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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

PDF file - 82K, GBM Patient Data (S1); EGFRvIII and CD133 expression in GBM Neurospheres or Normal Brain (NB) derived Neurospheres (S2); Percentage of co-localization of EGFRvIII+ with Markers of Differentiation and Stem/Progenitor Cells (S3).

PDF file - 3725K, Gating Tree for Fluorescence Activated Cell Sorting (FACS) Analysis (S1); Vessel Staining of EGFRvIII in GBM Xenografts (S2); Characterization and Affinity Analysis of the Recombinant Antibodies (S3); Antibody Dependent Cellular Cytotoxicity Induced by BsAb (S4); Limiting Dilution Analysis of Unsorted Tumor Spheres Before and After BsAb Induced ADCC (S5).

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII+/CD133+ defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII+ cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII− cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII+/CD133+ coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII+/CD133+ population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population. Cancer Res; 74(4); 1238–49. ©2013 AACR.

To investigate family-basedBlood samples and survey questionnaires were collected from 282 families including 772 individuals. The recruited families were from 10 selected communities in the greater Zhengzhou area with different living standards, and the family members' general data,Among the 772 individuals examined,In our study sample from the general public of central China

Exosomes play important roles in intercellular communication through signaling pathways affecting tumor microenvironment modulation and tumor proliferation, including those in glioblastoma (GBM). As yet, however, limited studies have been conducted on the inhibitory effect of human bone marrow-derived mesenchymal stem cell (hBMSC)-derived exosomes on GBM development. Therefore, we set out to assess the role of hBMSC secreted exosomes, in particular those carrying microRNA-34a (miR-34a), in the development of GBM. Microarray-based expression analysis was employed to identify differentially expressed genes and to predict miRNAs regulating MYCN expression. Next, hBMSCs were transfected with a miR-34a mimic or inhibitor after which exosomes were isolated. Proliferation, apoptosis, migration, invasion and temozolomide (TMZ) chemosensitivity of exosome-exposed GBM cells (T-98G, LN229 and A-172) were measured in vitro. The mechanism underlying MYCN regulation was investigated using lentiviral transfections. The in vivo inhibitory effect of exosomal miR-34a was measured in nude mice xenografted with GBM cells through subcutaneous injection of hBMSCs with an upregulated miR34a content. We found that poorly-expressed miR-34a specifically targeted and negatively regulated the expression of MYCN in GBM cells. In addition we found that miR-34a was delivered to T-98G, LN229 and A-172 GBM cells via hBMSC-derived exosomes. Exogenous overexpression of miR-34a in hBMSC-derived exosomes resulted in inhibition of GBM cell proliferation, invasion, migration and tumorigenesis in vitro and in vivo, while promoting the chemosensitivity of GBM cells to TMZ by silencing MYCN. From our data we conclude that hBMSC-derived exosomes overexpressing miR-34a may be instrumental for the therapeutic targeting and clinical management of GBM.

Significant progresses have been made in adoptive cell therapy with CAR-T cells for cancers, especially for hematological malignancies. However, the treatment of solid tumors still poses a tremendous challenge and remains an unmet medical need. Several factors are held responsible for the inadequate responses: tumor heterogeneity, inefficient homing of T cells to tumor tissues, immunosuppressive microenvironment and the shortage of specific antigens shortage. Mesothelin is a cell-surface glycoprotein highly expressed in many types of solid tumors. As such, it has attracted much attention as a molecular target in cancer immunotherapy. Here, we delineate the barriers imposed by solid tumors on CARs, outline the rationale of mesothelin as a target for immunotherapy, summarize the preclinical and clinical results of mesothelin-targeted therapies, and extrapolate the expected results of CAR-T cells directed against mesothelin for solid tumors.

Adoptive cellular immunotherapy employing chimeric antigen receptors-modified T (CAR-T) cells has demonstrated promising antitumor effects in hematologic cancers. However, CAR-T therapy confront many challenges in solid tumors like immunosuppressive microenvironment, molecular heterogeneity, etc. The cancer genome atlas (TCGA) of hepatocellular carcinoma (HCC) revealed many genetic characteristic and molecular tumorigenesis. EGFRvIII is a tumor specific antigen widely expressed in a variety of cancers including HCC and an ideal therapeutic target for cancer therapy. The liver cancer cell line SMMC7721 express high level EGFRvIII and widely applied in HCC investigations. Herein, we developed EGFRvIII CAR-T cells by piggyBac transposon system, and detected its specific killing effect against SMMC7721 cells in vitro and in vivo. Results indicated that transduction efficiency of CAR reached 53.1%. Expression of CAR protein was verified by immunoblotting as a band of approximate 57KD. The killing effect of CAR-T cells against SMMC7721 was positively correlated with E/T ratio (E:T=5:1, 10:1, 20:1, 40:1), and exceeded 50% at 20:1 ratio. Significant increase in IFN-γ and TNF-α secretion were detected in the co-culture supernatant of CAR-T cells and SMMC7721, comparable to the level of exogenous EGFRvIII-expressing U87 cells. The killing activity and cytokine secretion were both dependent on the expression level of EGFRvIII in target cells. In HCC xenograft models, CAR-T cells could effectively suppress the growth of SMMC7721. In conclusion, EGFRvIII CAR-T cells demonstrated specific antitumor effect against SMMC7721 in vitro and in vivo, providing basis for immunotherapy of HCC in future clinical use.

Malignant gliomas are aggressive primary neoplasms that originate in the glial cells of the brain or the spine with notable resistance to standard treatment options. We carried out the study with the aim to shed light on the sensitization of resveratrol to temozolomide (TMZ) against glioma through the Wnt signaling pathway. Initially, glioma cell lines with strong resistance to TMZ were selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the glioma cells were subjected to resveratrol, TMZ, Wnt signaling pathway inhibitors, and activators. Cell survival rate and inhibitory concentration at half maximum value were detected by MTT, apoptosis by flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, in vitro proliferation by hanging drop method and β-catenin translocation into nuclei by TOP/FOP-FLASH assay. The expressions of the Wnt signaling pathway-related and apoptosis-related factors were determined by western blot analysis. Nude mice with glioma xenograft were established to detect tumorigenic ability. Glioma cell lines T98G and U138 which were highly resistant to TMZ were selected for subsequent experiments. Resveratrol increased the efficacy of TMZ by restraining cell proliferation, tumor growth, and promoting cell apoptosis in glioma cells. Resveratrol inhibited Wnt2 and β-catenin expressions yet elevated GSK-3β expression. Moreover, the Wnt signaling pathway participates in the sensitivity enhancing of resveratrol to TMZ via regulating O 6 -methylguanine-DNA methyltransferase (MGMT) expression. Resveratrol sensitized TMZ-induced glioma cell apoptosis by repressing the activation of the Wnt signaling pathway and downregulating MGMT expression, which may confer new thoughts to the chemotherapy of glioma.

Aims Our study aims to investigate the effect of microRNA-21 (miR-21) on the proliferation, senescence, and apoptosis of glioma cells by targeting SPRY1 via the PTEN/PI3K/AKT signaling pathway. Methods Glioma tissues and brain tissues were collected for this study after surgical decompression for traumatic brain injury. RT-qPCR was employed to measure mRNA levels of miR-21, SPRY1, PTEN, PI3K, and AKT, and Western blotting was conducted to determine protein levels of SPRY1, PTEN, PI3K, AKT, p-AKT, Caspase-3, Caspase-9, P53, GSK3, and p-GSK3. Human glioma U87 cells were assigned into the blank, negative control (NC), miR-21 mimics, miR-21 inhibitors, siRNA-SPRY1, and miR-21 inhibitors + siRNA-SPRY1 groups, with human HEB cells serving as the normal group. Cell proliferation, cell cycle, and apoptosis were determined by MTT and flow cytometry, respectively. Results Compared with control group, an increased expression of miR-21, PI3K, AKT, p-AKT, P53, and p-GSK3, and a decreased expression of SPRY1, PTEN, Caspase-3, and Caspase-9 were observed in the glioma group, and no significant differences were found in the expression of GSK3. SPRY1 was verified to be the target gene of miR-21. Compared with the blank and NC groups, levels of PI3K, AKT, p-AKT, P53, and p-GSK3 increased while levels of SPRY1, PTEN, Caspase-3, and Caspase-9 decreased in the miR-21 mimics and siRNA-SPRY1 groups; the miR-21 inhibitors group reversed the tendency; furthermore, the miR-21 inhibitors group showed decreased cell proliferation but promoted apoptosis, which were opposite to the results of the miR-21 mimics and siRNA-SPRY1 groups. Conclusion MicroRNA-21 might promote cell proliferation and inhibit cell senescence and apoptosis of human glioma cells by targeting SPRY1 via the PTEN/PI3K/AKT signaling pathway.

Noninvasive diagnosis of Helicobacter pylori (H. pylori) infection is very attractive. This study investigated the single strand DNA (ssDNA) acquisition method from H. pylori in dental plaque, and the integration of our previously developed 43-mer H. pylori DNA biosensor with the obtained target ssDNA (tDNA). Dental plaque samples were collected from 34 patients/volunteers, whose gastric H. pylori infection statuses were tested with the 13C urea breath test (UBT). The samples were treated with colony polymerase chain reaction (PCR) to obtain double strand DNA (dsDNA) of 104 basepairs (bp) long. A blocker ssDNA was designed and used in thermal treatment of the dsDNA to release the 104-mer tDNA, which contains the 43-mer DNA sequence in the middle. PCR primers were designed, and the tDNA releasing and detection conditions with the biosensor were optimized. The limit of detection with the biosensor was 12 fM dsDNA. The dental plaque detection results correlated quite well with the UBT results, with a sensitivity of 100%, and specificity of 97%. These results indicate that the residence of H. pylori in dental plaque is highly associated with gastric H. pylori infection, and detection of dental plaque samples with our DNA biosensor is promisingly applicable in noninvasive diagnosis of H. pylori infection.

Exosomes play important roles in intercellular communication through signaling pathways affecting tumor microenvironment modulation and tumor proliferation, including those in glioblastoma (GBM). As yet, however, limited studies have been conducted on the inhibitory effect of human bone marrow-derived mesenchymal stem cell (hBMSC)-derived exosomes on GBM development. Therefore, we set out to assess the role of hBMSC secreted exosomes, in particular those carrying microRNA-34a (miR-34a), in the development of GBM.Microarray-based expression analysis was employed to identify differentially expressed genes and to predict miRNAs regulating MYCN expression. Next, hBMSCs were transfected with a miR-34a mimic or inhibitor after which exosomes were isolated. Proliferation, apoptosis, migration, invasion and temozolomide (TMZ) chemosensitivity of exosome-exposed GBM cells (T-98G, LN229 and A-172) were measured in vitro. The mechanism underlying MYCN regulation was investigated using lentiviral transfections. The in vivo inhibitory effect of exosomal miR-34a was measured in nude mice xenografted with GBM cells through subcutaneous injection of hBMSCs with an upregulated miR34a content.We found that poorly-expressed miR-34a specifically targeted and negatively regulated the expression of MYCN in GBM cells. In addition we found that miR-34a was delivered to T-98G, LN229 and A-172 GBM cells via hBMSC-derived exosomes. Exogenous overexpression of miR-34a in hBMSC-derived exosomes resulted in inhibition of GBM cell proliferation, invasion, migration and tumorigenesis in vitro and in vivo, while promoting the chemosensitivity of GBM cells to TMZ by silencing MYCN.From our data we conclude that hBMSC-derived exosomes overexpressing miR-34a may be instrumental for the therapeutic targeting and clinical management of GBM.

Adoptive immunotherapy using chimeric antigen receptors-modified T cells (CAR-T) is a promising approach for cancer treatment. However, CARs currently applied in the clinics cannot be effectively regulated and the safety of CAR-T cell therapies remains a major concern. To improve the safety of CAR-T cells, we designed a synthetic splitting CAR (ssCAR) that can regulate T cell functions exogenously. Epidermal growth factor receptor variant III (EGFRvIII) was used as a molecular target for ssCAR. Our results indicate that both EGFRvIII and small molecule are needed for the activation of the ssCAR-T cells. AP21967 dose-dependently increased the expression of T cell activation, production of cytokines and extent of cell lysis. In conclusion, the gene switch designed in this study allows for temporal and spatial control over engineered T cells in a dose-and time-dependent manner by AP21967. Our work demonstrates the feasibility and improved safety profile of this novel treatment approach.

Noninvasive diagnosis of

Objective: The objective of this study was to evaluate the clinical value of endoscopic submucosal dissection (ESD) in the treatment of early esophageal cancer and precancerous lesions. Materials and Methods: We retrospectively analyzed 58 patients who suffered from early esophageal and precancerous lesions and received ESD in the First Affiliated Hospital of Zhengzhou University from February 2012 to January 2016. The clinical efficacy and safety of ESD in treating the early esophageal cancer and precancerous lesions was evaluated by analyzing the operation successful rate, postoperative pathology, complications, and follow-up data of patients who received ESD. Results: For the 58 patients, ESD was successfully completed in 56 cases with a success rate of 96.6%, whereas ESD was unsuccessful in 2 cases. Invasive lesions were observed in the esophageal muscular layer of 1 patient. Consequently, surgery was terminated and this patient was transferred to thoracotomy surgical intervention involving radical resection of esophageal cancer. Esophageal perforation was observed during the annular incision of the esophageal mucosa in another patient with early-stage cancer. This perforation was occluded with an endoscopic titanium clip and surgery was terminated. Intraoperative blood loss in 56 patients was ranged from 10 to 90 mL with an average of 28.3 ± 17.2 mL. The diameter of ESD resection lesion was varied from 2 to 6.0 cm with an average of 3.4 ± 1.1 cm. For the 56 patients, enbloc resection was performed in 50 patients, with an enbloc resection rate of 89.3%. Complete lesion resection was performed in 49 patients, with a complete resection rate of 87.5%. For all patients, 36 manifested with severe atypical hyperplasia confirmed by postoperative pathology, 11 showed moderate atypical hyperplasia, 2 showed carcinoma insitu, and 7 presented with esophageal squamous cell carcinoma. In these 7 patients, 6 patients whose lesions limited to their mucosa were in the early stage of cancer while 1 patient with esophageal cancer involving the incisal edge, and the submucosal layer was subjected to additional surgical treatment. In addition, 1 patient experienced postoperative delayed hemorrhage (1.79%), 6 patients suffered from fever (10.71%), 33 patients reported substernal burning pain (58.93%) that mostly lasted 1–2 days before spontaneous remission, 1 patient was observed intraoperative perforation (1.79%), and 3 patients showed postoperative esophageal stenosis (5.36%), received multiple balloon dilatations, and consumed fluids afterward. Follow-up visits were facilitated for 49 patients for more than 1 year, and their median follow-up time was 36 months. Of these patients, recurrence was observed in 3 patients, with a recurrence rate of 6.1% (3/49). Of these 3 patients, 2 received surgical treatment and 1 underwent another endoscopic lesion resection. No patient died of esophageal cancer during follow-up. Conclusion: ESD was safe and reliable for the treatment of early esophageal cancer and precancerous lesions, and its recurrence and complication rates were low. Complete pathological information could be obtained after operation, which could be applied to assess patients' condition accurately.

// Yan Zheng 1, * , Xiu-Ling Li 1, * , Xiang-Dong Sun 1 , Bing-Yong Zhang 1 , Puja Gupta 2 , Albert J Wong 2 , Tian-Fang Li 3 and Shuang-Yin Han 1 1 Translational Research Center, People’s Hospital of Henan Province, Zhengzhou University, Zhengzhou 450003, China 2 Brain Tumor Research Laboratories, Department of Neurosurgery, Stanford University Medical Center, Stanford, CA 94305, USA 3 The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450003, P.R. China * These authors contribute equally to this work Correspondence to: Shuang-Yin Han, email: hansy001@henu.edu.cn Tian-Fang Li, email: tfli@zzu.edu.cn Keywords: chimeric antigen receptor; regulation; glioblastoma Received: July 14, 2017 Accepted: October 30, 2017 Published: January 02, 2018 ABSTRACT Adoptive immunotherapy using chimeric antigen receptors-modified T cells (CAR-T) is a promising approach for cancer treatment. However, CARs currently applied in the clinics cannot be effectively regulated and the safety of CAR-T cell therapies remains a major concern. To improve the safety of CAR-T cells, we designed a synthetic splitting CAR (ssCAR) that can regulate T cell functions exogenously. Here, ssCAR was constructed by splitting recognition and key signaling module into distinct parts, mimicking the natural dual signal input of T cell activation. These separated parts form heterodimer upon stimulation by a small molecule with subsequent activation of ssCAR-T cells. Epidermal growth factor receptor variant III (EGFRvIII), a tumor specific antigen highly expressed in glioblastoma and other human malignancies, was used as a molecular target for ssCAR. Our results indicate that both EGFRvIII and small molecule are needed for the activation of the ssCAR-T cells. AP21967 dose-dependently increased the expression of T cell activation marker CD69, production of cytokines IFN-γ and TNF-α, and extent of cell lysis. When co-cultured with EGFRvIII + U87 in the presence of 500 nM AP21967, ssCAR-T cells showed a significant increase in cytokine production and targeted cells killing capacity to a level comparable to conventional CAR. Increasing the duration of treatment induced a simultaneous up-regulation of apoptotic rate of tumor cells. In conclusion, the gene switch designed in this study allows for temporal and spatial control over engineered T cells in a dose-and time-dependent manner by AP21967. Our work demonstrates the feasibility and improved safety profile of this novel treatment approach.

T-cell therapy using genetically engineered T cells modified with either T cell receptor or chimeric antigen receptor holds great promise for cancer immunotherapy. The concerns about its toxicities still remain despite recent successes in clinical trials. Temporal and spatial control of the engineered therapeutic T cells may improve the safety profile of these treatment regimens. To achieve these goals, numerous approaches have been tested and utilized including the incorporation of a suicide gene, the switch-mediated activation, the combinatorial antigen recognition, etc. This review will summarize the toxicities caused by engineered T cells and novel strategies to overcome them.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation widely expressed in glioma. However, its role and molecular mechanism in glioma have not been completely elucidated. Immunohistochemistry analyses of EGFRvIII, enhancer of zeste homolog 2 (EZH2) and aplysia ras homolog I (ARHI) were performed in tumor tissues from patients with glioma. Regulatory mechanisms among EGFRvIII, EZH2 and ARHI were examined by western blot and chromatin immunoprecipitation (ChIP). Cell proliferation and migration of glioma cells were examined. EGFRvIII and EZH2 expression were upregulated, while ARHI was downregulated in glioma tissues. EZH2 knockdown increased ARHI expression in glioma cell lines. ChIP assay suggested that EZH2 was enriched in the ARHI promoter. Furthermore, ectopic expression of EGFRvIII upregulated EZH2, suppressed ARHI expression, and promoted glioma cell proliferation. Additionally, treatment with 3-deazaneplanocin A (DZNep, an inhibitor of EZH2) inhibited expression of EZH2, increased protein level of ARHI, and partially abrogated the promoting effects of ARHI knockdown on glioma cell proliferation and migration. In summary, EGFRvIII-mediated epigenetic suppression of ARHI promoted glioma cell proliferation and migration via upregulating EZH2.

Chronic exposure to nickel compounds is associated with increased incidence of certain types of human cancer, including lung and nasal cancers. Despite intensive investigation, the oncogenic processes remain poorly understood. Apoptosis resistance is a key feature for tumor cells to escape physiological surveillance and acquire growth advantage over normal cells. Although NiCl2 exposure induces transformation of human lung epithelial cells, little information is available with regard to its molecular mechanisms, it is also not clear if the transformed cells are apoptosis resistant and tumorigenic. We explored the apoptosis resistance properties of nickel chloride‑transformed human lung epithelial cells and the underlying mechanisms. The results showed that transformed BEAS-2B human lung epithelial cells are resistant to NiCl2-induced apoptosis. They have increased Bcl-2, Bcl-xL and catalase protein levels over the passage matched non‑transformed counterparts. The mechanisms of apoptosis resistance are mitochondria‑mediated and caspase-dependent. Forced overexpression of Bcl-2, Bcl-xL and catalase proteins reduced NiCl2-induced cell death; siRNA‑mediated knockdown of their expression sensitized the cells to nickel-induced apoptosis, suggesting that Bcl-2, Bcl-xl and catalase protein expression plays a critical role in apoptosis resistance. Akt also participates in this process, as its overexpression increases Bcl-xL protein expression levels and attenuates NiCl2-induced apoptosis. Furthermore, transformed cells are tumorigenic in a xenograft model. Together, these results demonstrate that nickel-transformed cells are apoptosis‑resistant and tumorigenic. Increased expression of Bcl-2, Bcl-xL and catalase proteins are important mechanisms contributing to transformed cell oncogenic properties.

Glioblastoma (GBM) is one of the most devastating brain tumors with poor prognosis and high mortality. Although radical surgical treatment with subsequent radiation and chemotherapy can improve the survival, the efficacy of such regimens is insufficient because the GBM cells can spread and destroy normal brain structures. Moreover, these non-specific treatments may damage adjacent healthy brain tissue. It is thus imperative to develop novel therapies to precisely target invasive tumor cells without damaging normal tissues. Immunotherapy is a promising approach due to its capability to suppress the growth of various tumors in preclinical model and clinical trials. Adoptive cell therapy (ACT) using T cells engineered with chimeric antigen receptor (CAR) targeting an ideal molecular marker in GBM, e.g. epidermal growth factor receptor type III (EGFRvIII) has demonstrated a satisfactory efficacy in treating malignant brain tumors. Here we summarize the recent progresses in immunotherapeutic strategy using CAR-modified T cells oriented to EGFRvIII against GBM.

Background To evaluate plasma chaperonin containing TCP1 complex subunit 3 (CCT3) and IQ-motif-containing GTPase-activating protein-3 (IQGAP3) as biomarker for hepatocellular carcinoma (HCC) screening and diagnosis. Methods Blood samples were collected from 126 HCC patients with HCC, 88 patients with cirrhosis and 50 healthy volunteers to detect plasma α-fetoprotein (AFP), CCT3 and IQGAP3 levels. Plasma AFP, CCT3 and IQGAP3 protein levels were measured by enzyme linked immunosorbent assay (ELISA). Results In the plasma of HCC patients, both CCT3 and IQGAP3 were significantly higher than in patients with cirrhosis and in healthy controls (P

The mammalian Nit1 protein is homologous to plant and bacterial nitrilases. In flies and worms, Nit1 is fused to the 5' end of Fhit, suggesting that Nit1 may functionally interact with the Fhit pathway. Fhit has been shown to play a role of a tumor suppressor. Somatic loss of Fhit in human tissues is associated with a wide variety of cancers. Deletion of Fhit results in a predisposition to induced and spontaneous tumors in mice. It has been suggested that Nit1 collaborates with Fhit in tumor suppression. Similar to mice lacking Fhit, Nit1-deficient mice are more sensitive to carcinogen-induced tumors. It was previously shown that ectopic expression of Nit1 or Fhit led to caspase activation and apoptosis, and that both proteins may play a role in DNA damage-induced apoptosis. In this study, we analyzed the physiological function of Nit1 in T cells using Nit1-knockout mice. Nit1-deficient T cells can undergo apoptosis induced by DNA damage due to irradiation and chemical treatment. However, apoptosis induced by Fas or Ca(++) signals appeared to be compromised. Additionally, Nit1 deficiency resulted in T cell hyperproliferative responses induced by TCR stimulation. The expressions of T cell activation markers were elevated in Nit1(-/-) T cells. There was a spontaneous cell cycle entry and enhanced cell cycle progression in Nit1(-/-) T cells. These data indicate that Nit1 is a novel negative regulator in primary T cells.

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.

c-Jun NH2-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and have been implicated in the formation of several human tumors, especially gliomas. We have previously shown that a 55 kDa JNK isoform is constitutively active in 86% of human brain tumors and then showed that it is specifically a JNK2 isoform and likely to be either JNK2α2 or JNK2β2. Notably, we found that only JNK2 isoforms possess intrinsic autophosphorylation activity and that JNK2α2 has the strongest activity. In the present study, we have further explored the contribution of JNK2 isoforms to brain tumor formation. Analysis of mRNA expression by reverse transcription-PCR revealed that JNK2α2 is expressed in 91% (10 of 11) of glioblastoma tumors, whereas JNK2β2 is found in only 27% (3 of 11) of tumors. Both JNK2α2 and JNK2β2 mRNAs are expressed in normal brain (3 of 3). Using an antibody specific for JNK2α isoforms, we verified that JNK2α2 protein is expressed in 88.2% (15 of 17) of glioblastomas, but, interestingly, no JNK2α2 protein was found in six normal brain samples. To evaluate biological function, we transfected U87MG cells with green fluorescent protein–tagged versions of JNK1α1, JNK2α2, and JNK2α2APF (a dominant-negative mutant), and derived cell lines with stable expression. Each cell line was evaluated for various tumorigenic variables including cellular growth, soft agar colony formation, and tumor formation in athymic nude mice. In each assay, JNK2α2 was found to be the most effective in promoting that phenotype. To identify effectors specifically affected by JNK2α2, we analyzed gene expression. Gene profiling showed several genes whose expression was specifically up-regulated by JNK2α2 but down-regulated by JNK2α2APF, among which eukaryotic translation initiation factor 4E (eIF4E) shows the greatest change. Because AKT acts on eIF4E, we also examined AKT activation. Unexpectedly, we found that JNK2α2 could specifically activate AKT. Our data provides evidence that JNK2α2 is the major active JNK isoform and is involved in the promotion of proliferation and growth of human glioblastoma tumors through specific activation of AKT and overexpression of eIF4E. (Cancer Res 2006; 66(20): 10024-31)

The WWOX (WW domain containing oxidoreductase) gene at the common fragile site, FRA16D, is altered in many types of cancer, including lung cancer. We have examined the tumor suppressor function of WWOX in preclinical lung cancer models. The WWOX gene was expressed in lung cancer cell lines through recombinant adenovirus (Ad) infection (Ad- WWOX ), and through a drug [ponasterone A, (ponA)]-inducible system. After WWOX restoration in vitro , endogenous Wwox protein-negative cell lines (A549, H460, and H1299) underwent apoptosis through activation of the intrinsic apoptotic caspase cascade in A549 and H460 cells. Ectopic expression of Wwox caused dramatic suppression of tumorigenicity of A549, H460, and H1299 cells in nude mice after Ad- WWOX infection and after ponA induction of Wwox expression in H1299 lung cancer cells. Tumorigenicity and in vitro growth of U2020 (Wwox-positive) lung cancer cells was unaffected by Wwox overexpression. This study confirms that WWOX is a tumor suppressor gene and is highly effective in preventing growth of lung cancer xenografts, whether introduced through viral infection or by induction of a silent WWOX transgene.

DNA hypermethylation is associated with decreased expression of tumor suppressor genes. We previously observed decreased Fhit expression and Fhit promoter region hypermethylation in rodent tumors induced by various carcinogens, and noted that the 5' regulatory regions in the promoter, exon 1, and intron 1 were differentially methylated, depending on the tissue of origin. Because different carcinogens were used for induction of tumors of the different organs, we could not conclude that the methylation patterns were tissue-specific. To determine if in rat tissues: (1) Fhit methylation status is related to expression levels and (2) Fhit methylation patterns were tissue- or carcinogen-specific, we examined Fhit methylation status and expression levels in DMBA- and MNU-induced benign and malignant mammary tumors. Fhit intron 1 was methylated in 3/9 DMBA and all of MNU-induced benign mammary tumors, in association with reduced Fhit expression levels; Fhit promoter and intron 1 were methylated in all DMBA and MNU-induced carcinomas in association with highly reduced Fhit expression levels. Treatment of rat cancer cells in vitro with the DNA methyltransferase inhibitor, 5'-Aza-2'deoxycytidine, for 4 d, increased Fhit expression and altered the methylation status. Before treatment, both promoter and intron 1 regions were methylated; after treatment, only intron 1 remained methylated. Thus, in carcinogen-exposed rat tissues there is an overall association of Fhit expression with regulatory region methylation, and hypermethylation patterns did not vary with carcinogen. The specific patterns of hypermethylated CpGs in the Fhit regulatory regions thus appear to be tissue-specific.

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.

BACKGROUND FHIT and WWOX are a tumor suppressor and a candidate suppressor that encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3–24.1, respectively. Reduced or absent Fhit expression has been reported in two-thirds of invasive breast tumors in association with adverse prognostic factors. Loss of 16q has been reported frequently in low-grade, invasive breast tumors. METHODS Expression of Fhit and Wwox was evaluated by immunohistochemical staining in 97 archived breast carcinoma specimens. Expression levels were analyzed for correlations with each other, as well as with various patient and tumor characteristics. RESULTS Reduced Fhit and Wwox expression in tumors was observed in 54.6% and 63.2% of specimens, respectively. Fhit and Wwox expression were correlated strongly (P = 0.001). Reduced Fhit staining was seen more frequently in premenopausal patients (P = 0.010), estrogen receptor (ER)-negative or scantly ER-positive tumors (P = 0.058 borderline), high-grade tumors (P = 0.005), and tumors with metastases (P = 0.041). Reduced Wwox staining was more common in tumors with less favorable ER status (P = 0.033). Wwox expression in normal tissue was reduced in 32.9% of specimens, especially in patients with higher stage disease (P = 0.033). Severely reduced Wwox staining (extent < 10%) in normal tissue was found only in postmenopausal women, but reduced Wwox staining (11–75%) was more common in premenopausal women (P = 0.012). Tumor status, lymph node status, and intensity of Fhit expression in tumors were related independently to survival (P = 0.003, P < 0.001, and P = 0.046, respectively). CONCLUSIONS The strong correlation observed between Fhit and Wwox expression was consistent with the common elevated susceptibility of fragile sites to DNA damage. Reduced Fhit expression is associated with adverse prognostic factors. The current results suggest that Wwox also has an important and complex association with steroid hormone expression and breast carcinogenesis. Cancer 2004. © 2004 American Cancer Society.

Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit−/− cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit−/− cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression. © 2004 Wiley-Liss, Inc.

Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. These mutants clustered in beta-strands in the DNA-binding domain, particularly in one of the two beta-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.

In China, more than 80% of the esophageal cancer occurs in farmer village, and most of the esophageal cancer patients die at village home. Therefore, the cause of death for esophageal cancer patients has not been well characterized. The present study was thus undertaken to determine the causes of death in patients with esophageal squamous cell carcinoma (ESCC) to provide more information for further prevention and accurate treatment after ESCC diagnosed. All the 8,838 ESCC patients with a detailed cause of death in this study were derived from the ESCC database (1973-2015) in Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University, China. All the patients were categorized by age (0.05) and the cause of death by ESCC recurrence and metastasis, cardiovascular disease, different infections, second cancer, accident, suicide and uncertain cause was 96% (5485) 1.5% (84), 0.8% (44), 0.6% (34), 0.3% (18), 0.2% (16), 0.6% (32), respectively in males and 96.5% (3015), 1.1% (37), 0.8% (24) 0.6% (18), 0.2% (7), 0.2% (6) and 0.6% (18), respectively in females. The present results demonstrate that recurrence and metastasis are the major cause of death both in male and female for ESCC (96%). It is noteworthy that there are 0.2 percent of the ESCC patients have suicide which call for more attention of psychotherapy on cancer patients. [Supported by the Science and Technology Major Projects of Henan Province of China (161100311300), and Correspondence to: Li Dong Wang, the National Key Research and Development Program of China and Correspondence to: Li Dong Wang. Email: ldwang2007@126.com] Note: This abstract was not presented at the meeting. Citation Format: Xiang Yang Zhang, Jian Po Wang, Rui Ping Xu, Ya Qin Hou, Xiu Jian Chen, Ai Li Li, Xi Chen, Shuang Yin Han, Wei Xing Zhao, Li Sun, Min Wang, Wen Ting Fu, Li Dong Wang. Analysis of causes of death in patients with esophageal squamous cell carcinoma in China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3273. doi:10.1158/1538-7445.AM2017-3273

A 64-year-old man with neurofibromatosis type 1 (NF1) developed a primary malignant melanoma of the anus. Genetic analysis of the resected tumor confirmed loss of heterozygosity (LOH) of the NF1 gene. Anorectal malignant melanoma in NF1 is extremely rare, and genetic studies of the NF1 gene in such patients have not been reported. The allelic loss detected in the present patient supports the previously raised idea that NF1 can function as a tumor suppressor gene in the development of malignant melanoma in patients with NF1.

BACKGROUND β-catenin (CTNNB1) is known to be a member of the cadherin-catenin superfamily and to function in cell-cell adhesion. However, it also has been reported that CTNNB1 plays an important role in carcinogenesis. In the current study, the authors observed expression of the CTNNB1 protein in primary pituitary adenomas to investigate the role of CTNNB1 in the development of pituitary adenomas. METHODS A total of 37 pituitary adenomas were analyzed. Expression of CTNNB1 and the cell proliferation marker Ki-67 were observed immunohistochemically. In addition, the authors performed direct sequencing to detect somatic mutations of exon 3 of the CTNNB1 gene. RESULTS Twenty-one of 37 pituitary adenomas (57%) demonstrated abnormal nuclear accumulation of CTNNB1. It is interesting to note that tumors with an accumulation of CTNNB1 in the nucleus showed a statistical tendency toward an association with increased immunoreactivity of Ki-67 (P < 0.05) whereas no significant correlation was detected between the status of CTNNB1 and other clinicopathologic features. Missense mutations in exon 3 of the CTNNB1 gene also were detected in the cases with abnormal nuclear accumulation of the CTNNB1 protein. CONCLUSIONS The results of the current study suggest that up-regulation of the Wnt signaling pathway, including accumulation of mutant CTNNB1 in the nuclei, plays an important role in the tumorigenesis and development of adenoma in the pituitary gland. Cancer 2001;91:42–8. © 2001 American Cancer Society.

AimsIt has already been reported that loss of heterozygosity (LOH) on chromosome 1p is frequent in a variety of human cancers. This finding implies the presence of some important tumour suppressor genes in this region.p73, a candidate tumour suppressor gene identified recently in chromosome band 1p36.33, encodes a protein highly homologous to p53. To investigate the role of thep73gene in human carcinogenesis, we studied genetic alterations of this gene in various human cancers.MethodsWe analysed the entire coding exons as well as their surrounding exon-intron boundaries of thep73gene in 185 cases of various types of tumours (47 breast cancers, 43 colorectal cancers, 31 gastric cancers, 23 neuroblastomas, 21 lung cancer cell lines, and 20 pancreatic cancer cell lines); they are known as a group of tumours with frequent LOHs in the 1p region. PCR-SSCP analysis was performed and tumours in which aberrant migrating sized bands were observed were subjected to direct sequencing analyses.ResultsOf the 185 cases, only one somatic mis-sense mutation of glutamine from arginine at codon 269 in exon 7 was found in one breast cancer. In addition, several polymorphisms were found at codons 137, 336, 349, and 610, as well as in introns 6, 8, and 9. Monoallelic expression was also observed in pancreatic cancer cell lines.ConclusionsOur results suggest that inactivation of thep73gene does not play a major role in the tumour types analysed in the present study.

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII+/CD133+ defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII+ cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII− cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII+/CD133+ coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII+/CD133+ population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population. Cancer Res; 74(4); 1238–49. ©2013 AACR.

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. Conclusions Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

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.

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.

To investigate the distribution of hepatitis delta virus (HDV) marker among hepatitis B virus (HBV) infected patients and to reveal its clinical significance.To collect the clinical data and sera samples of HBV infected patients and to detect HDAg, Anti-HDV as well as HBV infection markers by means of enzyme-linked immunosorbnent assay. These data combined with clinical diagnostic results and biochemical index were then analyzed.462 samples of HBV infected patients were collected including 210 HBV carriers without symptom, 175 chronic HBV infections, 35 acute HBV infections and 42 liver fibrosis. The HDV infection rate was 4.8% overall. The highest infection rate of 9.5% was found in the group of liver fibrosis whereas the lower rate of 6.9% was found in HBV chronic carriers. HDV infection rate was 7.8% among the population of 40-60 years old, obviously higher than any other age groups.HDV infection was significantly higher in the chronic HBV patients and liver fibrosis patients. Because HDV infection was highly associated with the progress of liver disease, we suggest the screen of HDV markers among hepatitis patients and discriminate whether the patient was co-infected with HDV.

Epigenetic changes involved in cancer development, unlike genetic changes, are reversible. DNA methyltransferase and histone deacetylase inhibitors show antiproliferative effects in vitro, through tumor suppressor reactivation and induction of apoptosis. Such inhibitors have shown activity in the treatment of hematologic disorders but there is little data concerning their effectiveness in treatment of solid tumors. FHIT, WWOX and other tumor suppressor genes are frequently epigenetically inactivated in lung cancers. Lung cancer cell clones carrying conditional FHIT or WWOX transgenes showed significant suppression of xenograft tumor growth after induction of expression of the FHIT or WWOX transgene, suggesting that treatments to restore endogenous Fhit and Wwox expression in lung cancers would result in decreased tumorigenicity. H1299 lung cancer cells, lacking Fhit, Wwox, p16(INK4a) and Rassf1a expression due to epigenetic modifications, were used to assess efficacy of epigenetically targeted protocols in suppressing growth of lung tumors, by injection of 5-aza-2-deoxycytidine (AZA) and trichostatin A (TSA) in nude mice with established H1299 tumors. High doses of intraperitoneal AZA/TSA suppressed growth of small tumors but did not affect large tumors (200 mm(3)); lower AZA doses, administered intraperitoneally or intratumorally, suppressed growth of small tumors without apparent toxicity. Responding tumors showed restoration of Fhit, Wwox, p16(INKa), Rassf1a expression, low mitotic activity, high apoptotic fraction and activation of caspase 3. These preclinical studies show the therapeutic potential of restoration of tumor suppressor expression through epigenetic modulation and the promise of re-expressed tumor suppressors as markers and effectors of the responses.

The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.

The FHIT and WWOX genes encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3, respectively. Reduced Fhit and Wwox expression has been reported in approximately two-thirds of invasive breast tumors. Expression of these fragile gene products, as well as ErbB2 and p53, were evaluated immunohistochemically in 44 pure and 31 adjacent-to-invasive ductal carcinoma in-situ (DCIS) cases. Reduced Fhit and Wwox expression were observed in (i) 70% and 68% of pure DCIS; (ii) 52% and 55% of DCIS adjacent-to-invasive tumor cases; and (iii) 20% and 50% of adjacent normal tissue in pure DCIS cases. Reduced Wwox expression in adjacent normal tissue was observed in 30% of cases in the DCIS adjacent-to-invasive group. Reduced Fhit and Wwox expression was observed in 61% of adjoining invasive tumors. In all normal, pure DCIS, and DCIS adjacent-to-invasive lesions, Fhit and Wwox expression was positively associated (P = 0.034, P = 0.042, P = 0.004, respectively) and in the invasive component there was a positive trend toward association (P = 0.075). Fhit and Wwox were more frequently reduced in high-grade lesions in the DCIS adjacent-to-invasive (P = 0.025, P = 0.004, respectively). In the pure DCIS group, there was a statistically significant negative association between Fhit and ErbB2 expression in DCIS (P = 0.035). In summary, reduced Fhit and Wwox expression in in-situ breast cancer was associated, which may contribute to the high-grade DCIS-invasive tumor pathway.

This study aimed to (a) determine if DNA methylation is a mechanism of WWOX (WW domain containing oxidoreductase) and FHIT (fragile histidine triad) inactivation in lung, breast and bladder cancers; (b) examine distinct methylation patterns in neoplastic and adjacent tissues and (c) seek correlation of methylation patterns with disease status. Protein expression was detected by immunohistochemistry, and methylation status by methylation-specific PCR (MSP) and sequencing, in lung squamous cell carcinomas and adjacent tissues, invasive breast carcinomas, adjacent tissues and normal mammary tissues and bladder transitional cell carcinomas. Wwox and Fhit expression was reduced in cancers in association with hypermethylation. Differential patterns of WWOX and FHIT methylation were observed in neoplastic vs adjacent non-neoplastic tissues, suggesting that targeted MSP amplification could be useful in following treatment or prevention protocols. WWOX promoter MSP differentiates DNA of lung cancer from DNA of adjacent lung tissue. WWOX and FHIT promoter methylation is detected in tissue adjacent to breast cancer and WWOX exon 1 MSP distinguishes breast cancer DNA from DNA of adjacent and normal tissue. Differential methylation in cancerous vs adjacent tissues suggests that WWOX and FHIT hypermethylation analyses could enrich a panel of DNA methylation markers.

FHIT and WWOX are a tumor suppressor and a candidate suppressor that encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3-24.1, respectively. Reduced or absent Fhit expression has been reported in two-thirds of invasive breast tumors in association with adverse prognostic factors. Loss of 16q has been reported frequently in low-grade, invasive breast tumors.Expression of Fhit and Wwox was evaluated by immunohistochemical staining in 97 archived breast carcinoma specimens. Expression levels were analyzed for correlations with each other, as well as with various patient and tumor characteristics.Reduced Fhit and Wwox expression in tumors was observed in 54.6% and 63.2% of specimens, respectively. Fhit and Wwox expression were correlated strongly (P = 0.001). Reduced Fhit staining was seen more frequently in premenopausal patients (P = 0.010), estrogen receptor (ER)-negative or scantly ER-positive tumors (P = 0.058 borderline), high-grade tumors (P = 0.005), and tumors with metastases (P = 0.041). Reduced Wwox staining was more common in tumors with less favorable ER status (P = 0.033). Wwox expression in normal tissue was reduced in 32.9% of specimens, especially in patients with higher stage disease (P = 0.033). Severely reduced Wwox staining (extent10%) in normal tissue was found only in postmenopausal women, but reduced Wwox staining (11-75%) was more common in premenopausal women (P = 0.012). Tumor status, lymph node status, and intensity of Fhit expression in tumors were related independently to survival (P = 0.003, P0.001, and P = 0.046, respectively).The strong correlation observed between Fhit and Wwox expression was consistent with the common elevated susceptibility of fragile sites to DNA damage. Reduced Fhit expression is associated with adverse prognostic factors. The current results suggest that Wwox also has an important and complex association with steroid hormone expression and breast carcinogenesis.