Previous studies have shown that Arf
(−/−) and Ink4a
(−/−) mice are more prone to spontaneous tumors than wild-type animals, but each less so than Ink4a
(−/−) mice 
. The in vivo
carcinogenesis studies reported here evaluated the contribution of heterozygous mutations of Arf
, as well as a both tumor suppressor genes, to the induction of MM by asbestos, a well-established cause of this malignancy. In vivo
genetic models were used to investigate the relative impact of Arf
deficiency in a common genetic background. By analogy, our findings suggest that p14(ARF)
, like p16(INK4A)
, is an important target of 9p21 deletions in human MM. Moreover, the data indicate that co-deletion of Arf
can cooperate to accelerate tumorigenesis.
In previous studies of heterozygous Arf
mice, spontaneous tumors exhibited loss of the residual wild-type Arf
, consistent with a classical two-hit tumor suppressor gene. We found asbestos-induced MMs from heterozygous Ink4a
mice required biallelic inactivation of the predisposing tumor suppressor genes, and MMs were detected faster in the doubly deficient model. The importance of these tumor suppressors is in accordance with an investigation using conditional knockout mice 
, in which adeno-Cre-mediated homozygous excision of Ink4a
was sufficient to induce MM in the absence of asbestos exposure.
Notably, while tumor cells from Ink4a(+/−)-deficient mice acquired loss of Arf or p53 expression, loss of p16(Ink4a) was observed in only 3 of 6 MMs from Arf(+/−) mice. Similarly, in our earlier studies of Arf-deficient mice in a different (C57Bl/6) genetic background, all 11 MMs analyzed showed loss of Arf, although loss of p16(Ink4a) was identified in only two tumors. Collectively, these data imply that inactivation of Arf or p53 may be crucial for MM pathogenesis, whereas inactivation of p16(Ink4a) is not.
Since MMs analyzed here retained expression of p15Ink4b, loss of this gene is not critical for induction of MM by asbestos. In addition, our deletion mapping studies of human MMs revealed that deletions of p15INK4B
occur less frequently than losses of CDKN2A/ARF
and never occurred in the absence of a homozygous loss in the CDKN2A/ARF
Also similar to our earlier study of Arf
-deficient mice in a different (C57Bl/6) background, we rarely observed loss of Nf2 in MMs arising in Ink4a
- or Ink4a
-deficient mice. Inactivation of the NF2
tumor suppressor gene is postulated to facilitate cell cycle progression and tissue invasion/metastasis 
, and Nf2
-deficient mice are predisposed to asbestos-induced MM and contribute to its invasiveness and spreading 
. However, NF2/merlin loss does not appear to be required for development of MM in mice having this genotype and/or background.
Lastly, we found retention of functional p53 expression in MM cells that exhibit loss of Arf expression. Only one of the 20 asbestos-induced MMs showed loss of p53 expression (), and that single sample retained expression of Arf, consistent with our previous work showing a reciprocal pattern of inactivation of Tp53 in asbestos-induced MMs from Nf2
(+/−) mice that had retained expression of Arf 
. Interestingly, in MM cells with loss of Arf, the p53 pathway appeared to remain functional based on response to DNA damage (). These results from genetic model systems suggest that Arf loss can contribute to MM pathogenesis via p53-independent pathway(s), as previously noted in human MM cells 
, and that an intact p53 pathway remains a potential target for the treatment of this highly aggressive, chemo-resistant malignancy.
In summary, this is the first report directly assessing the relative importance of Ink4a and Arf in the susceptibility to asbestos-induced MM. Collectively, these in vivo data indicate that both Cdkn2A/Arf gene products suppress asbestos carcinogenicity. Furthermore, while Arf inactivation appears to be critical for MM pathogenesis and genomic instability (), the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis. Thus, future therapeutic approaches for MM should consider targeting pathways cooperatively regulated by both tumor suppressor genes.