bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from RIP1 Activates PI3K-Akt via a Dual Mechanism Involving NF-κB–Mediated Inhibition of the mTOR-S6K-IRS1 Negative Feedback Loop and Down-regulation of PTEN
Authors: Park Seongmi , Zhao Dawen , Hatanpaa Kimmo J. , Mickey Bruce E. , Saha Debabrata , Boothman David A. , Story Michael D. , Wong Eric T. , Burma Sandeep , Georgescu Maria-Magdalena , Rangnekar Vivek M. , Chauncey Sandili S. , Habib Amyn A.
Published: March 2023

Abstract
Therapeutic inhibition of mammalian target of rapamycin (mTOR) in cancer is complicated by the existence of a negative feedback loop linking mTOR to the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Thus, mTOR inhibition by rapamycin or TSC1/2 results in increased PI3K-Akt activation. The death domain kinase receptor interacting protein 1 (RIP1) plays a key role in nuclear factor-κB (NF-κB) activation and also activates the PI3K-Akt pathway through unknown mechanisms. RIP1 has recently been found to be overexpressed in glioblastoma multiforme, the most common adult primary malignant brain tumor, but not in grade II to III glioma. Our data suggest that RIP1 activates PI3K-Akt using dual mechanisms by removing the two major brakes on PI3K-Akt activity. First, increased expression of RIP1 activates PI3K-Akt by interrupting the mTOR negative feedback loop. However, unlike other signals that regulate mTOR activity without affecting its level, RIP1 negatively regulates mTOR transcription via a NF-κB–dependent mechanism. The second mechanism used by RIP1 to activate PI3K-Akt is down-regulation of cellular PTEN levels, which appears to be independent of NF-κB activation. The clinical relevance of these findings is highlighted by the demonstration that RIP1 levels correlate with activation of Akt in glioblastoma multiforme. Thus, our study shows that RIP1 regulates key components of the PTEN-PI3K-Akt-mTOR pathway and elucidates a novel negative regulation of mTOR signaling at the transcriptional level by the NF-κB pathway. Our data suggest that the RIP1-NF-κB status of tumors may influence response to treatments targeting the PTEN-PI3K-mTOR signaling axis. [Cancer Res 2009;69(10):4107–11]
Data from Endoplasmic Reticulum Stress Triggers XBP-1–Mediated Up-regulation of an EBV Oncoprotein in Nasopharyngeal Carcinoma
Authors: Hsiao Jenn-Ren , Chang Kung-Chao , Chen Chaio-Wei , Wu Shih-Yi , Su Ih-Jen , Hsu Mei-Chi , Jin Ying-Tai , Tsai Sen-Tien , Takada Kenzo , Chang Yao
Published: March 2023

Abstract
Endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) plays multiple roles in cancer development, but its specific roles for virus-associated cancers have not been fully understood. It is still unknown whether ER stress/UPR occurs in and contributes to nasopharyngeal carcinoma (NPC), an epithelial malignancy closely associated with EBV. Here, we report that UPR proteins are frequently detected in NPC biopsies. In addition, we reveal that, in EBV-infected NPC cells, ER stress inducers up-regulate a potent EBV oncoprotein latent membrane protein 1 (LMP1), and the ER stress-induced LMP1 enhances production of interleukin-8. ER stress triggers LMP1 expression at a transcriptional level, activating a distal LMP1 promoter TR-L1. TR-L1 contains an ER stress-responsive element, which is targeted by an UPR protein XBP-1. Ectopic expression of XBP-1 induces LMP1 expression, and knockdown of XBP-1 blocks ER stress-triggered up-regulation of LMP1 in NPC cells. Furthermore, XBP-1 significantly correlates with LMP1 expression in NPC tumor biopsies. Therefore, this study not only provides a novel clue linking ER stress/UPR to EBV-associated NPC but also suggests that ER stress/UPR can promote virus-associated cancer in a unique way by driving expression of a viral oncogene. [Cancer Res 2009;69(10):4461–7]
Data from Rearrangements and Amplification of IER3 (IEX-1) Represent a Novel and Recurrent Molecular Abnormality in Myelodysplastic Syndromes
Authors: Steensma David P. , Neiger Jessemy D. , Porcher Julie C. , Keats J. Jonathan , Bergsagel P. Leif , Dennis Thomas R. , Knudson Ryan A. , Jenkins Robert B. , Santana-Davila Rafael , Kumar Rajiv , Ketterling Rhett P.
Published: March 2023

Abstract
IER3 (formerly IEX-1) encodes a 27-kDa glycoprotein that regulates death receptor–induced apoptosis, interacts with NF-κB pathways, and increases expression rapidly in response to cellular stresses such as irradiation. Animal models, gene expression microarray experiments, and functional studies in cell lines have suggested a potential role for IER3 in oncogenesis, but, to date, no abnormalities of IER3 at the DNA level have been reported in patients with neoplasia. Here, we describe breakpoint cloning of a t(6;9)(p21;q34) translocation from a patient with a myelodysplastic syndrome (MDS), facilitated by conversion technology and array-based comparative genomic hybridization, which revealed a rearrangement translocating the IER3 coding region away from critical flanking/regulatory elements and to a transcript-poor chromosomal region, markedly decreasing expression. Using split-signal and locus-specific fluorescence in situ hybridization (FISH) probes, we analyzed 204 patients with diverse hematological malignancies accompanied by clonal chromosome 6p21 abnormalities, and found 8 additional patients with MDS with IER3 rearrangements (translocations or amplification). Although FISH studies on 157 additional samples from patients with MDS and a normal-karyotype were unrevealing, and sequencing the IER3 coding and proximal promoter regions of 74 MDS patients disclosed no point mutations, reverse transcription-PCR results suggested that dysregulated expression of IER3 is common in MDS (61% >4-fold increase or decrease in expression with decreased expression primarily in early MDS and increased expression primarily in later MDS progressing toward leukemia), consistent with findings in previous microarray experiments. These data support involvement of IER3 in the pathobiology of MDS. [Cancer Res 2009;69(19):7518–23]
Data from Role of MUTYH and MSH2 in the Control of Oxidative DNA Damage, Genetic Instability, and Tumorigenesis
Authors: Russo Maria Teresa , De Luca Gabriele , Casorelli Ida , Degan Paolo , Molatore Sara , Barone Flavia , Mazzei Filomena , Pannellini Tania , Musiani Piero , Bignami Margherita
Published: March 2023

Abstract
Mismatch repair is the major pathway controlling genetic stability by removing mispairs caused by faulty replication and/or mismatches containing oxidized bases. Thus, inactivation of the Msh2 mismatch repair gene is associated with a mutator phenotype and increased cancer susceptibility. The base excision repair gene Mutyh is also involved in the maintenance of genomic integrity by repairing premutagenic lesions induced by oxidative DNA damage. Because evidence in bacteria suggested that Msh2 and Mutyh repair factors might have some overlapping functions, we investigated the biological consequences of their single and double inactivation in vitro and in vivo. Msh2−/− mouse embryo fibroblasts (MEF) showed a strong mutator phenotype at the hprt gene, whereas Mutyh inactivation was associated with a milder phenotype (2.9 × 10−6 and 3.3 × 10−7 mutation/cell/generation, respectively). The value of 2.7 × 10−6 mutation/cell/generation in Msh2−/−Mutyh−/− MEFs did not differ significantly from Msh2−/− cells. When steady-state levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG) were measured in MEFs of different genotypes, single gene inactivation resulted in increases similar to those observed in doubly defective cells. In contrast, a synergistic accumulation of 8-oxoG was observed in several organs of Msh2−/−Mutyh−/− animals, suggesting that in vivo Msh2 and Mutyh provide separate repair functions and contribute independently to the control of oxidative DNA damage. Finally, a strong delay in lymphomagenesis was observed in Msh2−/−Mutyh−/− when compared with Msh2−/− animals. The immunophenotype of these tumors indicate that both genotypes develop B-cell lymphoblastic lymphomas displaying microsatellite instability. This suggests that a large fraction of the cancer-prone phenotype of Msh2−/− mice depends on Mutyh activity. [Cancer Res 2009;69(10):4372–9]
Data from Interaction of E-cadherin and PTEN Regulates Morphogenesis and Growth Arrest in Human Mammary Epithelial Cells
Authors: Fournier Marcia V. , Fata Jimmie E. , Martin Katherine J. , Yaswen Paul , Bissell Mina J.
Published: March 2023

Abstract
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual-function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two nonmalignant human mammary epithelial cell lines that form polarized, growth-arrested structures (acini) when cultured in three-dimensional laminin-rich extracellular matrix gels (lrECM). As acini begin to form, PTEN accumulates both in the cytoplasm and at cell-cell contacts where it colocalizes with the E-cadherin/β-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function–blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in Skbr-3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in three-dimensional lrECM, indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus seems to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells. [Cancer Res 2009;69(10):4545–52]
Data from Activation of Src by Protein Tyrosine Phosphatase 1B Is Required for ErbB2 Transformation of Human Breast Epithelial Cells
Authors: Arias-Romero Luis E. , Saha Sayanti , Villamar-Cruz Olga , Yip Shu-Chin , Ethier Stephen P. , Zhang Zhong-Yin , Chernoff Jonathan
Published: March 2023

Abstract
Protein tyrosine phosphatase (PTP) 1B plays a major role in inhibiting signaling from the insulin and leptin receptors. Recently, PTP1B was found to have an unexpected positive role in ErbB2 signaling in a mouse model of breast cancer, but the mechanism underlying this effect has been unclear. Using human breast epithelial cells grown in a three-dimensional matrix, we found that PTP1B, but not the closely related enzyme T-cell PTP, is required for ErbB2 transformation in vitro. Activation of ErbB2, but not ErbB1, increases PTP1B expression, and increased expression of PTP1B activates Src and induces a Src-dependent transformed phenotype. These findings identify a molecular mechanism by which PTP1B links an important oncogenic receptor tyrosine kinase to signaling pathways that promote aberrant cell division and survival in human breast epithelial cells. [Cancer Res 2009;69(11):4582–8]
Data from Biological Significance of Prolactin in Gynecologic Cancers
Authors: Levina Vera V. , Nolen Brian , Su YunYun , Godwin Andrew K. , Fishman David , Liu Jinsong , Mor Gil , Maxwell Larry G. , Herberman Ronald B. , Szczepanski Miroslaw J. , Szajnik Marta E. , Gorelik Elieser , Lokshin Anna E.
Published: March 2023

Abstract
There is increasing evidence that prolactin (PRL), a hormone/cytokine, plays a role in breast, prostate, and colorectal cancers via local production or accumulation. Elevated levels of serum PRL in ovarian and endometrial cancers have been reported, indicating a potential role for PRL in endometrial and ovarian carcinogenesis. In this study, we show that serum PRL levels are significantly elevated in women with a strong family history of ovarian cancer. We show dramatically increased expression of PRL receptor in ovarian and endometrial tumors as well as in endometrial hyperplasia, signifying the importance of PRL signaling in malignant and premalignant conditions. PRL mRNA was expressed in ovarian and endometrial tumors, indicating the presence of an autocrine loop. PRL potently induced proliferation in several ovarian and endometrial cancer cell lines. Binding of PRL to its receptor was followed by rapid phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, mitogen-activated protein kinase/ERK kinase 1, signal transducer and activator of transcription 3, CREB, ATF-2, and p53 and activation of 37 transcription factors in ovarian and endometrial carcinoma cells. PRL also activated Ras oncogene in these cells. When human immortalized normal ovarian epithelial cells were chronically exposed to PRL, a malignant transformation occurred manifested by the acquired ability of transformed cells to form clones, grow in soft agar, and form tumors in severe combined immunodeficient-beige mice. Transformation efficiency was diminished by a Ras inhibitor, providing proof that PRL-induced transformation uses the Ras pathway. In summary, we present findings that indicate an important role for PRL in ovarian and endometrial tumorigenesis. PRL may represent a risk factor for ovarian and endometrial cancers. [Cancer Res 2009;69(12):5226–33]
Data from MUC1-C Oncoprotein Functions as a Direct Activator of the Nuclear Factor-κB p65 Transcription Factor
Authors: Ahmad Rehan , Raina Deepak , Joshi Maya Datt , Kawano Takeshi , Ren Jian , Kharbanda Surender , Kufe Donald
Published: March 2023

Abstract
Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies. The mucin 1 (MUC1) oncoprotein is overexpressed in human carcinomas and, like NF-κB, blocks cell death and induces transformation. The present studies show that MUC1 constitutively associates with NF-κB p65 in carcinoma cells. The MUC1 COOH-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-κB p65 and, importantly, blocks the interaction between NF-κB p65 and its inhibitor IκBα. We show that NF-κB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-κB–mediated transcriptional activation. Studies in nonmalignant epithelial cells show that MUC1-C interacts with NF-κB in the response to tumor necrosis factor-α stimulation. Moreover, tumor necrosis factor-α induces the recruitment of NF-κB p65-MUC1-C complexes to NF-κB target genes, including the promoter of the MUC1 gene itself. We also show that an inhibitor of MUC1-C oligomerization blocks the interaction with NF-κB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-κB p65 promoter occupancy and expression of NF-κB target genes. These findings indicate that MUC1-C is a direct activator of NF-κB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-κB pathway. [Cancer Res 2009;69(17):7013–21]
Data from The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells
Authors: François Violaine , Ottaviani Sabrina , Renkvist Nicolina , Stockis Julie , Schuler Gerold , Thielemans Kris , Colau Didier , Marchand Marie , Boon Thierry , Lucas Sophie , van der Bruggen Pierre
Published: March 2023

Abstract
Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti–MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 × 10−6 to 2 × 10−3 among the CD4+ blood T lymphocytes of the patients. The CD4+ blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4+ T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25+ phenotype in the resting state. These CD25+ clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-β but none of cytokines IFN-γ, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25− clones had a significant but lower suppressive activity. Most of the CD25− clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4+ T cells that may exert regulatory T-cell function in vivo. [Cancer Res 2009;69(10):4335–45]
Data from Differentiation of Lung Adenocarcinoma, Pleural Mesothelioma, and Nonmalignant Pulmonary Tissues Using DNA Methylation Profiles
Authors: Christensen Brock C. , Marsit Carmen J. , Houseman E. Andres , Godleski John J. , Longacker Jennifer L. , Zheng Shichun , Yeh Ru-Fang , Wrensch Margaret R. , Wiemels Joseph L. , Karagas Margaret R. , Bueno Raphael , Sugarbaker David J. , Nelson Heather H. , Wiencke John K. , Kelsey Karl T.
Published: March 2023

Abstract
Pathologic differentiation of tissue of origin in tumors found in the lung can be challenging, with differentiation of mesothelioma and lung adenocarcinoma emblematic of this problem. Indeed, proper classification is essential for determination of treatment regimen for these diseases, making accurate and early diagnosis critical. Here, we investigate the potential of epigenetic profiles of lung adenocarcinoma, mesothelioma, and nonmalignant pulmonary tissues (n = 285) as differentiation markers in an analysis of DNA methylation at 1413 autosomal CpG loci associated with 773 cancer-related genes. Using an unsupervised recursively partitioned mixture modeling technique for all samples, the derived methylation profile classes were significantly associated with sample type (P < 0.0001). In a similar analysis restricted to tumors, methylation profile classes significantly predicted tumor type (P < 0.0001). Random forests classification of CpG methylation of tumors—which splits the data into training and test sets—accurately differentiated mesothelioma from lung adenocarcinoma over 99% of the time (P < 0.0001). In a locus-by-locus comparison of CpG methylation between tumor types, 1266 CpG loci had significantly different methylation between tumors following correction for multiple comparisons (Q < 0.05); 61% had higher methylation in adenocarcinoma. Using the CpG loci with significant differential methylation in a pathway analysis revealed significant enrichment of methylated gene-loci in Cell Cycle Regulation, DNA Damage Response, PTEN Signaling, and Apoptosis Signaling pathways in lung adenocarcinoma when compared with mesothelioma. Methylation profile–based differentiation of lung adenocarcinoma and mesothelioma is highly accurate, informs on the distinct etiologies of these diseases, and holds promise for clinical application. [Cancer Res 2009;69(15):6315–21]
Data from Stress-Activated Mitogen-Activated Protein Kinases c-Jun NH2-Terminal Kinase and p38 Target Cdc25B for Degradation
Authors: Uchida Sanae , Yoshioka Katsuji , Kizu Ryoichi , Nakagama Hitoshi , Matsunaga Tsukasa , Ishizaka Yukihito , Poon Randy Y.C. , Yamashita Katsumi
Published: March 2023

Abstract
Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress–activated Chk1 or Chk2, which triggers its SCFβ-TrCP–mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress–induced cell cycle checkpoint. [Cancer Res 2009;69(16):6438–44]
Data from Quantitative Assessment of the Complex Dynamics of G1, S, and G2-M Checkpoint Activities
Authors: Ubezio Paolo , Lupi Monica , Branduardi Davide , Cappella Paolo , Cavallini Edoardo , Colombo Valentina , Matera Giada , Natoli Claudia , Tomasoni Daniela , D'Incalci Maurizio
Published: March 2023

Abstract
Although studies of cell cycle perturbation and growth inhibition are common practice, they are unable to properly measure the activity of cell cycle checkpoints and frequently convey misinterpretation or incomplete pictures of the response to anticancer treatment. A measure of the strength of the treatment response of all checkpoints, with their time and dose dependence, provides a new way to evaluate the antiproliferative activity of the drugs, fully accounting for variation of the cell fates within a cancer cell line. This is achieved with an interdisciplinary approach, joining information from independent experimental platforms and interpreting all data univocally with a simple mathematical model of cell cycle proliferation. The model connects the dynamics of checkpoint activities at the molecular level with population-based flow cytometric and growth inhibition time course measures. With this method, the response to five drugs, characterized by different molecular mechanisms of action, was studied in a synoptic way, producing a publicly available database of time course measures with different techniques in a range of drug concentrations, from sublethal to frankly cytotoxic. Using the computer simulation program, we were able to closely reproduce all the measures in the experimental database by building for each drug a scenario of the time and dose dependence of G1, S, and G2-M checkpoint activities. We showed that the response to each drug could be described as a combination of a few types of activities, each with its own strength and concentration threshold. The results gained from this method provide a means for exploring new concepts regarding the drug–cell cycle interaction. [Cancer Res 2009;69(12):5234–40]
Data from Mutagenicity and Potential Carcinogenicity of Thiopurine Treatment in Patients with Inflammatory Bowel Disease
Authors: Nguyen Truc , Vacek Pamela M. , O'Neill Patrick , Colletti Richard B. , Finette Barry A.
Published: March 2023

Abstract
The thiopurines azathioprine and 6-mercaptopurine (6-MP) are effective immune modulators and cytotoxic agents extensively used in the treatment of autoimmune diseases, graft rejection, and cancer. There is compelling epidemiologic evidence that thiopurine treatment increases the risk for a variety of tumors by mechanisms that are unclear. We investigated the in vivo mutagenicity of long-term thiopurine treatment by determining the frequency and spectra of somatic mutation events at the hypoxanthine phosphoribosyltransferase (HPRT) locus in peripheral T lymphocytes as well as the prevalence of mutant clonal proliferation in a cross-sectional analysis of data from 119 children and adults with inflammatory bowel disease (IBD). ANOVA and regression were performed to assess relationships among the frequency and spectra of HPRT mutations with disease, duration of illness, duration of treatment, and total therapeutic dose of azathioprine and 6-MP. We observed a significant increase in the frequency of somatic mutations in 56 subjects treated with thiopurines for IBD compared with 63 subjects not treated with thiopurines. This increase was related to both total dose (P < 0.001) and duration of treatment (P < 0.001). Comparative mutation spectra analysis of 1,020 mutant isolates revealed a significant increase in the proportion of all transitions (P < 0.001), particularly G:C to A:T transitions (P < 0.001). Combined analyses of two signatures for mutant clonality, HPRT mutation, and T-cell receptor β CDR3 region unique gene sequence also showed a significant thiopurine-dependent increase in mutant cell clonal proliferation (P < 0.001). These findings provide in vivo evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention. [Cancer Res 2009;69(17):7004–23]
Data from Epithelial to Mesenchymal Transition Contributes to Drug Resistance in Pancreatic Cancer
Authors: Arumugam Thiruvengadam , Ramachandran Vijaya , Fournier Keith F. , Wang Huamin , Marquis Lauren , Abbruzzese James L. , Gallick Gary E. , Logsdon Craig D. , McConkey David J. , Choi Woonyoung
Published: March 2023

Abstract
A better understanding of drug resistance mechanisms is required to improve outcomes in patients with pancreatic cancer. Here, we characterized patterns of sensitivity and resistance to three conventional chemotherapeutic agents with divergent mechanisms of action [gemcitabine, 5-fluorouracil (5-FU), and cisplatin] in pancreatic cancer cells. Four (L3.6pl, BxPC-3, CFPAC-1, and SU86.86) were sensitive and five (PANC-1, Hs766T, AsPC-1, MIAPaCa-2, and MPanc96) were resistant to all three agents based on GI50 (50% growth inhibition). Gene expression profiling and unsupervised hierarchical clustering revealed that the sensitive and resistant cells formed two distinct groups and differed in expression of specific genes, including several features of “epithelial to mesenchymal transition” (EMT). Interestingly, an inverse correlation between E-cadherin and its transcriptional suppressor, Zeb-1, was observed in the gene expression data and was confirmed by real-time PCR. Independent validation experiment using five new pancreatic cancer cell lines confirmed that an inverse correlation between E-cadherin and Zeb-1 correlated closely with resistance to gemcitabine, 5-FU, and cisplatin. Silencing Zeb-1 in the mesenchymal lines not only increased the expression of E-cadherin but also other epithelial markers, such as EVA1 and MAL2, and restored drug sensitivity. Importantly, immunohistochemical analysis of E-cadherin and Zeb-1 in primary tumors confirmed that expression of the two proteins was mutually exclusive (P = 0.012). Therefore, our results suggest that Zeb-1 and other regulators of EMT may maintain drug resistance in human pancreatic cancer cells, and therapeutic strategies to inhibit Zeb-1 and reverse EMT should be evaluated. [Cancer Res 2009;69(14):5820–8]
Data from Identification of Inhibitors of ABCG2 by a Bioluminescence Imaging–Based High-Throughput Assay
Authors: Zhang Yimao , Byun Youngjoo , Ren Yunzhao R. , Liu Jun O. , Laterra John , Pomper Martin G.
Published: March 2023

Abstract
ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)–based assay was developed to allow high-throughput compound screening. This assay exploits our finding that d-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of d-luciferin and enhance bioluminescence signal by increasing intracellular d-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of ≥5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing. [Cancer Res 2009;69(14):5867–75]
Data from Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death
Authors: Piao Longzhen , Li Yuwen , Kim Soung Jung , Byun Hee Sun , Huang Song Mei , Hwang Soon-Kyung , Yang Keum-Jin , Park Kyeong Ah , Won Minho , Hong Janghee , Hur Gang Min , Seok Jeong Ho , Shong Minho , Cho Myung-Haing , Brazil Derek P. , Hemmings Brian A. , Park Jongsun
Published: March 2023

Abstract
Leucine zipper/EF hand–containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis. [Cancer Res 2009;69(8):3397–404]
Data from Host-Derived Angiopoietin-2 Affects Early Stages of Tumor Development and Vessel Maturation but Is Dispensable for Later Stages of Tumor Growth
Authors: Nasarre Patrick , Thomas Markus , Kruse Karoline , Helfrich Iris , Wolter Vivien , Deppermann Carleen , Schadendorf Dirk , Thurston Gavin , Fiedler Ulrike , Augustin Hellmut G.
Published: March 2023

Abstract
The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2–deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2–deficient mice. Yet, tumor growth in wild-type and Ang-2–deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2–deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2–deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately α-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies. [Cancer Res 2009;69(4):1324–33]
Data from Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest
Authors: Bailet Olivier , Fenouille Nina , Abbe Patricia , Robert Guillaume , Rocchi Stéphane , Gonthier Nadège , Denoyelle Christophe , Ticchioni Michel , Ortonne Jean-Paul , Ballotti Robert , Deckert Marcel , Tartare-Deckert Sophie
Published: March 2023

Abstract
Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation–mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated β-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity. [Cancer Res 2009;69(7):2748–56]
Data from Characterization of ERG, AR and PTEN Gene Status in Circulating Tumor Cells from Patients with Castration-Resistant Prostate Cancer
Authors: Attard Gerhardt , Swennenhuis Joost F. , Olmos David , Reid Alison H.M. , Vickers Elaine , A'Hern Roger , Levink Rianne , Coumans Frank , Moreira Joana , Riisnaes Ruth , Oommen Nikhil Babu , Hawche George , Jameson Charles , Thompson Emilda , Sipkema Ronald , Carden Craig P. , Parker Christopher , Dearnaley David , Kaye Stan B. , Cooper Colin S. , Molina Arturo , Cox Michael E. , Terstappen Leon W.M.M. , de Bono Johann S.
Published: March 2023

Abstract
Hormone-driven expression of the ERG oncogene after fusion with TMPRSS2 occurs in 30% to 70% of therapy-naive prostate cancers. Its relevance in castration-resistant prostate cancer (CRPC) remains controversial as ERG is not expressed in some TMPRSS2-ERG androgen-independent xenograft models. However, unlike these models, CRPC patients have an increasing prostate-specific antigen, indicating active androgen receptor signaling. Here, we collected blood every month from 89 patients (54 chemotherapy-naive patients and 35 docetaxel-treated patients) treated in phase I/phase II clinical trials of an orally available, highly specific CYP17 inhibitor, abiraterone acetate, that ablates the synthesis of androgens and estrogens that drive TMPRSS2-ERG fusions. We isolated circulating tumor cells (CTC) by anti–epithelial cell adhesion molecule immunomagnetic selection followed by cytokeratin and CD45 immunofluorescence and 4′,6-diamidino-2-phenylindole staining. We used multicolor fluorescence in situ hybridization to show that CRPC CTCs, metastases, and prostate tissue invariably had the same ERG gene status as therapy-naive tumors (n = 31). We then used quantitative reverse transcription–PCR to show that ERG expression was maintained in CRPC. We also observed homogeneity in ERG gene rearrangement status in CTCs (n = 48) in contrast to significant heterogeneity of AR copy number gain and PTEN loss, suggesting that rearrangement of ERG may be an earlier event in prostate carcinogenesis. We finally report a significant association between ERG rearrangements in therapy-naive tumors, CRPCs, and CTCs and magnitude of prostate-specific antigen decline (P = 0.007) in CRPC patients treated with abiraterone acetate. These data confirm that CTCs are malignant in origin and indicate that hormone-regulated expression of ERG persists in CRPC. [Cancer Res 2009;69(7):2912–8]
Data from 14-3-3ζ Overexpression Defines High Risk for Breast Cancer Recurrence and Promotes Cancer Cell Survival
Authors: Neal Christopher L. , Yao Jun , Yang Wentao , Zhou Xiaoyan , Nguyen Nina T. , Lu Jing , Danes Christopher G. , Guo Hua , Lan Keng-Hsueh , Ensor Joe , Hittelman Walter , Hung Mien-Chie , Yu Dihua
Published: March 2023

Abstract
The ubiquitously expressed 14-3-3 proteins are involved in numerous important cellular functions. The loss of 14-3-3σ is a common event in breast cancer; however, the role of other 14-3-3s in breast cancer is unclear. Recently, we found that 14-3-3ζ overexpression occurs in early stage breast diseases and contributes to transformation of human mammary epithelial cells. Here, we show that 14-3-3ζ overexpression also persisted in invasive ductal carcinoma and contributed to the further progression of breast cancer. To examine the clinical effect of 14-3-3ζ overexpression in advanced stage breast cancer, we performed immunohistochemical analysis of 14-3-3ζ expression in primary breast carcinomas. 14-3-3ζ overexpression occurred in 42% of breast tumors and was determined to be an independent prognostic factor for reduced disease-free survival. 14-3-3ζ overexpression combined with ErbB2 overexpression and positive lymph node status identified a subgroup of patients at high risk for developing distant metastasis. To investigate whether 14-3-3ζ overexpression causally promotes breast cancer progression, we overexpressed 14-3-3ζ by stable transfection or reduced 14-3-3ζ expression by siRNA in cancer cell lines. Increased 14-3-3ζ expression enhanced anchorage-independent growth and inhibited stress-induced apoptosis, whereas down-regulation of 14-3-3ζ reduced anchorage-independent growth and sensitized cells to stress-induced apoptosis via the mitochondrial apoptotic pathway. Transient blockade of 14-3-3ζ expression by siRNA in cancer cells effectively reduced the onset and growth of tumor xenografts in vivo. Therefore, 14-3-3ζ overexpression is a novel molecular marker for disease recurrence in breast cancer patients and may serve as an effective therapeutic target in patients whose tumors overexpress 14-3-3ζ. [Cancer Res 2009;69(8):3425–32]
Data from Aurora A Regulates Prometaphase Progression by Inhibiting the Ability of RASSF1A to Suppress APC-Cdc20 Activity
Authors: Song Su Jung , Song Min Sup , Kim Soon Jung , Kim So Yeon , Kwon Seung Hae , Kim Jhin Gook , Calvisi Diego F. , Kang Dongmin , Lim Dae-Sik
Published: March 2023

Abstract
The Aurora (Ipl) kinase family plays important roles in the regulation of mitosis and tumorigenesis. The tumor suppressor RASSF1A controls mitotic progression by regulating anaphase-promoting complex (APC)-Cdc20 activity and microtubule stability, but the mechanism by which this action is regulated has not been previously established. Here, we show that Aurora A and B associate with and phosphorylate RASSF1A on serine 203 in vivo at different times and in different subcellular compartments during mitosis. Notably, both depletion of Aurora A by RNA interference and expression of a nonphosphorylatable RASSF1A (S203A) mutant gene led to a marked delay in prometaphase progression. This is likely because of the failure of RASSF1A to dissociate from Cdc20, constitutive inhibition of APC-Cdc20, and accumulation of mitotic cyclins. In contrast, the delay in prometaphase progression caused by Aurora A depletion was largely normalized by phosphomimetic RASSF1A (S203D). Finally, RASSF1A phosphorylation on serine 203 was up-regulated in Aurora A–overexpressing human tumors. These findings indicate that Aurora A plays a critical role in RASSF1A-APC-Cdc20 regulatory mechanisms that control normal prometaphase progression and that are involved in tumorigenesis. [Cancer Res 2009;69(6):2314–23
Data from Heat Shock Protein 90 Inhibitors: New Mode of Therapy to Overcome Endocrine Resistance
Authors: Wong Cynthie , Chen Shiuan
Published: March 2023

Abstract
Aromatase inhibitors are important drugs to treat estrogen receptor α (ERα)–positive postmenopausal breast cancer patients. However, development of resistance to aromatase inhibitors has been observed. We examined whether the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) can inhibit the growth of aromatase inhibitor–resistant breast cancers and the mechanisms by which 17-DMAG affects proliferation. Aromatase inhibitor–responsive MCF-7aro and aromatase inhibitor–resistant LTEDaro breast epithelial cells were used in this study. We observed that 17-DMAG inhibited proliferation in both MCF-7aro and LTEDaro cells in a dose-dependent manner. 17-DMAG induced apoptosis and G2 cell cycle arrest in both cell lines. Although inhibition of HSP90 decreased the levels of ERα, the ERα transcriptional activity was not affected when cells were treated with 17-DMAG together with estradiol. Moreover, detailed mechanistic studies suggested that 17-DMAG inhibits cell growth via degradation of HSP90 client proteins AKT and HER2. Collectively, results from this study provide data to support that HSP90 inhibitors may be an effective therapy to treat aromatase inhibitor–resistant breast cancers and that improved efficacy can be achieved by combined use of a HSP90 inhibitor and an AKT inhibitor. [Cancer Res 2009;69(22):8670–7]
Data from Antitumor Efficacy of the Novel RAF Inhibitor GDC-0879 Is Predicted by BRAFV600E Mutational Status and Sustained Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway Suppression
Authors: Hoeflich Klaus P. , Herter Sylvia , Tien Janet , Wong Leo , Berry Leanne , Chan Jocelyn , O'Brien Carol , Modrusan Zora , Seshagiri Somasekar , Lackner Mark , Stern Howard , Choo Edna , Murray Lesley , Friedman Lori S. , Belvin Marcia
Published: March 2023

Abstract
Oncogenic activation of the BRAF serine/threonine kinase has been associated with initiation and maintenance of melanoma tumors. As such, development of pharmacologic agents to target RAF proteins or their effector kinases is an area of intense investigation. Here we report the biological properties of GDC-0879, a highly selective, potent, and orally bioavailable RAF small-molecule inhibitor. We used extracellular signal-regulated kinase (ERK)-1/2 and mitogen-activated protein kinase/ERK kinase (MEK)-1/2 phosphorylation as biomarkers to explore the relationship between tumor outcome and pharmacodynamic inhibition of the RAF-MEK-ERK pathway. In GDC-0879–treated mice, both cell line– and patient-derived BRAFV600E tumors exhibited stronger and more sustained pharmacodynamic inhibition (>90% for 8 hours) and improved survival compared with mutant KRAS–expressing tumors. Despite the involvement of activated RAF signaling in RAS-induced tumorigenesis, decreased time to progression was observed for some KRAS-mutant tumors following GDC-0879 administration. Moreover, striking differences were noted for RAF and MEK inhibition across a panel of 130 tumor cell lines. Whereas GDC-0879–mediated efficacy was associated strictly with BRAFV600E status, MEK inhibition also attenuated proliferation and tumor growth of cell lines expressing wild-type BRAF (81% KRAS mutant, 38% KRAS wild type). The responsiveness of BRAFV600E melanoma cells to GDC-0879 could be dramatically altered by pharmacologic and genetic modulation of phosphatidylinositol 3-kinase pathway activity. These data suggest that GDC-0879–induced signaling changes are dependent on the point of oncogenic activation within the RAS network. Taken together, these studies increase our understanding of the molecular determinants for antitumor efficacy resulting from RAF pathway inhibition and have implications for therapeutic intervention in the clinic. [Cancer Res 2009;69(7):3042–51]
Data from Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma
Authors: Singh Rajesh R. , Cho-Vega Jeong Hee , Davuluri Yogesh , Ma Shuguang , Kasbidi Fatan , Milito Cristiane , Lennon Patrick A. , Drakos Elias , Medeiros L. Jeffrey , Luthra Rajyalakshmi , Vega Francisco
Published: March 2023

Abstract
Deregulation of the sonic hedgehog (SHH) signaling pathway has been implicated in several cancers but has not been explored in T-cell lymphomas. Here, we report that the SHH/GLI1 signaling pathway is activated in anaplastic lymphoma kinase (ALK)–positive anaplastic large cell lymphoma (ALCL). We show that SHH, but not its transcriptional effector GLI1, is amplified in ALK+ ALCL tumors and cell lines, and that SHH and GLI1 proteins are highly expressed in ALK+ ALCL tumors and cell lines. We also show that inhibition of SHH/GLI1 signaling with cyclopamine-KAAD, as well as silencing GLI1 gene expression by small interfering (si)RNA, decreased cell viability and clonogenicity of ALK+ ALCL cells. Transfection of wild-type or mutant NPM-ALK into 293T cells showed that only wild-type NPM-ALK increased GLI1 protein levels and activated SHH/GLI1 signaling as shown by increase of CCND2 mRNA levels. Inhibition of ALK tyrosine kinase and phosphatidylinositol 3-kinase (PI3K)/AKT or forced expression of pAKT down-regulated or up-regulated SHH/GLI1 signaling, respectively. Inhibition of GSK3β in 293T cells also increased protein levels of GLI1. In conclusion, the SHH/GLI1 signaling pathway is activated in ALK+ ALCL. SHH/GLI1 activation is the result of SHH gene amplification and is further mediated by NPM-ALK through activation of PI3K/AKT and stabilization of GLI1 protein. There is a positive synergistic effect between the SHH/GLI1 and PI3K/AKT pathways that contributes to the lymphomagenic effect of NPM-ALK. [Cancer Res 2009;69(6):2550–8]
Data from RGS17, an Overexpressed Gene in Human Lung and Prostate Cancer, Induces Tumor Cell Proliferation Through the Cyclic AMP-PKA-CREB Pathway
Authors: James Michael A. , Lu Yan , Liu Yan , Vikis Haris G. , You Ming
Published: March 2023

Abstract
We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth. [Cancer Res 2009;69(5):2108–16]