Hypoxia Dynamically Regulates DBC1 Ubiquitination and Stability by SIAH2 and OTUD5 in Breast Cancer Progression

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted the competitive binding of SIAH2 with OTUD5 to DBC1, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin–proteasome pathway. Siah2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of Siah2/DBC1. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.


Introduction
The occurrence and development of tumors is modulated by the dual regulation of genetic instability and the tumor microenvironment (Singleton, Macann, and Wilson 2021). Hypoxic stress or low oxygen tension, a major hallmark of the tumor microenvironment, plays an essential role in the progression and metastasis of many solid tumors (Cheng et al. 2020;Lee et al. 2019). Moreover, our previous studies have documented that hypoxic stress attenuates tumorigenesis and progression by modulating the Hippo signaling pathway and mitochondrial biogenesis (Ma et al. 2015;Ma et al. 2019). To extensively understand the critical roles of hypoxic stress in tumorigenesis or tumor development, more precise mechanisms need to be further explored.
Deleted in Breast Cancer 1 (DBC1; also known as CCAR2) is a nuclear protein containing multifunctional domains and plays a critical role in a variety of cancers. Importantly, DBC1 cooperates with lots of epigenetic and transcriptional factors to regulate cell activities. DBC1 mediates p53 function not only by inhibiting the deacetylase activity of SIRT1, but also by inhibition of p53 ubiquitination and degradation (Qin et al. 2015;Akande et al. 2019) (Kim, Chen, and Lou 2008;Zhao et al. 2008). In addition, DBC1 can specifically inhibit deacetylase HDAC3 activity and alter its subcellular distribution, resulting in cell senescence (Chini et al. 2010). Most also upregulated, and genes associated with DNA repair and apoptosis were downregulated ( Figure 1H, I and Figure 1-figure supplement 1A, B). Collectively, these results suggest that hypoxia-induced DBC1 degradation contributes to tumor progression by regulating multiple pathways.

SIAH2 interacts with DBC1 and regulates its stability
To identify the E3 ligases that potentially ubiquitinates DBC1 to contribute its degradation, several ubiquitin E3 ligases previously reported to be involved in the hypoxia response were screened, and SIAH2 was finally identified to be responsible for the stability of DBC1 ( Figure   2A). To confirm our hypothesis, LC-MS/MS analysis of proteins bound to SIAH2 RM (a SIAH2 enzyme inactive mutant) was performed. DBC1 was validated as one of the substrates of SIAH2 ( Figure 2B). To further explore the relationship between SIAH2 and DBC1, we carried out a Co-IP assay and found that exogenously expressed Flag-SIAH2 RM  , and the Co-IP assay showed that the 1-230 amino acids at the N-terminus of DBC1 were necessary for binding to SIAH2 ( Figure 2E), and the fulllength SIAH2 was required for their interaction ( Figure 2F). Importantly, the N-terminus (1-461) of the DBC1 protein rather than the C-terminus (462-923) was pulled down by SIAH2 in vitro ( Figure 2G, H). These results suggest that the N-terminus of DBC1 is crucial for its interaction with SIAH2. In line with the fact that SIAH2, as an E3 ligase, mediates substrate degradation by the ubiquitin-proteasome pathway, we found that SIAH2, rather than SIAH2 RM , reduced the protein level of endogenous DBC1 in a dose-dependent manner ( Figure 2I, J) and had no effect  1F), suggesting that SIAH2 mediated DBC1 degradation through the proteasome pathway rather than lysosomes. These results reveal that SIAH2 promotes DBC1 degradation by directly interacting with DBC1.

SIAH2 is responsible for DBC1 ubiquitination and degradation under hypoxic stress
Next, we checked whether SIAH2 ubiquitinated DBC1, and the results showed that ectopic expression of SIAH2, but not SIAH2 RM , dramatically increased the ubiquitination level of DBC1 ( Figure 3A In vitro ubiquitination assay further revealed that DBC1 was directly ubiquitinated by SIAH2 rather than the E3 ligase-dead mutant SIAH2 RM ( Figure 3C). To identify the sites of DBC1 ubiquitinated by SIAH2, we performed MS analysis, and the results showed that K287 of DBC1 was the potential site ubiquitinated by SIAH2 ( Figure 3D). Next, we found that ectopic expression of SIAH2 did not increase the ubiquitination level of the K287R mutant of DBC1, and the mutant did not degrade ( Figure 3E, Figure 3-figure supplement 1C, D). In conclusion, these results demonstrate that SIAH2 ubiquitinates DBC1 to mediate its degradation. To further understand the mechanism underlying hypoxia-induced DBC1 degradation, we examined whether SIAH2 was responsible for DBC1 ubiquitination and degradation in response to hypoxia. Our results showed that under hypoxic stress, both SIAH2 deletion and K287R mutation of DBC1 failed to increase DBC1 ubiquitination ( Figure  together, these results demonstrate that hypoxia-induced DBC1 degradation was dependent on SIAH2-mediated DBC1 ubiquitination.

OTUD5 regulates the deubiquitination and stability of DBC1
Strikingly, we also observed that the ubiquitination level of DBC1 was sharply decreased when returned to normal conditions after hypoxic stress ( Figure 4A). Therefore, we screened the deubiquitinating enzymes (DUBs) plasmids library to identify the candidate responsible for Furthermore, we confirmed that endogenous DBC1 could directly interact with OTUD5 ( Figure   4B), which was further verified by an in vitro pull-down assay using purified His-tagged DBC1 to pull down OTUD5 from MDA-MB-231 cell lysates ( Figure 4C). We next tested whether DBC1 deubiquitination was dependent on OTUD5 enzyme activity. The ubiquitination assay showed that only ectopic expression of wild-type OTUD5 but not inactive mutant C224S led to DBC1 ubiquitination level decrease under hypoxia ( Figure 4D). Consistently, hypoxia-induced endogenous DBC1 degradation was specifically inhibited by expression of wild-type OTUD5 rather than the inactive mutant ( Figure 4E). Moreover, the CHX assay also confirmed that ectopic expression of wild-type OTUD5 obviously prolonged the half-life of DBC1 ( Figure 4F, G and Supplementary file 4), indicating that deubiquitination of DBC1 was beneficial for its stability.
Domain mapping analysis further revealed that the N-terminal region (amino acid residues 1-230) of DBC1, which binds with SIAH2, was also necessary for its interaction with OTUD5 ( Figure 4H). Interestingly, we found that either hypoxia treatment or overexpression of SIAH2 promoted the interaction between DBC1 and SIAH2, but inhibited the interaction of DBC1 with OTUD5 ( Figure 4I, Figure 4-figure supplement 1D). Overall, our results suggest that SIAH2 and OTUD5 competitively interact with DBC1 in response to hypoxia ( Figure 4J), cooperatively regulating reversible DBC1 ubiquitination and stability to orchestrate DBC1 function.

Hypoxia regulates tumor progression via the SIAH2-DBC1 axis
As suggested in Figure 1, hypoxia-induced DBC1 degradation might regulate pathways associated with tumor cell growth, such as SIRT1 and p53 signaling pathways. Therefore, we examined whether SIAH2-mediated DBC1 ubiquitination was responsible for tumor progression.
Our results showed that knockout of Siah2 attenuated the reduction of p53 pathway activity caused by hypoxia ( Figure 5A). In addition, deletion of SIAH2 promoted cell apoptosis under hypoxia, which could be rescued by simultaneous knockout of DBC1 ( Figure  Immunohistochemical analysis revealed that the number of Ki67-positive proliferative cells in Siah2 knockout-implanted tumors was decreased compared with that in both wild-type and Siah2/DBC1 double knockout xenografts ( Figure 5M). Furthermore, the EdU assays that using cells isolated from the xenografts of animals also showed similar results ( Figure 5N, O and Supplementary file 5). Collectively, these results demonstrate the critical role of the SIAH2-DBC1 axis in promoting tumor progression.

Correlation of SIAH2 and DBC1 expression with tumor progression in breast cancer
To evaluate whether the mechanism by which the SIAH2-DBC1 axis regulates tumor progression is relevant to human tumorigenesis and tumor progression. We analyzed RNA sequencing data in TCGA (Cancer Genome Atlas) using TIMER, and the results demonstrated that the expression level of SIAH2 was significantly higher in most tumors than in adjacent normal tissues ( Figure 6A). In particular, the expression of SIAH2 in breast cancers was higher than that in normal samples ( Figure 6B). Analyzing the data from TCGA, we found that SIAH2 was positively correlated with tumor stage and number of lymph nodes, indicative of tumor malignancy ( Figure 6C, D). To further explore the clinical relevance of SIAH2-mediated DBC1 degradation, we analyzed the expression of SIAH2 and DBC1 in breast cancer tissue microarrays of patients. The IHC results showed that DBC1 and SIAH2 were negatively correlated in this cohort, coincident with our finding that DBC1 was the substrate of SIAH2 ( Figure 6E, F). Statistical analyses of IHC suggested that SIAH2 was significantly upregulated in human breast tumor tissues ( Figure 6G), which further validated our results from TCGA analyses. Furthermore, the upregulation of SIAH2 was found to be positively correlated with clinical stage and the percentage of the Ki67-positive cell population ( Figure 6H, I), and lowlevel DBC1 expression was detrimental to the survival rate of breast cancer patients ( Figure   6J). These data reveal that under pathological conditions, SIAH2-mediated DBC1 ubiquitination and degradation are beneficial for tumor progression.

Discussion
It has been documented that DBC1, a specific nuclear protein containing multifunctional domains, participates in the positive and negative regulation of multiple signaling pathways.
Several findings have suggested that DBC1 acts as a natural and endogenous inhibitor of SIRT1, and DBC1 deletion increases the SIRT1-p53 interaction and represses p53 transcriptional activity to inhibit apoptosis and promote tumorigenesis (Kim, Chen, and Lou 2008;Zhao et al. 2008;Noh et al. 2013;Qin et al. 2015;Akande et al. 2019). Given the importance of DBC1 function, the regulatory mechanisms by which DBC1 protein stability is regulated remain unclear. Here, we showed that the protein level of DBC1 was degraded under hypoxic stress, which was dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5. Additionally, knockout of Siah2 promoted apoptosis and reduced cell migration and tumor proliferation, whereas double knockout of DBC1 and Siah2 partially rescued cell proliferation and tumorigenesis. Importantly, DBC1 is negatively correlated with SIAH2 expression levels in human breast tumors, suggesting that the SIAH2-DBC1 axis pathway may play a key role in human breast cancer. These results provide novel insights into the metastatic mechanisms of human breast cancer. An abundance of evidence has shown that SIAH2 mediates the ubiquitination and degradation of substrates, including PI3K, LATS2, Spry2, ACK1 and TYK2 (Chan et al. 2017;Ma et al. 2016;Buchwald et al. 2013;Nadeau et al. 2007;Muller et al. 2014), in response to hypoxic stress to modulate multiple signaling pathways, such as the Hippo pathway, Ras signaling pathway and STAT3 pathway. In general, DBC1 activity is regulated by multiple However, it remains unclear whether DBC1 function can be regulated by ubiquitination modification. Our group demonstrated for the first time that SIAH2 can also ubiquitinate DBC1 at Lys287 by binding to the N-terminus of DBC1, resulting in DBC1 degradation by the ubiquitin-proteasome pathway, and that deletion of SIAH2 will block DBC1 degradation in response to hypoxic stress. Collectively, we identify a novel mechanism by which SIAH2 regulates DBC1 protein stability in response to hypoxic stress, contributing to tumorigenesis and tumor progression.
It has been reported that DBC1 cooperates with SIRT1 to mediate different physiological functions within mammalian cells. For example, stimulation of DBC1 transcription inhibits SIRT1 activity, contributing to TGF-β-induced epithelial-mesenchymal transition ).
Additionally, SIRT7 represses DBC1 transcription to promote thyroid tumorigenesis by binding to the promoter of DBC1 (Li et al. 2019). Recently, DBC1 was found to play a role in upregulating glucose homeostasis-related genes, which are implicated in Type 2 diabetes pathogenesis (Basu et al. 2020). Strikingly, our findings reveal that SIAH2-mediated DBC1 ubiquitination under hypoxia regulates cell proliferation and tumorigenesis. We also found that the decrease in p53 acetylation was accompanied by DBC1 degradation; thus, we further validated that the p53 signaling pathway is the downstream mechanism contributing to DBC1regulated breast tumor progression.
In general, ubiquitination is a dynamic and reversible process cooperatively regulated by E3 ligases and DUBs (Li and Reverter 2021). In this study, we identified that the deubiquitinase OTUD5 could specifically cleave the polyubiquitin chains of DBC1 once hypoxic stress was removed. If OTUD5 was overexpressed under hypoxia, the ubiquitination and degradation of DBC1 would be inhibited. Interestingly, we further confirmed that SIAH2 and OTUD5 competitively bind to DBC1 at the same N-terminal region, and hypoxia promotes the interaction of DBC1 with SIAH2 rather than OTUD5, resulting in ubiquitination and degradation of DBC1 to promote tumor progression. It is well known that OTUD5 controls cell survival and cell proliferation by deubiquitinating substrates Guo et al. 2021;Cho et al. 2021;Li et al. 2020;de Vivo et al. 2019;Park et al. 2015;Luo et al. 2013;Kayagaki et al. 2007).
Based on our findings and others', we speculate that the activation of OTUD5-mediated DBC1 deubiquitination may suppress tumor growth, which may provide a novel target for clinical tumor therapy. Moreover, we still need to further investigate the details of how the OTUD5-DBC1 complex dissociates under hypoxia to modulate DBC1 stability.
Taken together, our study revealed that hypoxia stimulates SIAH2 to ubiquitinate DBC1 and inhibit OTUD5-mediated DBC1 deubiquitination, resulting in DBC1 degradation through the ubiquitin-proteasome pathway. Our results further address the importance of the SIAH2-DBC1 axis in promoting tumor cell survival and migration. In conclusion, we uncovered a complete and detailed dynamic regulatory mechanism by which DBC1 protein ubiquitination and stability are regulated under hypoxic stress. It is of great significance to deeply understand the function of SIAH2 and DBC1 and the role of hypoxia in regulating tumorigenesis and tumor progression, which provides a solid theoretical basis for cancer treatment.

Construction of plasmids
The expression plasmids for human SIAH2, DBC1, SIRT1 and ubiquitin were generated by amplifying the corresponding cDNA by PCR and cloning it into pcDNA4-TO-Myc-His-B, pCMV-3xFLAG, pEGFP-C1, pRK5-HA, pGEX4T1 or PET28a expression vectors. Site-specific mutants and special domain deletion mutants were generated using TransStart® FastPfu DNA Polymerase (Transgene, Cat# AP221-11) according to the manufacturer's protocols.

Stable knockdown and knockout cells generation
To generate HeLa and MDA-MB-231 cell lines with stable knockdown of Siah2 and DBC1, oligonucleotides were cloned into pLKO.1, and then the acquired plasmid was cotransfected into HEK293T cells with lentiviral packaging plasmids psPAX2 and pMD2. G for lentivirus production. After infection, MDA-MB-231 cells were selected with 2.5 µg/ml puromycin in culture medium. The single oligonucleotide pair used was as follows: SIAH2 #1 (5′- To generate Siah2 and DBC1 knockout HeLa and MDA-MB-231 cell lines, oligonucleotides were cloned into Lenti-CRISPR, and then the acquired plasmid was cotransfected into HEK293T cells with lentiviral packaging plasmids psPAX2 and pMD2. G for lentivirus production.
After infection, stable HeLa and MDA-MB-231 cells were selected with 2.5 µg/mL puromycin, and then single clones were picked. The knockout clones were confirmed by sequencing the edited genomic regions after PCR amplification and by western blotting.

Coimmunoprecipitation and pull-down assay
After the described treatment, cells were collected and lysed in 0.8 mL IP lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 10% glycerol) containing protease inhibitors (1:100, Roche) for 45 min on a rotor at 4 °C. After centrifugation at 12,000 g for 10 min, the supernatant was immunoprecipitated with 1.5 µg of specific antibodies overnight at 4 °C. Protein A/G agarose beads (15-30 µL, Santa Cruz) were washed and then added for another 2 h. The precipitants were washed seven times with wash buffer, and the immune complexes were boiled with loading buffer and analysed. GST-SIAH2 and His-DBC1 proteins were generated in E. coli and incubated in vitro overnight. Then, 25 µL GST beads (GE Healthcare) were added to the system for 2 h on a rotor at 4 °C.

Ubiquitination assay
In vivo ubiquitination assays Cells were transiently transfected with plasmids expressing HA-

Flow cytometry
After the described treatment, cells were collected and washed twice with prewarmed FBSfree DMEM and then stained with PI and Annexin V (Thermo Fisher) for 20 min at 37 °C. After staining, cells were washed twice with prewarmed PBS for analysis with a flow cytometer (BD Calibur).

Western blotting
Cells were collected and washed with PBS and then lysed in 1% SDS lysis buffer or NP-40 lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 10% glycerol) containing protease inhibitors (1:100, Roche) for 45 min on a rotor at 4 °C. After centrifugation at 12,000 g for 10 min, the supernatant was boiled with loading buffer. Cell lysates containing equivalent protein quantities were subjected to 6% or 10% SDS-PAGE, transferred to nitrocellulose membranes, and then incubated with 5% milk for 2 h at room temperature.
Then, the membranes were probed with related primary antibodies at 4 °C, followed by the

RNA extraction and real-time PCR
RNA samples were extracted with TRIzol reagent (Sigma T9424), reverse transcription PCR was performed with a Reverse Transcriptase kit (Promega A3803), real-time PCR was performed using Powerup SYBR Green PCR master mix (A25743) and a Step-One Plus realtime PCR machine (Applied Biosystems). Human Actin expression was used for normalization.

Quantification and statistical analysis
For quantitative analyses of western blots, real-time PCR results or flow cytometry data, values were obtained from three independent experiments. The quantitative data are presented as the means ± SEM. Student's t test was performed to assess whether significant differences existed between groups. Multiple comparisons were performed with one-way analysis of variance (ANOVA) and Tukey's post-hoc test. P values < 0.05 were considered statistically significant. The significance level is presented as *P < 0.05, **P < 0.01 and ***P < 0.001. "ns" indicates that no significant difference was found. All analyses were performed using Prism 8.0 (GraphPad Software, Inc., La Jolla, CA).

Tissue microarrays and immunohistochemistry.
The breast cancer tissue microarrays were purchased from US Biomax. These tissue microarrays consisted of 100 analysable cases of invasive breast carcinoma and 10 analysable cases of normal breast tissue. For antigen retrieval, the slides were rehydrated and then treated with 10 mM sodium citrate buffer (pH 6.0) heated for 3 min under pressure. in 100 μL PBS) were injected subcutaneously into the armpit of six-to eight-week-old female BALB/c nude mice. Tumor size was measured every 3-5 days one week after the implantation, and tumor volume was also analysed by using the formula V = 0.5×L×W2 (V: volume, L: length, W: width). The mice were then sacrificed, and the subcutaneous tumors were surgically removed, weighed and photographed. No statistical method was used to predetermine the sample size for each group. The experiments were not randomized.

RNA-sequencing analysis
RNA was extracted in biological triplicates using the miRNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA quality control was performed using a fragment analyser and standard or high-sensitivity RNA analysis kits (Labgene; DNF-471-0500 or DNF-472-0500

Differential expression analysis of two conditions/groups (two biological replicates per condition)
was performed using the DESeq2 R package (1.30.1). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P value of 0.05 and absolute log2FoldChange of 0.5 were set as the threshold for significantly differential expression.     Under normoxic conditions, the deubiquitinase OTUD5 contacts DBC1 to form a complex. In response to hypoxia, the E3 ubiquitin ligase SIAH2 competitively binds and ubiquitinates DBC1

Figure legends
with OTUD5, resulting in the degradation of DBC1 through the ubiquitin-proteasome system.