Oncometabolite L‐2‐hydroxyglurate directly induces vasculogenic mimicry through PHLDB2 in renal cell carcinoma

Abstract Metabolism reprograming is a hallmark of cancer and plays an important role in tumor progression. The aberrant metabolism in renal cell carcinoma (RCC) leads to accumulation of the oncometabolite l‐2‐hydroxyglurate (L‐2HG). L‐2HG has been reported to inhibit the activity of some α‐ketoglutarate‐dependent dioxygenases such as TET enzymes, which mediate epigenetic alteration, including DNA and histone demethylation. However, the detailed functions of L‐2HG in renal cell carcinoma have not been investigated thoroughly. In our study, we found that L‐2HG was significantly elevated in tumor tissues compared to adjacent tissues. Furthermore, we demonstrated that L‐2HG promoted vasculogenic mimicry (VM) in renal cancer cell lines through reducing the expression of PHLDB2. A mechanism study revealed that activation of the ERK1/2 pathway was involved in L‐2HG‐induced VM formation. In conclusion, these findings highlighted the pathogenic link between L‐2HG and VM and suggested a novel therapeutic target for RCC.

recent years, tyrosine kinase inhibitors (TKIs), such as sunitinib and pazopanib, have been administered in clinical treatment, especially in metastatic renal cell carcinoma (mRCC); these drugs significantly prolong the overall survival (OS) rate and progression-free survival (PFS) time. 5 However, most patients eventually develop acquired resistance after 6 to 15 months of angiogenesis-target therapy. 6,7 Thus, there is an urgent need to investigate the molecular mechanisms of RCC and explore new treatment strategies for it.
RCC is increasingly recognized as a metabolically solid tumor, characterized by dysregulation of cellular energetics and metabolic reprograming. 8 Previous studies reported that many genes such as VHL, MET, FH, SDH, FLCN, TSC1 and TSC2 were involved in pathways responding to metabolic stress. 9 L-2-Hydroxyglurate dehydrogenase (L2HGDH) is an FAD-dependent enzyme that oxidizes L-2-hydroxyglutarate (L-2HG) to alpha-ketoglutarate (α-KG). Interestingly, a recent report provided evidence that reduced mRNA and protein expression of L2HGDH in RCC promoted L-2HG accumulation and correlated with reduced 5-hydroxymethylcytosine. 10 2HG is a chiral molecule including D-and L-enantiomers. Although IDH1/2 mutants exclusively produce D-2HG, 11 L-2HG is induced by MDH1/MDH2 and LDHA during hypoxia. 12 L2HGDH and D2HGDH are important enzymes that oxidize 2HG to α-KG, preventing the accumulation of 2HG. 13,14 Previous studies demonstrated that both L-2HG and D-2HG were elevated in RCC, but L-2HG was the predominant enantiomer present in RCC, partly due to the low expression of L2HGDH. 10,15 Due to the structural similarity to α-KG, 2HG competitively inhibits some α-KG-dependent dioxygenases such as ten eleven translocation enzymes (TETs) and the Jumonji family of histone lysine demethylases, which are responsible for DNA and histone hypermethylation. 16,17 Recently, more evidence showed nonoxygenase enzymes were also implicated, including the DNMT1. 18 Inhibition of these enzymatic processes played an important role in tumor progression. Furthermore, elevated 2HG levels also resulted in changes to redox metabolism, potentially contributing to an increased risk of cancer. 19 Previous studies linked the D-2HG to the leukemia, brain tumors and colorectal cancer progression. [20][21][22][23] In contrast, Chen et al demonstrated D-2HG exerted a broad antileukemic activity through FTO/m6A/MYC/ CEBPA signaling. 24 However, more investigations are needed into 2HGrelated targets as well as the effects on tumor progression.
Vasculogenic mimicry (VM) is a new tumor vascular paradigm independent of endothelial cells (ECs), which has emerged as an important vasculogenic mechanism in tumors in addition to classic angiogenesis. 25 Periodic acid-Schiff (PAS) staining and CD34 immunohistochemistry (IHC) have been used to evaluate the presence of VM. 26 VM describes the specific capacity of aggressive cancer cells to form vessel-like networks, providing adequate nutrition supplements for tumor growth, and it is therefore associated with tumor metastasis and poor prognosis. 27 32 and VM drove some tumor cells to distant metastases in human cancers. 33 In fact, VM was composed of cancer cells, and the mechanism of channel formation was not the same as vessels formed by ECs. In this way, VM was thought to account for resistance to the antiangiogenesis therapy in some tumors. 34 Therefore, it is of great importance to explore the underlying mechanisms of VM formation so as to deepen the understanding of tumor neovascularization. Key molecular regulators of VM were identified in other cancer types, including MAPK, 35 vascular endothelial (VE)-cadherin 36 and MMPs. 37 To date, the presence of VM in RCC and its relationship with 2HG have not yet been elucidated.
In our study, we reported a role for L-2HG in promoting VM formation in RCC through a PHLDB2/MAPK pathway; however, the detailed mechanisms of PHLDB2 associated with RCC need further investigation. Thus, our study adds evidence that the oncometabolite L-2HG is a potential therapeutic target in RCC.

| IHC and PAS staining
The tumor tissues were fixed in 4% neutral-buffered paraformaldehyde, embedded in paraffin, cut into 5-μm sections and used for IHC.
In brief, the tissues were deparaffinized and rehydrated, and the samples were subjected to citrate-mediated high-temperature antigen retrieval, then incubated overnight with the primary antibodies CD34   41 We also explored the DNA methylation and gene expression in the Wanderer by the TCGA methylation arrays (450K Infinium chip).   Figure S1A,S1B). Consistent with the previous study, 10 we also observed high levels of L-2HG in tumor tissues compared to adjacent tissues (P < .01, Figure 1B), while R-2HG did not increase significantly (P > .05, Figure S1C). In addition, L-2HG was the dominant enantiomer in tumor tissues and at a level about 3-fold more than R- 2HG (P < .05 Figure 1C). When seeded on the Matrigel surface, RCC cells tended to form loops and networks as shown by the red arrows in Figure 1D. The vessel-like structures formed by the tumor cells are called VM; the process is independent of angiogenesis and is associated with tumor progression. 42 Thereafter, we examined the effect of

L-2HG on the VM in three typical RCC cell lines (786-O, A-498 and
OS-RC-2), by altering the L-2HG level. As shown in Figure 1D and  Figure 1E).

Interestingly, both angiogenesis and VM presented commonly in RCC.
However, the tumors with high L-2HG levels exhibited more VM structures than those with low L-2HG levels (P < .05, Figure 1E and Figure S1F). Collectively, both in vitro and in vivo data suggested that L-2HG promoted VM in RCC.

| Transcriptome analysis revealed the expression profile of RCC cells after L-2HG treatment
To gain mechanistic insights into VM formation by L-2HG, we  Table 2). In total, 187 differentially expressed genes (DEGs) including 62 upregulated genes and 125 downregulated genes (jfold changej ≥ 2, P < .05) were found (Figure 2A). A heat map of the DEGs is presented in Figure 2B, ZBTB38 and SIK1), consistent with the RNA-seq results that had changed most obviously ( Figure S2A). In addition, reducing the L-2HG level by overexpressing L2HGDH reversed these genes' expression ( Figure S2B). These results suggested that PHLDB2, ZBTB38 and SIK1 were downregulated by L-2HG. Analysis of these genes in the RCC using the TCGA data implied PHLDB2 expression was associated with the survival of RCC patients.
Therefore, PHLDB2 was the focus of our study, while ZBTB38 and

| PHLDB2 was downregulated by L-2HG and associated with progression in RCC
To investigate the regulatory mode of the VM formation by PHLDB2, we analyzed the relationship between PHLDB2 expression level and the OS rate on the UCSC Xena platform, including 517 RCC patients from the TCGA database. The results indicated that PHLDB2 was downregulated in tumor tissues compared to adjacent tissues and the expression level of PHLDB2 was negatively associated with the OS rate (P < .001, Figure 3F,G). In our own tissue samples from Sir Run Run Shaw Hospital, we also observed downregulation of PHLDB2 in tumor tissues compared to normal tissues (**P < .01, Figure 3H). Furthermore, the tumors with high L-2HG levels were correlated with low expression of PHLDB2 (**P < .01, Figure 3I).
Previous studies indicated that L-2HG induced the DNA methylation by inhibiting the TETs' function. 10,43 Therefore, we analyzed whether low expression of PHLDB2 was caused by the DNA methylation. In analysis of the TCGA data in the Wanderer, 44 the methylation levels of PHLDB2 promoter regions were significantly different between tumor and normal tissues ( Figure 3J). More, cg01303385, cg21857668 and cg22874988 were associated with the downregulation of PHLDB2 (Table 2 and Figure S3A, S3B and S3C). In addition, high cg21857668 levels correlated with poor OS in RCC patients based on the TCGA data while cg01303385 and cg22874988 showed no significance ( Figure S3D, S3E and S3F). Furthermore, we treated the RCC cell lines with decitabine (0.5 μM), a DNA methyltransferase inhibitor, and confirmed methylation changes affected the PHLDB2 expression ( Figure 3K). Overall, these results suggested that PHLDB2 played an important role in RCC progression and was regulated by L-2HG.

| PHLDB2 was associated with VM formation through MAPK pathway
It was reported that PHLDB2 (also known as LL5β), a PH domaincontaining protein, played an important role in mediating cell migration by forming a complex with its partners, such as CLASPS and Prickle 1. 45,46 However, whether and how PHLDB2 was implicated in RCC progression remained largely unknown. In our study, we found that PHLDB2 at both mRNA and protein levels was downregulated by L-2HG ( Figure 3A Figure 5B). In addition, the effect of PHLDB2 knockdown on ERK1/2 phosphorylation was reversed by restoring PHLDB2 expression (si#2 vs si#2 + OE, Figure 5C). Although statistically significant differences for ERK1/2 phosphorylation were not obtained caused by the limited number of repeated experiments, the change trend of ERK1/2 phosphorylation in each group was consistent ( Figure S4A, S4B, S4C and S4D).
Consistent with a previous study, 47 we also treated RCC cells (786-O and A-498) with U0126 (1 μM), an effective drug that inhibited ERK phosphorylation, and observed obvious reduction in VM formation compared to the control group ( Figure 5D). Overall, these results suggested that L-2HG functioned through reducing the expression of PHLDB2 and activating the ERK1/2 pathway to alter RCC VM formation ( Figure 5E).

| DISCUSSION
In our study, we showed that L-2HG was elevated in RCC consistent with previous study. 10 We also demonstrated that L-2HG, rather than R-2HG, contributed to the VM formation through reducing the mRNA and protein levels of PHLDB2. Our findings provide a new perspective on the important role of L-2HG in RCC.
2HG is the by-product of ongoing cellular metabolism produced by enzymes such as fumarate and succinate. In recent years, these oncometabolites were increasingly associated with many tumors including brain tumors, 20 acute myeloid leukemia, 21 colorectal cancer, 22 and head and neck squamous cell carcinoma. 48 Cheng et al measured the contents of D-2HG and L-2HG and observed a 30-fold increase of L-2HG in RCC tissues compared to adjacent normal tissues. 15 Similarly, we also found that L-2HG was dominant and elevated in RCC compared to the adjacent tissues. More evidence suggested that L2HGDH deficiency was the cause of the elevated level of L-2HG. 10,49 Our data also supported the hypothesis that L2HGDH reduced the cellular level of L-2HG, although there were some other factors such as hypoxia and MDH enzymes implicated in L-2HG accumulation. 12 Nevertheless, mechanisms of L-2HG accumulation in RCC warrant further investigation.
A previous study showed that 2HG activated HIF1α and/or VEGF signaling to stimulate the angiogenesis. 50 Sunil et al reported L-2HG as an oncometabolite promoting a migratory phenotype in RCC. 49 However, the role of L-2HG in VM formation remained unknown. In our study, we showed that L-2HG promoted VM in RCC cell lines and that diminishing the L-2HG via L2HGDH transfection inhibited VM formation. The RCC tissues with high L-2HG levels also exhibited more VM. Collectively, these findings suggested that the oncometabolite L-2HG produced by RCC was indeed associated with VM formation.
Our transcriptome analysis revealed that many relevant genes changed after L-2HG treatment. Through KEGG analysis of these DEGs, we found that many tumor-relevant pathways were involved, including apoptosis, cell cycle, phosphatidylinositol signaling and AMPK signaling. Furthermore, we found that PHLDB2 at both mRNA and protein levels were significantly downregulated in several RCC cell lines after L-2HG treatment. We also observed that PHLDB2  [51][52][53] Our data add the evidence that the oncometabolite L-2HG can contribute to VM formation. Therefore, targeting L-2HG is a novel strategy for RCC patients especially with high L-2HG levels. In addition, some small molecules targeting 2HG have been approved for glioma and leukemia. 54 AG-221, a potent and specific inhibitor of mutant IDH2 that leads to the production of R-2HG, has already shown significant survival benefits in aggressive leukemia. 55,56 However, targeting L-2HG for RCC patients' survival benefits requires more preclinical research.
The present study had limitations. The number of patients involved was insufficient to analyze the relationship between L-2HG and OS rate. In addition, other potential mechanisms of VM formation caused by the L-2HG, such as redox metabolism, were not examined.
Our future work would involve more patients and confirm the relationship between L-2HG and prognosis. Further studies of the molecular pathways that regulate 2HG-induced VM formation are our future research goal.