Previous evidence has implicated BAP1
as a tumor suppressor gene: the BAP1
gene is located on human chromosome 3 (3p21.3); deletions in 3p are detected in almost 100% of small-cell lung cancers (SCLC), renal cell carcinomas (RCC), over 90% of non-small-cell lung cancer (NSCLC) cell lines and more than 80% of breast carcinomas (21
). Our study shows, for the first time, that BAP1 exhibits another key characteristic of tumor suppressors, the ability to inhibit tumor growth in an animal model. This is not a trivial extension of the growth suppression studies. First, growth in soft agar is an incomplete model for tumorigenesis. Any number of factors provided in the milieu of the whole animal can affect tumor growth and the results we have presented make it clear that the growth properties observed in cell culture are mirrored in the whole animal. Second, since the cell population injected into the animals was not subject to selection by antibiotic, the strength of the effects seen is even more impressive. At the MOI used we anticipate virtually complete transfection of the cells with BAP1 expression vectors, but certainly a few cells will remain untransfected or express lower levels of BAP1 than the bulk pool of cells. The nearly complete suppression of tumor growth by wild-type BAP1 re-expression makes it clear that this is a powerful tumor suppressor. The partial effects of expressing catalytically dead BAP1, or the NLS mutants may be due to statistical variation (p values indicate the differences are not statistically significant) or a small dominant negative effect due to expression of the inactive or mislocalized protein.
Like BAP1, other deubiquitinating enzymes have recently been shown to be bona fide
tumor suppressors, including the tumor-suppressor gene, CYLD
) and the herpesvirus-associated ubiquitin-specific protease (HAUSP, or ubiquitin-specific protease 7) (23
). The UPS is an integral part of normal cellular functions including cell cycle progression, signal transduction, response to extracellular stress and DNA repair and mutations that affect UPS components have been found to either enhance the effect of oncoproteins or reduce the function or amount of tumor suppressors (4
Although BAP1 has been hypothesized to function in BRCA1-mediated processes (3
), our results, and others’, support the possibility of BRCA1-independent functions of BAP1. First, Mallery et al
) have shown that BAP1 does not appear to function in the deubiquitination of the BRCA1/BARD1 complex. Secondly, we have shown that BAP1-mediated growth suppression is independent of wild-type BRCA1 (). Because there is no evidence that BRCA1 is a biologically relevant substrate for BAP1, we suspect that there are other in vivo
targets of BAP1 deubiquitinating activity.
Deubiquitinating activity of BAP1 appears to be important in cancer pathogenesis. Missense mutations that abolish the deubiquitinating activity of BAP1 (A95D and G178V) have been identified in cancer cell lines (16
). We have shown that these cancer-associated BAP1 mutants are not only defective in deubiquitinating activity ( and ) but are also defective in tumor suppressor activity (), implying that DUB activity of BAP1 is important in cancer pathogenesis and further supporting BAP1 as a genuine tumor suppressor.
BAP1 is a nuclear localized DUB. Although two nuclear localization signals had previously been predicted (3
), we have shown, for the first time, that BAP1 contains a functional classical NLS () and that localization to the nucleus is required for BAP1-mediated tumor suppression (). The NLS2 motif of BAP1 is a hydrophobic PY-NLSs which uses karyopherin β 2 as an import carrier (24
). The identification of a functional classical NLS in BAP1 is consistent with BAP1 having a nuclear substrate and supports a mechanism whereby BAP1 must enter the nucleus and its deubiquitinating activity is involved in functions that lead to tumor suppression. These functions may include DNA damage repair, regulation of apoptosis and/or senescence or cell cycle regulation.
Several gene expression array studies have implicated BAP1 as a cell-cycle regulator (25
) and our preliminary studies support a role for BAP1 in cell-cycle regulation (). Our data also imply that BAP1-mediated tumor suppression results from an increase in cell death by apoptosis. () However, other characteristics of apoptosis such as cleavage of caspase-3, a molecular marker of apoptosis were not observed. This may be related to the relatively small portion of the cell population that is undergoing cell death at any time. Most studies on apoptosis inflict catastrophic insult on the cells and all cells undergo a synchronized apoptosis where molecular markers are evident by 24 to 72 hours. In the experiments reported here, the presence of BAP1 is speeding the transition to S phase and some of those cells have accumulated some degree of DNA damage. Therefore, only a portion of these cells will undergo apoptosis at each division, leading to a low level of apoptotic markers detectable at any time. It is also prudent to note that expression of BAP1 increased the population of cells that stain positive for both AnnexinV and PI, an indication of late apoptosis and/or necrosis. More in-depth analysis of BAP1-mediated cell death is warranted in order to determine the specific mechanism of BAP1-mediated growth inhibition. For example, other studies have described cases of alternative (non-apoptotic) programs of cell death which also exhibit features characteristic of necrosis (28
). We hypothesize that expression of BAP1 in NCI-H226 cells induces early exit out of G1, thereby bypassing the G1-S checkpoint and causing an accumulation of un-repaired DNA damage and eventual induction of cell death.
In summary, we have provided strong evidence that BAP1 fulfills three major criteria for a genuine tumor suppressor: i) it is mutated in cancer, ii) these cancer-associated mutations inactivate a key growth suppressive activity of the protein, and iii) restoration of the protein expression in mutant tumor cells antagonizes tumor growth. We have shown that BAP1 has a functional classical NLS and that deubiquitinating activity and nuclear localization are both required for its tumor suppressor activity. BAP1 acts as a cell-cycle regulator and this may be one of the mechanisms through which it carries out its tumor suppressor function. BAP1-mediated tumor suppression appears to be a complex sequence of events that may activate multiple pathways, thereby regulating the cell cycle in a manner that results in apoptosis, necrosis or both. The identification of the deubiquitinating enzyme BAP1 as a tumor suppressor may further our understanding of how the UPS functions in cancer development and/or progression as well as help identify potential new targets for anti-cancer drugs.