腫瘤微環境變化及細胞能量代謝對人類惡性腫瘤進展之影響

Effects of microenvironmental alteration and cellular energy metabolism on progression of human malignancies

張淑娟(Shu-Jyuan Chang)

指導教授 ： 關皚麗

高雄醫學大學/醫學院/醫學研究所博士班/博士(2020年)

摘要

腫瘤快速增生下，透過細胞適應性及代謝重組，以因應微環境變化，進而影響周邊非腫瘤細胞行為。本研究將分別針對腫瘤微環境調節及能量代謝變化對腫瘤發展之影響進行探討，我們透過三陰性乳癌及神經膠質瘤其完整進程模式作為研究對象。自噬為一種支持腫瘤存活及調節細胞代謝的適應性機轉，過去研究指出自噬在腫瘤發展過程似乎存在矛盾角色，除了防止受損胞器與蛋白質累積以抑制腫瘤，另一方面又可透過供應營養物質而增進已形成的腫瘤生長。因此我們藉由觀察在三陰性乳癌組織中自噬指標LC3，以評估細胞新陳代謝對腫瘤發展的重要性。研究結果指出，不論在癌細胞或癌幹細胞群中LC3表現低者，其具有高度淋巴結浸潤 (p=0.0005) 及高死亡風險 (P<0.0001)，顯示自噬體形成的缺陷可能導致細胞新陳代謝異常，驅使腫瘤走向惡性發展。癌細胞除了產生環境適應性外，其可經由代謝彈性調整來滿足快速增殖的營養需求。乳酸去氫酶 (LDH) 是腫瘤代謝中重要的一環，不僅參與乳酸代謝，更可轉換乳酸成丙酮酸以供產能之用。我們透過GEO資料庫分析及檢體染色觀察膠質瘤中LDH表現，發現惡性度越高的膠質瘤中LDHA及LDHB表現較正常或良性組織為高，其中LDHB又與IDH1突變具顯著關聯性 (p=0.0291)。我們更進一步從人類膠質母細胞瘤U-87 MG中，發現LDHB可透過調節VAP-1及integrin β5等移動蛋白，而參與惡性膠質瘤細胞遷移及侵犯表現；此與臨床檢體中LDHB表現於高侵犯性膠質瘤區域，並與腫瘤惡性度呈正比的趨勢相符，因此透過抑制LDHB將有利於降低膠質瘤侵襲性及促進標靶治療的發展。然而，不穩定的微環境不僅改變腫瘤細胞生理機能，亦會透過調節來改變周圍非腫瘤細胞的行為。我們發現高惡性腫瘤組織中，微環境調節因子-血管黏附蛋白 (VAP-1)表現量相較高，且與腫瘤侵犯性及惡性度呈正相關，進一步觀察到VAP-1與膠質瘤組織中腫瘤巨噬細胞免疫調節密切相關，VAP-1蛋白存在於腫瘤血管及M2巨噬細胞周邊，且病人惡性臨床特徵及存活率明顯受其影響。以上證據顯示，細胞代謝調節可改善腫瘤細胞的異質性和適應性，從而促進腫瘤進展過程中的M2活性，此將可作為未來癌症治療具潛力的策略方向。

關鍵字

細胞自噬 ； 腫瘤代謝 ； 乳酸去氫酶 ； 腫瘤巨噬細胞 ； 血管黏附蛋白-1 ； 人類惡性腫瘤發展

並列摘要

Rapidly growing tumors adapt to the variable microenvironment through cellular adaptation and metabolic reprogramming, which further affects the behavior of non-tumor cells surrounding malignant tissues. This study will discuss the effects of microenvironmental regulation and altered metabolism on tumor progression. Our research objects are triple negative breast cancer (TNBC) and gliomas which show as a complete progression model. Autophagy is an adaptive mechanism that supports tumor survival and regulates cell metabolism. Previous studies indicated that autophagy seems to have a contradictory role in tumor development. Malignant cells prevent the accumulation of damaged organelles and proteins to suppress tumor surival via autophagy. On the other hand, autophagy promotes the established tumors growth through sufficient nutrients and energy supplies. We observed the autophagy marker, LC3, is expressed in the TNBC tissues to assess the importance of cell metabolism for tumor development. These results indicated that low LC3 expression in cancer cells or cancer stem cell populations was correlated with highly lymphnode infiltration (p=0.0005) and the high risk of death (p<0.0001). It indicated that the deficiency of autophagosome synthesis may lead to abnormal cell metabolism, driving tumors to malignant development. Cancer cells produce environmental adaptability and display metabolic flexibility to supply the nutritional requirements for rapid proliferation. Lactate dehydrogenase (LDH) is an important part of tumor metabolism. LDH not only participates in lactate metabolism, but also converts lactate to pyruvate serving as an energy source for tumor cells. We analyzed the performance of LDH in gliomas through GEO database analysis and immunohistochemistry staining. High levels of LDHA and LDHB were found in high malignant areas of gliomas. LDHB is significantly associated with IDH1 mutations (p =0.0291). We further found LDHB participates in the migration and invasion of human glioblastoma U-87 MG cells by regulating mobile proteins such as VAP-1 and integer β5. Above data were consistent with LDHB expression in clinical specimens, which is manifested in highly aggressive gliomas and is related to tumor malignancy. Therefore, LDHB blocking would be beneficial to attenuate glioma invasiveness and promote the development of targeted therapy. However, cancer cells not only change their physiology via erratic microenvironment, but also alter the behavior of peripheral non-tumor cells through regulatory stimulation. A microenvironmental regulator, vascular adhesion protein-1 (VAP-1), expressed at relatively high levels in malignant glioma tissues. It was positively correlated with tumor aggressiveness and malignancy. We further observed that VAP-1 and tumor-associated macrophages (TAMs) have close relationship in gliomas. VAP-1 protein exists in tumor blood vessels and presents around M2 macrophages. The clinical characteristics and survival rate of glioma patients are significantly influenced by VAP-1 expression. The above evidence shows that cellular metabolic regulation improves the heterogeneity and adaptability of tumor cells, which promote M2 activivies during tumor progression. It will be the next promising strategies for the future cancer treatment therapy.

並列關鍵字

autophagy ； tumor metabolism ； lactate dehydrogenase ； tumor-associated macrophages ； vascular adhesion protein-1 ； progression of human malignancies

參考文獻

References

1. Hanahan D & Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646-674.

2. Hanahan D & Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57-70.

3. Qu Y, et al. (2019) Tumor microenvironment-driven non-cell-autonomous resistance to antineoplastic treatment. Molecular cancer 18(1):69.

4. Baghban R, et al. (2020) Tumor microenvironment complexity and therapeutic implications at a glance. Cell communication and signaling : CCS 18(1):59.