Non‐SMC condensin I complex subunit H participates in anti‐programmed cell death‐1 resistance of clear cell renal cell carcinomas

Abstract Non‐SMC condensin I complex subunit H (NCAPH) is reported to play an important role and be a poor prognostic factor in various cancers. However, the function and regulatory mechanism of NCAPH in clear cell renal cell carcinoma (ccRCC) remain unknown. The roles of NCAPH on ccRCC growth were detected in vitro and in vivo assays. The regulatory mechanism of NCAPH was explored by immunoprecipitation assay, ubiquitination assay, ChIP assay, RIP assay, luciferase reporter assay and RNA pull‐down assay. The role of NCAPH in immunoregulation also was explored by flow cytometry, T cell‐mediated tumour cell killing assay and immune‐competent mouse model. In this research, we displayed that NCAPH was upregulated in ccRCC and patients with elevated NCAPH expression had an undesirable prognosis. Functionally, NCAPH depletion restrained ccRCC growth in vitro and in vivo. The elevated NCAPH was attributed to FOXP3‐mediated transcription, FUS‐mediated transcription splicing and METTL3‐mediated m6A modification. Moreover, YTHDC1 promoted NCAPH mRNA nuclear export, and IGF2BP3 enhanced NCAPH mRNA stability in an m6A‐dependent manner. NCAPH increased PD‐L1 expression by inhibiting the degradation of β‐catenin in ccRCC cells, which further facilitated aerobic glycolysis and immune tolerance of ccRCC. Collectively, our findings display the vital function of NCAPH in ccRCC and uncover that NCAPH may be regarded as a potential therapeutic target to reverse the immune tolerance of ccRCC.

Non-SMC condensin I complex subunit H (NCAPH) is one of the non-SMC subunits in condensin I. 9 NCAPH plays a crucial effect on mitotic chromosome architecture and segregation. 10 NCAPH is increased in colon cancerous tissues and NCAPH depletion inhibits colon cancer growth, and migration, and induces cell apoptosis and cell cycle arrest. 11 Wang et al. indicate that NCAPH is elevated in cervical cancer and positively correlated to prognosis. 12 NCAPH inhibition restrains the proliferation, colony formation, migration, invasion and EMT process of cervical cancer cells by the PI3K/AKT/SGK pathway. Ogura et al. suggest that NCAPH is an undesirable prognostic factor in ER-positive breast cancer patients and NCAPH depletion suppresses the growth of breast cancer cells. 13 Xiong et al. display that NCAPH is elevated in non-small cell lung cancer (NSCLC) and implicated in NSCLC progression by binding to β-catenin protein and activating Wnt signalling. 14 NCAPH is also reported to play an oncogene in bladder cancer, 15 hepatocellular carcinoma, 16 pancreatic cancer, 17 lung cancer 18,19 and oral squamous cell carcinoma. 20 However, the function of NCAPH in ccRCC was barely reported.
N6-methyladenosine (m6A), an epigenetic modification of RNA, is related to RNA transcription splicing, transport, translation and degradation. As a reversible process, m6A modification is triggered by m6A methyltransferases and excised by m6A demethylases. The function of m6A modification is controlled by these proteins binding to m6A sites. There is mounting evidence indicating that m6A modification plays a key role in many human cancers, including ccRCC. [21][22][23] al. indicate that METTL3 promotes tumorigenesis via regulating HHLA2 mRNA expression in an m6A-dependent manner in ccRCC. 24 Besides, Shi et al. show that METTL3 promotes ABCD1 translation in an m6A-dependent manner in ccRCC. 25 However, the function and mechanism of m6A modification in ccRCC still need more exploration.
The presence of dysfunctional CD8+ T cells in the tumour microenvironment is a hallmark of low antitumor immune function. 26 The prolonged presence of tumour antigens and/or inhibitory TME drives antitumor effector CD8+ T cells into a state of impaired function known as 'T-cell exhaustion'. 27 Exhausted CD8+ T cells express high levels of inhibitory receptors, such as programmed cell death-1 (PD1) 27,28 and the function of partially exhausted CD8+ T cells could be partially reversed by anti-PD1 antibodies. 29, 30 Wang et al. indicated that TOX promotes promote CD8+ T cell exhaustion by inhibiting PD1 degradation. 31 Rong et al. showed that Matrix Gla protein promoted NF-κB phosphorylation, thereby activating PD-L1 expression to promote CD8+ T cell exhaustion. 32 However, the molecular mechanisms that regulate CD8+ T-cell exhaustion in TME remain largely unknown.
Here, NCAPH is found to be increased in ccRCC cell lines and tissues and accelerates ccRCC cell growth in vitro and in vivo. Elevated NCAPH level is induced by FOXP3-mediated transcription and FUSmediated transcription splicing. The m6A modification of NCAPH increased its nuclear export and mRNA stability. Besides, NCAPH promotes aerobic glycolysis and PD-L1 expression by inhibiting the degradation of β-catenin. NCAPH inhibits the T cell response and induces resistance to anti-PD-1 therapy. Thus, NCAPH may be regarded as an oncogene and a latent immunomodulator in ccRCC.

| RNA extraction and qRT-PCR
Total RNA extraction and detection of target genes were executed as described previously. 33 For the subcellular fractionation of RNA, cytoplasmic & nuclear RNA purification kit (Amyjet Scientific) was employed following the manual. The primers are listed in Table S2.

| Western blotting and immunoprecipitation
Total protein was extracted and the target protein was detected as described previously. 33 For immunoprecipitation (IP), the lysate was precleared by protein A/G-agarose, and IP was carried out by incubating the supernatants with antibodies against NCAPH or β-catenin and protein A/G-agarose overnight at 4 C. After centrifugation, the complexes were washed and resuspended. The targeting proteins were detected by Western blotting. Rabbit IgG isotype was used as a negative control. All antibody information was listed as follows: NCAPH (PA5-80842, Invitrogen),

| Ubiquitination assay
For the ubiquitination assay, the indicated cells were transfected with various constructs together with Myc-ubiquitin and Flag-β-catenin, treated with MG132, and lysed using RIPA lysis buffer. Ubiquitination was assessed by IP with an antibody against the Flag tag, followed by western blotting with an anti-Myc antibody.

| ChIP assay
ChIP assay was carried out as described previously. 33 Lysates were immunoprecipitated with control IgG (ab172730, Abcam) and antibodies against FOXP3 (ab215206, Abcam). The immunoprecipitated DNA fragment was analysed by qRT-PCR. The primers are listed in Table S2.

| Luciferase reporter assay
The promoter of NCAPH (À2000 $ 0) with mutated FOXP3 binding site was generated with QuickMutation™Plus Site-Directed Mutagenesis Kit (Beyotime). Wild-type and mutant promoter were synthesized and subcloned into a pGL3-Basic luciferase reporter vector (Promega). All luciferase assays were analysed after 48 h of transfection using Dual-Luciferase Kit (Promega). The luciferase activities were detected by a Dual-Luciferase Reporter Assay System (Promega) following the manual.
The expression of NCAPH mRNA in cells was determined by qRT-PCR.

| Flow cytometry
To measure apoptosis, ccRCC cells were double-stained with annexin V-FITC and PI (Beyotime) following the manual. Then stained cells were measured using a flow cytometer (Beckman Coulter).
To assess the level of membrane PD-L1 in ccRCC cells and membrane PD-1 in CD8-T cells, cells were stained with Alexa Fluor ® 488 Anti-PD-L1 antibody (ab209959, Abcam) or Alexa Fluor ® 488 Anti-PD1 antibody (ab220300, Abcam) and then measured using a flow cytometer.

| Enzyme-linked immunosorbent assay
The level of IFN-γ and TNF-α produced by CD8+ T cells were measured by enzyme-linked immunosorbent assay (ELISA) kits (eBioscience) following the manual.

| Biotinylated RNA pull-down assay
Pull-down assay was carried out as previously described. 33 The biotinylated NCAPH probe, biotinylated pre-NCAPH probe and biotinylated NC probe were produced by GenePharma. The precipitated proteins were identified by Western blotting.

| Immunohistochemistry
The slides were deparaffinized, rehydrated, antigen retrieval and endogenous peroxidase blocking. After blocking with 10% goat serum, the tissues were incubated with antibodies against Ki67 or CD8 at 4 C overnight. After washing, the slides were incubated with a secondary antibody and HRP-conjugated streptavidin in sequence. Then, DAB (Yeasen Biotechnology Co., Ltd.) was carried out to visualize targeting proteins. Haematoxylin was carried out to counterstain the slides.

| Statistics
The data are presented as the mean ± SD. Data analysis was conducted using GraphPad Prism 7.0. Student's t-tests and one-way ANOVA with Tukey's post hoc test were used for difference analysis.
Correlations analyses were evaluated by Pearson correlation coefficient. The survival data were analysed using the log-rank test. All experiments were repeated at least three times. Differences with p < 0.05 were considered significant.

| Elevated NCAPH level is related to poor prognosis in ccRCC patients
The level of NCAPH was found to be increased in a variety of tumours, including Kidney Renal Clear Cell Carcinoma (KIRC) by analysing The Cancer Genome Atlas (TCGA) data using TIMER (http:// timer.cistrome.org/) ( Figure S1A). CcRCC patients with elevated NCAPH expression had short overall survival time and disease-free survival time ( Figure S1B,C). NCAPH protein level was elevated in Clinical Proteomic Tumour Analysis Consortium (CPTAC, http:// ualcan.path.uab.edu/analysis-prot.html) ccRCC samples when compared to the normal tissues ( Figure S1D). We also confirmed the elevated NCAPH level in 87 paired ccRCC tissues from our hospital ( Figure 1A). Increased NCAPH expression was closely related to large tumour size, higher histologic grades and advanced TNM stages (Table S1) and poor prognosis in ccRCC patients ( Figure 1B). Receiver Operating Characteristic curve displayed that NCAPH may be used as a diagnostic indicator for ccRCC patients ( Figure 1C). Finally, compared with HK-2 cells, NCAPH mRNA and protein expression in ccRCC cell lines were significantly higher ( Figure S1E,F). Collectively, our data indicate that NCAPH may play an oncogenic role in ccRCC.

| NCAPH accelerates ccRCC cells growth in vitro and in vivo
To confirm the role of NCAPH in ccRCC, we constructed NCAPH overexpressed or depleted ccRCC cells ( Figure 1D; Figure S1G,H). NCAPH-depleted xenografts ( Figure 1K). Taken together, our data strongly verify that NCAPH accelerates ccRCC cell growth in vitro and in vivo, and targeting NCAPH may be an underlying therapeutic strategy for ccRCC.

| FOXP3 promotes the transcription and FUS facilitate the maturation of NCAPH
To explore the underlying mechanism for the overexpression of  Figure S2D). However, there was no noteworthy association between STAT4 level and prognosis in KIRC ( Figure S2E). Therefore, we chose FOXP3 as a further research object. Luciferase reporter assays showed that the overexpression of FOXP3 increased the luciferase activity of the wild-type but not the mutant construct ( Figure 2B). FOXP3 depletion plays the opposite role. ChIP assays indicated that FOXP3 could occupy the promoter of NCAPH ( Figure 2C). FOXP3 overexpression increased the enrichment of FOXP3 in NCAPH promoter, and upregulated the mRNA and protein expression of NCAPH ( Figure 2D-H). These results affirm that FOXP3 promotes the transcription of NCAPH.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are reported to control the alternative splicing of RNAs and assist mRNAs transport, translation and stabilization. Here, we filtrated the hnRNPs which were predicted to binding to NCAPH by using ENCORI (The Encyclopedia of RNA Interactomes, https://starbase.sysu.edu.cn/) database and 12 hnRNPs were screened out ( Figure 2I). The change of 12 hnRNPs expression and the correlation between hnRNPs and NCAPH in KIRC were analysed. Nine hnRNPs were found to be up-regulated and only one hnRNPs was found to be down-regulated in ccRCC samples. Next, correlation analysis indicated that seven upregulated hnRNPs were found to be positively correlated to NCAPH expression in ccRCC samples. Among the seven filtrated hnRNPs, FUS was selected for its highest R-value. FUS was predicted to binding to the 3 0 UTR and introns of NCAPH. RIP and Pulldown assays affirmed that FUS could bind to precursor NCAPH (pre-NCAPH), but not NCAPH mRNA in both ccRCC cell lines ( Figure 2J,K). Moreover, FUS overexpression significantly reduced the pre-NCAPH levels, enhanced NCAPH mRNA levels and increased NCAPH protein expression in ccRCC cells (Figure 2L,M). In short, FUS promotes the maturation of NCAPH mRNA and enhances the expression of NCAPH.
3.4 | YTHDC1 induces NCAPH mRNA nuclear export and IGF2BP3 enhances NCAPH mRNA stability in an m6A-dependent manner By analysing the sequence of NCAPH mRNA, multiple potential sites for m6A were found ( Figure 3A). Moreover, m6A writer METTL3 and m6A reader IGF2BP1, IGF2BP2, IGF2BP3 and YTHDC1 were predicted to binding to NCAPH by ENCORI ( Figure 3B). The protein level of IGF2BP1 and IGF2BP2 was downregulated, whereas IGF2BP3 protein level was upregulated in CPTAC ccRCC samples. There was no change in METTL3 and YTHDC1 protein expression. Correlation analysis showed that METTL3, IGF2BP1, IGF2BP2 and IGF2BP3 expression were correlated to NCAPH expression. Then, RIP assays were used to verify the m6A modification of NCAPH mRNA and the binding between NCAPH mRNA and m6A-related proteins. As shown in

| NCAPH promotes PD-L1 expression by inhibiting the degradation of β-catenin
It is reported that NCAPH can inhibit ubiquitin-mediated β-catenin protein degradation in NSCLC. 14 We then wondered whether NCAPH could regulate β-catenin protein stability in ccRCC. To test the idea, co-IP assays were carried out, and found that endogenous NCAPH proteins interact with β-catenin proteins physically ( Figure 4A,B).
PD-L1 is reported to be a target gene of β-catenin in glioblastoma. 34 Here, we found that β-catenin overexpression significantly increased the mRNA and protein level of PD-L1, as well as the membrane PD-L1 in ccRCC cells ( Figure 4E-G). NCAPH depletion has the opposite effect. β-catenin overexpression could rescue the decreased PD-L1 mRNA, PD-L1 protein and membrane PD-L1 level mediated by NCAPH knockdown. In other word, NCAPH regulates PD-L1 expression by inhibiting the degradation of β-catenin.

| NCAPH promotes aerobic glycolysis by enhancing β-catenin
As β-catenin is closely related to aerobic glycolysis in various tumours, we next explored whether NCAPH participated in aerobic glycolysis of ccRCC cells. NCAPH depletion obviously inhibited glucose consumption, lactate production, and ECAR in ccRCC cells ( Figure 5A-C).

| NCAPH inhibits the T cell response against ccRCC
By using TISIDB, we found that NCAPH expression was significantly correlated to multiple TILs (tumour-infiltrating lymphocytes), immunoinhibitors, immunostimulators, MHCs, chemokines and receptors ( Figure S4A-F), indicating the potential immune regulatory function of NCAPH. In addition, NCAPH level was positively related to the infiltrated CD8 T cells, Tregs and macrophages in TIMER ( Figure S4G).
Here, we aimed to explore the influence of NCAPH on CD8 T cells. T cell-mediated killing assays indicated that NCAPH depletion sensitized Caki-1 cells to CD8 T cells-mediated cytolysis, whereas overexpression decreased the cytolysis of CD8 T cells ( Figure 6A). Moreover, the protein level of perforin, granzyme B and secreted IFN-γ and TNF-α from cocultured CD8 T cells was decreased by NCAPH overexpression and increased by NCAPH depletion (Figure 6B-D). As increased PD1 is closely related to CD8 T cell exhaustion, we then detected PD1 levels in cocultured CD8 T cells. NCAPH overexpression increased and NCAPH depletion inhibited the PD1 level in cocultured CD8 T cells ( Figure 6E).

| DISCUSSION
Multiple types of biomarkers for ccRCC have been proposed and experimentally evaluated in the past decades. [35][36][37] In this study, we identified that NCAPH was highly expressed in ccRCC and significantly correlated with clinical stages and prognosis. Overexpression of NCAPH could accelerate ccRCC cell growth and the dysfunction of CD8+ T cells in vitro and in vivo. Mechanistically, NCAPH enhanced PD-L1 expression by inhibiting the degradation of β-catenin.
Transcriptional control is an important gene expression. Wang et al. find that HPV E7 increases NCAPH gene expression via E2F1-mediated transcription activation in cervical carcinoma. 12 Moreover, Octamer transcription factor 1 (OCT1) is identified to be an activating transcription factor for NCAPH in breast cancer 13 and MYB Proto-Oncogene Like 2 (MYBL2) also is identified to be an activating transcription factor for NCAPH in NSCLC. 19 Here, we found that transcription factor FOXP3, which was increased in ccRCC, could bind to NCAPH promoter and promote NCAPH transcription.
FUS, an RNA-binding protein, was recently reported to be a novel regulator of the biogenesis of RNA. 38 Satoshi et al. indicate that FUS regulates precursor mRNA splicing by interacting with beta-catenin. 39 Yang et al. suggest that FUS directly binds to precursor RHOBTB3 mRNA to promote the biogenesis of circRHOBTB3. 40 In this study, we identified that NCAPH was a target for the alternative splicing factor FUS, which promoted the maturation of NCAPH mRNA by binding to precursor NCAPH. Therefore, our research revealed a new target of FUS.
The gene expression regulation network is very complex and is involved in multiple signalling pathways. In this study, we found that the abnormal expression of NCAPH in ccRCC is related to transcriptional control, alternative splicing and m6A modification. First, the transcription factor FOXP3 increased the transcription of NCAPH gene. Then, the mature NCAPH was generated by precursor NCAPH with the help of FUS. Besides, the m6A modification induced by METTL3 further enhanced NCAPH mRNA stability in an IGF2BP3-dependent manner. Our study initially revealed the molecular mechanism of the abnormal expression of NCAPH in ccRCC, further expanded the regulatory mechanism of NCAPH, and laid a foundation for the treatment of ccRCC targeting NCAPH.
ccRCC is highly immune infiltrated and immune checkpoint blockade therapy and combination regimens have significantly improved the prognosis of ccRCC patients. 41,42 The infiltration of exhaustionphenotype CD8+ T cells is negatively correlated to ccRCC patient prognosis. 43 Li et al. indicate that NCAPH is positively correlated to the infiltration of acquired immunocytes and negatively associated with the infiltration of innate immunocytes in lung adenocarcinoma. 18 In this study, we identified NCAPH as an immunoregulation factor. Elevated NCAPH expression was positively related to the infiltrated CD8 T cells, Tregs and macrophages in ccRCC. NCAPH overexpression tolerated ccRCC cells to CD8 T cells-mediated cytolysis and promoted CD8 T cell exhaustion by increasing PD1 overexpression. Moreover, we also found that NCAPH promoted PD-L1 expression by inhibiting the degradation of β-catenin in ccRCC cells, resulting in immune tolerance of ccRCC. Besides, NCAPH overexpression promoted resistance to anti-PD-1 therapy in vivo. In short, our findings revealed for the first time the function of NCAPH in immune regulation, and targeting NCAPH may be an effective treatment for ccRCC.
Metabolic reprogramming is one of the hallmarks of a wide variety of tumours, including ccRCC. Glycolysis produces large amounts of energy needed by tumour cells and increases the production of lactic acid. 44 The release of lactic acid can form an acidic extracellular microenvironment, which further promotes aggressiveness of tumour cells. 45 Moreover, glycolysis also plays a crucial role in tumour immune escape. The acidic extracellular microenvironment damages the recognition of immune cells to cancer cells. 46 Production of lactic acid also blunts tumour immunosurveillance by T cells. [47][48][49] In our findings, we found that NCAPH promoted aerobic glycolysis by stabilizing β-catenin protein and upregulated NCAPH in ccRCC cells would induce CD8 T cells exhaustion by enhancing PD1 expression. Considering that PD1 could be increased by lactic acid, [49][50][51] we inferred that NCAPH regulated CD8 T cells exhaustion by enhancing glycolysis and the release of lactic acid in ccRCC.

| CONCLUSIONS
In summary, our study indicated that NCAPH could serve as an important prognostic predictor and an immunomodulator for ccRCC patients. NCAPH promoted the growth and anti-PD-1 resistance of ccRCC ( Figure 7L). Targeting NCAPH could regard as an effective therapeutic strategy for ccRCC.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
The datasets used and analysed during the current study are available from the corresponding author upon reasonable request.