High expression of ACKR3 in human CRC specimens
To investigate the role of ACKR3 in colorectal cancer (CRC), we firstly analyzed the data of ACKR3 gene expression issued in the Oncomine database. An analysis of a Hong Colorectal dataset indicated that the mRNA levels of ACKR3 were significantly higher in colorectal cancers than in normal tissues (p = 7.50E-5, Fold Change = 2.427) (Fig. 1A). Importantly, the increased ACKR3 was associated with the progression of clinical stages of colorectal cancer as indicated in the GEPIA database (Fig. 1B). F value = 2.23, Pr (>F) = 0.084. We then performed the immunohistochemistry assay (IHC) to determine ACKR3 levels in human CRC tissues and their paracancerous tissues (Fig. 1C). Of the 60 CRC specimens, 57 cases were determined higher level of ACKR3 expression than their paracancerous tissues (Supplementary, Table S4). Western blotting assay further identified higher levels of ACKR3 in human fresh colonic cancer tissues than their paracancerous tissues (Fig. 1D). In vitro cultured cells, the colonic cancer cell lines but not human normal colonic cell line demonstrated higher levels of ACKR3 (Fig. 1E).
Increased ACKR3 in intestinal epithelium cells conferred colorectal tumorigenesis in Villin-ACKR3 mice
Villin-ACKR3 mice demonstrated the increased severity of colorectal tumorigenesis as compared to their WT littermates, showing a significant weight loss (Fig. 2A) and an high disease activity index (DAI index) (Fig. 2B). AOM/DSS induced shorter colorectal length in Villin-ACKR3 mice showed than WT mice (Fig. 2C,D). Importantly, Villin-ACKR3 mice developed more colorectal tumors than WT mice (Fig. 2E). These colorectal tumors in Villin-ACKR3 mice demonstrated higher levels of ACKR3 (Fig. 2F), with significantly increased CXCL12 (Fig. 2G) than those in their WT littermates. Microscopic analysis of colonic tissues showed the exacerbated colonic damage with higher dysplasia in Villin-ACKR3 mice than WT mice (Fig. 2H). These symptoms of colorectal tumorigenesis in Villin-ACKR3 mice might associate with the upregulation of signaling of pluripotent potential and the ERK signaling in the intestinal epithelium cells (Supplementary Figure 1 and Figure 2).
ACKR3 induced β-arr1 translocation into the nucleus to interact with NOLC1
As the down-stream signal of GPCR, β-arr1 might respond to the activated ACKR3. Human colon cancer cells HCT116 exposed to CXCL12 to activation of ACKR3 demonstrated an increase of β-arr1 in the nucleus with time dependent manner (p < 0.05 vs. 0 h) (Fig. 3A). Since CXCL12 is the ligand of both ACKR3 and CXCR4, we thus used AMD3100, the specific CXCR4 inhibitor, to prevent action of the CXCL12-activated CXCR4 on ACKR3, and then analyzed the expression of β-arr1 in the nucleus. An increase of β-arr1 had remained in the nucleus in the absence of CXCR4 (Fig. 3B). Nuclear β-arr1 was clearly seen in the immunofluorescence staining cells (Fig. 3C). Nuclear β-arr1 was also seen in colorectal cancer of Villin-ACKR3 mice (Fig. 3D). Reversely, silencing of ACKR3 significantly prevented the translocation of β-arr1 into the nucleus (Fig. 3E).
As β-arr1 was translocated into the nucleus, we next investigated the functions of nuclear β-arr1. We firstly had searched any information regarding the interaction of β-arr1 with nuclear proteins issued in the HitPredict database. We then observed a specific indication that nuclear β-arr1 might interact with NOLC1. Herein, a strong interaction of nuclear β-arr1 with NOLC1 was identified in HCT116 cells with activated ACKR3 by CXCL12 (Fig. 3F, i and ii). Since NOLC1 levels varied during the cell cycles [18-20], we thus identified the interaction of nuclear β-arr1 with NOLC1 more obviously in the interphase and the telophase than in the prophase and the metaphase (Fig. 3G). Knockdown of ACKR3 significantly prevented the interaction of nuclear β-arr1 with NOLC1 (Fig. 3H).
The interaction of nuclear β-arr1 with NOLC1 resulted in the promotion of NOLC1 in the nucleolus
It is known that NOLC1 is a phosphorylated nucleolus protein functions as a regulator of RNA polymerase I [18-20]. However, still little has been known its roles in the tumorigenesis. The Cancer Genome Atlas (TCGA) reported high levels of NOLC1 in colorectal cancers (Fig. 4A). Our results showed the increased NOLC1 levels in colorectal cancer grown in Villin-ACKR3 mice (Fig. 4B) as well as human colonic cancer tissues (Fig. 4C). We further performed the Immunofluorescence assay to mark the colocalization of NOLC1 with Fibrillarin, the rRNA methyl-transferase, and UBF1, the nucleolar transcription factor 1. NOLC1 was exactly identified in the nucleolus (Fig. 4D). Reversely, NOLC1 was not presented in the nucleolus of cancer cells knockdown of β-arr1 (Fig. 4E) or ACKR3 (Fig. 4F). These results indicated that activation of ACKR3 resulted in the increase of NOLC1 in the nucleolus through the induction of nuclear translocation of β-arr1.
The increased nucleolus NOLC1 promoted the synthesis of rRNA of ribosome biogenesis in cancer cells with ACKR3 activation
To investigate the nucleolus NOLC1-promoted in colonic tumorigenesis, we analyzed the level of nuclear AgNOR protein, a marker of cell proliferation, in colorectal cancer tissues of Villin-ACKR3 mice. More deeply and globally staining of AgNOR proteins was measured in colorectal cancer of Villin-ACKR3 than that of WT mice (Fig. 5A). To illustrate the role of NOLC1 in the nucleolus, we employed cell model silenced NOLC1 to analyze the levels of synthesis of rRNA of ribosome biogenesis. We designed 3 types of siRNAs to target NOLC1. The levels of NOLC1 were significantly reduced by 51.2%, 49.7%, 65.1%, respectively, in si1-, si2-, and si3-treated HCT116 cells (Fig. 5B). Silence of NOLC1 by si3 resulted in a significant reduction of nucleoli (Fig. 5C). We then analyzed the levels of precursor rRNA 45S, 36S, and 32S rRNA by RT-qPCR assay. As shown in Fig. 5D, the expression levels of pre-rRNA 45S, 36S, and 32S were down-regulated in cancer cells with silenced NOLC1 (siNOLC1 cells) as compared with control WT cells. These results indicated that silencing gene of NOLC1 resulted in the reduction of rRNA synthesis. We further analyzed the nucleolar size of cancer cells with silenced NOLC1 under the transmission electron microscopy. Silencing of NOLC1 gene resulted in the nucleolus smaller than control cells (Fig. 5E). Western blotting analysis demonstrated a higher level of nucleolar NOLC1 in colonic cancer tissues in Villin-ACKR3-AD than WT-AD mice. These colonic cancer tissues with elevated NOLC1 exhibited higher level of POLR1A (RPA194), the largest subunit of RNA Pol I, and higher level of UBF1, the transcription initiation factor of rRNA transcription, in Villin-ACKR3-AD than WT mice (Fig. 5F). These results suggest that the ACKR3-activated nucleolar NOCL1 is associated with the synthesis of rRNA of ribosome biogenesis.
The interaction of nucleolar NOLC1 with Fibrillarin led to the increase of Fibrillarin and resulted in the promotion of rRNA transcription
It wa reported that Fibrillarin plays an important role in the tumorigenesis through inducing rRNA transcription [21]. As we identified the function of nucleolar NOLC1 in promoting synthesis of rRNA of ribosome biogenesis, we wanted to know whether Fibrillarin was involved in the NOCL1-activated synthesis of rRNA. Analysis of correlation data issued by GEPIA database indicated a correlation of nucleolar NOLC1 to Fibrillarin in CRC tissues (correlation coefficient R = 0.4, Fig. 6A). Further immunofluorescence analysis indicated a strong colocalization of NOLC1 with Fibrillarin in nucleolus of HCT116 cells (Fig. 6B). Western blotting analysis indicated a significant higher Fibrillarin in Villin-ACKR3 mice than WT mice (Fig. 6C). Conversely, knockdown of nucleolar NOLC1 significantly reduced Fibrillarin levels (Fig. 6D). The interaction of nucleolar NOLC1 with Fibrillarin was clearly seen in colorectal cancer cells of Villin-ACKR3 mice but not obviously in colorectal cancer cells of WT mice (Fig. 6E). These results indicated that the interaction of NOLC1 with Fibrillarin induced the upregulation of Fibrillarin and further promoted rRNA transcription in colorectal cancer cells of Villin-ACKR3 mice. We conclude that activation of ACKR3 promotes Colitis and colorectal tumorigenesis through the increase of ribosome biogenesis by the interaction of nuclear NOLC1 with Fibrillarin (Fig. 6F).