Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

Abstract

SummaryMesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard.

Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats

“…The early genetic event at the Cdkn2a locus by exposure to either long asbestos or long carbon nanotubes was the hypermethylation of Cdkn2a , whereas deletion of Cdkn2a occurs at a late stage of the malignant transformation 40 . The homozygous deletion of Cdkn2a/2b was not observed in the epithelioid subtype (EM) (0/6), but it was observed in the sarcomatoid subtype (SM) (4/5) in our study 36 and EM (1/5) in another study 41 when iron saccharate was used for rat MM carcinogenesis.…”

Clay-induced DNA double-strand breaks underlay genetic diversity, antibiotic resistance and could be a molecular basis for asbestos-induced cancer

“…Another example of a potentially dangerous material are the carbon nanotubes (CNTs), a novel industrial material with many applications. The genetic alterations provoked by these nanotubes in rat malignant mesothelioma were similar to those induced by asbestos [39].…”

Beyond Stanton and Pott hypothesis; carbon nanotubes-induced malignant mesothelioma as a disease of gene loss

“…Recent investigations have revealed that depletion of p19 (ARF) and epigenetic silencing of p16 with CDKN2A hypermethylation are frequently associated with mesothelioma progression due to either amosite asbestos or long-shaped CNTs exposure. 3 Mechanistically, the degree of genotoxic oxidative stress measured by 8-OHdG and the activation of pro-oncogenic signal pathways regulated by Akt, ERK1/2, and Src kinase are quite similar in the pleura between asbestos and CNTs exposure. This fact is consistent with Dr Fukushima's review article 1 stating that exposure to MWCNTs results in asbestos-like pathologic alterations including lung carcinoma and pleural mesothelioma.…”

“…Importantly, malignant mesothelioma is considered to be a "disease of gene loss" rather than being associated with driver mutations. 3 Genetic analyses have clarified the depletions and/or loss-of-functional mutations of CDKN2A, NF2, BAP1 genes, and both p53-dependent DNA repair pathway and PI3K/Akt/mTOR signal transduction are involved in mesothelioma development. Alterations in CDKN2A locus with inactivation of p16 (INK4a) are reported in more than a half of malignant mesothelioma patients.…”

“…This fact is consistent with Dr Fukushima's review article 1 stating that exposure to MWCNTs results in asbestos-like pathologic alterations including lung carcinoma and pleural mesothelioma. Remarkably, the sustained activation of pro-oncogenic signal pathways in CNTs-induced lesions occurs largely in non-mesothelial stromal cells, 3 suggesting that the presence of oxidative DNA damage in the chronically inflammatory microenvironment contributes to the hypermethylation and silencing of CDKN2A-coding tumor suppressor molecules (Figure 1).…”