(IFN) Pathway in Multiple Cancer Cell Types

1 Title: Hypoxia induces transcriptional and translational downregulation of the type

2 I interferon (IFN) pathway in multiple cancer cell types

3 Ana Miar1, Esther Arnaiz2, Esther Bridges1, Shaunna Beedie1, Adam P Cribbs3, Damien

4 J. Downes4, Robert Beagrie4, Jan Rehwinkel5, Adrian L. Harris1‡

5 1 Department of Medical Oncology, Molecular Oncology Laboratories, Weatherall 6 Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, 7 OX3 9DS, UK

8 2 Onkologia Molekularraren Taldea, Onkologia Arloa, Molecular Oncology Group, 9 Biodonostia Instituto de Investigación Sanitaria, Spain

10 3 Botnar Research Center, Nuffield Department of Orthopaedics, Rheumatology and 11 Musculoskeletal Sciences, NIHR Oxford BRU, University of Oxford, OX3 7DQ, UK

12 4 Medical Research Council (MRC) Molecular Hematology Unit, MRC Weatherall 13 Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK

14 5 Medical Research Council Human Immunology Unit, Medical Research Council 15 Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, 16 University of Oxford, Oxford OX3 9DS, UK

18 ‡To whom correspondence should be addressed: Adrian L. Harris Department of

19 Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John

20 Radcliffe Hospital, OX3 9DS, UK. E-mail: [email protected]. Phone: +44

21 (0)1865 222457 (PA).

22 Short title: Hypoxia effect on type I IFN on cancer

23 This work has been supported by Breast Cancer Now (grant reference 2015MayPR479).

24 Disclosure statement: Authors have nothing to declare.

25 Keywords: hypoxia, IFN, breast cancer

26 bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

27 Abstract:

28 Hypoxia is a common phenomenon in solid tumours and is considered a hallmark of

29 cancer. Increasing evidence shows that hypoxia promotes local immune suppression.

30 Type I IFN is involved in supporting cytotoxic T lymphocytes by stimulating the

31 maturation of dendritic cells (DCs) and enhancing their capacity to process and present

32 antigens. However, there is little information about the relationship between hypoxia and

33 the type I interferon (IFN) pathway, which comprises the sensing of double-stranded

34 RNA and DNA (dsRNA/dsDNA), followed by IFNα/β secretion and transcription

35 activation of IFN-stimulated genes (ISGs). The aims of this study were to determine both

36 the effect and mechanisms of hypoxia on the I IFN pathway in breast cancer.

37 There was a downregulation of the type I IFN pathway expression at mRNA and protein

38 level in cancer cell lines under hypoxia in vitro and in vivo in xenografts. This pathway

39 was suppressed at each level of signalling, from the dsRNA sensors (RIG-I, MDA5), the

40 adaptor (MAVS), transcription factors (IRF3, IRF7, STAT1) and several ISGs (RIG-I,

41 IRF7, STAT1, ADAR-p150). There was also lower IFN secretion under hypoxic

42 conditions. HIF1 and HIF2 regulation of gene expression did not explain most of the

43 effects. However, ATAC-Seq data revealed that in hypoxia peaks with STAT1 and IRF3

44 motifs had decreased accessibility.

45 Thus hypoxia leads to an overall 50% downregulation of the type I IFN pathway due to

46 repressed transcription and lower chromatin accessibility in a HIF1/2α-independent

47 manner, which could contribute to immunosuppression in hypoxic tumours.

50 bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

51 INTRODUCTION:

52 Oxygen can only diffuse 100-180µm from the nearest capillary and consequenctly, poorly

53 vascularized tumours or those that grow quickly suffer from hypoxia, low pH and nutrient

54 starvation. Hypoxia is closely linked to the hallmarks of cancer as it induces the switch

55 to glycolytic metabolism, promotes proliferation, enhances resistance to apoptosis,

56 induces unlimited replication potential and genomic instability, reduces immuno-

57 surveillance, and induces angiogenesis and migration to less hypoxic areas (1).

58 This hypoxia response is driven by a family of dimeric hypoxia-inducible transcription

59 factors (HIF-1, HIF-2, HIF-3) which are composed of an oxygen-sensitive α-subunit

60 (HIF-1α, HIF-2α, HIF-3α) and a constitutively expressed β-subunit (HIF-1β) (2). Under

61 well oxygenated conditions, HIF-1/2α are bound by the von Hippel-Lindau (VHL)

62 protein which targets them for proteasomal degradation (3). VHL binding is dependent

63 upon hydroxylation of specific proline residues by prolyl hydroxylase PHD2, which uses

64 O2 as a substrate, and as a consequence its activity is inhibited under hypoxia (4).

65 Hypoxia induces an immunosuppressive microenvironment; it inhibits the ability of

66 macrophages to phagocytose dead cells, present antigens to T cells, inhibits the anti-

67 tumour effects of macrophages (5), and favours their differentiation to M2 type which is

68 more immunosuppressive (6). T-cell-mediated immune functions are downregulated by

69 low oxygen levels, resulting in impairment of cytokine expression and less T-cell

70 activation (7). Moreover, the cytotoxic potential of natural killer (NK) cells is reduced

71 under hypoxia resulting in a decreased anti-tumour response that allows metastasis (8).

72 Type I interferons (IFNs) comprise multiple IFNα’s (encoded by 13 genes), IFNβ

73 (encoded by one gene) and other less studied IFNs (IFNε, IFNκ, IFNω). They are

74 produced by most cell types and their transcription is upregulated after the activation of

75 pattern recognition receptors (PRR) which sense viral/bacterial dsRNA ( through RIG-I, bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

76 MDA5, or TLRs) and dsDNA/unmethylated DNA ( through cGAS, DDX41, IFI16, or

77 TLRs). Type I IFNs bind to the heterodimeric IFNα/β receptor composed of IFNAR1 and

78 IFNAR2, triggering the JAK-STAT pathway and activating the transcription of IFN-

79 stimulated genes (ISGs) (9) (Supplementary figure 1). Type I IFNs are involved in all

80 three phases of the cancer immunoediting process by which the immune system

81 eliminates the malignant cells, moulds the immunogenic phenotypes of developing

82 tumours and selects those clones with reduced immunogenicity (10). Type I IFNs support

83 cytotoxic T lymphocytes by stimulating the maturation of dendritic cells (DCs) and

84 enhancing their capacity to process and present antigens (11). Endogenous IFNα/β is

85 required for the lymphocyte-dependent rejection of highly immunogenic sarcomas in

86 immunocompetent hosts (10), and they are crucial for the innate immune response to

87 transformed cells as Ifnar1-/- CD8+ dendritic cells are deficient in antigen cross-

88 presentation, and fail to reject highly immunogenic tumours (12). Moreover, type I IFNs

89 boost immune effector functions by enhancing perforin 1 and granzyme B expression

90 (13), and promoting survival of memory T cells.

91 Type I IFN is involved in the success of current anticancer treatments both directly

92 (tumour cell inhibition) and indirectly (anti-tumour immune responses to the nucleic acids

93 and proteins released by dying cells, recently named immunogenic cell death, ICD) (14)

94 (9). In mouse models, IFNAR1 neutralization using antibodies blocked the therapeutic

95 effect of antibodies against human epidermal growth factor receptor 2 (HER2) or

96 epidermal growth factor receptor (EGFR) (15, 16). Moreover, radiotherapy, via DNA

97 damage, induces intratumoural production of IFNβ which enhances the cross-priming

98 capacity of tumour-infiltrating dendritic cells (17). Chemotherapeutic agents such as

99 anthracyclines, bleomycin or oxalaplatin induce ICD, and the activation of an IFN-

100 dependent program is required for successful therapy (18, 19). bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

101 Tumour hypoxia creates resistance to many cancer treatments as oxygen is essential for

102 ROS formation to kill cells during ionizing radiation (20, 21), and HIF1α upregulation

103 mediates resistance to chemotherapy by upregulating anti-apoptotic and pro-survival

104 genes (22).

105 Thus we investigated the regulation of the type I IFN pathway under hypoxia at basal

106 levels and upon activation of IFN signalling using the dsRNA mimic

107 polyinosinic:polycytidylic acid (poly I:C). We found there was substantial HIF-

108 independent downregulation of IFN signalling which could affect all the before

109 mentioned therapies and contribute to treatment resistance.

110

111

112

113

114 bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

115 MATERIAL AND METHODS

116 Cell culture and transfection

117 All cell lines used are listed in supplementary table 1. They were cultured in DMEM low

118 glucose medium (1g/l) supplemented with 10% FBS no longer than 20 passages. They

119 were mycoplasma tested every 3 months and authenticated during the course of this

120 project. Cells were subjected to 1% or 0.1% hypoxia for the periods specified in each

121 experiment using an InVivO2 chamber (Baker).

122 Transfection of poly I:C (P1530, Sigma) was performed in Optimem reduced serum

123 (11058021, Thermo Fisher Scientific) medium for 6h unless it is otherwise indicated.

124 Lipofectamine 2000 (11668019, Thermo Fisher Scientific) was used following the

125 manufacturer’s instructions.

126 Western blot and RT-qPCR

127 Methods detailed in Supplementary materials.

128 IFN bioassay

129 Performed as previously described (23) and detailed in Supplementary Materials.

130 Single Cell sequencing and analysis

131 Single-cell RNA-seq libraries were prepared as per the Smart-seq2 protocol by Picelli

132 et al (24) with minor technical adaptations detailed in Supplementary material.

133 scRNA-seq data were deposited in Gene Expression Omnibus under SuperSeries

134 accession number GSE134038 and detailed in Supplementary materials.

135 Xenograft growth and IHC

136 Procedures were carried out under a Home Office license. Xenograft experiments were

137 performed as described in (25) and detailed in Supplementary materials.

138 ATAC-seq and analysis bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

139 Samples were prepared and analysed as previously described (26) with minor

140 modifications detailed in Supplementary materials. ATAC-seq data is available in the

141 Gene Expression Omnibus (GSE133327).

142 RESULTS

143 Hypoxia causes downregulation of the type I IFN pathway in unstimulated cells both at

144 mRNA and protein level

145 MCF7 cells were subjected to normoxia, 1% hypoxia or 0.1% hypoxia for 48h. This lead

146 to a decrease of both protein (figure 1A) and RNA (figure 1B) of members of the type I

147 IFN pathway. The effect was seen from dsRNA sensors (RIG-I [encoded by DDX58],

148 MDA5 [encoded by IFIH1]), adaptor MAVS, transcription factors triggering IFNα/β

149 production (IRF3, IRF7) and transcription factors involved in ISGs’ activation (STAT1,

150 STAT2), to downregulation of downstream ISGs (exemplified by ADAR-p150). This

151 inhibitory effect was oxygen concentration-dependent and 0.1% hypoxia caused a greater

152 downregulation of the pathway than 1% hypoxia.

153 This finding was confirmed in other breast cancer types including oestrogen receptor

154 positive (ER+; T47D), HER2+ (BT474) and triple negative (TN; MDA-MB-453, MDA-

155 MB-231, HCC1187, MDA-MB-468) cells. In general, a downregulation of the type I

156 pathway was observed in all cell lines in hypoxia. However, TN cell lines seemed to be

157 more resistant to the effect of hypoxia, showing a lower or no downregulation of some

158 transcripts such as DDX58, IRF3 or MX1 (figure 1C).

159

160 Single cell transcriptomic analysis of the effects of hypoxia on the type I IFN pathway

161 Next, we performed a single cell RNA-Seq experiment using MCF7 cells subjected to

162 0.1% hypoxia for 72h. We found that most of the genes in the type I IFN pathway (such

163 as DDX58, IRF3 or MX1) are significantly less expressed in hypoxia (figure 2A). bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

164 We also observed a decrease in the expression of several genes associated with the DNA

165 sensing branch (IFI16, TMEM173 [encodes STING], DDX41 and MB21D1 [encodes

166 cGAS]) in MCF7 cells exposed to hypoxia. Next, we investigated the expression of a

167 number of well described ISGs(27). This revealed a clear subpopulation of cells in which

168 ISGs were downregulated following exposure to hypoxic conditions, e.g. DDX58, IFIH1,

169 STAT1, STAT2, IFIT1, IFIT3 or MX2. In contrast, HIF1α targets such those involved in

170 glycolysis, were upregulated as expected (PDK1, PGK1, P4HA1, NDRG1, CA9; figure

171 2B).

172

173 Reoxygenation after hypoxia leads to recovery of the type I IFN pathway

174 A time course in 0.1% hypoxia up to 48h showed that type I IFN downregulation at

175 protein level was obvious at 48h in hypoxia (figure 3A), whereas at RNA level, there was

176 a significant decrease from 16h onwards in IFIH1, MAVS, STAT1 and ADAR (figure 3B).

177 MCF7 cells were incubated in normoxia or 0.1% hypoxia for 48h and subjected to

178 reoxygenation for 8h, 16h or 24h. At protein level (figure 3C), hypoxia caused the

179 downregulation of the pathway but reoxygenation up to 24h did not reverse this effect

180 except for RIG-I. However, at RNA level (figure 3D), reoxygenation caused a gradual

181 upregulation of type I IFN pathway levels. Thus RNA expression responded more rapidly

182 to O2 tension than protein.

183

184 Hypoxia downregulates the type I IFN pathway at protein level upon stimulus with a

185 dsRNA mimetic

186 MCF7 cells were incubated in normoxia or 0.1% hypoxia for 48h and transfected with

187 poly I:C (mimetic of dsRNA) in the last 6h of the incubation to determine if the

188 downregulation caused by hypoxia would persist upon activation. Poly I:C activated the bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

189 type I IFN pathway as shown using pIRF3-Ser386 and pSTAT1-Tyr701; however

190 hypoxia led to lower activation (lower levels of pIRF3 and pSTAT1) and to lower

191 expression of RIG-I, MDA5, MAVS and IRF7 (figure 4A). These findings suggested that

192 production/secretion of IFNs was lower in hypoxia than in normoxia.

193 To investigate this, the stimulation of IFN-stimulated response element (ISRE) by

194 potential IFNs present in the supernatant from normoxic or hypoxic cells was measured

195 using indicator cells (HEK293-3C11, hereafter 3C11) (23). 3C11 cells harbour a stably

196 integrated luciferase reporter under control of an ISRE, allowing the measurement of type

197 I IFNs in supernatant samples. 3C11 cells were incubated with supernatant from MCF7

198 cells previously treated with different concentrations of poly I:C and normoxia or

199 hypoxia, as described in the Supplementary Materials section. At all poly I:C

200 concentrations, hypoxic supernatants showed lower activation of ISRE confirming the

201 lower production/secretion of IFNs in these conditions (figure 4B).

202 A time course in hypoxia was performed using MCF7 cells cultured for 6h, 24h and 48h

203 in normoxia or 0.1% hypoxia and transfected with poly I:C in the last 6h of incubation.

204 pIRF3 and pSTAT1 were induced upon poly I:C transfection and their expression was

205 downregulated when the cells were exposed to 48h hypoxia but not at shorter incubations.

206 RIG-I, MDA5 and MAVS were downregulated at 24h, suggesting that hypoxia

207 diminishes the sensitivity of this pathway, particularly effecting late signalling in the

208 amplification phase (figure 4C).

209 The reponse to hypoxia was analysed in a panel of breast cancer cell lines which were

210 incubated in normoxia or 0.1% hypoxia for 48h and transfected with poly I:C during the

211 last 6h. Again, the activation of the pathway was lower in hypoxia and the expression of

212 MDA5, RIG-I, STAT1 and IRF7 was also decreased, showing a general effect in breast

213 cancer cell lines (figure 4D). bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

214 We assessed cell lines from other cancer types or normal tissues e.g. HepG2

215 hepatocarcinoma, HKC8, a non-transformed proximal renal tubular cell line and

216 fibroblasts. The pSTAT1-Tyr701 response to poly I:C was reduced in all cell lines in

217 hypoxia (supplementary figure 2). Interestingly, pIRF3 expression in HepG2 was higher

218 in hypoxia than in normoxia compared to IRF3 expression which was lower. This

219 different response may contribute to the higher viral replication under hypoxia in

220 hepatocarcinoma (28). In HKC8 cells and fibroblast, IRF3 expression was higher in

221 hypoxia or did not change but pIRF3 was reduced. Thus, hypoxia causes a general

222 downregulation of the type I IFN pathway in cancer, and similarly in normal cell lines.

223

224 Type I IFN downregulation is partially dependent on HIF1α

225 To determine the role of HIF1α in the results observed under hypoxic conditions, we used

226 MCF7-HIF1α-KO and MCF7-WT cells. In MCF7-WT, hypoxia suppressed RIG-I,

227 MDA5 and IRF3 expression. Interestingly, in MCF7-HIF1α KO cells in normoxia, RIG-

228 I and STAT1 levels were higher than in MCF7-WT cells, supporting a partial role for

229 HIF1α in suppressing them. However, in hypoxia, STAT1 and RIG-I levels were reduced,

230 indicating a HIF1α-independent mechanism to further regulate them maybe compatible

231 with residual HIF2 effect.

232 Poly I:C in both cell lines in normoxia induced IRF3, pIRF3, IRF7 and pSTAT1. This

233 induction was higher in MCF7-HIF1α-KO cells, apart from pSTAT1, which was still

234 induced but to lower level than the MCF7-WT cells (figure 5A).

235 The effect of poly I:C in hypoxia was attenuated for all the above proteins to similar

236 residual levels in both cell lines, which excluded HIF1α as the main mechanism.

237 Higher IRF3, pIRF3 and IRF7 induction in MCF7-HIF1α-KO than in MCF7-WT

238 suggested a possible increase in IFN production. Using the supernatant from these cells bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

239 to treat 3C11 cells as described above, there was no ISRE activation in normoxic

240 supernatants from WT or MCF7-HIF1α-KO at basal level. For both, ISRE activation was

241 similarly increased by poly I:C in normoxia. However hypoxic MCF7-WT supernatant

242 caused a significantly lower ISRE stimulation and this decrease was not noted for hypoxic

243 MCF7-HIF1α-KO supernatants which triggered significantly higher ISRE activation than

244 MCF7-WT (figure 5B).

245 IFNAR1 and IFNAR2 expression was determined and they were significantly

246 downregulated in MCF7-WT cell in hypoxia compared to normoxia. Interestingly,

247 MCF7-HIF1α-KO cells showed in normoxia significantly lower expression of both

248 compared with MCF7-WT and comparable to hypoxic MCF7-WT levels. Moreover,

249 hypoxia did not further decrease IFNAR1 and IFNAR2 levels at mRNA level (figure 5C).

250 This shows that HIF1α is unlikely to explain the downregulation in hypoxia, as IFNAR1

251 and IFNAR2 were lower without HIF1α, and they would be expected to be higher.

252 These data suggest a complex mechanism in which HIF1α has a basal role increasing

253 STAT1 and responsiveness to poly I:C, but the effect of hypoxia downregulating many

254 IFN genes persists. Finally, the hypoxia downregulation of IFN secretion is lost, implying

255 a role for HIF1α in this last step.

256 Some ISGs associated with chronic anti-viral response such MDA5, MX1 or STAT1 can

257 be transcribed by unphosphorylated STAT1, and this effect is driven by higher IRF9

258 expression (29). Both at mRNA and protein levels, MCF7-HIF1α-KO cells showed

259 higher IRF9 expression (figure 5D) associated with significantly higher ISG expression

260 even in hypoxia compared to MCF7-WT cells (figure 5E).

261 However, as for all the other cell lines, hypoxic MCF7-HIF1α-KO cells showed lower

262 ISG expression than normoxic MCF7-HIF1α-KO cells. These data suggest that HIF1α

263 upregulation in hypoxia is partly responsible for the type I IFN pathway downregulation bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

264 observed in this condition but other HIF1α-independent mechanisms cause a further

265 decrease.

266

267 Type I IFN downregulation and role of HIF2α

268 The converse experiment was performed with renal cancer cells, which showed vHL loss

269 or mutations leading to stabilization of HIF1α/HIF2α in normoxia. RCC4-EV (empty

270 vector, mutant vHL), RCC4-vHL (transfected with wild type vHL and, as a result,

271 displaying lower levels of HIF1α/HIF2α in normoxia), 786-0 parental (vHL mutation and

272 HIF1α deficient) and 786-0-HIF2α-KO (deficient in both HIF1α and HIF2α) were used.

273 In RCC4 cells, poly I:C strongly induced MDA5 in normoxia and this was reduced in

274 hypoxia, showing that high HIF1α/HIF2α alone was not enough to suppress normoxic

275 levels to those obtained in hypoxia. Poly I:C induced similar changes in RIG-I, IRF3 and

276 pIRF3 in both cell lines in normoxia, again showing no effect of high HIF1α/HIF2α in

277 this circumstance. Much higher STAT1 and pSTAT1 was detected in the vHL corrected

278 cell line in normoxia and both decreased in hypoxia, clearly linking suppression of

279 STAT1 to HIF1α/2α (Supplementary figure 3A). In this model pSTAT1 was upregulated

280 and STAT1 downregulated in RCC4-wild type vHL cells, also pointing to a role of

281 HIF1α/2α in suppressing phosphorylation.

282 In 786-0 cells (Supplementary figure 3B), poly I:C did not affect RIG-I or MDA5 levels

283 but it induced pIRF3 and pSTAT1 expression in both cell lines. As observed in other

284 HIF1α-KO cell lines, STAT1 level was highly expressed in both cell lines. However,

285 hypoxia led to downregulation of MDA5, IRF3, pIRF3, STAT1, pSTAT1 and ADAR

286 expression, again showing that constant deficiency of HIF1α/HIF2α still allowed a further

287 suppression of IFN response in hypoxia. bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

288 In general and despite of the differences observed in these models, there is a partial role

289 of HIF1/2α in regulating some members of the pathway, but more importantly, it is clear

290 that there is a HIF1/2α-independent downregulation of the type I IFN pathway.

291

292 Type I IFN pathway is also downregulated in hypoxic areas in vivo

293 To confirm that hypoxic areas in vivo also showed lower expression of type I IFN

294 pathway, xenograft experiments were conducted using MCF7 and MDA-MB231 cells.

295 Avastin, a VEGF antagonist, was used to create tumour hypoxia by reducing

296 angiogenesis, as described previously (30-32). Immunohistochemistry was performed

297 using antibodies against IRF3, IRF7 and ADAR in 5 control and 5 avastin-treated mice

298 for each cell line. Avastin treatment decreased the percentage of CD31-positive cells

299 (vessel marker) and, consequently, the proportion of necrosis was significantly higher

300 (25). Moreover, the percentage of CA9+ cells was greater in Avastin-treated xenografts

301 compared to PBS. Interestingly, IRF3, IRF7 and ADAR were significantly

302 downregulated in avastin-treated mice in the case of MCF7 xenografts (figure 6A), and

303 IRF3 and IRF7 also showed significantly lower expression in MDA-MB231 xenografts

304 (figure 6B).

305

306 ATAC-Seq data shows lower chromatin accessibility on STAT1 and IRF3 containing

307 promoters

308 To investigate how hypoxia may downregulate the type I IFN pathway independently of

309 HIF1α/HIF2α we performed ATAC-seq on MCF7 cells cultured for 48h in normoxia or

310 0.1% hypoxia. We found 5,577 peaks (7.4%) with differential accessibility (figure 7A).

311 Peaks showing increased accessibility during hypoxia (n = 2,439) were enriched for

312 promoters, whereas those with decreased accessibility (n = 3,138) tended to be intergenic bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

313 or intronic (figure 7B), showing a switch in regulatory networks driving gene expression.

314 Consistent with HIF1/HIF2 driving an active hypoxia response, motif analysis showed

315 enrichment for HIF1 sites in distal peaks with increased accessibility (figure 7C).

316 Conversely, peaks with differential decreased accessibility showed higher levels of

317 STAT1, IRF3, FOXA1 and GATA3 motifs, than unchanged peaks. FOXA1 and GATA3

318 motifs were present more frequently in distal peaks with decreased accessibility and less

319 frequently present in peaks with differential increase accessibility. IRF3 motifs were

320 significantly less represented in peaks with increased accessibility both at promoters and

321 distal sites. STAT1 motifs only showed significantly less presence in increased accessible

322 peaks at promoters. The observed loss of accessibility at FOXA1 and GATA3 sites is

323 consistent with their roles downstream in ER signaling and the degradation of ER under

324 low O2 conditions (33). Therefore, hypoxia appears to drive global changes in chromatin

325 accessibility, partially through the shutdown of both type I IFN and ER signalling

326 responsive promoters and enhancers, although globally most decreases are in intergenic

327 regions and introns.

328 DISCUSSION

329 We describe a new mechanism of hypoxic immunosuppression via downregulation of the

330 type I IFN pathway. This occurred at oxygen tensions commonly found in tumours and

331 in more severe hypoxia present in many cases (1% vs 0.1%). The type I IFN pathway is

332 downregulated at RNA level in less than 16h under hypoxia whereas protein level is

333 downregulated after a 48h exposure. Reoxygenation gradually reverses the levels and it

334 takes 24h to reach normoxic levels again. The observation that RNA recovers more

335 quickly than protein after reoxygenation points to de novo transcription or stabilisation

336 occurring during the reoxygenation phase, and RNA changes leading to the protein

337 expression changes rather than protein degradation. However, there was no overshoot and

338 upregulation above the normoxic baseline after reoxygenation. bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

339 Furthermore, we observed lower activation of the type I IFN pathway and less

340 production/secretion of interferons under hypoxia in all cell lines tested when they were

341 activated with poly I:C. This suggests that endogenous activators of the pathway in a

342 hypoxic tumour microenvironment would lead to lower stimulation and consequently to

343 lower immune response. This hypothesis is supported by lower presence of T-cells and

344 NK cells in hypoxic areas in lung and dermal tumours (34) and in prostatic tumours (35).

345 In both studies the immune infiltration increased when the hypoxic areas were reduced

346 using respiratory hyperoxia (34) or the hypoxia-activated prodrug TH-302 (35),

347 respectively.

348 We observed that the type I IFN downregulation in hypoxia is independent of HIF1α and

349 HIF2α, although some members (RIG-I, and mainly STAT1) are regulated by HIF1α as

350 observed in MCF7-HIF1α-KO cells. Moreover, HIF2α seems not to play an important

351 role in the type I IFN downregulation as observed in 786-0 HIF2α-KO cells. Previous

352 work reported that RIG-I harbours hypoxic response elements in its 3’UTR and RIG-I

353 expression was upregulated during hypoxia in human myotubes (36). However, a

354 different study in tumour cells supported our finding that RIG-I and MDA5 are

355 downregulated under hypoxia and they required functional IFNAR1 to respond to

356 stimulus (37). Recently, type I IFN signalling was shown to be inhibited by high lactate

357 levels generated during the metabolic switch to glycolysis occurring in hypoxia (38). As

358 lactate production requires HIF1α, this result could explain the partial effect we observed

359 in HIF1α-KO cells. However, the inhibitory effect seen in hypoxia needed 16h to be

360 significant and it could be a consequence of the HIF1α-independent general transcription

361 inhibition occurring during hypoxia that leads to 42% and 65% decrease in total RNA

362 transcription after 24h and 48h exposure to 0.2% hypoxia, respectively (39). bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

363 Apart from the transcriptional inhibition, translation blockade also occurs in hypoxia as

364 a quick and reversible mechanism upon reoxygenation via phosphorylation of eIF2α to

365 adapt to changing environmental conditions. This mechanism is also HIF1α-independent

366 and peaks after 2h in hypoxia but often recovers after 24 hours (40, 41).

367 We investigated each step in the type I IFN pathway, from the sensors (RIG-I, MDA5),

368 transcription factors (IRF3, IRF7, STAT1), ISGs (MX1, ADAR) and interferon release,

369 and each stage was reduced under hypoxic conditions, although there was heterogeneity.

370 We therefore studied the chromatin accessibility in hypoxia related to these genes by

371 ATAC-Seq. Overall we found that 48h exposure to hypoxia led to global changes rather

372 than changes in specific pathways and only 7% of the type I IFN pathway were associated

373 with differentially accessible peaks. Interestingly, hypoxia caused decreased accessibility

374 at STAT1 and IRF3 containing promoters, suggesting lower transcription from those

375 genes but the downregulation and lower activation observed in hypoxia cannot be

376 explained only by this. However, a recent paper showed that 1h exposure to hypoxia

377 caused a rapid and HIF1α-independent induction of histone methylation, and the locations

378 of H3K4me3 and H3K36me3 predict the transcriptional response hours later (42). These

379 methylation markers are normally related to active transcription and they were

380 significantly less present in type I IFN genes after hypoxia, potentially indicating that the

381 expression of genes in this pathway are repressed by histone modifications that occurred

382 in hypoxia due to decreased enzyme activity of oxygen-requiring JmjC-containing

383 enzymes, and particularly KDM5A, that would affect transcription and translation of this

384 pathway later on (42).

385 Here, we propose a model in which hypoxia downregulates every single step in the type

386 I IFN pathway. However, there is a partial effect of HIF1α as its deletion increased the

387 levels of MDA5, RIG-I, IRF3, IRF7, STAT1 and IFN secretion upon activation in bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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 normoxia by upregulating IRF9 and not by increasing phosphorylation of STAT1.

389 Conversely, hypoxia is still able to decrease the IFN response when HIF1α is deleted

390 potentially by specifically decreasing the transcription of this pathway (figure 7D).

391 Supporting our work, ISG15 (a typical ISG increased by IFNαβ) was reported to decrease

392 in hypoxia and interact and modify HIF1α via ISGylation, thus reducing its levels and

393 affecting its biological impact on EMT and cancer stemness acquisition (43). In the same

394 line, response to type I IFN pathway was reported to be incompatible with induction of

395 pluripotency (44).

396 All together, these data suggest that the relevance of this hypoxic downregulation of the

397 IFN response is the potential effect on enhancing tumour growth by decreasing the

398 cytotoxicity of T lymphocytes and the capacity of dendritic cells (DCs) to process and

399 present antigens (11). As a result, this downregulation could contribute to the radio- and

400 chemo-resistance observed in hypoxic areas as these therapies rely on intact type I IFN

401 signalling (14). This provides a further rational for targeting the hypoxic tumour

402 population in combination with check point inhibitor therapy (45) and selection of

403 hypoxic patients for study of IFN response inducers is warranted.

404

405

406 ACKNOWLEDGEMENTS

407 We thank Dr Dimitrios Anastasiou and Dr Fiona Grimm (The Francis Crick Institute,

408 UK) for giving us MCF7-WT and MCF7-HIF1α-KO cells.

409 We also thank Matthew Gosden (University of Oxford, UK) for his technical advice

410 performing the ATAC-Seq experiments.

411 AUTHOR CONTRIBUTION

412 AM and ALH designed the experiments, analysed the data and wrote the manuscript. AM

413 and EA performed the experiments. EB performed the animal experiments and stained bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

414 the samples. SB performed the single cell experiments and APC analysed them. DD and

415 RB analysed ATAC-Seq data, and JR provided reagents and advice. This research was

416 supported by funding from Cancer Research UK (ALH, EB, SB), Breast Cancer Research

417 Foundation (ALH), and Breast Cancer Now (ALH, AM).

418 bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

419 References:

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520 41. Koumenis C, Naczki C, Koritzinsky M, Rastani S, Diehl A, Sonenberg N, et al., Regulation 521 of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and 522 phosphorylation of the translation initiation factor eIF2alpha. Mol Cell Biol 2002; 22: 7405-16. 523 42. Batie M, Frost J, Frost M, Wilson JW, Schofield PRocha S, Hypoxia induces rapid 524 changes to histone methylation and reprograms chromatin. Science 2019; 363: 1222-1226. 525 43. Yeh YH, Yang YC, Hsieh MY, Yeh YCLi TK, A negative feedback of the HIF-1alpha 526 pathway via interferon-stimulated gene 15 and ISGylation. Clin Cancer Res 2013; 19: 5927-39. 527 44. Eggenberger J, Blanco-Melo D, Panis M, Brennand KJtenOever BR, Type I interferon 528 response impairs differentiation potential of pluripotent stem cells. Proc Natl Acad Sci U S A 529 2019; 116: 1384-1393. 530 45. Li Y, Patel SP, Roszik JQin Y, Hypoxia-Driven Immunosuppressive Metabolites in the 531 Tumor Microenvironment: New Approaches for Combinational Immunotherapy. Front 532 Immunol 2018; 9: 1591.

533

534 bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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.

535 Figure 1

A B

0.015 ** 0.006 * 0.08 *** 0.008

n n

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Normoxia ER+ HER2+ TN 0.1% hypoxia

Figure 1. Type I interferon (IFN) pathway expression in breast cancer cells. A) Western-blot showing protein levels of different genes

involved in the pathway in MCF7 cells cultured in normoxia, 0.1% and 1% hypoxia for 48h. B) qPCR data showing mRNA expression of

genes involved in the pathway in the same experiment (n=10). C) mRNA expression of genes in the type IFN pathway in different breast

cancer cell lines cultured in normoxia or 0.1% hypoxia for 24h (n=3). * p<0.05, ** p<0.01, *** p<0.001.

p=0.085 MAVS p=0.001 RIG-I p=0.0022 ADAR p=0.0.044 IFI16

CGAS p=0.024 DHX58 NS MDA5 p=1.39E-07 p=0.0003 STAT2

p=0.99 p=9.25E-19 DDX41 NS STAT1 NS IFIT3 NS MX2

p=0.93 p=5.07e-06 IFNAR1 p=2.76E-05 IRF3 p=8.79E-11 STING NS IRF7

p=0.099 p=0.0007 MX1 p=1.34E-14 IFNAR2 NS IFIT1 p=0.008 ISG15

Figure 2. Single-cell analysis of type I IFN pathway in MCF7. A) Violin plots showing expression (log counts) of type IFN

bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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. genes in normoxia (orange) and 0.1% hypoxia (blue) after 72h. B) Heatmap correlating mRNA expression type I IFN genes

previously published with hypoxia target genes. Figure 3

A B n 0.0025 * n 0.04

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T I

N 48h H 48h H+8h H+16h H+24h S N 48h H 48h H+8h H+16h H+24h N 48h H 48h H+8h H+16h H+24h

Figure 3. Type I IFN over time and during reoxygenation in MCF7 cells. A) Western-blot showing protein levels of different genes involved in the

pathway in MCF7 cells cultured in normoxia for 48h or 0.1% hypoxia for 4h, 8h, 16h, 24h and 48h. B) qPCR data showing mRNA expression of

genes involved in the pathway in the same experiment (n=3). C) Western-blot showing protein changes in MCF7 when exposed to normoxia or 0.1%

hypoxia for 48h and after reoxygenation for 8h, 16h and 24h. D) qPCR data showing mRNA expression changes for the same experiment (n=3). *

p<0.05, ** p<0.01, *** p<0.001.

A B hypoxia normoxia POLY I:C, 100 Poly I:C 0 10ng 20ug 0 10ng 20ug IVT-RNA *** Normoxia 80 * 0.1% hypoxia

MDA5 ) e

n ### g

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C Normoxia 0.1% hypoxia 48h 6h 24h 48h 6h 24h 48h 6h 24h 48h Poly I:C - + + + - - - + + + RIG-I

MDA5

IRF3

pIRF3 Ser386 pSTAT1 Tyr701

STAT1

actin

D BT474 MDA-453 T47D MDA-468 MDA-231 normoxia hypoxia normoxia hypoxia normoxia hypoxia normoxia hypoxia normoxia hypoxia Poly I:C 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug 0 10ng 20ug MDA5

RIG-I pSTAT1 Ser727 STAT1

pIRF3 Ser386 IRF3

IRF7

actin

bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. 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 4. Type I IFN activation by poly I:C in breast cancer cells. A) Western-blot showing protein levels of MCF7 cells cultured in

normoxia or 0.1% hypoxia for 48h and transfected with poly I:C in the last 6h. B) IFN bioassay showing Interferon Stimulated Response

Element (ISRE) activation via luciferase activity in HEK293 cells using supernatants of MCF7 cells previously treated with normoxia or

0.1% hypoxia for 24h and transfected with different concentrations of poly I:C (n=3). C) Western-blot showing protein changes in different

breast cancer cells when exposed to normoxia or 0.1% hypoxia for 48h and transfected with 20ug/ml poly I:C in the last 6h of the treatment.

* p<0.05, ** p<0.01, *** p<0.001 in normoxia and normoxia vs hypoxia, # p<0.05, ## p<0.01, ### p<0.001 in hypoxia samples. Figure 5 A MCF7-WT HIF1α-KO B Norm Hyp Norm Hyp Poly I:C - + - + - + - + * HIF1a 50 ### Normoxia

) *** e * 0.1% hypoxia RIG-I n

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STAT1 poly I:C concentration ADAR-p150 ADAR-p110

actin n

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** o

i ***

0.055 i 0.0008 s

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s e

0.050 e r

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p 0.0006 x

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F I WT HIF1a-KO I WT HIF1a-KO D

n 0.20 o MCF7-WT i *** Normoxia HIF1α-KO s *** *** s 0.1% hypoxia

Norm Hyp Norm Hyp e

r 0.15

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n s

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x 5

e 0.010 e bioRxiv preprint doi: https://doi.org/10.1101/715151; this version posted July 25, 2019. The copyright holder for this preprint (which was 1 * r

0.2 A

l D

v 0.005

0.1 a

r A

T 0.0 0.000

S WT HIF1a-KO WT HIF1a-KO

Figure 5. HIF1α-independency of type I IFN downregulation in hypoxia. A) Western-blot showing protein changes in different MCF7-WT vs MCF7-HIF1α-KO cells when exposed to normoxia or 0.1% hypoxia for 48h and transfected with 0.5ug/ml poly I:C in the last 6h of the treatment B) ISRE activation via luciferase activity in 3C11 cells using supernatants of MCF7-WT and MCF7-HIF1α-KO cells previously treated with normoxia or 0.1% hypoxia for 24h and transfected with poly

I:C (n=3). C) IFNAR1 and IFNAR2 mRNA expression in MCF7-WT and MCF7-HIF1α-KO cells. D) IRF9 protein and mRNA levels in MCF7-WT and MCF7-HIF1α-

KO cells in the same as experiment as in A). E) mRNA expression in MCF7-WT and MCF7-HIF1α-KO cells cultured in normoxia or 0.1% hypoxia for 48h. * p<0.05,

** p<0.01, *** p<0.001 in normoxia and normoxia vs hypoxia, # p<0.05, ## p<0.01, ### p<0.001 in hypoxia samples. Figure 6 MCF7 A Control Avastin treated

80 80 60 ***

IRF3 ***

*** g

n n

60 i 60

n i

i 40

40 40

A R

R 20

IRF7

20 20 A

% % %

0 0 0 Control Avastin Control Avastin Control Avastin ADAR ADAR

MDA-MB-231 B Control Avastin treated

100 80 IRF3 60

*** **

g g

80 n

n i

i 60

n i

i 40

a t

a 60

R 7

3 40

F F

IRF7 D

20 R

A 20

% %

0 0 0 Control Avastin Control Avastin Control Avastin ADAR ADAR

Figure 6. Downregulation of the type I IFN staining in hypoxic tumours in vivo. A) IRF3, IRF7 and ADAR staining in MCF7

xenografts from control and avastin-treated mice and their respective quantification. B) IRF3, IRF7 and ADAR staining in MDA-MB-231

xenografts from control and avastin-treated mice and their respective quantification. n=5 per group, ** p<0.01, *** p<0.001.

↓O D 2 ↓transcription ↑HIF1α ↑ chromatin condensation

Type I IFN pathway

Figure 7. ATAC-seq reveals dynamics of chromatin accessibility during hypoxia. A) MA plot of differentially accessible peaks during

hypoxia with peaks significantly more (red) or less (blue) accessible during hypoxia. B) Annotation of all ATAC-seq peaks, and peaks with

significant changes in accessibility. C) Percent of ATAC-seq peaks containing HIF1, STAT1, IRF3, FOXA1 and GATA3 transcription factor

binding motifs. Peaks are divided into transcription start site (TSS) associated and TSS-distal peaks (all other categories in B). The number of

peaks that are unchanged, decreased and increased in A) are shown graphically and following the same colour-code in C). Number of

differentially accessible peaks in each class is shown in the HIF-1 graph and are identical for the others. P-values are for a Fisher’s exact test

with Bonferroni multiple test correction. D) Schematic representation of the hypoxia-dependent and HIF1α-independent mechanism by which

hypoxia downregulates the type I IFN pathway.