Our data indicate that 2-HG, produced by IDH1R132H in gliomas, inhibited the proline hydroxylase EGLN2 activity, resulting in reduced CDH1 protein levels by ubiquitin-proteasome pathway and finally arrested the cell cycle. EGLN2 could stabilize CDH1, inhibiting the degradation of CDH1. Consequently, sustained low levels of CDH1 induced by 2-HG halted the cell cycle, impeding tumor development. These findings offer a theoretical basis for the improved prognosis observed in patients with IDH1R132H mutations(Fig. 5).
Mutations in the IDH1 are common in various tumors. Most cancer-associated IDH1 mutations occur at the well-known site R132, which is characterized by reducing α-KG to the carcinogenic metabolite 2-HG. Glioblastoma patients with IDH1R132H mutations have some unique clinical characteristics, such as markedly better clinical prognosis and younger age[28], but the reason remains unclear. This study focuses on this phenomenon to explore the mechanism of IDH1R132H mutation and 2-HG in it.
We observed that IDH1R132H and 2-HG increased CDH1 proteasomal degradation, resulting in elevated levels of CDH1 substrates such as VHL and Cyclin A2. These observations align with the overexpression of various CDH1 substrates, including DNA replication factors[29], mitotic kinases[30], and mitotic and S phase cyclins[31], which are commonly observed in a broad range of human malignancies.
The sole structural distinction between 2-HG and α-KG lies in the substitution of hydroxyl groups for oxygen atoms bonded to C2 in α-KG. This similarity implies that 2-HG might inhibit prolyl hydroxylases (PHD), which catalyze proline hydroxylation using α-KG and oxygen. Our findings suggested that both α-KG and oxygen independently increased CDH1 expression, regardless of HIF-1α. Furthermore, we observed that hypoxia enhanced CDH1 phosphorylation levels. Efficient cell-cycle progression requires a significant reduction in APC/CCDH1 activity as cells transition from G1 to S-phase. This reduced activity is achieved primarily through the hyperphosphorylation of CDH1 by cyclin-dependent kinases (CDK) in late G1, which inhibits the interaction of CDH1 with the APC/C[32]. We showed that after mutating four phosphorylation sites to alanine, hypoxia failed to increase CDH14A degradation. Recent reports have also emphasized elevated CyclinD-CDK4/6 activity in human cancer cells, attributing it to the inactivation of APC/CCDH1 through hyperphosphorylation of CDH1[33,34].
Furthermore, our findings indicated that EGLN2 positively regulated CDH1 protein levels. 2-HG disrupted the cell cycle by interfering with EGLN2-mediated hydroxylation of CDH1. Inhibition of EGLN2 has been shown to disrupt the cell cycle; for instance, EGLN2 regulates cyclin D1 protein levels independently of HIF[35]. Loss of EGLN2 inhibits mitotic progression by inducing mitotic spindle disorganization[36]. We propose that the E3 ligase preferentially targets un-hydroxylated CDH1, which is produced when EGLN2 is inhibited, for degradation, thereby linking α-KG inhibition and hypoxia to aneuploidy. Our findings demonstrated that CDH1 prolyl hydroxylation by EGLN2 inhibited its proteasomal degradation, contrasting with HIF-1α, whose prolyl hydroxylation by EGLN2 promotes its proteasomal degradation[37]. To our knowledge, this is the first example that prolylhydroxylation stabilizes a protein, and it makes physiologic sense given that high oxygen and KG signals would facilitate cell proliferation, in which high CDH1 levels and smooth chromosome segregation are required.
In this study, we demonstrated that both 2-HG and IDH1R132H knockin inhibited HeLa proliferation, with IDH1R132H knockin cells showing greater sensitivity to 2-HG treatment. Additionally, Pianka et al.[38] proposed a distinct mechanism for 2-HG-mediated inhibition of IDH1mut glioma growth, suggesting that 2-HG can reduce glioma growth by inhibiting the m6A demethylase, FTO. Additionally, EGLN2 knockout showed no response to 2-HG supplementation in our study. Moreover, our results indicated that both 2-HG and exogenous overexpression of IDH1R132H promoted cell apoptosis. These findings are consistent with 2-HG's inhibition of mTOR signaling and ATP synthase, resulting in growth arrest and tumor suppression[39,40]. Apoptosis and cell cycle arrest are well-known mechanisms for inhibiting cell growth, while chromosomal aneuploidy is frequently observed in solid tumors[41], indicative of aberrant cell cycle progression. Our study uncovered that 2-HG induced cell cycle arrest, notably impeding exit from the M phase, and diminished CDH1 levels. APC/CCDH1, a key regulator of the cell cycle, governs the stability of M and G1 phases[42]. The precise segregation of chromosomes during the M phase is crucial to prevent the acquisition of abnormal karyotypes by daughter cells[43]. Our findings implied that dysregulation of CDH1 by 2-HG may potentially trigger aneuploidy in pathological samples from patients with IDH1 mutations. Further investigation into the specific mechanisms involved will be pursued in our follow-up studies.
We observed that 2-HG increased DNA synthesis. This aligns with previous studies indicating that 2-HG contributes to metabolic reprogramming, including nucleotide synthesis utilization and DNA repair capacity[44,45]. The degradation of CDH1 by ubiquitination induced by 2-HG leads to cell cycle arrest, which could be rescued by CDH1 overexpression. This disruption can result in errors during DNA replication in S phase and missegregation of sister chromatids, ultimately leading to chromosome aneuploidy.
These results provide insights into why IDH1 mutation carriers exhibit longer survival rates: lower CDH1 levels and a slower cell division process induced by 2-HG delay tumor development, contributing to prolonged survival. We speculate that cell cycle arrest triggered by 2-HG may represent a suppressive mechanism against tumor development within the body.
In summary, our study elucidates that the accumulation of the metabolite 2-HG, induced by IDH1R132H mutation in glioblastoma patients, leads to the phosphorylation and subsequent degradation of CDH1 through inhibition of hydroxylation by EGLN2, ultimately causing cell cycle arrest at the M phase and inhibiting cell proliferation. This provides a theoretical basis for the extended survival observed in IDH1R132H mutation patients and sheds light on the molecular mechanism underlying oxygen's regulation of the cell cycle.