signaling axis. This was highly gratifying because, although bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

454 GLIS2 is a relatively understudied protein, it had already been linked to EMT, primary cilia, and 455 stemness in other contexts. With regard to EMT, GLIS2 was found by others to repress EMT in 456 the kidney (21). We did not observe evidence of increased Mesenchymal-Epithelial Transition 457 (MET) when GLIS2 was ectopically activated in M-like mammary cells, leading us to conclude that 458 GLIS2 acts downstream of EMT in the mammary epithelium. However, our data do not rule out 459 the possibility that GLIS2 inactivation helps reinforce EMT through a positive feedback loop. 460 With regard to primary cilia, GLIS2 was shown by others to localize to the primary cilium in incisor 461 tooth epithelial stem cells undergoing differentiation, and in kidney cells (21, 23). However, the 462 role of the primary cilium in regulating GLIS2 had not been previously addressed. Our data show 463 that GLIS2 is ubiquitinated and inhibited in a cilium-dependent and BBS11-dependent fashion. 464 Notably, several members of the BBS family of proteins are known to bind to GLIS2, including 465 BBS11, and these various BBS proteins have been found to localize to primary cilia or centriolar 466 satellites (25, 26, 33). Thus, we speculate that primary cilia serve as cell signaling platforms where 467 BBS11 induces GLIS2 ubiquitination and consequent inactivation. Finally, and most importantly, 468 our data show that GLIS2 regulates stemness of basal MaSCs. The signaling pathway(s) 469 downstream of GLIS2 that control(s) MaSC-stemness remain to be investigated, but it is intriguing 470 to note that GLIS2 is known to repress two key stem cell signaling pathways, Wnt and Hedgehog 471 (34, 35). This intersects with our prior finding that Hedgehog signaling acts downstream of primary 472 ciliogenesis in basal MaSCs and MaTICs (11). 473 474 As mentioned, prior reports revealed that GLIS2 controls normal development of both the kidney 475 and the incisor tooth epithelium (21-23). Our own data show that GLIS2 also plays a critical role 476 in normal mammary gland development, which is consistent with our discovery of its role in MaSC 477 regulation. More specifically, we found a major morphogenetic defect at puberty in Glis2-deficient 478 female mice. We detected only rudimentary trees in Glis2-deficient animals, in which the normal 479 bilayer structure of the mammary epithelium is disrupted and an excess of basal MaSCs was 480 detected. These findings suggest that there is an inappropriate persistence or expansion of the 481 SC compartment and/or a failure of engagement of the differentiation program at the appropriate 482 developmental time. 483 484 Our prior work revealed a critical role for primary cilia in MaTICs of the more mesenchymal HMLER 485 cell line, which yield tumors that display the hallmarks of claudin-low human breast cancers (11). 486 Here, we found that primary cilia control GLIS2 in these MaTICs, thereby enabling their self- 487 renewal and tumorigenic capacities both in vitro and in vivo. To directly address the relevance of bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

488 these findings in human breast tumors, we established a biobank of claudin-low breast tumor 489 samples. Subsequently, we found that claudin-low breast tumors are enriched in tumor cells that 490 display an M-like phenotype and assemble primary cilia. Significantly, we found that a GLIS2 491 signature identifies claudin-low tumors from other breast cancer subtypes as effectively as the 492 previously identified claudin-low signature. In conclusion, by establishing molecular mechanisms 493 by which EMT induces ciliogenesis and thereby controls MaSC and MaTIC biology, our findings 494 reveal a novel aspect of the cell biology critical to the development of the mammary gland and the 495 formation of claudin-low breast cancers. 496 497 498 Materials and Methods

499 500 Animals. Transgenic and wild-type mice were housed and handled in accordance with protocols 501 approved by the Animal Care Committees of the Massachusetts Institute of Technology (USA), 502 the University of Iowa (USA) and the University of Rennes (France). Slug-IRES-YPF and Glis2 503 mutant mice were generated and selected as previously described (Guo et al., 2012, Kim et al., 504 2008). C57BL/6J and NOD SCID animals were obtained from the Jackson laboratory (stock 505 numbers 000664 and 001303).

506 Human samples. Patient reduction mammoplasty tissue samples and breast tumor samples were 507 obtained in compliance with all relevant laws. Breast cancer patients (n = 62) were diagnosed and 508 treated at the Centre Eugène Marquis between 2013 and 2018, none received chemotherapy, 509 endocrine therapy, or radiation therapy prior to surgery. Tissues were collected by a pathologist 510 after resection by a surgeon. Some fragments were paraffin-embedded (normal tissues and 511 tumors), other fragments were flash-frozen (tumors), and other fragments were dissociated within 512 2 h after surgical resection (tumors). Briefly, breast tumor pieces were weighed and cut into small 513 fragments (< 2 mm3), which were then dissociated with a tumor dissociation kit (Human) using a 514 mechanical–chemical–mechanical cycle in a gentleMACS Dissociator (Miltenyi Biotec), according 515 to the manufacturer’s recommendations. Next, a cycle of mechanical–chemical–mechanical 516 dissociation was performed and dissociated cells were resuspended in RPMI. Macroscopic pieces 517 were removed using a Corning® cell strainer (70 μm). Tumor cells were then washed twice in 518 RPMI (20 ml) and counted using a hemocytometer. Viable cells were then cryopreserved.

519 Primary mouse mammary epithelial cell isolation and FACS. Mammary glands from 10-16 520 weeks old C57BL/6J females were minced and dissociated using collagenase/hyaluronidase bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

521 (STEMCELL Technologies) diluted (1:10) in DMEM/F-12 medium at 37°C for 5 h under constant 522 agitation. The dissociated glands were spun down at 450 x g for 5 min and resuspended in 523 ammonium chloride solution (STEMCELL Technologies) diluted (4:1) in HBSS buffer 524 supplemented with 10 mM HEPES and 2 % FBS (HF buffer). The samples were spun down and 525 resuspended in warm Trypsin-EDTA (Stem Cell Technologies) by pipetting for 3 min. Trypsin was 526 inactivated by addition of HF buffer. The digested samples were spun down and further digested 527 with dispase solution (STEMCELL Technologies) supplemented with 0.1 mg/mL DNase I for 1 528 min. Cell suspensions were diluted with HF buffer and filtered through a 40 µm cell strainer to 529 collect single cells. To separate various cell populations, single cells were stained with a Live- 530 Dead fixable violet dye and antibodies against TER-119, CD31, CD45 (PE/Cy7, Biolegend); CD24 531 (PE, Biolegend); CD49f (APC, Biolegend). Stained cells were sorted on a FACSAria II and FACS 532 Fusion sorters.

533 2D cell culture and treatments. Primary mammary epithelial cells were cultured in EpiCult-B 534 mouse medium (STEMCELL Technologies) supplemented with proliferation supplements, 10 535 ng/mL recombinant human epidermal growth factor, 10 ng/mL recombinant human basic fibroblast 536 growth factor, 4 μg/mL heparin, and penicillin/streptomycin. HMLE cells were cultured in a 1:1 537 mixture of DMEM/F-12 supplemented with 10 % FBS, 0.01 mg/mL insulin, 0.48 µg/mL 538 hydrocortisone and complete MEGM supplemented with bovine pituitary hormone (Lonza). For 539 activating tetracycline-inducible Snail and Zeb1 expression, cells were grown with 1 µg/mL 540 doxycycline hyclate (Sigma-Aldrich) in media. For all ciliogenesis assays, cells were grown until 541 high confluence and DMEM/F12 was used to serum starve the cells. For FGFR1 inhibition, cells 542 were treated with SU5402 (Sigma-Aldrich) in DMEM/F12 medium.

543 Mammary organoid culture. Matrigel organoid culture was performed as described previously 544 (Guen et al., 2017). Briefly, freshly isolated mammary epithelial cells or transduced cells were 545 cultured in complete Epicult-B medium (STEMCELL Technologies) containing 5 % matrigel 546 (Corning), 5 % heat-inactivated FBS, 10 ng/mL EGF, 10 ng/mL FGF, 4 µg/mL heparin, 5 µM Y- 547 27632. Cells were seeded at 2000 cells/well in 96-well ultralow attachment plates (Corning). 548 Organoids were counted 7-14 days after seeding. For FGFR1 inhibition, SU5402 was added to 549 the medium at 10 µM.

550 Mammosphere and tumorsphere culture. Cells were cultured in complete Mammocult medium 551 (STEMCELL Technologies) containing 4 µg/mL heparin, 0.48 µg/mL hydrocortisone, 1% methyl 552 cellulose. Non-transformed cells were seeded at 1000 cells/well and transformed cells at 150 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

553 cells/well in 96-well ultralow attachment plates (Corning). Spheres were counted 7-14 days after 554 seeding.

555 Mammary gland whole-mount analysis. Glands were fixed in Carnoy’s fixative (100% EtOH, 556 chloroform, glacial acetic acid; 6:3:1) for 4 hours, washed in 70% ethanol for 15 min and gradually 557 rehydrated in 70%, 35%, 15% ethanol baths for 5 min each, and stained with carmine alum 558 overnight at room temperature. Glands were washed with sequential 5 min washes in 50%, 70%, 559 95%, and 100% ethanol, cleared in xylene overnight and stored in methyl salicylate until analysis 560 using a Nikon macroscope. Images were processed with ImageJ.

561 Tumorigenesis assay. For orthotopic cell implantations, tumor cells were resuspended in a 1:1 562 mixture of complete MEGM medium with matrigel. Cells in 20 µL were injected bilaterally into 563 inguinal mammary fat pads of 8-weeks-old NOD SCID females. Tumors were resected 8 weeks 564 post-implantation and their mass was determined to establish the tumor burden per animal.

565 Immunohistochemistry, Immunofluorescence and image analysis. 5 µm paraffin tissue 566 sections of formalin-fixed mammary glands and breast tumors were stained following the RUO 567 DISCOVERY Universal staining procedure (Roche) using a Discovery ULTRA staining module. 568 Cells fixed in 4% paraformaldehyde for 10 min on glass coverslips were permeabilized with 0.3% 569 Triton X100 for 10 min and blocked with 5% goat serum for 1 h before staining 1 h with primary 570 antibodies. Organoids and spheres fixed in 4% paraformaldehyde for 30 min were permeabilized 571 with 0.3% Triton X100 for 30 min and blocked with 5% goat serum for 1 h 30 before staining 572 overnight at 4°C with primary antibodies. The following primary antibodies were used: Arl13b 573 (NeuroMab 73- 287, 1:100), YFP (Cell Signaling 2956, 1:100), Slug (Cell Signaling 9585, 1:100), 574 acetylated- (Cell Signaling 5335, 1:500), g-tubulin (Sigma T5326, 1:50), SMA (Abcam 575 ab21027, 1:100), E-cadherin (Cell Signaling 3195, 1:200), Cep170 (Fisher Scientific 10383523, 576 1:200), Krt8 (DSHB TROMA-I-s, 1 :1000), Krt14 (Biolegend 905301, 1:1000), Claudin 4 (Fisher 577 Scientific 10303233, 1:200), Claudin 7 (Fisher Scientific 10537403, 1:200), Large T (Santa Cruz 578 sc-20800, 1:200), VIM (Dako M0725, 1:200), TSPAN8 (Personal gift from Jane Visvader, 1:500). 579 The following secondary antibodies were used: anti-mouse 488 (Life Technologies A11001, 580 1:500), anti-mouse 546 (Life Technologies A11003, 1:500), anti-mouse IgG1 647 (Life 581 Technologies A21240, 1:500), anti-mouse IgG2A 488 (Life Technologies A21131, 1:500), anti- 582 rabbit 488 (Life Technologies A21206, 1:500), anti-rabbit 546 (Life Technologies A11010, 1:500), 583 anti-rabbit 647 (Life Technologies A31573, 1:500), anti-rat 568 (Life Technologies A11077, 1:500). 584 Mounted coverslips with cells were examined using Deltavision Olympus X71 microscopes. Z- bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

585 stacks were deconvolved (Softworx) and processed with ImageJ. Organoids and spheres were 586 embedded in low-melting point agarose and analyzed using a Light sheet Z1 Zeiss microscope. 587 Z-stacks were analyzed with Imaris and ImageJ.

588 Ubiquitination assay. HMLE variants were washed with PBS prior to protein extraction with lysis 589 buffer (PBS, protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail (Sigma- 590 Aldrich), 0.1 % NP-40, 100 μM PR-619). The pull-down of ubiquitinated proteins from protein 591 extracts was performed with TUBE 2 agarose beads (LifeSensors), following the manufacturer’s 592 instructions. Briefly, protein extracts were incubated for 30 minutes on uncoupled agarose to 593 remove non-specific binding and 2 hours on equilibrated TUBE 2 agarose on a rotating wheel. 594 Beads were washed 3 times with lysis buffer. Proteins were heat-denatured and eluted in laemmli 595 buffer and analyzed by western blot.

596 Western blot experiments and cell cycle analysis. These experiments were conducted using 597 standard procedures as described previously (36, 37). Western blots were performed using the 598 primary antibodies against Snail (Cell Signaling 3879, 1:500), Twist (Abcam ab50887, 1:200), 599 Zeb1 (Santa Cruz Biotechnology sc-25388, 1:300), E-cadherin (Cell Signaling 3195, 1:1000), 600 Vimentin (Dako M0725, 1:1000), Fibronectin (BD Biosciences 610077, 1:1000), N-cadherin (BD 601 Biosciences 610920, 1:1000), KIF3A (Proteintech 13930-1- AP, 1:1000), IFT20 (Proteintech 602 13615-1-AP, 1:100), FGFR1 (Abcam ab76464, 1:1000), BBS11 (Proteintech 10326-1-AP, 603 1:1000), HA (Roche 11867423001, 1:1000), RFP (Rockland 600-401-379S, 1:1000), AKT (Cell 604 Signaling 9272, 1:1000), pAKT (Cell Signaling 4060, 1:1000), MEK (Cell Signaling 9122, 1:1000), 605 pMEK (Cell Signaling 9121, 1:1000), Ub (P4D1) (Santa cruz sc-8017, 1:300), (Sigma-Aldrich 606 20-33, 1:1000) and secondary antibodies HRP-coupled anti-mouse or anti-rabbit (GE Healthcare 607 and Jackson Laboratories, 1:5000). The cell cycle analysis was performed using HMLE cells that 608 were fixed in 70% ethanol for 1 h on ice. DNA was stained with Propidium Iodide/RNAse staining 609 buffer (BD Biosciences) for 15 min. DNA content of 30,000 cells for each condition was determined 610 using a BD Fortessa X20 flow cytometer and the FlowJo software.

611 Quantitative Real Time-PCR. Total RNA was isolated using the PicoPure RNA Isolation Kit 612 (Thermo Fisher Scientific) for mouse cells or the RNeasy kit (Qiagen) for human cells, and cDNAs 613 were generated with random primers and SuperScript III Reverse Transcriptase (Life 614 Technologies). Real-time quantitative PCR reactions were performed with StepOnePlus Real- 615 Time PCR system (Life Technologies) and 7900HT Fast Real-Time PCR System (Applied 616 Biosystem) using SYBR Green PCR Master Mix (Life Technologies) and gene-specific primers. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

617 Relative expression levels were normalized to GAPDH. Primers used for qPCR analysis are the 618 following:

619 Cdh1_fw CAGTGAAGCGGCATCTAAAGC

620 Cdh1_rev TTGGATTCAGAGGCAGGGTCG

621 Zeb1_fw CACAGTGGAGAGAAGCCATAC

622 Zeb1_rev CACTGAGATGTCTTGAGTCCTG

623 Slug_fw CTCACCTCGGGAGCATACAG

624 Slug_rev GACTTACACGCCCCAAGGATG

625 Snai1_fw CACACTGGTGAGAAGCCATTC

626 Snai1_rev CTTGTGGAGCAAGGACATGCG

627 Krt14_fw CAGCAAGACAGAGGAGCTGAACC

628 Krt14_rev CCAGGGATGCTTTCATGCTGAG

629 Krt18_fw GGTCTCAGCAGATTGAGGAGAG

630 Krt18_rev CCAAGTCAATCTCCAAGGTCTGG

631 FGFR1_fw GGAGGCTACAAGGTCCGTTA

632 FGFR1_rev TGCAGGTGTAGTTGCCCTTG

633 CDH1_fw CAGTCAAAAGGCCTCTACGG

634 CDH1_rev CAGAAACGGAGGCCTGATGG

635 VIM_fw GGAAATGGCTCGTCACCTTC

636 VIM_rev GAAATCCTGCTCTCCTCGCC

637 GLI1_fw CACAAGTGCACGTTTGAAGGG

638 GLI1_rev CATGTATGGCTTCTCACCCG

639 CDH11_fw CGCAGAGCGTATACCAGATG

640 CDH11_rev CCTCCTGTGTTTCATAGTCCG bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

641 TRIM32_fw CAGTTAACGTGGAAGATTCC

642 TRIM32_rev GAGGCACTGCTGGATATTGG.

643

644 Plasmids, lentivirus production, CRISPR mutations. KIF3A, IFT20, TRIM32 guide RNAs were 645 selected from http://crispr.mit.edu/ and https://chopchop.cbu.uib.no/ websites and cloned into 646 lentiCRIPRV2 plasmids containing, either a puromycin-resistance gene, or a blasticidin-resistance 647 gene designed to replace the puromycin one. GLIS2 full-length and C-terminal (c. 1_444del) were 648 amplified from a Precision LentiORF GLIS2 plasmid (Horizon Discovery) and cloned in frame with 649 3xHA or dTomato tags in a pEF_BSD lentiviral plasmid through GIBSON cloning. The following 650 primers were used:

651 CRISPR

652 sgCTL_fw GCTGATCTATCGCGGTCGTC

653 sgCTL_rev GACGACCGCGATAGATCAGC

654 sgKIF3A_fw GAAATCAATGTGCTACAAAC

655 sgKIF3A_rev GTTTGTAGCACATTGATTTC

656 sgIFT20_fw CCAGCAGACCATAGAGCTGA

657 sgIFT20_rev TCAGCTCTATGGTCTGCTGG

658 sgTRIM32#1_fw CACCGGCGGACACCATTGATGCTAC

659 sgTRIM32#1_rev AAACGTAGCATCAATGGTGTCCGCC

660 sgTRIM32#2_fw CACCGAACTCGTCTGCGGGAACTTA

661 sgTRIM32#2_rev AAACTAAGTTCCCGCAGACGAGTTC

662 sgTRIM32#3_fw CACCGGTCTGCCCCGGCAATTCTGC

663 sgTRIM32#3_rev AAACGCAGAATTGCCGGGGCAGACC bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

664 sgTrim32#1_fw CACCGGCGGACGCCATTGATGCTGC

665 sgTrim32#1_rev AAACGCAGCATCAATGGCGTCCGCC

666 sgmTrim32#3_fw CACCGGGCTGCCTCGGCAGTTCTGC

667 sgmTrim32#3_rev AAACGCAGAACTGCCGAGGCAGCCC

668 HA-GLIS2 cloning:

669 1_GTGTCGTGAgccaccatggtgTACCCATACGATGTTCCTGAC

670 2_GTCAGGAACATCGTATGGGTAcaccatggtggcTCACGACAC

671 3_GACGTTCCAGATTACGCTCACTCCCTGGACGAGCCG

672 4_CGGCTCGTCCAGGGAGTGAGCGTAATCTGGAACGTC

673 5_GACGTTCCAGATTACGCTTTCCTTACCCCTCCCAAGGAC

674 6_GTCCTTGGGAGGGGTAAGGAAAGCGTAATCTGGAACGTC

675 7_CTCAAACCGGCTGTGGTGAACGGATCCGGCGCAACAAACTTC

676 8_GAAGTTTGTTGCGCCGGATCCGTTCACCACAGCCGGTTTGAG

677 GLIS2Cter cloning:

678 1_ CTCAAACCGGCTGTGGTGAACGGATCCGGCGCAACAAACTTC

679 2_GAAGTTTGTTGCGCCGGATCCGTTCACCACAGCCGGTTTGAG

680 3_ctctggttatgtgtgggagggctaagaattcgttccggagtcgtcg

681 4_cgacgactccggaacgaattcttagccctcccacacataaccagag

682 5_ggatctggagcaacaaacttcACCTTCCTTACCCCTCCCAAGGAC

683 6_GTCCTTGGGAGGGGTAAGGAAGGTgaagtttgttgctccagatcc bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

684 Lentiviruses were produced in 293FT cells after their transfection with lentiCRISPR or 685 pEF_BSD_HA-GLIS2 or pEF_BSD_dTomGLIS2Cter (transfer) in combination with psPAX2 686 (packaging) and pMD2.G (envelope) plasmids, using Lipofectamine 2000 (Life Technologies) 687 according to the manufacturer’s instructions. Supernatants containing lentiviruses were collected 688 48 and 72 h post-transfection. Primary mammary epithelial cells, HMLE and HMLER cells growing 689 in a monolayer were transduced with the supernatants-containing viruses in the presence of 8 690 µg/mL polybrene. Transduced cells were selected with 2 µg/mL puromycin (GIBCO) or 8 µg/mL 691 blasticidin (GIBCO). HMLE KIF3A-/- and IFT20-/- clones were selected by FACS.

692 Gene expression and ChIP-sequencing analyses. For gene expression analysis in HMLE and 693 HMLER cells, total RNA was extracted from highly confluent cells. 25 ng for each of the two 694 biological replicates per condition was used to generate libraries for whole-transcriptome analysis 695 following High Throughput 3’ Digital Gene Expression library preparation. Libraries were 696 sequenced on an Illumina Nextseq500. Raw sequence reads were collapsed by sequence identity 697 and UMI (Unique Molecular Identifier) and aligned to the (UCSC hg19 build) using 698 TopHat v. 2.0.4. Genic read counts were quantified using the End Sequence Analysis Toolkit 699 (ESAT v1). Count normalization and differential analyses were conducted using the DESeq 700 package (R/Bioconductor) and genes with q-value < 0.05 and absolute fold change greater than 701 2 were tagged as differentially expressed. Row-normalized heatmaps for differentially expressed 702 genes were created using Heatplus (R/Bioconductor). Gene set enrichment analyses (GSEA) 703 were performed with the GSEA platform of the Broad Institute 704 (http://www.broadinstitute.org/gsea/index.jsp). For gene expression analysis in breast tumors, 705 total RNA was extracted from frozen sections of breast tumor samples using the Nucleospin RNA 706 set for Nucleozol kit according to the manufacturer’s protocol (Macherey-Nagel). Libraries were 707 prepared using a modified version of the Takara SMARTer Stranded Total RNA-Seq Kit - Pico 708 Input Mammalian kit (Hendricks et al., in preparation). In brief, 100 ng of RNA at 10 ng/μL was 709 sonicated using RL230 Covaris sonicator (Covaris Inc) and resulting material was confirmed using 710 a Fragment Analyzer (Agilent). 10 ng of each sonicated sample was prepared using the pico input 711 kit as for FFPE samples using a 1:8 volume reduction on the STP MosquitoHV. Final libraries 712 were validated by Fragment Analyzer and qPCR prior to sequencing on a NextSeq500 with 50nt 713 single end reads. Reads were mapped to the human genome (USCS hg19 build) using TopHat v. 714 2.1.0 and transcript abundance was determined using HTSeqCount v. 0.9.1. Molecular subtyping, 715 PAM50 and Claudin-low, was performed using the Genefu v.2.20.0 R package 716 (http://www.pmgenomics.ca/bhklab/software/genefu). For gene expression analysis in mouse bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

717 luminal cells and MaSC-basal cells, raw sequencing data for GSE60450 were downloaded from 718 the GEO/SRA repository (ftp-trace.ncbi.nlm.nih.gov/sra) and reads were mapped to the UCSC 719 mm9 mouse genome build (genome.ucsc.edu) using RSEM. Targeted pairwise differential 720 expression analyses between basal and luminal samples were conducted using EBSeq v1.4.0 721 with median normalization. All RNA-seq analyses were conducted in the R Statistical 722 Programming language. For gene expression analysis across breast tumor subtypes the pipeline 723 from Fougner et al (30) was used on the TCGA-BRCA Dataset 724 (github.com/clfougner/ClaudinLow). For ChIP-sequencing analysis, previously reported genome- 725 wide murine Snail binding sites elucidated using ChIP-seq were obtained from the Gene 726 Expression Omnibus (GSE61198). Mouse mm10 (NCBI Build 38/GRCm38 assembly) loci were 727 translated to corresponding mm9 (NCBI Build 37 assembly) loci using the UCSC liftOver utility 728 (command-line version timestamped 20170321). Peaks were annotated and labeled with 729 corresponding genomic features using R package ChIPseeker (ver. 1.22.1) and UCSC mm9 730 knownGene annotation. Peaks within +/- 3000bp of transcription sites were annotated as 731 promoter-associated peaks. Mouse gene symbols were mapped to human equivalents using 732 homology information from the Mouse Genome Informatics portal (MGI, informatics.jax.org) and 733 promoter peaks from ChIP-seq analysis were subsequently associated with the 47 candidate 734 ciliogenesis inducers along with differential gene expression results from Snail v. CTL RNA-seq 735 analysis.

736 Statistical analysis. Prism was used to analyze data, draw graphs and perform statistical 737 analyses. Data are presented as the mean ± standard errors of mean (s.e.m.). Statistical analyses 738 were carried out by Student’s t-test unless otherwise specified. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 739 were considered significant.

740 Data availability. RNA-sequencing data have been deposited in the Gene Expression Omnibus 741 (GEO) under accession code GSE160549. Previously published data that were reanalyzed are 742 available under the accession numbers GSE60450 and GSE61198.

743 744 Supplementary Materials 745 746 Fig. S1: EMT program activation and ciliogenesis. 747 Fig. S2: EMT programs induce FGFR1 expression and FGF signaling controls MaSC stemness. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

748 Fig. S3: GLIS2 inhibition occurs at the post-transcriptional level and TRIM32 knockout does not 749 affect the M-like status of HMLE cells. 750 Fig. S4: GLIS2Cter expression does not affect the M-like status of HMLE cells or primary 751 ciliogenesis but affects cilium-signaling. 752 Fig. S5: EMT-dependent primary ciliogenesis results in GLIS2 inhibition in transformed cells and 753 GLIS2Cter expression does not affect EMT and ciliogenesis but affects ciliary signaling. 754 Fig. S6: Claudin-low tumors express low levels of epithelial markers, display primary cilia, and 755 express high levels of GLIS2 target genes. 756 Table S1: Snail direct target genes. 757

758 References

759

760 1. N. Y. Fu, E. Nolan, G. J. Lindeman, J. E. Visvader, Stem Cells and the Differentiation 761 Hierarchy in Mammary Gland Development. Physiol Rev 100, 489-523 (2020). 762 2. S. A. Mani et al., The epithelial-mesenchymal transition generates cells with properties of 763 stem cells. Cell 133, 704-715 (2008). 764 3. W. Guo et al., Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 765 148, 1015-1028 (2012). 766 4. X. Ye et al., Distinct EMT programs control normal mammary stem cells and tumour- 767 initiating cells. Nature 525, 256-260 (2015). 768 5. M. M. Wilson, R. A. Weinberg, J. A. Lees, V. J. Guen, Emerging Mechanisms by which 769 EMT Programs Control Stemness. Trends Cancer 6, 775-780 (2020). 770 6. M. Nassour et al., Slug controls stem/progenitor cell growth dynamics during mammary 771 gland morphogenesis. PLoS One 7, e53498 (2012). 772 7. S. Phillips et al., Cell-state transitions regulated by SLUG are critical for tissue regeneration 773 and tumor initiation. Stem Cell Reports 2, 633-647 (2014). 774 8. N. Harbeck et al., Breast cancer. Nat Rev Dis Primers 5, 66 (2019). 775 9. E. L. McCoy et al., Six1 expands the mouse mammary epithelial stem/progenitor cell pool 776 and induces mammary tumors that undergo epithelial-mesenchymal transition. J Clin 777 Invest 119, 2663-2677 (2009). 778 10. A. P. Morel et al., EMT inducers catalyze malignant transformation of mammary epithelial 779 cells and drive tumorigenesis towards claudin-low tumors in transgenic mice. PLoS Genet 780 8, e1002723 (2012). bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

781 11. V. J. Guen et al., EMT programs promote basal mammary stem cell and tumor-initiating 782 cell stemness by inducing primary ciliogenesis and Hedgehog signaling. Proc Natl Acad 783 Sci U S A, (2017). 784 12. V. J. Guen, C. Prigent, Targeting Primary Ciliogenesis with Small-Molecule Inhibitors. Cell 785 Chem Biol, (2020). 786 13. B. Elenbaas et al., Human breast cancer cells generated by oncogenic transformation of 787 primary mammary epithelial cells. Genes Dev 15, 50-65 (2001). 788 14. J. M. Neugebauer, J. D. Amack, A. G. Peterson, B. W. Bisgrove, H. J. Yost, FGF signalling 789 during embryo development regulates cilia length in diverse epithelia. Nature 458, 651- 790 654 (2009). 791 15. A. Honda et al., FGFR1-mediated protocadherin-15 loading mediates cargo specificity 792 during intraflagellar transport in inner ear hair-cell kinocilia. Proc Natl Acad Sci U S A 115, 793 8388-8393 (2018). 794 16. A. D. Jenks et al., Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms 795 in Cancer. Cell Rep 23, 3042-3055 (2018). 796 17. A. C. Pond et al., Fibroblast growth factor receptor signaling is essential for normal 797 mammary gland development and stem cell function. Stem Cells 31, 178-189 (2013). 798 18. B. T. Spike et al., A mammary stem cell population identified and characterized in late 799 embryogenesis reveals similarities to human breast cancer. Cell Stem Cell 10, 183-197 800 (2012). 801 19. S. C. Goetz, K. V. Anderson, The primary cilium: a signalling centre during vertebrate 802 development. Nat Rev Genet 11, 331-344 (2010). 803 20. A. J. Firestone et al., Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic 804 . Nature 484, 125-129 (2012). 805 21. M. Attanasio et al., Loss of GLIS2 causes nephronophthisis in humans and mice by 806 increased apoptosis and fibrosis. Nat Genet 39, 1018-1024 (2007). 807 22. Y. S. Kim et al., Kruppel-like protein Glis2 is essential for the maintenance of 808 normal renal functions. Mol Cell Biol 28, 2358-2367 (2008). 809 23. D. Singer et al., Prominin-1 controls stem cell activation by orchestrating ciliary dynamics. 810 EMBO J 38, (2019). 811 24. P. Holmfeldt et al., Functional screen identifies regulators of murine hematopoietic stem 812 cell repopulation. J Exp Med 213, 433-449 (2016). 813 25. H. Ramachandran et al., Interaction with the Bardet-Biedl gene product TRIM32/BBS11 814 modifies the half-life and localization of Glis2/NPHP7. J Biol Chem 289, 8390-8401 (2014). bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

815 26. J. F. Reiter, M. R. Leroux, Genes and molecular pathways underpinning . Nat 816 Rev Mol Cell Biol 18, 533-547 (2017). 817 27. F. Zhang et al., Characterization of Glis2, a novel gene encoding a Gli-related, Kruppel- 818 like transcription factor with transactivation and repressor functions. Roles in kidney 819 development and neurogenesis. J Biol Chem 277, 10139-10149 (2002). 820 28. N. Y. Fu et al., Identification of quiescent and spatially restricted mammary stem cells that 821 are hormone responsive. Nat Cell Biol 19, 164-176 (2017). 822 29. A. Prat et al., Phenotypic and molecular characterization of the claudin-low intrinsic 823 subtype of breast cancer. Breast Cancer Res 12, R68 (2010). 824 30. C. Fougner, H. Bergholtz, J. H. Norum, T. Sorlie, Re-definition of claudin-low as a breast 825 cancer phenotype. Nat Commun 11, 1787 (2020). 826 31. M. Rozycki et al., The fate of the primary cilium during myofibroblast transition. Mol Biol 827 Cell 25, 643-657 (2014). 828 32. X. Liu et al., Wdpcp promotes epicardial EMT and epicardium-derived cell migration to 829 facilitate coronary artery remodeling. Sci Signal 11, (2018). 830 33. Y. H. Kim et al., A complex of BBS1 and NPHP7 is required for cilia motility in zebrafish. 831 PLoS One 8, e72549 (2013). 832 34. Y. S. Kim, H. S. Kang, A. M. Jetten, The Kruppel-like zinc finger protein Glis2 functions as 833 a negative modulator of the Wnt/beta-catenin signaling pathway. FEBS Lett 581, 858-864 834 (2007). 835 35. B. Li et al., Increased hedgehog signaling in postnatal kidney results in aberrant activation 836 of nephron developmental programs. Hum Mol Genet 20, 4155-4166 (2011). 837 36. V. J. Guen et al., CDK10/cyclin M is a protein kinase that controls ETS2 degradation and 838 is deficient in STAR syndrome. Proc Natl Acad Sci U S A 110, 19525-19530 (2013). 839 37. V. J. Guen et al., STAR syndrome-associated CDK10/Cyclin M regulates actin network 840 architecture and ciliogenesis. Cell Cycle 15, 678-688 (2016). 841

842 Acknowledgments

843 844 General: We thank Xavier Pinson, Roselyne Viel and Aurore Nicolas for help in development of 845 light sheet microscopy, immunohistochemistry methods, and processing of breast tumor samples, 846 respectively; Julia Fröse for providing HMLE cells; Feng Zhang for providing the LentiCRISPR v2 847 plasmid (Addgene plasmid no. 52961); The Swanson and Biosit Biotechnology Centers, including 848 MRic, H2P2, CRB Santé, Arche, the MIT BioMicro Center and Flow cytometry core facilities for bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

849 technical support. Funding: This work was supported by Fondation MIT, the Koch Institute 850 Support (core) Grant P30-CA14051 from the National Cancer Institute, Fondation ARC, 851 Cancéropôle Grand Ouest, Université de Rennes 1. M.M.W. was supported by the David H. Koch 852 Graduate Fellowship. A.M.J. research was supported by the Intramural Research Program of the 853 NIEHS, NIH Z01-ES-101585. V.J.G. was supported by Postdoctoral Fellowships from the Koch 854 Institute and Fondation ARC. Author contributions: M.M.W., C.C., J.A.L., V.J.G. designed 855 research; M.M.W., C.C., S.M., M.L.G., A.S., Q.D., T.E.C., S.M., V.J.G. performed research, Q.D., 856 F.G., P.T., A.M.J., J.E.V., R.A.W., M.A., C.P. contributed new reagents/analytic tools, M.M.W., 857 C.C., M.L.G, A.S., A.B., J.A.L., V.J.G., analyzed data. M.M.W., J.A.L., V.J.G. wrote the paper. 858 Competing interests: The authors declare no conflict of interest. Data and materials 859 availability: All data needed to evaluate the conclusions in the paper are present in the paper 860 and/or the Supplementary Materials. 861

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878 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424695; this version posted December 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1: EMT programs induce primary ciliogenesis at intermediate 879 Figures transition states in the mammary gland A Human mammary gland DNA DNA Ecad Arl13b Slug Slug Slug gTub Tub Slug g Ecad Arl13b DNA DNA Tub g Ecad Arl13b DNA

800 *** p ≤ 0.001 100 *** p ≤ 0.001 600 80 60 400 40 200 20 0 % Ciliated cells 0 Ecad signal intensity

HMLE Slug - Slug + HMLE Slug - Slug + B + Snail + Zeb1 C Day D + Snail + Zeb1 Day 0 Day 0 4 4 0 0 8 8 12 12 Snail/

0 32 - 190 - 30 + Snail

80 - 80 - % Ciliated cells

Day Snail Zeb1 7 3 + Zeb1 HMLE + *** 8 4 Ecad Vim 4 Ecad Vim 30 ** ** 3 ** 3 ** 20 * ** 2 * 2 8 4 10 * levels levels

1 *** 1 *** % Ciliated cells *** *** ** ** 0 12 Relative protein 0 Relative protein 0 0 2 3 4 Ecad 0 4 8 0 4 8 Day 12 12 DNA Arl13b 880 Day Day 881 Fig. 1: EMT programs induce primary ciliogenesis at intermediate transition states in the 882 mammary gland. (A) Mammary gland sections from healthy human patients were stained for the 883 indicated proteins (inset: 3X, n = 4), E-cadherin expression and the percentage of ciliated cells 884 were quantified. Representative results from 3 independent experiments are shown. (B-D) 885 Epithelial-like HMLE cells were treated with 1 µg/mL of doxycycline to induce Snail or Zeb1 886 expression over 12 days. (B) The morphology of the cells was examined by brightfield microscopy, 887 (C) their expression of the indicated proteins was analyzed by western blot and protein levels were 888 quantified (n = 3, mean ± SEM), (D) their ability to form primary cilia was determined and the 889 percentage of ciliated cells was quantified at the indicated time points (n = 3, mean ± SEM). 890 Representative results from 3 independent experiments are shown. All scale bars: Brightfield 50 891 µm, Immunofluorescence 15 µm. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 892 Figure 2: EMT-TFs activate the expression of positive regulators of cilium assembly to promote primary ciliogenesis HMLE CTLCTLSNAILSnail Day DMSO SU5402 MaSC-Basal cells A C D E F med high CTL Snail HHIPHHIP DNA Lin- CD24 CD49f

MAP1BMAP1B 4 0 8 GLI2GLI2 12 Arl13b FGFR1FGFR1 RGS9RGS9 SPATA6SPATA6 FGFR1 Cep170 PCDHB15PCDHB15 GLI1GLI1 3 3 100 - ROM1ROM1 SYNE1SYNE1 46 - TTLL1TTLL1 Zeb1 FUZ FUZ Actin CEP112CEP112 PARVAPARVA ASAP1ASAP1 EHD3EHD3 SPATA7SPATA7 RAB8BRAB8B FGFR1

CCDC28BCCDC28B + HMLE 100 - CEP170CEP170 2 2 CIB2CIB2 46 - DZIP1LDZIP1L PKD2PKD2 Snail BBIP1BBIP1 Actin S5_E2 S7_E2 S6_M2 S8_M2 RILPL2RILPL2 C21orf2C21orf2 ARL2BPARL2BP Mammary organoid INVSINVS 15 ** S7_E2 S5_E2 S8_M2 S6_M2 CEP19CEP19 PRKACBPRKACB GLI3 −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5 GLI3 1 1 **p ≤ 0.01 BBS9BBS9 60 8 ***p ≤ 0.001 NEK1NEK1 DZIP1DZIP1 10 1.5 1.0 0.5 0.0 0.5 − 1.0 − 1.5 − RAB23RAB23 6 B GABARAPGABARAP * ARL3ARL3 40 IFT52IFT52 4 NDE1NDE1 5 EMT Cilium IFT122IFT122 47 TULP3TULP3 20 ICK ICK 2 signature gene set GSNGSN levels (+ Snail) 0 0 Relative FGFR1 AK1 % Ciliated cells AK1 Cilia length (µm) IFT20IFT20 0 0 0 HIF1AHIF1A PRKAR1APRKAR1A 0 4 8 12 Day DMSOSU5402 DMSOSU5402 G Z-projection Single plan H DMSO DNA Krt8 Krt14 Krt8 Krt14

1.5 * p ≤ 0.05 1.0

0.5

0.0 Ciliogenesis index

DMSOSU5402

DNA Arl13b Slug gTub DNA Arl13b Slug gTub SU5402 *** p ≤ 0.001 256

m2) 64 µ 16 4 1

Cilia size ( 0.25

DMSOSU5402

893 DNA Arl13b Slug 894 Fig. 2: EMT-TFs activate the expression of positive regulators of cilium assembly to promote primary ciliogenesis. (A) Control 895 (CTL) and Snail-expressing (Snail) HMLE cells were grown until high-confluence and serum starved for 24h prior RNA extraction. 896 Gene expression was analyzed by RNA-sequencing. Heat-maps showing the differential expression (DE) genes (q-value ≤ 0.05, 897 Fold-change ≥ 2) between samples are displayed. (B) Venn diagram displaying the overlap between the EMT signature (upregulated 898 genes in Snail-expressing cells relative to CTL cells defined in A) and a curated cilium gene set from MSigDB. 47 genes were found 899 in both gene sets. (C) Heat-map showing the differential expression of the 47 genes between CTL and Snail-expressing HMLE cells. 900 (D) Epithelial-like HMLE cells were treated with doxycycline to induce Snail and Zeb1 expression as described in the legend of Fig. 1 901 and FGFR1 protein levels were analyzed by western blot and quantified (n = 3, mean ± SEM). Representative results from 3 902 independent experiments are shown. (E) Snail-expressing HMLE cells were grown until high-confluence and serum starved in the 903 presence of DMSO or of the FGFR1 inhibitor SU5402 (10 µM) for 24h. Treated cells were stained for the indicated proteins and the 904 percentage of ciliated cells and cilia length was measured (n = 3, mean ± SEM). Representative results from 3 independent experiments 905 are shown (inset: 10X, Scale bar: 15 µm). (F-H) MaSC-enriched basal cells (Lin- CD24med CD49fhigh) from C57BL/6J adult females (≥ 906 10 weeks old) were isolated by FACS using the indicated cell surface markers and plated in 3D. (F) Morphology of mammary organoids 907 was examined by brightfield microscopy. (G) Organoids were stained for the indicated proteins and analyzed by light sheet microscopy. 908 Representative images are shown. Scale bars: upper and lower left panels 100 µm, upper right panel 50 µm and lower right panel 5 909 µm. (H) Organoids were treated with DMSO or with SU5402 (10 µM), stained for the indicated proteins, and analyzed by light sheet 910 microscopy. Ciliogenesis index (Arl13b particles/Slug particles) as well as cilia size were determined for all conditions (n = 3, mean ± 911 SEM). Representative results from 3 independent experiments are shown. Representative images are shown. Scale bar: 50 µm (inset: 912 2.5X). 913 Figure 3: Primary cilia control a BBS11-GLIS2 signaling axis

Enriched in M vs E HMLE + Snail DMSO CilA Depleted in M CilA vs DMSO A 80 B C ** p ≤ 0.01 DMSO CilA 60 FDR = 0.000 FDR = 0.008 40 20 % Ciliated cells 0

CilA DNA Arl13b gTub DMSO S14_M2_TRT S16_M2_TRT D S15_M2_CTR S13_M2_CTR + HA-GLIS2 S16_M2_TRT S14_M2_TRT S13_M2_CTR −1.0 −0.5 S15_M2_CTR 0.0 0.5 1.0 1.5

1.5 1.0 0.5 0.0 0.5 − 1.0 − SNL + HA-GLIS2 CilA CilA E F G SNL + sgCTL sgKIF3A SNL + + SNL CTL + DMSO + CTL DMSO + SNL DMSO + SNL + SNL CTL DMSO + CTL sgKIF3AsgIFT20 Long exp. sgCTLsgKIF3AsgIFT20 sgCTL 135 - 135 - Long exp. 100 - HA-GLIS2ub 100 - sgCTL sgKIF3A sgIFT20 HA-GLIS2ub 80 - 80 - 100 - 58 - HA-GLIS2 KIF3A 58 - Short exp. 80 - Arl13b HA-GLIS2 135 - sgIFT20 60 **p ≤ 0.01 135 - Short exp. 100 - HA-GLIS2ub 22 - IFT20

DNA 40 100 - 80 - 17 - HA-GLIS2ub 80 - 58 - HA-GLIS2 46 - 20 Actin 58 - 46 - Actin HA-GLIS2 % Ciliated cells 0 46 - Actin INPUTS TUBE sgCTL sgIFT20 INPUTS TUBE sgCKIF3A H I J K + HA-GLIS2

SNL + sgCTL sgTRIM32#1 CTL SNL sgCTL sgCTL 80 - 58 - BBS11 CTLCTLSNLSNL SNL+ + sgTRIM32CTLSNLSNLSNL + + sgTRIM32 46 - Actin 135 - Long exp. 100 - HA-GLIS2ub Arl13b sgTRIM32#3 n.s. p > 0.05 80 - 3 * p ≤ 0.05 sgCTL sgTRIM32#1 sgTRIM32#2 sgTRIM32#3 60 80 - 58 - HA-GLIS2 Short exp. 2 BBS11 DNA 40 135 - 58 - 100 - HA-GLIS2 46 - 20 ub 1 Actin 80 -

% Ciliated cells 58 - HA-GLIS2 0 0 (mean grey value) 46 - Actin Relative BBS11 levels CTL SNL sgCTL 914 sgTRIM32#1sgTRIM32#3 INPUTS TUBE 915 Fig. 3: Primary cilia control a BBS11-GLIS2 signaling axis. (A, B) CTL and Snail-expressing HMLE cells were grown 916 until high confluence and serum starved for 24h with DMSO and Ciliobrevin A (CilA). (A) Cells were stained for the 917 indicated proteins and the percentage of ciliated cells was determined. Scale bar: 15 µm (n = 3 mean ± SEM). 918 Representative results from 3 independent experiments are shown. (B) Gene expression was analyzed by RNA- 919 sequencing in both conditions. Heat-maps showing the differentially expressed genes (q-value ≤ 0.05, Fold-change ≥ 920 2) between samples are displayed. (C) A GSEA analysis shows significant enrichment of GLIS2 target genes in the list 921 of upregulated genes in Snail-expressing cells (M) relative to CTL (E) cells (left panel) and depletion in the list of Snail- 922 expressing (M) CilA-treated relative to DMSO-treated cells (right panel) (False discovery rate (FDR) ≤ 0.05). (D) CTL 923 and Snail-expressing (SNL) HMLE cells that express HA-GLIS2 were treated as described above. Ubiquitinated proteins 924 were purified by TUBE pull-down and analyzed by western blot. Representative results from 3 independent experiments 925 are shown. (E) KIF3A and IFT20 knockouts were validated by western blot of extracts from the indicated cells. (F) The 926 impact on primary ciliogenesis was assessed as described in A. Scale bar: 15 µm. (G) The impact on HA-GLIS2 927 ubiquitination was analyzed as described in D. (H) BBS11 expression levels were determined in the indicated cells by 928 western blot analysis and quantified (n = 3 mean ± SEM). Representative results from 3 independent experiments are 929 shown. (I) TRIM32 knockout was validated by western blot of extracts from the indicated cells. (J) The impact on primary 930 ciliogenesis was assessed as described in A. Scale bar: 15 µm. (K) The impact of TRIM32 knockout on HA-GLIS2 931 ubiquitination was analyzed, as described in D, in the indicated cells. Figure 4: GLIS2 is a central regulator of basal MaSC and mammary gland development

Mammospheres 30 p ≤ 0.001 30 30 *** p ≤ 0.001 A B Arl13b C p ≤ 0.001 D CTL Snail *** 20 *** 20 20 DNA

10 g 10 10 Tub Organoids HMLE

Mammospheres 0 Mammospheres 0 0 CTL CTL CTL Snail E F H&E GLIS2Cter GLIS2Cter DNA TSPAN8 Glis2+/+ +/+

* p = 0.0249 Glis2 5 4 *** p ≤ 0.001 80 3 60 2 40 Glis2-/- 1 20 Ductal length (mm) 0 0

Glis2-/- Glis2+/+Glis2-/- TSPAN8 signal intensity/duct Glis2+/+ - / - Glis2

932 933 Fig. 4: GLIS2 is a central regulator of basal MaSC and mammary gland development. (A-C) 934 Mammospheres from Snail-expressing HMLE cells were examined for morphology by brightfield 935 microscopy or for ciliated cells by immunofluorescence for the indicated proteins by light sheet 936 microscopy. Mammosphere-forming capacity was quantified for the indicated cells (n = 3 mean ± 937 SEM). Representative results from 3 independent experiments are shown. Scale bars: 100 µm. 938 (D) Organoid-forming capacity was determined for sorted MaSC-enriched basal cells in which 939 GLIS2Cter was overexpressed (n = 3 mean ± SEM). Representative results from 3 independent 940 experiments are shown. (E) Mammary glands from Glis2+/+ or Glis2-/- female mice (4 weeks old, n 941 ≥ 5) were stained with Carmine Alum and ductal length was analyzed after whole mount 942 preparation. Scale bar: 1 mm. (F) Paraffin sections from the mammary glands were stained with 943 H&E or for the indicated protein. TSPAN8 signal intensity was measured per duct in all ducts of 944 the sections analyzed. Representative images are shown. Scale bars: H&E 100 µm (inset: 2X), 945 immunofluorescence 50 µm. 946 947 948 949 950 951 952 953 Figure 5: The primary cilium-GLIS2 axis controls claudin-low MaTIC stemness and tumorigenicity Tumorspheres 250 p ≤ 0.001 A *** 200 shCTL shEcad 150 100 50 Tumorspheres 0 HMLER

shCTLshEcad B DNA Arl13b gTub Cilia/GLI1/ TSPAN8/ in TS

200 p ≤ 0.001 C *** 150 100 50

Tumorspheres 0

CTL GLIS2Cter D CTL GLIS2Cter 2.0 * p = 0.0164 1.5 1.0 0.5

Tumor Burden (g) Burden Tumor 0.0

CTL 954 GLIS2Cter 955 Fig. 5: The primary cilium-GLIS2 axis controls claudin-low MaTIC stemness and 956 tumorigenicity. (A-C) Tumorspheres from shEcad-expressing HMLER cells were examined for 957 morphology by brightfield microscopy or for ciliated cells by immunofluorescence for the indicated 958 proteins and light sheet microscopy. Tumorsphere-forming capacity was quantified for the 959 indicated cell variants (n = 3 mean ± SEM). Representative results from 3 independent 960 experiments are shown. Scale bars: Brightfield image, 300 µm; and immunofluorescence image, 961 100 µm. (D) Bilateral orthotopic implantations were conducted with HMLER shEcad control (CTL) 962 or GLIS2Cter-expressing cells. Representative mice are shown. Tumor burden per mouse (mean 963 ± SEM) was determined 8 weeks post-implantation with two sites of implantation per mouse and 964 four mice per cell type. 965 966 967 968 969 970 971 972 973 974 row min row max

Type Claudin BL1 Claudin BL2 Others IM LAR Lum rowM min row max MSL TypeUNS Claudin BL1 Claudin FigureBL2 Others 6: Primary cilia mark claudin-low tumor cells in E-M states and high IM LAR Lum M expressionMSL of GLIS2 target genes is a hallmark of claudin-low tumors UNS id A row min row max B Type Claudin DNA Ecad

BL1 IDClaudinSplID#14 SplID#02 SplID#04 SplID#61 SplID#26 SplID#58 SplID#01 SplID#05 SplID#03 SplID#48 SplID#45 SplID#50 SplID#37 SplID#55 SplID#54 SplID#10 SplID#11 SplID#20 SplID#62 SplID#08 SplID#21 SplID#52 SplID#41 SplID#43 SplID#32 SplID#51 SplID#15 SplID#18 SplID#24 SplID#36 SplID#57 SplID#40 SplID#42 SplID#53 SplID#59 SplID#23 SplID#63 SplID#64 SplID#66 SplID#12 SplID#09 SplID#22 SplID#27 SplID#30 SplID#49 SplID#13 SplID#06 SplID#28 SplID#19 SplID#07 SplID#38 SplID#16 SplID#34 SplID#35 SplID#25 SplID#47 SplID#39 SplID#29 SplID#31 SplID#56 SplID#33 SplID#46 SplID#44 BL2 Others H&E Claudin 4 Claudin 7

IM UNS BL1 BL1 UNS M LAR IM BL1 BL1 BL1 BL1 UNS BL1 BL1 UNS BL1 BL2 IM IM UNS IM M M M BL1 BL1 IM IM UNS BL1 M LAR IM LAR UNS LAR Lum Lum Lum BL2 BL2 BL2 BL2 BL2 MSL UNS BL2 M M M UNS IM IM MSL M IM IM UNS IM UNS MSL LAR LAR LAR Type Lum id M

MSL Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin UNSClaudin id B ID SplID#14 SplID#02 SplID#04 SplID#61 SplID#26 SplID#58 SplID#01 SplID#05 SplID#03 SplID#48 SplID#45 SplID#50 SplID#37 SplID#55 SplID#54 SplID#10 SplID#11 SplID#20 SplID#62 SplID#08 SplID#21 SplID#52 SplID#41 SplID#43 SplID#32 SplID#51 SplID#15 SplID#18 SplID#24 SplID#36 SplID#57 SplID#40 SplID#42 SplID#53 SplID#59 SplID#23 SplID#63 SplID#64 SplID#66 SplID#12 SplID#09 SplID#22 SplID#27 SplID#30 SplID#49 SplID#13 SplID#06 SplID#28 SplID#19 SplID#07 SplID#38 SplID#16 SplID#34 SplID#35 SplID#25 SplID#47 SplID#39 SplID#29 SplID#31 SplID#56 SplID#33 SplID#46 SplID#44 CDH1 CLDN3

UNS BL1 BL1 UNS M LAR IM BL1 BL1 BL1 BL1 UNS BL1 BL1 UNS BL1 BL2 IM IM UNS IM M M M BL1 BL1 IM IM UNS BL1 M LAR IM LAR UNS LAR Lum Lum Lum BL2 BL2 BL2 BL2 BL2 MSL UNS BL2 M M M UNS IM IM MSL M IM IM UNS IM UNS MSL LAR LAR CLDN4 Type EPCAM MKI67 CD44

Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin ITGA6 Claudin idSNAI1 Luminal id ALDH1A1CDH1 ESR1CLDN3 PGRCLDN4 ERBB2EPCAM

ID SplID#14 SplID#02 SplID#04 SplID#61 SplID#26 SplID#58 SplID#01 SplID#05 SplID#03 SplID#48 SplID#45 SplID#50 SplID#37 SplID#55 SplID#54 SplID#10 SplID#11 SplID#20 SplID#62 SplID#08 SplID#21 SplID#52 SplID#41 SplID#43 SplID#32 SplID#51 SplID#15 SplID#18 SplID#24 SplID#36 SplID#57 SplID#40 SplID#42 SplID#53 SplID#59 SplID#23 SplID#63 SplID#64 SplID#66 SplID#12 SplID#09 SplID#22 SplID#27 SplID#30 SplID#49 SplID#13 SplID#06 SplID#28 SplID#19 SplID#07 SplID#38 SplID#16 SplID#34 SplID#35 SplID#25 SplID#47 SplID#39 SplID#29 SplID#31 SplID#56 SplID#33 SplID#46 SplID#44 KRT19MKI67 GATA3CD44 UNS BL1 BL1 UNS M LAR IM BL1 BL1 BL1 BL1 UNS BL1 BL1 UNS BL1 BL2 IM IM UNS IM M M M BL1 BL1 IM IM UNS BL1 M LAR IM LAR UNS LAR Lum Lum Lum BL2 BL2 BL2 BL2 BL2 MSL UNS BL2 M M M UNS IM IM MSL M IM IM UNS IM UNS MSL LAR KRT18LAR Type ITGA6 CLDN7SNAI1 OCLNALDH1A1 MUC1ESR1 Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Others Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin Claudin KRT14Claudin Claudin PGR

id low KRT17ERBB2 - KRT5KRT19CDH1 HIF1AGATA3CLDN3 ITGB1KRT18CLDN4 VIMCLDN7EPCAM MMEOCLNMKI67 THY1MUC1CD44

ZEB1KRT14ITGA6 Claudin ZEB2KRT17SNAI1 SNAI2KRT5ALDH1A1 TWIST1HIF1AESR1 TWIST2ITGB1PGR VIMERBB2 ClaudinMMEKRT19 -low THY1GATA3 C KRT18 ZEB1 ZEB2CLDN7 Luminal B #1 (BT31) #2SNAI2 (BT90)OCLN #3 (BT166) TWIST1MUC1 TWIST2KRT14 KRT17 KRT5 HIF1A ITGB1 VIM MME

Vim THY1 ZEB1 ZEB2 SNAI2 TWIST1

Ecad TWIST2 Arl13b Arl13b DNA

D ClaudinLow

OCLN ClaudinLow ClaudinLow CDCA3 CDH1 LAD1 S100A6 OCLN S100A4 ISG15 EPCAM IFITM3 CASP4 PSMB8 CDH1 PSMB9 ITGA6 ADAM8 HLA−G LSP1 EPCAM LTB CLDN7 ClaudinLow IRF1 HLA−DMB HLA−DRA ITGA6 CD74 CLDN4 OCLN HLA−DMA CCL5 Claudin-low HLA−DQA1 matrix_21 ClaudinLow HLA−DQB1 CLDN3 CDH1 CLDN7 HLA−DRB1 4 CCR2 ClaudinLow VCAM1 FCER1G MUC1 EPCAM CLDN4 AIF1 2 Other LILRB3 FCGR2A matrix_16 ClaudinLow matrix_21 ClaudinLow ICAM1 TWIST2 ITGA6 0 C3 CLDN3 CXCL10 4 4 TNFSF13B ClaudinLow −2 ClaudinLow CXCL14 VIM CLDN7 Gene SYCP1 2 MUC1 2 BOK Other Other BBC3 −4 PYCARD THY1 CLDN4 expression IGFBP2 0 0 MGP TWIST2 FGFR1 matrix_21 ClaudinLow PDGFRA SNAI2 CLDN3 CMTM3 −2 4 −2 FBN1 VIM LUM ClaudinLow DCN MUC1 PDGFRL −4 TWIST1 2 Other−4 COL3A1 THY1 COL1A1 SPARC 0 COL1A2 MME TWIST2 ADAMTS2 POSTN SNAI2 NID1 −2 SPON1 SNAI1 VIM CXCL12 DPT TWIST1−4 CFH SPARCL1 ALDH1A1 THY1 MFAP4 COL14A1 LTBP2 MME OGN ZEB1 SNAI2 TSC22D1 CIDEC IGSF10 SNAI1 COL15A1 ZEB2 TWIST1 COL4A1 MATN2 CX3CL1 ALDH1A1 CCL21 ITGB1 MME CCL2 SOCS3 PF4 VIM SNAI1 ZEB1 EFEMP2 MFAP2 COL18A1 MMP14 ALDH1A1 ZEB2 TGFBI SERPINE1 ANGPTL4 OSMR TLR2 ZEB1 ITGB1 975 TGFBR2 ZEB2 976 Fig. 6: Primary cilia mark claudin-low tumor cells in E-M states and high expressionITGB1 of GLIS2 target 977 genes is a hallmark of claudin-low tumors. (A) Human breast tumor fragments from 62 independent 978 patients were flash frozen after surgery. RNA was extracted from sections of frozen tumors and gene 979 expression was analyzed by RNA-sequencing. Expression of a subset of genes was analyzed to 980 discriminate claudin-low tumors from other molecular subtypes. The Heat-map showing the differential 981 expression of the genes is displayed. (B-C) Paraffin sections of breast tumors were stained with H&E or for 982 the indicated proteins. Scale bar: 100 µm (B), 20 µm (C). (D) Heat maps showing the differential expression 983 of GLIS2 target genes (left panel), and the reference list of genes used to mark claudin-low tumors (right 984 panel), in breast cancers are displayed, along with their ability to segregate claudin-low tumors from other 985 breast tumors. 986 SupplementaFigurery MaterialsS1: EMT program activation and ciliogenesis 987 A Mouse mammary gland Duct Terminal-end bud DNA Arl13b DNA Arl13b YFP SMA YFP Sma YFP mouse YFP - IRES - Slug

B HMLE + TetO-Snail + TetO-Zeb1

E-like cells E-like cells

988 989 Fig. S1: EMT program activation and ciliogenesis. (A) Mammary gland sections from Slug- 250419_DS.fcs 250419_DZ.fcs P2 P2 990 IRES-YFP mice (8 weeks8735 old) were stained for the 8358indicated proteins. Representative images of 991 a duct and of a remaining terminal-end bud (TEB) are shown. Scale bar: 15 µm (inset: 1.5X). (B) 992 Epithelial-like (E-like, CD44lo CD24hi) cells from the indicated un-induced HMLE variants were 993 isolated by FACS using the indicated cell surface markers. 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 Figure S2: EMT programs induce FGFR1 expression and FGF signaling 1015 controls MaSC stemness A HMLE B HMLE D CTL Snail Tub CTL Snail g Arl13b 245 - FN Ecad DNA 135 - Ncad

135 - Ecad Tub

g 100 - Arl13b 58 -

tub Vim -

DNA Ac 180 - Zeb1 *** p ≤ 0.001 50 40 32 - Snail 30 20 25 - Twist 10 22 -

Ciliated cells (%) 0 46 - Actin CTL Snail

Gene Peak_Locus Peak_score C Fgfr1 chr8:26628989-26629969 967,08 Syne1 chr10:5151497-5152354 878,99 Candidate Rom1 chr19:9002887-9005351 811,54 ciliogenesis Spata6 chr4:111392399-111393074 344,24 regulators Hhip chr8:82581190-82582094 288,3 chr13:100217303-100217763, 68.57, Map1b chr13:100284691-100287186 1094.65

Id1 chr2:152561661-152562935 808,16 Known chr12:34641660-34643001, 663,31, Snail target Twist1 chr12:34643028-34644057 192,88 genes Zeb1 chr18:5591087-5592397 580,58

Clonal Mixed E F SU5402 (µM) DMSO SU5402 H 1.5 I 128 *** (10 µM) 0.1 DMSO 1 64 10 ** *** 1.0 + DMSO 32 58 - + SU5402 16 ** pAKT dTom 97.27 % 8 0.5 CTL +Snail DMSOSnail 4 135 - 58 - AKT Ecad 2 0.0 1 100 - (Short exp.) Phosphorylated/Total Phosphorylated/Total Relative protein levels 135 - J 46 - pMEK 20 AKT **p ≤ 0.01 MEK (Long exp.) 300 100 - *p ≤ 0.05 15 Relative RNA levels Relative RNA 0.03 46 - G

Fold change Snail/CTL MEK 200 58 - Vim *** 10 n.s. 46 - Actin 5 p > 0.05 100 VIM 46 - 1500 cells FGFR1CDH1 % G2/M cells Actin 0 Organoids per 0

+ FBS DMSO10 µM20 µM -FBS +DMSO 1016 -FBS + SU5402 1017 Fig. S2: EMT programs induce FGFR1 expression and FGF signaling controls MaSC stemness. (A) Control (CTL) and Snail-expressing HMLE cells 1018 were grown until high-confluence, serum starved for 24 h, and stained for the indicated proteins. The percentage of ciliated cells was measured (n = 3, 1019 mean ± SEM). Representative results from 3 independent experiments are shown (Scale bar: 15 µm). (B) Western blot analysis of the protein expression 1020 levels of EMT markers in CTL or Snail-expressing cells. (C) Snail direct target genes were identified by CHIP-sequencing analysis using mammary tumor 1021 cells (GSE61198). Genes that were identified in Fig. 2C as putative ciliogenesis inducers were found in the list (Fgfr1, Syne1, Rom1, Spata6), along with 1022 known Snail direct targets (Id1, Id2, Twist1, Zeb1, Vtn). TSS/Peak coordinates are indicated for each gene, as well as the peak score, which is a normalized 1023 value of the read counts that were identified for each gene. (D) Snail-binding motifs associated to Snail dependent regulation of transcription (E-boxes or 1024 a binding motif associated to Snail-dependent activation of transcription38) were found in close proximity (within the 400 bp upstream) or in the Fgfr1 1025 promoter (mm9 genome). (E) Relative levels of the indicated gene transcripts were determined by real-time qPCR analysis of CTL and Snail-expressing 1026 HMLE cells (n = 3, mean ± SEM). Representative results of 3 independent experiments are shown. (F) Western blot analysis of MEK and AKT 1027 phosphorylation in Snail-expressing HMLE cells treated with DMSO or SU5402 at the indicated concentrations. Phosphorylated and total protein levels 1028 were quantified (n = 3, mean ± SEM). Representative results of 3 independent experiments are shown. (G) Snail-expressing HMLE cells were exposed 1029 to serum or serum-starved with DMSO or SU5402 (10 µM). The percentage of cells in G2/M was determined by FACS analysis of the DNA content in 1030 each variant (n = 3, mean ± SEM). Representative results of 3 independent experiments. (H) Western blot analysis of the protein expression levels of 1031 EMT markers in CTL or Snail-expressing cells treated with DMSO or with SU5402 (10 µM). (I) MaSC-enriched basal cells that expressed dTomato 1032 (dTom) were mixed (1:1) with cells that did not express dTom and plated in 3D. The clonality of the organoid resulting from the co-culture was quantified 1033 by brightfield and fluorescence microscopy. Scale bar, 100 µm. (J) Organoid-forming capacity was determined for MaSC-enriched basal cells treated with 1034 SU5402 at the indicated concentrations 10 days after plating (n = 3, mean ± SEM). Representative results of 3 independent experiments. ** p ≤ 0.01, *** 1035 p ≤ 0.001. Figure S3: GLIS2 inhibition occurs at the post-transcriptional level and TRIM32 knockout does not affect the M-like status of HMLE cells A B CTL Snail Snail+DMSO Snail+CilA 300 1000 3

100 1 10

1 normalized read counts Relative RNA levels Relative RNA 0.1 CTLSNL

GLIS2 SNL + CilA GLIS2 GLI1 SNL + DMSO CDH11 C + HA-GLIS2 CilA CilA SNL + DMSO + SNL + SNL + SNL CTL DMSO + CTL DMSO + CTL DMSO + SNL 245 - Ubiquitinated 190 - proteins 135 - INPUTS TUBE D E 1000 n.s. p > 0.05 1.0 100 TRIM32 10 0.5 RNA levels RNA normalized read counts 1 Relative 0.0

CTLSNL CTL SNL

TRIM32 SNL + CilA SNL + DMSO F G sgCTL sgTRIM32#1 sgTRIM32#3 sgCTL sgTRIM32#1 sgTRIM32#3 130 - Ecad

Vim 55 - Actin 1036 43 - 1037 Fig. S3: GLIS2 inhibition occurs at the post-transcriptional level and TRIM32 knockout does 1038 not affect the M-like status of HMLE cells. (A) Expression of GLIS2 was identified in the 1039 indicated HMLE variants by RNA-sequencing as described in the legend of Fig. 3 (mean ± SEM). 1040 (B) Relative levels of the indicated gene transcripts in the indicated cells were determined by real- 1041 time qPCR analysis (n = 3, mean ± SEM). Representative results of 3 independent experiments. 1042 (C) CTL and Snail-expressing (SNL) HMLE cells with or without HA-GLIS2 were treated or not 1043 with DMSO and Ciliobrevin A (CilA). Ubiquitinated proteins were purified by TUBE pull-down and 1044 analyzed by western blot. (D) Expression of TRIM32 was identified by RNA-sequencing as 1045 described in the legend of Fig. 3 (mean ± SEM). (E) Relative levels of TRIM32 were determined 1046 in the indicated cells by real-time qPCR analysis (n = 3, mean ± SEM). Representative results of 1047 3 independent experiments. (F) The impact of TRIM32 knockout in Snail-expressing HMLE cells 1048 on morphology was examined by brightfield microscopy. (G) The impact on expression of EMT 1049 markers was determined by western blot analysis. 1050 1051 1052 1053 1054 1055 Figure S4: GLIS2Cter expression does not affect the M-like status of HMLE cells or primary ciliogenesis but affects cilium-signaling

A B 1.5

CTLGLIS2Cter 1.0 130 - Ecad dTOM- 72 - GLIS2Cter 0.5 *** 55 - Vim *** 0.0

Actin Fold change GLIS2Cter/CTL GLI1 43 - CDH11 C CTL GLIS2Cter

D CTL GLIS2Cter 40 n.s.p > 0.05 30 20 10

DNA Arl13b % Ciliated cells 0 E CTL GLIS2Cter CTL 1.5×106 n.s GLIS2Cter p > 0.05 1.0×106

5.0×105 Cell number 0.0 0 2 4 Day 1056 1057 Fig. S4: GLIS2Cter expression does not affect the M-like status of HMLE cells or primary 1058 ciliogenesis but affects cilium-signaling. (A) GLIS2Cter expression and its impact on 1059 expression of EMT markers in Snail-expressing HMLE cells were determined by western blot 1060 analysis. (B) Relative levels of GLI1 and CDH11 were determined in the indicated variants by real- 1061 time qPCR analysis (n = 3, mean ± SEM). Representative results of 3 independent experiments. 1062 (C-E) The impact of GLIS2Cter expression on morphology was examined by brightfield 1063 microscopy (C), on ciliogenesis was assessed by immunofluorescence microscopy (D), on 1064 proliferation in 2-dimension was analyzed by counting the total cell number per well (3 wells/line) 1065 at the indicated time points (E). All scale bars: Brightfield 100 µm, Immunofluorescence 15 µm. 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 Figure S5: EMT-dependent primary ciliogenesis results in GLIS2 inhibition in transformed cells and GLIS2Cter expression does not affect EMT and ciliogenesis but affects ciliary signaling

HMLER + Claudin-low-like tumors (HMLER + shEcad) A HMLER + B H&E DNA shCTL shEcad Arl13b

shCTL shEcad Large T

245 - Ecad

135 - FN 100 - 46 - Actin

80 DMSO CilA shCTL shEcad+DMSO shCTL shEcad C ** p ≤ 0.01 D ** p ≤ 0.01 60 Ecad 40

shEcad shEcad+CilA

Arl13b 20 % Ciliated cells DNA S3_E1 S1_E1 0 S4_M1 S2_M1 S9_M1_CTR S10_M1_TRT S12_M1_TRT S11_M1_CTR S1_E1 S3_E1 CilA S2_M1 S4_M1 S9_M1_CTR S10_M1_TRT S12_M1_TRT shCTLshEcad DMSO −1.0 −0.5 0.0 0.5 1.0 1.5 −1.5 −1.0 −0.5 S11_M1_CTR 0.0 0.5 1.0 1.5 shEcad + E F1.5 1.0 0.5 0.0 0.5 − 1.0 − G −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5 Enriched in shEcad vs shCTL Depleted in shEcad+CilA vs DMSO

CTL GLIS2Cter FDR = 0.05 FDR = 0.008 80 - dTOM- CTL GLIS2Cter GLIS2Cter Vim 55 -

43 - Actin

I J 1.5

CTL GLIS2Cter 1×106 CTL GLIS2Cter 60 n.s p > 0.05 1.0 H n.s. p > 0.05 8×105 40 6×105 Arl13b 4×105 20 0.5 *** 2×105 Cell number *** DNA % Ciliated cells 0 0 CTL 0 1 2 4 0.0 Day Fold change GLIS2Cter/CTL GLI1 1082 GLIS2Cter CDH11 1083 Fig. S5: EMT-dependent primary ciliogenesis results in GLIS2 inhibition in transformed cells and 1084 GLIS2Cter expression does not affect EMT and ciliogenesis but affects ciliary signaling. (A) shControl 1085 (CTL)- and sh-Ecadherin (Ecad)-expressing HMLER cells were examined for morphology by brightfield 1086 microscopy (scale bar: 100 µm) and for their expression of EMT markers by western blot analysis. (B) Sections 1087 of tumors arising from HMLER + shEcad cells that were orthotopically transplanted in the fat pad of female mice, 1088 as described in the legend of Fig. 5, were stained with H&E as well as for the indicated proteins. Representative 1089 images are shown. Scale bars: 15 µm. (C, D) shCTL- and shEcad-expressing HMLE cells were grown until high 1090 confluence and serum starved for 24h without or with DMSO or Ciliobrevin A (CilA). (C) Cells were stained for the 1091 indicated proteins and the percentage of ciliated cells was determined (n = 3 mean ± SEM). Representative results 1092 from 3 independent experiments are shown. Scale bar: 15 µm. (D) Gene expression in the indicated HMLER 1093 variants was analyzed by RNA-sequencing. Heat-maps showing the differentially expressed genes (q-value ≤ 1094 0.05, Fold-change ≥ 2) between samples are displayed. (E) A GSEA analysis shows significant enrichment of 1095 GLIS2 target genes in the list of upregulated genes in shEcad- versus (vs) shCTL-expressing cells (left panel) 1096 and depletion in the list of shEcad CilA-treated vs DMSO-treated cells (right panel) (False discovery rate (FDR) 1097 ≤ 0.05). (F-J) The impact of GLIS2Cter expression in HMLER + shEcad cells on expression of EMT markers and 1098 GLIS2 targets, on morphology, on ciliogenesis and cell proliferation was examined as described in the legend of 1099 Fig. S4. Scale bars: Brightfield 50 µm, Immunofluorescence 15 µm. Figure S6: Claudin-low tumors express low levels of epithelial markers, display primary cilia, and express high levels of GLIS2 target genes A 200 C 150

100 FDR = 0.001 E-index 50 0

Luminal Claudin-low

B Luminal B Claudin-low DNA Arl13b Tub g Ecad Vim Merge

1100 1101 Fig. S6: Claudin-low tumors express low levels of epithelial markers, display primary cilia, and 1102 express high levels of GLIS2 target genes. (A-B) Paraffin sections of breast tumors were stained as 1103 described in the legend of Fig. 6B-C. (A) Ecadherin, Claudin 4 and Claudin 7 signal intensities were 1104 measured and used to define an Epithelial-index (E-index = (Ecadherin signal intensity + Claudin 4 1105 signal intensity + Claudin 7 signal intensity)/3). (B) Scale bar: 20 µm. (C) A GSEA analysis shows 1106 significant enrichment of GLIS2 target genes (FDR<0.05) in the list of upregulated genes in claudin- 1107 low cancers versus other breast cancers defined by Prat et al29. Supplementary Table 1

Candidate ciliogenesis inducers with Snail v. CTL RNA-seq differential gene expression results and associated promoter peaks (+/- 3kbp) from Ye et al. GSE61198 (baseMean = Mean gene expression across samples; padj = adjusted p-value; TSS_dist = Distance of peak from transcription start site of gene; HUGO = Human Genome Organization/Human Committee).

Gene baseMean baseMeanE2baseMeanM2foldChange..M2.E2.log2FoldChange p-value padj Peak_Locus Peak_score TSS_dist HUGO_symbolMouse_symbol HHIP 33,2322775 0,36739044 66,0971645 179,909863 7,49113047 1,14E-06 1,08E-05chr8:82581190-82582094 288,3 0 HHIP Hhip MAP1B 696,670724 31,3460161 1361,99543 43,450352 5,44129596 4,99E-80chr13:100217303-100217763,chr13:100284691-1002871861,46E-77 68.57,1094.65 -2881,0 MAP1B Map1b GLI2 13,6893718 0,95594003 26,4228036 27,6406497 4,78871962 0,00055653 0,00298486 - - - - - FGFR1 182,046344 13,9680856 350,124602 25,0660407 4,64766223 3,30E-49 4,00E-47chr8:26628989-26629969 967,08 0 FGFR1 Fgfr1 RGS9 15,0084026 1,2127509 28,8040542 23,7510062 4,56991673 3,01E-06 2,66E-05 - - - - - SPATA6 24,8683436 2,20434266 47,5323444 21,563047 4,43048915 4,89E-09 6,50E-08chr4:111392399-111393074 344,24 0 SPATA6 Spata6 PCDHB15 6,92276693 0,84536046 13,0001734 15,3782605 3,94282042 0,00467907 0,0195211 - - - - - GLI1 30,7814666 7,27650542 54,2864277 7,46050811 2,89927389 4,22E-05 0,000295 - - - - - ROM1 39,3642609 9,92316637 68,8053554 6,93381053 2,79364841 5,41E-08 6,22E-07chr19:9002887-9005351 811,54 0 ROM1 Rom1 SYNE1 134,740619 42,9991795 226,482058 5,26712511 2,39701573 1,29E-10 2,06E-09chr10:5151497-5152354 878,99 0 SYNE1 Syne1 TTLL1 56,0516361 18,3411192 93,762153 5,11212821 2,35392402 1,65E-08 2,03E-07chr15:83340984-83341547 389,4 0 TTLL1 Ttll1 FUZ 14,9755133 5,07216276 24,8788638 4,90498137 2,29424766 0,00403358 0,01717569chr7:52150597-52151812 75,41 0 FUZ Fuz CEP112 48,4688785 20,3599545 76,5778025 3,76119714 1,91119193 1,90E-05 0,00014271chr11:108286427-108286794 419,03 0 CEP112 Cep112 PARVA 344,578028 148,444453 540,711603 3,64251806 1,86493613 2,12E-18 6,38E-17chr7:119570608-119572212 1588,34 0 PARVA Parva ASAP1 294,257016 127,563628 460,950404 3,61349399 1,85339449 1,55E-16chr15:64143186-64144290,chr15:64212826-642165004,05E-15 127.2,85.76 0,0 ASAP1 Asap1 EHD3 19,9150939 9,04215418 30,7880335 3,40494454 1,7676313 0,00750788 0,02923174 - - - - - SPATA7 50,2577779 23,299078 77,2164778 3,31414306 1,72863588 7,75E-05 0,00050774 - - - - - RAB8B 127,856636 60,4592865 195,253985 3,2295119 1,69131613 4,92E-06 4,16E-05chr9:66766875-66767995 993,37 0 RAB8B Rab8b CCDC28B 133,911378 64,6504371 203,172319 3,14262871 1,65197183 5,36E-09 7,09E-08 - - - - - CEP170 273,638225 133,805642 413,470808 3,09008502 1,62764653 1,17E-11 2,09E-10 - - - - - CIB2 24,8852135 12,4948995 37,2755275 2,9832595 1,57688948 0,00900328 0,03407317chr9:54407616-54408430 628,13 0 CIB2 Cib2 DZIP1L 29,9267224 15,3234435 44,5300013 2,90600487 1,53903712 0,00753286 0,02931552chr9:99532602-99533020 66,28 2340 DZIP1L Dzip1l PKD2 179,006736 93,5316752 264,481797 2,82772437 1,4996415 6,06E-09 7,95E-08chr5:104887764-104889627 849,1 0 PKD2 Pkd2 BBIP1 204,936653 109,928887 299,944418 2,72853138 1,44812464 3,03E-09 4,11E-08 - - - - - RILPL2 130,602113 70,4537563 190,75047 2,70745635 1,43693808 5,21E-07 5,17E-06chr5:124927672-124928586 322,8 0 RILPL2 Rilpl2 C21orf2 68,4160087 37,1958602 99,6361571 2,67868942 1,42152732 0,00011531 0,00072777 - - - - - ARL2BP 225,570614 124,781609 326,359619 2,61544647 1,38705724 2,47E-09 3,41E-08chr8:97189964-97191328 710,71 0 ARL2BP Arl2bp INVS 54,2290488 30,3187726 78,1393251 2,57725885 1,36583745 0,00111643 0,00554467chr4:48291980-48293202 961,25 0 INVS Invs CEP19 61,0195571 34,3244157 87,7146986 2,55546079 1,35358346 0,00069257 0,00362462chr16:32099007-32100345 1188,92 0 CEP19 Cep19 PRKACB 117,656213 66,5943445 168,718082 2,53351968 1,34114303 6,92E-06 5,69E-05chr3:146475485-146476580 884,49 0 PRKACB Prkacb GLI3 117,219506 68,6881076 165,750905 2,41309464 1,2708845 2,03E-05 0,0001518chr13:15554405-15556571 631,33 0 GLI3 Gli3 BBS9 38,7971559 22,9709637 54,6233481 2,37793019 1,24970636 0,00821325 0,03157102chr9:22279772-22280586 492,63 0 BBS9 Bbs9 NEK1 295,999785 176,673087 415,326483 2,35081918 1,23316358 5,99E-05 0,00040251chr8:63471208-63472731 1219,21 0 NEK1 Nek1 DZIP1 160,662471 97,2768831 224,048059 2,3031994 1,20363932 9,92E-06 7,91E-05 - - - - - RAB23 134,867381 83,6085089 186,126254 2,22616401 1,15455989 5,21E-05 0,00035491chr1:33776383-33777461 897,16 0 RAB23 Rab23 GABARAP 2588,67289 1633,26481 3544,08097 2,16993653 1,11765284 1,79E-13 3,74E-12chr11:69804207-69805537 985,15 0 GABARAP Gabarap ARL3 954,370788 603,468947 1305,27263 2,16294912 1,11299973 8,46E-11 1,38E-09chr19:46647205-46648515 1301,37 0 ARL3 Arl3 IFT52 248,627258 158,153709 339,100806 2,14412174 1,10038682 8,92E-07 8,56E-06chr2:162842567-162843527 798,53 0 IFT52 Ift52 NDE1 169,690466 108,302221 231,078712 2,13364703 1,09332153 4,21E-05 0,00029422 - - - - - IFT122 52,6859535 34,1425327 71,2293743 2,08623581 1,06090224 0,01092902 0,04014183chr6:115851028-115851446 62,39 -1202 IFT122 Ift122 TULP3 223,81195 145,979675 301,644225 2,06634399 1,04708045 9,58E-06 7,67E-05chr6:128305242-128306816 899,44 0 TULP3 Tulp3 ICK 142,376045 93,8029836 190,949106 2,03564 1,02548245 0,00250384chr9:77956004-77957130,chr9:77960803-779615780,01127444 983.94,368.99 0,0 ICK Ick GSN 1137,41215 751,96355 1522,86076 2,02517896 1,0180494 7,70E-10 1,12E-08chr2:35111441-35112246 559,03 0 GSN Gsn AK1 589,244641 392,717974 785,771308 2,00085395 1,00061586 7,78E-05 0,00050975chr2:32484191-32485622 220,98 0 AK1 Ak1 IFT20 397,67839 267,904338 527,452443 1,96880889 0,97732308 5,98E-07 5,87E-06 - - - - - HIF1A 646,692907 436,14135 857,244464 1,96551981 0,97491091 7,93E-08 8,91E-07chr12:75008273-75009778 1483,46 0 HIF1A Hif1a 1108 PRKAR1A 943,201202 636,459752 1249,94265 1,96389897 0,97372072 7,29E-09 9,45E-08chr11:109511449-109513326 615,97 0 PRKAR1A Prkar1a 1109 Table S1: Snail direct target genes. Candidate ciliogenesis inducers with Snail v. CTL RNA-seq 1110 differential gene expression results and associated promoter peaks (+/- 3kbp) from Ye et al. 1111 GSE61198 (baseMean = Mean gene expression across samples; padj = adjusted p-value; 1112 TSS_dist = Distance of peak from transcription start site of gene; HUGO = Human Genome 1113 Organization/Human Gene Nomenclature Committee).