Mutually exclusive inactivation of DMP1 and ARF/p53 in lung cancer

Abstract

Dmp1 (Dmtf1) is activated by oncogenic Ras-Raf signaling and induces cell-cycle arrest in an Arf, p53-dependent fashion. The survival of K-ras(LA) mice was shortened by approximately 15 weeks in both Dmp1(+/-) and Dmp1(-/-) backgrounds, the lung tumors of which showed significantly decreased frequency of p53 mutations compared to Dmp1(+/+). Approximately 40% of K-ras(LA) lung tumors from Dmp1(+/+) mice lost one allele of the Dmp1 gene, suggesting the primary involvement of Dmp1 in K-ras-induced tumorigenesis. Loss of heterozygosity (LOH) of the hDMP1 gene was detectable in approximately 35% of human lung carcinomas, which was found in mutually exclusive fashion with LOH of INK4a/ARF or that of P53. Thus, DMP1 is a pivotal tumor suppressor for both human and murine lung cancers.

Figure 1. Tumor-free survival in cohorts of (A) Dmp1^+/+, Dmpl^+/−, and Dmp1^−/−; K-ras^LA2/+ mice and (B) Dmp1^+/+, Dmp1^+/−, and Dmp1^−/−; K-ras^LA1/+ mice

A: Statistically significant difference in survival was found between Dmp1^+/+ vs. Dmp1^+/−; K-ras^LA2/+ (P< 0.005) and Dmp1^+/+ vs. Dmp1^−/−; K-ras^LA2/+ mice (P< 0.005). There were no significant differences in survival between Dmp1^+/− vs. Dmp1^−/−; K-ras^LA2/+ (P= 0.38). B: Significant difference of survival was found between Dmp1^+/+ vs. Dmp1^−/−; K-ras^LA1/+ (P< 0.001) and Dmp1^+/+ vs. Dmp1^+/−; K-ras^LA1/+ mice (P= 0.001), but not between Dmp1^+/− vs. Dmp1^−/−; K-ras^LA1/+ (P= 0.13). C: Retention of the Dmp1 wild-type allele in lung tumors from Dmp1^+/−; K-ras^LA/+ mice as examined by genomic DNA PCR. D: Expression of the Dmp1 mRNA in lung tumors from Dmp1^+/+, Dmp1^+/−, and Dmp1^−/−; K-ras^LA/+ mice. Real-time PCR was conducted to quantitate the Dmp1 mRNA expression in K-ras^LA/+ lung tumors. Black bars show the average level of Dmp1 expression in the lung of each genotype. Gray bars are the samples that showed higher level of Dmp1 expression in K-ras^LA lung tumors than in their normal lung controls. The means Dmp1^+/− SD for three experiments are shown. E: Well-differentiated lung adenocarcinoma found in a Dmp1^+/−; K-ras^LA1/+ mouse. H&E stain. F: Detection of the Dmp1 protein in the nuclei of Dmp1^+/−; K-ras^LA1/+ mouse tumor cells with the Dmp1-specific antibody, RAX. G: Negative staining of the lung tumor section of Dmp1^−/−; K-ras^LA1/+ mouse with RAX antibody. Scale bar in E, F, and G is 100 µm.

Figure 2. Pathological examination of tumors found in K-ras^LA/+ mice

A: Multiple lung adenomas and adenocarcinomas in a Dmp1^+/+; K-ras^LA1/+ mouse (42-week-old). B: Advanced lung adenocarcinoma in a Dmp1^+/−; K-ras^LA1/+ mouse (39-week-old). C: Disseminated lung adenocarcinoma in a Dmp1^−/−; K-ras^LA1/+ mouse (38-week-old). D: Leg metastasis of lung adenocarcinoma in a Dmp1^+/−; K-ras^LA1/+ mouse (40-week-old). E: Cholangiocarcinoma of the liver in a Dmp1^+/−; K-ras^LA1/+ mouse (40-week-old). F: Lung nodule number and size (mean +/− SEM) in K-ras^LA mice. Ranges were compared with unpaired Student’s t-tests. G: Well-differentiated adenocarcinoma found in a Dmp1^+/+; K-ras^LA2/+ mouse. H: Poorly differentiated adenocarcinoma in a Dmp1^+/−; K-ras^LA2/+ mouse. The tumor cells are very pleomorphic and are invading into blood vessels. I: Intrabronchial invasion of a Dmp1^−/−; K-ras^LA2/+ lung carcinoma. J: Liver metastasis of lung adenocarcinoma in a Dmp1^+/−; K-ras^LA2/+ mouse. Scale bar in G, H, I, and J is 100 µm.

Figure 3

Analysis of the Arf-Mdm2-p53 pathway in K-ras^LA/+ lung tumors. A: Western blotting of lung tumors for p53, Mdm2, p19^Arf, and p16^Ink4a. Tumor cells were resected from the center of well-circumscribed lung carcinomas under the light microscope and proteins were extracted. The control cell lysates were obtained from spontaneously immortalized p53-mutant MEFs for p53, p19^Arf, p16^Ink4a, and Actin; dm3T3 cells for Mdm2. The p53 mutations were detected in 4 of 11 Dmp1^+/+; K-ras^LA/+ mice (36 %) while they were not found in the Dmp1^+/− and Dmp1^−/−; K-ras^LA/+ lung tumors. B: Detection of the p19^Arf Exon1β genomic DNA by semi-quantitative-PCR. The Arf gene was not homozygously deleted in K-ras^LA lung tumors regardless of the Dmp1 genotype. C: Quantification of the Dmp1 genomic DNA by real-time PCR. The black bars show the relative copy number of the Dmp1 genomic DNA in Dmp1^+/+, Dmp1^+/−, and Dmp1^−/− mice tails, with β-actin as an internal control (mean +/− SEM). The Dmp1 locus is deleted in 5 of 12 randomly chosen lung tumors from Dmp1^+/+; K-ras^LA/+ mice. The Dmp1 gene was hemizygously deleted in 5 of 8 cases of K-ras^LA/+ lung tumors with wild-type p53 (#465, #381, #154, #395, and #205). D: Quantification of the Dmp1 genomic DNA in lung tumors from p53^+/− or p53^−/−; K-ras^LA mice (mean +/− SEM). The Dmp1 gene was not deleted in any one of these 14 lung tumors from p53-knockout mice.

Figure 4. Loss of heterozygosity (LOH) analysis of the hDMP1, INK4a/ARF, and P53 loci in human non-small cell lung carcinomas

A: Genomic locus of the hDMP1 gene. The two different primer sets were designed to amplify the dinucleotide repeat sequences located on the 5’ and 3’ end of the hDMP1 gene. The non-coding exons were colored silver and the coding exons were colored gold. B: Genomic structure of the human INK4a/ARF locus. The two sets of PCR primers were designed to detect the dinucleotide repeats within 500 bps of Exon 1β (#33647) and those between Exon 1β and Exon 1α (#27251). The inverted triangles shown in red indicate the location of high-affinity hDMP1-binding sites. C: Genomic structure of the human P53 gene and the location of the PCR primers used for LOH analyses. D–I: Representative patterns of LOH for hDMP1, INK4a/ARF, and P53 in human non-small cell lung carcinoma. Genomic DNA was extracted from lung carcinomas and their normal counterparts and PCR was conducted with 6-FAM-labeled primers that amplify the dinucleotide repeats within (or close to) each locus. The area peaks of the PCR products were quantitated by ABI 3700 DNA analyzer. The qLOH values were determined through the following equation: qLOH = Area Peak 1/Area Peak 2 (normal tissue) divided by Area Peak 1’/Area Peak 2’ (tumor tissue). The arrows indicate the peak that was lost in tumor cells. The sample was considered to have LOH when the value was >2.0 or <0.5. D: LOH analysis of NSCLC with 5’ hDMP1 primer set, #92465. E: LOH analysis of NSCLC with 3’ hDMP1 primer set, #198004. F: LOH analysis with INK4a/ARF 5’ probe, #33647. G: LOH analysis with INK4a/ARF 3’ probe, #27251. H: LOH analysis with P53 5’ #158111 primers. I: LOH analysis with P53 3’ #89737 primers.

Figure 5. Summary of the results for qLOH values and promoter hypermethylation in 51 cases of human non-small cell lung cancer

The positive results for LOH (qLOH >2.0 or <0.5) were shown in bold red characters. When one of the two markers (5’ or 3’) showed qLOH value >2.0 or <0.5, the sample was found to be positive for LOH for the tumor suppressor locus. Cases of mutually exclusive inactivation of hDMP1 and INK4a/ARF or hDMP1 and P53 are shown “yes” in bold blue characters. Abbreviations: LC adeno, adenocarcinoma of the lung; LC squam, squamous cell carcinoma of the lung; LC adenosq, adenosquamous carcinoma of the lung; hDMP1 Met, human DMP1 promoter hypermethylation; ARF Met, p14^ARF promoter hypermethylation; INK4a Met, p16^INK4a promoter hypermethylation. Exclusive of DMP1 LOH, LOH of INK4a/ARF (or P53) not overlapping with that of hDMP1 in the same sample. Del, homozygous deletion; single, LOH was not evaluated due to a single peak result; partial, weak promoter methylation. N.D., not determined. N.D.*, not determined due to positive signals from histologically normal tissue (Holst et al., 2003).

Figure 6. Detailed mapping of the chromosomal 7q21 region deleted in human NSCLC

A: Genomic structure of the human hDMP1 locus and the primers used for the LOH analyses. GRM3: glutamate receptor 3; KIAA1324L: KIAA1324-like; MGC4175: Mammalian Gene Collection 4175; CROT: carnitine O-octanoyltransferase; ABCB4: ATP-binding cassette, sub-family B (MDR/TAP), member 4; ABCB1: ATP-binding cassette, sub-family B (MDR/TAP), member 1. B: Summary of the qLOH and real-time PCR values with 9 different markers (7 LOH and 2 real-time PCR) in the 20 hDMP1 LOH(+) samples and 9 hDMP1 LOH(−) samples. The positive results for LOH (qLOH >2.0 or <0.5) are shown in bold red. In real-time PCR, the samples were found to have hemizygous deletion of hDMP1 when the genomic DNA level was 0.25–0.65 (H.D. in bold red, DNA in the normal lung = 1.00). The borderline cases (0.66–0.75) are shown in pink. Samples that showed point mutation for K-Ras at codon 12 or 13 are shown in bold ID. H.D., hemizygous deletion; no del, no deletion as studied by genomic DNA real-time PCR; N.D., not done; single, single peak in LOH analysis. Note that there are only two genes at the hDMP1 locus between markers #69164 and #251945 (hDMP1 and MGC4175) and this locus is selectively deleted in 15 of 19 NSCLC samples.

Figure 7. Proliferation assay of human non-small cell lung cancer cell lines by overexpressing Dmp1:ER

A: H460; hDMP1^+/−, ARF⁺, p16^del, P53⁺, Rb⁺ B: H1299; hDMP1⁺, ARF⁺, p16^Met, P53^del, Rb⁺ C: A549; hDMP1⁺, ARF^del, p16^del, P53⁺, Rb⁺ D: H358; hDMP1⁺, ARF⁺, p16^Met, P53^del, Rb⁺ Pink lines show the growth curves of Dmp1:ER virus-infected cells treated with 2µM 4-HT, blue lines show those of mock-infected cells with 4-HT (mean +/− SEM). Activation of Dmp1:ER by 4-HT inhibited the growth of H460 cells with wild-type ARF and P53, but had little effects on other lung cancer cell lines that showed deletion of ARF or P53. E: Western blotting analyses of H460 and H1299 cells expressing activated Dmp1:ER or empty vector with specific antibodies to Dmp1, p14^ARF, p53, hDM2, and p21^Cip1. The numbers show hours after addition of 4-HT.