Transforming Growth Factor β Signaling in Colorectal Cancer Cells With Microsatellite Instability Despite Biallelic Mutations in TGFBR2
Transforming Growth Factor β Signaling in Colorectal Cancer Cells With Microsatellite Instability Despite Biallelic Mutations in TGFBR2
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DOI：
10.1053/j.gastro.2015.02.052
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发表时间：
2015-06-01
期刊：
影响因子：
29.4
通讯作者：
Morreau, Hans
中科院分区：
文献类型：
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作者：
de Miranda, Noel F. C. C.;van Dinther, Maarten;Morreau, Hans
背景与目的：大多数具有高水平微卫星不稳定性（MSI-H）的结直肠癌（CRC）细胞在编码转化生长因子β受体II（TGFBR 2）的基因中的微卫星序列处积累突变。因此，TGF β信号传导被认为在这些肿瘤中是有缺陷的，尽管已经报道具有TGFBR 2突变的CRC细胞对TGF β保持敏感。我们研究了TGF β信号传导如何在MSI-H CRC细胞中继续。方法：我们对32例MSI-H结肠癌组织和6个细胞系（HCT 116、LS 180、LS 411 N、RKO、SW 48和SW 837）的TGFBR 2基因的10-腺嘌呤微卫星序列进行了测序。在所有CRC细胞系中，通过SMAD 2磷酸化和通过使用TGF β-响应性报告构建体检测TGF β信号传导的激活。使用短发夹RNA在CRC细胞中敲低TGFBR 2的转录物。全长和突变形式的TGFBR 2在LS 411 N细胞中表达，其不响应于TGF β，并测量其活性。结果：SMAD 2在大多数MSI-H CRC组织中被磷酸化（在44%的MSI-H肿瘤中强检测，在34%的MSI-H肿瘤中弱检测）。MSI-H细胞中SMAD 2的磷酸化需要TGFBR 2-甚至是编码移码突变的形式。转录和翻译的TGFBR 2与1个核苷酸的缺失在其微卫星序列仍然产生全长TGFBR 2蛋白。然而，蛋白质表达需要保存TGFBR 2微卫星序列;细胞中，该序列被替换为同义的nonmicrosatellite序列不产生功能性TGFBR 2蛋白。结论：尽管在TGFBR 2基因中存在移码突变，但TGF β信号传导在一些MSI-H CRC细胞中保持活跃，因为突变的基因仍然表达功能性蛋白质。在结直肠肿瘤中重新激活TGF β信号转导的策略可能是不必要的，应该评估微卫星不稳定性的其他区域突变的功能影响。
BACKGROUND & AIMS: Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor beta receptor II (TGFBR2). TGF beta signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGF beta. We investigated how TGF beta signaling might continue in MSI-H CRC cells. METHODS: We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGF beta signaling was detected by SMAD2 phosphorylation and through use of a TGF beta-responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGF beta, and their activities were measured. RESULTS: SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2-even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein. CONCLUSION: TGF beta signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGF beta signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated.