Tumor Suppressor Alterations Cooperate To Drive Aggressive Mesotheliomas With Enriched Cancer Stem Cells Via A P53-Mir-34A-C-Met Axis

Malignant mesothelioma is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced malignant mesothelioma compared with asbestosexposed Nf2+/- or wild-type mice. Ascites from some Nf2 +/-; Cdkn2a+/- mice exhibited large tumor spheroids, and tail vein injections of malignant mesothelioma cells established from these mice, but not from Nf2+/- or wildtype mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2+/-;Cdkn2a+/- mice. Intraperitoneal injections of malignant mesothelioma cells derived from Nf2+/-; Cdkn2a +/- mice into severe combined immunodeficient mice produced tumors that penetrated the diaphragm and pleural cavity and harbored increased cancer stem cells (CSC). Malignant mesothelioma cells from Nf2+/-; Cdkn2a+/- mice stained positively for CSC markers and formed CSC spheroids in vitro more efficiently than counterparts from wild-type mice. Moreover, tumor cells from Nf2+/-;Cdkn2a+/- mice showed elevated c-Met expression/activation, which was partly dependent on p53-mediated regulation of miR-34a and required for tumor migration/invasiveness and maintenance of the CSC population. Collectively, these studies demonstrate in vivo that inactivation of Nf2 and Cdkn2a cooperate to drive the development of highly aggressive malignant mesotheliomas characterized by enhanced tumor spreading capability and the presence of a CSC population associated with p53/miR-34a-dependent activation of c-Met. These findings suggest that cooperativity between losses of Nf2 and Cdkn2a plays a fundamental role in driving the highly aggressive tumorigenic phenotype considered to be a hallmark of malignant mesothelioma. © 2014 American Association for Cancer Research

Synthetic Secoisolariciresinol Diglucoside (LGM2605) Prevents Asbestos-Induced Inflammation and Genotoxic Cell Damage in Human Mesothelial Cells

Although alveolar macrophages play a critical role in malignant transformation of mesothelial cells following asbestos exposure, inflammatory and oxidative processes continue to occur in the mesothelial cells lining the pleura that may contribute to the carcinogenic process. Malignant transformation of mesothelial cells following asbestos exposure occurs over several decades; however, amelioration of DNA damage, inflammation, and cell injury may impede the carcinogenic process. We have shown in an in vitro model of asbestos-induced macrophage activation that synthetic secoisolariciresinol diglucoside (LGM2605), given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage. Therefore, it was hypothesized that LGM2605 could also be effective in reducing asbestos-induced activation and the damage of pleural mesothelial cells. LGM2605 treatment (50 µM) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 µg/cm2). Supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure for reactive oxygen species (ROS) generation, DNA damage (oxidized guanine), inflammasome activation (caspase-1 activity) and associated pro-inflammatory cytokine release (IL-1β, IL-18, IL-6, TNFα, and HMGB1), and markers of oxidative stress (malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-iso-PGF2α). Asbestos induced a time-dependent ROS increase that was significantly (p < 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% ± 1.0%. Although levels of inflammasome-activated cytokines, IL-1β and IL-18, reached 29.2 pg/mL ± 0.7 pg/mL and 43.9 pg/mL ± 0.8 pg/mL, respectively, LGM2605 treatment significantly (p < 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values (3.8 pg/mL ± 0.2 pg/mL and 5.4 pg/mL ± 0.2 pg/mL, respectively). Furthermore, levels of IL-6 and TNFα in asbestos-exposed mesothelial cells were high (289.1 pg/mL ± 2.9 pg/mL and 511.3 pg/mL ± 10.2 pg/mL, respectively), while remaining undetectable with LGM2605 pretreatment. HMGB1 (a key inflammatory mediator and initiator of malignant transformation) release was reduced 75.3% ± 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2α, markers of oxidative cell injury, were significantly (p < 0.001) reduced by 80.5% ± 0.1% and 76.6% ± 0.3%, respectively. LGM2605, given preventively, reduced ROS generation, DNA damage, and inflammasome-activated cytokine release and key inflammatory mediators implicated in asbestos-induced malignant transformation of normal mesothelial cells

Novel LRRK2 mutations and other rare, non-BAP1-related candidate tumor predisposition gene variants in high-risk cancer families with mesothelioma and other tumors

There is irrefutable evidence that germline BRCA1-associated protein 1 gene (BAP1) mutations contribute to malignant mesothelioma (MM) susceptibility. However, BAP1 mutations are not found in all cases with evidence of familial MM or in other high-risk cancer families affected by various cancers, including MM. The goal of this study was to use whole genome sequencing (WGS) to determine the frequency and types of germline gene variants occurring in 12 MM patients who were selected from a series of 141 asbestos-exposed MM patients with a family history of cancer but without a germline BAP1 mutation. WGS was also performed on two MM cases, a proband and sibling, from a previously reported family with multiple cases of MM without the inheritance of a predisposing BAP1 mutation. Altogether, germline DNA sequencing variants were identified in 21 cancer-related genes in 10 of the 13 probands. Germline indel, splice site and missense mutations and two large deletions were identified. Among the 13 MM index cases, 6 (46%) exhibited one or more predicted pathogenic mutations. Affected genes encode proteins involved in DNA repair (ATM, ATR, BRCA2, BRIP1, CHEK2, MLH3, MUTYH, POLE, POLE4, POLQ and XRCC1), chromatin modification (ARID1B, DNMT3A, JARID2 and SETD1B) or other cellular pathways: leucine-rich repeat kinase 2 gene (LRRK2) (two cases) and MSH4. Notably, somatic truncating mutation or deletions of LRRK2 were occasionally found in MMs in The Cancer Genome Atlas, and the expression of LRRK2 was undetectable or downregulated in a majority of primary MMs and MM cell lines we examined, implying that loss of LRRK2 expression is a newly recognized tumor suppressor alteration in MM

An asbestos-exposed family with multiple cases of pleural malignant mesothelioma without inheritance of a predisposing BAP1 mutation

We report a family with domestic exposure to asbestos and diagnosis of multiple cancers, including eight pleural malignant mesotheliomas and several other lung or pleural tumors. DNA sequence analysis revealed no evidence for an inherited mutation of BAP1. Sequence analysis of other potentially relevant genes, including TP53, CDKN2A, and BARD1, also revealed no mutation. DNA microarray analysis of tissue from two mesotheliomas revealed multiple genomic imbalances, including consistent losses of overlapping segments in 2q, 6q, 9p, 14q, 15q, and 22q, but no losses of chromosome 3 harboring the BAP1 locus. However, the results of immunohistochemical analysis demonstrated loss of nuclear BAP1 staining in three of six mesotheliomas tested, suggesting that somatic alterations of BAP1 occurred in a subset of tumors from this family. Since mesothelioma could be confirmed in only a single generation, domestic exposure to asbestos may be the predominant cause of mesothelioma in this family. Given the existence of unspecified malignant pleural tumors and lung cancers in a prior generation, we discuss the possibility that some other tumor susceptibility or modifier gene(s) may contribute to the high incidence of mesothelioma in this family. Because the incidence of mesothelioma in this family is higher than that expected even in workers heavily exposed to asbestos, we conclude that both asbestos exposure and genetic factors have played a role in the high rate of mesothelioma and potentially other pleural or lung cancers seen in this family.

Germline Mutations in <i>Mtap</i> Cooperate with <i>Myc</i> to Accelerate Tumorigenesis in Mice

<div><p>Objective</p><p>The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (<i>MTAP</i>) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in <i>Mtap (Mtap^lacZ)</i> could accelerate tumorigenesis development in two different mouse cancer models, <i>Eμ-myc</i> transgenic and <i>Pten^+/−</i>.</p><p>Methods</p><p><i>Mtap Eμ-myc</i> and <i>Mtap Pten</i> mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of <i>Mtap^lacZ/+</i> and Mtap<i>^+/+</i> mice.</p><p>Results</p><p>Survival in both models was significantly decreased in <i>Mtap^lacZ/+</i> compared to <i>Mtap^+/+</i> mice. In <i>Eµ-myc</i> mice, <i>Mtap</i> mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control <i>Eµ-myc Mtap^+/+</i> mice. Surprisingly, examination of <i>Mtap</i> status in lymphomas in <i>Eµ-myc Mtap^lacZ/+</i> and <i>Eµ-myc Mtap^+/+</i> animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of <i>Mtap</i> may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched <i>Mtap^+/+</i> and <i>Mtap^lacZ/+</i> animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.</p><p>Conclusion</p><p>Our findings show that germline inactivation of a single <i>Mtap</i> allele alters gene expression and enhances lymphomagenesis in <i>Eµ-myc</i> mice.</p></div

Types of tumors in <i>Mtap^+/+ Pten^+/−</i> and <i>Mtap^lacZ/+ Pten^+/−</i> animals.

<p>Types of tumors in <i>Mtap^+/+ Pten^+/−</i> and <i>Mtap^lacZ/+ Pten^+/−</i> animals.</p

FACS Analysis of <i>Em-myc Mtap^+/+</i> and <i>Em-myc Mtap^lacZ/+</i> mice.

<p>FACS Analysis of <i>Em-myc Mtap^+/+</i> and <i>Em-myc Mtap^lacZ/+</i> mice.</p

Mtap effect on tumor formation in <i>Eµ-Myc</i> and <i>Pten</i>^+/− mice.

<p>A. Tumor-free survival of <i>Eµ-myc Mtap^+/+</i> and <i>Eµ-myc Mtap^lacZ/+</i> mice. Sibling <i>Eµ-myc Mtap^lacZ/+</i> (n = 48) and <i>Eµ-myc Mtap^+/+</i> (n = 42) were monitored daily for tumor formation and/or death for 368 days (p<0.001) B. Tumor-free survival of <i>Mtap^+/+ Pten^+/−</i> and <i>Mtap^lacZ/+ Pten^+/−</i> mice. Sibling of <i>Mtap^+/+ Pten^+/+</i> (n = 32) and <i>Mtap^lacZ/+ Pten^+/−</i> (n = 32) were monitored daily and followed for 450 days (p<0.031). Tick marks show censored observations (deaths with no detectable tumor).</p

Histogram of P-values between <i>Mtap^+/+</i> and <i>Mtap^lacZ/+</i> livers.

<p>Line shows theoretical distribution of the null hypothesis (no differences in gene expression, P<0.0001).</p