背景:溶血磷脂酸受体3(LPAR3)与Gαi/o和Gα11/q信号传导偶联。以前,我们报道LPAR3在致癌物诱导的转化细胞中高度甲基化.这里,我们证明LPAR3表现出恶性转化活性,尽管在转化细胞中被下调。
方法:使用CRISPR/Cas9系统建立了NIH3T3和Bhas42细胞中的LPAR3敲除(KO)。进行RT-PCR和DNA测序以确认LPAR3的KO。通过WST-1测定进一步检查LPAR3KO的细胞效应,免疫印迹分析,transwell迁移试验,集落形成试验,伤口擦伤,体外细胞转化试验,和自噬测定。
结果:在具有LPAR3下调的v-H-ras转化细胞(Ras-NIH3T3)中,LPAR3的异位表达显着增强了迁移。特别是,Bhas42(v-Ha-ras转染的Balb/c3T3)和NIH3T3细胞中的LPAR3敲除(KO)引起细胞存活率降低,转换后的焦点,和殖民地的形成。LPAR3KO通过破坏自噬体与溶酶体的融合,导致LC3-II和自噬体的大量积累和自噬通量的抑制。相反,LPAR3下调后,Ras-NIH3T3细胞的自溶酶体成熟正常。在Ras-NIH3T3细胞中,MEK和ERK的基础磷酸化明显增加,而在LPAR3KO细胞中显著降低,提示MEK信号增强参与Ras-NIH3T3细胞的自噬体-溶酶体融合。
结论:在Ras转化细胞中,LPAR3的反常下调通过自噬诱导与MEK激活发挥协同的促肿瘤活性。我们的发现对癌症化疗方法的发展具有重要意义。
BACKGROUND: Lysophosphatidic acid receptor 3 (LPAR3) is coupled to Gαi/o and Gα11/q signaling. Previously, we reported that LPAR3 is highly methylated in carcinogen-induced transformed cells. Here, we demonstrate that LPAR3 exhibits malignant transforming activities, despite being downregulated in transformed cells.
METHODS: The LPAR3 knockout (KO) in NIH 3 T3 and Bhas 42 cells was established using the CRISPR/Cas9 system. Both RT-PCR and DNA sequencing were performed to confirm the KO of LPAR3. The cellular effects of LPAR3 KO were further examined by WST-1 assay, immunoblotting analysis, transwell migration assay, colony formation assay, wound scratch assday, in vitro cell transformation assay, and autophagy assay.
RESULTS: In v-H-ras-transformed cells (Ras-NIH 3 T3) with LPAR3 downregulation, ectopic expression of LPAR3 significantly enhanced the migration. In particular, LPAR3 knockout (KO) in Bhas 42 (v-Ha-ras transfected Balb/c 3 T3) and NIH 3 T3 cells caused a decrease in cell survival, transformed foci, and colony formation. LPAR3 KO led to the robust accumulation of LC3-II and autophagosomes and inhibition of autophagic flux by disrupting autophagosome fusion with lysosome. Conversely, autolysosome maturation proceeded normally in Ras-NIH 3 T3 cells upon LPAR3 downregulation. Basal phosphorylation of MEK and ERK markedly increased in Ras-NIH 3 T3 cells, whereas being significantly lower in LPAR3 KO cells, suggesting that increased MEK signaling is involved in autophagosome-lysosome fusion in Ras-NIH 3 T3 cells.
CONCLUSIONS: Paradoxical downregulation of LPAR3 exerts cooperative tumor-promoting activity with MEK activation through autophagy induction in Ras-transformed cells. Our findings have implications for the development of cancer chemotherapeutic approaches.