In this work, we present a comprehensive analysis of the single and combinatorial functions of the DOK family members Dok1, Dok2, and Dok3 in lung tumor suppression. We find that either heterozygous loss (in the case of Dok2) or complete loss of a single Dok gene can promote tumorigenesis. In addition, Dok1, Dok2, and Dok3 have overlapping functions and can cooperate in lung tumor suppression, because combinatorial knockout of all three Dok genes strongly promotes lung tumor formation in vivo.
We elucidated the cell biological changes that result from Dok
inactivation in vivo
, specifically the perturbations of the AT2 and BASC cellular compartments that occur in the lungs of Dok
TKO mice prior to frank tumorigenesis. BASC expansions are associated with lung tumorigenesis in a number of mouse models18,26–29
, but the role of BASCs as cancer-initiating cells for lung cancer remains to be definitively established. Nonetheless, our data demonstrate that combinatorial loss of Dok1
, and Dok3
perturbs BASC and AT2 cell signaling pathways and homeostasis.
single and compound mutant mice develop a range of immune30–34
phenotypes, several pieces of evidence support a cell autonomous mechanism of lung tumorigenesis in Dok
mutant mice. First, the kinetics of the CML-like myeloproliferative disorder and lung phenotype are different in Dok
TKO mice, with the onset of lung cancer (as early as 6 weeks) preceding the onset of leukemia, in which the expansion of the myeloid compartment becomes apparent at approximately 1 year of age with incomplete penetrance (see Supplementary Fig. 6
). Second, the immune phenotypes of the Dok133,34
, and Dok3 32
KO animals are distinct, owing to the cell lineage-dependent expression of these genes in the hematopoietic compartment. In contrast, all three single KO genotypes develop lung cancer. Third, all three Dok
genes are expressed in bronchioalveolar stem cells (BASCs) and these cells exhibit aberrant activation of Erk and Akt in vivo
TKO mice. Fourth, enforced overexpression of DOK2
inhibits the growth of human lung cancer cells in vitro
and after xenograft. Altogether, these facts indicate that Dok
KO mice likely develop lung cancer due to a cell-autonomous deregulation of Erk and Akt signaling resulting from loss of Dok
expression in lung epithelial and progenitor cells.
Importantly, we show that DOK2 is the target of frequent copy number loss in human lung cancer, and this genomic loss is accompanied by a downregulation of DOK2 mRNA expression, supportive of a tumor suppressive role for this gene. Moreover, heterozygous loss of DOK2 alone can promote lung tumorigenesis, as demonstrated by the fact that Dok2 heterozygous mice also develop lung cancer, and this occurs in the absence of LOH. These data suggest that DOK2 is a haploinsufficient human lung tumor suppressor.
Recent genome-wide analyses of copy number variation in human lung cancer have provided comprehensive descriptions of the genetic landscape of human cancer19,20,35
. However, the success of this approach has been limited to loci subject to focal deletion and focal amplification, which represent only a portion of the genome20
. Other regions of the genome, including 8p, are consistently targeted by broad-scale deletions20
, and it is now believed that regions of broad losses may encompass multiple, rather than single, tumor suppressors19
. Based on these observations, many groups have proposed that at least two to three tumor suppressors may reside on 8p, including at least one tumor suppressor in the 8p21.3 locus, and that the simultaneous compound loss of these tumor suppressors may be required for tumorigenesis19,36–39
. In this context, our findings identify DOK2
as an 8p21.3 tumor suppressor, and the possible cooperative tumorigenic effect of compound haploinsufficiency of DOK2
and other putative 8p tumor suppressors, such as DUSP4
should be investigated (Supplementary Figure 7
Our work emphasizes the key role of mouse molecular genetics in identifying and validating candidate tumor suppressor genes in broad-scale deleted regions. This in vivo approach in the mouse proves even more necessary and decisive when tumor suppressor haploinsufficiency and compound haploinsufficiency are suggested to represent the driving forces underlying human tumorigenesis.