PHOSPHATE exporter XPR1/SLC53A1 is required for the tumorigenicity of epithelial ovarian cancer

Abstract Ovarian cancer is the fifth most common cause of cancer‐related death in women. Ovarian clear cell carcinoma (OCCC) is a chemotherapy‐resistant epithelial ovarian cancer with poor prognosis. As a basis for the development of therapeutic agents that could improve the prognosis of OCCC, we performed a screen for proteins critical for the tumorigenicity of OCCC using the CRISPR/Cas9 system. Here we show that knockdown of the phosphate exporter XPR1/SLC53A1 induces the growth arrest and apoptosis of OCCC cells in vitro. Moreover, we show that knockdown of XPR1/SLC53A1 inhibits the proliferation of OCCC cells xenografted into immunocompromised mice. These results suggest that XPR1/SLC53A1 plays a critical role in the tumorigenesis of OCCC cells. We speculate that XPR1/SLC53A1 might be a promising molecular target for the therapeutic treatment of OCCC.


| INTRODUC TI ON
Epithelial ovarian cancer has one of the worst prognoses among gynecologic malignancies. 1,2 Approximately 60% of epithelial ovarian cancer cases are diagnosed at stages III and IV, and their 5-year survival rate is less than 30%. 3,4 Epithelial ovarian cancer is classified into four major histological subtypes: serous, clear cell, endometrioid, and mucinous. 5 Ovarian clear cell carcinoma is less sensitive to conventional platinum-based chemotherapy and has worse prognosis than other subtypes. [6][7][8][9][10] The incidence of OCCC in ovarian cancer is higher in Asia, especially in Japan (25% of ovarian cancers), than in North America and Australia (5%). 11,12 Mutations in various oncogenes and tumor suppressor genes, including K-ras, p53, ARID1A, and PIK3CA, have been shown to be involved in the development of OCCC. 13,14 In particular, ARID1A and PIK3CA are mutated in approximately half of the cases of OCCC.
Human xenotropic and polytropic retrovirus receptor 1/solute carrier 53A1 (hereafter called XPR1) was originally identified as a receptor for xenotropic and polytropic murine leukemia viruses. [15][16][17] As a multipass transmembrane protein, XPR1 functions as an inorganic phosphate exporter. 18 In cooperation with the phosphate importer SLC20A2, XPR1 regulates cellular phosphate homeostasis in an inositol polyphosphate-dependent manner: inositol pyrophosphate (PP-InsP) signaling molecules such as 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP 8 ) interact with the N-terminal SYG1-Pho81-XPR1 (SPX) domain of XPR1 and regulate its phosphate exporter activity. 19,20 In addition, XPR1 has been reported to regulate cAMP levels through an interaction with G protein β subunits, although its role in phosphate regulation remains to be elucidated. 21 Mutations in XPR1 have been identified in patients with primary familial brain calcification, a genetic disease characterized by cerebral calcium phosphate deposition and associated with neuropsychiatric disorders. 22,23 Furthermore, it has been reported that XPR1 expression is enhanced in tongue squamous cell carcinoma tissues compared to normal tongue tissues and correlates with poor prognosis. 24 We attempted to identify novel molecular targets critical for the proliferation and tumorigenicity of OCCC using the CRISPR/

| CRISPR-CAS9 screening
CRISPR-CAS9 screening was carried out using TKO CRISPR Library Version 3 (lentiCRISPRv2; Addgene #90294) following the protocols provided in https://www.addge ne.org/poole d-libra ry/moffa t-crisp r-knock out-tkov3/. 25 The OCCC cell lines OVISE, ES2, TOV21G, and   JHOC5 were transfected with lentiCRISPRv2 at an MOI of 0.3 followed by selection with puromycin for 2 days. A sample of 2 × 10 7 cells was then pelleted and frozen under liquid nitrogen as a Day 0 sample. Cells were then passaged until they had undergone eight doublings. To maintain sufficient sgRNA coverage, the total number of cells was maintained above 2 × 10 7 for the duration of the culture period. Cells cultured for eight cell doublings from Day 0 were taken as a final time point. Genomic DNA was extracted using the Blood and Cell Culture DNA MidiKit (#13343; Qiagen). One hundred twenty five micrograms of genomic DNA from each sample was split into 2.5 μg fractions and sgRNA sequences were amplified using Herculase II fusion DNA polymerase. Reactions were measured for fragment size using the Agilent 2200 Tapestation and quantified using the KAPA SYBR Fast qPCR Kit (#7959362001; KAPA Biosystems). To generate and analyze sgRNA count data, MAGeCK (version 0.5.9) was used ("mageck count" and "mageck mle --norm-method control --control-sgrna --cnv-norm" command). 26
Silencer select siRNA negative control (4390843; Thermo Fisher Scientific) was used as a control.

| Human ovarian cancer samples
Ovarian cancer tissues were prepared as described previously 11  Informed consent was obtained from all subjects involved in the study.

| RNA sequencing analysis
Total RNA was extracted using TRIsure reagent (Bioline). For ovarian cancer tissue dataset, cDNA libraries were prepared using the Illumina TruSeq Stranded Total RNA and the Ribo-Zero Gold LT Sample Prep Kit. All libraries were sequenced using an Illumina HiSeq 2500 to create single-end 65 bp reads, which were aligned to the human reference genome build hg38 using STAR. 27 For XPR1 knockdown dataset, sequencing library construction (mRNA-seq) and Illumina sequencing (paired-end 150 bp reads) were undertaken by AnnoRoad Gene Technology. RSEM 28 was used to calculate transcripts per kilobase million (TPM, Ensembl gene annotation GRCh38). For differential expression analyses, we applied the count data to edgeR. 29 The count data were fitted with a general linear model. Gene Ontology enrichment analysis and GSEA were undertaken using the R package "clusterProfiler". 30 The ovarian normal tissue dataset from GTEx in UCSC Xena (https://xena.ucsc.edu/) was combined with our ovarian cancer tissue dataset (see above), and then normalized by the quantile method using the R package "limma".

| Cell viability assay
Cells (1 × 10³ cells) were transfected with siRNA (10 nM) and seeded into 96-well plates. After 24 h, fresh medium was replaced. One, 4, and 6 days after transfection, cell viability was assessed indirectly by measuring the intracellular levels of ATP using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). Luminescence was measured on a Mithras LB 940 (Berthold).
Real-time PCR was carried out using a LightCycler480 (Roche). The results were normalized against the values detected for GAPDH.
Primers used for quantitative RT-PCR are described in Table S1.

| Genome-wide CRISPR/CAS9 screens using OCCC cell lines
To identify genes critical for the proliferation of OCCC, we previously performed CRISPR/CAS9 screens against the OCCC cell lines OVISE, ES2, TOV21G, and JHOC5 ( Table 1) using the TKOv3 sgRNA library. From a list of genes selected in this screen (Data S1), we focused on genes that encode membrane proteins overexpressed in cancer cells, which could be suitable targets for Ab drug development. We observed that depletion of 22 genes encoding the SLC family of membrane proteins resulted in a significant decrease in the proliferation of OCCC cell lines ( Figure 1A). Furthermore, we analyzed data from the Cancer Dependency Map (https://depmap.  We observed that ZIP10/SLC39A10, GLUT1/SLC2A1, and XPR1/ SLC53A1, but not PHC/SLC25A3 or MDU1/SLC3A2, were upregulated in ovarian cancers compared to normal tissue ( Figure 1C). As many studies have already been done on SLC39A10 and SLC2A1, we decided to focus on XPR1.

| Effect of XPR1 knockdown on gene expression profiles of OCCC cell lines
To clarify the effect of XPR1 knockdown on gene expression profiles of OCCC cells, we undertook RNA sequencing and GSEA using OVISE, ES2, TOV21G, and JHOC5 cells transfected with siRNA targeting XPR1 (Figure 2A,B). The GSEA revealed that the p53 pathway was activated in all cell lines. Inconsistent with a previous report, 24 nuclear factor-κB signaling was enhanced in OVISE, ES2, and TOV21G cells. Inflammatory response, epithelial-mesenchymal transition, and K-ras signaling were also activated in all of these cell lines. Gene Ontology overrepresentation analysis revealed that expression of genes involved in oxidative stress and other stress responses was enhanced in all cell lines ( Figure 2C). The GO biological process analysis indicated that the genes upregulated in common in at least three cell lines ( Figure 2D) were enriched for those involved in stress response function ( Figure 2E).

| Knockdown of XPR1 suppresses the growth of OCCC cells
We next attempted to examine the effects of RNAi-mediated knockdown of XPR1 on the growth and tumorigenicity of OCCC cells. As OVISE and ES2, but not TOV21G or JHOC5, were tumorigenic in mice, we used OVISE and ES2 in subsequent experiments. We found that siRNA knockdown of XPR1 resulted in a significant decrease in the proliferation of both cell lines in vitro ( Figure 3A,B). Consistent with a previous report, 18 the intracellular phosphate level was also decreased ( Figure S1). Furthermore, FACS analysis revealed that knockdown of XPR1 induced the accumulation of sub-G 1 cells ( Figure 3C). We therefore measured the changes in the expression of marker genes indicating p53- To further clarify the significance of p53 in XPR1 knockdowninduced growth suppression, we compared the effects of siRNA against XPR1 on the proliferation of the colon tumor cell lines HCT116 (p53 +/+ ) and a derivative, HCT116 (p53 −/− ), in which p53 is disrupted by homologous recombination. 33 We found that knockdown of XPR1 caused both a marked inhibition of the proliferation and an increase in the sub-G 1 population of both cell lines ( Figure 4A-D). However, knockdown of XPR1 induced upregulation of PUMA, NOXA, and GADD45 only in HCT116 (p53 +/+ ) cells and not in HCT116 (p53 −/− ) cells ( Figure 4E). These results suggest that knockdown of XPR1 can suppress the proliferation of cancer cells through both p53-dependent and -independent mechanisms.
We next investigated the effects of siRNA-mediated knockdown of XPR1 on the growth of various cancer cell lines ( Figures   S2A,B). Immunoblotting analysis was also undertaken to examine XPR1 protein expression in these cell lines. We considered XPR1 to be three bands between 63 and 75 kDa ( Figure S3A), because exogenously expressed XPR1 migrated to the same position ( Figure   S3B). Moreover, the intensity of these three bands was decreased in TOV21G cells transfected with siRNAs targeting XPR1 ( Figure S3C).
Furthermore, we examined the expression levels of XPR1 in various cancer tissues and corresponding normal tissues ( Figure S4). For example: in lung cancer, XPR1 expression was higher than normal tissue and XPR1 knockdown was effective against H1299 but not A549 cells; in renal cancer, XPR1 expression was not much different from normal tissue, but XPR1 knockdown was effective against 786O cells; and in colon cancer, XPR1 knockdown was effective against HCT116 ( Figure 4C) but not DLD1 cells. These results suggest that the sensitivity of cancer cells to XPR1 knockdown cannot simply be explained by XPR1 expression levels.

| Knockdown of XPR1 suppresses the tumorigenicity of OCCC cells
Finally, we examined the effects of an shRNA targeting XPR1 on the tumorigenicity of OCCC cells. We infected OVISE and ES2 cells with a lentivirus expressing an shRNA targeting XPR1 and transplanted these into immunodeficient mice. The growth of these tumor cells was significantly retarded compared to tumor cells infected with control virus ( Figure 5). These results suggest that XPR1 is required for the tumorigenicity of OCCC cells in vivo.
In the present study, we identified the transmembrane protein polyphosphate-dependent manner. 19,20 We speculate that XPR1 knockdown induces p53-dependent and -independent apoptosis by dysregulating phosphate homeostasis. In this regard, it is interesting to note that high extracellular phosphate induces apoptosis along with high levels of intracellular phosphate, reactive oxygen species generation, mitochondrial membrane depolarization, and caspase activation. 34 Further investigation is required to clarify the mechanisms underlying apoptosis induced by XPR knockdown.
In conclusion, we have shown that XPR1 is critical for the proliferation and tumorigenicity of epithelial ovarian cancers, especially OCCC. Our results suggest that drugs such as mAbs that inhibit XPR1 function might be useful for the therapeutic treatment of OCCC.