B Signaling Via Down-Regulation of Fas-Associated Factor 1 in Asbestos-Induced Mesotheliomas from Arf Knockout Mice

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

Deborah A. Altomarea,1, Craig W. Mengesa,1, Jianming Peia, Lili Zhanga, Kristine L. Skele-Stumpa, Michele Carboneb, Agnes B. Kanec, and Joseph R. Testaa,2

aHuman Genetics Program, Fox Chase Cancer Center, Philadelphia, PA 19111; bDepartment of Pathology, Cancer Research Center of Hawaii, Honolulu, HI 96813; and cDepartment of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912

Edited by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, and approved January 5, 2009 (received for review September 4, 2008)

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) human MM cell lines has been shown to induce G1-phase cell cycle and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of arrest and apoptotic cell death (8), suggesting that ARF, like INK4A, cyclin dependent kinase 4) and ARF (alternative reading frame) is an important target of 9p21 deletions in MM. here, that are translated from alternatively spliced mRNAs. Human Arf knockout mice represent an invaluable resource to test the ARF is implicated as a tumor suppressor gene, mainly in association relevance of ARF to MM pathogenesis. The Arf mouse model used with the simultaneous deletion of INK4A. However, questions here was generated by replacing the exon 1␤ of Arf with a remain as to whether loss of ARF alone is sufficient to drive Pgk-Neomycin cassette, leaving Ink4a sequences intact (9). Arf null tumorigenesis. Here, we report that mice deficient for Arf are (Ϫ/Ϫ) mice are highly prone to tumors, developing undifferentiated susceptible to accelerated asbestos-induced malignant mesotheli- sarcomas, carcinomas, and tumors of the nervous system within 1 oma (MM). MMs arising in Arf (؉/؊) mice consistently exhibit year of age; heterozygous Arf (ϩ/Ϫ) mice develop tumors at a lower biallelic inactivation of Arf, but, unexpectedly, do not acquire rate (20%) and longer latency. additional recurrent genetic alterations that we previously iden- Here, we show that Arf (ϩ/Ϫ) mice exposed to asbestos are tified in asbestos-induced MMs arising in Nf2 (؉/؊) mice. Array predisposed to MM, with a significantly shorter tumor latency CGH analysis was used to detect a recurrent deletion at chromo- compared with wild-type littermates. The murine MMs exhibit some 4C6 in MMs from Arf (؉/؊) mice. A candidate gene in this biallelic inactivation of the remaining wild-type Arf allele, although region, Faf1 (FAS-associated factor 1), was further explored, be- they do not exhibit the typical profile of tumor suppressor gene cause it encodes a protein implicated in tumor cell survival and in inactivation observed in human MMs. Array (a)CGH analysis led the pathogenesis of some human tumor types. We confirmed us to identify hemizygous loss of the Faf1 (FAS-associated factor 1) hemizygous loss of Faf1 and down-regulation of Faf1 protein in a locus, resulting in aberrant TNF-␣-induced NF-␬B signaling, a ؉ ؊ series of MMs from Arf ( / ) mice, and we then showed that Faf1 pathway previously implicated in asbestos-induced oncogenesis and ␣ ␬ regulates TNF- -mediated NF- B signaling, a pathway previously MM cell survival (10, 11). implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a Results significant role in driving MM pathogenesis, and implicate Faf1 as .Arf (؉/؊) Mice Are Susceptible to Asbestos-Induced MM ␣ ␬ After a key component in the TNF- /NF- B signaling node that has now repeated injections of crocidolite asbestos, we found markedly been independently implicated in asbestos-induced oncogenesis. accelerated MM development in asbestos-treated Arf (ϩ/Ϫ) mice, compared with wild-type littermates (Fig. 1A), whereas none of the array-CGH ͉ tumor suppressors control TiO2-treated mice developed MM. The median latency for detection of MM in Arf (ϩ/Ϫ) mice was 42 weeks after initial he CDKN2A (INK4a/ARF) locus encodes 2 distinct proteins asbestos exposure, compared with 56 weeks in wild-type mice. A Ttranslated from alternatively spliced mRNAs; p16(INK4A), log-rank test demonstrated that Arf (ϩ/Ϫ) mice had decreased designated as INK4A (inhibitor of cyclin dependent kinase 4) here, survival times, compared with wild-type mice (P ϭ 4.63E-13). ␣ is encoded by exons 1 , 2, and 3. The alternate product p14(ARF), Epithelial, mixed, and sarcomatoid histologies were observed in dubbed ARF (alternative reading frame protein) here, is specified asbestos-treated Arf (ϩ/Ϫ) and wild-type mice, although epithelial ␤ by exons 1 , 2, and 3 (1, 2). Amino acid sequences of INK4A and morphology predominated (Fig. 1B). The growth and histological ␣ ␤ ARF are unrelated, because exons 1 and 1 show no homology features of the MMs in this series of Arf (ϩ/Ϫ) mice are similar to and exon 2/3 sequences are translated in different reading frames. asbestos-induced tumors in previous chronic carcinogenicity assays The CDKN2A locus is among the most commonly mutated in wild-type and Tp53-deficient (ϩ/Ϫ) mice (12, 13). Epithelial genomic sites in human cancer (2). Point mutations or deletions MMs frequently presented with tumor ascites, spheroids, and specifically affecting exon 1␣ of INK4A are not uncommon, although intragenic mutations affecting exon 1␤ of ARF are seldom, if ever, observed (2, 3). Overall, the high frequency of concurrent Author contributions: D.A.A., C.W.M., A.B.K., and J.R.T. designed research; D.A.A., C.W.M., INK4A and ARF loss has made it difficult to assess the contribution J.P., L.Z., K.L.S.-S., and A.B.K. performed research; D.A.A., C.W.M., J.P., M.C., A.B.K., and of ARF to human tumorigenesis. J.R.T. analyzed data; and D.A.A., C.W.M., and J.R.T. wrote the paper. We previously demonstrated that the CDKN2A locus is homozy- The authors declare no conflict of interest. gously deleted in most malignant mesothelioma (MM) cell lines and This article is a PNAS Direct Submission. in many MM tumor specimens (4). FISH analysis revealed a high Freely available online through the PNAS open access option. incidence (Ϸ50 to 75%) of homozygous deletions in frozen MM Data deposition: Microarray data reported in this paper has been deposited in the Gene specimens or MM cells cultured for Յ5 days (5, 6). Reexpression Expression Omnibus (GEO) database (accession no. GSE12419). of INK4A in MM cells resulted in cell cycle arrest, cell death, as well 1D.A.A. and C.W.M contributed equally to this work. as tumor suppression and regression (7). In contrast to the estab- 2To whom correspondence should be addressed. E-mail: [email protected]. lished role of INK4A, the involvement of ARF in MM is less This article contains supporting information online at www.pnas.org/cgi/content/full/ understood. However, adenovirus-mediated transfer of ARF in 0808816106/DCSupplemental.

3420–3425 ͉ PNAS ͉ March 3, 2009 ͉ vol. 106 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808816106 Downloaded by guest on September 23, 2021 Fig. 1. Arf (ϩ/Ϫ) mice exhibit decreased MM latency compared with wild-type littermates. (A) Comparison of survival in asbestos-treated Arf (ϩ/Ϫ) and wild-type mice, depicted by Kaplan–Meier survival curves. Arf (ϩ/Ϫ) mice showed signiﬁcantly shorter survivals than wild-type mice based on the log-rank test (P ϭ 4.63e-13). One Arf (ϩ/Ϫ) mouse and 5 wild-type mice had no ob- vious tumors and were excluded as censored observa- tions. (B) Summary of MMs arising in asbestos-treated Arf (ϩ/Ϫ) and wild-type mice. (C) Representative histo- pathology of MMs from Arf (ϩ/Ϫ) 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.

diffuse peritoneal seeding on the serosal lining (Fig. 1C). In general, band C6, which was identified in all 4 MM cultures. This region is the MMs in Arf (ϩ/Ϫ) mice did not show extensive invasion of the distal to a homozygous loss of the Cdk2na locus observed in MM lymphatics and skeletal muscle, unlike tumors from Tp53 (ϩ/Ϫ)or cells from wild-type mouse 104 (Fig. 3A), identified in Fig. 2 as Nf2 (ϩ/Ϫ) mice. Overall, 8 of 25 MMs from the asbestos-treated Arf having loss of Ink4a/Arf DNA, RNA, and protein. Closer exami- (ϩ/Ϫ) mice exhibited invasion into the lymphatics or muscle tissue, and the incidence of invasion was equivalent to that of MMs from wild-type mice (9 of 22 MMs).

MMs from Arf (؉/؊) Mice Exhibit Biallelic Inactivation. MM cells from ascites or peritoneal lavage were cultured. We found biallelic inactivation of Arf in MMs from Arf (ϩ/Ϫ) mice, demonstrated by the loss of the wild-type allele in all 11 primary MM cultures analyzed, along with the fact that the mutant knockout allele was retained (not deleted) in 10 of the same 11 tumors (Fig. 2A). In comparison, loss of both wild-type Arf alleles was observed in 3 of 7 MMs from asbestos-treated wild-type mice, and down-regulation of Arf expression was observed in 2 additional MMs in this series, bringing the total to 5 of 7 (71%) MMs from wild-type littermates with loss of functional Arf (Fig. 2A). Immunoblot analysis of a subset of tumor cell lysates from Arf (ϩ/Ϫ) and wild-type mice confirmed the RT-PCR results (Fig. 2B). Our previous deletion mapping of human MM cell lines uncov- ered frequent homozygous deletions of chromosome 9p21 (4), which contains INK4A, ARF, and p15(INK4B), referred to here as INK4B. We found that MM cells from asbestos-treated Arf (ϩ/Ϫ) mice seldom had homozygous deletions of Ink4a or Ink4b.InArf (ϩ/Ϫ) mice, absence of p53 was not observed in any of the 11 MMs from Arf (ϩ/Ϫ)mice(Fig.2A and B). One of the MMs from a wild-type mouse showed loss of Tp53, but retention of Arf (wild- Fig. 2. MMs arising in Arf (ϩ/Ϫ) exhibit biallelic inactivation of Arf, but do not type mouse 132). Overall, the reciprocal association between Arf acquire other tumor suppressor losses typical of human MMs. (A) Composite of

and Tp53 loss was similar to that observed in previous studies of genomic PCRs and RT-PCRs for p19Arf exon 1␤, p16Ink4a exon 1␣, p15Ink4b, Nf2, MEDICAL SCIENCES Nf2-deficient (ϩ/Ϫ) mice and human MMs. and p53 from primary MM cells (passage Յ5); ␤-actin and 18S protein were controls for template DNA and RNA, respectively. Open circles indicate absence, A Recurrent Deletion in MM Cells from Arf-Deficient Mice Implicates and solid circles indicate retention of at least 1 allele. Note that MMs from all Arf ϩ Ϫ Faf1. We performed aCGH on primary MM cells from 4 Arf (ϩ/Ϫ) ( / ) mice were conﬁrmed to retain the mutant knockout allele. (B) Immunoblot of MM cells showing retention of residual p16Ink4a in MMs from all Arf (ϩ/Ϫ) mice to identify recurrent genomic imbalances connected with MM mice except tumor 124 (shown in A to have loss of p16Ink4a, p19Arf, and in these mice. Several inconsistent genomic changes, primarily loss p15Ink4b). Only the MM from wild-type mouse 132 retained Arf protein; this or gain of whole chromosomes, were observed. The only recurrent tumor instead had loss of p53. Expression of Nf2 (merlin) was observed in all MMs; change was a focal loss of an Ϸ2-Mb segment in chromosome 4, ␤-actin was a loading control.

Altomare et al. PNAS ͉ March 3, 2009 ͉ vol. 106 ͉ no. 9 ͉ 3421 Downloaded by guest on September 23, 2021 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 (ϩ/Ϫ) 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 Ϫ2 are con- sidered 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 (ϩ/Ϫ) mouse 128 hybridized to Agilent 244K chip. Line corresponding to the moving average was calculated with preset linear algo- rithm, 1-Mb window. Other genes in vicinity are Dmrta2 and Elavl4/HuD.

nation of the chromosome 4C6 deletion revealed that it was a copy from wild-type mice (Fig. 4B). Frequent down-regulation of FAF1 number variant (CNV), i.e., a germ-line polymorphism (Fig. 3B). protein expression was also observed in 7 of 7 human MM cell lines However, a hemizygous region of loss was detected in the distal when compared with normal mesothelial cells, and loss of FAF1 shoulder of the CNV peak. A candidate target gene located in the staining occurred in 12 of 14 human MM tumors [supporting region of hemizygous loss (Fig. 3C), Faf1, was further pursued, information (SI) Fig. S1]. because it encodes a factor that regulates cell survival (14, 15). Allelic loss of Faf1 in MMs from Arf (ϩ/Ϫ) mice was validated Faf1 Correlates with TNF-␣-Induced Nuclear p65 and NF-␬B Activity in by semiquantitative genomic PCR (Fig. 4A). Interestingly, immu- MM Cells. Previous reports have implicated Faf1 as a negative noblot analysis showed that Faf1 was down-regulated in most (6 of regulator of NF-␬B through inhibition p65/RelA nuclear localiza- 7) MMs from Arf (ϩ/Ϫ) mice, as well as a subset (4 of 7) of MMs tion (14, 15). To evaluate whether Faf1 down-regulation correlates with increased nuclear p65 localization, MM cells were stimulated with TNF-␣, a known inducer of NF-␬B in mesothelial cells. Nuclear p65 levels were evaluated by cellular fractionation, fol- lowed by immunoblot analysis (Fig. 5B). At all times tested, Faf1-negative MM cells from 2 Arf (ϩ/Ϫ) mice exhibited increased nuclear p65, compared with Faf1-positive MM cells from a wild- type mouse (Fig. 5 B and C). Importantly, there was no difference in total p65 protein between the 3 MM cultures, suggesting that Faf1 status correlates specifically with TNF-␣-induced nuclear p65 levels (Fig. 5A). The cyclic increase and decrease of p65 in the nucleus over time in all 3 MM cultures (Fig. 5 A and B) is consistent with other reports in which a bimodal, temporal-regulated signal has been observed that is indicative of the strength and duration of NF-␬B signaling in response to stimuli (16, 17). Increased nuclear p65 levels were also detected in Faf1-negative versus Faf1-positive Fig. 4. Conﬁrmation of Faf1 down-regulation in asbestos-induced mouse MM MM cells by using immunofluorescence analysis to monitor p65 cells. (A) Semiquantitative genomic PCR of Faf1/Gapdh levels in MM cells from ␣ wild-type and Arf (ϩ/Ϫ) mice used for aCGH analysis. (B) Immunoblot of Faf1 localization in cells stimulated with TNF- (Fig. S2). Together, levels in MMs from wild-type and Arf (ϩ/Ϫ) mice. Asterisk(s) indicate cell lines these data correlate decreased expression of Faf1 with increased used for semiquantitative genomic PCR analysis in A. TNF-␣-induced nuclear p65 in MM cells.

3422 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808816106 Altomare et al. Downloaded by guest on September 23, 2021 seen in control cells (Fig. 6A). NF-␬B activity was reduced in MM cells reexpressing Faf1, suggesting that Faf1 levels directly regulate NF-␬BsignalinginMMcells. To address whether Faf1 loss is sufficient to increase NF-␬B signaling, siRNA was used to knock down Faf1 in Faf1-positive MM 104 cells. Using 3 different RNAi sequences, we were able to knock down Faf1 levels by up to 63% (Fig. 6B). TNF-␣-induced NF-␬B luciferase activity was evaluated to determine whether knock down of Faf1 is sufficient to up-regulate NF-␬B signaling. As shown in Fig. 6B, higher NF-␬B activity was observed with all 3 siRNA used when compared with cells nucleofected with control siRNA, independent of TNF-␣ treatment. Together, these data implicate Faf1 as an important regulator of the NF-␬B pathway, whose loss may contribute to aberrant NF-␬B signaling and tumor progression in our mouse model of asbestos-induced MM. Discussion A paradigm for MM tumor progression is undefined, although it is clear that recurrent loss/inactivation of several tumor suppressor genes is likely to be involved in a multistep genetic progression cascade (18). In particular, the CDKN2A/ARF and CDKN2B loci on human chromosome 9 and the NF2 locus on chromosome 22 are known to be frequently inactivated in human MMs (4). There is considerable interest in employing genetically defined mouse models to better understand the significance of specific tumor suppressor genes in the pathogenesis of MM. Asbestos- treated Nf2 (ϩ/Ϫ) mice have been shown to have increased susceptibility and accelerated onset of asbestos-induced MM, compared with control mice, and the tumors consistently show loss of the remaining wild-type Nf2 allele (19, 20). We reported that Nf2 (ϩ/Ϫ) mice exposed to asbestos fibers recapitulate molecular features of human MM, including activation of Akt signaling and biallelic inactivation of the tumor suppressor genes Ink4a, Arf, Ink4b, and Nf2 (20). Similar inactivation of tumor suppressor genes in MMs from Nf2-deficient mice was reported by others (21). Mice lacking Arf are highly prone to tumor development, with Fig. 5. Faf1-deﬁcient MMs exhibit more TNF-␣-induced p65 nuclear accumu- undifferentiated sarcomas predominating over lymphomas, unlike lation and NF-␬B activity. (A) Immunoblot of Faf1, p65, and actin in MMs from Tp53-null mice (9). We report here that Arf (ϩ/Ϫ) mice have a wild-type mouse 104 (Faf1-positive) and Arf (ϩ/Ϫ) mice 110 and 129 (Faf1- markedly increased susceptibility to asbestos-induced MM, com- negative). (B) Immunoblot of p65 and lamin B in nuclear extracts from TNF-␣- pared with wild-type littermates. Interestingly, biallelic inactivation treated (0, 0.5, 1, and 4 h) MM cells from tumors 104, 110, and 129. (C) Semiquan- of Arf was not accompanied by loss of Ink4a, providing further titation of nuclear p65 by immunoblotting (3 independent experiments) for TNF-␣-treated cells. (D) Luciferase assay of NF-␬B activity in MM cells treated with support for a significant independent role of Arf loss/inactivation in TNF-␣ for 24 h. MM, and is not simply a bystander effect that accompanies loss of Ink4a. These findings are in keeping with the initial characterization of Arf mice, where spontaneous tumors were found to retain We next examined whether loss of Faf1, as a consequence of expression of Ink4a and Ink4b (9). These findings also are consis- increased nuclear p65, correlates with increased NF-␬B activity. tent with those of Sharpless et al. (22), who reported that homozy- Using a NF-␬B-luciferase reporter construct, we evaluated relative gous deletions of Ink4a and Arf have significant and nonredundant NF-␬B activity in Faf1-positive versus Faf1-negative MM cells roles in suppressing malignant transformation in vivo. ϩ Ϫ stimulated with TNF-␣. As shown in Fig. 5D, we observed increased Inactivation of Tp53 was rarely observed in MMs from Arf ( / ) NF-␬B-luciferase activity in both TNF-␣-stimulated and TNF-␣- mice. This finding is consistent with the reciprocal association between inactivation of Arf and Tp53 observed in MMs from unstimulated Faf1-deficient cells, compared with that observed in ϩ Ϫ Faf1-expressing cells. humans and Nf2 ( / ) mice (20), i.e., inactivation of Arf and To determine whether Faf1 directly regulates NF-␬B signaling in retention of p53, or vice versa, is sufficient to promote MM tumorigenesis. In contrast to MMs arising in humans and in Nf2 MM cells, we reexpressed Faf1 in Faf1-deficient cells, and evaluated ϩ Ϫ ␬ ␣ ( / ) mice, inactivation of Ink4a, Ink4b, and Nf2 was not fre- nuclear p65 levels and NF- B activity in response to TNF- .MM ϩ Ϫ ϩ Ϫ quently observed in MMs from Arf ( / ) mice. Although Arf 110 and 129 cells from Arf ( / ) mice were nucleofected with Faf1 deficiency predisposes to accelerated onset of asbestos-induced or empty vector (Fig. 6A). Faf1-positive MM 104 cells from MM, the fact that MMs seen in Arf (ϩ/Ϫ) mice were not highly wild-type mice were included as a control. Reexpression of Faf1 invasive suggests that additional somatic genetic alterations are MEDICAL SCIENCES resulted in reduced nuclear p65 levels in Faf1-deficient cells, with required for extensive invasion and metastasis. levels similar to those observed in Faf1-positive cells (Fig. S3). Variation in DNA copy number is increasingly recognized as a Collectively, these data directly link Faf1 with control of nuclear p65 factor contributing to genetic diversity (23, 24). CNVs theoretically levels during TNF-␣ stimulated NF-␬B signaling. could affect gene expression or function that contributes to disease We next evaluated whether reexpression of Faf1 reduces activa- susceptibility. In our study, aCGH revealed a striking recurrent tion of NF-␬B activity in Faf1-deficient MM cells. Consistent with CNV in chromosome 4C6, with a less prominent copy number loss nuclear p65 levels, reexpression of Faf1 in MM 110 and 129 cells near this CNV. The general location of the CNV in band 4C6, in resulted in reduced NF-␬B luciferase activity, compared with that a region that has synteny to human chromosome 1p32.3-1p33, was

Altomare et al. PNAS ͉ March 3, 2009 ͉ vol. 106 ͉ no. 9 ͉ 3423 Downloaded by guest on September 23, 2021 Fig. 6. NF-␬B activity is regulated by reexpression or knock down of Faf1 in MM cells. (A) reexpression in Faf1-deﬁcient MM cells reduces NF-␬B activity. (Left) Immunoblot of Faf1, p65, and ␤-actin levels in MM cells, from wild-type mouse 104 and Arf (ϩ/Ϫ) mice 110 and 129, nucleofected with pcDNA3.1-Faf1 or pcDNA3.1 alone. (Right) Luciferase assay comparing relative NF-␬B activity in MM cells treated with TNF-␣ for 24 h. (B) Knock down of Faf1 increases NF-␬B activity in Faf1-positive MM cells treated with TNF-␣ for 24 h. (Left) Immunoblot of Faf1 and ␤-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/␤-actin levels. (Right) Luciferase assay of relative NF-␬B activity in TNF-␣-treated Faf1-positive MM 104 cells nucleofected with siControl or siFaf1 no. 1, 2, or 3.

intriguing given that several CGH studies have implicated loss divide rather than undergo apoptosis (10). NF-␬B has been shown in/near this chromosomal region in human tumors, including MM. to be a constitutive survival factor in transformed human mesothe- In particular, D1S427-FAF1 was frequently lost in human uterine lial cells, as well as MM tumor cells (11). Inhibition of NF-␬B cervix carcinomas (25). This finding and the fact that Faf1 encodes activity in MM cells through the pleiotropic actions of Bortezomib, a factor involved in the regulation of cell survival led us to pursue an inhibitor of the 20S proteasome that also inhibits I␬B degrada- the possible involvement of this gene in MMs from Arf (ϩ/Ϫ) mice. tion, induces cell cycle blockage and apoptosis in vitro, as well as Recurrent loss and down-regulation of Faf1 was observed in our tumor growth inhibition in vivo (11). Also, therapeutic targeting of MMs. Other known candidate genes located in the deleted region, TNF-␣/NF-␬B signaling decreases drug resistance and increases i.e., Dmrta2 (Doublesex- and mab-3-related transcription factor cytotoxicity in MM cells (29). Collectively, our findings validate a A2) and Elavl4/HuD (embryonic lethal, abnormal vision, Drosophi- role for the TNF-␣/NF-␬B pathway in MM pathogenesis, and the la-like 4), were not pursued at this time. Arf (ϩ/Ϫ) model described here should prove useful for preclinical FAF1 is evolutionarily conserved and involved in various bio- testing of therapies targeting TNF-␣/NF-␬BsignalinginMM logical processes. Although FAF1 was identified as a member of the tumors. Fas death-inducing signaling complex (DISC) (26–28), we did not The mouse model used here targets the p53 pathway through Arf find that Faf1 loss contributed to attenuation of Fas receptor/DISC- deficiency to accelerate MM development. Similarly, Tp53- mediated caspase cleavage in our mouse MM cells. Thus, we believe deficient mice exhibit accelerated MM after asbestos exposure (12, loss of Faf1 leading to deregulation of the NF␬B pathway may be 13), and hamsters infected with SV40, to inhibit p53 via large T a more important activity than its contribution to Fas receptor- antigen expression, also show markedly increased incidence and mediated apoptosis in this murine model of MM. NF-␬B activation accelerated development of MM (30). Therefore, the investigations induced by TNF-␣, interleukin-1␤, or lipopolysaccharide was presented here, together with these former studies, demonstrate shown to be inhibited by FAF1 overexpression by preventing that genetic or viral alterations that impair the p53 pathway, directly translocation of the RelA (p65) subunit of NF-␬B into the nucleus or indirectly, increase the risk of asbestos-mediated carcinogenesis. and decreasing its DNA-binding activity on TNF-␣ treatment (14). These findings may have implications for cancer prevention, be- Like human FAF1, we showed that mouse Faf1 regulates NF-␬B cause now 3 independent lines of research indicate that an impaired activation induced by TNF-␣, preventing translocation of p65 into p53 pathway leads to an increased risk of developing MM after the nucleus. Both restoration of Faf1 in MM cells and knock down asbestos exposure. of Faf1 with siRNA in Faf1-positive MM cells had the predicted Materials and Methods effect on NF-␬B signaling. Also, although we originally identified ϩ Ϫ Animals. Arf (ϩ/Ϫ) breeding pairs were a gift from C. Sherr (St. Jude Children’s down-regulation of Faf1 in MMs from Arf ( / ) mice, some MMs Research Hospital, Memphis, TN). Mice were housed and treated according to from wild-type mice also showed down-regulation of Faf1. We also guidelines established by the National Institutes of Health Guide for the Care and observed Faf1 down-regulation in human MM cell lines and Use of Laboratory Animals. Male Arf (ϩ/Ϫ) and wild-type littermates were tumors; thus, reduced Faf1 expression may have implications in genotyped as described (31). human as well as mouse MM. Several groups have shown that TNF-␣ signaling is important in Treatment of Mice. Union Internationale Contre le Cancer (UICC)-grade crocido- human MM pathogenesis. TNF-␣ has been shown to inhibit asbes- lite asbestos was obtained from SPI Supplies, and TiO2 particles were obtained tos-induced cytotoxicity via a NF-␬B-dependent pathway (10). A from Aldrich Chemicals. We used methodology described previously (20), inject- ϩ Ϫ model was proposed whereby exposure to asbestos leads to the ing Arf ( / ) mice i.p. with crocidolite asbestos or control TiO2. Although not a ␣ ﬁber, TiO2 has been used in previous i.p. injection and inhalation studies (32), accumulation of macrophages and release of TNF- , whereas because of low toxicity and poor solubility. For details, see SI Materials and asbestos simultaneously induces human mesothelial cells to secrete Methods. A total of 26 Arf (ϩ/Ϫ) and 27 wild-type mice treated with asbestos, and ␣ ␣ ␬ TNF- . TNF- then activates NF- B to promote mesothelial cell 13 Arf (ϩ/Ϫ) mice and 18 wild-type littermates treated with TiO2 completed the survival and permits cells with asbestos-induced DNA damage to protocol. Other than MMs in Arf (ϩ/Ϫ) mice, 1 rhabdomyosarcoma and 1 lung

3424 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808816106 Altomare et al. Downloaded by guest on September 23, 2021 adenoma were found in the asbestos-treated group, and 1 hemangiosarcoma plasmid was from Stratagene. Stealth siRNAs against Mus musculus Faf1 (oligo ID was found in the TiO2-treated group. Two Arf (ϩ/Ϫ) and 1 wild-type mouse did MSS204189, MSS204190, and MSS204188) were from Invitrogen; 400 pmol of not complete asbestos treatments, and were not analyzable for cause of death. each siRNA molecule were nucleofected into mouse MM cells by using Amaxa Cell Line Nucleofector Kit R with program T-20. Primary Cell Cultures. Primary murine MM cells were isolated from ascitic fluid and/or lavage of the peritoneal cavity, as previously described (20). Early passage Luciferase Reporter Assays. MM cells were nucleofected with 1 ␮gofPathDetect cells from asbestos-treated mice were evaluated for mesothelial markers. MMs pNF-␬B-Luc Cis and 0.1 ␮g of pRenilla-Luc (Promega). At 24-h postnucleofection, from both wild-type mice and Arf (ϩ/Ϫ) mice typically expressed a panel of cells were treated or not with 50 ng/mL mouse TNF-␣ (Sigma) for 24 h before markers as assessed by RT-PCR, including WT1, cytokeratins 18 and 19, N- being harvested for Dual-Luciferase Reporter Assay (Promega). For Faf1 reexpres- cadherin, and E-cadherin. sion, 1 ␮g of pcDNA3.1-FAF1 or empty plasmid were nucleofected into Faf1- deficient MM cells, along with pNF-␬B-Luc Cis and pRenilla-Luc. To determine PCR and RT-PCR. Primers for genomic DNA and RT-PCR for the analysis of Arf, whether knock down of Faf1 increases NF-␬B activity, Stealth siRNAs against Mus Ink4a,Ink4b,Nf2, and p53, as well as Wt1,Krt18 (cytokeratin 18), Krt19,Cdh1, and musculus Faf1 were nucleofected into MM cells as per manufacturer’s instruc- Cdh2 were described previously (20). Primers for murine ␤-actin were purchased tions. After 48 h, cells were treated or not with 50 ng/mL mouse TNF-␣ for 24 h from Clontech Laboratories. Primer sets for Faf1 and Gapdh and corresponding before luciferase assay. Cells were harvested in 1X lysis buffer (Promega) and PCR conditions are described in SI Materials and Methods. freeze-thawed once before clarifying the lysate by centrifugation at 14,000 ϫ g for 10 min; 20 ␮L of sample was used for both luciferase assay and Bio-Rad protein ␬ Western Blottings. Immunoblot analysis of protein lysates was performed as assay according to the manufacturer’s protocols. NF- B luciferase values were described elsewhere (33), and in more detail online (SI Materials and Methods); normalized by both Renilla luciferase activity (transfection efficiency) and protein 15–30 ␮g of protein/sample was subjected to immunoblot analysis. Antibodies concentration. included anti-Arf from Abcam, anti-FAF1 (Ab1) from Cell Signaling Technology, anti-FAF1 (Ab2) from BioVision, and anti-Ink4a (M-156), anti-Nf2 (H-260), anti- Cell Fractionation. Cell fractions were isolated with NE-PER Nuclear and Cyto- NF-␬B p65 (C-20), anti-lamin B (C20), and anti-␤-actin (I-19) from Santa Cruz plasmic Extraction Reagents (Pierce Biotechnology), following the manufactur- Biotechnology, and anti-p53 (NCL-p53–505) from Novocastra. er’s recommendations. Nuclear protein extracts were quantitated by using a Bio-Rad protein assay, and equal concentrations for each treatment were sub- Array-Based CGH. Array CGH analysis was performed by using 44K and/or 244K jected to immunoblot analysis. Nuclear p65 levels were semiquantitated by Genomic DNA Arrays (Agilent), according to the manufacturer’s instructions (SI densitometry analysis and normalized to relative lamin B levels as a measure of Materials and Methods). Slides were scanned by using an Agilent scanner. Data nuclear extraction efficiency. were extracted by using Feature Extraction Software from Agilent. Output files were imported into Agilent CGH Analytics for DNA Copy Number Analysis. ACKNOWLEDGMENTS. We thank Alexander J. Olson, Matthew K. Hoelzle, and Norma Messier for technical assistance. This work was supported by the National Cancer Institute Grants CA-06927 and CA-114047 (to J.R.T.) and CA-009035 (to Plasmids, siRNA, and Nucleofection. FAF1 cDNA (clone ID 2958207) was from C.W.M), by the National Institute of Environmental Health Sciences Grant ES- Open Biosystems. A 1950-bp fragment encoding FAF1 was excised with EcoRI and 03721 (to A.B.K), by an appropriation from the Commonwealth of Pennsylvania XhoI, and subcloned into pcDNA3.1. The pcDNA3.1-FAF1 plasmid was nucleo- (Fox Chase Cancer Center), and by a gift from Local no. 14 Mesothelioma Fund of fected into mouse MM cells by using an Amaxa Nucleofector II and Cell Line the International Association of Heat and Frost Insulators and Allied Workers in Nucleofector Kit R with program U-030. PathDetect pNF␬B-Luc Cis reporter memory of Hank Vaughan and Alice Haas.

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