Transdifferentiation of Melanoma Cells by the Reprogramming Factors Attenuates Malignant Nature In?Vitro and In?Vivo
Transdifferentiation of Melanoma Cells by the Reprogramming Factors Attenuates Malignant Nature In?Vitro and In?Vivo
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重编程因子对黑色素瘤细胞的转分化可减弱体外和体内的恶性性质
DOI：
10.1016/j.jid.2018.06.179
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发表时间：
2019
影响因子：
6.5
通讯作者：
Sano Shigetoshi
中科院分区：
文献类型：
--
作者：
Takaishi Mikiro;Sano Shigetoshi
预防转移是战胜包括恶性黑色素瘤在内的癌症的最终目的。已经表明，肿瘤细胞的恶性与获得性间充质性质，即上皮-间充质转化（EMT）有关，不仅在上皮来源的癌症中，而且在恶性黑素瘤中（Pearlman et al.，2017年）。因此，抑制恶性黑色素瘤中的间充质性质可能具有治疗益处。我们以前已经建立了重编程因子引入的癌细胞（RIC）从鳞状细胞癌（SCC）线。细胞经历间充质-上皮转化（MET）并在体外和体内减弱其恶性（Takaishi等人，2016年）。在这项研究中，我们调查了黑色素瘤细胞是否是这种情况。以前的报道表明，诱导多能干细胞是通过引入重编程基因从成熟的黑色素细胞和黑色素瘤细胞系中产生的（Bernhardt et al.，2017年，Utikal等人，2009年）。在此，我们将OCT 3/4、SOX 2、KLF 4、c-MYC和LIN 28引入到A2058细胞（一种人黑素瘤细胞系）中，如先前所报道的（Takaishi et al.，2016年）。与SCC RIC类似，黑素瘤RIC显示MET样转分化，细胞形态发生变化（图1a），但它们与诱导多能干细胞完全不同。逆转录PCR分析显示，与亲本细胞相比，RIC中黑素细胞标记基因（MLANA、MITF、TYR、KIT、SOX 9和PAX 3）下调（图1 B，参见补充材料和方法以及在线补充表S1）。RIC中的MITF蛋白水平降低（图1a和c，参见补充材料和方法以及补充表S2）。MITF不仅对黑素细胞中的黑素生成具有重要作用，而且对黑色素瘤细胞的生长和存活也具有重要作用（Wellbrock和Arozarena，2015）。据报道，SOX 2（重编程因子之一）的过表达减少了MeWo细胞中MITF的量（Cimadamore等人，2012年）。MITF基因由几种转录因子激活，包括SOX 10、PAX 3、LEF 1、CREB 1等（Vachtenheim和Ondrusova，2015）。在本研究中，PAX 3在黑素瘤RIC中下调（图1 B）。因此，黑色素瘤RIC中MITF的下调可能是由于SOX 2增加和PAX 3减少。此外，包括SNAI 2和ZEB 2在内的间充质特征分子在黑素瘤RIC中减少（图1 B和c）。这些形态和分子的变化在源自小鼠黑素瘤B16/BL 6的RIC中大量重现（参见在线补充图S1 a-c）。RIC细胞团块的颜色是苍白的，而亲本和模拟对照是黑色的，表明RIC中黑素生成的下调（参见在线补充图S2 a）。总的来说，这些数据表明，将重编程因子强制表达到黑素瘤细胞中导致MET样转分化。
Prevention of metastasis is the ultimate aim to conquer cancers including malignant melanoma. It has been shown that malignancy of tumor cells is linked to acquired mesenchymal properties, namely epithelial-mesenchymal transition (EMT), not only in cancers of epithelial origin but also in malignant melanomas (Pearlman et al., 2017). Thus, suppression of the mesenchymal natures in malignant melanomas could be of therapeutic benefit. We have previously established reprogramming factor-introduced cancer cells (RICs) from squamous cell carcinoma (SCC) lines. The cells underwent mesenchymal-epithelial transition (MET) and attenuated their malignancy in vitro and in vivo (Takaishi et al., 2016). In this study, we investigated whether this was the case with melanoma cells.Previous reports showed that induced pluripotent stem cells were generated from mature melanocytes and melanoma cell lines by introduction of reprogramming genes (Bernhardt et al., 2017, Utikal et al., 2009). Here we introduced OCT3/4, SOX2, KLF4, c-MYC, and LIN28 into A2058 cells, a human melanoma line, as reported previously (Takaishi et al., 2016). Similar to SCC RICs, melanoma RICs showed the MET-like transdifferentiation with changes in cell morphology (Figure 1 a), but they were completely different from induced pluripotent stem cells. Reverse transcription PCR analysis showed down-regulation of melanocyte signature genes (MLANA, MITF, TYR, KIT, SOX9, and PAX3) in RICs compared with parental cells (Figure 1 b, see Supplementary Materials and Methods, and Supplementary Table S1 online). MITF protein level was reduced in the RICs (Figure 1 a and c, see Supplementary Materials and Methods, and Supplementary Table S2). MITF has significant roles not only for melanogenesis in melanocytes but also for growth and survival of melanoma cells (Wellbrock and Arozarena, 2015). It has been reported that overexpression of SOX2, one of the reprogramming factors, reduced the amount of MITF in MeWo cells (Cimadamore et al., 2012). The MITF gene is activated by several transcription factors including SOX10, PAX3, LEF1, CREB1, and others (Vachtenheim and Ondrusova, 2015). In this study, PAX3 was down-regulated in melanoma RICs (Figure 1 b). Therefore, the down-regulation of MITF in melanoma RICs could be due to increased SOX2 and decreased PAX3. Additionally, the mesenchymal signature molecules, including SNAI2 and ZEB2, were reduced in the melanoma RICs (Figure 1 b and c). These changes in the morphology and molecules were largely reproduced in RICs derived from mouse melanomas B16/BL6 (see Supplementary Figure S1 a–c online). The color of RIC cell pellet was pale, whereas the parental and mock control were black, indicating down-regulation of melanogenesis in the RICs (see Supplementary Figure S2 a online). Collectively, the data indicated that forced expression of the reprogramming factors into melanoma cells led to MET-like transdifferentiation.