CHUK/IKKalpha loss in lung epithelial cells enhances NSCLC growth associated with HIF up-regulation

During the natural progression of Non-small-cell lung cancer (NSCLC), tumor cells evolve progressively through the accumulation of mutations, some of which involve oncogenic (K-RAS, EGFR) or tumor suppressor (P53) genes. These mutations alter cell signaling pathways to promote tumor growth and survival in the tumor microenvironment. Herein we show that CHUK (IKK-alpha) acts as a prominent tumor suppressor in two independent NSCLC models. Using a novel transgenic mouse strain, where IKK-alpha gene is ablated using tamoxifen in alveolar type II epithelial cells, loss of IKK-alpha increased the number and size of lung tumors in response to the chemical carcinogen urethane. Furthermore, IKK-alpha knock-down in three human NSCLC lines (showing independent K-Ras or p53 mutations and status) promoted their growth as xenografts in immunocompromised mice. Transcriptomic and functional studies of IKK-alpha knock-down tumors, relative to their wild type counterparts, suggested that the loss of IKK-alpha promoted the activation of HIF-1 alpha and higher tumor cell growth and survival under hypoxic conditions. Together, these results suggest that IKK-alpha acts as a tumor suppressor by suppressing the activity of HIF-1 alpha and tumor cell growth/survival under hypoxic conditions. Overall design: Tumor RNA samples were sequenced in quadruplicate RNAseq was performed in Human and mouse WT and CHUK-Knockdown NSCLC tumors or tumor xenografts. For human tumor xenografts, three independent human Wt or CHUK-knockdown NSCLC lines (H1437, A549 and H1299), which differ either in their p53 or K-Ras functional status were obtained from the Sanger (UK) cell line database, which provides the status of their K-Ras and p53, EGFR, ARF and p16 alleles. These three human NSCLC lines (IKK WT and IKK KD) and H1299 and A549 (p52WT and p52KD) were grown as tumor xenografts by subcutaneous transplantation into either side (Left side for WT and right side for KD cells) of immune compromised 5 week old NSG (NOD-SCID-IL2Rgamma) mice (2 x 10^6 cells per injection in 200 µl of PBS). Mice were sacrificed 3 weeks and part of the dissected tumors was immediately snap frozen in liquid nitrogen prior to RNA extraction with the TRI-Reagent (Merck) protocol, according to the manufacturer's instructions. For mouse tumors, Mice with IKKa allele containing LoxP recombination sites and a ROSA-fLacz Cre-inducible LacZ reporter gene were crossed with Sftpc-CreERT2 mice (Rock et al., 2011), which harbor a tamoxifen-inducible CreERT2 recombinase under the control of the Sftpc gene promoter that is only active in alveolar type II (AT-II) lung epithelial cells. To induce either IKK or IKK deletion in AT-II lung epithelial cells six week-old male or female mice were injected intraperitoneally (i.p.) for 5 consecutive days with 2mg tamoxifen (Sigma) dissolved in corn oil. To induce NSCLCs, one week after tamoxifen administration mice received weekly i.p. urethane injections (1g/kg) (Sigma) dissolved in PBS for 12 consecutive weeks, and were sacrificed 6 months after the first urethane injection.

非小细胞肺癌（Non-small-cell lung cancer, NSCLC）的自然进程中，肿瘤细胞会随着突变的累积逐步演化，部分突变涉及致癌基因（K-RAS、EGFR）或抑癌基因（P53）。这些突变会改变细胞信号通路，从而在肿瘤微环境中促进肿瘤的生长与存活。
本文证实，CHUK（IKK-alpha）在两种独立的非小细胞肺癌模型中发挥显著的抑癌作用。研究使用一种新型转基因小鼠品系，该品系可在肺泡II型上皮细胞中通过他莫昔芬诱导敲除IKK-α基因，结果显示，IKK-α缺失会增加化学致癌物氨基甲酸乙酯（urethane）诱导的肺部肿瘤数量并增大肿瘤体积。
此外，在三株携带不同K-Ras或p53突变状态的人非小细胞肺癌细胞系中敲低IKK-α，可促进这些细胞在免疫缺陷小鼠体内形成异种移植瘤。
对IKK-α敲低肿瘤与其野生型对照进行转录组学与功能学研究后发现，IKK-α缺失会激活缺氧诱导因子-1α（HIF-1 alpha），并在缺氧条件下增强肿瘤细胞的生长与存活能力。
综上，上述结果表明，IKK-α通过抑制HIF-1α的活性，在缺氧条件下遏制肿瘤细胞生长与存活，从而发挥抑癌作用。
实验整体设计：本研究对人源及小鼠源野生型与CHUK（IKK-alpha）敲低的非小细胞肺癌肿瘤或肿瘤异种移植瘤进行了四次重复的RNA测序（RNAseq）。
对于人源肿瘤异种移植瘤，我们从英国桑格（Sanger）细胞系数据库获取了三株独立的野生型或CHUK敲低的人非小细胞肺癌细胞系（H1437、A549与H1299），这些细胞系在p53或K-Ras功能状态上存在差异，该数据库同时提供了其K-Ras、p53、EGFR、ARF及p16等位基因的状态。将这三株人非小细胞肺癌细胞系（含IKK野生型与IKK敲低型）以及H1299与A549（含p52野生型与p52敲低型）通过皮下移植至5周龄免疫缺陷NSG（NOD-SCID-IL2Rgamma）小鼠的两侧背部（左侧接种野生型细胞，右侧接种敲低型细胞），每处注射2×10^6个细胞，悬浮于200 μL磷酸盐缓冲液（PBS）中。接种3周后处死小鼠，剥离肿瘤后立即将部分组织置于液氮中快速冷冻，随后按照制造商说明书，采用TRI-Reagent（Merck）试剂盒提取总RNA。
对于小鼠肿瘤模型，我们将携带LoxP重组位点IKKα等位基因及ROSA-fLacz Cre诱导型LacZ报告基因的小鼠，与Sftpc-CreERT2小鼠（Rock等，2011）杂交。后者在仅活跃于肺泡II型（AT-II）肺上皮细胞的Sftpc基因启动子调控下，表达他莫昔芬诱导型CreERT2重组酶。为在AT-II肺上皮细胞中诱导敲除IKK-α，对6周龄的雌雄小鼠连续5天腹腔注射2 mg溶于玉米油的他莫昔芬（Sigma）。为诱导非小细胞肺癌，在他莫昔芬给药1周后，每周向小鼠腹腔注射1 g/kg溶于磷酸盐缓冲液的氨基甲酸乙酯（Sigma），连续注射12周，于首次注射氨基甲酸乙酯6个月后处死小鼠。