Apolipoprotein L1 is a tumor suppressor in clear cell renal cell carcinoma metastasis

The 5-year survival rate of kidney cancer drops dramatically from 93% to 15% when it is metastatic. Metastasis constitutes for 30% of kidney cancer cases, in which clear cell renal cell carcinoma (ccRCC) is the most prominent subtype. By sequencing mRNA of ccRCC patient samples, we found that apolipoprotein L1 (APOL1) was highly expressed in tumors compared to their adjacent normal tissues. This gene has been previously identified in a large body of kidney disease research and was reported as a potential prognosis marker in many types of cancers. However, the molecular function of APOL1 in ccRCC, especially in metastasis, remained unknown. In this study, we modulated the expression of APOL1 in various renal cancer cell lines and analyzed their proliferative, migratory, and invasive properties. Strikingly, APOL1 overexpression suppressed ccRCC metastasis both in vitro and in vivo. We then explored the mechanism by which APOL1 alleviated ccRCC malignant progression by investigating its downstream pathways. APOL1 overexpression diminished the activity of focal adhesive molecules, Akt signaling pathways, and EMT processes. Furthermore, in the upstream, we discovered that miR-30a-3p could inhibit APOL1 expression. In conclusion, our study revealed that APOL1 play a role as a tumor suppressor in ccRCC and inhibit metastasis, which may provide novel potential therapeutic approaches for ccRCC patients.


Introduction
According to the SEER database, the 5-year survival rate of kidney cancer is 93% when it is confined to primary site, but dramatically drops down to 15% if it metastasizes to distant parts of the body (1).Notably, kidney cancer is the top ten most common cancers found in males, incidence and mortality rates are 7.8 and 3.0 for every 100,000 males, respectively (2).Clear cell renal cell carcinoma (ccRCC), characterized by loss of chromosome 3p and VHL mutations (3), accounts for 70% of all renal malignancies and is associated with poor survival rates (4).It is important to note that VHL loss by itself does not lead to ccRCC nor triggers distant metastasis (5,6).Discovering the mechanism behind further cellular dysregulation is critical to understand ccRCC tumorigenesis.
In ccRCC, apolipoprotein L1 (APOL1) is transcriptionally upregulated in a distinct metastatic tumor cell cluster and is associated with poor survival (7).This gene has been shown to be highly expressed in tumor tissues (8)(9)(10)(11)(12)(13) and is found to be a potential prognosis marker for many types of cancers via bioinformatic analyses (14)(15)(16)(17)(18)(19)(20)(21)(22)(23).Apolipoprotein L gene family is only found in a few higher primates and human beings, and APOL1 is the only secreted member due to its N-terminal signal peptide (24).APOL1 is secreted into the serum by the liver, bound to highdensity lipoproteins, complexed to IgM, and is expressed for intracellular transport in several organs (25).Their structures and functions are similar to the Bcl-2 gene family (B3-only proteins) (26).The molecular functions of APOL1 include chloride channel activity, lipid binding, and other biological processes (27)(28)(29).APOL1 is also known to play a role as an oncogene in pancreatic cancer (8).In the Human Protein Atlas database, APOL1 notably has the highest RNA expression level in many kidney cancer cell lines compared to other cancer types (30).The role of APOL1 in ccRCC metastasis remains unknown.
The critical process to metastasis may include mechanisms such as epithelial to mesenchymal transition (EMT) and the expression status of focal adhesion molecules.To invade, resist apoptosis, and to disseminate, carcinoma cells must lose their epithelial phenotypes, detach from epithelial sheets, while gaining the mesenchymal characteristics, which is known as the EMT.In EMT, the decreased expression of E-cadherin, a marker of epithelial cells, and upregulation of N-cadherin, a marker of mesenchymal cells, are known to regulate invasion and metastasis.Moreover, some pleiotropically acting transcriptional factors orchestrate the EMT, such as Snail and Slug (31).Focal adhesions are the contact sites of cell cytoskeleton to extracellular matrix (ECM) through molecules such as integrins.One hallmark of focal adhesions is the clustering of integrins, which affects the organization of the cytoskeleton underlying the plasma membrane.Focal adhesion kinase (FAK) is a key cytoplasmic non-receptor tyrosine kinase that play a key role in cell adhesion and migration process for cancer metastasis (32).
By sequencing mRNA of ccRCC patient samples, we found that APOL1 was highly expressed in renal tumors compared to adjacent normal tissues.The suppression of APOL1 induced the activation of focal adhesion proteins, Akt, and its downstream targets, and initiated the EMT process.These results proposed that the role of APOL1 is as a tumor suppressor to inhibit tumor dissemination, which may provide novel potential therapeutic approaches against renal cancer metastasis.

Human patient samples and RNA and microRNA sequencing
Human specimens were collected after obtaining informed consent in accordance with the Declaration of Helsinki and was approved by Institutional Review Board of Chungbuk National University Hospital.
Total RNA and microRNA (miRNA) from patient samples were extracted by miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and was sent to Macrogen (South Korea) for sequencing and analysis.

microRNA transfection
Negative control miRNA and miR-30a (Bioneer, South Korea) were transfected to primary renal cancer cells using Lipofectamine RNAiMAX (Invitrogen).The cells were incubated for 3 days.

Protein extraction and western blotting
Total protein from cell lysates were extracted by RIPA Lysis and Extraction Buffer (89900, Thermo Scientific) supplemented with Halt Protease Inhibitor Cocktail (78410, Thermo Scientific) and Halt Phosphatase Inhibitor Cocktail (78420, Thermo Scientific).Protein concentrations were measured by Pierce BCA Protein Assay Kit (23225, Thermo Scientific).

Proliferation assay
Cells were stained with CellTrace CFSE reagent (C34554, Invitrogen) and were incubated for 0, 1, 3, 5 days.Each day, cells were harvested and fixed with 4% paraformaldehyde.Analysis was completed using an FACS Canto II Flow Cytometer (BD Life Sciences, USA) with 488−nm excitation and a 530/30-nm bandpass emission filter.The results were analyzed by FlowJo software version 10 (BD Life Sciences).

Wound healing assay
The cells were seeded on a 6-well plate at 80% confluency.After attaching overnight, a scratch was made by using the 200 µL tip.Images of four random microscopic fields under a light microscope (bright field) were taken at 0 hour and indicated hours with a 10X objective lens.The percentage of wound healing was calculated by TScratch program version 1.0 (ETH Zürich, Switzerland).

Migration and invasion assay
For transwell experiments, 100 µL of 0.4 mg/mL Matrigel (356230, Corning) and 750 µL of complete media (10% FBS) were respectively loaded into the upper chamber and lower chamber of Transwell with 8.0 µm Pore Polycarbonate Membrane Insert (3422, Corning), and incubated for 2 hours in the CO 2 incubator.After 2 hours, 1 x 10 5 cells suspension in serum free media were added onto Matrigel-coated cell culture insert and incubate for indicated hours (16h, 18h or 24h).Then, the medium in upper chamber was removed and the insert was washed twice with PBS.Cells were fixed with 4% paraformaldehyde for 2 min at RT, and were washed twice with PBS.
Cells were then permeablized with 100% methanol for 20 min at RT, and washed twice with PBS.After that, cells were stained with 0.4% crystal violet and incubated at room temperature for 15 min, and washed twice with PBS.Non-invaded cells were scrapped off with cotton swabs.Invasive cells were counted in four random microscopic fields under the Leica DMi8 microscope (bright field) with a 20X objective lens.For migration assay, the same protocol was performed without Matrigel-coated on the insert.

In vivo metastasis assays
The mice experiment protocol was approved by the Institutional Animal Care and Use Committee of Soonchunhyang University (SCH20-0049) and performed in accordance with the Animal Facility of Soonchunhyang Institute of Medi-Bio Science guidelines.12-week-old male BALC/c nude mice (Nara Biotech, South Korea) were housed in specific pathogen free conditions.The mice were intravenously injected via the tail vein with 2 x 10 6 cells in 100 mL of 1X PBS of control or APOL1 overexpressing Caki-1 cells.After 8 weeks, these mice were euthanized by carbon dioxide inhalation, and their lungs were perfused and dissected for visual inspection of the metastatic nodules.The lungs were fixed in 4% paraformaldehyde for 24 hours and then were processed and embedded into a paraffin block.Paraffin sections (5 mM) were mounted on microscope slides and stained with Harris hematoxylin (YD Diagostics, South Korea) and eosin (BBC Bichemical, USA), and were imaged with Leica DMi8 microscope (bright field) with a 20X and 40X objective lens.7-weekold male BALC/c nude mice (Nara Biotech, South Korea) were subcutaneously injected on the right flank with 10 7 cells in 100 mL of 1X PBS of A-498, 786-O and Caki-1 cells.After 8 weeks, these mice were euthanized, and their tumors were collected for subsequent analysis.For immunohistochemistry, the tumors were fixed in 4% paraformaldehyde for 24 hours and then were processed and embedded into a paraffin block.Paraffin sections (5 mM) were mounted on microscope slides and proceeded with primary antibody APOL1 (sc-390440, Santa Cruz) and counterstained with Harris hematoxylin (YD Diagostics, South Korea).Images were taken by Leica DMi8 microscope (bright field) with a 20X objective lens.

Statistical analysis
Each experiment was performed in triplicates.Statistical analyses were performed using GraphPad Prism 9 version 9.3.0.All tests were two-tailed unpaired t tests.The p-value <0.05 was considered to be statistically significant.

APOL1 is highly expressed in clear cell renal cell carcinoma
To characterize the molecular mechanisms related to malignant properties of ccRCC, we first performed RNA sequencing (RNA-seq, GSE252600) on 9 pairs of tumors and adjacent normal tissues, with 3 of them having metastatic sites (Table 1).The hierarchical clustering heatmap and the multidimensional analysis of the RNAseq revealed the distinguished expression profiles of tumor from the normal tissues.The tumors were grouped together rather than with their normal adjacent tissues (Figures 1A, B).There were 2390 differentially expressed genes (DEG) obtained from the RNA-seq analysis, and among them 459 candidate genes overlapped with the TCGA-KIRC dataset (Supplementary Figure S1A).Among the top 20 upregulated genes that were highly expressed in ccRCC compared to normal tissues (Supplementary Figure S1B), TGFBI, PLOD2, TMEM45A, FCGR1C, HIST1H2BH, CCL5 and APOL1 were closely correlated to poor survival rate in ccRCC patients.Furthermore, TGFBI, PLOD2 and APOL1 had clear tumor progression patterns in the TCGA-KIRC dataset (Figure 1C, Supplementary Figures S1C, D).In an effort to identify which of the three genes we should investigate in, we also sequenced for differential expressions in microRNA expression (GSE252629), to see which gene was the most regulated.We overlapped differentially expressed miRNAs of tumors and adjacent normal tissues with miRNAs targeting these 3 genes.APOL1 and TGFBI were targeted by 28 and 24 miRNAs, respectively, whereas PLOD2 were targeted by 13 miRNAs (data not shown).We focused on the novelty of APOL1 as a potential cancer marker whose low expression strongly associated to poor survival rate at metastatic stage of ccRCC (Figure 1D), considering that TGFBI had already been identified as oncogene in RCC (39).
After realizing the potential of APOL1 as a candidate marker, we then examined its expression in various renal cell lines.We utilized immortalized proximal tubule epithelial cell line from normal adult human kidney HK-2, clear cell renal cell carcinoma cell lines A-498 and 786-O, and the metastatic cell line Caki-1.Both APOL1 transcript and protein levels were not detected in HK-2 but were robustly expressed in primary cancer cells and retained less expression in the metastatic cells (Figures 1E, F).To mimic the condition of primary and metastatic stages of ccRCC, we subcutaneously injected the primary cancer A-498 and 786-O cells and metastatic Caki-1 cells to nude mice, respectively.As a  S1E, F).Furthermore, moderate level of APOL1 were observed in ccRCC patients at late stages (Supplementary Figure S1G).These observations were consistent with the fluctuating expression of APOL1 protein in ccRCC from Clinical Proteomic Tumor Analysis Consortium (CPTAC) (Supplementary Figure S1H).

miR-30a-3p is downregulated in ccRCC and suppresses APOL1 expression
To further investigate the miRNA that could regulate APOL1 expression and ccRCC metastasis, we analyzed small RNA sequencing data (miRNA-seq) of 9 patient samples.Hierarchical clustering heatmap and the multidimensional scaling of miRNAseq revealed the distinguished expression profiles of the tumor and normal tissues (Figures 2A, B).There were 103 differently expressed miRNAs, and among them, 28 miRNAs were predicted to target the 3'UTR region of APOL1 by TargetScan, where 16 were upregulated and 12 were downregulated miRNAs (Supplementary Figure S2A).Due to the high expression of APOL1 in ccRCC, we focused on the miRNAs that had attenuated expression in ccRCC (Supplementary Figure S2B), and therefore examined the correlation between APOL1 and these 12 downregulated miRNAs.We found that miR-30a-3p expression was negatively correlated with APOL1 expression, and this was statistically significant in both miRNAseq and the TCGA-KIRC dataset (Figures 2C, D, Supplementary Figures S2C, D, E, F).In in vivo model, the expression of miR-30a-3p was low in tumors of primary cancer cells and high in tumor of metastatic cells, which negatively correlated with APOL1 expression (Supplementary Figure S2G).Moreover, the low level of miR-30a-3p in ccRCC (Figure 2C, Supplementary Figures S2H, I) was associated with poor survival rates (Figure 2E).To validate whether the miR-30a-3p targeting 3'UTR of APOL1 regulated its expression in vitro, we transfected this miRNA to the high APOL1 expression renal cells, A-498 and 786-O (Supplementary Figure S2J).As expected, APOL1 protein expression was reduced (Figure 2F).Furthermore, we treated A-498 cells with the top 3 significantly downregulated miRNAs.As a result, these miRNAs did not specifically suppress APOL1 expression in renal cancer cells as the effect of miR-30a-3p (Supplementary Figure S2K).In conclusion, we found that the miRNAs and RNA worked in cohort to actively regulate ccRCC, and miR-30a-3p regulated APOL1 expression, which is in turn a potential hallmark of ccRCC.

APOL1 suppresses ccRCC metastasis in vitro and in vivo
To assess the role of APOL1 in tumor progression, we generated a stable knockdown of APOL1 in the primary cancer A-498 and 786-O cells and overexpressed APOL1 in the metastatic Caki-1 cells by lentiviral transfection.The renal cancer cells were efficiently knocked down and overexpressed at both transcript and protein levels (Figures 3A-C, Supplementary Figure S3A).The knockdown and overexpression of APOL1 did not affect the cell proliferation rate (Supplementary Figure S3B).Interestingly, the results from the wound healing and transwell assays showed that APOL1 knockdown promoted cell migration and invasion (Figures 3D-G).In coherence with these findings, APOL1 overexpression obstructed cell motility (Figures 3H, I).
To verify the function of APOL1 expression in metastatic progression in vivo, we intravenously injected the APOL1 overexpressing Caki-1 cells into the tail vein of nude mice.In line with the in vitro results, the number of lung metastatic nodules in the mice injected with APOL1 overexpressing Caki-1 cells were reduced compared to its control plasmids (Figures 3J-L).There was no difference in the weight between control and experimental mouse groups (Supplementary Figure S3C).Taken together, we found that APOL1 was critical in the progression of ccRCC, but was shown to block metastasis at later stages of cancer.

Suppression of APOL1 facilitates FAK activation and EMT via the Akt signaling pathway
To gain insights into the mechanism by which APOL1 ameliorated renal malignant progression, we investigated the tumorigenic pathways by GSEA and gene enrichment analyses of 212 upregulated genes in ccRCC.As a result, Akt signaling pathway was shown to be the most significantly activated pathway by analysis with Wikipathways, Reactome, PID, KEGG database, among several other pathways in ccRCC.Namely, focal adhesion-Akt signaling pathway, negative regulation of the PI3K/Akt network, NF-kappa B signaling pathway were shown to be regulated (Figures 4A, B).The top terms for GO analysis were the intracellular functions of APOL1 rather than its secreted function, and was shown to be involved in regulation of cellular process and binding functions (Supplementary Figure S4).
To determine whether APOL1 dependent Akt pathway could be involved in cell migration of tumor cells, we checked the Akt pathway in the APOL1 knockdown or overexpressing cells.Akt phosphorylation and the downstream targets including nuclear factor kappa light chain enhancer of activated B cells (NFkB) and glycogen synthase kinase 3 (GSK3b) were enhanced in the APOL1 knockdown A-498 and 786-O cells and downregulated in the APOL1 overexpressing Caki-1 cells (Figure 4C).Consistent with our findings, APOL1 suppression by miR-30a activated the Akt signaling pathway (Figure 4D).
Elevation of many integrin molecules may trigger metastasis in renal cell carcinoma (40, 41), moreover, integrins also activate Akt via FAK, paxillin, and integrin-linked kinase (42).Because APOL1 is known to interact with integrin aVb3 to activate integrins in podocytes to dysregulate of actin cytoskeleton (43), we checked the expression of several focal adhesion proteins in APOL1 knocked down and overexpressing cells.In agreement with the increased cell migration in APOL1 knockdown cells, focal adhesion proteins integrin aVb3, p-FAK, kindlin 1, vinculin, a-actinin, p-Talin was upregulated.Consistent with these findings, APOL1 overexpression downregulated these focal adhesion proteins (Figure 4E).
We hypothesized that the EMT could also influence the migration and invasion of tumor cells.Thus, we examined the EMT related protein expression in APOL1 knockdown or overexpressing cells.Mesenchymal marker N-cadherin and APOL1 overexpression in renal cell carcinoma reduces the metastasis in vitro and in vivo.transcriptional repressors of E-cadherin (Snail and Slug) upregulated while epithelial marker E-cadherin was downregulated in APOL1 knockdown cells.On the other hand, APOL1 overexpression inhibited EMT and showed increased Ecadherin (Figure 4F).
In summary, suppression of APOL1 activated tumorigenic pathways, especially that of Akt, in which FAK contributed to increase tumor aggressiveness through the expression of adhesion molecules and EMT processes.

Discussion
ccRCC is the most prominent subtype of kidney cancer, where inactivation of VHL tumor suppressor gene is regarded as the governing event for progressing through HIF transcription factor activation in conventional ccRCC.APOL1, found as a tumor suppressor in our study, is one of the upregulated downstream genes of HIF (44).Similarly, transcriptome sequencing analysis of ccRCC against adjacent normal tissues in our research demonstrated that APOL1 was highly expressed in cancer samples with high mortality rates.Furthermore, adenosine to inosine RNA editing events is notably found in the 3'UTR region of APOL1 across 33 cancer types from TCGA dataset (45).This event leads to the upregulation of APOL1, and is associated with poor overall survival of lung adenocarcinoma (46).This is how APOL1 expression is increased to promote renal cancer progression through cell metabolic alterations (47).
In our study, the mRNA expression of APOL1 is increased according to the stages of cancer, however, the protein level is dropped at the later stages (Supplementary Figures S1C, G, H).This phenomenon infers that APOL1 may be modulated during its transcriptional or translational processes to be reduced in its expression in later cancer stages.We found that miR-30a-3p targets APOL1 and found evidence that it may affect ccRCC metastasis via the Akt signaling pathway.However, this finding may be contradictory with the previous studies of miR-30a as a tumor suppressor in renal cell carcinoma and other various carcinomas (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58).Our discovery revealed the connection between miR-30a-3p and APOL1 as the miRNA to be the inhibitor of the tumor suppressor.
One of our important findings was that APOL1 was highly expressed in primary renal cancer cells and retained lower expression patterns in metastatic cells.This was the same as drop APOL1 protein expression in more aggressive tumor grades (Supplementary Figure S1H).Consistently, the downregulation of APOL1 promoted EMT and cell motility of non-metastatic cells, whereas the upregulation of APOL1 suppressed cell migration in metastatic cells.These observations correspond to the previous inference that EMT process arise when knocking out APOL1 (47).Altogether, deregulation of APOL1 protein may be a hallmark of advanced renal cancer.
Our functional enrichment analysis showed that the Akt signaling was the major underlying pathway pertaining to ccRCC progression.The PI3K/Akt signaling pathway was reported to be activated in various renal cancer cell lines regardless of the VHL deficiency (59).Hence, the phosphorylation of Akt may inhibit GSK3b in RCC cells by facilitating GSK3b ubiquitination (60).Subsequently, NFkB is constituently activated in the signaling cascades initiated by phosphatidylinositol 3-kinase (PI3K) and Akt (61).Both NFkB activation and GSK3b degradation may lead to Snail or Slug mediated EMT (62)(63)(64).Additionally, Akt is the downstream of FAK (65) and integrin aVb3, and promotes EMT by upregulation of Slug expression in RCC (66).FAK localizes into integrin receptor cell adhesion sites and to the ECM.When activated, it recruits important components of the cytoskeleton, such as kindlin 1, vinculin, a-actinin, and talin to the cell membrane, providing both an anchorage for attachment and transduce intracellular signals (32).Our study revealed that integrin aVb3 activating FAK plays as an intermediating role between APOL1 and Akt signaling pathway, facilitating EMT.Altogether, APOL1 is an upstream regulator of these signaling pathways which ultimately leads to ccRCC tumor progression.
In pancreatic cancer, APOL1 is known to promote proliferation and inhibit apoptosis (8); however, in renal cell carcinoma, it does not alter proliferation and instead inhibit cell migration and invasion.APOL1 therefore is involved in diverse cellular processes and may have different roles depending on cancer types.Even though APOL1 is thought to be dispensable (67), its risk alleles have been proved to worsen kidney disease via cytotoxic effect on podocytes (68), furthermore, according to our research, APOL1 has a critical role in ccRCC.To further understand the related molecular mechanisms, direct inhibitory interaction between APOL1 and integrin molecules may need to be elucidated.
In summary, renal cancer cells may disseminate to distant organs through activation of focal adhesion proteins and trigger Akt downstream target genes to promote EMT.Our study revealed the intracellular function of APOL1 as a tumor suppressor in renal cancer cell EMT and metastasis through FAK/Akt/GSK3b and NFkB, which may be potentially targeted to control ccRCC metastasis (Figure 5).

1 APOL1
FIGURE 1 APOL1 is highly expressed in clear cell renal cell carcinoma.(A-C) Heat map of hierarchical clustering (A), multidimensional scaling (B) and APOL1 mRNA expression (C) in RNA-seq of 9 patient ccRCC tumors compared to their adjacent normal tissues.(D) Kaplan-Meier survival analysis based on the APOL1 expression at stage 4 and grade 4 in the TCGA-KIRC dataset.(E, F) qRT-PCR analysis of APOL1 mRNA (E) and western blot analysis of APOL1 protein (F) in A-498, 786-O (primary cancer) and Caki-1 (metastatic) cells compared to HK-2 (normal) cells.Actin served as an internal control.
FIGURE 2 miR-30a-3p suppresses APOL1 expression.(A-C) Heat map of hierarchical clustering (A), multidimensional scaling (B) and miR-30a-3p expression (C) in miRNA-seq of 9 patient ccRCC tumors compared to their adjacent normal tissues.(D) Pearson correlation analysis between miR-30a-3p and APOL1 in patient ccRCC tumors.(E) Kaplan-Meier survival analysis based on the miR-30a expression in the TCGA-KIRC dataset.(F) Western blot analysis of APOL1 protein in control and miR-30a transfected A-498 and 786-O cells.Actin served as an internal control.

4 APOL1
FIGURE 4 APOL1 suppression activates Akt signaling pathway in clear cell renal cell carcinoma.(A) GSEA of RNA-seq data from patient ccRCC tumors compared to their adjacent normal tissues.(B) Gene enrichment analysis of 212 upregulated genes in patient ccRCC tumors.(C, D) Western blot analysis of Akt, NFĸB, GSK3b protein in control and APOL1 knockdown/overexpressing renal cells (C), and miR-30a transfected A-498 and 786-O cells (D).(E) Western blot analysis of focal adhesion proteins in control and APOL1 knockdown A-498 cells, APOL1 knockdown 786-O cells and APOL1 overexpressing Caki-1 cells.(F) Western blot analysis of EMT markers in control and APOL1 knockdown A-498 cells, APOL1 knockdown 786-O cells and APOL1 overexpressing Caki-1 cells.Actin served as an internal control.

TABLE 1
The stage and metastatic status of 9 ccRCC human samples.