Activated TNF-alpha/NF-kappaB signaling via down-regulation of Fas-associated factor 1 in asbestos-induced mesotheliomas from Arf knockout mice

Abstract

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of cyclin dependent kinase 4) and ARF (alternative reading frame) here, that are translated from alternatively spliced mRNAs. Human ARF is implicated as a tumor suppressor gene, mainly in association with the simultaneous deletion of INK4A. However, questions remain as to whether loss of ARF alone is sufficient to drive tumorigenesis. Here, we report that mice deficient for Arf are susceptible to accelerated asbestos-induced malignant mesothelioma (MM). MMs arising in Arf (+/-) mice consistently exhibit biallelic inactivation of Arf, but, unexpectedly, do not acquire additional recurrent genetic alterations that we previously identified in asbestos-induced MMs arising in Nf2 (+/-) mice. Array CGH analysis was used to detect a recurrent deletion at chromosome 4C6 in MMs from Arf (+/-) mice. A candidate gene in this region, Faf1 (FAS-associated factor 1), was further explored, because it encodes a protein implicated in tumor cell survival and in the pathogenesis of some human tumor types. We confirmed hemizygous loss of Faf1 and down-regulation of Faf1 protein in a series of MMs from Arf (+/-) mice, and we then showed that Faf1 regulates TNF-alpha-mediated NF-kappaB signaling, a pathway previously implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a significant role in driving MM pathogenesis, and implicate Faf1 as a key component in the TNF-alpha/NF-kappaB signaling node that has now been independently implicated in asbestos-induced oncogenesis.

Fig. 1.

Arf (+/−) mice exhibit decreased MM latency compared with wild-type littermates. (A) Comparison of survival in asbestos-treated Arf (+/−) and wild-type mice, depicted by Kaplan–Meier survival curves. Arf (+/−) mice showed significantly shorter survivals than wild-type mice based on the log-rank test (P = 4.63e-13). One Arf (+/−) mouse and 5 wild-type mice had no obvious tumors and were excluded as censored observations. (B) Summary of MMs arising in asbestos-treated Arf (+/−) and wild-type mice. (C) Representative histopathology of MMs from Arf (+/−) mice treated with asbestos. (Top) Noninvasive epithelial MM growing on the abdominal surface of the diaphragm. Malignant cells exfoliate from the surface of the tumor. (Middle) Bloody tumor ascites. Malignant epithelial MM cells grow in suspension within the peritoneal cavity. (Bottom) MMs grow as solid tumor spheroids within the peritoneal cavity and may attach to the parietal and visceral serosal linings. Sections were stained with hematoxylin and eosin.

Fig. 2.

MMs arising in Arf (+/−) exhibit biallelic inactivation of Arf, but do not acquire other tumor suppressor losses typical of human MMs. (A) Composite of genomic PCRs and RT-PCRs for p19Arf exon 1β, p16Ink4a exon 1α, p15Ink4b, Nf2, and p53 from primary MM cells (passage ≤5); β-actin and 18S protein were controls for template DNA and RNA, respectively. Open circles indicate absence, and solid circles indicate retention of at least 1 allele. Note that MMs from all Arf (+/−) mice were confirmed to retain the mutant knockout allele. (B) Immunoblot of MM cells showing retention of residual p16Ink4a in MMs from all Arf (+/−) mice except tumor 124 (shown in A to have loss of p16Ink4a, p19Arf, and p15Ink4b). Only the MM from wild-type mouse 132 retained Arf protein; this tumor instead had loss of p53. Expression of Nf2 (merlin) was observed in all MMs; β-actin was a loading control.

Fig. 3.

Array CGH of murine MMs reveals recurrent genomic loss in chromosome 4, band C6, near a CNV distal to the Cdk2na (Ink4/Arf) locus. (A) Chromosome 4 view of representative MMs from Arf (+/−) mice showing CNV (arrow) distal to homozygous deletion of the Ink4a/Arf locus observed in MM cells from wild-type littermate 104. Dots indicate oligonucleotides on CGH Analytics scatter plot with negative values. (B) Log2 ratios confirming CNV (arrow) in chromosome 4. Log2 ratios of 2 to −2 are considered normal variances (Upper). Approximate base pair locations in chromosome 4 are indicated. Plot of CNVs in the region indicates that there actually may be 2 separate peaks in this region (Lower). Normal chromosome copy number is 2, and values of 0 indicate deletion of the region. (C) Representative view of proximal side of the peak in band 4C6 indicating a hemizygous loss of Faf1 (Agilent CGH Analytics 3.4 gene view). Profile corresponds to genomic DNA from MM cells from Arf (+/−) mouse 128 hybridized to Agilent 244K chip. Line corresponding to the moving average was calculated with preset linear algorithm, 1-Mb window. Other genes in vicinity are Dmrta2 and Elavl4/HuD.

Fig. 4.

Confirmation of Faf1 down-regulation in asbestos-induced mouse MM cells. (A) Semiquantitative genomic PCR of Faf1/Gapdh levels in MM cells from wild-type and Arf (+/−) mice used for aCGH analysis. (B) Immunoblot of Faf1 levels in MMs from wild-type and Arf (+/−) mice. Asterisk(s) indicate cell lines used for semiquantitative genomic PCR analysis in A.

Fig. 5.

Faf1-deficient MMs exhibit more TNF-α-induced p65 nuclear accumulation and NF-κB activity. (A) Immunoblot of Faf1, p65, and actin in MMs from wild-type mouse 104 (Faf1-positive) and Arf (+/−) mice 110 and 129 (Faf1-negative). (B) Immunoblot of p65 and lamin B in nuclear extracts from TNF-α-treated (0, 0.5, 1, and 4 h) MM cells from tumors 104, 110, and 129. (C) Semiquantitation of nuclear p65 by immunoblotting (3 independent experiments) for TNF-α-treated cells. (D) Luciferase assay of NF-κB activity in MM cells treated with TNF-α for 24 h.

Fig. 6.

NF-κB activity is regulated by reexpression or knock down of Faf1 in MM cells. (A) reexpression in Faf1-deficient MM cells reduces NF-κB activity. (Left) Immunoblot of Faf1, p65, and β-actin levels in MM cells, from wild-type mouse 104 and Arf (+/−) mice 110 and 129, nucleofected with pcDNA3.1-Faf1 or pcDNA3.1 alone. (Right) Luciferase assay comparing relative NF-κB activity in MM cells treated with TNF-α for 24 h. (B) Knock down of Faf1 increases NF-κB activity in Faf1-positive MM cells treated with TNF-α for 24 h. (Left) Immunoblot of Faf1 and β-actin levels in MM cells from wild-type mouse nucleofected with siControl or siFaf1 (40 pmol each). Relative knock down is expressed as the KD (knock down) ratio and was calculated by densitometry of Faf1 levels/β-actin levels. (Right) Luciferase assay of relative NF-κB activity in TNF-α-treated Faf1-positive MM 104 cells nucleofected with siControl or siFaf1 no. 1, 2, or 3.