biogenesis: a novel mechanism of colorectal tumorigenesis

Atypical chemokine receptor 3 (ACKR3), previously known as C-X-C chemokine receptor type 7 (CXCR7), has emerged as a key player in several biologic processes. Its atypical “intercepting receptor” signaling properties have established ACKR3 as the main regulator in pathophysiological processes in many diseases. However, much less is known the underlying mechanisms of ACKR3 in promoting tumorigenesis. We found, in both human and animal model, that activation of ACKR3 promotes colorectal tumorigenesis through the NOLC1-induced perturbations of rRNA biogenesis. As compared with adjacent non-neoplastic tissue, human colonic cancer tissues demonstrated higher expression of ACKR3, and high ACKR3 expression was associated with increased severity of colonic cancer. Villin-ACKR3 transgenic mice demonstrated the characteristics of ACKR3-induced colorectal cancer, showing the nuclear β-arrestin-1-activated perturbation of rRNA biogenesis. Activation of ACKR3 induced nuclear translocation of β-arrestin-1 (β-arr1), leading to the interaction of β-arr1 with nucleolar and coiled-body phosphoprotein 1 (NOLC1). As the highly phosphorylated protein in the nucleolus, NOLC1 further interacted with Fibrillarin, a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation, leading to the increase of methylation in Histone H2A, resulting in the promotion of rRNA transcription of ribosome biogenesis. Conclusion: ACKR3 promotes colorectal tumorigenesis through the perturbation of rRNA biogenesis by nuclear β-arr1-induced interaction of NOLC1 with Fibrillarin. HIGH LIGHTS ACKR3 is an atypical G protein-coupled receptor (GPCR) ACKR3 promotes colorectal tumorigenesis ACKR3 induces nuclear translocation of β-arr1 Nuclear β-arr1 interacts with NOLC1 to activate Fibrillarin Interaction of NOLC1 to Fibrillarin leads to perturbation of rRNA biogenesis

less is known the underlying mechanisms of ACKR3 in promoting tumorigenesis. We Chemokine receptor ACKR3, formerly named as CXCR7/RDC1, is an atypical G 50 protein-coupled receptor (GPCR) (Bachelerie et al., 2014;Balabanian et al., 2005;51 Burns et al., 2006). Unlike classical GPCRs, ACKR3 usually fails to activate 52 canonical Gi protein-mediated signaling due to the lack of a conserved DRYLAIV 53 structure (Cancellieri et al., 2013). ACKR3 is once regarded as a scavenger receptor 54 and decoy receptor (Luker et al., 2012;Meyrath et al., 2020). During the 55 pathophysiological processes, ACKR3 could be activated by endogenous ligands 56 CXCL11/I-TAC and CXCL12/SDF-1, resulting in the development of numerous 57 diseases, such as cancers, chronic inflammation, and cardiovascular disorders 58 (Sanchez-Martin et al., 2013). ACKR3 preferentially triggers the β-arrestin-dependent 59 signaling, leading to the internalization via the AKT or ERK1/2 signaling pathways 60 (Becker et al., 2019;Li et al., 2019;Rajagopal et al., 2010). At present, still little is 61 known about mechanism of the ACKR3-induced β-arrestin-dependent signaling in 62 tumorigenesis. ACKR3 was found overexpression in many cancers. Activation of 63 ACKR3 has been considered to promote cancer growth through the key processes, 64 such as proliferation, anti-apoptosis, angiogenesis, etc (Miao et al., 2007;Sun et al., 65 2010;Wang et al., 2008). ACKR3 has thus considered as a potential therapeutic target 66 for the clinical management of cancers. However, these ongoing efforts have been less 67 encouraging in animal models because ACKR3 could participate in a complex 68 signaling network, interacting with additional targets and signaling pathways through 69 various crosstalk and compensatory signaling mechanisms. Further investigating the 70 6 mechanisms of ACKR3 could help us to fully understand the role of ACKR3 in 71 colorectal tumorigenesis (Salanga et al., 2009). 72 As a "crosslinker" receptor, ACKR3 is likely to bind with CXCR4, a classic 73 chemokine receptor for CXCL12, to form the ACKR3/CXCR4 heterodimer. In the 74 ACKR3/CXCR4 heterodimer, ACKR3 might function as modulating the CXCR4 75 signaling to an active and independent signaling receptor (Koch and Engele, 2020). 76 Previously, we revealed a mechanism of ACKR3/CXCR4 heterodimer in promoting 77 colorectal tumorigenesis. ACKR3 was found to modulate or just assist the CXCR4 tumorigenesis independent of CXCR4 signaling. As yet the mechanism of ACKR3, 83 only known an atypical G protein-coupled receptor, has not been fully investigated.

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In the present study, we uncovered a new mechanism of ACKR3 in the promotion 85 of colorectal tumorigenesis through the NOLC1-induced perturbations of rRNA 86 biogenesis. Since β-arr2 has a strong nuclear export signal (NES) in its C terminus, 87 this specific NES excludes it from sustained presence in the nucleus (Meier, 1996;88 Scott et al., 2002). Thus, in the present study, we identified β-arr1 as a downstream 89 signaling of activated ACKR3 in the pathophysiological processes of colorectal 90 tumorigenesis. The ACKR3-induced nuclear β-arr1 interacted with NOLC1 to form 91 the β-arr1-NOLC1 complex. NOLC1 is a highly phosphorylated nucleolus coiled 92 7 protein and a binding protein for RNA polymerase I. NOLC1 is essential for small 93 nucleolar riboprotein synthesis and a critical transcription factor for an array of gene 94 transcriptions (Kim et al., 2003;Meier, 1996)

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High expression of ACKR3 in human CRC specimens 105 To investigate the role of ACKR3 in colorectal cancer (CRC), we firstly analyzed the 106 data of ACKR3 gene issued in the Oncomine database. An analysis of a Hong 107 Colorectal dataset indicated that the mRNA levels of ACKR3 were significantly 108 higher in colorectal cancers than in normal tissues (P = 7.50E-5, Fold Change = 2.427) 109 ( Fig 1A). Importantly, the increased ACKR3 was associated with the progression of 110 clinical stages of colorectal cancer as indicated in the GEPIA database ( Fig 1B). F 111 value = 2.23, Pr (>F) = 0.084. We then performed the immunohistochemistry assay 112 (IHC) to determine ACKR3 levels in human CRC tissues and their paracancerous 113 tissues ( Fig 1C). Of the 60 CRC specimens, 57 cases were identified higher ACKR3 114 8 expression than their paracancerous tissues (Supplementary, Table S4). Western 115 blotting assay further identified higher levels of ACKR3 in human fresh colonic 116 cancer tissues than their paracancerous tissues ( Fig 1D). In vitro cultured cells, the 117 cancer cell lines but not human normal colonic cell line demonstrated higher levels of 118 ACKR3 ( Fig 1E).  Figure S1 and Figure S2).

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As the down-stream signal of GPCR, β-arr1 might respond to the activated ACKR3.

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Human colon cancer cells HCT116 exposed to CXCL12 to activation of ACKR3 139 demonstrated an increase of β-arr1 in the nucleus with time dependent manner (P < 140 0.05 vs. 0 h) (Fig 3A). Since CXCL12 is the ligand of both ACKR3 and CXCR4, we 141 thus used AMD3100, the specific CXCR4 inhibitor, to prevent action of the 142 CXCL12-activated CXCR4 on ACKR3, and then analyzed the expression of β-arr1 in 143 the nucleus. An increase of β-arr1 had remained in the nucleus in the absence of 144 CXCR4 ( Fig 3B). Nuclear β-arr1 was clearly seen in the immunofluorescence staining 145 cells ( Figure 3C). Nuclear β-arr1 was also seen in colorectal cancer of Villin-ACKR3 146 mice ( Fig 3D). Reversely, silencing of ACKR3 significantly prevented the 147 translocation of β-arr1 into the nucleus (Fig 3E).

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As β-arr1 was translocated into the nucleus, we next investigated the functions of 149 nuclear β-arr1. We had searched any information regarding the interaction of β-arr1 150 with nuclear proteins issued in the HitPredict database. We observed a specific 151 indication that nuclear β-arr1 might interact with NOLC1. Herein, a strong interaction 152 of nuclear β-arr1 with NOLC1 was identified in HCT116 cells with activated ACKR3 153 by CXCL12 (Fig 3F, i and ii). Since NOLC1 levels varied during the cell cycles (Pai 154 et al., 1995), we thus identified the interaction of nuclear β-arr1 with NOLC1 more 155 obviously in the interphase and the telophase than in the prophase and the metaphase 156 ( Fig 3G). Knockdown of ACKR3 significantly prevented the interaction of nuclear 157 β-arr1 with NOLC1 ( Fig 3H). The interaction of nuclear β-arr1 with NOLC1 resulted in the promotion of 160 NOLC1 in the nucleolus 161 NOLC1 is a highly phosphorylated nucleolus protein functions as a regulator of RNA 162 polymerase I (Kim et al., 2003;Meier, 1996). Still little has been known its roles in    It is known that, as an atypical GPCR, targeting ACKR3 does not lead to typical 223 G-protein-coupled receptor-mediated calcium mobilization and chemotaxis, but rather 224 13 the recruitment of β-arrestins and the internalization of GPCR (Nguyen et al., 2020). 225 ACKR3 has long been considered as a scavenger receptor and decoy receptor (Luker 226 et al., 2012;Meyrath et al., 2020). ACKR3 is upregulated in the inflammatory cells 227 and the malignant cells. Even though ACKR3 was found to function in the 228 ACKR3/CXCR4 heterodimer in tumorigenesis (Koch and Engele, 2020), much less is 229 known about the mechanism of ACKR3. Since Villin-ACKR3 mice developed more 230 exacerbated colorectal cancer than Villin-CXCR4 mice, thus we hypothesize that 231 ACKR3 play a crucial role independent of CXCR4. In the present study, our clinical 232 data showed that high ACKR3 was associated with increased severity of clinical 233 stages. ACKR3 has been received attention in diagnostics, targeting drug design, and 234 management of patients.

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To unveil the mechanism of ACKR3 in colorectal tumorigenesis, we firstly 236 answered the question that how the activated ACKR3 signaling be translocated into 237 the cytoplasm and then to the nucleus. We have understood that GPCRs could interact 238 with β-arrestins (β-arr1 and β-arr2) to function as scaffolds for a multiple kinases that 239 connect GPCRs to the effector pathways. These complex signaling network and  (Scott et al., 2002). In the present study, we revealed that activation of 247 ACKR3 induced nuclear translocation of β-arr1 but not β-arr2 into the nucleus.

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How NOLC1 was identified as the downstream effector of nuclear β-arr1? We 251 firstly searched for the clues about the interaction related to the nuclear β-arr1 in the 252 Hitpredict database. An interesting link of β-arr1 to NOLC1 was found in the database. with pre-rRNA processing. NOLC1 also plays an essential role in rDNA transcription 281 and further inducing rRNA in ribosome biogenesis (Chen et al., 1999;Meier, 2005).

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Thus, ribosome biogenesis is identified as the downstream effector of nuclear NOLC1.

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In the present study, we found that colorectal cancer grown in Villin-ACKR3 mice  In conclusion, ACKR3 promotes colorectal tumorigenesis through the perturbation 302 of rRNA biogenesis by nuclear β-arr1-induced interaction of NOLC1 with Fibrillarin.

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We envision that future approaches to treat colorectal cancer should use ACKR3 304 inhibitors for preventing the ACKR3-activated NOLC1 and Fibrillarin in the 305 nucleolus.