ELOVL5‐mediated fatty acid elongation promotes cellular proliferation and invasion in renal cell carcinoma

Abstract Renal cell carcinoma (RCC) features altered lipid metabolism and accumulated polyunsaturated fatty acids (PUFAs). Elongation of very long–chain fatty acid (ELOVL) family enzymes catalyze fatty acid elongation, and ELOVL5 is indispensable for PUFAs elongation, but its role in RCC progression remains unclear. Here, we show that higher levels of ELOVL5 correlate with poor RCC clinical prognosis. Liquid chromatography/electrospray ionization‐tandem mass spectrometry analysis showed decreases in ELOVL5 end products (arachidonic acid and eicosapentaenoic acid) under CRISPR/Cas9‐mediated knockout of ELOVL5 while supplementation with these fatty acids partially reversed the cellular proliferation and invasion effects of ELOVL5 knockout. Regarding cellular proliferation and invasion, CRISPR/Cas9‐mediated knockout of ELOVL5 suppressed the formation of lipid droplets and induced apoptosis via endoplasmic reticulum stress while suppressing renal cancer cell proliferation and in vivo tumor growth. Furthermore, CRISPR/Cas9‐mediated knockout of ELOVL5 inhibited AKT Ser473 phosphorylation and suppressed renal cancer cell invasion through chemokine (C‐C motif) ligand‐2 downregulation by AKT‐mTOR‐STAT3 signaling. Collectively, these results suggest that ELOVL5‐mediated fatty acid elongation promotes not only cellular proliferation but also invasion in RCC.


| INTRODUC TI ON
Fatty acids (FAs) comprising lipids, such as triglycerides, sphingolipids, and phospholipids, are important structural, energy, and signaling sources. FA length is a crucial functional indicator, and the rate-limiting steps of de novo long-chain fatty acid (LC-FA) synthesis are regulated by the conserved elongation of very long-chain fatty acid enzyme family (ELOVLs). 1 A total of seven ELOVLs are currently recognized and categorized into saturated and monounsaturated FA-specific (ELOVL1, ELOVL3, ELOVL6, and ELOVL7) and polyunsaturated fatty acid (PUFA)-specific activities (ELOVL2, ELOVL4, and ELOVL5). 2 Altered lipid metabolism is a key cancer biomarker because turnover is accelerated within the tumor microenvironment. 2,3 Renal cell carcinoma (RCC), roughly 2% of total adult malignancies, is usually found as clear cell RCC (ccRCC), with papillary RCC (pRCC) and chromophobe RCC (chRCC) following. 4 If RCC is localized, partial or radical nephrectomy are usually curative, 5,6 while unresectable or metastatic RCC cases receive drugs targeting vascular endothelial growth factor (VEGF), immune checkpoints, and mammalian target of rapamycin (mTOR). 4,5 These therapies have somewhat improved prognoses, but outcomes remain dismal. 5 ELOVL5 elongates linoleic acid (18:2, n-6) and α-linolenic acid Here, we show that inhibition of ELOVL5 alters lipid metabolism and ablates cellular proliferation in favor of apoptosis, ostensibly from enhanced endoplasmic reticulum (ER) stress. Furthermore, we show that ELOVL5-mediated lipid metabolism is linked to AKT activation, which may contribute to cellular invasion. This is the first demonstration linking ELOVL5-mediated PUFA elongation and renal cancer progression.

| Patients and RCC samples
Tumor specimens from 40 patients who underwent either radical or partial nephrectomies were obtained from the University of Tsukuba Hospital under protocols approved by the Ethics Committee of the University of Tsukuba (approval number: H28-104). All patients gave written, informed consent. Tumor stages were assigned according to the TNM staging of the Union for International Cancer Control. 9 Pathological grades were classified by the Fuhrman grading system. 10

| Immunohistochemistry
Immunohistochemistry was conducted as described previously. 11 Primary antibodies can be found in Table S1. The labeling index for Ki-67 was calculated as the percentage of positive tumor nuclei divided by the total number of tumor cells examined.

| FA preparation
AA (Wako, Cat #194625) and EPA (Cayman Chemical, Cat #90110) were dissolved in ethanol and mixed vigorously to a final concentration of 10 mM, filter-sterilized, and added to the culture medium with 0.5% FA-free BSA (Wako, Cat #017-15141) in phosphate-buffered saline to 10 μM final concentration.

| Western blot analysis
Western blot analysis was performed as described previously. 8,12 The primary antibodies used are listed in Table S1. β-actin was used as an internal control.

| Nude mouse xenograft assays
Female 6 to 8-week-old Balb/c nude (nu/nu) mice were purchased from Charles River Laboratories Japan. For subcutaneous xenograft assays, 1 × 10 7 cells were injected subcutaneously into the flank.
Orthotopic xenografts were performed as described previously. 15 Briefly, a 2-cm incision was made on the left flank to expose the kidney before 1 × 10 6 cells were injected into the renal parenchyma.  in corresponding normal and tumor tissues, we excluded one control sample without available data from a corresponding primary tumor. For survival analysis, 530 patients were divided into two groups (low and high expression) using expression cutoff values that yielded maximal survival differences between groups at the lowest log-rank P value.

| Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) analysis of FA metabolites
Fatty acid metabolites were purified from lipid fractions by solidphase extraction with Oasis HLB columns (Waters Corporation).
Purification and liquid chromatography were performed as described previously. 17,18 Briefly, FA metabolites were extracted from 4 × 10 7 cells with internal standards. FA metabolites were then separated using a high-performance liquid chromatography system (Nexera LC-30 AD, Shimadzu Corporation) equipped with an XBridge C18 column (particle size 3.5 μm, length 150 mm, inner diameter 1.0 mm; Waters) and analyzed on a triple quadrupole mass spectrometer (LCMS-8040; Shimadzu). Mass spectrometry analyses were carried out in negative ion mode while FA metabolites were analyzed via multiple reaction monitoring, as previously reported. 17,18 For quantification, calibration curves were prepared for each compound and deuterated internal standards were used to monitor recoveries. Data analysis was performed with LabSolutions software (Shimadzu Corporation).

| Statistical analysis
Data were expressed as mean ± SD, except for subcutaneous xenograft assays (mean ± SE). Categorical variables were compared between cohorts using Pearson's chi-squared test for two or more variables. Continuous variables were compared using the Student Additional materials and methods are detailed in Data S1. Our RNA-sequencing data are available at GSE192403.

| ELOVL5 is overexpressed in RCC and higher levels of ELOVL5 expression correlate with poor clinical prognoses in RCC patients
We confirmed significant elevation of ELOVL5 mRNA and protein expression levels in RCC compared with control tissue ( Figure 1A-C). In immunohistochemical analysis, 30 of 40 ccRCC cases showed significantly higher clinical staging coupled to higher ELOVL5 staining intensities ( Table 1). Patient characteristics and ELOVL5 staining statuses are summarized in Table 1.
Next, we explored the tumor progression role of ELOVL5 in RCC patients using TCGA database (KIRC cohort). As shown in Figure 1D, overall survival (OS) was negatively associated with increased ELOVL5 expression, linking ELOVL5 and RCC disease progression.

| CRISPR/Cas9-mediated knockout of ELOVL5 reduces RCC proliferation and invasion ability
To examine whether ELOVL5 regulates proliferative and invasive ability, we knocked out ELOVL5 expression with two independ-

| CRISPR/Cas9-mediated knockout of ELOVL5 reduces tumor growth and invasion in vivo
We used a nude mouse xenograft model to examine the tumor progression role of ELOVL5 in RCC, finding that CRISPR/Cas9mediated knockout of ELOVL5 in ACHN and 786-O cell lines suppressed tumor growth after subcutaneous implantation ( Figure 3A). We next calculated the Ki-67 index in xenograft tumors infected with sgControl or sgELOVL5 and found significant differences among tumors in the two groups ( Figure 3B).
Furthermore, we performed orthotopic xenograft assays in ACHN cell lines to examine whether ELOVL5 regulates renal cancer cell invasion in vivo. As shown in Figure 3C, sgControl cells infiltrated into normal kidney parenchyma whereas sgELOVL5 cells stopped well before. These results further support the capacity of ELOVL5 in RCC proliferative and invasive abilities.

| ELOVL5 elongation of polyunsaturated FAs is critical for cellular proliferation of and invasion by RCC
To examine the alteration of lipid metabolism under CRISPR/Cas9mediated knockout of ELOVL5, we examined total FA levels between ACHN/sgControl and ACHN/sgELOVL5 cells by LC/ESI-MS/MS analysis. As expected, AA and EPA levels were reduced in sgELOVL5 cells compared with sgControl cells ( Figure 4A) and, furthermore, we found that AA or EPA supplementation partially reversed the

| CRISPR/Cas9-mediated knockout of ELOVL5 inhibits lipid droplet (LD) formation and induces the unfolded protein response (UPR)
As a clear biomarker of ccRCC is intracellular LDs that are related to disease progression, [19][20][21] we examined whether ELOVL5 promotes LD formation by live-cell staining with Lipi-Green probe. As shown in Figure 5A LD functions to protect against ER stress, maintain ER homeostasis, and control protein quality. 19,20,24 As decreased LD disrupts ER homeostasis, inducing the UPR and cell death, 24 we next examined the expression of UPR sensor proteins and target genes. As shown in Figure 5C, the phosphorylation of UPR sensor proteins PERK and IRE-1, as well as ATF6 expression, were increased in sgELOVL5 cells of both cell lines. Furthermore, the UPR target gene CHOP was increased in sgELOVL5 cells of both cell lines ( Figure 5D,E). We next examined whether PUFAs affect the expression of CHOP and observed that AA or EPA supplementation partially reversed its expression ( Figure 5F). These results suggest that ELOVL5-mediated lipid metabolism maintains ER homeostasis through LD formation to promote cellular proliferation.  found that the activity of caspases 3 and 7 were significantly elevated in sgELOVL5-treated ACHN and 786-O cell lines ( Figure 6A).
As depolarized mitochondrial membrane potential is a crucial earlystage checkpoint in apoptosis, 28 we assessed such changes using a JC-1 assay that disassociates into monomers under membrane

| ELOVL5 promotes cellular invasion through CCL2 regulation
The  (Table S3). Among these four genes, CCL2 was remarkably downregulated in both ACHN and 786-O cell lines ( Figure S1), similar to previous reports that CCL2 promotes tumor migration and metastasis, and strengthening our assertion that CCL2 is a crucial cellular invasion gene associated with The Cancer Genome Atlas data analysis revealed that increased CCL2 expression significantly correlated with poor prognosis, and this was also positively correlated with ELOVL5 expression ( Figure S2A,B). Immunohistochemical analyses revealed that 29 of 40 ccRCC cases showed significantly higher CCL2 staining intensities ( Figure S3) and 23 of 29 ccRCC cases (79.3%) were coupled to higher ELOVL5 staining intensities. The protein levels of CCL2 were significantly decreased in sgELOVL5 cells of both cell lines ( Figure 7D,E). To firmly establish that CCL2 mediates cellular invasion by RCC cells, we next subjected ACHN and 786-O cells to CCL2-siRNA and observed significant decreases in CCL2 mRNA expression ( Figure 7F). A Matrigel invasion assay using these same siRNA-treated cells resulted in significant suppression of cellular invasion by siRNA-treated cells of both cell lines ( Figure 7G). Taken together, these results suggest that ELOVL5 promotes cellular invasion via CCL2 regulation.

| ELOVL5-mediated lipid metabolism promotes CCL2 upregulation through the phosphorylation of AKT Ser473 in RCC cells
To identify how ELOVL5 regulates CCL2 expression in RCC cells, we performed upstream regulator analysis using IPA in ACHN/ sgControl and ACHN/sgELOVL5 cells. As shown in Figure 8A, AKT was identified as a potential kinase in ACHN cells. As AKT-mTOR-STAT3 signaling triggers CCL2 transcription, [30][31][32] we confirmed that the phosphorylation of AKT Ser473, mTOR Ser2448, and STAT3 Thr705 was significantly decreased by ELOVL5 ablation in ACHN and 786-O cell lines ( Figure 8B). To further investigate this, we examined whether AA or EPA affects CCL2 expression and, as shown in Figure 8C, the mRNA expression of CCL2 was partially reversed with AA or EPA supplementation into the medium. Next, we examined whether AKT Ser473 phosphorylation is involved in altered lipid metabolism by ELOVL5 and saw a partial trend to increase after AA or EPA treatment ( Figure 8D,E). Taken together, these results suggest that alteration of lipid metabolism by ELOVL5 promotes renal cancer invasion through the upregulation of CCL2 by AKT-mTOR-STAT3 signaling.

| DISCUSS ION
Here, we determined that ELOVL5 is essential in regulating RCC cancer progression. We established that it is overexpressed in    showed that the mRNA expression of pro-apoptotic genes regulated by CHOP was increased by ELOVL5 knockout, leading to the conclusion that, collectively, ELOVL5 knockout inhibits LD formation and promotes ER stress-induced apoptosis.
Previous studies connected CCL2 with poor prognoses in patients with diverse cancers, including RCC. 29,44,45 The role of CCL2 in cancer is to stimulate cancer cell migration, activate vascular endothelial cells (resulting in angiogenesis), and recruit leukocytes. 29 We demonstrated that CCL2 is associated with cellular invasion in vitro and its expression is decreased by ELOVL5 knockout via ablation of AKT-mTOR-STAT3 signaling. [30][31][32][46][47][48] Phosphatidylinositol lipids (PI) are essential for phosphorylation of AKT, while acyl chain composition, as regulated by ELOVL5, mediates the biological activity of PI. 49 Regulation by endogenously synthesized FA on AKT activation is poorly understood but previous reports showed that the inhibition of SCD1 alters PI acyl chain composition from monounsaturated to more saturated acyl chains, resulting in an AKT phosphorylation decrease. 50,51 This was demonstrated by Rueda-Rincin et al and Xu et al who tied oleic acid (OA) and AA administration to increased AKT phosphorylation. 34,51 Our results are in line with these reports indicating that MUFA and PUFA are important FA in AKT phosphorylation. Future studies into direct binding assays will reveal any potential synergy between stearoyl-CoA desaturases and the ELOVL family of chain elongators.
In conclusion, this is the first demonstration linking ELOVL5mediated PUFA elongation and renal cancer progression. Further analysis is necessary to elucidate the upstream role of ELOVL5 in the signaling pathway but our results suggest that ELOVL5 is a novel target molecule for suppressing RCC proliferation and invasion potential.

ACK N OWLED G M ENTS
We are thankful for the skillful technical assistance of Mrs. Noriko Kunita and Mrs. Naoko Ueki (University of Tsukuba). We would like to thank LIPIDOME LAB Co., Ltd. for lipid analysis and Dr. Kazuhiro Yoshikawa (Aichi Medical University) for kindly providing the SKRC52 cell line.

D I SCLOS U R E
The authors have no conflict of interest.

E TH I C A L A PPROVA L
Approval of the research protocol by an Institutional Reviewer Board: Tumor specimens were obtained from the University of Tsukuba Hospital under protocols approved by the Ethics Committee of the University of Tsukuba (Approval Number: H28-104).

I N FO R M ED CO N S ENT
All patients gave written, informed consent.

R EG I S TRY A N D TH E R EG I S TR ATI O N N U M B E R O F TH E S TU DY/ TR I A L
n/a.

A N I M A L S TU D I E S
All animal experiments were approved by the Institutional Animal Care and Use Committee, University of Tsukuba (Approval Number: 21-361).