Abstract：
BACKGROUND: The presence of distant metastasis at the time of initial diagnosis is a prevalent issue in non-small cell lung cancer (NSCLC), affecting around 30-40 % of the patients. Acidic tumor microenvironment (TME) provides favorable conditions that increase the invasiveness and aggressiveness of NSCLC. The activity of the glycolytic enzyme lactate dehydrogenase (LDHA) increases intracellular lactate accumulation, which creates an acidic TME. However, it is not yet known whether LDHA is involved in enhancing the metastatic potential of NSCLC and if LDHA is a druggable therapeutic target for NSCLC.
OBJECTIVE: We aimed to investigate the molecular mechanisms underlying the enhanced NSCLC metastasis in acidic TME, and to explore whether sulforaphane (SFN), an active compound in Raphani Semen, can serve as a LDHA inhibitor to inhibit NSCLC metastasis in the acidic TME.
METHODS: To mimic the acidic TME, NSCLC cells were cultured in acidic medium (pH 6.6), normal medium (pH 7.4) served as control. Western blotting, bioinformatic analysis, luciferase assay and rescue experiments were used to explore the mechanism and investigate the anti-metastatic effect of SFN both in vitro and in vivo.
RESULTS: Acidic environment increases the expression of LDHA which in turn increases the production of lactic acid that contributes to the acidity of TME. Interestingly, elevated LDHA expression results from increased c-Myc expression, which transactivates LDHA. c-Myc expression is directly regulated by miR-7-5p. In vitro study shows that overexpression of miR-7-5p reverses the acidic pH-enhanced c-Myc and LDHA expressions and also abolishes the enhanced NSCLC cell migration. More importantly, SFN significantly inhibits NSCLC growth and metastasis by reducing lactate production via the miR-7-5p/c-Myc/LDHA axis. Besides, it also regulates the expressions of monocarboxylate transporter 1 (MCT1) and MCT4 that transport lactate across cell membrane.
CONCLUSIONS: The miR-7-5p/c-Myc/LDHA axis is involved in the enhanced NSCLC metastasis in the acidic TME. SFN, a novel LDHA inhibitor, reduces lactate production by targeting the miR-7-5p/c-Myc/LDHA axis, and hence inhibits NSCLC metastasis. Our findings not only delineate a novel mechanism, but also support the clinical translation of SFN as a novel therapeutic agent for treating metastatic NSCLC.
OBJECTIVE: We aimed to investigate the molecular mechanisms underlying the enhanced NSCLC metastasis in acidic TME, and to explore whether sulforaphane (SFN), an active compound in Raphani Semen, can serve as a LDHA inhibitor to inhibit NSCLC metastasis in the acidic TME.
METHODS: To mimic the acidic TME, NSCLC cells were cultured in acidic medium (pH 6.6), normal medium (pH 7.4) served as control. Western blotting, bioinformatic analysis, luciferase assay and rescue experiments were used to explore the mechanism and investigate the anti-metastatic effect of SFN both in vitro and in vivo.
RESULTS: Acidic environment increases the expression of LDHA which in turn increases the production of lactic acid that contributes to the acidity of TME. Interestingly, elevated LDHA expression results from increased c-Myc expression, which transactivates LDHA. c-Myc expression is directly regulated by miR-7-5p. In vitro study shows that overexpression of miR-7-5p reverses the acidic pH-enhanced c-Myc and LDHA expressions and also abolishes the enhanced NSCLC cell migration. More importantly, SFN significantly inhibits NSCLC growth and metastasis by reducing lactate production via the miR-7-5p/c-Myc/LDHA axis. Besides, it also regulates the expressions of monocarboxylate transporter 1 (MCT1) and MCT4 that transport lactate across cell membrane.
CONCLUSIONS: The miR-7-5p/c-Myc/LDHA axis is involved in the enhanced NSCLC metastasis in the acidic TME. SFN, a novel LDHA inhibitor, reduces lactate production by targeting the miR-7-5p/c-Myc/LDHA axis, and hence inhibits NSCLC metastasis. Our findings not only delineate a novel mechanism, but also support the clinical translation of SFN as a novel therapeutic agent for treating metastatic NSCLC.
摘要：
背景：在初始诊断时存在远处转移是非小细胞肺癌（NSCLC）的普遍问题，影响约30-40%的患者。酸性肿瘤微环境（TME）提供了增加NSCLC侵袭性和侵袭性的有利条件。糖酵解酶乳酸脱氢酶（LDHA）的活性增加了细胞内乳酸的积累，产生酸性TME。然而,目前尚不清楚LDHA是否参与增强NSCLC的转移潜能,以及LDHA是否是NSCLC的可药物治疗靶点.
目的：我们旨在研究酸性TME中NSCLC转移增强的分子机制，并探讨萝卜硫素(SFN)是否，Raphani精液中的一种活性化合物，可以作为LDHA抑制剂抑制酸性TME中的NSCLC转移。
方法：为了模拟酸性TME，NSCLC细胞在酸性培养基（pH6.6）中培养，正常培养基（pH7.4）作为对照。西方印迹,生物信息学分析，通过荧光素酶实验和拯救实验来探讨其机制，并研究SFN在体外和体内的抗转移作用。
结果：酸性环境增加了LDHA的表达，进而增加了有助于TME酸度的乳酸的产生。有趣的是,c-Myc表达增加导致LDHA表达升高，它能反式激活LDHA。c-Myc表达直接受miR-7-5p调控。体外研究表明，miR-7-5p的过表达逆转了酸性pH增强的c-Myc和LDHA表达，并消除了增强的NSCLC细胞迁移。更重要的是,SFN通过miR-7-5p/c-Myc/LDHA轴减少乳酸产生显著抑制NSCLC生长和转移。此外,它还调节跨细胞膜转运乳酸的单羧酸转运蛋白1（MCT1）和MCT4的表达。
结论：miR-7-5p/c-Myc/LDHA轴参与酸性TME增强的NSCLC转移。SFN,一种新型LDHA抑制剂，通过靶向miR-7-5p/c-Myc/LDHA轴来减少乳酸的产生，并因此抑制NSCLC转移。我们的发现不仅描绘了一种新的机制，但也支持SFN作为治疗转移性NSCLC的新型治疗剂的临床翻译。
目的：我们旨在研究酸性TME中NSCLC转移增强的分子机制，并探讨萝卜硫素(SFN)是否，Raphani精液中的一种活性化合物，可以作为LDHA抑制剂抑制酸性TME中的NSCLC转移。
方法：为了模拟酸性TME，NSCLC细胞在酸性培养基（pH6.6）中培养，正常培养基（pH7.4）作为对照。西方印迹,生物信息学分析，通过荧光素酶实验和拯救实验来探讨其机制，并研究SFN在体外和体内的抗转移作用。
结果：酸性环境增加了LDHA的表达，进而增加了有助于TME酸度的乳酸的产生。有趣的是,c-Myc表达增加导致LDHA表达升高，它能反式激活LDHA。c-Myc表达直接受miR-7-5p调控。体外研究表明，miR-7-5p的过表达逆转了酸性pH增强的c-Myc和LDHA表达，并消除了增强的NSCLC细胞迁移。更重要的是,SFN通过miR-7-5p/c-Myc/LDHA轴减少乳酸产生显著抑制NSCLC生长和转移。此外,它还调节跨细胞膜转运乳酸的单羧酸转运蛋白1（MCT1）和MCT4的表达。
结论：miR-7-5p/c-Myc/LDHA轴参与酸性TME增强的NSCLC转移。SFN,一种新型LDHA抑制剂，通过靶向miR-7-5p/c-Myc/LDHA轴来减少乳酸的产生，并因此抑制NSCLC转移。我们的发现不仅描绘了一种新的机制，但也支持SFN作为治疗转移性NSCLC的新型治疗剂的临床翻译。
