effect of PPA2 knockdown on gene expression under normoxia or hypoxia of DLD1 colerectal cancer cells

Metastatic colorectal cancer (mCRC) is the major cause of death in patients with CRC. Hypoxia is a hallmark of solid tumors that promotes cell metabolic adaptation and metastasis. However, the mechanism linking mCRC to hypoxia-regulated metabolic adaptation remains unclear. Here, we found that inorganic pyrophosphatase 2 (PPA2) suppresses mCRC progression via its phosphatase function. PPA2 is expressed at low levels in mCRC specimens and lowly expressed PPA2 mediates better responses to hypoxia to enhance CRC glycolysis and metastasis by promoting hypoxia-inducible factor-1a (HIF-1a) signaling. Mechanistically, PPA2 decreases HIF-1a stability through noncanonical ubiquitin-mediated proteasomal degradation via recruitment of E3 ligase neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), which inhibits glycolysis and metastasis-related gene expression. Furthermore, PPA2 directly interacts with NEDD4 and dephosphorylates NEDD4 at the T758 residue, leading to the increased interaction between NEDD4 and HIF-1a. Under hypoxic stress, NAD-dependent protein deacetylase sirtuin-5 (SIRT5) promotes the dissociation of PPA2 and NEDD4 by inducing PPA2 desuccinylation at the K176 residue, which ultimately contributes to the improved stability of HIF-1a under hypoxic conditions. Our findings reveal the suppressive role of PPA2 in HIF-1a-dependent mCRC, suggesting that the SIRT5-PPA2-NEDD4-HIF-1a axis can be assessed for integrated prognosis evaluation and represents a potential therapeutic target. Overall design: PPA2-depleted DLD1 cells were establised by shRNA, and were incobated under normoxia (HO) or hypoxia (LO) for 12h before sample collection.

转移性结直肠癌（Metastatic colorectal cancer, mCRC）是结直肠癌（colorectal cancer, CRC）患者的主要致死原因。缺氧是实体瘤的标志性特征，可促进细胞代谢适应与肿瘤转移。然而，连接mCRC与缺氧调控代谢适应的具体机制仍不明确。本研究发现，无机焦磷酸酶2（inorganic pyrophosphatase 2, PPA2）通过其磷酸酶活性抑制mCRC的进展。PPA2在mCRC组织中呈低表达状态，低水平的PPA2可通过激活缺氧诱导因子-1α（hypoxia-inducible factor-1α, HIF-1α）信号通路，增强CRC细胞对缺氧的应答，进而促进结直肠癌的糖酵解与转移过程。
从机制层面来看，PPA2可通过招募E3泛素连接酶神经前体细胞表达发育下调基因4（neural precursor cell-expressed developmentally downregulated gene 4, NEDD4），介导非经典泛素化依赖的蛋白酶体降解途径，降低HIF-1α的蛋白稳定性，从而抑制糖酵解及转移相关基因的表达。进一步研究显示，PPA2可直接与NEDD4相互结合，并对NEDD4的T758位点进行去磷酸化修饰，由此增强NEDD4与HIF-1α的相互作用能力。在缺氧应激条件下，烟酰胺腺嘌呤二核苷酸（NAD）依赖的蛋白去乙酰化酶沉默信息调节因子5（NAD-dependent protein deacetylase sirtuin-5, SIRT5）可通过诱导PPA2的K176位点发生去琥珀酰化修饰，促进PPA2与NEDD4的解离，最终提高缺氧环境下HIF-1α的蛋白稳定性。
本研究揭示了PPA2在HIF-1α依赖型mCRC中的抑癌作用，提示SIRT5-PPA2-NEDD4-HIF-1α信号轴可用于整合性预后评估，同时也代表了潜在的治疗靶点。整体实验设计：通过短发夹RNA（small hairpin RNA, shRNA）构建PPA2敲低的DLD1细胞系，在样本收集前，将细胞分别置于常氧（HO）与缺氧（LO）环境中培养12小时。