以蛋白基因體學探討C1QTNF7在肺腺癌中的免疫介導腫瘤抑制活性

Abstract

肺癌是全球癌症相關死亡的首要原因，突顯出對新型預後標誌與治療靶點的迫切需求。人類蛋白質組計畫 (Human Proteome Project) 旨在系統性地建立人類蛋白質體資訊，為疾病的診斷與治療提供更多可能。肺癌的高死亡率促使我們進行研究，而完成蛋白質組圖譜的目標激勵我們探索功能未知蛋白在癌症進展中的作用，這一探索為基於蛋白的精準醫療鋪展了道路。通過分析台灣癌症登月計畫（Taiwan Cancer Moonshot）所提供的大規模組織蛋白基因體學數據，我們篩選出一個位於四號染色體的功能未知蛋白，其稱作C1q and TNF-related protein 7（C1QTNF7），該蛋白在我們的肺腺癌研究族群中出現異常表現，然而其於癌症進程中的角色仍然有待探索。

藉由分析台灣癌症登月計畫的研究族群，我們發現C1QTNF7之蛋白表現在86%的肺腺癌患者腫瘤組織中顯著下調，並與患者的整體存活期、復發率、病理分化、腫瘤期數等臨床病理特徵均密切相關，C1QTNF7高表現的患者，傾向具有較佳的臨床結果，暗示其可能的臨床應用前景。為了闡明C1QTNF7之調控機制，我們建立了C1QTNF7過度表現（overexpression）與敲落 (knockdown) 之穩定肺癌細胞株，以免疫螢光染色觀察其細胞內定位，並透過收集條件培養基（conditioned medium），驗證C1QTNF7的分泌蛋白特性。接著，我們從RNA定序分析結果中，選擇具有顯著變化的差異表現基因（differentially expressed genes）進行路徑分析，發現富集的路徑主要涉及腫瘤轉移與免疫訊息傳遞。我們利用體外功能性實驗，進一步驗證了C1QTNF7對腫瘤細胞爬行與侵襲能力的抑制效果。此外，為了探討C1QTNF7與免疫訊息傳遞的關聯性，我們利用肺腺癌研究族群進行免疫細胞富集預測分析，並建立Jurkat T細胞共同培養模型、THP-1細胞衍生的巨噬細胞分化模型、與植入人類周邊血單核細胞的免疫系統擬人化小鼠模型，發現體外系統中C1QTNF7對上述兩類免疫細胞之活化與分化存在顯著影響。

總而言之，我們的研究結果揭露了C1QTNF7在肺腺癌中的腫瘤抑制活性，C1QTNF7可能在腫瘤微環境中的腫瘤轉移和免疫調節執行重要功能。以上發現為深入研究C1QTNF7的分子訊息網絡提供了更深的見解，C1QTNF7的免疫介導作用也突顯了其在肺腺癌預後和治療策略之應用潛力。

Lung cancer remains the primary contributor to cancer-associated mortality, highlighting the unmet need for novel prognostic markers and therapeutic strategies. The Human Proteome Project is dedicated to systematically characterizing human proteome, providing advancements in disease diagnosis and treatment. The significant fatality rate of lung cancer prompts our research. At the same time, the essential goal of completing the human proteome map drives our investigation into the involvement of uncharacterized proteins in cancer progression. This exploration paves the way for protein-driven precision medicine. By leveraging the tissue proteogenomic data from the Taiwan Cancer Moonshot cohort, we identified an uncharacterized protein, complement 1q and tumor necrosis factor-related protein 7 (C1QTNF7) encoded from chromosome 4. C1QTNF7 was found to be dysregulated in the lung adenocarcinoma cohort; nevertheless, its contribution to cancer progression remains to be elucidated.

In our study, C1QTNF7 was significantly downregulated in 86% of the tumor samples compared with paired normal tissues adjacent to the tumor (NAT). In the follow-up analysis of the Taiwan Cancer Moonshot cohort, clinicopathological characteristics of the 89 patients revealed that C1QTNF7 expression was associated with overall survival, recurrence, pathology differentiation, and tumor stage. Patients with high C1QTNF7 expression demonstrated favorable clinical outcomes, suggesting the potential prognostic value of C1QTNF7. To uncover the regulatory mechanisms of C1QTNF7, we established C1QTNF7-overexpressing and C1QTNF7-knockdown stable lung cancer cell lines. Immunofluorescence staining was performed to characterize the intracellular localization of C1QTNF7, and the conditioned medium was collected to validate its role as a secreted protein.

Furthermore, the differentially expressed genes identified by RNA sequencing analysis were subjected to pathway enrichment analysis. The analysis revealed an enrichment in metastasis- and immune signaling-related pathways. Upon further validation by in vitro functional assays, C1QTNF7 inhibited tumor cell migration and invasion. Additionally, to explore the correlation between C1QTNF7 and immune signaling, we estimated the immune infiltrated profiles of lung adenocarcinoma cohorts, as well as established Jurkat T cell co-culture, macrophage differentiation, and peripheral blood mononuclear cell-derived humanized mouse models. Our findings revealed that C1QTNF7 regulated T cell activation and macrophage differentiation in vitro.

In conclusion, our study unveiled the tumor suppressive activity of C1QTNF7 in lung adenocarcinoma. C1QTNF7 may exert a crucial influence on tumor metastasis and immune modulation within the tumor microenvironment. These findings provide insights for delving deeper into the molecular signaling network of C1QTNF7. The immune-mediated role of C1QTNF7 also highlights promising prognostic and therapeutic strategies for lung adenocarcinoma.