bluuebunny/crossref_metadata_2025 huggingface dataset:
Progression of Barrett's Metaplasia to Adenocarcinoma Is Associated with the Suppression of the Transcriptional Programs of Epidermal Differentiation
Authors: Kimchi Erik T. , Posner Mitchell C. , Park James O. , Darga Thomas E. , Kocherginsky Masha , Karrison Theodore , Hart John , Smith Kerrington D. , Mezhir James J. , Weichselbaum Ralph R. , Khodarev Nikolai N.
Published: July 2017

We did expressional profiling on 24 paired samples of normal esophageal epithelium, Barrett's metaplasia, and esophageal adenocarcinomas. Matching tissue samples representing the three different histologic types were obtained from each patient undergoing esophagectomy for adenocarcinoma. Our analysis compared the molecular changes accompanying the transformation of normal squamous epithelium with Barrett's esophagus and adenocarcinoma in individual patients rather than in a random cohort. We tested the hypothesis that expressional profiling may reveal gene sets that can be used as molecular markers of progression from normal esophageal epithelium to Barrett's esophagus and adenocarcinoma. Expressional profiling was done using U133A GeneChip (Affymetrix), which represent approximately two thirds of the human genome. The final selection of 214 genes permitted the discrimination of differential gene expression of normal esophageal squamous epithelium, Barrett's esophagus, and adenocarcinoma using two-dimensional hierarchical clustering of selected genes. These data indicate that transformation of Barrett's esophagus to adenocarcinoma is associated with suppression of the genes involved in epidermal differentiation, including genes in 1q21 loci and corresponding to the epidermal differentiation complex. Correlation analysis of genes concordantly expressed in Barrett's esophagus and adenocarcinoma revealed 21 genes that represent potential genetic markers of disease progression and pharmacologic targets for treatment intervention. PCR analysis of genes selected based on DNA array experiments revealed that estimation of the ratios of GATA6 to SPRR3 allows discrimination among normal esophageal epithelium, Barrett's dysplasia, and adenocarcinoma.
Genistein Inhibits p38 Map Kinase Activation, Matrix Metalloproteinase Type 2, and Cell Invasion in Human Prostate Epithelial Cells
Authors: Huang Xiaoke , Chen Shan , Xu Li , Liu Yueqin , Deb Dilip K. , Platanias Leonidas C. , Bergan Raymond C.
Published: July 2017

Epidemiologic studies associate consumption of genistein, in the form of dietary soy, with lower rates of metastatic prostate cancer. We have previously shown that genistein inhibits prostate cancer cell detachment in vitro, that it is well tolerated in an older cohort of men with prostate cancer, and that it alters cell signaling in that same cohort. We have also shown that p38 mitogen-activated protein kinase (MAPK) is necessary for transforming growth factor β (TGF-β)–mediated increases in prostate cancer adhesion. Although cell invasion is closely linked to metastatic behavior, little is known about how this process is regulated in prostate cancer or what effect, if any, genistein has on associated processes. We now show that genistein inhibits matrix metalloproteinase type 2 (MMP-2) activity in six of seven prostate cell lines tested, blocks MMP-2 induction by TGF-β, and inhibits cell invasion. Efficacy was seen at low nanomolar concentrations, corresponding to blood concentrations of free genistein attained after dietary consumption. Inhibition of p38 MAPK by either SB203580 or dominant-negative construct blocked induction of MMP-2 and cell invasion by TGF-β. Genistein exerted similar effects and was found to block activation of p38 MAPK by TGF-β. This study shows that p38 MAPK is necessary for TGF-β–mediated induction of MMP-2 and cell invasion in prostate cancer and that genistein blocks activation of p38 MAPK, thereby inhibiting processes closely linked to metastasis, and does so at concentrations associated with dietary consumption. Any potential causal link to epidemiologic findings will require further investigation.
Combined Genetic Assessment of Transforming Growth Factor-β Signaling Pathway Variants May Predict Breast Cancer Risk
Authors: Kaklamani Virginia G. , Baddi Lisa , Liu Junjian , Rosman Diana , Phukan Sharbani , Bradley Ciarán , Hegarty Chris , McDaniel Bree , Rademaker Alfred , Oddoux Carole , Ostrer Harry , Michel Loren S. , Huang Helen , Chen Yu , Ahsan Habibul , Offit Kenneth , Pasche Boris
Published: July 2017

There is growing evidence that common variants of the transforming growth factor-β (TGF-β) signaling pathway may modify breast cancer risk. In vitro studies have shown that some variants increase TGF-β signaling, whereas others have an opposite effect. We tested the hypothesis that a combined genetic assessment of two well-characterized variants may predict breast cancer risk. Consecutive patients (n = 660) with breast cancer from the Memorial Sloan-Kettering Cancer Center (New York, NY) and healthy females (n = 880) from New York City were genotyped for the hypomorphic TGFBR1*6A allele and for the TGFB1 T29C variant that results in increased TGF-β circulating levels. Cases and controls were of similar ethnicity and geographic location. Thirty percent of cases were identified as high or low TGF-β signalers based on TGFB1 and TGFBR1 genotypes. There was a significantly higher proportion of high signalers (TGFBR1/TGFBR1 and TGFB1*CC) among controls (21.6%) than cases (15.7%; P = 0.003). The odds ratio [OR; 95% confidence interval (95% CI)] for individuals with the lowest expected TGF-β signaling level (TGFB1*TT or TGFB1*TC and TGFBR1*6A) was 1.69 (1.08-2.66) when compared with individuals with the highest expected TGF-signaling levels. Breast cancer risk incurred by low signalers was most pronounced among women after age 50 years (OR, 2.05; 95% CI, 1.01-4.16). TGFBR1*6A was associated with a significantly increased risk for breast cancer (OR, 1.46; 95% CI, 1.04-2.06), but the TGFB1*CC genotype was not associated with any appreciable risk (OR, 0.89; 95% CI, 0.63-1.21). TGFBR1*6A effect was most pronounced among women diagnosed after age 50 years (OR, 2.20; 95% CI, 1.25-3.87). This is the first study assessing the TGF-β signaling pathway through two common and functionally relevant TGFBR1 and TGFB1 variants. This approach may predict breast cancer risk in a large subset of the population.
Evidence for the Presence of Disease-Perturbed Networks in Prostate Cancer Cells by Genomic and Proteomic Analyses: A Systems Approach to Disease
Authors: Lin Biaoyang , White James T. , Lu Wei , Xie Tao , Utleg Angelita G. , Yan Xiaowei , Yi Eugene C. , Shannon Paul , Khrebtukova Irina , Lange Paul H. , Goodlett David R. , Zhou Daixing , Vasicek Thomas J. , Hood Leroy
Published: July 2017

Prostate cancer is initially responsive to androgen ablation therapy and progresses to androgen-unresponsive states that are refractory to treatment. The mechanism of this transition is unknown. A systems approach to disease begins with the quantitative delineation of the informational elements (mRNAs and proteins) in various disease states. We employed two recently developed high-throughput technologies, massively parallel signature sequencing (MPSS) and isotope-coded affinity tag, to gain a comprehensive picture of the changes in mRNA levels and more restricted analysis of protein levels, respectively, during the transition from androgen-dependent LNCaP (model for early-stage prostate cancer) to androgen-independent CL1 cells (model for late-stage prostate cancer). We sequenced >5 million MPSS signatures, obtained >142,000 tandem mass spectra, and built comprehensive MPSS and proteomic databases. The integrated mRNA and protein expression data revealed underlying functional differences between androgen-dependent and androgen-independent prostate cancer cells. The high sensitivity of MPSS enabled us to identify virtually all of the expressed transcripts and to quantify the changes in gene expression between these two cell states, including functionally important low-abundance mRNAs, such as those encoding transcription factors and signal transduction molecules. These data enable us to map the differences onto extant physiologic networks, creating perturbation networks that reflect prostate cancer progression. We found 37 BioCarta and 14 Kyoto Encyclopedia of Genes and Genomes pathways that are up-regulated and 23 BioCarta and 22 Kyoto Encyclopedia of Genes and Genomes pathways that are down-regulated in LNCaP cells versus CL1 cells. Our efforts represent a significant step toward a systems approach to understanding prostate cancer progression.
Mitochondrial Targeting of HumanO6-Methylguanine DNA Methyltransferase Protects against Cell Killing by Chemotherapeutic Alkylating Agents
Authors: Cai Shanbao , Xu Yi , Cooper Ryan J. , Ferkowicz Michael J. , Hartwell Jennifer R. , Pollok Karen E. , Kelley Mark R.
Published: July 2017

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O6-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34+ committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34+ cells were infected with oncoretroviral vectors that targeted O6-benzylguanine (6BG)–resistant MGMT (MGMTP140K) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent–induced cell killing during chemotherapy.
Anoxia Is Necessary for Tumor Cell Toxicity Caused by a Low-Oxygen Environment
Authors: Papandreou Ioanna , Krishna Chaya , Kaper Fiona , Cai Deli , Giaccia Amato J. , Denko Nicholas C.
Published: July 2017

Cells exposed to oxygen deprivation in vitro have been shown to reduce proliferation and/or engage in programmed cell death. There is considerable controversy in the literature as to the role of hypoxia-inducible factor-1 (HIF-1) and HIF-1 target genes in initiating these responses. We therefore examined the oxygen dependence and the role of the hypoxia-responsive transcription factor HIF-1 in making the cellular death decision. Oxygen concentrations as low as 0.5% did not alter the growth of HIF-1–proficient or HIF-1–deficient murine fibroblasts, or human tumor cells, despite the appropriate induction of HIF-1 target genes. Severe hypoxia (<0.01% oxygen) did induced apoptosis, resulting in decreased colony formation, chromatin condensation, DNA fragmentation, and caspase activation but also independent of HIF1α status. Transcriptional induction of HIF-1–dependent genes putatively involved in cell death like BNip3 and BNip3L was therefore disassociated from hypoxia-dependent toxicity. Likewise, forced overexpression of a nondegradable form of HIF-1α in several human tumor cell lines was not sufficient to induce apoptosis under normoxic conditions. Taken together, these findings indicate that additional molecular events are triggered by anoxia in a HIF-1–independent manner, and these changes are necessary for cell death observed in low-oxygen environments.
DNA Adducts and Lung Cancer Risk: A Prospective Study
Authors: Peluso Marco , Munnia Armelle , Hoek Gerard , Krzyzanowski Michal , Veglia Fabrizio , Airoldi Luisa , Autrup Herman , Dunning Alison , Garte Seymour , Hainaut Pierre , Malaveille Christian , Gormally Emmanuelle , Matullo Giuseppe , Overvad Kim , Raaschou-Nielsen Ole , Clavel-Chapelon Francoise , Linseisen Jacob , Boeing Heiner , Trichopoulou Antonia , Trichopoulos Dimitrios , Kaladidi Anna , Palli Domenico , Krogh Vittorio , Tumino Rosario , Panico Salvatore , Bueno-De-Mesquita H. Bas , Peeters Petra H. , Kumle Merethe , Gonzalez Carlos A. , Martinez Carmen , Dorronsoro Miren , Barricarte Aurelio , Navarro Carmen , Quiros J. Ramón , Berglund Goran , Janzon Lars , Jarvholm Bengt , Day Nicholas E. , Key Tim J. , Saracci Rodolfo , Kaaks Rudolf , Riboli Elio , Vineis Paolo
Published: July 2017

Objectives were to investigate prospectively the ability of DNA adducts to predict cancer and to study the determinants of adducts, especially air pollutants. DNA adducts were measured in a case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) investigation. Cases included newly diagnosed lung cancer (n = 115), upper respiratory cancers (pharynx and larynx; n = 82), bladder cancer (n = 124), leukemia (n = 166), and chronic obstructive pulmonary disease or emphysema deaths (n = 77) accrued after a median follow-up of 7 years among the EPIC former smokers and never-smokers. Three controls per case were matched for questionnaire analyses and two controls per case for laboratory analyses. Matching criteria were gender, age, smoking status, country of recruitment, and follow-up time. Individual exposure to air pollution was assessed using concentration data from monitoring stations in routine air quality monitoring networks. Leukocyte DNA adducts were analyzed blindly using 32P postlabeling technique. Adducts were associated with the subsequent risk of lung cancer, with an odds ratio (OR) of 1.86 [95% confidence interval (95% CI), 0.88-3.93] when comparing detectable versus nondetectable adducts. The association with lung cancer was stronger in never-smokers (OR, 4.04; 95% CI, 1.06-15.42) and among the younger age groups. After exclusion of the cancers occurring in the first 36 months of follow-up, the OR was 4.16 (95% CI, 1.24-13.88). A positive association was found between DNA adducts and ozone (O3) concentration. Our prospective study suggests that leukocyte DNA adducts may predict lung cancer risk of never-smokers. Besides, the association of DNA adduct levels with O3 indicates a possible role for photochemical smog in determining DNA damage.
Anticancer Activity of the Antibiotic Clioquinol
Authors: Ding Wei-Qun , Liu Bolin , Vaught Joshua L. , Yamauchi Hanako , Lind Stuart E.
Published: July 2017

Clioquinol, a metal chelator, has been used for many years as an antimicrobial agent and more recently as a potential treatment for Alzheimer's disease. Because it binds copper and zinc, metals essential for the activity of the enzyme superoxide dismutase-1 (SOD1), a potential target for anticancer drug development, we investigated its effects on human cancer cells. Treatment with clioquinol reduced the viability of eight different human cancer cell lines in a concentration-dependent manner, with IC50 values in the low micromolar range. Biochemical analysis revealed that clioquinol induced cancer cell death through apoptotic pathways that require caspase activity. Although clioquinol induced modest inhibition of SOD1 activity in treated cells, comparable inhibition by a known SOD1 inhibitor, diethyldithiocarbamate, did not result in cytotoxicity. The addition of copper, iron, or zinc did not rescue cells from cliquinol-induced cytotoxicity but enhanced its killing, arguing against metal chelation as its major mechanism of action. To test if clioquinol might act as an ionophore, a fluorescent probe was used to monitor intracellular zinc concentrations. The addition of clioquinol resulted in elevated levels of intracellular zinc, indicating that clioquinol acts as a zinc ionophore. In an in vivo xenografts mouse model, clioquinol inhibited tumor growth of xenografts over a 6-week period, without inducing visible toxicity. Our results show that clioquinol has anticancer effects both in vitro and in vivo. Transition metal ionophores may be a subclass of metal chelators with anticancer activity deserving of further development.
Activation of the Fas-FasL Signaling Pathway by MDA-7/IL-24 Kills Human Ovarian Cancer Cells
Authors: Gopalan Began , Litvak Anya , Sharma Sikha , Mhashilkar Abner M. , Chada Sunil , Ramesh Rajagopal
Published: July 2017

The tumor-suppressive activity of melanoma differentiation–associated gene-7 (mda-7), also known as interleukin 24 (IL-24), has been shown in a spectrum of human cancer cells in vitro and in vivo. However, mechanisms responsible for antitumor activity of mda-7 in human ovarian cancer cells have not been identified. We investigated the therapeutic activity and underlying mechanisms of adenovirus-mediated mda-7 gene (Ad-mda7) transfer in human ovarian cancer cells. Ad-mda7 treatment resulted in overexpression of MDA-7/IL-24 protein in both ovarian cancer and normal ovarian epithelial cells. However, Ad-mda7 significantly (P = 0.001) inhibited cell proliferation and induced apoptosis only in tumor cells and not in normal cells. Studies addressing the mechanism of action of Ad-mda7–induced tumor cell apoptosis revealed early activation of the transcription factors c-Jun and activating transcription factor 2, which in turn stimulated the transcription of an immediate downstream target, the death-inducer Fas ligand (FasL), and its cognate receptor Fas. Associated with the activation of Fas-FasL was the activation of nuclear factor κB and induction of Fas-associated factor 1, Fas-associated death domain, and caspase-8. Promoter-based reporter gene analyses showed that Ad-mda7 specifically activated the Fas promoter. Inhibition of Fas using small interfering RNA resulted in a significant decrease in Ad-mda7–mediated tumor cell death. Additionally, blocking of FasL with NOK-1 antibody abrogated Ad-mda7–mediated apoptosis. Collectively, these results show that Ad-mda7–mediated killing of human ovarian cancer cells involves activation of the Fas-FasL signaling pathway, a heretofore unrecognized mediator of MDA-7 apoptosis induction.
The Hydra Phenomenon of Cancer: Why Tumors Recur Locally after Microscopically Complete Resection
Authors: Höckel Michael , Dornhöfer Nadja
Published: July 2017

After surgical resection with microscopically clear margins, solid malignant tumors recur locally in up to 50%. Although the effect of a local tumor recurrence on the overall survival may be low in common cancers such as carcinoma of the breast or prostate, the affected patients suffer from exacerbated fear and the burden of the secondary treatment. With some tumor entities such as carcinoma of the uterine cervix or carcinoma of the head and neck, a local recurrence indicates incurability in the majority of cases. The pathomechanisms of local tumor spread and relapse formation are still unclear and comparatively little research has been devoted to their elucidation. Through the analysis of clinical and molecular data, we propose the concept of two pathogenetically and prognostically different local relapse types (i) in situ recurrences that arise in the residual organ/organ system not involved in the surgery for the primary tumor and (ii) scar recurrences that develop at the site of previous tumor resection. Whereas field cancerization, the monoclonal or multiclonal displacement of normal epithelium by a genetically altered but microscopically undistinguishable homologue, may explain the origin of in situ recurrences, most scar recurrences are regarded as the result of the interaction of minimal residual microscopically occult cancer with the surgical wound environment inside a developmentally defined tissue or organ compartment. The therapeutic implications derived from these concepts and areas of future research aimed to reduce local relapses are discussed in this perspective.
Stem and Progenitor-Like Cells Contribute to the Aggressive Behavior of Human Epithelial Ovarian Cancer
Authors: Bapat Sharmila A. , Mali Avinash M. , Koppikar Chaitanyananda B. , Kurrey Nawneet K.
Published: July 2017

The cellular mechanisms underlying the increasing aggressiveness associated with ovarian cancer progression are poorly understood. Coupled with a lack of identification of specific markers that could aid early diagnoses, the disease becomes a major cause of cancer-related mortality in women. Here we present direct evidence that the aggressiveness of human ovarian cancer may be a result of transformation and dysfunction of stem cells in the ovary. A single tumorigenic clone was isolated among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer. During the course of the study, yet another clone underwent spontaneous transformation in culture, providing a model of disease progression. Both the transformed clones possess stem cell–like characteristics and differentiate to grow in an anchorage-independent manner in vitro as spheroids, although further maturation and tissue-specific differentiation was arrested. Significantly, tumors established from these clones in animal models are similar to those in the human disease in their histopathology and cell architecture. Furthermore, the tumorigenic clones, even on serial transplantation continue to establish tumors, thereby confirming their identity as tumor stem cells. These findings suggest that: (a) stem cell transformation can be the underlying cause of ovarian cancer and (b) continuing stochastic events of stem and progenitor cell transformation define the increasing aggression that is characteristically associated with the disease.
SRC-3 Is Required for Prostate Cancer Cell Proliferation and Survival
Authors: Zhou Hai-Jun , Yan Jun , Luo Weiping , Ayala Gustavo , Lin Sue-Hwa , Erdem Halime , Ittmann Michael , Tsai Sophia Y. , Tsai Ming-Jer
Published: July 2017

Prostate cancer is the most common cancer in men in America. Currently, steroid receptor coactivators have been proposed to mediate the development and progression of prostate cancer, at times in a steroid-independent manner. Steroid receptor coactivator-3 (SRC-3, p/CIP, AIB1, ACTR, RAC3, and TRAM-1) is a member of the p160 family of coactivators for nuclear hormone receptors including the androgen receptor. SRC-3 is frequently amplified or overexpressed in a number of cancers. However, the role of SRC-3 in cancer cell proliferation and survival is still poorly understood. In this study, we show that SRC-3 is overexpressed in prostate cancer patients and its overexpression correlates with prostate cancer proliferation and is inversely correlated with apoptosis. Consistent with patient data, we have observed that reduction of SRC-3 expression by small interfering RNA decreases proliferation, delays the G1-S transition, and increases cell apoptosis of different prostate cancer cell lines. Furthermore, with decreased SRC-3 expression, proliferating cell nuclear antigen and Bcl-2 expression, as well as bromodeoxyuridine incorporation in prostate cancer cells are reduced. Finally, knockdown of SRC-3 with inducible short hairpin RNA expression in prostate cancer cells decreased tumor growth in nude mice. Taken together, these findings indicate that SRC-3 is an important regulator of prostate cancer proliferation and survival.
Dietary Genistein Reduces Metastasis in a Postsurgical Orthotopic Breast Cancer Model
Authors: Vantyghem Sharon A. , Wilson Sylvia M. , Postenka Carl O. , Al-Katib Waleed , Tuck Alan B. , Chambers Ann F.
Published: July 2017

Metastatic spread, not primary tumor burden, is the leading cause of breast cancer deaths. For patient prognosis to improve, new systemic adjuvant therapies that are capable of effectively inhibiting the outgrowth of seeded tumor cells after surgical treatment of the primary breast tumor are needed. To facilitate the preclinical development of such therapies, relevant animal models of breast cancer metastasis that can mimic the postsurgical adjuvant setting are required. Here we developed a preclinical xenograft model of breast cancer metastasis where the primary tumor was removed by surgical resection before systemic adjuvant treatment. We used this model to assess the antimetastatic effect of postsurgical dietary intervention with the soy isoflavone genistein. The anticancer activity of genistein has been established in vitro and in vivo, however, few studies have tested the potential of genistein as an antimetastatic therapy. Using our model, we tested the efficacy of adjuvant treatment with genistein to inhibit the outgrowth of metastases postsurgery. To establish primary tumors, human breast carcinoma cells, MDA-MB-435/HAL, were implanted into the mammary fat pad of female nude mice. Primary tumors were left to grow for 5 weeks before being surgically removed. Mice were then randomized into two diet groups: control soy-free diet versus genistein-supplemented diet. Five weeks later, metastatic burden was assessed. Genistein reduced the percent metastatic burden in the lungs by 10-fold. These results indicate that dietary intervention following cancer surgery can affect the outgrowth of seeded tumor cells. The availability of well-characterized, clinically relevant animal models for studying factors that regulate metastatic outgrowth postsurgery will provide an important tool for developing new systemic adjuvant therapies.
Differential Gene Up-Regulation by Hypoxia-Inducible Factor-1α and Hypoxia-Inducible Factor-2α in HEK293T Cells
Authors: Wang Victoria , Davis David A. , Haque Muzammel , Huang L. Eric , Yarchoan Robert
Published: July 2017

Cells exposed to hypoxia respond by increasing the level of hypoxia-inducible factor-1 (HIF-1). This factor then activates a number of genes by binding to hypoxia response elements in their promoter regions. A second hypoxia-responsive factor, HIF-2, can activate many of the same genes as HIF-1. Overexpression of HIFs accompanies the pathogenesis of many tumors. It is unclear, however, as to the respective role of these factors in responsiveness to hypoxia and other stresses. To address this issue, we used microarray technology to study the genes activated in HEK293T cells by hypoxia or transfection with the α chain of HIF-1 (or mutant HIF-1 resistant to degradation) or HIF-2. Fifty-six genes were found to be up-regulated at least 3-fold by either hypoxia or transfection. Of these, 21 were elevated both by transfection with HIF-1α and with HIF-2α, and 14 were preferentially activated by HIF-1α including several involved in glycolysis. Ten genes were preferentially activated by HIF-2α, including two (CACNA1A and PTPRZ1) implicated in neurologic diseases. Interestingly, most HIF-2α–responsive genes were not substantially activated by hypoxia. An additional 10 genes were up-regulated by hypoxia but minimally activated by HIF-1α or HIF-2α transfection. Ten of the genes were studied by quantitative real-time PCR and/or by Northern blot and the results paralleled those found with microarray technology. Although confirmation in other systems will be necessary, these results indicate that whereas some genes are robustly activated by both HIF-1 and HIF-2, others can be preferentially activated by one or the other factor.
Spontaneous Human Adult Stem Cell Transformation
Authors: Rubio Daniel , Garcia-Castro Javier , Martín María C. , de la Fuente Ricardo , Cigudosa Juan C. , Lloyd Alison C. , Bernad Antonio
Published: July 2017

Human adult stem cells are being evaluated widely for various therapeutic approaches. Several recent clinical trials have reported their safety, showing them to be highly resistant to transformation. The clear similarities between stem cell and cancer stem cell genetic programs are nonetheless the basis of a recent proposal that some cancer stem cells could derive from human adult stem cells. Here we show that although they can be managed safely during the standard ex vivo expansion period (6-8 weeks), human mesenchymal stem cells can undergo spontaneous transformation following long-term in vitro culture (4-5 months). This is the first report of spontaneous transformation of human adult stem cells, supporting the hypothesis of cancer stem cell origin. Our findings indicate the importance of biosafety studies of mesenchymal stem cell biology to efficiently exploit their full clinical therapeutic potential.
RedirectingIn vivoElicited Tumor Infiltrating Macrophages and Dendritic Cells towards Tumor Rejection
Authors: Guiducci Cristiana , Vicari Alain P. , Sangaletti Sabina , Trinchieri Giorgio , Colombo Mario P.
Published: July 2017

A hostile tumor microenvironment interferes with the development and function of the adaptive immune response. Here we report the mechanisms by which large numbers of tumor-infiltrating macrophages and dendritic cells (DC) can be redirected to become potent effectors and activators of the innate and adaptive immunity, respectively. We use adenoviral delivery of the CCL16 chemokine to promote accumulation of macrophages and DC at the site of preestablished tumor nodules, combined with the Toll-like receptor 9 ligand CpG and with anti–interleukin-10 receptor antibody. CpG plus anti–interleukin-10 receptor antibody promptly switched infiltrating macrophages infiltrate from M2 to M1 and triggered innate response debulking large tumors within 16 hours. Tumor-infiltrating DC matured and migrated in parallel with the onset of the innate response, allowing the triggering of adaptive immunity before the diffuse hemorrhagic necrosis halted the communication between tumor and draining lymph nodes. Treatment of B6>CXB6 chimeras implanted with BALB/c tumors with the above combination induced an efficient innate response but not CTL-mediated tumor lysis. In these mice, tumor rejection did not exceed 25%, similarly to that observed in CCR7-null mice that have DC unable to prime an adaptive response. The requirement of CD4 help was shown in CD40-KO, as well as in mice depleted of CD4 T cells, during the priming rather than the effector phase. Our data describe the critical requirements for the immunologic rejection of large tumors: a hemorrhagic necrosis initiated by activated M1 macrophages and a concomitant DC migration to draining lymph nodes for subsequent CTL priming and clearing of any tumor remnants.
Inhibition of HER-2/neu Kinase Impairs Androgen Receptor Recruitment to the Androgen Responsive Enhancer
Authors: Liu Yuanbo , Majumder Samarpan , McCall Wesley , Sartor Carolyn I. , Mohler James L. , Gregory Christopher W. , Earp H. Shelton , Whang Young E.
Published: July 2017

Advanced prostate cancer invariably recurs despite androgen deprivation therapy. The androgen receptor (AR) likely plays a key role in this progression and in the continued survival and proliferation of prostate cancer cells in the low androgen environment. Cross-talk with growth factor receptors, such as epidermal growth factor receptor (EGFR) family, has been postulated as a potential mechanism to activate AR in recurrent prostate cancer. We have investigated the role of HER-2/neu (ErbB-2) tyrosine kinase in AR function by characterizing the effect of inhibiting endogenous HER-2 activity in LNCaP cells. We used two independent methods, expression of intracellular single-chain antibody against HER-2 and treatment with a novel dual EGFR/HER-2 kinase inhibitor GW572016 (lapatinib). Expression of intracellular HER-2 antibody scFv-5R and treatment with GW572016 inhibited HER-2 signaling. This HER-2 inhibition led to impairment of AR-mediated functions, such as androgen-stimulated growth and the induction of endogenous prostate-specific antigen (PSA) mRNA and protein. Androgen-stimulated recruitment of AR and histone acetylation at the androgen responsive enhancer of the PSA gene, detected by chromatin immunoprecipitation analysis, were impaired by HER-2 inhibition. GW572016 was more potent in its ability to inhibit PSA expression and AR recruitment and histone acetylation than the EGFR-selective kinase inhibitor ZD1839 (gefitinib), consistent with the HER-2 kinase playing the major role in AR regulation. These results show that HER-2 signaling is required for optimal transcriptional activity of AR in prostate cancer cells and suggest that HER-2 inhibition may provide a novel strategy to disrupt AR function in prostate cancer.
IFN Unresponsiveness in LNCaP Cells Due to the Lack of JAK1 Gene Expression
Authors: Dunn Gavin P. , Sheehan Kathleen C.F. , Old Lloyd J. , Schreiber Robert D.
Published: July 2017

We reported previously that 23% of human lung adenocarcinoma cell lines were unresponsive to IFN-γ. To extend this finding to cancer cells derived from distinct tissues of origin, we assessed IFN-γ receptor signaling in the LNCaP human prostate adenocarcinoma cell line, which in previous experiments by others failed to induce a range of IFN-dependent biological responses. In this report, we show that LNCaP cells fail to respond to either IFN-γ or IFN-α because of an impairment in the proximal signaling events downstream of both IFN-γ and IFN-α/β receptors that lead to the activation of STAT1. Furthermore, we show that LNCaP insensitivity to the IFNs is a result of the absence of expression of the JAK1 kinase, an obligate component shared by both IFN-γ and IFN-α/β receptors. JAK1 was undetectable in LNCaP cells at both protein and message levels. Treatment of LNCaP cells with a combination of inhibitors of DNA methyltransferases and histone deacetylases induced expression of JAK1 message. These results identify the molecular basis for IFN insensitivity in the LNCaP cell line and suggest that epigenetic silencing of key immunologic signaling components may be one mechanism by which tumor cells evade immune detection and elimination.
Ceratamines, Structurally Simple Microtubule-Stabilizing Antimitotic Agents with Unusual Cellular Effects
Authors: Karjala Geoffrey , Chan Queenie , Manzo Emiliano , Andersen Raymond J. , Roberge Michel
Published: July 2017

Ceratamine A and ceratamine B are heterocyclic alkaloids recently identified in a screen for compounds that arrest cells in mitosis. Treatment of breast carcinoma MCF-7 cells causes a concentration-dependent block of cell cycle progression exclusively at mitosis. In vitro studies with purified tubulin indicate that the ceratamines directly stimulate microtubule polymerization in the absence of microtubule-associated proteins. Cells treated with ceratamines show a dense perinuclear microtubule network in interphase and multiple pillar-like tubulin structures in mitotic cells. The ceratamines do not compete with paclitaxel for binding to microtubules in vitro. Unlike other microtubule-stabilizing agents, the ceratamines have simple structures with no chiral centers, making them attractive drug leads.
Serum Soluble Epidermal Growth Factor Receptor Concentrations Decrease in Postmenopausal Metastatic Breast Cancer Patients Treated with Letrozole
Authors: Lafky Jacqueline M. , Baron Andre T. , Cora Elsa M. , Hillman David W. , Suman Vera J. , Perez Edith A. , Ingle James N. , Maihle Nita J.
Published: July 2017

Previous studies have implicated estrogen as a regulator of epidermal growth factor receptor (EGFR) expression in breast tumors. We therefore speculated that estrogen might modulate serologic soluble EGFR (sEGFR) concentrations in breast cancer patients. Accordingly, we measured serum sEGFR concentrations in postmenopausal women with metastatic breast cancer (MBC) treated with letrozole, an aromatase inhibitor that blocks estrogen synthesis. Serum specimens were obtained prior to and following 1 and 3 months of letrozole therapy. We report that sEGFR concentrations do not differ between MBC patients prior to letrozole treatment and age- and postmenopause-matched healthy women (P = 0.468). In contrast, however, sEGFR concentrations decreased significantly in 76% of MBC patients after both 1 month (P = 0.006) and 3 months (P = 0.003) of letrozole therapy versus pretreatment concentrations. Within the limitations of this study, we found no evidence for an association between pretreatment sEGFR concentrations or decreased treatment sEGFR concentrations and either progression-free or overall survival. Nonetheless, we conclude that future prospective studies are warranted to determine if baseline and/or longitudinal serum sEGFR concentrations may be useful for predicting disease progression and survival, and/or for monitoring responsiveness to aromatase inhibitors or other endocrine therapies in breast cancer patients.
A Novel Angiogenic Role for Prostaglandin F2α-FP Receptor Interaction in Human Endometrial Adenocarcinomas
Authors: Sales Kurt J. , List Tammy , Boddy Sheila C. , Williams Alistair R.W. , Anderson Richard A. , Naor Zvi , Jabbour Henry N.
Published: July 2017

Prostaglandins have been implicated in several neovascular diseases. In the present study, we found elevated FP receptor and vascular endothelial growth factor (VEGF) expression colocalized in glandular epithelial and vascular cells lining the blood vessels in endometrial adenocarcinomas. We investigated the signaling pathways activated by the FP receptor and their role in modulating VEGF expression in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with FP receptor cDNA in the sense or antisense orientations. Treatment of Ishikawa cells with prostaglandin F2α (PGF2α) rapidly induced transphosphorylation of the epidermal growth factor receptor (EGFR) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 via the FP receptor. Activation of EGFR-Ras-mitogen-activated protein kinase/ERK kinase (MEK) signaling via the FP receptor resulted in an increase in VEGF promoter activity, expression of VEGF mRNA, and secretion of VEGF protein. These effects of PGF2α on the FP receptor could be abolished by treatment of cells with a specific FP receptor antagonist, chemical inhibitors of c-Src, matrix metalloproteinase, and EGFR kinase or by inactivation of signaling with dominant-negative mutant isoforms of EGFR, Ras, or MEK or with small inhibitory RNA oligonucleotides targeted against the EGFR. Finally, we confirmed that PGF2α could potentiate angiogenesis in endometrial adenocarcinoma explants by transactivation of the EGFR and induction of VEGF mRNA expression.
The Proto-Oncogene ERG in Megakaryoblastic Leukemias
Authors: Rainis Liat , Toki Tsutomu , Pimanda John E. , Rosenthal Ester , Machol Keren , Strehl Sabine , Göttgens Berthold , Ito Etsuro , Izraeli Shai
Published: July 2017

Aneuploidy is one of the hallmarks of cancer. Acquired additions of chromosome 21 are a common finding in leukemias, suggesting a contributory role to leukemogenesis. About 10% of patients with a germ line trisomy 21 (Down syndrome) are born with transient megakaryoblastic leukemia. We and others have shown acquired mutations in the X chromosome gene GATA1 in all these cases. The gene or genes on chromosome 21 whose overexpression promote the megakaryoblastic phenotype are presently unknown. We propose that ERG, an Ets transcription factor situated on chromosome 21, is one such candidate. We show that ERG is expressed in hematopoietic stem cells, megakaryoblastic cell lines, and in primary leukemic cells from Down syndrome patients. ERG expression is induced upon megakaryocytic differentiation of the erythroleukemia cell lines K562 and UT-7, and forced expression of ERG in K562 cells induces erythroid to megakaryoblastic phenotypic switch. We also show that ERG activates the gpIb megakaryocytic promoter and binds the gpIIb promoter in vivo. Furthermore, both ERG and ETS2 bind in vivo the hematopoietic enhancer of SCL/TAL1, a key regulator of hematopoietic stem cell and megakaryocytic development. We propose that trisomy 21 facilitates the occurrence of megakaryoblastic leukemias through a shift toward the megakaryoblastic lineage caused by the excess expression of ERG, and possibly by other chromosome 21 genes, such as RUNX1 and ETS2, in hematopoietic progenitor cells, coupled with a differentiation arrest caused by the acquisition of mutations in GATA1.
Involvement of the RASSF1A Tumor Suppressor Gene in Controlling Cell Migration
Authors: Dallol Ashraf , Agathanggelou Angelo , Tommasi Stella , Pfeifer Gerd P. , Maher Eamonn R. , Latif Farida
Published: July 2017

We have previously shown that RASSF1A associates with the microtubules. This association alters the microtubule dynamics and seems essential for RASSF1A tumor suppressive function. Mutant variants of RASSF1A that do not associate fully with the microtubules have reduced ability to stabilize them and cause cell cycle arrest. Here we show that overexpression of RASSF1A diminished the ability of A549 non–small cell lung cancer cells to migrate either through a transwell filter or to close a wound. In addition, we employed gene knockdown as well as mouse embryonic fibroblasts (MEFs) from Rassf1a knockout mice to analyze RASSF1A function in controlling cell motility. A549 cells stably transfected with RASSF1A exhibited increased cell-cell adhesion and less refractive morphology compared with controls. Conversely, RASSF1A knockdown in HeLa caused loss of cell-cell adhesion and a more refractive morphology. RASSF1A-depleted HeLa cells as well as Rassf1a−/− MEFs displayed increased cell migration that could be partly phosphatidylinositol 3-kinase dependent. Time-lapse microscopy showed the RASSF1A-depleted cells are highly motile with fibroblast-like morphology and diminished cell-cell adhesion. Staining of the cytoskeleton in RASSF1A-depleted HeLa cells and MEFs show marked differences in terms of microtubules outgrowth and actin stress fibers formation. This observation was associated with increased activation of Rac1 in RASSF1A-knockdown cells and the Rassf1a−/− MEFs. In addition, expression of a dominant-negative variant of Rac1 in the RASSF1A-depleted HeLa cells reduced their ability to form lamellipodia and other protrusions. These findings represent a novel function for RASSF1A, which may help explain its tumor suppression ability independently of its effects on cell cycle and apoptosis.
Chaperon and Adjuvant Activity of hsp70: Different Natural Killer Requirement for Cross-Priming of Chaperoned and Bystander Antigens
Authors: Massa Chiara , Melani Cecilia , Colombo Mario P.
Published: July 2017

Heat shock proteins (HSP) convey both chaperoned propeptide and danger signal to dendritic cells (DC). However, few studies have compared the two activities. Using a murine inducible hsp70 secreted by cells distinct from those providing the tumor antigens, we showed that hsp70 exerts efficacious adjuvant effects toward DC cross-priming. Hsp70 induces DC maturation and phagocytosis of cellular debris both in vitro and in vivo, which are conducive to CTL response to chaperoned and nonchaperoned antigens. Whereas the ability of hsp70 to induce cross-presentation of chaperoned peptides is natural killer (NK) independent, the adjuvant activity requires NK cells at the site of DC-hsp70 interaction to induce CTL response and therapeutic effect against lung metastases. However, although bystander activity provides equal CTL induction, the best therapeutic efficacy rests on cell vaccine secreting hsp70 that combines chaperoned antigen and danger signal within the same cell.
Mechanism of Action of (−)-Epigallocatechin-3-Gallate: Auto-oxidation–Dependent Inactivation of Epidermal Growth Factor Receptor and Direct Effects on Growth Inhibition in Human Esophageal Cancer KYSE 150 Cells
Authors: Hou Zhe , Sang Shengmin , You Hui , Lee Mao-Jung , Hong Jungil , Chin Khew-Voon , Yang Chung S.
Published: July 2017

(−)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the phosphorylation of epidermal growth factor receptor (EGFR). We observed similar effects of EGCG in esophageal squamous cell carcinoma KYSE 150 cells and epidermoid squamous cell carcinoma A431 cells. Pretreatment of KYSE 150 cells with EGCG (20 μmol/L) for 0.5 to 24 hours in HAM's F12 and RPMI 1640 mixed medium at 37°C, before the addition of EGF, resulted in a decreased level of phosphorylated EGFR (by 32-85%). Prolonged treatment with EGCG (8 or 24 hours) also decreased EGFR protein level (both by 80%). EGCG treatment for 24 hours also caused decreased signals of HER-2/neu in esophageal adenocarcinoma OE19 cells. These effects of EGCG were prevented or diminished by the addition of superoxide dismutase (SOD, 5 units/mL), or SOD plus catalase (30 units/mL), to the cell culture medium. A similar phenomenon on inactivation of EGFR was observed in A431 cells as well. Under culture conditions for KYSE 150 cells, EGCG was unstable, with a half-life of ∼30 minutes; EGCG dimers and other oxidative products were formed. The presence of SOD in the culture medium stabilized EGCG and increased its half-life to longer than 24 hours and some EGCG epimerized to (+)-gallocatechin-3-gallate. A mechanism of superoxide radical-mediated dimerization of EGCG and H2O2 formation is proposed. The stabilization of EGCG by SOD in the culture medium potentiated the activity of EGCG in inhibiting KYSE 150 cell growth. The results suggest that in cell culture conditions, the auto-oxidation of EGCG leads to EGFR inactivation, but the inhibition of cell growth is due to other mechanisms. It remains to be determined whether the presently observed auto-oxidation of EGCG occurs in vivo. In future studies of EGCG and other polyphenolic compounds in cell culture, SOD may be added to stabilize EGCG and to avoid possible artifacts.
Effects of PS-341 on the Activity and Composition of Proteasomes in Multiple Myeloma Cells
Authors: Altun Mikael , Galardy Paul J. , Shringarpure Reshma , Hideshima Teru , LeBlanc Richard , Anderson Kenneth C. , Ploegh Hidde L. , Kessler Benedikt M.
Published: July 2017

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite enormous research efforts. The remarkable activity of the proteasome inhibitor bortezomib (PS-341, Velcade) observed in clinical trials of patients with relapsed refractory myeloma has led to investigations of the role of the ubiquitin-proteasome pathway in the pathogenesis of myeloma. Here we report a biochemical analysis of proteasome activity and composition in myeloma cells exposed to PS-341 in the presence or absence of cytokines present in the bone marrow milieu. We observed that the myeloma cell lines MM1.S, RPMI8226, and U266 contain active immunoproteasomes, the amount of which is enhanced by IFN-γ and tumor necrosis factor-α. Using a radiolabeled active site–directed probe specific for proteasome catalytic subunits, we show that PS-341 targets the β5 and β1 subunits in a concentration-dependent manner. Furthermore, PS-341 also targeted the corresponding catalytic subunits of the immunoproteasome, β5i and β1i, respectively. These data suggest that PS-341 targets both normal and immunoproteasome species to a similar extent in myeloma cells.