bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Integrated Genomic Profiling of Chronic Lymphocytic Leukemia Identifies Subtypes of Deletion 13q14
Authors: Ouillette Peter , Erba Harry , Kujawski Lisa , Kaminski Mark , Shedden Kerby , Malek Sami N.
Published: March 2023

Abstract
Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous illness with a variable clinical course. Loss of chromosomal material on chromosome 13 at cytoband 13q14 is the most frequent genetic abnormality in CLL, but the molecular aberrations underlying del13q14 in CLL remain incompletely characterized. We analyzed 171 CLL cases for loss of heterozygosity and subchromosomal copy loss on chromosome 13 in DNA from fluorescence-activated cell sorting–sorted CD19+ cells and paired buccal cells using the Affymetrix XbaI 50k SNP array platform. The resulting high-resolution genomic maps, together with array-based measurements of expression levels of RNA in CLL cases with and without del13q14 and quantitative PCR–based expression analysis of selected genes, support the following conclusions: (a) del13q14 is heterogeneous and composed of multiple subtypes, with deletion of Rb or the miR15a/miR16 loci serving as anatomic landmarks, respectively; (b) del13q14 type Ia deletions are relatively uniform in length and extend from breakpoints close to the miR15a/miR16 cluster to a newly identified telomeric breakpoint cluster at the ∼50.2 to 50.5 Mb physical position; (c) LATS2 RNA levels are ∼2.6-fold to 2.8-fold lower in cases with del13q14 type I that do not delete Rb, as opposed to del13q14 type II or all other CLL cases; (d) PHLPP RNA is absent in ∼50% of CLL cases with del13q14; and (e) ∼15% of CLL cases display marked reductions in miR15a/miR16 expression that are often but not invariably associated with bi-allelic miR15a/miR16 loss. These data should aid future investigations into biological differences imparted on CLL by different del13q14 subtypes. [Cancer Res 2008;68(4):1012–21]
Data from DNA Hypomethylation Arises Later in Prostate Cancer Progression than CpG Island Hypermethylation and Contributes to Metastatic Tumor Heterogeneity
Authors: Yegnasubramanian Srinivasan , Haffner Michael C. , Zhang Yonggang , Gurel Bora , Cornish Toby C. , Wu Zhijin , Irizarry Rafael A. , Morgan James , Hicks Jessica , DeWeese Theodore L. , Isaacs William B. , Bova G. Steven , De Marzo Angelo M. , Nelson William G.
Published: March 2023

Abstract
Hypomethylation of CpG dinucleotides in genomic DNA was one of the first somatic epigenetic alterations discovered in human cancers. DNA hypomethylation is postulated to occur very early in almost all human cancers, perhaps facilitating genetic instability and cancer initiation and progression. We therefore examined the nature, extent, and timing of DNA hypomethylation changes in human prostate cancer. Contrary to the prevailing view that global DNA hypomethylation changes occur extremely early in all human cancers, we show that reductions in 5meC content in the genome occur very late in prostate cancer progression, appearing at a significant extent only at the stage of metastatic disease. Furthermore, we found that, whereas some LINE1 promoter hypomethylation does occur in primary prostate cancers compared with normal tissues, this LINE1 hypomethylation is significantly more pronounced in metastatic prostate cancer. Next, we carried out a tiered gene expression microarray and bisulfite genomic sequencing–based approach to identify genes that are silenced by CpG island methylation in normal prostate cells but become overexpressed in prostate cancer cells as a result of CpG island hypomethylation. Through this analysis, we show that a class of cancer testis antigen genes undergoes CpG island hypomethylation and overexpression in primary prostate cancers, but more so in metastatic prostate cancers. Finally, we show that DNA hypomethylation patterns are quite heterogeneous across different metastatic sites within the same patients. These findings provide evidence that DNA hypomethylation changes occur later in prostate carcinogenesis than the CpG island hypermethylation changes and occur heterogeneously during prostate cancer progression and metastatic dissemination. [Cancer Res 2008;68(21):8954–67]
Data from Discovery and Validation of Protein Abundance Differences between Follicular Thyroid Neoplasms
Authors: Netea-Maier Romana T. , Hunsucker Stephen W. , Hoevenaars Brigiet M. , Helmke Steve M. , Slootweg Pieter J. , Hermus Ad R. , Haugen Bryan R. , Duncan Mark W.
Published: March 2023

Abstract
Distinguishing between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC) by cytologic features alone is not possible. Molecular markers may aid distinguishing FTA from FTC in patients with indeterminate cytology. The aim of this study is to define protein abundance differences between FTC from FTA through a discovery (proteomics) and validation (immunohistochemistry) approach. Difference gel electrophoresis (DIGE) and peptide mass fingerprinting were performed on protein extracts from five patients with FTC and compared with six patients with FTA. Individual gel comparisons (i.e., each FTC extract versus FTA pool) were also performed for the five FTC patients. Immunohistochemical validation studies were performed on three of the identified proteins. Based on DIGE images, 680 protein spots were matched on individual gels. Of these, 102 spots showed statistically significant differences in abundance between FTC and FTA in the individual gel analyses and were therefore studied further. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify 54 of these protein spots. Three candidates involved in protein folding (heat shock protein gp96, protein disulfide isomerase A3, and calreticulin) were studied by immunohistochemistry. Moderate calreticulin immunohistochemical staining was the best single marker with a high negative predictive value (88%); combining all three markers (any marker less than moderate staining) had the best positive predictive value (75%) while still retaining a good negative predictive value (68%). With DIGE, we identified 54 proteins differentially abundant between FTC and FTA. Three of these were validated by immunohistochemistry. These findings provide further insights into the diagnosis, prognosis, and pathophysiology of follicular-derived thyroid neoplasms. [Cancer Res 2008;68(5):1572–80]
Data from Regulation of Id1 Expression by Src: Implications for Targeting of the Bone Morphogenetic Protein Pathway in Cancer
Authors: Gautschi Oliver , Tepper Clifford G. , Purnell Phillip R. , Izumiya Yoshihiro , Evans Christopher P. , Green Tim P. , Desprez Pierre Y. , Lara Primo N. , Gandara David R. , Mack Philip C. , Kung Hsing-Jien
Published: March 2023

Abstract
Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)–induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1. [Cancer Res 2008;68(7):2250–8]
Data from A Novel RET Kinase–β-Catenin Signaling Pathway Contributes to Tumorigenesis in Thyroid Carcinoma
Authors: Gujral Taranjit S. , van Veelen Wendy , Richardson Douglas S. , Myers Shirley M. , Meens Jalna A. , Acton Dennis S. , Duñach Mireia , Elliott Bruce E. , Höppener Jo W.M. , Mulligan Lois M.
Published: March 2023

Abstract
The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RET's potent transforming and mitogenic signals are still not clear. Here, we show that nuclear localization of β-catenin is frequent in both thyroid tumors and their metastases from MEN 2 patients, suggesting a novel mechanism of RET-mediated function through the β-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, β-catenin and show that the interaction between RET and β-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, β-catenin escapes cytosolic down-regulation by the adenomatous polyposis coli/Axin/glycogen synthase kinase-3 complex and accumulates in the nucleus, where it can stimulate β-catenin–specific transcriptional programs in a RET-dependent fashion. We show that down-regulation of β-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumor growth in nude mice. Together, our data show that a β-catenin–RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma. [Cancer Res 2008;68(5):1338–46]
Data from CX3CR1 Is Expressed by Prostate Epithelial Cells and Androgens Regulate the Levels of CX3CL1/Fractalkine in the Bone Marrow: Potential Role in Prostate Cancer Bone Tropism
Authors: Jamieson Whitney L. , Shimizu Saori , D'Ambrosio Julia A. , Meucci Olimpia , Fatatis Alessandro
Published: March 2023

Abstract
We have previously shown that the chemokine fractalkine promotes the adhesion of human prostate cancer cells to bone marrow endothelial cells as well as their migration toward human osteoblasts in vitro. Thus, the interaction of fractalkine with its receptor CX3CR1 could play a crucial role in vivo by directing circulating prostate cancer cells to the bone. We found that although CX3CR1 is minimally detectable in epithelial cells of normal prostate glands, it is overexpressed upon malignant transformation. Interestingly, osteoblasts, stromal and mesenchymal cells derived from human bone marrow aspirates express the cell-bound form of fractalkine, whereas the soluble form of the chemokine is detected in bone marrow supernatants. To investigate the mechanisms regulating the levels of soluble fractalkine in the bone marrow, we focused on androgens, which play a critical role in both prostate cancer progression and skeletal metastasis. Here, we show that dihydrotestosterone dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide—an antagonist of the androgen receptor—as well as the wide-spectrum inhibitor of matrix metalloproteases, GM6001. However, dihydrotestosterone was unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells on a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. In conclusion, our results indicate that CX3CR1, fractalkine, and the enzymes responsible for its cleavage might represent suitable targets for therapies aiming to counteract skeletal secondary tumors from prostate adenocarcinoma. [Cancer Res 2008;68(6):1715–22]
Data from Inflammatory Signaling and Aryl Hydrocarbon Receptor Mediate Synergistic Induction of Interleukin 6 in MCF-7 Cells
Authors: Hollingshead Brett D. , Beischlag Timothy V. , DiNatale Brett C. , Ramadoss Preeti , Perdew Gary H.
Published: March 2023

Abstract
The pleiotropic cytokine interleukin 6 (IL-6) is involved in immune cell homeostasis. Additionally, IL-6 expression and signaling in tumor cells have been shown to elicit both protumor and antitumor properties. There is a plethora of mechanistic knowledge regarding how IL-6 signal transduction translates to biological responses. However, there is little understanding as to what factors control IL-6 expression within a tumor cell environment. The studies presented herein show that, in MCF-7 breast and ECC-1 endocervical cancer cells, the stimulation of aryl hydrocarbon receptor (AHR) activity, in combination with IL-1β or phorbol 12-myristate 13-acetate (PMA) treatment, results in a marked synergistic induction of IL-6 levels over what is seen without AHR activation. Chromatin immunoprecipitation experiments suggest that the regulation of IL-6 mRNA expression occurs at the chromatin level, as AHR presence on the IL-6 promoter was observed in response to treatment with AHR ligand. Synergistic induction of IL-6 expression was sustained for 72 hours, with accumulation of IL-6 protein reaching levels 4.8-fold above IL-1β treatment alone. In addition, transcriptional regulation of the prototypic AHR responsive gene Cyp1a1 was negatively regulated by PMA and IL-1β treatment. Silencing of RELA expression alleviated IL-1β–mediated repression of AHR transcriptional activity, whereas PMA-mediated repression was maintained. Additionally, small interfering RNA studies reveal that AHR and RELA are necessary for synergistic induction of IL-6. The findings presented here reveal the AHR as a potential therapeutic target for selective modulation of IL-6 expression in some tumor cell types. The data also suggest a possible previously unrecognized mechanism of AHR-mediated tumor promotion. [Cancer Res 2008;68(10):3609–17]
Data from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia
Authors: Akakura Shin , Huang Changhui , Nelson Peter J. , Foster Barbara , Gelman Irwin H.
Published: March 2023

Abstract
SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein that encodes metastasis-suppressor activity through the suppression of Src-mediated oncogenic signaling and vascular endothelial growth factor expression. SSeCKS expression is down-regulated in Src- and Ras-transformed fibroblasts, in human cancer cell lines and in several types of human cancer, including prostate. Normal human and mouse prostates express abundant SSeCKS in secretory epithelial cells and, to a lesser extent, in the surrounding mesenchyme. Here, we show that the loss of SSeCKS results in prostatic hyperplasia in the anterior and ventral lobes as well as increased levels of apoptosis throughout the prostate. Dysplastic foci were observed less frequently but were associated with the loss of E-cadherin staining and the loss of high molecular weight cytokeratin-positive basal epithelial cells. SSeCKS-null prostate tissues expressed significantly higher relative levels of AKTpoS473 compared with wild-type controls, suggesting that SSeCKS attenuates phosphatidylinositol-3-OH kinase signaling. The data suggest that SSeCKS-null mice have increased susceptibility for oncogenic transformation in the prostate. [Cancer Res 2008;68(13):5096–103]
Data from c-Jun NH2-Terminal Kinase 1 Is a Critical Regulator for the Development of Gastric Cancer in Mice
Authors: Shibata Wataru , Maeda Shin , Hikiba Yohko , Yanai Ayako , Sakamoto Kei , Nakagawa Hayato , Ogura Keiji , Karin Michael , Omata Masao
Published: March 2023

Abstract
c-Jun NH2-terminal kinase (JNK) links several cellular processes, including proliferation and survival, and is believed to be involved in carcinogenesis. However, the role of JNK in gastric tumorigenesis is unknown. Immunohistochemical analysis reveals that JNK is frequently activated in human gastric cancer tissue. We investigated whether JNK1, a major JNK isozyme, is involved in chemically induced gastric cancer development. Mice lacking JNK1 exhibited a marked decrease in gastric carcinogenesis induced by N-methyl-N-nitrosourea, relative to their wild-type counterparts. Impaired tumor development correlated with decreased tumor initiation, which is associated with the production of reactive oxygen species. We also found that lower levels of tumorigenesis were correlated with the decreased expression of cyclin D and CDK as well as decreased cell proliferation. Taken together, JNK seems to be involved in both tumor initiation and promotion and may be an attractive target for the prevention of gastric carcinogenesis. [Cancer Res 2008;68(13):5031–9]
Data from Maintenance of Intratumoral Androgens in Metastatic Prostate Cancer: A Mechanism for Castration-Resistant Tumor Growth
Authors: Montgomery R. Bruce , Mostaghel Elahe A. , Vessella Robert , Hess David L. , Kalhorn Thomas F. , Higano Celestia S. , True Lawrence D. , Nelson Peter S.
Published: March 2023

Abstract
Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription–PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59–0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03–0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment. [Cancer Res 2008;68(11):4447–54]
Data from Clinical and Biological Significance of Tissue Transglutaminase in Ovarian Carcinoma
Authors: Hwang Jee Young , Mangala Lingegowda S. , Fok Jansina Y. , Lin Yvonne G. , Merritt William M. , Spannuth Whitney A. , Nick Alpa M. , Fiterman Derek J. , Vivas-Mejia Pablo E. , Deavers Michael T. , Coleman Robert L. , Lopez-Berestein Gabriel , Mehta Kapil , Sood Anil K.
Published: March 2023

Abstract
Tissue type transglutaminase (TG2) is a unique multifunctional protein that plays a role in many steps in the cancer metastatic cascade. Here, we examined the clinical (n = 93 epithelial ovarian cancers) and biological (in vitro adhesion, invasion, and survival and in vivo therapeutic targeting) significance of TG2 in ovarian cancer. The overexpression of TG2 was associated with significantly worse overall patient survival in both univariate and multivariate analyses. Transfection of TG2 into SKOV3ip1 cells promoted attachment and spreading on fibronectin-coated surfaces and increased the in vitro invasive potential of these cells. Conversely, TG2 silencing with small interfering RNA (siRNA) of HeyA8 cells significantly decreased the invasive potential of the cells and also increased docetaxel-induced cell death. In vivo therapy experiments using chemotherapy-sensitive (HeyA8) and chemotherapy-resistant (HeyA8-MDR and RMG2) models showed significant antitumor activity both with TG2 siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine alone and in combination with docetaxel chemotherapy. This antitumor activity was related to decreased proliferation and angiogenesis and increased tumor cell apoptosis in vivo. Taken together, these findings indicate that TG2 overexpression is an adverse prognostic factor in ovarian carcinoma and TG2 targeting may be an attractive therapeutic approach. [Cancer Res 2008;68(14):5849–58]
Data from Signatures Associated with Rejection or Recurrence in HER-2/neu–Positive Mammary Tumors
Authors: Worschech Andrea , Kmieciak Maciej , Knutson Keith L. , Bear Harry D. , Szalay Aladar A. , Wang Ena , Marincola Francesco M. , Manjili Masoud H.
Published: March 2023

Abstract
We have previously shown T-cell–mediated rejection of the neu-overexpressing mammary carcinoma cells (MMC) in wild-type FVB mice. However, following rejection of primary tumors, a fraction of animals experienced a recurrence of a neu antigen-negative variant (ANV) of MMC (tumor evasion model) after a long latency period. In the present study, we determined that T cells derived from wild-type FVB mice can specifically recognize MMC by secreting IFN-γ and can induce apoptosis of MMC in vitro. Neu transgenic (FVBN202) mice develop spontaneous tumors and cannot reject it (tumor tolerance model). To dissect the mechanisms associated with rejection or tolerance of MMC tumors, we compared transcriptional patterns within the tumor microenvironment of MMC undergoing rejection with those that resisted it either because of tumor evasion/antigen loss recurrence (ANV tumors) or because of intrinsic tolerance mechanisms displayed by the transgenic mice. Gene profiling confirmed that immune rejection is primarily mediated through activation of IFN-stimulated genes and T-cell effector mechanisms. The tumor evasion model showed combined activation of Th1 and Th2 with a deviation toward Th2 and humoral immune responses that failed to achieve rejection likely because of lack of target antigen. Interestingly, the tumor tolerance model instead displayed immune suppression pathways through activation of regulatory mechanisms that included in particular the overexpression of interleukin-10 (IL-10), IL-10 receptor, and suppressor of cytokine signaling (SOCS)-1 and SOCS-3. These data provide a road map for the identification of novel biomarkers of immune responsiveness in clinical trials. [Cancer Res 2008;68(7):2436–46]
Data from Neutrophil Gelatinase–Associated Lipocalin: A Novel Suppressor of Invasion and Angiogenesis in Pancreatic Cancer
Authors: Tong Zhimin , Kunnumakkara Ajaikumar B. , Wang Huamin , Matsuo Yoichi , Diagaradjane Parmeswaran , Harikumar Kuzhuvelil B. , Ramachandran Vijaya , Sung Bokyung , Chakraborty Arup , Bresalier Robert S. , Logsdon Craig , Aggarwal Bharat B. , Krishnan Sunil , Guha Sushovan
Published: March 2023

Abstract
Neutrophil gelatinase–associated lipocalin (NGAL) is a 25-kDa secreted acute phase protein, which is also up-regulated in multiple cancers, including breast, lung, and pancreas. Recently, NGAL has been proposed as an early biomarker in pancreatic cancer (PaCa). However, its biological role in PaCa is unknown. In this study, we examined in vitro and in vivo the functional role of NGAL in PaCa. Well- to moderately differentiated PaCa cells (AsPC-1, BxPC-3, and Capan-2) expressed high levels of NGAL but moderately to poorly differentiated PaCa cells (PANC-1 and MIAPaCa-2) expressed undetectable NGAL levels. Immunohistochemistry of untreated tissue microarray showed specific NGAL staining in resected PaCa specimens (P = 0.0167). Stable NGAL overexpression (MIAPaCa-2 and PANC-1) significantly blocked PaCa cell adhesion and invasion in vitro and vice versa with stable PaCa clones (BxPC-3 and AsPC-1). Moreover, NGAL overexpression reduced focal adhesion kinase (FAK) tyrosine-397 phosphorylation in PaCa cells. Furthermore, NGAL overexpression potently decreased angiogenesis in vitro partly through reduced vascular endothelial growth factor (VEGF) production and vice versa. Stable NGAL overexpression or underexpression had no effect on PaCa cell survival, viability, and response to chemotherapeutic drugs. Finally, MIAPaCa-2 cells overexpressing NGAL reduced tumor volume (P = 0.012), local and distant metastasis (P = 0.002), and angiogenesis (P = 0.05) with no effect on K-67 proliferation index (P > 0.1) in an orthotopic nude mouse PaCa model. Collectively, our results suggest that NGAL reduces adhesion/invasion partly by suppressing FAK activation and inhibits angiogenesis partly by blocking VEGF production in PaCa cells. Thus, NGAL is a potential suppressor of invasion and angiogenesis in advanced PaCa. [Cancer Res 2008;68(15):6100–8]
Data from PIK3CA Mutations and Copy Number Gains in Human Lung Cancers
Authors: Yamamoto Hiromasa , Shigematsu Hisayuki , Nomura Masaharu , Lockwood William W. , Sato Mitsuo , Okumura Naoki , Soh Junichi , Suzuki Makoto , Wistuba Ignacio I. , Fong Kwun M. , Lee Huei , Toyooka Shinichi , Date Hiroshi , Lam Wan L. , Minna John D. , Gazdar Adi F.
Published: March 2023

Abstract
We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance. [Cancer Res 2008;68(17):6913–21]
Data from Optimized Clostridium-Directed Enzyme Prodrug Therapy Improves the Antitumor Activity of the Novel DNA Cross-Linking Agent PR-104
Authors: Liu Shie-Chau , Ahn G-One , Kioi Mitomu , Dorie Mary-Jo , Patterson Adam V. , Brown J. Martin
Published: March 2023

Abstract
We have previously shown that spores of the nonpathogenic clostridial strain C. sporogenes genetically engineered to express the E. coli–derived cytosine deaminase gene are effective in converting systemically injected nontoxic 5-fluorocytosine into the toxic anticancer drug 5-fluorouracil, thereby producing tumor-specific antitumor activity. To improve the expression of E. coli–derived genes with this system, we first replaced the original fdP promoter in the vector with one of two powerful endogenous clostridial promoters: that of the thiolase gene (thlP) and that for the clostridial transcription factor abrB310 (abrBP). These substitutions improved protein expression levels of the prodrug-activating genes by 2- to 3-fold in comparison with fdP-driven expression. However, despite these strong promoters, we found much higher expression of the nitroreductase (NTR) protein in the E. coli host compared with the clostridial host, which we hypothesized could be the result of different codon use between the two organisms. To test this, we constructed new expression vectors with an artificially synthesized NTR gene using optimized clostridial codons (sNTR). Results from both enzymatic assays and Western blots of cell extracts from clostridial transformants harboring plasmid constructs of thlP-sNTR and abrBP-sNTR showed that the expression and activity of the NTR gene product was increased by ∼20-fold compared with the original construct. In vivo studies with i.v. administered sNTR-expressing C. sporogenes spores in SiHa tumor–bearing mice showed significantly improved antitumor efficacy when combined with either 5-aziridinyl-2,4-dinitrobenzamide (CB1954) or the novel dinitrobenzamide mustard prodrug, PR-104. [Cancer Res 2008;68(19):7995–8003]
Data from Phosphatidylinositol 3-Kinase Hyperactivation Results in Lapatinib Resistance that Is Reversed by the mTOR/Phosphatidylinositol 3-Kinase Inhibitor NVP-BEZ235
Authors: Eichhorn Pieter J.A. , Gili Magüi , Scaltriti Maurizio , Serra Violeta , Guzman Marta , Nijkamp Wouter , Beijersbergen Roderick L. , Valero Vanesa , Seoane Joan , Bernards René , Baselga José
Published: March 2023

Abstract
Small molecule inhibitors of HER2 are clinically active in women with advanced HER2-positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach, we performed a genome wide loss-of-function short hairpin RNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumor suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we show that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that phosphatidylinositol 3-kinase (PI3K)–induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance, which can be effectively reversed by NVP-BEZ235. [Cancer Res 2008;68(22):9221–30]
Data from Direct Presentation of a Melanocyte-Associated Antigen in Peripheral Lymph Nodes Induces Cytotoxic CD8+ T Cells
Authors: Schuler Prisca , Contassot Emmanuel , Irla Magali , Hugues Stéphanie , Preynat-Seauve Olivier , Beermann Friederich , Donda Alena , French L.E. , Huard Bertrand
Published: March 2023

Abstract
Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8+ T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8+ T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8+ T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8+ T cells, independently of bone marrow–derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer. [Cancer Res 2008;68(20):8410–8]
Data from Class I PI3 Kinase Inhibition by the Pyridinylfuranopyrimidine Inhibitor PI-103 Enhances Tumor Radiosensitivity
Authors: Prevo Remko , Deutsch Eric , Sampson Oliver , Diplexcito Julie , Cengel Keith , Harper Jane , O'Neill Peter , McKenna W. Gillies , Patel Sonal , Bernhard Eric J.
Published: March 2023

Abstract
Cell signaling initiated at the epidermal growth factor receptor (EGFR), RAS oncoproteins, or PI3K contributes to a common pathway that promotes tumor survival after radiation-induced DNA damage. Inhibition of signaling at the level of EGFR, RAS, and PI3K has been tested, but clinical applicability has been shown only at the level of the EGFR or by inhibiting RAS indirectly with prenyltransferase inhibitors. Inhibition of PI3K with LY294002 or wortmannin lacks specificity and has shown unacceptable toxicity in preclinical studies. We previously showed that inhibiting class I PI3K expression with siRNA resulted in enhanced radiation killing of tumor cells. Here, we tested the possibility of achieving specific tumor cell radiosensitization with a pharmacologic inhibitor of class I PI3K, the pyridinylfuranopyrimidine inhibitor PI-103. Our results show that inhibiting PI3K activity reduces phosphorylation of AKT at serine 473. Reduced survival is seen in cells with AKT activation and seems preferential for tumor cells over cells in which AKT activity is not elevated. Reduced survival is accompanied by persistence of DNA damage as evidenced by persistence of γH2AX and Rad 51 foci after irradiation in the presence of the inhibitor. Reduced survival does not result from cell cycle redistribution during the PI-103 treatment intervals tested, although combining PI-103 treatment with radiation enhances the G2-M delay observed after irradiation. These results indicate that pharmacologic inhibitors with enhanced specificity for class I PI3K may be of benefit when combined with radiotherapy. [Cancer Res 2008;68(14):5915–23]
Data from Reduced Eukaryotic Initiation Factor 2Bε-Subunit Expression Suppresses the Transformed Phenotype of Cells Overexpressing the Protein
Authors: Gallagher James W. , Kubica Neil , Kimball Scot R. , Jefferson Leonard S.
Published: March 2023

Abstract
Eukaryotic initiation factor 2B (eIF2B), a five-subunit guanine nucleotide exchange factor, plays a key role in the regulation of mRNA translation. Expression of its ε-subunit is specifically up-regulated in certain conditions associated with increased cell growth. Therefore, the purpose of the present study was to examine the effect of repressing eIF2Bε expression on growth rate, protein synthesis, and other characteristics of two tumorigenic cell lines that display up-regulated expression of the ε-subunit. Experiments were designed to compare spontaneously transformed fibroblasts to transformed mouse embryonic fibroblasts infected with a lentivirus containing a short hairpin RNA directed against eIF2Bε. Cells expressing the short hairpin RNA displayed a reduction in eIF2Bε abundance to 30% of the value observed in uninfected transformed mouse embryonic fibroblasts, with no change in the expression of any of the other four subunits. The repression of eIF2Bε expression was accompanied by reductions in guanine nucleotide exchange factor activity and global rates of protein synthesis. Moreover, repressed eIF2Bε expression led to marked reductions in cell growth rate in culture, colony formation in soft agar, and tumor progression in nude mice. Similar results were obtained in MCF-7 human breast cancer cells in which eIF2Bε expression was repressed through transient transfection with a small interfering RNA directed against the ε-subunit. Overall, the results support a role for eIF2Bε in the regulation of cell growth and suggest that it might represent a therapeutic target for the treatment of human cancer. [Cancer Res 2008;68(21):8752–60]
Data from Analysis of the Mechanisms Mediating Tumor-Specific Changes in Gene Expression in Human Liver Tumors
Authors: Acevedo Luis G. , Bieda Mark , Green Roland , Farnham Peggy J.
Published: March 2023

Abstract
There is widespread interest in efficient characterization of differences between tumor and normal samples. Here, we show an effective methodology for genome-scale characterization of tumors. Using matched normal and tumor samples from liver cancer patients, as well as non–cancer-related normal liver tissue, we first determined changes in gene expression as monitored on RNA expression arrays. We identified several hundred mRNAs that were consistently changed in the tumor samples. To characterize the mechanisms responsible for creation of the tumor-specific transcriptome, we performed chromatin immunoprecipitation on microarray experiments to assay binding of RNA polymerase II, H3me3K27, and H3me3K9 and DNA methylation in 25,000 promoter regions. These experiments identified changes in active and silenced regions of the genome in the tumor cells. Finally, we used a “virtual comparative genomic hybridization” method to identify copy number alterations in the tumor samples. Through comparison of RNA polymerase II binding, chromatin structure, DNA methylation, and copy number changes, we suggest that the major contributor to creation of the liver tumor transcriptome was changes in gene copy number. [Cancer Res 2008;68(8):2641–51]
Data from Bioselection of a Gain of Function Mutation that Enhances Adenovirus 5 Release and Improves Its Antitumoral Potency
Authors: Gros Alena , Martínez-Quintanilla Jordi , Puig Cristina , Guedan Sonia , Molleví David G. , Alemany Ramon , Cascallo Manel
Published: March 2023

Abstract
Genetic bioselection of a mutagenized Ad5wt stock in human tumor xenografts led us to isolate AdT1, a mutant displaying a large-plaque phenotype in vitro and an enhanced systemic antitumor activity in vivo. AdT1 phenotype correlates with an increased progeny release without affecting total viral yield in different human tumors and cancer-associated fibroblasts. An approach combining hybrid Ad5/AdT1 recombinants and sequencing identified a truncating insertion in the endoplasmic reticulum retention domain of the E3/19K protein (445A mutation) which relocates the protein to the plasma membrane and is responsible for AdT1's enhanced release. E3/19K-445A phenotype does not correlate with the protein's ability to interact with MHC-I or induce apoptosis. Intracellular calcium measurement revealed that the 445A mutation induces extracellular Ca2+ influx, deregulating intracellular Ca2+ homeostasis and inducing membrane permeabilization, a viroporin-like function. E3/19K-445A mutants also display enhanced antitumoral activity when injected both intratumorally and systemically in different models in vivo. Our results indicate that the inclusion of mutation 445A in tumor-selective adenoviruses would be a very powerful tool to enhance their antitumor efficacy. [Cancer Res 2008;68(21):8928–10]
Data from Loss of RhoB Expression Promotes Migration and Invasion of Human Bronchial Cells Via Activation of AKT1
Authors: Bousquet Emilie , Mazières Julien , Privat Maud , Rizzati Virginie , Casanova Anne , Ledoux Adeline , Mery Eliane , Couderc Bettina , Favre Gilles , Pradines Anne
Published: March 2023

Abstract
Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Previously, we reported an inverse correlation between RhoB expression and the progression of the lung cancer, occurring between preinvasive and invasive tumors. Herein, we mimicked the loss of RhoB observed throughout lung oncogenesis with RNA interference in nontumoral bronchial cell lines and analyzed the consequences on both cell transformation and invasion. Down-regulation of RhoB did not modify the cell growth properties but did promote migration and invasiveness. Furthermore, RhoB depletion was accompanied by modifications of actin and cell adhesion. The specific activation of the Akt1 isoform and Rac1 was found to be critical for this RhoB-mediated regulation of migration. Lastly, we showed that RhoB down-regulation consecutive to K-RasV12 cell transformation is critical for cell motility but not for cell proliferation. We propose that RhoB loss during lung cancer progression relates to the acquisition of invasiveness mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT and Rac1 pathways rather than to tumor initiation. [Cancer Res 2009;69(15):6092–99]
Data from An A13 Repeat within the 3′-Untranslated Region of Epidermal Growth Factor Receptor (EGFR) Is Frequently Mutated in Microsatellite Instability Colon Cancers and Is Associated with Increased EGFR Expression
Authors: Yuan Ziqiang , Shin Joongho , Wilson Andrew , Goel Sanjay , Ling Yi-He , Ahmed Naseem , Dopeso Higinio , Jhawer Minaxi , Nasser Shannon , Montagna Cristina , Fordyce Kenneth , Augenlicht Leonard H. , Aaltonen Lauri A. , Arango Diego , Weber Thomas K. , Mariadason John M.
Published: March 2023

Abstract
Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3′-untranslated region (3′-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3′-UTR mutant MSI cell lines. Cell lines with an EGFR 3′-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3′-UTR mutations were more sensitive to EGFR inhibition than EGFR 3′-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors. [Cancer Res 2009;69(19):7811–8]
Data from Impaired Turnover of Prolactin Receptor Contributes to Transformation of Human Breast Cells
Authors: Plotnikov Alexandr , Varghese Bentley , Tran Thai H. , Liu Chengbao , Rui Hallgeir , Fuchs Serge Y.
Published: March 2023

Abstract
Signaling by polypeptide hormone prolactin (PRL) is mediated by its cognate receptor (PRLr). PRLr is commonly stabilized in human breast cancer due to decreased phosphorylation of residue Ser349, which when phosphorylated recruits the βTrcp E3 ubiquitin ligase and facilitates PRLr degradation. Here, we show that an impaired PRLr turnover results in an augmented PRL signaling and PRL-induced transcription. Human mammary epithelial cells harboring degradation-resistant PRLr display accelerated proliferation and increased invasive growth. Conversely, a decrease in PRLr levels achieved by either pharmacologic or genetic means in human breast cancer cells dramatically reduced transformation and tumorigenic properties of these cells. Consequences of alteration of PRLr turnover for homeostasis of mammary cells and development of breast cancers, as well as the utility of therapies that target PRLr function in these malignancies, are discussed. [Cancer Res 2009;69(7):3165–72]
Data from Androgen-Stimulated UDP-Glucose Dehydrogenase Expression Limits Prostate Androgen Availability without Impacting Hyaluronan Levels
Authors: Wei Qin , Galbenus Robert , Raza Ashraf , Cerny Ronald L. , Simpson Melanie A.
Published: March 2023

Abstract
UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronate, an essential precursor for production of hyaluronan (HA), proteoglycans, and xenobiotic glucuronides. High levels of HA turnover in prostate cancer are correlated with aggressive progression. UGDH expression is high in the normal prostate, although HA accumulation is virtually undetectable. Thus, its normal role in the prostate may be to provide precursors for glucuronosyltransferase enzymes, which inactivate and solubilize androgens by glucuronidation. In this report, we quantified androgen dependence of UGDH, glucuronosyltransferase, and HA synthase expression. Androgen-dependent and androgen-independent human prostate cancer cell lines were used to test the effects of UGDH manipulation on tumor cell growth, HA production, and androgen glucuronidation. Dihydrotestosterone (DHT) increased UGDH expression ∼2.5-fold in androgen-dependent cells. However, up-regulation of UGDH did not affect HA synthase expression or enhance HA production. Mass spectrometric analysis showed that DHT was converted to a glucuronide, DHT-G, at a 6-fold higher level in androgen-dependent cells relative to androgen-independent cells. The increased solubilization and elimination of DHT corresponded to slower cellular growth kinetics, which could be reversed in androgen-dependent cells by treatment with a UDP-glucuronate scavenger. Collectively, these results suggest that dysregulated expression of UGDH could promote the development of androgen-independent tumor cell growth by increasing available levels of intracellular androgen. [Cancer Res 2009;69(6):2332–9]
Data from Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells
Authors: Gosselin Karo , Martien Sébastien , Pourtier Albin , Vercamer Chantal , Ostoich Peter , Morat Luc , Sabatier Laure , Duprez Laurence , T'Kint de Roodenbeke Claire , Gilson Eric , Malaquin Nicolas , Wernert Nicolas , Slijepcevic Predrag , Ashtari Marjan , Chelli Fazia , Deruy Emeric , Vandenbunder Bernard , De Launoit Yvan , Abbadie Corinne
Published: March 2023

Abstract
Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age. [Cancer Res 2009;69(20):7917–24]