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The expression of the WWOX tumor suppressor gene is lost or reduced in a large fraction of various cancers, including prostate cancer. We previously reported that Wwox overexpression induced apoptosis and suppressed prostate cancer growth in vitro and in vivo. In this study, pathways through which Wwox contributes to control of prostate cancer cell growth have been investigated. We found that Wwox interacts with Ap2γ and prevents it from entering the nucleus to bind the ERBB2 promoter region to activate transcription of ERBB2, a mediator of androgen receptor activity and prostate cancer cell growth at limiting androgen concentration. Ectopic expression of Wwox reduced ErbB2 protein expression in vitro and expression of Wwox protein inversely correlated with expression of ErbB2 protein in prostate cancer tissues. Furthermore, Wwox suppressed Ap2γ/ErbB2–induced prostate cancer cell growth and suppressed prostate-specific antigen secretion through interaction with Ap2γ and down-modulation of ErbB2, an effect that required functional androgen receptor. (Mol Cancer Res 2007;5(9):957–65)

Supplementary Figures S1-S2 from Wwox Suppresses Prostate Cancer Cell Growth through Modulation of ErbB2-Mediated Androgen Receptor Signaling

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.

Supplementary Data Figure 1 from Inhibition of Breast Cancer Cell Growth In vitro and In vivo: Effect of Restoration of Wwox Expression

Supplementary Figure 2 from Fhit Modulates the DNA Damage Checkpoint Response

Supplementary Figure 3 from Fhit-Deficient Hematopoietic Stem Cells Survive Hydroquinone Exposure Carrying Precancerous Changes

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2′-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues. (Cancer Res 2006; 66(13): 6477-81)

Supplementary Figure 1 from A Role for the WWOX Gene in Prostate Cancer

Supplementary Figure 2 from Fhit-Deficient Hematopoietic Stem Cells Survive Hydroquinone Exposure Carrying Precancerous Changes

The fragile FHIT gene is among the first targets of DNA damage in preneoplastic lesions, and recent studies have shown that Fhit protein is involved in surveillance of genome integrity and checkpoint response after genotoxin exposure. We now find that Fhit-deficient hematopoietic cells, exposed to the genotoxin hydroquinone, are resistant to the suppression of stem cell in vitro colony formation observed with wild-type (Wt) hematopoietic cells. In vivo–transplanted, hydroquinone-exposed, Fhit-deficient bone marrow cells also escaped the bone marrow suppression exhibited by Wt-transplanted bone marrow. Comparative immunohistochemical analyses of bone marrow transplants showed relative absence of Bax in Fhit-deficient bone marrow, suggesting insensitivity to apoptosis; assessment of DNA damage showed that occurrence of the oxidized base 8-hydroxyguanosine, a marker of DNA damage, was also reduced in Fhit-deficient bone marrow, as was production of intracellular reactive oxygen species. Treatment with the antioxidant N-acetyl-l-cysteine relieved hydroquinone-induced suppression of colony formation by Wt hematopoietic cells, suggesting that the decreased oxidative damage to Fhit-deficient cells, relative to Wt hematopoietic cells, accounts for the survival advantage of Fhit-deficient bone marrow. Homology-dependent recombination repair predominated in Fhit-deficient cells, although not error-free repair, as indicated by a higher incidence of 6-thioguanine–resistant colonies. Tissues of hydroquinone-exposed Fhit-deficient bone marrow–transplanted mice exhibited preneoplastic alterations, including accumulation of histone H2AX-positive DNA damage. The results indicate that reduced oxidative stress, coupled with efficient but not error-free DNA damage repair, allows unscheduled long-term survival of genotoxin-exposed Fhit-deficient hematopoietic stem cells carrying deleterious mutations. [Cancer Res 2008;68(10):3662–70]

In preneoplastic lesions, the DNA damage checkpoint is induced and loss of heterozygosity at the FRA3B/FHIT common chromosome fragile region precedes or is coincident with activation of the checkpoint response in these early stages. Introduction of exogenous Fhit into cells in vitro led to modulation of expression of checkpoint proteins Hus1 and Chk1 at mid-S checkpoint, a modulation that led to induction of apoptosis in esophageal cancer cells but not in noncancerous primary cultures. Mutation of the conserved Fhit tyrosine 114 resulted in failure of this function, confirming the importance of this residue. The results suggest that the DNA damage–susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein that is necessary for protecting cells from accumulation of DNA damage through its role in modulation of checkpoint proteins, and inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. (Cancer Res 2006; 66(23): 11287-92)

Supplementary Figure Legend from A Role for the WWOX Gene in Prostate Cancer

Supplementary Figure 1 from Fhit-Deficient Hematopoietic Stem Cells Survive Hydroquinone Exposure Carrying Precancerous Changes

Supplementary Figure 3 from Fhit Modulates the DNA Damage Checkpoint Response

Supplementary Figure 1 from Fhit Modulates the DNA Damage Checkpoint Response

// Stefano Volinia 1 , Teresa Druck 2 , Carolyn A. Paisie 3 , Morgan S. Schrock 2 and Kay Huebner 2 1 Department of Morphology, Surgery & Experimental Medicine, University of Ferrara, 44121 Ferrara, Italy 2 Department of Cancer Biology & Genetics, The Ohio State University Comprehensive Cancer Center & Wexner Medical Center, Biomedical Research Tower, Columbus, OH 43210, USA 3 University of Washington, Department of Biomedical Informatics & Medical Education, Center for Infectious Disease Research, Seattle, WA 98109, USA Correspondence to: Kay Huebner, email: kay.huebner@osumc.edu Keywords: exome sequences; mutational signatures; data mining; clock-like signatures; cancer genes Received: September 07, 2017 Accepted: October 25, 2017 Published: November 06, 2017 ABSTRACT The FHIT gene is located at the fragile FRA3B locus where activation by carcinogen-induced and endogenous replication stress causes FHIT deletions even in normal cells over a lifetime. Our lab has shown that loss of FHIT expression causes genome instability and provides single-strand DNA substrates for APOBEC3B hypermutation, in line with evidence that FHIT locus deletions occur in many cancers. Based on these biological features, we hypothesized that FHIT loss drives development of COSMIC mutational signature 5 and here provide evidence, including data mining of >6,500 TCGA samples, that FHIT is the cancer-associated gene with copy number alterations correlating most significantly with signature 5 mutation rate. In addition, tissues of Fhit-deficient mice exhibit a mutational signature strongly resembling signature 5 (cosine similarity value = 0.89). We conclude that FHIT loss is a molecular determinant for signature 5 mutations, which occur in all cancer types early in cancer development, are clock-like, and accelerated by carcinogen exposure. Loss of FHIT caretaker function may be a predictive and preventive marker for cancer development.

FHIT is a genome caretaker gene that is silenced in >50% of cancers. Loss of Fhit protein expression promotes accumulation of DNA damage, affects apoptosis and epithelial-mesenchymal transition, though molecular mechanisms underlying these alterations have not been fully elucidated. Initiation of genome instability directly follows Fhit loss and the associated reduced Thymidine Kinase 1 (TK1) protein expression. The effects on TK1 of Fhit knockdown and Fhit induction in the current study confirmed the role of Fhit in regulating TK1 expression. Changes in Fhit expression did not impact TK1 protein turnover or transcription from the TK1 promoter, nor steady-state levels of TK1 mRNA or turnover. Polysome profile analysis showed that up-regulated Fhit expression resulted in decreased TK1 RNA in non-translating messenger ribonucleoproteins and increased ribosome density on TK1 mRNA. Fhit does not bind RNA but its expression increased luciferase expression from a transgene bearing the TK1 5’-UTR. Fhit has been reported to act as a scavenger decapping enzyme, and a similar result with a mutant (H96) that binds but does not cleave nucleoside 5’,5’-triphosphates suggests the impact on TK1 translation is due to its ability to modulate the intracellular level of cap-like molecules. Consistent with this, cells expressing Fhit mutants with reduced activity toward cap-like dinucleotides exhibit DNA damage resulting from TK1 deficiency, whereas cells expressing wild-type Fhit or the H96N mutant do not. The results have implications for the mechanism by which Fhit regulates TK1 mRNA, and more broadly, for its modulation of multiple functions as tumor suppressor/genome caretaker.

In this study, loss of expression of the fragile site-encoded Wwox protein was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. WWOX gene deletions occur in a variety of human cancer types, and reduced Wwox protein expression can be detected early during cancer development. We found that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox-knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). Cancer cells that survive radiation recur more rapidly in a xenograft model of irradiated breast cancer cells; Wwox-deficient cells exhibited significantly shorter tumor latencies vs Wwox-expressing cells. This Wwox effect has important consequences in human disease: in a cohort of cancer patients treated with radiation, Wwox deficiency significantly correlated with shorter overall survival times. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR) and decreased non-homologous end-joining (NHEJ) repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. Upon silencing of Rad51, a protein critical for HDR, Wwox-deficient cells were resensitized to radiation. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for WWOX, in which Brca1-Wwox interaction supports NHEJ as the dominant DSB repair pathway in Wwox-sufficient cells. Taken together, the experimental results suggest that reduced Wwox expression, a common occurrence in cancers, dysregulates DSB repair, enhancing efficiency of likely mutagenic repair, and enabling radiation and cisplatin treatment resistance.

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.

Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.

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.

The FHIT gene is located at the fragile FRA3B locus where activation by carcinogen-induced and endogenous replication stress causes FHIT deletions even in normal cells over a lifetime. Our lab has shown that loss of FHIT expression causes genome instability and provides single-strand DNA substrates for APOBEC3B hypermutation, in line with evidence that FHIT locus deletions occur in many cancers. Based on these biological features, we hypothesized that FHIT loss drives development of COSMIC mutational signature 5 and here provide evidence, including data mining of >6,500 TCGA samples, that FHIT is the cancer-associated gene with copy number alterations correlating most significantly with signature 5 mutation rate. In addition, tissues of Fhit-deficient mice exhibit a mutational signature strongly resembling signature 5 (cosine similarity value = 0.89). We conclude that FHIT loss is a molecular determinant for signature 5 mutations, which occur in all cancer types early in cancer development, are clock-like, and accelerated by carcinogen exposure. Loss of FHIT caretaker function may be a predictive and preventive marker for cancer development.

Expression of Fhit and Wwox protein is frequently lost or reduced in many human cancers. In this report, we provide data that further characterizes the molecular consequences of Fhit loss in the initiation of DNA double-strand breaks (DSBs), and of Wwox loss in altered repair of DSBs. We show that loss of Fhit initiates mild genome instability in early passage mouse kidney cells, confirming that DNA damage associated with Fhit-deficiency is not limited to cancer cells. We also demonstrate that the cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation in early passage mouse kidney cells before extensive genome instability occurs. As for consequences of Wwox loss in cancer, we show in a small panel of breast cancer cells and mouse embryonic fibroblasts that Wwox expression predicts response to radiation and mitomycin C, all agents that cause DSBs. In addition, loss of Wwox significantly reduced progression free survival in a cohort of ovarian cancer patients treated with platin-based chemotherapies. Finally, stratification of a cohort of squamous lung cancers by Fhit expression reveals that Wwox expression is significantly reduced in the low Fhit-expressing group, suggesting that loss of Fhit is quickly succeeded by loss of Wwox. We propose that Fhit and Wwox loss work synergistically in cancer progression and that DNA damage caused by Fhit could be targeted early in cancer initiation for prevention, while DNA damage caused by Wwox loss could be targeted later in cancer progression, particularly in cancers that develop resistance to genotoxic therapies.

Elk is a member of the eph family of receptor tyrosine kinases. Elk is expressed only in the brain and testes of the developing and adult rat, and the interaction of elk with its ligand(s) has been suggested to play a role in the development or maintenance of the nervous system. The mouse gene Eplg2 encodes a potential elk ligand that is highly conserved among rat, mouse, and human. Eplg2 has been mapped to the central portion of the mouse X chromosome, tightly linked to the androgen receptor (Ar) locus. Linkage conservation between the mouse and the human X chromosomes suggested that the human homologue (EPLG2) would map near human AR, in the interval Xq11-q12. In the present study, we have confirmed this prediction and have localized EPLG2 to a 200-kb interval in Xq12 by somatic cell hybrid analysis, two-color fluorescence in situ hybridization (FISH), and yeast artificial chromosome (YAC) hybridization. 12 refs., 1 fig.

We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion gene was discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5’-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5’-end of the fly Fhit gene, this gene washailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions.

Zinc deficiency is associated with high incidences of esophageal and other cancers in humans and leads to a highly proliferative hyperplastic condition in the upper gastrointestinal tract in laboratory rodents. Zn replenishment reduces the incidence of lingual, esophageal and forestomach tumors in Zn-deficient rats and mice. While previous animal studies focused on Zn deficiency, we have investigated the effect of Zn supplementation on carcinogenesis in Zn-sufficient mice of wild-type and tumor suppressor-deficient mouse strains. All mice received N-nitrosomethylbenzylamine and half the mice of each strain then received Zn supplementation. At killing, mice without Zn supplementation had developed more tumors than Zn-supplemented mice: wild-type C57BL/6 mice developed an average of 7.0 versus 5.0 tumors for Zn supplemented (P < 0.05); Zn-supplemented Fhit−/− mice averaged 5.7 versus 8.0 for control mice (P < 0.01); Zn-supplemented Fhit−/−Nit1−/− mice averaged 5.4 versus 9.2 for control mice (P < 0.01) and Zn-supplemented Fhit−/−Rassf1a−/− (the murine gene) mice averaged 5.9 versus 9.1 for control mice (P < 0.01). Zn supplementation reduced tumor burdens by 28% (wild-type) to 42% (Fhit−/−Nit1−/−). Histological analysis of forestomach tissues also showed significant decreases in severity of preneoplastic and neoplastic lesions in Zn-supplemented cohorts of each mouse strain. Thus, Zn supplementation significantly reduced tumor burdens in mice with multiple tumor suppressor deficiencies. When Zn supplementation was begun at 7 weeks after the final carcinogen dose, the reduction in tumor burden was the same as observed when supplementation began immediately after carcinogen dosing, suggesting that Zn supplementation may affect tumor progression rather than tumor initiation.

The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit−/−Nit1−/− mice and observed that double knockout mice develop more spontaneous and carcinogen-induced tumors than Fhit−/− mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney-derived double-deficient cells appear not to activate the pChk2 pathway and when treated with H2O2, show little evidence of DNA damage, compared with wild type and Fhit−/− cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas. J. Cell. Biochem. 107: 1097–1106, 2009. © 2009 Wiley-Liss, Inc.

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G2/M or sub-G1 fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.

Inadequate dietary Zn consumption increases susceptibility to esophageal and other cancers in humans and model organisms. Since Zn supplementation can prevent cancers in rodent squamous cell carcinoma (SCC) models, we were interested in determining if it could have a preventive effect in a rodent skin cancer model, as a preclinical basis for considering a role for Zn in prevention of human nonmelanoma skin cancers, the most frequent cancers in humans. We used the 7,12‐dimethyl benzanthracene carcinogen/phorbol myristate acetate tumor promoter treatment method to induce skin tumors in Zn‐sufficient wild‐type and Fhit (human or mouse protein) knockout mice. Fhit protein expression is lost in >50% of human cancers, including skin SCCs, and Fhit‐deficient mice show increased sensitivity to carcinogen induction of tumors. We hypothesized that: (1) the skin cancer burdens would be reduced by Zn supplementation; (2) Fhit −/−(Fhit, murine fragile histidine triad gene) mice would show increased susceptibility to skin tumor induction versus wild‐type mice. 30 weeks after initiating treatment, the tumor burden was increased ~2‐fold in Fhit −/− versus wild‐type mice (16.2 versus 7.6 tumors, P

The expression of the WWOX tumor suppressor gene is lost or reduced in a large fraction of various cancers, including prostate cancer. We previously reported that Wwox overexpression induced apoptosis and suppressed prostate cancer growth in vitro and in vivo. In this study, pathways through which Wwox contributes to control of prostate cancer cell growth have been investigated. We found that Wwox interacts with Ap2γ and prevents it from entering the nucleus to bind the ERBB2 promoter region to activate transcription of ERBB2, a mediator of androgen receptor activity and prostate cancer cell growth at limiting androgen concentration. Ectopic expression of Wwox reduced ErbB2 protein expression in vitro and expression of Wwox protein inversely correlated with expression of ErbB2 protein in prostate cancer tissues. Furthermore, Wwox suppressed Ap2γ/ErbB2–induced prostate cancer cell growth and suppressed prostate-specific antigen secretion through interaction with Ap2γ and down-modulation of ErbB2, an effect that required functional androgen receptor. (Mol Cancer Res 2007;5(9):957–65)

FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.

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.

In preneoplastic lesions, the DNA damage checkpoint is induced and loss of heterozygosity at the FRA3B/FHIT common chromosome fragile region precedes or is coincident with activation of the checkpoint response in these early stages. Introduction of exogenous Fhit into cells in vitro led to modulation of expression of checkpoint proteins Hus1 and Chk1 at mid-S checkpoint, a modulation that led to induction of apoptosis in esophageal cancer cells but not in noncancerous primary cultures. Mutation of the conserved Fhit tyrosine 114 resulted in failure of this function, confirming the importance of this residue. The results suggest that the DNA damage–susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein that is necessary for protecting cells from accumulation of DNA damage through its role in modulation of checkpoint proteins, and inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. (Cancer Res 2006; 66(23): 11287-92)

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2′-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues. (Cancer Res 2006; 66(13): 6477-81)

To identify functions of the fragile tumour suppressor gene, FHIT, matched pairs of Fhit-negative and -positive human cancer cell clones, and normal cell lines established from Fhit -/- and +/+ mice, were stressed and examined for differences in cell cycle kinetics and survival. A larger fraction of Fhit-negative human cancer cells and murine kidney cells survived treatment with mitomycin C or UVC light compared to matched Fhit-positive cells; approximately 10-fold more colonies of Fhit-deficient cells survived high UVC doses in clonigenic assays. The human cancer cells were synchronised in G1, released into S and treated with UVC or mitomycin C. At 18 h post mitomycin C treatment approximately 6-fold more Fhit-positive than -negative cells had died, and 18 h post UVC treatment 3.5-fold more Fhit-positive cells were dead. Similar results were obtained for the murine -/- cells. After low UVC doses, the rate of DNA synthesis in -/- cells decreased more rapidly and steeply than in +/+ cells, although the Atr-Chk1 pathway appeared intact in both cell types. UVC surviving Fhit -/- cells appear transformed and exhibit5-fold increased mutation frequency. This increased mutation burden could explain the susceptibility of Fhit-deficient cells in vivo to malignant transformation.

We describe the cloning and characterization of a human 5-HT6 serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5-HT6 cDNA encodes a 440-amino-acid polypeptide whose sequence diverges significantly from that published for the rat 5-HT6 receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5-HT6 amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5-HT6 receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35-p36. This localization overlaps that established for the serotonin 5-HT1D alpha receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5-HT6 receptor also reveals an Rsal restriction fragment length polymorphism within the coding region.

It has been suggested that delayed DNA replication underlies fragility at common human fragile sites, but specific sequences responsible for expression of these inducible fragile sites have not been identified. One approach to identify such cis-acting sequences within the large nonexonic regions of fragile sites would be to identify conserved functional elements within orthologous fragile sites by interspecies sequence comparison. This study describes a comparison of orthologous fragile regions, the human FRA3B / FHIT and the murine Fra14A2 / Fhit locus. We sequenced over 600 kbp of the mouse Fra14A2 , covering the region orthologous to the fragile epicenter of FRA3B , and determined the Fhit deletion break points in a mouse kidney cancer cell line (RENCA). The murine Fra14A2 locus, like the human FRA3B , was characterized by a high AT content. Alignment of the two sequences showed that this fragile region was stable in evolution despite its susceptibility to mitotic recombination on inhibition of DNA replication. There were also several unusual highly conserved regions (HCRs). The positions of predicted matrix attachment regions (MARs), possibly related to replication origins, were not conserved. Of known fragile region landmarks, five cancer cell break points, one viral integration site, and one aphidicolin break cluster were located within or near HCRs. Thus, comparison of orthologous fragile regions has identified highly conserved sequences with possible functional roles in maintenance of fragility.

We have sequenced 870 kilobases of the FHIT / FRA3B locus, from FHIT intron 3 to intron 7. The locus is AT rich (61.5%) and Alu poor (6.2%), and it apparently does not harbor other genes. In a detailed analysis of the 308-kilobase region between FHIT exon 5 and the telomeric end of intron 3, a region known to encompass a human papillomavirus-16 integration site and two clusters of aphidicolin-induced chromosome 3p14.2 breakpoints, we have precisely mapped 10 deletion and translocation endpoints in cancer-derived cell lines relative to positions of specific repetitive elements, regions of high genome flexibility and aphidicolin-induced breakpoints. Conclusions are ( i ) that aphidicolin-induced breakpoint clusters fall close to high-flexibility sequences, suggesting that these sequences contribute directly to aphidicolin-induced fragility; ( ii ) that 9 of the 10 FHIT allelic deletions in cancer cell lines resulted in loss of exons, with 7 deletion endpoints near long interspersed nuclear elements or long terminal repeat elements; and ( iii ) that cancer-specific deletions encompass multiple high-flexibility genomic regions, suggesting that fragile breaks may occur at these regions, whereas repair of the breaks involves homologous pairing of flanking sequences with concomitant deletion of the damaged fragile sequence.

BACKGROUND The origins of and interrelations between low grade and high grade neuroendocrine lung tumors, typical and atypical carcinoids, and small cell lung carcinoma (SCLC) have not been elucidated. Karyotypic and molecular genetic studies have demonstrated deletions in 3p in 100% of SCLCs and the candidate lung tumor suppressor gene, FHIT, at 3p14.2 is not expressed in the majority of SCLCs. Similar studies of typical and atypical carcinoids could clarify the interrelations among these tumors. METHODS For molecular genetic analyses, archival carcinoids and paired normal cells were microdissected from paraffin sections, deparaffinized, and DNA prepared. Oligonucleotide primer pairs for 12 microsatellite markers mapping between 3p14.2 and 3p21.3 were used to amplify allelic DNA fragments from 13 typical and 6 atypical carcinoids. In addition, an independent series of archival sections of carcinoids and SCLCs was tested by immunohistochemistry for expression of Fhit protein. RESULTS Of the six atypical carcinoids examined, three had lost an allele at all informative markers, whereas one had lost alleles in two distinct regions and two showed allele loss in a subregion of the chromosome region tested. Of the 13 typical carcinoids, 3 showed allele loss at only 1 or 2 loci each. Typical carcinoids, similar to normal lung epithelia, were strongly positive for the cytoplasmic Fhit protein, SCLCs were uniformly negative, and atypical carcinoids appeared to express an intermediate level of Fhit protein. CONCLUSIONS Loss of heterozygosity at 3p14.2-p21.3 is significantly more extensive in all atypical carcinoids. Atypical carcinoids, which exhibit clinicopathologic features intermediate between typical carcinoids and small cell carcinomas and have been considered well differentiated neuroendocrine carcinomas, also are intermediate between typical carcinoids and SCLC on the basis of extent of loss of 3p alleles and reduced expression of Fhit protein. Cancer 1998;83:1109-1117. © 1998 American Cancer Society.

The hypothesis that chromosomal fragile sites may be “weak links” that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the FHIT gene, which encompasses the common fragile site, FRA3B . Sequence analysis of 276 kb of the FRA3B / FHIT locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in FHIT gene internal deletions, probably as a result of carcinogen-induced damage at FRA3B fragile sites.

The ALL-1 gene is involved in human acute leukemia through chromosome translocations or internal rearrangements. ALL-1 is the human homologue of Drosophila trithorax. The latter is a member of the trithorax group (trx-G) genes which together with the Polycomb group (Pc-G) genes act as positive and negative regulators, respectively, to determine the body structure of Drosophila. We have cloned a novel human gene, ALR, which encodes a gigantic 5262 amino acid long protein containing a SET domain, five PHD fingers, potential zinc fingers, and a very long run of glutamines interrupted by hydrophobic residues, mostly leucine. The SET motif, PDH fingers, zinc fingers and two other regions are most similar to domains of ALL-1 and TRX. The first two motifs are also found in other trx-G and Pc-G proteins. The ALR gene was mapped to chromosome band 12q12-13, adjacent to the VDR gene. This region is involved in duplications and translocations associated with cancer. The analysis of ALR expression showed that its approximately 18 kb long mRNA is expressed, like ALL-1, in most adult tissues, including a variety of hematopoietic cells, with the exception of the liver. Whole mount in situ hybridization to early mouse embryos indicates expression in multiple tissues. Based on similarities in structure and expression pattern, ALR is likely to play a similar role to ALL-1 and trx, although its target genes have yet to be identified.

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-dimethylbenz[a]anthracene, 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., PLoS ONE, 8(11), e80730; 2013

cDNAs encoding the human homolog of BAG1, a Bcl-2-binding protein with anti-apoptotic function, were cloned. DNA sequence analysis of human BAG1 cDNAs predicts a protein with an additional 55 amino acids at its NH2-terminus compared to the mouse protein. Immunoblot assays using monoclonal antibodies raised against bacterially produced h-BAG1 protein confirmed the larger size of the human protein ({approximately}34 kDa) compared to mouse. PCR analysis of DNA form human x rodent somatic cell hybrids using human BAG1-specific primers localized the gene to human chromosome 9. Cosmid clones of h-BAG1 were obtained and used for fluorescence in situ hybridization analysis of normal metaphase chromosomes, thus localizing h-BAG1 to 9p12, a region associated with hereditary disorders that may involve developmental dysregulation of programmed cell death. 15 refs., 4 figs.

The receptor protein tyrosine phosphatase gamma gene, PTP gamma (locus name PTPRG), was previously mapped to chromosome region 3p14.2, within a 2- to 4-Mb region centromeric to the 3p14.2 breakpoint of the t(3;8) familial renal cell carcinoma (RCC)-associated constitutional chromosome translocation. Because of its chromosomal position, its enzymatic properties as a receptor phosphatase, which might oppose a growth activating kinase activity, its homozygous deletion in murine L cells, and its transcriptional activity in numerous normal tissues, including kidney, the PTP gamma gene was an attractive tumor suppressor gene candidate for renal cell carcinoma. To determine whether the PTP gamma gene was a target of loss of heterozygosity or mutation in RCCs and to determine its map position relative to the t(3;8) break at 3p14.2, we have isolated YAC and lambda genomic clones for the PTP gamma gene and other 3p14.2 markers and determined the relative positions of the t(3;8) break, a 3p14.2 de novo break possibly in a fragile site, and the 5' end of the PTP gamma gene. Additionally, the genomic structure, position of the proximal promotor, and intron-exon border sequences of the 30-exon 780-kb PTP gamma gene have been determined, which will facilitate analysis of the PTP gamma gene in tumors.

A 200–300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B , is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of the histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5′, 5′′′ P 1 , P 4 -tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5′ untranslated exons centromeric to the renal carcinoma–associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in ∼50% of esophageal, stomach, and colon carcinomas.

The phospholipase C {gamma}-2 (Plcg2) gene encodes an enzyme that plays a crucial role in intracellular signal transduction pathways. This enzyme is important because of its role in the generation of second messengers following the hydrolysis of phosphatidylinositol 4,5-bisphosphate. We have now determined the chromosomal location of this gene in the mouse and human genomes. An interspecific backcross involving AEJ/Gn and Mus spretus mice was used to localize the gene in mouse. A rodent/human somatic cell hybrid panel was used to map PLCG2 in the human genome. Our results position Plcg2 in the central region of mouse chromosome 8. We also show that PLCG2 maps to the long arm of human chromosome 16, in the region q22-qter. Plcg2 does not map near its most closely related family member, Plcg1, in either genome, indicating that the mammalian Plcg genes belong to a dispersed family. 29 refs., 3 figs., 2 tabs.

Common fragile sites predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Chromosomal fragile sites not only are susceptible to DNA instability in cancer cells, but may also be associated with genes that contribute to the neoplastic process. FRA7G is a common fragile site containing the candidate tumor suppressor genes CAV1, CAV2, and TESTIN (TES). The human gastric cancer cell line GTL-16 has an amplification of this genomic region and was used to seek evidence for the suppressor candidacy of one of these genes. Our results demonstrate that CAV1, CAV2, and TESTIN are coamplified with the MET oncogene and overexpressed in GTL-16. Somatic mutation was not detected in the coding regions of these genes, although they were each overexpressed. The results show that CAV1, CAV2, and TESTIN are not tumor suppressor genes in this gastric cancer.

The region surrounding the ZNF35 zinc finger protein gene on 3p21 is of particular interest, as this region of chromosome 3 is frequently involved in rearrangements and/or deletions associated with various human tumors including lung and renal carcinoma. We have analyzed yeast artificial chromosomes (YACs), identified by PCR screening, using oligonucleotides derived from the ZNF35 gene. PFGE and Southern blot/hybridization analysis revealed that the clones cover 750-kb including the ZNF35 gene. The use of specific somatic cell hybrids have allowed us to locate the YAC contig telomeric to the D3F15S2 locus, in a region which is frequently deleted in lung carcinomas. In addition, we have developed a novel cDNA hybridization protocol allowing the isolation of transcribed sequences present in the overlapping YAC clones. Using the cDNA hybridization selection, we have isolated and characterized one transcribed sequence (D3S1362E) from the 3p21 YAC contig and the corresponding cDNA has been isolated. DNA sequencing analysis indicated that the D3S1363E cDNA codes for a putative transcription factor. Northern blot analysis indicated that the D3S1362E sequence hybridized to multiple transcripts in skeletal muscle, and weakly hybridizing transcripts of similar sizes were detected in other tissues.