Expression profiling by high throughput sequencing
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.
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.