KLF4 regulates adult lung tumor-initiating cells and represses K-Ras-mediated lung cancer

Abstract

Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. To identify novel factors that contribute to lung cancer pathogenesis, we analyzed a lung cancer database from The Cancer Genome Atlas and found that Krüppel-like Factor 4 (KLF4) expression is significantly lower in patients' lung cancer tissue than in normal lung tissue. In addition, we identified seven missense mutations in the KLF4 gene. KLF4 is a transcription factor that regulates cell proliferation and differentiation as well as the self-renewal of stem cells. To understand the role of KLF4 in the lung, we generated a tamoxifen-induced Klf4 knockout mouse model. We found that KLF4 inhibits lung cancer cell growth and that depletion of Klf4 altered the differentiation pattern in the developing lung. To understand how KLF4 functions during lung tumorigenesis, we generated the K-ras(LSL-G12D/+);Klf4(fl/fl) mouse model, and we used adenovirus-expressed Cre to induce K-ras activation and Klf4 depletion in the lung. Although Klf4 deletion alone or K-ras mutation alone can trigger lung tumor formation, Klf4 deletion combined with K-ras mutation significantly enhanced lung tumor formation. We also found that Klf4 deletion in conjunction with K-ras activation caused lung inflammation. To understand the mechanism whereby KLF4 is regulated during lung tumorigenesis, we analyzed KLF4 promoter methylation and the profiles of epigenetic factors. We found that Class I histone deacetylases (HDACs) are overexpressed in lung cancer and that HDAC inhibitors induced expression of KLF4 and inhibited proliferation of lung cancer cells, suggesting that KLF4 is probably repressed by histone acetylation and that HDACs are valuable drug targets for lung cancer treatment.

Figure 1

Klf4 is a putative tumor suppressor for lung cancer. (a) Schematic diagram and table list of mutational sites in the KLF4 gene domains. ZF, zinc finger domain; ‘*', Mutational sites, details of which are explained in the table below. (b) KLF4 expression in normal and lung tumor tissues. The P value was calculated based on a linear mixed model. (c) Top: Morphology of lung cancer cells with ectopic KLF4 expression. Middle and bottom: growth curve of lung cancer cell lines A549 and H460. ‘Control', vector-virus-infected cells. (d and e) Western blot (d), semi-quantitative RT-PCR and quantification (e) of expression of KLF4 target genes in lung cancer cell lines with the effect of overexpression of KLF4. Data are represented as mean±S.D

Figure 2

Klf4 ablation altered the differentiation pattern in mouse lung. (a) Genetic model of the Rosa-Cre⁺;Klf4^fl/fl mouse. LoxP elements allow Cre-induced Klf4 deletion (top); and Rosa26 drives CreER^T2 expression in mouse lung (bottom). (b–d) Immunofluorescent staining for KLF4 (red) in wild-type mouse lung (b) and in Klf4-deleted mouse lung (c). (d) Statistical analysis of KLF4-positive cells in mouse lung. Data are represented as mean±S.D. (e–g) Immunofluorescent staining for SP-C in wild-type (e) and Klf4-deleted (f) mouse lung. (g) Statistical analysis of SP-C-positive cells in mouse lung. Data are represented as mean±S.D. Scale bars for (b and c): 30 μm. Scale bars for (e and f): 100 μm

Figure 3

Establishing the genetic model of the K-ras^LSL-G12D/+;Klf4^fl/fl mouse. (a) LoxP elements allow Cre-induced Klf4 deletion or G12D mutation-mediated K-ras activation. (b) H&E staining and KLF4 immunofluorescent staining in normal tissue from wild-type mice (top) and in tumor tissue from K-ras^LSL-G12D/+;Klf4^fl/fl mice (bottom) tissue in the lung, indicating loss of Klf4 expression in lung tumor. Scale bars: 100 μm

Figure 4

Klf4 deletion facilitates lung tumor formation and progression. (a) H&E staining of the lung tissue from wild-type mice (control), the K-ras^LSL-G12D/+mice, Klf4^fl/fl mice and K-ras^LSL-G12D/+;Klf4^fl/fl mice by 8 weeks post infection. Scale bars from left to right: 2 mm and 100 μm. (b) IHC staining of SP-C (top) and immunofluorescent staining of KLF4 (red, bottom) showing that SP-C tends to increase in tumor tissue from K-ras^LSL-G12D/+;Klf4^fl/fl mice than in normal tissue from wild-type mice. Scale bars: 100 μm. (c) Statistical analysis of the percentage of tumor area by 8 weeks post infection (based on H&E staining). Data are represented as mean±S.D

Figure 5

Multiple mechanisms of KLF4 downregulation in lung cancer. (a) Correlation between KLF4 expression and methylation in lung adenocarcinoma patients. Pearson's correlation coefficient was −0.03 (P=0.8, 95% confidence interval −0.27 to 0.21). (b) HDAC expression in normal lung and lung tumor tissues from TCGA lung adenocarcinoma samples. P values were calculated based on linear mixed models. (c) Semi-quantitative RT-PCR (left) and real-time PCR (right) testing expression of KLF4 and target gene p21^Cip1/WAF1 in HEK293T and A549 cell lines when treated with HDAC inhibitors. Data are represented as mean±S.D. (d) Growth curve of A549 and H460 cell lines when treated with HDAC inhibitors