Molecular analysis of a multistep lung cancer model induced by chronic inflammation reveals epigenetic regulation of p16 and activation of the DNA damage response pathway

Abstract

The molecular hallmarks of inflammation-mediated lung carcinogenesis have not been fully clarified, mainly due to the scarcity of appropriate animal models. We have used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers and genetic alterations. We analyzed markers of DNA damage response (DDR), proliferative stress, and telomeric stress: gamma-H2AX, p16, p53, and TERT. Lung cancer-related epigenetic and genetic alterations, including promoter hypermethylation status of p16(CDKN2A), APC, CDH13, Rassf1, and Nore1A, as well as mutations of Tp53, epidermal growth factor receptor, K-ras, N-ras, and c-H-ras, have been also studied. Our results showed DDR pathway activation in preneoplastic lesions, in association with inducible nitric oxide synthase and p53 induction. p16 was also induced in early tumorigenic progression and was inactivated in bronchiolar dysplasias and tumors. Remarkably, lack of mutations of Ras and epidermal growth factor receptor, and a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16(CDKN2A), CDH13, and APC, but not in Rassf1 and Nore1A, were clearly observed. These data suggest the existence of a specific molecular signature of inflammation-driven lung carcinogenesis that shares some, but not all, of the molecular landmarks of chemically induced lung cancer.

Keywords: DNA damage response; animal model; inflammation; lung cancer; preneoplastic lesions.

Figure 1

Oxidative stress and DDR in silica-induced lesions measured by immunohistochemistry in serial consecutive sections. The figures in this plate show the activation of the iNOS/p53/γ-H2AX pathway in the multistep progression from normal tissue to tumors. Low levels of iNOS and p53 proteins were found in morphologically normal bronchial epithelial cells, whereas γ-H2AX was completely absent (A). In normal bronchioli, iNOS expression was also found in smooth muscle cells (arrow; A). A clear increase in the coexpression of iNOS/p53/γ-H2AX was observed from hyperplastic (B and C) to dysplastic (D) bronchiolar cells. In tumors, colocalization was still present in some areas, although γ-H2AX levels were reduced compared to hyperplastic and advanced preneoplastic tissues (E and F). Counterstaining by Harris hematoxylin. Original magnifications, × 420 (C, E, and F); × 630 (A, B, and D).

Figure 2

p53, p16, and TERT nuclear expression by immunohistochemistry in the multistep progress to lung cancer. (A) Normal lung epithelial cells showed low levels of nuclear protein expression for p53, p16, and TERT. (B and C) Hyperplastic bronchiolar (B) and alveolar (C) cells showed a significant increase for p53, p16, and TERT nuclear immunostaining. (D) DB at 10 to 12 months showed a higher percentage of positive nuclear cells for p53 and TERT. However, p16 was significantly decreased in these advanced preneoplastic lesions. (E and F) p53 and TERT overexpression was commonly observed in AC (E) and SCC (F), whereas p16 overexpression was observed in SCC (F) and in a subset of more fibrotic AC. Loss of p16 protein expression (< 25% positive nuclei) was detected in 44% of AC (p16; E). Counterstaining by Harris hematoxylin. Original magnifications, × 420 (p53-TERT; B) (p16; D, E, and F); × 560 (p16; B); × 700 (A and C) (p53-TERT; D).

Figure 3

Differential expression for p53, p16, and TERT between different stages of cancer progression. Graphs show the mean percentage of nuclear protein expression and its 95% confidence interval for p53, p16, and TERT immunostaining in all the histologic structures studied. TUMOR, AC and SCC.

Figure 4

Mutational analysis for Ras (n = 23), Tp53, and EGFR (n = 32) in tumor DNA extracts. (A) No mutations were found in K-ras, N-ras, c-H-ras, or EGFR in any of the silica-induced tumors analyzed after microdissection. For Tp53, five mutations were found in three tumors of 32 cases analyzed (9%). (B) Electropherogram of the DNA sequence of the tumor (mt) versus the corresponding adjacent normal tissue (wt) showing the mutations found in exon 5 in AC and exon 6 in SCC.

Figure 5

DNA global hypomethylation analysis in controls (n = 6); preneoplasia-containing lungs on months 3 to 4 (n = 5), months 6 to 8 (n = 5), and months and 10 to 12 (n = 5) after treatment; and in tumors (n = 9). A clear global genomic hypomethylation with an average loss of 25% in mC DNA content only occurs in tumors. Results are expressed as mean ± S.D. Fisher's exact test was applied, and differences were considered significant when P < .05.

Figure 6

Bisulfite genomic sequencing of the p16(CDKN2A), H-cadherin (CDH13), and APC gene promoters in normal tissues (n = 6); preneoplasia-containing tissues on months 3 to 4 (n = 5), months 6 to 8 (n = 5), and months 10 to 12 (n = 5); and nine representative lung tumors. Vertical bars show the distribution of CpG islands at p16, H-cadherin, and APC. The vertical arrow indicates the transcriptional start point. Black dots indicate methylated CpG islands; white dots indicate unmethylated CpG islands. The position of the bisulfite sequencing primers is represented with white horizontal arrows. We observed H-cadherin promoter hypermethylation in seven of nine silica-induced tumors (T1, T2, T3, T4, T5, T6, and T9), and APC promoter hypermethylation in five of nine tumors (T2, T3, T4, T5, and T9). Hypermethylation in the p16 promoter was also an important event in six of nine tumors studied (T1, T2, T3, T5, T8, and T9).

Figure 7

Summary of the molecular events found in the silica-induced lung carcinogenesis model. DDR, p16, TERT, and p53 overexpressions are observed from early preneoplastic lesions. p16 inactivation occurs in dysplastic bronchiolar lesions and tumors. No mutations were observed in tumors, with the only exception of Tp53 mutations with a very low frequency. Epigenetic alterations were found in tumors in the tumor-suppressor genes p16(CDKN2A), APC, and CDH13.