結合公開資料庫分析與小鼠模型建立以探討乳癌腦轉移之分子機制

Abstract

乳癌腦轉移（Breast cancer brain metastasis, BCBM）是晚期乳癌患者發病率與死亡率的主要原因，其中以 HER2 陽性乳癌與三陰性乳癌患者的發生率更高。儘管全身性療法的進步能夠顯著延長患者的存活率，但血腦屏障（blood-brain barrier, BBB）會阻擋大多數全身性治療藥物的進入，使腦部成為癌細胞逃避藥物攻擊的「庇護所」，導致乳癌腦轉移的發生率上升。因此，深入了解乳癌腦轉移的分子機制並尋找潛在的治療靶點至關重要。 本研究分為兩個部分：第一個部分透過建立乳癌腦轉移小鼠模型及公開資料庫分析探討乳癌腦轉移之分子機制；第二部分則著重於抗氧化酶 GPX4 在乳癌腦轉移中的作用。 在本研究的第一部分中，我們成功建立乳癌腦轉移小鼠模型，並分離出具腦轉移與脊髓轉移潛力的轉移衍生細胞株。這些轉移衍生細胞展現出較高的遷移能力與惡性特徵，其中腦轉移衍生細胞更顯示出明確的腦轉移傾向。透過公開資料庫分析與棕櫚酸氧化壓力試驗，指出脂肪酸代謝會參與乳癌腦轉移的調控過程。進一步結合RNA 定序分析與藥理抑制遷移試驗，顯示腦轉移衍生細胞可能上調脂肪酸合成，以增強其遷移能力。綜上所述，本研究提出脂肪酸合成可能為乳癌腦轉移之驅動力，並能夠作為潛在的代謝治療靶點。 本研究第二部分探討癌細胞於轉移過程中面臨氧化壓力之調控機制，在轉移過程中，癌細胞內的活性氧（reactive oxygen species, ROS）水平會顯著升高。為了應對這種壓力並在轉移過程中存活，癌細胞會上調其抗氧化系統。GPX4是細胞內重要的抗氧化酶，能夠清除脂質過氧化物，抑制鐵死亡的發生。近年來多項研究指出，GPX4 不僅與乳癌治療抗藥性有關，更與腫瘤遠端轉移密切相關。 實驗室先前研究發現，對Trastuzumab具抗藥性的乳癌細胞相較其親代細胞，展現出更強的遷移能力，並伴隨GPX4表現的上調，暗示GPX4 與抗藥性乳癌細胞之轉移潛力提升相關。臨床上，對Trastuzumab 具抗藥性的乳癌患者常以腦部作為疾病復發的初始轉移部位。透過公開資料庫分析，發現BCBM樣本中 GPX4 表現量及其相關代謝途徑呈現上調趨勢，暗示 GPX4 可能參與乳癌腦轉移的調控過程。在功能性層面上，抑制 GPX4 會影響乳癌細胞的遷移行為。然而，受限於目前建立之GPX4過度表現系統，本研究未能更精確釐清 GPX4 在乳癌腦轉移中的實際調控角色。綜上所述，本研究指出GPX4可能在BCBM過程中扮演關鍵調控角色，而其促進 BCBM 發生的具體分子機制，將於討論部分進一步闡述與探討。

Breast cancer brain metastasis (BCBM) is a major cause of morbidity and mortality in breast cancer patients, particularly those with HER2-positive and triple-negative subtypes. Despite advancements in systemic therapies that significantly prolong patient survival, the incidence of BCBM has increased, largely because the blood-brain barrier (BBB) impedes the entry of most therapeutic drugs. Therefore, understanding the molecular mechanisms underlying BCBM is crucial for developing effective and novel therapeutic strategies. This study is divided into two parts. The first part is to investigate the molecular mechanisms of BCBM through the establishment of a mouse model and public database analysis. The other is to investigate the role of GPX4 in BCBM. In the first part, we established a BCBM mouse model and isolated brain-metastasis and spinal-metastasis derived cells. These metastasis-derived cells enhanced migratory and aggressive characteristics, with brain metastasis-derived cells showing a distinct organotropism for the brain. Public database analysis revealed that fatty acid metabolism might be involved in breast cancer brain metastasis. Furthermore, integration of RNA sequencing with pharmacological migration inhibition assays demonstrated that brain metastasis-derived cells upregulated fatty acid synthesis to enhance their migratory ability. In conclusion, these findings suggest that fatty acid synthesis serves as a key metabolic driver of BCBM and represents a potential therapeutic target. For the second part of the thesis, during metastasis, cancer cells elevated intracellular reactive oxygen species (ROS), leading to oxidative stress. Consequently, cancer cells frequently upregulate their antioxidant systems to adapt oxidative stress, facilitating their survival and metastasis. GPX4 reduces lipid peroxides thereby inhibiting ferroptosis. Recent studies indicate that GPX4 plays a key role not only in therapeutic resistance in breast cancer but also in distant metastasis. Our previous work observed that Trastuzumab-resistant breast cancer cells exhibited enhanced migratory capacity and accompanied by increased GPX4 expression. Clinically, trastuzumab-resistant breast cancer patients frequently develop brain metastasis as an early site of disease relapse. Further analysis of public datasets revealed that GPX4 expression and GPX4-associated metabolic pathways were upregulated in BCBM samples. Functionally, suppression of GPX4 affected the migratory behavior of breast cancer cells. However, owing to limitations of current GPX4 overexpression system, the precise regulatory role of GPX4 in BCBM could not be fully elucidated in this study. In conclusion, this study suggests that GPX4 may play a key regulatory role in the development of breast cancer brain metastasis.