Low expression of the E3 ubiquitin ligase CBL confers chemoresistance in human pancreatic cancer and is targeted by epidermal growth factor receptor inhibition.
Low expression of the E3 ubiquitin ligase CBL confers chemoresistance in human pancreatic cancer and is targeted by epidermal growth factor receptor inhibition.
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DOI：
10.1158/1078-0432.ccr-14-0610
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Donahue TR
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文献类型：
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作者：
Kadera BE;Toste PA;Wu N;Li L;Nguyen AH;Dawson DW;Donahue TR
CBL是一种泛素连接酶，在60%的人胰腺导管腺癌(PDAC)中表达降低，并与总生存期缩短有关。我们试图确定低CBL对临床上更具侵袭性的PDAC有多大直接影响。对人PDAC进行CBL、pEGFR和EGFR染色。在PDAC细胞(PANC-1、L3.6pl、ASPC-1)中通过瞬时转染(SiRNA)或稳定敲除(ShRNA)建立CBL-LOW模型。用四甲基偶氮唑盐比色法和流式细胞仪检测细胞存活率和细胞凋亡率。免疫印迹和磷酸化受体酪氨酸激酶(PRTK)阵列用于检测信号转导。NOD-SCID-IL2Rγ缺陷型小鼠双侧皮下接种PDAC或PDACCBL-Low细胞，给予吉西他滨+厄洛替尼治疗4周≥。15例肿瘤中有12例CBL与pEGFR蛋白表达呈负相关。CBL基因敲除通过上调pEGFR(Y1068)、PERK和PAKT增加PDAC对吉西他滨和5-FU的耐药性。PDACCBL-Low细胞的PRTK阵列显示了额外的激活的酪氨酸激酶，但都比EGFR的幅度低得多。EGFR抑制剂erlotinib在体外和体内均可消除低CBL引起的化疗耐药增加。厄洛替尼+吉西他滨处理的PDACCBL-Low细胞在PARP、Caspase3和Annexin V/PI被切割后出现更多的凋亡。低CBL通过应激诱导的EGFR激活导致PDAC的化疗耐药，这种激活可被EGFR抑制有效地消除。这些结果提示泛素化失调是PDAC中EGFR过度激活的关键机制，低CBL可能定义了PDAC肿瘤可能对厄洛替尼治疗有反应。
Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDACs) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC. Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2Rγnull mice were subcutaneously implanted with PDAC or PDACCBL-low cells on opposite flanks and treated with gemcitabine ± erlotinib for ≥4 weeks. There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-FU by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDACCBL-low cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib + gemcitabine treated PDACCBL-low cells exhibited greater apoptosis by cleaved PARP, Caspase 3 and Annexin V/PI. Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment.