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Esophageal squamous cell carcinoma (ESCC) is a deadly disease with few prevention or treatment options. ESCC development in humans and rodents is associated with Zn deficiency (ZD), inflammation, and overexpression of oncogenic microRNAs: miR-31 and miR-21. In a ZD-promoted ESCC rat model with upregulation of these miRs, systemic antimiR-31 suppresses the miR-31-EGLN3/STK40-NF-κB–controlled inflammatory pathway and ESCC. In this model, systemic delivery of Zn-regulated antimiR-31, followed by antimiR-21, restored expression of tumor-suppressor proteins targeted by these specific miRs: STK40/EGLN3 (miR-31), PDCD4 (miR-21), suppressing inflammation, promoting apoptosis, and inhibiting ESCC development. Moreover, ESCC-bearing Zn-deficient (ZD) rats receiving Zn medication showed a 47% decrease in ESCC incidence vs. Zn-untreated controls. Zn treatment eliminated ESCCs by affecting a spectrum of biological processes that included downregulation of expression of the two miRs and miR-31-controlled inflammatory pathway, stimulation of miR-21-PDCD4 axis apoptosis, and reversal of the ESCC metabolome: with decrease in putrescine, increase in glucose, accompanied by downregulation of metabolite enzymes ODC and HK2. Thus, Zn treatment or miR-31/21 silencing are effective therapeutic strategies for ESCC in this rodent model and should be examined in the human counterpart exhibiting the same biological processes.

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 Figure S1 from Protein Expression Profiling Identifies Cyclophilin A as a Molecular Target in Fhit-Mediated Tumor Suppression

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 S1 SNAI1 induces mesenchymal genes prior to repression of epithelial genes, concomitant with miR-424 upregulation. (A) Phase contrast images of HMLE Empty Vector and HMLE SNAI1-inducible cells after 4 and 12 days of SNAI1 induction. Bar-100 μm. Epithelial and mesenchymal markers, by qPCR, after (B) 4 and (C) 12 days of SNAI1 induction, two-tailed unpaired t-test. (D) Mature and (E) primary miR-424 levels, by qPCR, after 4 days of SNAI1 induction, two-way ANOVA, Bonferroni multiple comparison post-test. SEM shown, representative images/graphs of n{greater than or equal to}3. #p

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]

Supplementary Figure Legends and Supplementary Materials and Methods

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

Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that miR-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, miR-424 increases motility, decreases adhesion, and induces a growth arrest, changes associated with a complete EMT that can be reversed when miR-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient miR-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, miR-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, miR-424 decreases tumor initiation and is posttranscriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT–MET axis. Next-generation RNA sequencing revealed miR-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK–ERK signaling is critical for miR-424–mediated decreases in tumor-initiating phenotypes. These findings suggest miR-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT–MET axis. Cancer Res; 75(9); 1908–21. ©2015 AACR.

MicroRNA microarray table results after (Table S1) 4 days and (Table S2) 12 days of TWIST1 induction.

Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by progressive narrowing of pulmonary arteries, resulting in right heart failure and death. BMPR2 (bone morphogenetic protein receptor type 2) mutations account for most familial PAH forms whereas reduced BMPR2 is present in many idiopathic PAH forms, suggesting dysfunctional BMPR2 signaling to be a key feature of PAH. Modulating BMPR2 signaling is therapeutically promising, yet how BMPR2 is downregulated in PAH is unclear. Objectives: We intended to identify and pharmaceutically target BMPR2 modifier genes to improve PAH. Methods: We combined siRNA high-throughput screening of >20,000 genes with a multicohort analysis of publicly available PAH RNA expression data to identify clinically relevant BMPR2 modifiers. After confirming gene dysregulation in tissue from patients with PAH, we determined the functional roles of BMPR2 modifiers in vitro and tested the repurposed drug enzastaurin for its propensity to improve experimental pulmonary hypertension (PH). Measurements and Main Results: We discovered FHIT (fragile histidine triad) as a novel BMPR2 modifier. BMPR2 and FHIT expression were reduced in patients with PAH. FHIT reductions were associated with endothelial and smooth muscle cell dysfunction, rescued by enzastaurin through a dual mechanism: upregulation of FHIT as well as miR17-5 repression. Fhit(−/−) mice had exaggerated hypoxic PH and failed to recover in normoxia. Enzastaurin reversed PH in the Sugen5416/hypoxia/normoxia rat model, by improving right ventricular systolic pressure, right ventricular hypertrophy, cardiac fibrosis, and vascular remodeling. Conclusions: This study highlights the importance of the novel BMPR2 modifier FHIT in PH and the clinical value of the repurposed drug enzastaurin as a potential novel therapeutic strategy to improve PAH.

// 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.

Significance ESCC is a deadly disease with few prevention or treatment options. In a dietary Zn deficiency-promoted rat ESCC model with miR-31–controlled inflammation pathway up-regulation, systemic antimiR-31 reduces miR-31 level and inflammation, suppressing ESCC development. We identified Egln3 as a direct miR-31 target and developed a constitutive miR-31 knockout rat model. Interactions of oncogenic miR-31, EGLN3 down-regulation, and inflammation occur in rat and human ESCC tissue. Since miR-31 genetic knockout prevents Zn deficiency-associated ESCC by eliminating or greatly reducing the entire miR-31-EGLN3/STK40-NF-κB–controlled inflammatory process, we conclude that miR-31 and downstream signaling proteins should be tested as prognostic and therapeutic targets and inexpensive Zn supplementation should be implemented for ESCC prevention in deficient populations., MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden following N-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P = 0.038) and miR-31 gene knockout abrogated development of ESCC (P = 1 × 10−6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB–controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31–associated EGLN3/NF-κB–controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31−/− rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development.

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.

Significance Prostate cancer in man is associated with Zn loss, citrate metabolite reduction, overexpression of the miR-183-96-182 cluster, and regulation of Zn homeostasis through Zn transporter suppression. Our mechanistic study shows that a low-Zn diet upregulates this miR cluster in Zn-deficient middle-aged rat prostate, with ZIP1 mRNA/protein downregulation and a citrate-oxidizing metabolic phenotype, linking citrate reduction directly to prostatic Zn loss. The findings of this study show that the transcriptional and metabolic signal pathways induced by Zn deficiency in rats and almost certainly in men are critical for the development of human and rat prostate cancer and provide a strong rationale for including Zn supplementation in clinical trials to reduce the prostate cancer burden in the human population., Prostate cancer is a leading cause of cancer death in men over 50 years of age, and there is a characteristic marked decrease in Zn content in the malignant prostate cells. The cause and consequences of this loss have thus far been unknown. We found that in middle-aged rats a Zn-deficient diet reduces prostatic Zn levels (P = 0.025), increases cellular proliferation, and induces an inflammatory phenotype with COX-2 overexpression. This hyperplastic/inflammatory prostate has a human prostate cancer-like microRNA profile, with up-regulation of the Zn-homeostasis–regulating miR-183-96-182 cluster (fold change = 1.41–2.38; P = 0.029–0.0003) and down-regulation of the Zn importer ZIP1 (target of miR-182), leading to a reduction of prostatic Zn. This inverse relationship between miR-182 and ZIP1 also occurs in human prostate cancer tissue, which is known for Zn loss. The discovery that the Zn-depleted middle-aged rat prostate has a metabolic phenotype resembling that of human prostate cancer, with a 10-fold down-regulation of citric acid (P = 0.0003), links citrate reduction directly to prostatic Zn loss, providing the underlying mechanism linking dietary Zn deficiency with miR-183-96-182 overexpression, ZIP1 down-regulation, prostatic Zn loss, and the resultant citrate down-regulation, changes mimicking features of human prostate cancer. Thus, dietary Zn deficiency during rat middle age produces changes that mimic those of human prostate carcinoma and may increase the risk for prostate cancer, supporting the need for assessment of Zn supplementation in its prevention.

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.

FHIT, located at FRA3B, is one of the most commonly deleted genes in human cancers, and loss of FHIT protein is one of the earliest events in cancer initiation. However, location of FHIT at a chromosomal fragile site, a locus prone to breakage and gap formation under even mild replication stress, has encouraged claims that FHIT loss is a passenger event in cancers. We summarize accumulated evidence that FHIT protein functions as a genome “caretaker” required to protect the stability of genomes of normal cells of most tissues from agents causing intrinsic and extrinsic DNA damage. FHIT loss leads to intracellular replication stress and subsequent genome instability, which provides an opportunistic mutational landscape in preneoplasias for selection of a variety of other cancer-driving mutations. We also review evidence showing that FHIT loss leads to enhanced activation of other common fragile sites, including the FRA16D/WWOX locus, and creates optimal single-stranded DNA substrates for the hypermutator enzyme, APOBEC3B.

Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that miR-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, miR-424 increases motility, decreases adhesion, and induces a growth arrest, changes associated with a complete EMT that can be reversed when miR-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient miR-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, miR-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, miR-424 decreases tumor initiation and is posttranscriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT–MET axis. Next-generation RNA sequencing revealed miR-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK–ERK signaling is critical for miR-424–mediated decreases in tumor-initiating phenotypes. These findings suggest miR-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT–MET axis. Cancer Res; 75(9); 1908–21. ©2015 AACR.

Background FHIT is a genome caretaker/tumor suppressor that is silenced in >50% of cancers. Although it was identified more than 20 years ago, questions remain as to how FHIT loss contributes to cancer, and conversely, how FHIT acts to maintain genome integrity and suppress malignancy. Fhit belongs to the histidine triad family of enzymes that catalyze the degradation of nucleoside 5′,5′-triphosphates, including the m7GpppN ‘caps’ that are generated when mRNAs undergo 3′-5′ decay. This raised the possibility that Fhit loss might affect changes in the translation of cancer-associated mRNAs, possibly as a consequence of increased intracellular concentrations of these molecules. Results Ribosome profiling identified several hundred mRNAs for which coding region ribosome occupancy changed as a function of Fhit expression. While many of these changes could be explained by changes in mRNA steady-state, a subset of these showed changes in translation efficiency as a function of Fhit expression. The onset of malignancy has been linked to changes in 5’-UTR ribosome occupancy and this analysis also identified ribosome binding to 5′-untranslated regions (UTRs) of a number of cancer-associated mRNAs. 5’-UTR ribosome occupancy of these mRNAs differed between Fhit-negative and Fhit-positive cells, and in some cases these differences correlated with differences in coding region ribosome occupancy. Conclusions In summary, these findings show Fhit expression impacts the translation of a number of cancer associated genes, and they support the hypothesis that Fhit’s genome protective/tumor suppressor function is associated with post-transcriptional changes in expression of genes whose dysregulation contributes to malignancy. Electronic supplementary material The online version of this article (10.1186/s12943-017-0749-x) contains supplementary material, which is available to authorized users.

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.

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1 -KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1 -KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable “repair enzyme” to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.

WWOX, the WW domain-containing oxidoreductase gene at chromosome region 16q23.3–q24.1, spanning chromosomal fragile site FRA16D, encodes the 46 kDa Wwox protein, a tumor suppressor that is lost or reduced in expression in a wide variety of cancers, including breast, prostate, ovarian, and lung. The function of Wwox as a tumor suppressor implies that it serves a function in the prevention of carcinogenesis. Indeed, in vitro studies show that Wwox protein interacts with many binding partners to regulate cellular apoptosis, proliferation, and/or maturation. It has been reported that newborn Wwox knockout mice exhibit nascent osteosarcomas while Wwox+/− mice exhibit increased incidence of spontaneous and induced tumors. Furthermore, absence or reduction of Wwox expression in mouse xenograft models results in increased tumorigenesis, which can be rescued by Wwox re-expression, though there is not universal agreement among investigators regarding the role of Wwox loss in these experimental models. Despite this proposed tumor suppressor function, the overlap of the human WWOX locus with FRA16D sensitizes the gene to protein-inactivating deletions caused by replication stress. The high frequency of deletions within the WWOX locus in cancers of various types, without the hallmark protein inactivation-associated mutations of “classical” tumor suppressors, has led to the proposal that WWOX deletions in cancers are passenger events that occur in early cancer progenitor cells due to fragility of the genetic locus, rather than driver events which provide the cancer cell a selective advantage. Recently, a proposed epigenetic cause of chromosomal fragility has suggested a novel mechanism for early fragile site instability and has implications regarding the involvement of tumor suppressor genes at chromosomal fragile sites in cancer. In this review, we provide an overview of the evidence for WWOX as a tumor suppressor gene and put this into the context of fragility associated with the FRA16D locus.

// Catherine E. Waters 1, 2 , Joshua C. Saldivar 3 , Zaynab A. Amin 2 , Morgan S. Schrock 1, 2 , Kay Huebner 2 1 Biomedical Sciences Graduate Program, Ohio State University Wexner Medical Center, Columbus, Ohio 2 Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University Wexner Medical Center, Columbus, 43210, Ohio 3 Department of Chemical and Systems Biology, Stanford University School of Medicine, Standford, 94305, California Correspondence to: Kay Huebner, e-mail: kay.huebner@osumc.edu Keywords: FHIT, replication stress, DNA double strand breaks, APOBEC deaminase, mutations Received: August 14, 2014 Accepted: October 23, 2014 Published: October 31, 2014 ABSTRACT APOBEC cytidine deaminase activity is a major source of hypermutation in cancer. But previous studies have shown that the TC context signature of these enzymes is not observed in sizable fractions of cancers with overexpression of APOBEC, suggesting that cooperating factors that contribute to this mutagenesis should be identified. The fragile histidine triad protein (Fhit) is a tumor suppressor and DNA caretaker that is deleted or silenced in >50% of cancers. Loss of Fhit protein activity causes replication stress through reduced Thymidine Kinase 1 expression, increased DNA breaks, and global genome instability in normal and cancer cells. Using data from The Cancer Genome Atlas (TCGA), we show that FHIT -low/ APOBEC3B -high expressing lung adenocarcinomas display significantly increased numbers of APOBEC signature mutations. Tumor samples in this cohort with normal FHIT expression do not exhibit APOBEC hypermutation, despite having high APOBEC3B expression. In vitro , silencing Fhit expression elevates APOBEC3B-directed C > T mutations in the TP53 gene. Furthermore, inhibition of Fhit loss-induced DNA damage via thymidine supplementation decreases the TP53 mutation burden in FHIT -low/ APOBEC3B -high cells. We conclude that APOBEC3B overexpression and Fhit-loss induced DNA damage are independent events that, when occurring together, result in a significantly increased frequency of APOBEC-induced mutations that drive cancer progression.

The fragile FHIT gene, encompassing the chromosomal fragile site FRA3B, is an early target of DNA damage in precancerous cells. While vulnerable to DNA damage itself, FHIT protein expression is essential to protect from DNA damage-induced cancer initiation and progression by modulating genome stability, oxidative stress and levels of accumulating DNA damage. Thus, FHIT, whose expression is lost or reduced in many human cancers, is a tumor suppressor and genome caretaker whose loss initiates genome instability in preneoplastic lesions. Ongoing studies are seeking more detailed understanding of the role of FHIT in the cellular response to oxidative damage. This review discusses the relationship between FHIT, reactive oxygen species production, and DNA damage in the context of cancer initiation and progression.

Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.

Cell survival after DNA damage relies on DNA repair, the abrogation of which causes genomic instability and development of cancer. However, defective DNA repair in cancer cells can be exploited for cancer therapy using DNA-damaging agents. DNA double-strand breaks are the major lethal lesions induced by ionizing radiation (IR) and can be efficiently repaired by DNA homologous recombination, a system that requires numerous factors including the recombinase RAD51 (RAD51). Therapies combined with adjuvant radiotherapy have been demonstrated to improve the survival of triple-negative breast cancer patients; however, such therapy is challenged by the emergence of resistance in tumor cells. It is, therefore, essential to develop novel therapeutic strategies to overcome radioresistance and improve radiosensitivity. In this study we show that overexpression of microRNA 155 (miR-155) in human breast cancer cells reduces the levels of RAD51 and affects the cellular response to IR. miR-155 directly targets the 3′-untranslated region of RAD51. Overexpression of miR-155 decreased the efficiency of homologous recombination repair and enhanced sensitivity to IR in vitro and in vivo. High miR-155 levels were associated with lower RAD51 expression and with better overall survival of patients in a large series of triple-negative breast cancers. Taken together, our findings indicate that miR-155 regulates DNA repair activity and sensitivity to IR by repressing RAD51 in breast cancer. Testing for expression levels of miR-155 may be useful in the identification of breast cancer patients who will benefit from an IR-based therapeutic approach.

// Pierluigi Gasparini 1,* , Luciano Cascione 1,8,* , Matteo Fassan 1,2,* , Francesca Lovat 1 , Gulnur Guler 3 , Serdar Balci 4 , Cigdem Irkkan 5 , Carl Morrison 6 , Carlo M. Croce 1 , Charles L. Shapiro 7 , Kay Huebner 1 1 Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Wexner Medical Center and Comprehensive Cancer Center, Columbus, Ohio, USA 2 ARC-NET Research Centre, University and Hospital Trust of Verona, Verona, Italy 3 Department of Pathology, Hacettepe University, Ankara,Turkey 4 Department of Pathology, Yildirim Beyazit University, Ankara Ataturk Research and Training Hospital, Ankara, Turkey 5 Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital of the Ministry of Health 6 Department of Pathology, Roswell Park Cancer Institute, Basic Science Building, Elm and Carlton Streets Buffalo, NY 7 Division of Medical Oncology and the Breast Program James Cancer Hospital and Ohio State University Comprehensive Cancer Center, Columbus, Ohio, USA 8 IOR, Institute of Oncology Research, Bellinzona, Switzerland * These authors contributed equally to the work Correspondence: Pierluigi Gasparini, email: // Keywords : Triple Negative breast cancer, microRNA, five markers, prognosis, treatments, outcome Received : December 11, 2013 Accepted : January 19, 2014 Published : January 21, 2014 Abstract Triple Negative Breast Cancers (TNBC) is a heterogeneous disease at the molecular and clinical level with poor outcome. Molecular subclassification of TNBCs is essential for optimal use of current therapies and for development of new drugs. microRNAs (miRNA) are widely recognized as key players in cancer progression and drug resistance; investigation of their involvement in a TNBC cohort may reveal biomarkers for diagnosis and prognosis of TNBC. Here we stratified a large TNBC cohort into Core Basal (CB, EGFR and/or CK5, 6 positive) and five negative (5NP) if all markers are negative. We determined the complete miRNA expression profile and found a subset of miRNAs specifically deregulated in the two subclasses. We identified a 4-miRNA signature given by miR-155, miR-493, miR-30e and miR-27a expression levels, that allowed subdivision of TNBCs not only into CB and 5NP subgroups (sensitivity 0.75 and specificity 0.56; AUC=0.74) but also into high risk and low risk groups. We tested the diagnostic and prognostic performances of both the 5 IHC marker panel and the 4-miRNA expression signatures, which clearly identify worse outcome patients in the treated and untreated subcohorts. Both signatures have diagnostic and prognostic value, predicting outcomes of patient treatment with the two most commonly used chemotherapy regimens in TNBC: anthracycline or anthracycline plus taxanes. Further investigations of the patients’ overall survival treated with these regimens show that regardless of IHC group subdivision, taxanes addition did not benefit patients, possibly due to miRNA driven taxanes resistance. TNBC subclassification based on the 5 IHC markers and on the miR-155, miR-493, miR-30e, miR-27a expression levels are powerful diagnostic tools. Treatment choice and new drug development should consider this new subtyping and miRNA expression signature in planning low toxicity, maximum efficacy therapies.