bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Mirk/Dyrk1B Maintains the Viability of Quiescent Pancreatic Cancer Cells by Reducing Levels of Reactive Oxygen Species
Authors: Deng Xiaobing , Ewton Daina Z. , Friedman Eileen
Published: March 2023

Abstract
The kinase Mirk/dyrk1B mediated the clonogenic growth of pancreatic cancer cells in earlier studies. It is now shown that Mirk levels increased 7-fold in SU86.86 pancreatic cancer cells when over a third of the cells were accumulated in a quiescent G0 state, defined by Hoechst/Pyronin Y staining. Depletion of Mirk by a doxycycline-inducible short hairpin RNA increased the G0 fraction to ∼50%, suggesting that Mirk provided some function in G0. Mirk reduced the levels of reactive oxygen species (ROS) in quiescent cultures of SU86.86 cells and of Panc1 cells by increasing transcription of the antioxidant genes ferroxidase, superoxide dismutase (SOD)2, and SOD3. These genes were functional antioxidant genes in pancreatic cancer cells because ectopic expression of SOD2 and ferroxidase in Mirk-depleted cells lowered ROS levels. Quiescent pancreatic cancer cells quickly lost viability when depleted of Mirk because of elevated ROS levels, exhibiting up to 4-fold less colony-forming activity and 4-fold less capability for dye exclusion. As a result, reduction of ROS by N-acetyl cysteine led to more viable cells. Mirk also destabilizated cyclin D1 and D3 in quiescent cells. Thus, quiescent pancreatic cancer cells depleted of Mirk became less viable because they were damaged by ROS, and had increased levels of G1 cyclins to prime cells to escape quiescence. [Cancer Res 2009;69(8):3317–24]
Data from Reactivation of Suppressed RhoB is a Critical Step for the Inhibition of Anaplastic Thyroid Cancer Growth
Authors: Marlow Laura A. , Reynolds Lisa A. , Cleland Alan S. , Cooper Simon J. , Gumz Michelle L. , Kurakata Shinichi , Fujiwara Kosaku , Zhang Ying , Sebo Thomas , Grant Clive , McIver Bryan , Wadsworth J. Trad , Radisky Derek C. , Smallridge Robert C. , Copland John A.
Published: March 2023

Abstract
Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of the disease for which new therapeutic options are desperately needed. Previously, we showed that the high-affinity peroxisome proliferator–activated receptor γ (PPARγ) agonist, RS5444, inhibits cell proliferation of ATC cells via induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). We show here that up-regulation of RhoB is a critical step in PPARγ-mediated activation of p21-induced cell stasis. Using multiple independently derived ATC cell lines, we found that treatment with RS5444 leads to the up-regulation of RhoB and subsequent activation of p21, and that silencing of RhoB by RNAi blocks the ability of RS5444 to induce p21 and to inhibit cell proliferation. Our results show that transcriptional regulation of RhoB by the nuclear transcription factor PPARγ is responsible for the induction of p21 mRNA and protein. We further implicate RhoB as a key signaling effector for the growth inhibition of ATC, as treatment with a histone deacetylase inhibitor shown to increase RhoB expression in lung cancer cells caused the up-regulation of RhoB in ATC cells accompanied by increased expression of p21 and inhibition of cell proliferation; this effect occurred even in ATC cells that were unresponsive to RS5444 due to a lack of expression of PPARγ. Our results implicate RhoB as a novel intermediate in critical signaling pathways and as an additional target for therapeutic intervention in ATC. [Cancer Res 2009;69(4):1536–44]
Data from Presence of the Carcinogen N′-Nitrosonornicotine in the Urine of Some Users of Oral Nicotine Replacement Therapy Products
Authors: Stepanov Irina , Carmella Steven G. , Briggs Anna , Hertsgaard Louise , Lindgren Bruce , Hatsukami Dorothy , Hecht Stephen S.
Published: March 2023

Abstract
N′-nitrosonornicotine (NNN) is a strong carcinogen present in unburned tobacco and cigarette smoke. We here analyze data obtained in two studies, in which a biomarker of exposure to NNN—the sum of NNN and its pyridine-N-glucuronide, called total NNN—was quantified in the urine of people who had stopped smoking and used various nicotine replacement therapy (NRT) products. In 13 of 34 nicotine gum or lozenge users from both studies, total NNN at one or more time points after biochemically confirmed smoking cessation was comparable with, or considerably higher than, the baseline levels. For most of the subjects who used the nicotine patch as a smoking cessation aid, urinary total NNN at all post–quit time points was <37% of their mean baseline levels. These results indicate that endogenous formation of significant amounts of NNN may occur sporadically in some users of oral NRT. Given the carcinogenicity of NNN and the frequent use of nicotine gum as a smoking cessation aid, further studies are needed so that preventive measures can be developed. [Cancer Res 2009;69(21):8236–40]
Data from Tobacco Smoke Induces Polycomb-Mediated Repression of Dickkopf-1 in Lung Cancer Cells
Authors: Hussain Mustafa , Rao Mahadev , Humphries Ashley E. , Hong Julie A. , Liu Fang , Yang Maocheng , Caragacianu Diana , Schrump David S.
Published: March 2023

Abstract
Limited information is available about epigenetic mechanisms by which cigarette smoke enhances the initiation and progression of lung cancer. To examine this issue, A549 and Calu-6 lung cancer cells were cultured in normal media with or without tobacco smoke condensate (TSC) under clinically relevant exposure conditions. Ten-day TSC exposure dramatically increased the tumorigenicity of lung cancer cells in nude mice. Microarray and quantitative reverse transcription-PCR (RT-PCR) experiments revealed that this phenomenon coincided with diminished expression of Dickkopf-1 (Dkk-1). Western blot, chromatin immunoprecipitation, methylation-specific PCR, and pyrosequencing experiments showed that repression of Dkk-1 coincided with decreased H4K16Ac, increased H3K27me3, and recruitment of SirT1, EZH2, SUZ12, and Bmi1 without DNA hypermethylation within the Dkk-1 promoter despite prolonged TSC exposures. Removal of TSC from culture media resulted in loss of promoter-associated polycomb repressor complexes and reexpression of Dkk-1. siRNA-mediated knockdown of EZH2 and SirT1 partially abrogated TSC-mediated inhibition of Dkk-1 expression. Western blot and quantitative RT-PCR array experiments showed that TSC exposure as well as knockdown of Dkk-1 activated Wnt signaling and significantly up-regulated Wnt5a in lung cancer cells. Knockdown of Dkk-1 recapitulated the dramatic protumorigenic effects of TSC exposure in Calu-6 cells. Despite the transient nature of Dkk-1 repression following TSC exposure in vitro, Dkk-1 remained silenced in tumor xenografts derived from TSC-treated Calu-6 cells. Collectively, these data provide evidence that cigarette smoke directly engages polycomb machinery to activate a signaling network implicated in maintenance of cancer stem cells. [Cancer Res 2009;69(8):3570–8]
Data from Low-Density Lipoprotein Receptor-Related Protein 1 Promotes Cancer Cell Migration and Invasion by Inducing the Expression of Matrix Metalloproteinases 2 and 9
Authors: Song Heesang , Li Yonghe , Lee Jiyeon , Schwartz Alan L. , Bu Guojun
Published: March 2023

Abstract
The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor involved in the metabolism of various extracellular ligands, including proteinases, that play critical roles in tumor invasion. Although several studies have shown an increased expression of LRP1 in cancer cells, its function in tumor development and progression remains largely unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and MMP9 and thereby promotes the migration and invasion of human glioblastoma U87 cells. Knockdown of LRP1 expression greatly decreased U87 cell migration and invasion, which was rescued by the forced expression of a functional LRP1 minireceptor. Inhibition of ligand binding to LRP1 by a specific antagonist, receptor-associated protein, also led to reduced cancer cell migration and invasion. Because MMPs play critical roles in cancer cell migration and invasion, we examined the expression of several MMPs and found that the expression of functional MMP2 and MMP9 was selectively decreased in LRP1 knockdown cells. More importantly, decreased cell migration and invasion of LRP1 knockdown cells were completely rescued by exogenous expression of MMP2 or MMP9, suggesting that these MMPs are likely downstream targets of LRP1-mediated signaling. We further show that the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in LRP1-silenced cells, suggesting that ERK is a potential mediator of LRP1-regulated MMP2 and MMP9 expression in U87 cells. Together, our data strongly suggest that LRP1 promotes glioblastoma cell migration and invasion by regulating the expression and function of MMP2 and MMP9 perhaps via an ERK-dependent signaling pathway. [Cancer Res 2009;69(3):879–86]
Data from DNA Cross-Links in Human Tumor Cells Exposed to the Prodrug PR-104A: Relationships to Hypoxia, Bioreductive Metabolism, and Cytotoxicity
Authors: Singleton Rachelle S. , Guise Christopher P. , Ferry Dianne M. , Pullen Susan M. , Dorie Mary J. , Brown J. Martin , Patterson Adam V. , Wilson William R.
Published: March 2023

Abstract
PR-104, currently in clinical trial, is converted systemically to the dinitrobenzamide nitrogen mustard prodrug PR-104A, which is reduced selectively in hypoxic cells to cytotoxic hydroxylamine (PR-104H) and amine (PR-104M) metabolites. Here, we evaluate the roles of this reductive metabolism, and DNA interstrand cross-links (ICL), in the hypoxic and aerobic cytotoxicity of PR-104. Using a panel of 9 human tumor cell lines, cytotoxicity was determined by clonogenic assay after a 2-hour aerobic or hypoxic exposure to PR-104A. PR-104H and PR-104M were determined by high performance liquid chromatography/mass spectrometry, and ICL with the alkaline comet assay. Under hypoxia, the relationship between ICL and cell killing was similar between cell lines. Under aerobic conditions, there was a similar relationship between ICL and cytotoxicity, except in lines with very low rates of aerobic reduction of PR-104A (A2780, C33A, H1299), which showed an ICL-independent mechanism of PR-104A cytotoxicity. Despite this, in xenografts from the same lines, the frequency of PR-104–induced ICL correlated with clonogenic cell killing (r2 = 0.747) with greatest activity in the fast aerobic metabolizers. In addition, changing levels of hypoxia in SiHa tumors modified both ICL frequency and tumor growth delay in parallel. We conclude that both aerobic and hypoxic nitroreduction of PR-104A contribute to the monotherapy antitumor activity of PR-104 in human tumor xenografts, and that ICL are responsible for its antitumor activity and represent a broadly applicable biomarker for tumor cell killing by this novel prodrug. [Cancer Res 2009;69(9):3884–91]
Data from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation
Authors: Mukhopadhyay Nishit K. , Kim Jayoung , Cinar Bekir , Ramachandran Aruna , Hager Martin H. , Di Vizio Dolores , Adam Rosalyn M. , Rubin Mark A. , Raychaudhuri Pradip , De Benedetti Arrigo , Freeman Michael R.
Published: March 2023

Abstract
The regulation of androgen receptor (AR) expression in prostate cancer is still poorly understood. The activation of the epidermal growth factor receptor (EGFR) in prostate cancer cells was previously shown to lower AR expression by a rapamycin-sensitive, posttranscriptional mechanism involving the AR mRNA 5′-untranslated region (5′-UTR). In a search for an intermediate within the EGFR/phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway that regulates AR at this site, we identified the nucleic acid–binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNP-K), by mass spectrometric analysis of Akt immune complexes from lipid raft–enriched subcellular fractions. We show here that hnRNP-K is a novel inhibitor of AR mRNA translation that regulates androgen-responsive gene expression and prostate cancer cell proliferation. A functional hnRNP-K binding site involved in down-regulating AR protein levels was identified in the AR mRNA 5′-UTR. Further analysis revealed that hnRNP-K is also able to inhibit AR translation in the absence of the 5′-UTR, consistent with the presence of additional predicted hnRNP-K binding sites within the AR open reading frame and in the 3′-UTR. Immunohistochemical analysis of a human prostate cancer tissue microarray revealed an inverse correlation between hnRNP-K expression and AR protein levels in organ-confined prostate tumors and a substantial decline in cytoplasmic hnRNP-K in metastases, despite an overall increase in hnRNP-K levels in metastatic tumors. These data suggest that translational inhibition of AR by hnRNP-K may occur in organ-confined tumors but possibly at a reduced level in metastases. HnRNP-K is the first protein identified that directly interacts with and regulates the AR translational apparatus. [Cancer Res 2009;69(6):2210–8]
Data from Identification of SULF2 as a Novel Transcriptional Target of p53 by Use of Integrated Genomic Analyses
Authors: Chau B. Nelson , Diaz Robert L. , Saunders Matthew A. , Cheng Chun , Chang Aaron N. , Warrener Paul , Bradshaw Jeffrey , Linsley Peter S. , Cleary Michele A.
Published: March 2023

Abstract
Microarray analysis has been useful for identifying the targets of many transcription factors. However, gene expression changes in response to transcription factor perturbation reveal both direct transcriptional targets and secondary gene regulation. By integrating RNA interference, gene expression profiling, and chromatin immunoprecipitation technologies, we identified a set of 32 direct transcriptional targets of the tumor suppressor p53. Of these 32 genes, 11 are not currently associated with the core p53 pathway. From among these novel pathway members, we focused on understanding the connection between p53 and SULF2, which encodes an extracellular heparan sulfate 6-O-endosulfatase that modulates the binding of growth factors to their cognate receptors and that has been shown to function as a tumor suppressor. Genetic and pharmacologic perturbation of p53 directly influences SULF2 expression, and similar to silencing of TP53, RNA interference–mediated suppression of SULF2 results in an impaired senescence response of cells to genotoxic stress. Thus, our integrated genomic approach has led to the identification of a novel mediator of p53 network biology. [Cancer Res 2009;69(4):1368–74]
Data from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice
Authors: Vincent François , Bonnin Philippe , Clemessy Maud , Contrerès Jean-Olivier , Lamandé Noël , Gasc Jean-Marie , Vilar José , Hainaud Patricia , Tobelem Gérard , Corvol Pierre , Dupuy Evelyne
Published: March 2023

Abstract
Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]
Data from Mitogen-Activated Protein Kinase Inhibition Induces Translocation of Bmf to Promote Apoptosis in Melanoma
Authors: VanBrocklin Matthew W. , Verhaegen Monique , Soengas Maria S. , Holmen Sheri L.
Published: March 2023

Abstract
Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF have been identified in ∼85% of melanomas, and components of this pathway have been developed as molecular targets for therapeutic intervention. We and others have shown that inhibition of this pathway with specific small molecule MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors induces a wide range of apoptotic responsiveness in human melanoma cells both in vitro and in vivo. To define the molecular mechanism underlying variable apoptotic sensitivity of melanoma cells to MEK inhibition, we examined the expression and subcellular localization of Bcl-2 family members in a comprehensive set of human melanoma cell lines. Whereas the proapoptotic protein Bim was activated and localized to the mitochondrial membrane in all cell lines regardless of apoptotic sensitivity, Bmf activation and cytosolic translocation was exclusive to sensitive cells. In resistant cells, Bmf remained sequestered to the cytoskeleton through dynein light chain 2 (DLC2) binding. Overexpression of Bmf in resistant cells did not enhance apoptosis, whereas expression of mutant BmfA69P, which has decreased binding to DLC2, promoted cell death. Expression of BmfA69P mutants possessing the Bcl-2 homology 3 (BH3) domain mutation L138A, which impairs BH3 interactions, did not enhance apoptosis in resistant cells. RNA interference targeting Bim and Bmf provided protection from apoptosis induced by MEK inhibition. These results show a novel role for Bmf in promoting apoptosis and provide insight into the mechanism of apoptotic resistance to MEK inhibition in melanoma. [Cancer Res 2009;69(5):1985–94]
Data from Persistence of Betapapillomavirus Infections as a Risk Factor for Actinic Keratoses, Precursor to Cutaneous Squamous Cell Carcinoma
Authors: Plasmeijer Elsemieke I. , Neale Rachel E. , de Koning Maurits N.C. , Quint Wim G.V. , McBride Penelope , Feltkamp Mariet C.W. , Green Adele C.
Published: March 2023

Abstract
Human papillomaviruses from the β genus (βPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We assessed the extent to which βPV infections persisted long-term in a subtropical Australian community and whether βPV persistence is positively associated with actinic keratoses, precursor for SCC. Eyebrow hairs were collected from 171 participants of the community-based Nambour Skin Cancer Study in 1996 and 2003. Hair samples were tested for the presence of DNA from 25 different βPV types and assessed in relation to actinic keratosis presence in 2007. In 1996, a total of 413 βPV infections were found in 73% of participants, increasing to 490 infections among 85% in 2003. Of the total βPV infections detected, 211 (30%) were found to persist. Age was significantly associated with βPV persistence: those ages >60 years had 1.5-fold (95% confidence interval, 1.1-1.9) increased risk of type-specific viral persistence than those ages <40 years. After accounting for actinic keratoses at baseline, persistence of βPV DNA resulted in a 1.4-fold (95% confidence interval, 1.0-1.9) increase in risk of having actinic keratoses on the face in 2007. In conclusion, persistent βPV infections in this population were associated with an increased occurrence of actinic keratosis. Additional studies are needed to determine the possible association of βPV persistence with SCC. [Cancer Res 2009;69(23):8926–31]
Data from Strongly Enhanced Antitumor Activity of Trastuzumab and Pertuzumab Combination Treatment on HER2-Positive Human Xenograft Tumor Models
Authors: Scheuer Werner , Friess Thomas , Burtscher Helmut , Bossenmaier Birgit , Endl Josef , Hasmann Max
Published: March 2023

Abstract
The human epidermal growth factor receptor (HER) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of HER2 is associated with aggressive disease and poor prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven survival benefit for women with HER2-positive early and metastatic breast cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization inhibitor that binds to a different epitope on HER2 than trastuzumab and inhibits HER2 dimer formation with other HER family members such as HER3 and HER1. We investigated the antitumor activity of these agents alone and in combination in HER2-positive breast and non–small cell lung cancer xenografts. Our data show that the combination of trastuzumab and pertuzumab has a strongly enhanced antitumor effect and induces tumor regression in both xenograft models, something that cannot be achieved by either monotherapy. The enhanced efficacy of the combination was also observed after tumor progression during trastuzumab monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we show by in vitro assay that both trastuzumab and pertuzumab potently activate antibody-dependent cellular cytotoxicity. However, our data suggest that the strongly enhanced antitumor activity is mainly due to the differing but complementary mechanisms of action of trastuzumab and pertuzumab, namely inhibition of HER2 dimerization and prevention of p95HER2 formation. [Cancer Res 2009;69(24):9330–6]
Data from CDC25B Mediates Rapamycin-Induced Oncogenic Responses in Cancer Cells
Authors: Chen Run-qiang , Yang Qing-kai , Lu Bing-wen , Yi Wei , Cantin Greg , Chen Yan-ling , Fearns Colleen , Yates John R. , Lee Jiing-Dwan
Published: March 2023

Abstract
Because the mammalian target of rapamycin (mTOR) pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anticancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an as yet unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin. Among these, rapamycin regulated four kinases and four phosphatases. A siRNA-dependent screen of these proteins showed that AKT induction by rapamycin was attenuated by depleting cellular CDC25B phosphatase. Rapamycin induces the phosphorylation of CDC25B at Serine375, and mutating this site to Alanine substantially reduced CDC25B phosphatase activity. Additionally, expression of CDC25B (S375A) inhibited the AKT activation by rapamycin, indicating that phosphorylation of CDC25B is critical for CDC25B activity and its ability to transduce rapamycin-induced oncogenic AKT activity. Importantly, we also found that CDC25B depletion in various cancer cell lines enhanced the anticancer effect of rapamycin. Together, using rapamycin phosphoproteomics, we not only advance the global mechanistic understanding of the action of rapamycin but also show that CDC25B may serve as a drug target for improving mTOR-targeted cancer therapies. [Cancer Res 2009;69(6):OF2663–8]
Data from Epidermal Growth Factor Receptor Signaling Synergizes with Hedgehog/GLI in Oncogenic Transformation via Activation of the MEK/ERK/JUN Pathway
Authors: Schnidar Harald , Eberl Markus , Klingler Stefan , Mangelberger Doris , Kasper Maria , Hauser-Kronberger Cornelia , Regl Gerhard , Kroismayr Renate , Moriggl Richard , Sibilia Maria , Aberger Fritz
Published: March 2023

Abstract
Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC. [Cancer Res 2009;69(4):1284–92]
Data from Activation of Ribosomal RNA Transcription by Hepatitis C Virus Involves Upstream Binding Factor Phosphorylation via Induction of Cyclin D1
Authors: Raychaudhuri Santanu , Fontanes Vanessa , Barat Bhaswati , Dasgupta Asim
Published: March 2023

Abstract
Hepatitis C virus (HCV) causes chronic infection in humans leading to liver cirrhosis and hepatocellular carcinoma. rRNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis, and changes in Pol I transcription rate are associated with profound alterations in the growth rate of the cell. Because rRNA synthesis is intimately linked to cell growth and frequently up-regulated in many cancers, we hypothesized that HCV might have the ability to activate rRNA synthesis in infected cells. We show here that rRNA promoter–mediated transcription is significantly (10- to 12-fold) activated in human liver–derived cells following infection with type 2 JFH-1 HCV or transfection with the subgenomic type 1 HCV replicon. Further analysis revealed that HCV nonstructural protein 5A (NS5A) was responsible for activation of rRNA transcription. Both the NH2-terminal amphipathic helix and the polyproline motifs of NS5A seem to be essential for rRNA transcription activation. The NS5A-dependent activation of rRNA transcription seems to be due to hyperphosphorylation and consequent activation of upstream binding factor (UBF), a Pol I DNA binding transcription factor. We further show that hyperphosphorylation of UBF occurs as a result of up-regulation of both cyclin D1 and cyclin-dependent kinase 4 by the HCV NS5A polypeptide. These results suggest that the endoplasmic reticulum–associated NS5A is able to transduce signals into the nucleoplasm via UBF hyperphosphorylation leading to rRNA transcription activation. These results could, at least in part, explain a mechanism by which HCV contributes to transformation of liver cells. [Cancer Res 2009;69(5):2057–64]
Data from LY303511 Enhances TRAIL Sensitivity of SHEP-1 Neuroblastoma Cells via Hydrogen Peroxide–Mediated Mitogen-Activated Protein Kinase Activation and Up-regulation of Death Receptors
Authors: Shenoy Kirthan , Wu Yongfei , Pervaiz Shazib
Published: March 2023

Abstract
We recently reported that LY294002 (LY29) and LY303511 (LY30) sensitized tumor cells to drug-induced apoptosis independent of the phosphoinositide 3-kinase/Akt pathway. Here, we investigated the mechanism of LY30-induced sensitization of human neuroblastoma cells to TRAIL-mediated apoptosis. We provide evidence that LY30-induced increase in intracellular H2O2 up-regulates the expression of TRAIL receptors (DR4 and DR5) in SHEP-1 cells by activating mitogen-activated protein kinases, resulting in a significant amplification of TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Involvement of the death receptors was further confirmed by the ability of blocking antibodies against DR4 and/or DR5 to inhibit LY30-induced TRAIL sensitization. Pharmacologic inhibition of c-Jun NH2 terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation by SP600125 and PD98059, respectively, blocked LY30-induced increase in sensitization to TRAIL-mediated death. Finally, small interfering RNA–mediated gene silencing of JNK and ERK inhibited LY30-induced increase in surface expression of DR4 and DR5, respectively. These data show that JNK and ERK are two crucial players involved in H2O2-mediated increase in TRAIL sensitization of tumor cells upon exposure to LY30 and underscore a novel mode of action of this inactive analogue of LY29. Our findings could have implications for the use of LY30 and similar compounds for enhancing the apoptotic sensitivity of neuroblastoma cells that often become refractory to chemotherapy. [Cancer Res 2009;69(5):1941–50]
Data from Identification of Potential Driver Genes in Human Liver Carcinoma by Genomewide Screening
Authors: Woo Hyun Goo , Park Eun Sung , Lee Ju-Seog , Lee Yun-Han , Ishikawa Tsuyoshi , Kim Yoon Jun , Thorgeirsson Snorri S.
Published: March 2023

Abstract
Genomic copy number aberrations and corresponding transcriptional deregulation in the cancer genome have been suggested to have regulatory roles in cancer development and progression. However, functional evaluation of individual genes from lengthy lists of candidate genes from genomic data sets presents a significant challenge. Here, we report effective gene selection strategies to identify potential driver genes based on systematic integration of genome scale data of DNA copy numbers and gene expression profiles. Using regional pattern recognition approaches, we discovered the most probable copy number–dependent regions and 50 potential driver genes. At each step of the gene selection process, the functional relevance of the selected genes was evaluated by estimating the prognostic significance of the selected genes. Further validation using small interference RNA–mediated knockdown experiments showed proof-of-principle evidence for the potential driver roles of the genes in hepatocellular carcinoma progression (i.e., NCSTN and SCRIB). In addition, systemic prediction of drug responses implicated the association of the 50 genes with specific signaling molecules (mTOR, AMPK, and EGFR). In conclusion, the application of an unbiased and integrative analysis of multidimensional genomic data sets can effectively screen for potential driver genes and provides novel mechanistic and clinical insights into the pathobiology of hepatocellular carcinoma. [Cancer Res 2009;69(9):4059–66]
Data from Clinical Predictors for Germline Mutations in Head and Neck Paraganglioma Patients: Cost Reduction Strategy in Genetic Diagnostic Process as Fall-Out
Authors: Neumann Hartmut P.H. , Erlic Zoran , Boedeker Carsten C. , Rybicki Lisa A. , Robledo Mercedes , Hermsen Mario , Schiavi Francesca , Falcioni Maurizio , Kwok Pingling , Bauters Catherine , Lampe Karen , Fischer Markus , Edelman Emily , Benn Diana E. , Robinson Bruce G. , Wiegand Stefanie , Rasp Gerd , Stuck Boris A. , Hoffmann Michael M. , Sullivan Maren , Sevilla Maria A. , Weiss Marjan M. , Peczkowska Mariola , Kubaszek Agata , Pigny Pascal , Ward Robyn L. , Learoyd Diana , Croxson Michael , Zabolotny Dmitry , Yaremchuk Svetlana , Draf Wolfgang , Muresan Mihaela , Lorenz Robert R. , Knipping Stephan , Strohm Michael , Dyckhoff Gerhard , Matthias Christoph , Reisch Nicole , Preuss Simon F. , Eβer Dirk , Walter Martin A. , Kaftan Holger , Stöver Timo , Fottner Christian , Gorgulla Harald , Malekpour Mahdi , Zarandy Masoud Motasaddi , Schipper Jörg , Brase Christoph , Glien Alexander , Kühnemund Matthias , Koscielny Sven , Schwerdtfeger Peter , Välimäki Matti , Szyfter Witold , Finckh Ulrich , Zerres Klaus , Cascon Alberto , Opocher Giuseppe , Ridder Gerd J. , Januszewicz Andrzej , Suarez Carlos , Eng Charis
Published: March 2023

Abstract
Multiple genes and their variants that lend susceptibility to many diseases will play a major role in clinical routine. Genetics-based cost reduction strategies in diagnostic processes are important in the setting of multiple susceptibility genes for a single disease. Head and neck paraganglioma (HNP) is caused by germline mutations of at least three succinate dehydrogenase subunit genes (SDHx). Mutation analysis for all 3 costs ∼US$2,700 per patient. Genetic classification is essential for downstream management of the patient and preemptive management of family members. Utilizing HNP as a model, we wanted to determine predictors to prioritize the most heritable clinical presentations and which gene to begin testing in HNP presentations, to reduce costs of genetic screening. Patients were tested for SDHB, SDHC, and SDHD intragenic mutations and large deletions. Clinical parameters were analyzed as potential predictors for finding germline mutations. Cost reduction was calculated between prioritized gene testing compared with that for all genes. Of 598 patients, 30.6% had SDHx germline mutations: 34.4% in SDHB, 14.2% SDHC, and 51.4% SDHD. Predictors for an SDHx mutation are family history [odds ratio (OR), 37.9], previous pheochromocytoma (OR, 10.9), multiple HNP (OR, 10.6), age ≤40 years (OR, 4.0), and male gender (OR, 3.5). By screening only preselected cases and a stepwise approach, 60% cost reduction can be achieved, with 91.8% sensitivity and 94.5% negative predictive value. Our data give evidence that clinical parameters can predict for mutation and help prioritize gene testing to reduce costs in HNP. Such strategy is cost-saving in the practice of genetics-based personalized health care. [Cancer Res 2009;69(8):3650–6]
Data from Improved Expression and Reactivity of Transduced Tumor-Specific TCRs in Human Lymphocytes by Specific Silencing of Endogenous TCR
Authors: Okamoto Sachiko , Mineno Junichi , Ikeda Hiroaki , Fujiwara Hiroshi , Yasukawa Masaki , Shiku Hiroshi , Kato Ikunoshin
Published: March 2023

Abstract
Adoptive T-cell therapy using lymphocytes genetically engineered to express tumor antigen-specific TCRs is an attractive strategy for treating patients with malignancies. However, there are potential drawbacks to this strategy: mispairing of the introduced TCR α/β chains with the endogenous TCR subunits and competition of CD3 molecules between the introduced and endogenous TCRs can impair cell surface expression of the transduced TCR, resulting in insufficient function and potential generation of autoreactive T cells. In addition, the risk of tumor development following the infusion of cells with aberrant vector insertion sites increases with the vector copy number in the transduced cells. In this study, we developed retroviral vectors encoding both small interfering RNA constructs that specifically down-regulate endogenous TCR and a codon-optimized, small interfering RNA–resistant TCR specific for the human tumor antigens MAGE-A4 or WT1. At low copy numbers of the integrated vector, the transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR and reduced expression of endogenous TCRs. In consequence, the vector-transduced lymphocytes showed enhanced cytotoxic activity against antigen-expressing tumor cells. Therefore, our novel TCR gene therapy may open a new gate for effective immunotherapy in cancer patients. [Cancer Res 2009;69(23):9003–11]
Data from Zerumbone Abolishes RANKL-Induced NF-κB Activation, Inhibits Osteoclastogenesis, and Suppresses Human Breast Cancer–Induced Bone Loss in Athymic Nude Mice
Authors: Sung Bokyung , Murakami Akira , Oyajobi Babatunde O. , Aggarwal Bharat B.
Published: March 2023

Abstract
Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) has emerged as a major mediator of bone resorption, commonly associated with cancer and other chronic inflammatory diseases. Inhibitors of RANKL signaling thus have potential in preventing bone loss. In the present report, the potential of zerumbone, a sesquiterpene derived from subtropical ginger, to modulate osteoclastogenesis induced by RANKL and breast cancer was examined. We found that zerumbone inhibited RANKL-induced NF-κB activation in mouse monocyte, an osteoclast precursor cell, through inhibition of activation of IκBα kinase, IκBα phosphorylation, and IκBα degradation. Zerumbone also suppressed RANKL-induced differentiation of these cells to osteoclasts. This sesquiterpene also inhibited the osteoclast formation induced by human breast tumor cells and by multiple myeloma cells. Finally, we examined whether zerumbone could prevent human breast cancer–induced bone loss in animals. We found that zerumbone decreased osteolysis in a dose-dependent manner in MDA-MB-231 breast cancer tumor-bearing athymic nude mice. These results indicate that zerumbone is an effective blocker of RANKL-induced NF-κB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss. [Cancer Res 2009;69(4):1477–84]
Data from Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects
Authors: Kimura Richard H. , Cheng Zhen , Gambhir Sanjiv Sam , Cochran Jennifer R.
Published: March 2023

Abstract
There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity (∼10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or 64Cu-1,4,7,10-tetra-azacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC50, ∼20 nmol/L) 64Cu-DOTA–conjugated knottin peptides was 4.47% ± 1.21% and 4.56% ± 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC50, ∼0.4 μmol/L; 1.48 ± 0.53%ID/g) and c(RGDyK) (IC50, ∼1 μmol/L; 2.32 ± 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, 64Cu-DOTA–conjugated knottin peptides generated lower levels of nonspecific liver uptake (∼2%ID/g) compared with c(RGDyK) (∼4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. 64Cu-DOTA–conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers. [Cancer Res 2009;69(6):2435–42]
Data from Overexpression of the Cellular DEK Protein Promotes Epithelial Transformation In vitro and In vivo
Authors: Wise-Draper Trisha M. , Mintz-Cole Rachael A. , Morris Teresa A. , Simpson David S. , Wikenheiser-Brokamp Kathryn A. , Currier Mark A. , Cripe Timothy P. , Grosveld Gerard C. , Wells Susanne I.
Published: March 2023

Abstract
High levels of expression of the human DEK gene have been correlated with numerous human malignancies. Intracellular DEK functions have been described in vitro and include DNA supercoiling, DNA replication, RNA splicing, and transcription. We have shown that DEK also suppresses cellular senescence, apoptosis, and differentiation, thus promoting cell growth and survival in monolayer and organotypic epithelial raft models. Such functions are likely to contribute to cancer, but direct evidence to implicate DEK as an oncogene has remained elusive. Here, we show that in line with an early role in tumorigenesis, murine papilloma formation in a classical chemical carcinogenesis model was reduced in DEK knockout mice. Additionally, human papillomavirus E6/E7, hRas, and DEK cooperated in the transformation of keratinocytes in soft agar and xenograft establishment, thus also implicating DEK in tumor promotion at later stages. Finally, adenoviral DEK depletion via short hairpin RNA expression resulted in cell death in human tumor cells in vitro and in vivo, but did not significantly affect differentiated epithelial cells. Taken together, our data uncover oncogenic DEK activities as postulated from its frequent up-regulation in human malignancies, and suggest that the targeted suppression of DEK may become a strategic approach to the treatment of cancer. [Cancer Res 2009;69(5):1792–9]
Data from SCUBE2 Suppresses Breast Tumor Cell Proliferation and Confers a Favorable Prognosis in Invasive Breast Cancer
Authors: Cheng Chien-Jui , Lin Yuh-Charn , Tsai Ming-Tzu , Chen Ching-Shyang , Hsieh Mao-Chih , Chen Chi-Long , Yang Ruey-Bing
Published: March 2023

Abstract
Signal peptide-CUB-epidermal growth factor–like domain-containing protein 2 (SCUBE2), originally identified from the endothelium and several nonendothelial primary cell types, was recently shown to be expressed in invasive breast carcinomas. However, the protein localization and biological significance of SCUBE2 in breast cancer are unknown. In this report, we show by anti-SCUBE2 immunostaining that SCUBE2 is mainly expressed in vascular endothelial and mammary ductal epithelial cells in normal breast tissue. In addition, we observed positive staining for SCUBE2 in 55% (86 of 156) of primary breast tumors. Patients with positive SCUBE2 protein–expressing tumors had better prognosis than those with negative SCUBE2 protein–expressing tumors in terms of disease-free survival. Multivariate analysis confirmed SCUBE2 protein expression as an independent prognostic factor for disease-free survival. Furthermore, overexpression of ectopic SCUBE2 protein resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH terminal region of SCUBE2 directly bound to and antagonized bone morphogenetic protein activity. Together, our results show for the first time that altered SCUBE2 expression is important in breast cancer progression and SCUBE2 may serve as a useful prognostic marker. [Cancer Res 2009;69(8):3634–41]
Data from Neoadjuvant Vaccination Provides Superior Protection against Tumor Relapse following Surgery Compared with Adjuvant Vaccination
Authors: Grinshtein Natalie , Bridle Byram , Wan Yonghong , Bramson Jonathan L.
Published: March 2023

Abstract
Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine, we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly, neoadjuvant vaccination was far superior and provided ∼100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. [Cancer Res 2009;69(9):3979–85]
Data from Poly(ADP-Ribose) Polymerase-1 Inhibitor Treatment Regresses Autochthonous Brca2/p53-Mutant Mammary Tumors In vivo and Delays Tumor Relapse in Combination with Carboplatin
Authors: Hay Trevor , Matthews James R. , Pietzka Lucie , Lau Alan , Cranston Aaron , Nygren Anders O.H. , Douglas-Jones Anthony , Smith Graeme C.M. , Martin Niall M.B. , O’Connor Mark , Clarke Alan R.
Published: March 2023

Abstract
Germ-line heterozygosity of the BRCA2 gene in women predisposes to breast and ovarian cancers. Successful therapies targeted specifically at these neoplasms have thus far remained elusive. Recent studies in mice have shown that inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) targets cells lacking Brca2 and xenografts derived from BRCA2-deficient ES cells or Chinese hamster ovary cells. We set out to develop a more relevant preclinical model that will inform and accelerate translation into the clinic. As such, we conditionally deleted Brca2 and p53 within murine mammary epithelium and treated the resulting tumors in situ with a highly potent PARP-1 inhibitor (AZD2281) alone or in combination with carboplatin. Daily exposure to AZD2281 for 28 days caused significant regression or growth inhibition in 46 of 52 tumors. This response was shown to be specific to tumors lacking both Brca2and p53. AZD2281/carboplatin combination therapy for 28 days showed no advantage over carboplatin monotherapy. However, if PARP inhibitor treatment was continued, this significantly increased the time to tumor relapse and death in these mice. This preclinical study is the first to show in vivo hypersensitivity of spontaneously arising Brca2-deficient mammary tumors to PARP-1 inhibition monotherapy or combination therapy. As such, our data add substantial weight to the argument for the use of PARP inhibitors as therapeutic agents against human breast cancers in which BRCA2 is mutated. Moreover, the specificity that we have shown further suggests that PARP inhibitors will be generally effective against tumors caused by dysregulation of components of the homologous recombination pathway. [Cancer Res 2009;69(9):3850–55]
Data from Phosphorylation of TRAF2 within Its RING Domain Inhibits Stress-Induced Cell Death by Promoting IKK and Suppressing JNK Activation
Authors: Thomas Gregory S. , Zhang Laiqun , Blackwell Ken , Habelhah Hasem
Published: March 2023

Abstract
Tumor necrosis factor (TNF) receptor–associated factor 2 (TRAF2) is an adaptor protein that modulates the activation of the c-Jun NH2 terminal kinase (JNK)/c-Jun and IκB kinase (IKK)/nuclear factor-κB (NF-κB) signaling cascades in response to TNFα stimulation. Although many serine/threonine kinases have been implicated in TNFα-induced IKK activation and NF-κB–dependent gene expression, most of them do not directly activate IKK. Here, we report that protein kinase Cζ phosphorylates TRAF2 at Ser55, within the RING domain of the protein, after TNFα stimulation. Although this phosphorylation event has a minimal effect on induction of the immediate/transient phase of IKK and JNK activation by TNFα, it promotes the secondary/prolonged phase of IKK activation and inhibits that of JNK. Importantly, constitutive TRAF2 phosphorylation increased both basal and inducible NF-κB activation and rendered Ha-Ras-V12–transformed cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some malignant cancer cell lines and Hodgkin's lymphoma. These results reveal a new level of complexity in TNFα-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation is one of the events that are responsible for elevated basal NF-κB activity in certain human cancers. [Cancer Res 2009;69(8):3665–72]