DCAF13 promotes breast cancer cell proliferation by ubiquitin inhibiting PERP expression

Abstract Evolutionarily conserved DDB1‐and CUL4‐associated factor 13 (DCAF13) is a recently discovered substrate receptor for the cullin RING‐finger ubiquitin ligase 4 (CRL4) E3 ubiquitin ligase that regulates cell cycle progression. DCAF13 is overexpressed in many cancers, although its role in breast cancer is currently elusive. In this study we demonstrate that DCAF13 is overexpressed in human breast cancer and that its overexpression closely correlates with poor prognosis, suggesting that DCAF13 may serve as a diagnostic marker and therapeutic target. We knocked down DCAF13 in breast cancer cell lines using CRISPR/Cas9 and found that DCAF13 deletion markedly reduced breast cancer cell proliferation, clone formation, and migration both in vitro and in vivo. In addition, DCAF13 deletion promoted breast cancer cell apoptosis and senescence, and induced cell cycle arrest in the G1/S phase. Genome‐wide RNAseq analysis and western blotting revealed that loss of DCAF13 resulted in both mRNA and protein accumulation of p53 apoptosis effector related to PMP22 (PERP). Knockdown of PERP partially reversed the hampered cell proliferation induced by DCAF13 knockdown. Co‐immunoprecipitation assays revealed that DCAF13 and DNA damage‐binding protein 1 (DDB1) directly interact with PERP. Overexpression of DDB1 significantly increased PERP polyubiquitination, suggesting that CRL4DCAF13 E3 ligase targets PERP for ubiquitination and proteasomal degradation. In conclusion, DCAF13 and the downstream effector PERP occupy key roles in breast cancer proliferation and potentially serve as prognostics and therapeutic targets.


| INTRODUC TI ON
Breast cancer is one of the leading causes of cancer-related deaths among females worldwide. 1 In 2020, breast cancer surpassed lung cancer as the leading cause of global cancer incidence. 2 Although early stage breast cancer has a good prognosis following surgery and chemotherapy, approximately 90% of deaths among patients with breast cancer are due to recurrence and distant metastasis of the primary tumor. 3 The mechanisms underlying breast cancer development and recurrence should therefore be investigated to develop effective diagnostic and therapeutic modalities.
The cullin RING-finger ubiquitin ligase 4 (CRL4) is an important component of the ubiquitin-proteasome system that contributes to tumorigenesis through multiple mechanisms, including involvement in DNA damage and repair, cell cycle regulation, and the ubiquitination of oncoproteins and tumor suppressors. 4,5 CUL4 uses DNA damagebinding protein 1 (DDB1) as a linker to interact with DDB1-and CUL4associated factors (DCAFs), which serve as substrate receptors of the CRL4 E3 ligase. 6,7 Through different DCAFs, CRL4 E3 ligases recognize a number of substrates for ubiquitination and degradation, such as the tumor repressor LAST1/2, 8 CDT1, 9,10 dicer 1, 11 and NF2, 12 and contribute to the occurrence and development of tumors. DDB1-and CUL4-associated factor 13 has recently been identified as a substrate recognition receptor for CRL4 E3 ligases that mediates pre-implantation embryonic development and mammalian oocyte growth. 13,14 DCAF13 has also attracted growing interest in cancer research. In osteosarcoma cells, the CRL4B-DCAF13 E3 ligase complex specifically targets the tumor suppressor phosphatase and tensin homolog (PTEN) for degradation. 15 Moreover, DCAF13 is frequently amplified in hepatocellular carcinoma patients, and increased expression of DCAF13 reportedly defined a subgroup of hepatocellular carcinoma patients with significantly poor prognosis. 16,17 DCAF13 also acts as a novel RNA-binding protein (RBP) that contributes to triple-negative breast cancer (TNBC) metastasis. 18 Accordingly, DCAF13 is central in cancer biology and potentially serves as a biomarker for more robust staging of cancer patients.
In breast cancer, however, the exact role of DCAF13 is currently unclear.
In this study, we found that DCAF13 was overexpressed in malignant human breast tissue and breast cancer cell lines. Deletion of DCAF13 inhibited breast cancer cell growth, colony formation, and migration and inhibited the growth of human breast cancer xenografts in mice. We further observed that CRL4B-DCAF13 E3 ligase facilitates polyubiquitination of PERP, which acts as the transcriptional downstream protein of p53 and p63. Accordingly, several important functions of DCAF13 were unveiled in the context of breast cancer and PERP was identified as a novel substrate of CRL4 DCAF13 E3 ligase. reduced breast cancer cell proliferation, clone formation, and migration both in vitro and in vivo. In addition, DCAF13 deletion promoted breast cancer cell apoptosis and senescence, and induced cell cycle arrest in the G1/S phase. Genome-wide RNAseq analysis and western blotting revealed that loss of DCAF13 resulted in both mRNA and protein accumulation of p53 apoptosis effector related to PMP22 (PERP).

| Cell culture and stable cell line generation
Knockdown of PERP partially reversed the hampered cell proliferation induced by DCAF13 knockdown. Co-immunoprecipitation assays revealed that DCAF13 and DNA damage-binding protein 1 (DDB1) directly interact with PERP. Overexpression of DDB1 significantly increased PERP polyubiquitination, suggesting that CRL4 DCAF13 E3 ligase targets PERP for ubiquitination and proteasomal degradation. In conclusion, DCAF13 and the downstream effector PERP occupy key roles in breast cancer proliferation and potentially serve as prognostics and therapeutic targets.

| Cell proliferation, colony formation, and invasion assay
For cell growth curve analysis, cells were seeded in six-well plates at a density of 100,000 cells/well. At

| Antibodies, western blotting, and coimmunoprecipitation
Whole cell lysates were prepared with RIPA lysis buffer (Beyotime), denatured at 95 °C for 10 min, separated using SDS-PAGE, and transferred to PVDF membranes (Millipore). The target proteins were detected by primary antibodies and horseradish peroxidase-linked secondary antibodies (Proteintech) ( Table S2). The bands were revealed using an enhanced chemiluminescence detection kit (Millipore).
For co-immunoprecipitation experiments, the plasmid was transfected into 293A cells that were subsequently lysed in RIPA lysis buffer and centrifuged. The supernatants were immunoprecipitated with the respective antibodies for 3 h at 4 °C, followed by incubation with Protein A/G agarose beads (Thermo Fisher Scientific) for 1 h at 4 °C. The immunocomplexes were then washed six times with RIPA buffer containing 5% Tween 80 and assayed by Western blotting.

| Breast tumor biopsies
Paraffin-embedded human breast cancer tissue microarrays (TMAs; N = 75 patients) were purchased from Fanpu Biotech, Inc. Each specimen in these TMAs was represented in two cores on the slide, each measuring 1.5 mm in diameter. Tumor type, staging, and classification data are presented in Tables S3 and S7. Furthermore, fresh breast tumor tissue and adjacent nonneoplastic tissue (N = 10) were biopsied from patients who had been admitted to the Jiaxing University First Affiliated Hospital (Jiaxing, Zhejiang, China). All patients or their legal representatives signed an informed consent document. Tumor type, staging, and classification data are presented in Table S8. Approval was obtained from the institutional review board of the Jiaxing University First Affiliated Hospital Ethics Committee (Registration No. LS2021-KY-047).

| Quantitative real time PCR
Total RNA was extracted from cultured cells using TRIzol Reagent (Invitrogen Life Technologies). The extracted RNA was reversetranscribed into cDNA using PrimeScript RT Reagent Kit (Takara).
Real-time PCR analysis was performed using SYBR Green Master Mix kit (Takara) and an Applied 7300 Real-time PCR system (Eppendorf).
Relative mRNA levels of each gene were normalized to household gene levels (β-actin). Experiments were performed in triplicate. The primers are detailed in Table S4.

| RNA interference
The siRNA oligonucleotides corresponding to the target sequence of PERP and SLC22A18 were designed and synthesized by GenePharma (Table S5). A negative control siRNA (GenePharma) was included in the experiments. Breast cancer cells were seeded into six-well plates at a density of 500,000 cells/well. The siRNA sequences were transfected into cells using the GP-siRNA-Mate Plus reagent (GenePharma) according to the manufacturer's instructions.

| Flow cytometric analyses of apoptosis and cell cycle
For the apoptosis assay, cells were incubated with Annexin V-PE and propidium iodide (BD Biosciences) and analyzed using FC500 flow cytometer (Beckman Coulter). Cell cycle analysis was performed following propidium iodide staining using a CycleTest Plus DNA Reagent Kit (BD Biosciences) according to the manufacturer's instructions. The results were evaluated with a FACScan flow cytometer (BD Biosciences).
The number of cells in each phase were determined.

| Senescence-associated β-galactosidase activity assay
Cell senescence analysis was performed using a senescence βgalactosidase staining kit (Cell Signaling Technology). A total of 100,000 cells were stained with freshly prepared senescenceassociated β-galactosidase overnight at 37 °C and counterstained with eosin. The number of blue-stained senescent cells in randomly selected fields was expressed as a percentage of the total cells.

| Statistical analysis
All in vitro assays were performed at least three times. Statistical analysis was performed using GraphPad Prism software (GraphPad Software). Groups were compared using a nonparametric test unless otherwise noted. The log-rank test was applied to Kaplan-Meier analysis in Figure 1D. Data were considered significant at p < 0.05.

| DCAF13 is overexpressed in human breast cancer tissue and cells
To investigate DCAF13 expression patterns in breast cancer tissue, immunohistochemical analysis was performed on the breast cancer tissue microarrays. Based on staining intensity, we classified the samples into five groups with increasing staining intensity from the weakest (−) to the strongest (++++) ( Figure 1A). Positive DCAF13 staining was We further investigated DCAF13 expression using the data in TCGA database. The clinical data of 1085 breast cancer patients from TCGA database are presented in Tables S6 and S7. We found that DCAF13 expression was significantly elevated in breast cancer compared to healthy tissues (p = 1.1 × 10 −41 ; Figure 1C). The OS in the DCAF13 high-expression group was significantly worse than in the DCAF13 low-expression group (p = 0.016; Figure 1D Accordingly, breast cancer cell lines are comparable to human breast cancer tissue in terms of DCAF13 overexpression.

| DCAF13 deletion inhibits breast cancer cell proliferation, colony formation, and cell migration in vitro
We eliminated DCAF13 in breast cancer cell lines (4T1, 4T07, and MCF-7) by CRISPR/Cas9. Although it was very challenging to obtain complete DCAF13 knockout clones, two clones with a DCAF13 deletion (KO19# and KO31#) had significantly lower DCAF13 protein expression levels (Figures 2A and S1D,E). Compared wild-type cells, DCAF13 deletion led to a remarkable reduction in cell growth ( Figure 2B-D). Correspondingly, the expression of cell proliferation marker p-histone H3 was also diminished in DCAF13-deficient cells (Figure 2A,I). Colony formation assays further showed that DCAF13 deletion reduced the number of colonies formed ( Figures 2E-G and   S1D,E). Moreover, Transwell assays demonstrated that DCAF13 deletion markedly suppressed the migration capacity of 4T07 and 4T1 cells ( Figure 2H). These results demonstrate that DCAF13 is required for breast cancer cell proliferation and migration.

| DCAF13 deletion promotes breast cancer cell cycle, apoptosis, and senescence
To further elucidate the mechanism underlying the inhibition of cell proliferation by DCAF13 deletion, flow cytometry was employed to analyze the cell cycle in 4T1 and 4T07 cells. Compared to wild-type  Figure 3D). Also, an increased number of DCAF13 knockout cells exhibited elevated SAβ-galactosidase staining ( Figure 3E). Taken together, these results suggest that the deletion of DCAF13 inhibits breast cancer cell proliferation by arresting the cells in the G1 phase of the cell cycle, consequently promoting apoptosis and senescence.

| DCAF13 deletion suppresses tumor growth in vivo
To investigate the role of DCAF13 in breast cancer in vivo, we subcutaneously transplanted wild-type or DCAF13 knockout breast cancer (4T07 and 4T1) cells into both flanks of BALB/c mice. DCAF13 knockout xenograft-bearing animals exhibited a lower tumor volume and weight compared to the wild-type mice at the end of the experiment ( Figure 4A,B). The growth curves provide compelling evidence for stalled tumor cell growth in the DCAF13 knockout group ( Figure 4A,B). Accordingly, data attest that DCAF13 is important for breast cancer development.
In follow-up experiments it was found that levels of DCAF13 and p-histone H3 were significantly reduced in DCAF13-deficient tumors ( Figure 4C,D), corroborating the link between DCAF13 deletion and inhibited tumor growth. To elucidate the effect of DCAF13 deletion on apoptosis in vivo, the expression levels of cleaved caspase-3 and the DNA damage marker p-H2AX were examined. Both were highly expressed in DCAF13-deficient tumors ( Figure 4C,D), which is consistent with the increased p-H2AX associated with DCAF13 deletion in vitro (Figure 2A).

| Identification of DCAF13 downstream targets using RNAseq
To understand how DCAF13 deletion inhibits breast cancer growth, genome-wide RNAseq analysis was performed of wild-type and DCAF13-deficient 4T07 cells ( Figure 5A). A total of 449 differentially expressed genes were identified in the DCAF13 knockout group ( Figure 5B). Pathway analysis showed that the genes altered by DCAF13 knockout may play roles in cancer development ( Figure 5C).
Next, we determined whether DCAF13 deletion induced similar transcriptional dysregulation in vivo by qRT-PCR of xenograft biopsies. mRNA levels of ELK-1 and MMP13 were decreased, whereas those of PERP, SLC22A18, and p63 were increased in 4T07 and 4T1 DCAF13 knockout xenografts (Figures 5E and S2C). At the protein level, expression of p-H2AX, p-CHK1, GOLPH2, and nucleophosmin was increased in DCAF13 partial knockout cells, whereas the expression of β-catenin and Hp1 was decreased ( Figures 5F and S1F).

| PERP is an essential DCAF13 target gene in breast cancer
Both tumor suppressor genes SLC22A18 and PERP are reportedly related to the development of breast cancer. 20,21,[27][28][29] To demonstrate that the observed decrease in DCAF13 knockout tumor cell proliferation was due to DCAF13-dependent expression of SLC22A18 or PERP, we performed SLC22A18 and PERP knockdown experiments using RNA interference. Both SLC22A18 and PERP mRNA expressions were efficiently suppressed in 4T07 DCAF13 knockout cells ( Figure 6A,B). SLC22A18 inhibition did not rescue the cell proliferation defect caused by DCAF13-deletion ( Figure 6C). However, PERP inhibition partially rescued the induced DCAF13-deficient cell proliferation defects ( Figure 6D). To further confirm that PERP inhibition could recover the DCAF13 knockout breast cancer cell proliferation, we established both PERP-KO and DCAF13-KO breast cancer cells by using CRISPR/Cas9 technology ( Figure 6E). The results revealed that PERP deletion partially rescued cell proliferation and colony formation in the DCAF13-deficient cells ( Figure 6F,G).
Through online software analysis (http://ubibr owser.ncpsb.org. cn/ubibr owser/), PERP exhibits multiple ubiquitination sites. We hypothesized that PERP could be recognized and ubiquitinated by CRL4 DCAF13 E3 ubiquitin ligase. Co-immunoprecipitation showed that DCAF13 directly interacts with PERP ( Figure 6H F I G U R E 5 Identification of DCAF13 downstream targets by RNAseq. (A) Hierarchical clustering of gene expression profiles from three biologically independent samples based on Pearson correlation coefficient in wild-type and DCAF13 knockout 4T07 cells. Blue to red signifies weak to strong correlation, respectively. TD1, TD2, and TD3: DCA13 knockout 4T07 cells; T1, T2, and T3: wild-type 4T07 cells. (B) Gross overview of upregulated genes in wild-type and DCAF13 knockout 4T07 cells. (C) Differential signaling pathway regulation in DCAF13 knockout 4T07 cells compared to wild-type cells. (D) RT-PCR analysis of the expression of significantly dysregulated genes in wild-type and DCAF13 knockout 4T07 cells. Data are presented as mean ± SE (N = 3). *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.001 (nonparametric test). (E) RT-PCR analysis of the expression of ELK-1, MMP13, SLC22A18, PERP, and p63 in wild-type and DCAF13 knockout xenografts. (F) Immunofluorescence analysis of p-H2AX, p-CHK1, β-catenin, and GOLPH2 (green) levels in wild-type and DCAF13-deficient 4T07 cells. Cells were counterstained with DAPI (blue) DCAF13 were deleted and found that both the DCAF13 SOF △ and WD △ truncations interacted with PERP, suggesting that these two domains are not involved in the interaction ( Figure 6I). Given that DCAF13 functions as a substrate receptor protein for CRL4 E3 ubiquitin ligase complex, we further explored whether PERP acts as a substrate for CRL4 E3 ubiquitin ligase. We then examined the association between PERP and DDB1, the linker protein of CRL4 E3 ligase. PERP interacted with DDB1, suggesting that PERP could form complexes with CRL4 DCAF13 E3 ligase ( Figure 6H). Moreover, overexpression of DDB1 significantly increased the polyubiquitination of PERP ( Figure 6J), suggesting that CRL4 DCAF13 E3 ligase targets PERP for ubiquitination and proteasomal degradation.

| DISCUSS ION
Several studies support the possibility of DCAF13 being an oncogene. 14,16-18 DCAF13 has been reported to be overexpressed in hepatocellular carcinoma, and its overexpression is significantly associated with poor survival in patients with hepatocellular carcinoma. 16 DCAF13 was also significantly upregulated in human lung adenocarcinomas, and knockdown of DCAF13 inhibited human lung adenocarcinoma cell migration. 17 Through DCAF13, CRL4 E3 ligase specifically recognizes the tumor suppressor PTEN for degradation. 13,14 Consistent with these reports, our present work illustrates that DCAF13 is a potential oncogene in breast cancer. We found that DCAF13 was significantly upregulated in breast cancer tissues. DCAF13 deletion resulted in defects in cell proliferation, colony formation, and migration, induced cell cycle arrest at G1/S phase, and increased cell apoptosis. A recent study by Liu demonstrates that DCAF13 promotes triple-negative breast cancer metastasis. 18 Consistent with Liu's study, we found that a high expression of DCAF13 in breast cancer tissues was closely correlated with overall survival. In addition, our study not only revealed that DCAF13 promotes breast cancer metastasis, but also contributes to cell proliferation, apoptosis, and senescence. Importantly, we elucidate a new molecular mechanism by which CRL4 DCAF13 E3 ligase targets polyubiquitination and degradation of PERP.
P53 apoptosis effector related to PMP22 was identified as the transcriptional downstream protein of p63. 29 13 an inhibitor of PI3K-AKT signaling pathway, thereby maintaining the activity of AKT activity and promoting oocytes meiotic maturation, implying that DCAF13 deletion leads to the inactivation of AKT activity. F I G U R E 6 PERP is the key target of DCAF13 in breast cancer cell. (A) The mRNA expression level of SLC22A18 in wild-type or DCAF13 knockout 4T07 cells transfected with control siRNA (siCon) or SLC22A18 siRNAs. **p < 0.01, ***p < 0.001. (B) The mRNA expression level of PERP in 4T07 wild-type cells or DCAF13 knockout cells transfected with control siRNA (siCon) or PERP siRNAs. ***p < 0.001. (C) SLC22A18 deletion in DCAF13 knockout 4T07 cells did not affect cell proliferation. The error bars represent SD (n = 3). n.s (p > 0.05). Three replicates were included. (D) PERP deletion rescues cell proliferation defects in 4T07 DCAF13 KO cells by CCK8 assay. *p < 0.05, ***p < 0.001. (E) Western blotting analysis of the expression of PERP in wild-type, DCAF13 knockout, DCAF13 knockout and PERP knockout 4T07 cells. (F) PERP deletion partially rescues the cell growth defects in DCAF13 knockout 4T07 cells. The error bars represent SD. *p < 0.05. ***p < 0.001. Three replicates were included. (G) PERP deletion partially rescues the colony formation defects in DCAF13-deficient cells. Representative images from the colony formation assay (left panel) and the colony number analysis (right panel) were shown. All experiments were performed in triplicate. **p < 0.01, ***p < 0.001 (H) Co-immunoprecipitation result showing PERP interacts with DCAF13 and DDB1. 293T cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with anti-Flag beads. Input lysates was immunoblotted with antibodies against HA. (I) 293T cells transiently transfected with plasmids encoding the full-length Flag-DCAF13 or the DCAF13 two truncations (Flag-DCAF13 SOF △ and Flag-DCAF13 WD △ ) together with HA-PERP. Protein extracts were immunoprecipitated using anti-Flag beads. (J) Co-immunoprecipitation experiments showing PERP polyubiquitination. (K) A working model explaining how DCAF13 promotes breast cancer cell proliferation. In wild-type breast cancer cell, PERP, a tumor suppressor protein, is polyubiquitinated by CRL4DCAF13 and destabilized. This promotes the proliferation of breast cancer cell. When DCAF13 was knocked out, PERP could not be polyubiquitinated and became stabilized, resulting in the inhibition of breast cancer cell proliferation In summary, our study not only revealed critical roles of DCAF13 in the growth and metastasis of breast cancer but also highlighted the mechanism where CRL4 DCAF13 E3 ligase mediates PERP degradation and promotes breast cancer cell proliferation, as illustrated in Figure 6K. This study indicated that DCAF13 could be a potential target for breast cancer diagnosis and treatment. It may be expedient to silence the expression of DCAF13 for the purpose of treating breast cancer. In the field of cancer therapy, small interfering RNA (siRNA) intervention is considered as a powerful tool for gene silencing (knockdown), enabling the suppression of oncogenic factors in cancer. 35 Our findings provide evidence for future breast cancer therapy by inhibiting DCAF13 with siRNAs. Although siRNA intervention has been applied to the treatment of breast cancer, 36 siRNA-based therapies have not yet reached the clinic, but with further development of multiple targets, sophisticated delivery systems, and combination treatments, hopefully a breakthrough can be achieved.

D I SCLOS U R E
The authors declare no potential conflicts of interest.