缺乏PHRF1表現的小鼠肝癌細胞在低氧條件下影響HIF1α蛋白總量的機制探討

Abstract

肝細胞癌（Hepatocellular carcinoma, HCC）是全球最常見的原發性肝癌，其惡性進展與腫瘤內部缺氧微環境密切相關。缺氧可穩定並活化轉錄因子HIF1α，促進腫瘤細胞的存活、轉移與血管新生。近年研究指出，ERK訊號傳導可透過磷酸化作用提升HIF1α的穩定性，而DUSP6作為其負向調控因子，可能抑制此途徑活性。PHRF1為具有E3泛素連接酶活性的蛋白，可參與DNA修復與表觀遺傳調節，亦可能具腫瘤抑制功能，惟其在肝癌細胞低氧反應中的角色尚未明確。初步觀察發現，PHRF1缺失的小鼠肝癌細胞在低氧條件下會導致HIF1α顯著上升，顯示PHRF1可能參與HIF1α負向調控。因此本研究探討PHRF1於低氧環境中對HIF1α穩定性與表現的影響，並研究其是否透過ERK/DUSP6路徑進行調控。結果顯示，PHRF1缺失提升HIF1α與pERK蛋白量表現，同時抑制DUSP6，並促進細胞在正常氧氣環境下的EMT與遷移能力，但在低氧下此能力反而下降。儘管HIF1α上升，PHRF1缺失細胞的增殖能力仍顯著降低，顯示其可能具有HIF1α非依賴性的功能，或過度HIF1α活化引發細胞壓力反應，抑制細胞功能。綜合以上結果，我們推測PHRF1可能透過ERK/DUSP6途徑參與HIF1α的調控，影響肝癌細胞對低氧狀態下的適應行為。

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancers worldwide, with poor prognosis and strong association with hypoxic tumor microenvironments. Hypoxia stabilizes and activates the transcription factor HIF1α, promoting tumor cell survival, migration/invasion, metastasis, and angiogenesis. Recent studies suggest that ERK signaling enhances HIF1α stability via phosphorylation of HIF1α, while DUSP6 acts as a negative regulator to dephosphorylate ERK pathway. PHRF1 is an E3 ubiquitin ligase involved in DNA repair and epigenetic regulation. However, the role of PHRF1 in liver cancer remains unclear. Preliminary observations showed that PHRF1 knockout under hypoxic conditions leads to increased HIF1α protein expression, implying a negative regulatory role of PHRF1 in HIF1α regulation. This study aims to study how PHRF1 regulated HIF1α expression and stability under hypoxia condition, and whether ERK/DUSP6 pathway was also affected in the absence of PHRF1. Our results revealed that PHRF1 deficiency elevated HIF1α and phosphorylated ERK (pERK) levels while the amount of DUSP6 was decreased under hypoxia. The expression of epithelial–mesenchymal transition (EMT) markers and cell migration ability were elevated under normoxia but reduced under hypoxia. Despite elevated HIF1α levels, PHRF1-deficient cells exhibited impaired clonogenic formation, suggesting that PHRF1 may regulate this process through HIF1α-independent mechanisms or through stress responses triggered by excessive HIF1α activity. Collectively, these findings indicate that PHRF1 may modulate HIF1α through the ERK/DUSP6 axis and influence hypoxia adaptation and malignancy in HCC cells.