The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. Wwox protein contains two WW domains that are generally known to mediate protein-protein interaction. ...Here we show that Wwox physically interacts via its first WW domain with the p53 homolog, p73. The tyrosine kinase, Src, phosphorylates Wwox at tyrosine 33 in the first WW domain and enhances its binding to p73. Our results further demonstrate that Wwox expression triggers redistribution of nuclear p73 to the cytoplasm and, hence, suppresses its transcriptional activity. In addition, we show that cytoplasmic p73 contributes to the proapoptotic activity of Wwox. Our findings reveal a functional cross-talk between p73 and Wwox tumor suppressor protein.
Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a ...genome 'caretaker' whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit (-/-) MEFs escape senescence to become immortal more rapidly than Fhit (+/+) MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenzaanthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a 'mutator' phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.
The FHIT gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. Silencing of ...Fhit protein expression causes thymidine kinase 1 downregulation, resulting in dNTP imbalance, and spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, insertions/deletions, and single‐base substitutions. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome “caretaker” whose loss initiates genome instability in preneoplastic lesions. To follow the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, we established epithelial cell lines from kidney tissues of Fhit−/− and +/+ mouse pups early after weaning, and subjected cell cultures to nutritional and carcinogen stress, which +/+ cells did not survive. Through transcriptome profiling and protein expression analysis, we observed changes in the Trp53/p21 and survivin apoptotic pathways in −/− cells, and in expression of proteins involved in epithelial–mesenchymal transition. Some Fhit‐deficient cell lines showed anchorage‐independent colony formation and increased invasive capacity in vitro. Furthermore, cells of stressed Fhit−/− cell lines formed s.c. and metastatic tumors in nude mice. Collectively, we show that Fhit loss and subsequent thymidine kinase 1 inactivation, combined with selective pressures, leads to neoplasia‐associated alterations in genes and gene expression patterns in vitro and in vivo.
We followed the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, by establishing epithelial cell lines from kidney tissues of Fhit−/− and +/+ mice. Some Fhit‐deficient cell lines demonstrated anchorage‐independent colony formation, increased invasive capacity in vitro, and formed subcutaneous and metastatic tumors in nude mice. Deregulation of the Fhit‐TK1 pathway, combined with selective pressures, leads to neoplasia‐associated alterations in gene expression patterns promoting transformation in vitro and in vivo.
Purpose: The WWOX gene is down-regulated in breast cancer and loss of Wwox expression correlates with important clinical features of breast
cancer. Thus, we have examined the effect of restoration of ...Wwox expression in breast cancer-derived cells.
Experimental Design: Wwox protein expression was restored by the following: ( a ) infection with a recombinant adenovirus carrying WWOX cDNA (Ad- WWOX ) or ( b ) treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, to activate the endogenous WWOX gene, in breast cancer-derived cells in vitro and in vivo .
Results: Restoration of Wwox expression led to suppression of growth of Wwox-deficient breast cancer-derived cells, through activation
of the intrinsic caspase pathway, but did not affect growth of Wwox-sufficient MCF7 cells. Intratumoral Wwox restoration,
through Ad- WWOX infection or endogenous Wwox reactivation by 5-aza-2′-deoxycytidine injection, suppressed tumor growth in nude mice by inducing
apoptosis. Alteration of global methylation levels was not observed.
Conclusions: The results confirm that overexpression of exogenous Wwox inhibits breast cancer cell growth in vitro and in vivo and, perhaps more importantly, shows that restoration of endogenous Wwox expression, and likely other proteins, by treatment
with a de novo methyltransferase inhibitor, also inhibits breast cancer cell growth and reverses breast cancer xenograft growth.
Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit‐deficient mouse models are exquisitely ...susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit‐deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit‐deficient tissues and cultured cells, we found 300 to >1000 single‐base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the “age at diagnosis” signature identified in human cancers. The Fhit‐deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in −/− exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit‐deficient cells. Fhit‐deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen‐induced preneoplastic changes.
Due to absence of Fhit genome caretaker function, Fhit knockout mouse cells develop aneuploidy, allele copy number losses and −/− tissues develop >1000 single‐base substitutions in the 2% of the genome included in exomes. The mutation signature is characterized by increased C>T and T>C mutations, similar to the “age at diagnosis” signature identified in human cancers. The increase in T>C mutations in −/− exomes may be due to thymidine triphosphate imbalance, resulting from decreased expression of Thymidine Kinase 1 in Fhit‐deficient cells.
It was hypothesized as early as 1986, that the recently discovered common fragile sites could facilitate recombination events, such as deletions and translocations, that result in clonally expanded ...cancer cell populations with specific chromosome alterations in specific cancer types. A natural extension of this hypothesis is that the clonal expansion must be driven by alteration of genes at recombination breakpoints whose altered functions actually drive clonal expansion. Nevertheless, when the
FHIT gene was discovered at
FRA3B, the most active common chromosome fragile region, and proposed as an example of a tumor suppressor gene altered by chromosome translocations and deletions, a wave of reports suggested that the
FHIT gene was altered in cancer simply because it was in a fragile region and not because it had contributed to the clonal expansion, thus turning the original hypothesis upside down. Now, after nearly ten years and more than 500
FHIT reports, it is apparent that
FHIT is an important tumor suppressor gene and that there are genes at other fragile regions that contribute significantly to development of cancer. A second fragile gene with a demonstrated role in cancer development is the
WWOX gene on chromosome 16q; alterations to the
WWOX gene contribute to development of hormone responsive and other cancers. Results of our recent studies of these two fragile tumor suppressor genes were summarized at the first Fragilome meeting in Heidelberg, Feb. 2005.
The Tumor Spectrum in FHIT-Deficient Mice Zanesi, Nicola; Fidanza, Vincenzo; Fong, Louise Y. ...
Proceedings of the National Academy of Sciences - PNAS,
08/2001, Volume:
98, Issue:
18
Journal Article
Peer reviewed
Open access
Mice carrying one inactivated Fhit allele (Fhit +/- mice) are highly susceptible to tumor induction by N-nitrosomethylbenzylamine, with 100% of Fhit +/- mice exhibiting tumors of the ...forestomach/squamocolumnar junction vs. 25% of Fhit +/+ controls. In the current study a single N-nitrosomethylbenzylamine dose was administered to Fhit +/+, +/-, and -/- mice to compare carcinogen susceptibility in +/- and -/- Fhit-deficient mice. At 29 weeks after treatment, 7.7% of wild-type mice had tumors. Of the Fhit -/- mice 89.5% exhibited tumors (average 3.3 tumors/mouse) of the forestomach and squamocolumnar junction; half of the -/- mice had medium (2 mm diameter) to large (>2 mm) tumors. Of the Fhit +/- mice 78% exhibited tumors (average 2.4 tumors/mouse) and 22% showed medium to large tumors. Untreated Fhit-deficient mice have been observed for up to 2 years for spontaneous tumors. Fhit +/- mice (average age 21 mo) exhibit an average of 0.94 tumors of different types; Fhit -/- mice (average age 16 mo) also showed an array of tumors (average 0.76 tumor/mouse). The similar spontaneous and induced tumor spectra observed in mice with one or both Fhit alleles inactivated suggests that Fhit may be a one-hit tumor suppressor gene in some tissues.
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