Smyd1 And G6Pd Modulation Are Critical Events For Mir-206-Mediated Differentiation Of Rhabdomyosarcoma

ABSTRACTRhadomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS cells resemble fetal myoblasts but are unable to complete myogenic differentiation. In previous work we showed that miR-206, which is low in RMS, when induced in RMS cells promotes the resumption of differentiation by modulating more than 700 genes. To better define the pathways involved in the conversion of RMS cells into their differentiated counterpart, we focused on two miR-206 effectors emerged from the microarray analysis, SMYD1 and G6PD. SMYD1, one of the most highly upregulated genes, is a H3K4 histone methyltransferase. Here we show that SMYD1 silencing does not interfere with the proliferative block or with the loss anchorage independence imposed by miR-206, but severely impairs differentiation of ERMS, ARMS, and myogenic cells. Thus SMYD1 is essential for the activation of muscle genes. Conversely, among the downregulated genes, we found G6PD, the enzyme catalyzing the rate-limiting step of the pentose phosphate shunt. In this work, we confirmed that G6PD is a direct target of miR-206. Moreover, we showed that G6PD silencing in ERMS cells impairs proliferation and soft agar growth. However, G6PD overexpression does not interfere with the pro-differentiating effect of miR-206, suggesting that G6PD downmodulation contributes to - but is not an absolute requirement for - the tumor suppressive potential of miR-206. Targeting cancer metabolism may enhance differentiation. However, therapeutic inhibition of G6PD is encumbered by side effects. As an alternative, we used DCA in combination with miR-206 to increase the flux of pyruvate into the mitochondrion by reactivating PDH. DCA enhanced the inhibition of RMS cell growth induced by miR-206, and sustained it upon miR-206 de-induction. Altogether these results link miR-206 to epigenetic and metabolic reprogramming, and suggest that it may be worth combining differentiation-inducing with metabolism-directed approaches.

摘要 横纹肌肉瘤（Rhabdomyosarcoma, RMS）是儿童最常见的软组织肉瘤。RMS细胞类似胎儿成肌细胞，但无法完成肌源性分化。既往研究表明，在RMS中低表达的miR-206，当其在RMS细胞中被诱导表达时，可通过调控超过700个基因促进分化重启。为更清晰地界定参与RMS细胞向分化型细胞转化的通路，本研究聚焦于基因芯片分析筛选出的两个miR-206效应因子：SMYD1与G6PD。SMYD1是上调幅度最高的基因之一，属于H3K4组蛋白甲基转移酶。本研究证实，沉默SMYD1不会干扰miR-206介导的增殖阻滞与锚定非依赖性生长丧失，但会严重损害胚胎型横纹肌肉瘤（ERMS）、腺泡型横纹肌肉瘤（ARMS）以及肌源细胞的分化能力，表明SMYD1对肌肉基因的激活至关重要。反之，在下调基因中，我们发现G6PD——一种催化磷酸戊糖分流途径限速步骤的酶。本研究验证了G6PD是miR-206的直接靶标。进一步实验显示，在ERMS细胞中沉默G6PD可抑制细胞增殖与软琼脂生长能力。然而，过表达G6PD并不会干扰miR-206的促分化效应，提示G6PD的下调虽有助于miR-206发挥肿瘤抑制潜能，但并非其绝对必需条件。靶向肿瘤代谢或可增强分化治疗效果，但直接抑制G6PD易受副作用限制。作为替代方案，我们采用二氯乙酸盐（Dichloroacetate, DCA）联合miR-206，通过重新激活丙酮酸脱氢酶（PDH）以增加丙酮酸向线粒体的通量。DCA可增强miR-206对RMS细胞生长的抑制作用，并在miR-206表达被抑制后仍维持该抑制效果。综上，本研究结果将miR-206与表观遗传重编程及代谢重编程建立关联，提示将诱导分化疗法与代谢靶向疗法联合应用具有潜在研究价值。