Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.
PLoS One. 2020 Jun 3;15(6):e0233887. doi: 10.1371/journal.pone.0233887. eCollection 2020.
Increased cytoplasmic lipid droplets (LDs) and elevated AKT/mTOR signaling are characteristics of clear cell renal cell carcinoma (ccRCC). Lysophosphatidic acid (LPA), a potent lipid mitogen generated via autotaxin (elevated in ccRCC), can modulate tumor progression but its role in altering chemotherapeutic sensitivity to mTOR inhibitors is unclear and thus is the focus of the studies presented herein. Using malignant (A-498, 769-P and 786-O) and normal immortalized kidney (HK-2) cell lines, we investigated their cellular responsiveness to Temsirolimus (TEMS, mTOR inhibitor) in the absence or presence of LPA by monitoring alterations in AKT/mTOR pathway mediators (via western blotting), LDs (using LipidTOX and real-time PCR to assess transcript changes in modulators of LD biogenesis/turnover), mitochondrial networks (via immunofluorescence staining for TOM20 and TOM70), as well as cellular viability. We identified that TEMS reduced cellular viability in all renal cell lines, with increased sensitivity in the presence of an autophagy inhibitor. TEMS also altered activation of AKT/mTOR pathway mediators, abundance of LDs, and fragmentation of mitochondrial networks. We observed that these effects were antagonized by LPA. In HK-2 cells, LPA markedly increased LD size and abundance, coinciding with phospho-MAPK and phospho-S6 activation, increased diacylglycerol O-acetyltransferase 2 (DGAT2) mRNA (which produces triacylglycerides), and survival. Inhibiting MAPK partially antagonized LPA-induced LD changes. Collectively, we have identified that LPA can reverse the effects of TEMS by increasing LDs in a MAPK-dependent manner; these results suggest that LPA may contribute to the pathogenesis and chemotherapeutic resistance of ccRCC.
细胞质脂滴（LDs）增加和 AKT/mTOR 信号升高是透明细胞肾细胞癌（ccRCC）的特征。溶血磷脂酸（LPA）是一种通过自分泌酶（在 ccRCC 中升高）产生的有效的脂质有丝分裂原，可调节肿瘤进展，但它在改变 mTOR 抑制剂对化疗的敏感性方面的作用尚不清楚，因此是本文研究的重点。使用恶性（A-498、769-P 和 786-O）和正常永生化肾（HK-2）细胞系，我们通过监测 AKT/mTOR 通路调节剂的变化（通过 Western blot），LDs（使用 LipidTOX 和实时 PCR 评估 LD 生物发生/周转调节剂的转录变化），线粒体网络（通过免疫荧光染色 TOM20 和 TOM70），以及细胞活力，研究了它们在不存在或存在 LPA 的情况下对 Temsirolimus（mTOR 抑制剂）的细胞反应。我们发现 TEMS 降低了所有肾细胞系的细胞活力，在自噬抑制剂存在下敏感性增加。TEMS 还改变了 AKT/mTOR 通路调节剂的激活、LD 的丰度和线粒体网络的碎片化。我们观察到这些影响被 LPA 拮抗。在 HK-2 细胞中，LPA 明显增加了 LD 的大小和丰度，同时磷酸化-MAPK 和磷酸化-S6 激活、二酰基甘油 O-乙酰转移酶 2（DGAT2）mRNA（产生三酰基甘油）增加和存活。抑制 MAPK 部分拮抗了 LPA 诱导的 LD 变化。总的来说，我们已经确定 LPA 可以通过 MAPK 依赖性方式增加 LDs 来逆转 TEMS 的作用；这些结果表明 LPA 可能有助于 ccRCC 的发病机制和化疗耐药性。
