bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization
Authors: Novak Petr , Jensen Taylor J. , Garbe James C. , Stampfer Martha R. , Futscher Bernard W.
Published: March 2023

Abstract
The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16INK4A expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment. [Cancer Res 2009;69(12):5251–8]
Data from Reactivation of Androgen Receptor–Regulated TMPRSS2:ERG Gene Expression in Castration-Resistant Prostate Cancer
Authors: Cai Changmeng , Wang Hongyun , Xu Youyuan , Chen Shaoyong , Balk Steven P.
Published: March 2023

Abstract
It seems clear that androgen receptor (AR)–regulated expression of the TMPRSS2:ERG fusion gene plays an early role in prostate cancer (PC) development or progression, but the extent to which TMPRSS2:ERG is down-regulated in response to androgen deprivation therapy (ADT) and whether AR reactivates TMPRSS2:ERG expression in castration-resistant PC (CRPC) have not been determined. We show that ERG message levels in TMPRSS2:ERG fusion-positive CRPC are comparable with the levels in fusion gene–positive primary PC, consistent with the conclusion that the TMPRSS2:ERG expression is reactivated by AR in CRPC. To further assess whether TMPRSS2:ERG expression is initially down-regulated in response to ADT, we examined VCaP cells, which express the TMPRSS2:ERG fusion gene, and xenografts. ERG message and protein rapidly declined in response to removal of androgen in vitro and castration in vivo. Moreover, as observed in the clinical samples, ERG expression was fully restored in the VCaP xenografts that relapsed after castration, coincident with AR reactivation. AR reactivation in the relapsed xenografts was also associated with marked increases in mRNA encoding AR and androgen synthetic enzymes. These results show that expression of TMPRSS2:ERG, similarly to other AR-regulated genes, is restored in CRPC and may contribute to tumor progression. [Cancer Res 2009;69(15):6027–32]
Data from Nanoparticle-Delivered Suicide Gene Therapy Effectively Reduces Ovarian Tumor Burden in Mice
Authors: Huang Yu-Hung , Zugates Gregory T. , Peng Weidan , Holtz David , Dunton Charles , Green Jordan J. , Hossain Naushad , Chernick Michael R. , Padera Robert F. , Langer Robert , Anderson Daniel G. , Sawicki Janet A.
Published: March 2023

Abstract
There is currently no effective therapy for patients with advanced ovarian cancer. To address the need for a more effective treatment for this deadly disease, we conducted preclinical tests in ovarian tumor–bearing mice to evaluate the therapeutic efficacy of using a cationic biodegradable poly(β-amino ester) polymer as a vector for nanoparticulate delivery of DNA encoding a diphtheria toxin suicide protein (DT-A). The promoter sequences of two genes that are highly active in ovarian tumor cells, MSLN and HE4, were used to target DT-A expression to tumor cells. Administration of DT-A nanoparticles directly to s.c. xenograft tumors and to the peritoneal cavity of mice bearing primary and metastatic ovarian tumors resulted in a significant reduction in tumor mass and a prolonged life span compared to control mice. Minimal nonspecific tissue and blood chemistry toxicity was observed following extended treatment with nanoparticles. DT-A nanoparticle therapy suppressed tumor growth more effectively than treatment with clinically relevant doses of cisplatin and paclitaxel. Our findings suggest that i.p. administration of polymeric nanoparticles to deliver DT-A encoding DNA, combined with transcriptional regulation to target gene expression to ovarian tumor cells, holds promise as an effective therapy for advanced-stage ovarian cancer. [Cancer Res 2009;69(15):6184–91]
Data from Harnessing Competing Endocytic Pathways for Overcoming the Tumor-Blood Barrier: Magnetic Resonance Imaging and Near-Infrared Imaging of Bifunctional Contrast Media
Authors: Migalovich Helena Sheikhet , Kalchenko Vyacheslav , Nevo Nava , Meir Gila , Kohen Fortune , Neeman Michal
Published: March 2023

Abstract
Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)–based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells. [Cancer Res 2009;69(13):5610–7]
Data from Defective Repair of Oxidative DNA Damage in Triple-Negative Breast Cancer Confers Sensitivity to Inhibition of Poly(ADP-Ribose) Polymerase
Authors: Alli Elizabeth , Sharma Vandana B. , Sunderesakumar Preethi , Ford James M.
Published: March 2023

Abstract
Subtypes of breast cancer that represent the two major types of epithelial cells in the breast (luminal and basal) carry distinct histopathologic profiles. Breast cancers of the basal-like subtype, which include the majority of hereditary breast cancers due to mutations in the breast cancer susceptibility gene 1 (BRCA1), frequently assume triple-negative status, i.e., they lack expression of estrogen receptor-α and progesterone receptor, and lack overexpression or amplification of the HER2/NEU oncogene. Defects in DNA damage response pathways result in genome instability and lead to carcinogenesis, but may also be exploited for therapeutic purposes. We analyzed repair of oxidative DNA damage by the base-excision repair (BER) pathway, which when aberrant leads to genomic instability and breast carcinogenesis, in cell lines that represent the different subtypes of breast cancer and in the presence of BRCA1 deficiency. We found that basal-like and BRCA1-mutated breast cancer cells were defective in BER of oxidative DNA damage, and that this defect conferred sensitivity to inhibition of poly(ADP-ribose) polymerase, a DNA repair enzyme. The defect may be attributed, at least in part, to a novel role for BRCA1 in the BER pathway. Overall, these data offer preventive, prognostic, and therapeutic usefulness. [Cancer Res 2009;69(8):3589–96]
Data from Expansion of Highly Cytotoxic Human Natural Killer Cells for Cancer Cell Therapy
Authors: Fujisaki Hiroyuki , Kakuda Harumi , Shimasaki Noriko , Imai Chihaya , Ma Jing , Lockey Timothy , Eldridge Paul , Leung Wing H. , Campana Dario
Published: March 2023

Abstract
Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56+CD3- NK cells from peripheral blood (range, 5.1- to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3+ lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2–stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2–stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer. [Cancer Res 2009;69(9):4010–7]
Data from Lin−Sca-1+CD49fhigh Stem/Progenitors Are Tumor-Initiating Cells in the Pten-Null Prostate Cancer Model
Authors: Mulholland David J. , Xin Li , Morim Ashkan , Lawson Devon , Witte Owen , Wu Hong
Published: March 2023

Abstract
We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin−Sca-1+CD49fhigh (LSC) subpopulation overlaps with CK5+;p63+ cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model. [Cancer Res 2009;69(22):8555–62]
Data from Comparative Analyses of Chromosome Alterations in Soft-Tissue Metastases within and across Patients with Castration-Resistant Prostate Cancer
Authors: Holcomb Ilona N. , Young Janet M. , Coleman Ilsa M. , Salari Keyan , Grove Douglas I. , Hsu Li , True Lawrence D. , Roudier Martine P. , Morrissey Colm M. , Higano Celestia S. , Nelson Peter S. , Vessella Robert L. , Trask Barbara J.
Published: March 2023

Abstract
Androgen deprivation is the mainstay of therapy for progressive prostate cancer. Despite initial and dramatic tumor inhibition, most men eventually fail therapy and die of metastatic castration-resistant (CR) disease. Here, we characterize the profound degree of genomic alteration found in CR tumors using array comparative genomic hybridization (array CGH), gene expression arrays, and fluorescence in situ hybridization (FISH). Bycluster analysis, we show that the similarity of the genomic profiles from primary and metastatic tumors is driven by the patient. Using data adjusted for this similarity, we identify numerous high-frequency alterations in the CR tumors, such as 8p loss and chromosome 7 and 8q gain. By integrating array CGH and expression array data, we reveal genes whose correlated values suggest they are relevant to prostate cancer biology. We find alterations that are significantly associated with the metastases of specific organ sites, and others with CR tumors versus the tumors of patients with localized prostate cancer not treated with androgen deprivation. Within the high-frequency sites of loss in CR metastases, we find an overrepresentation of genes involved in cellular lipid metabolism, including PTEN. Finally, using FISH, we verify the presence of a gene fusion between TMPRSS2 and ERG suggested by chromosome 21 deletions detected by array CGH. We find the fusion in 54% of our CR tumors, and 81% of the fusion-positive tumors contain cells with multiple copies of the fusion. Our investigation lays the foundation for a better understanding of and possible therapeutic targets for CR disease, the poorly responsive and final stage of prostate cancer. [Cancer Res 2009;69(19):7793–802]
Data from A Double Hit to Kill Tumor and Endothelial Cells by TRAIL and Antiangiogenic 3TSR
Authors: Ren Bin , Song Keli , Parangi Sareh , Jin Taiguang , Ye Min , Humphreys Robin , Duquette Mark , Zhang Xuefeng , Benhaga Nordine , Lawler Jack , Khosravi-Far Roya
Published: March 2023

Abstract
As tumor development relies on a coordination of angiogenesis and tumor growth, an efficient antitumor strategy should target both the tumor and its associated vessels. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner. Additionally, thrombospondin-1, a naturally occurring inhibitor of angiogenesis, and a recombinant protein containing functional domains of thrombospondin-1, 3TSR, have been shown to be necessary and sufficient to inhibit tumor angiogenesis. Here, we show that a combination of a TRAIL receptor 2 agonist antibody, Lexatumumab, and 3TSR results in a significantly enhanced and durable tumor inhibition. We further observed that 3TSR induces apoptosis in primary endothelial cells by up-regulating the expression of TRAIL receptors 1 and 2 in a CD36 and Jun NH2-terminal kinase-dependent manner leading to the activation of both intrinsic and extrinsic apoptotic machineries. The modulation of these pathways is critical for 3TSR-induced apoptosis as disrupting either via specific inhibitors reduced apoptosis. Moreover, 3TSR attenuates the Akt survival pathway. These studies indicate that 3TSR plays a critical role in regulating the proapoptotic signaling pathways that control growth and death in endothelial cells and that a combination of TRAIL and 3TSR acts as a double hit against tumor and tumor-associated vessels. [Cancer Res 2009;69(9):3856–65]
Data from Inactivation of the Nijmegen Breakage Syndrome Gene Leads to Excess Centrosome Duplication via the ATR/BRCA1 Pathway
Authors: Shimada Mikio , Sagae Ruri , Kobayashi Junya , Habu Toshiyuki , Komatsu Kenshi
Published: March 2023

Abstract
Nijmegen breakage syndrome is characterized by genomic instability and a predisposition for lymphoma and solid tumors. Nijmegen breakage syndrome 1 (NBS1), the protein which is mutated in these patients, functions in association with BRCA1 and ATR as part of the cellular response to DNA double-strand breaks. We show here that NBS1 forms foci at the centrosomes via an interaction with γ-tubulin. Down-regulation of NBS1 by small interfering RNA induces supernumerary centrosomes, and this was confirmed with experiments using Nbs1 knockout mouse cells; the introduction of wild-type NBS1 (wt-NBS1) cDNA into these knockout mouse cells reduced the number of supernumerary centrosomes to normal levels. This phenotype in NBS1-deficient cells is caused by both centrosome duplication and impaired separation of centrioles, which have been observed in BRCA1-inhibited cells. In fact, supernumerary centrosomes were observed in Brca1 knockout mouse cells, and the frequency was not affected by NBS1 down-regulation, suggesting that NBS1 maintains centrosomes via a common pathway with BRCA1. This is consistent with findings that NBS1 physically interacts with BRCA1 at the centrosomes and is required for BRCA1-mediated ubiquitination of γ-tubulin. Moreover, the ubiquitination of γ-tubulin is compromised by either ATR depletion or an NBS1 mutation in the ATR interacting (FHA) domain, which is essential for ATR activation. These results suggest that, although centrosomes lack DNA, the NBS1/ATR/BRCA1 repair machinery affects centrosome behavior, and this might be a crucial role in the prevention of malignances. [Cancer Res 2009;69(5):1768–75]
Data from Triggering of Toll-like Receptor 4 Expressed on Human Head and Neck Squamous Cell Carcinoma Promotes Tumor Development and Protects the Tumor from Immune Attack
Authors: Szczepanski Miroslaw J. , Czystowska Malgorzata , Szajnik Marta , Harasymczuk Malgorzata , Boyiadzis Michael , Kruk-Zagajewska Aleksandra , Szyfter Witold , Zeromski Jan , Whiteside Theresa L.
Published: March 2023

Abstract
Toll-like receptors (TLR) expressed on inflammatory cells play a key role in host defense against pathogens, benefiting the host. TLR are also expressed on tumor cells. To evaluate the role of TLR in tumor cells, we investigated TLR4 signaling effects on human head and neck squamous cell carcinoma (HNSCC). Tumor tissues were obtained from 27 patients with laryngeal and 12 with oral cavity cancers. Normal mucosa was obtained from 10 patients with nonneoplastic disorders. Smears for bacteria were taken from all patients during surgery. TLR4 expression in tumors and HNSCC cell lines (PCI-1, PCI-13, and PCI-30) was detected by reverse transcription-PCR and immunohistochemistry. Cell growth, apoptosis, nuclear factor-κB (NF-κB) translocation, and MyD88 and IRAK-4 expression, as well as Akt phosphorylation were measured following tumor cell exposure to the TLR4 ligand lipopolysaccharide (LPS). Tumor cell sensitivity to NK-92–mediated lysis was evaluated in 4-hour 51Cr-release assays. Cytokine levels in HNSCC supernatants were measured in Luminex-based assays. TLR4 was expressed in all tumors, HNSCC cell lines, and normal mucosa. The TLR4 expression intensity correlated with tumor grade. LPS binding to TLR4 on tumor cells enhanced proliferation, activated phosphatidylinositol 3-kinase/Akt pathway, up-regulated IRAK-4 expression, induced nuclear NF-κB translocation, and increased production (P < 0.05) of interleukin (IL)-6, IL-8, vascular endothelial growth factor, and granulocyte macrophage colony-stimulating factor. TLR4 triggering protected tumor cells from lysis mediated by NK-92 cells. TLR4 ligation on tumor cells supports HNSCC progression. [Cancer Res 2009;69(7):3105–13]
Data from ARTEMIS Nuclease Facilitates Apoptotic Chromatin Cleavage
Authors: Britton Sébastien , Frit Philippe , Biard Denis , Salles Bernard , Calsou Patrick
Published: March 2023

Abstract
One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis. [Cancer Res 2009;69(20):8120–6]
Data from Caveolin-1 Regulates the Antagonistic Pleiotropic Properties of Cellular Senescence through a Novel Mdm2/p53-Mediated Pathway
Authors: Bartholomew Janine N. , Volonte Daniela , Galbiati Ferruccio
Published: March 2023

Abstract
We show that caveolin-1 is a novel binding protein for Mdm2. After oxidative stress, caveolin-1 sequesters Mdm2 away from p53, leading to stabilization of p53 and up-regulation of p21Waf1/Cip1 in human fibroblasts. Expression of a peptide corresponding to the Mdm2 binding domain of caveolin-1 is sufficient to up-regulate p53 and p21Waf1/Cip1 protein expression and induce premature senescence. Oxidative stress–induced activation of the p53/p21Waf1/Cip1 pathway and induction of premature senescence are compromised in caveolin-1 null mouse embryonic fibroblasts (MEF). We also show that reintroduction of caveolin-1 in oncogenic Ras (RasG12V)–transformed fibroblasts, which express residual levels of caveolin-1, is sufficient to promote cellular senescence. Moreover, caveolin-1 expression in MEFs is required for senescent fibroblast-induced stimulation of cell growth and tumorigenesis of both RasG12V-transformed fibroblasts and MDA-MB-231 breast cancer epithelial cells both in vitro and in vivo. Thus, our results propose caveolin-1 as a key mediator of the antagonistic pleiotropic properties of cellular senescence. [Cancer Res 2009;69(7):2878–86]
Data from Prediction of Clinical Outcome in Multiple Lung Cancer Cohorts by Integrative Genomics: Implications for Chemotherapy Selection
Authors: Broët Philippe , Camilleri-Broët Sophie , Zhang Shenli , Alifano Marco , Bangarusamy Dhinoth , Battistella Maxime , Wu Yonghui , Tuefferd Marianne , Régnard Jean-François , Lim Elaine , Tan Patrick , Miller Lance D.
Published: March 2023

Abstract
The role of adjuvant chemotherapy in patients with stage IB non–small-cell lung cancer (NSCLC) is controversial. Identifying patient subgroups with the greatest risk of relapse and, consequently, most likely to benefit from adjuvant treatment thus remains an important clinical challenge. Here, we hypothesized that recurrent patterns of genomic amplifications and deletions in lung tumors could be integrated with gene expression information to establish a robust predictor of clinical outcome in stage IB NSCLC. Using high-resolution microarrays, we generated tandem DNA copy number and gene expression profiles for 85 stage IB lung adenocarcinomas/large cell carcinomas. We identified specific copy number alterations linked to relapse-free survival and selected genes within these regions exhibiting copy number–driven expression to construct a novel integrated signature (IS) capable of predicting clinical outcome in this series (P = 0.02). Importantly, the IS also significantly predicted clinical outcome in two other independent stage I NSCLC cohorts (P = 0.003 and P = 0.025), showing its robustness. In contrast, a more conventional molecular predictor based solely on gene expression, while capable of predicting outcome in the initial series, failed to significantly predict outcome in the two independent data sets. Our results suggest that recurrent copy number alterations, when combined with gene expression information, can be successfully used to create robust predictors of clinical outcome in early-stage NSCLC. The utility of the IS in identifying early-stage NSCLC patients as candidates for adjuvant treatment should be further evaluated in a clinical trial. [Cancer Res 2009;69(3):1055–62]
Data from Induction of Prostaglandin E2 Pathway Promotes Gastric Hamartoma Development with Suppression of Bone Morphogenetic Protein Signaling
Authors: Oshima Hiroko , Itadani Hiraku , Kotani Hidehito , Taketo Makoto Mark , Oshima Masanobu
Published: March 2023

Abstract
Mutations in bone morphogenetic protein (BMP) receptor 1A (BMPR1A) are responsible for a subset of cases of juvenile polyposis (JP) syndrome that develops hamartomatous tumors in the gastrointestinal tract. Mouse genetic studies have shown that suppression of BMP signaling in the intestines causes JP-type hamartoma development. Here, we generated K19-Nog transgenic mice expressing noggin, a BMP antagonist, in gastric epithelium. However, inhibition of BMP signaling did not cause gastric phenotypes. We thus crossed K19-Nog with K19-C2mE mice that expressed Ptgs2 and Ptges in the stomach to generate compound transgenic mice. Expression of Ptgs2 and Ptges results in prostaglandin E2 (PGE2) biosynthesis, and both enzymes are induced in most human gastrointestinal tumors. Importantly, K19-Nog/C2mE compound mice developed gastric hamartomas that were morphologically similar to those found in JP with mucin-containing dilated cysts and inflammatory infiltration. Notably, treatment of K19-Nog/C2mE mice with a cyclooxygenase-2 inhibitor, celecoxib, significantly reduced tumor size with suppression of angiogenesis, suggesting that induction of the PGE2 pathway together with inhibition of BMP signaling is required for gastric hamartoma development. Moreover, microarray analyses revealed that canonical Wnt signaling target genes were not induced in K19-Nog/C2mE hamartomas, indicating that BMP inhibition and PGE2 induction lead to gastric hamartoma development independent of the Wnt/β-catenin pathway. These results, taken together, suggest that the PGE2 pathway is an effective preventive target against BMP-suppressed gastric hamartomas, as well as for Wnt/β-catenin–activated adenocarcinomas. [Cancer Res 2009;69(7):2729–33]
Data from PTEN Loss Contributes to Erlotinib Resistance in EGFR-Mutant Lung Cancer by Activation of Akt and EGFR
Authors: Sos Martin L. , Koker Mirjam , Weir Barbara A. , Heynck Stefanie , Rabinovsky Rosalia , Zander Thomas , Seeger Jens M. , Weiss Jonathan , Fischer Florian , Frommolt Peter , Michel Kathrin , Peifer Martin , Mermel Craig , Girard Luc , Peyton Michael , Gazdar Adi F. , Minna John D. , Garraway Levi A. , Kashkar Hamid , Pao William , Meyerson Matthew , Thomas Roman K.
Published: March 2023

Abstract
Clinical resistance to epidermal growth factor receptor (EGFR) inhibition in lung cancer has been linked to the emergence of the EGFR T790M resistance mutation or amplification of MET. Additional mechanisms contributing to EGFR inhibitor resistance remain elusive. By applying combined analyses of gene expression, copy number, and biochemical analyses of EGFR inhibitor responsiveness, we identified homozygous loss of PTEN to segregate EGFR-dependent and EGFR-independent cells. We show that in EGFR-dependent cells, PTEN loss partially uncouples mutant EGFR from downstream signaling and activates EGFR, thereby contributing to erlotinib resistance. The clinical relevance of our findings is supported by the observation of PTEN loss in 1 out of 24 primary EGFR-mutant non–small cell lung cancer (NSCLC) tumors. These results suggest a novel resistance mechanism in EGFR-mutant NSCLC involving PTEN loss. [Cancer Res 2009;69(8):3256–61]
Data from Constitutive Activation of the DNA Damage Signaling Pathway in Acute Myeloid Leukemia with Complex Karyotype: Potential Importance for Checkpoint Targeting Therapy
Authors: Cavelier Cindy , Didier Christine , Prade Naïs , Mansat-De Mas Véronique , Manenti Stéphane , Recher Christian , Demur Cécile , Ducommun Bernard
Published: March 2023

Abstract
Genomic instability in solid tumors participates in the oncogenetic process and is associated with the activation of the DNA damage response pathway. Here, we report the activation of the constitutive DNA damage and checkpoint pathway associated with complex karyotypes in samples from patients with acute myeloid leukemia (AML). We show that antagonizing CHK1 kinase with a small inhibitory compound or by RNA interference strongly reduces the clonogenic properties of high–DNA damage level AML samples, particularly those with complex karyotypes. Moreover, we observe a beneficial effect of CHK1 inhibition in high–DNA damage level AML samples treated with 1-β-d-arabinofuranosylcytosine. In contrast, CHK1 inhibition has no effect on the clonogenic properties of normal hematopoietic progenitors. All together, our results indicate that CHK1 inhibition may represent an attractive therapeutic opportunity in AML with complex karyotype. [Cancer Res 2009;69(22):8652–61]
Data from Vascular Cell Adhesion Molecule-1 Is a Regulator of Ovarian Cancer Peritoneal Metastasis
Authors: Slack-Davis Jill K. , Atkins Kristen A. , Harrer Christine , Hershey E. Daniel , Conaway Mark
Published: March 2023

Abstract
Ovarian cancers metastasize by attaching to and invading through the mesothelium, a single layer of mesothelial cells lining the peritoneal cavity. The presence of invasive peritoneal metastases is associated with a poor prognosis for ovarian cancer (5-year survival <25%). Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface receptor that mediates leukocyte attachment and extravasation across endothelial and mesothelial monolayers at sites of inflammation. Membranous VCAM-1 expression was observed on the mesothelium of 13 of 14 women with ovarian cancer compared with 6 of 15 who were cancer-free. Using a cell culture model system of mesothelial invasion, highly tumorigenic SKOV-3 and ES-2 cells were 2.5 to 3 times more efficient in transmigration through the mesothelial monolayer compared with poorly tumorigenic OVCAR-3 cells. Blocking antibodies to, or small interfering RNA knockdown of, VCAM-1 or its ligand α4β1 integrin significantly decreased, but did not completely inhibit, transmigration of SKOV-3 cells through mesothelial monolayers. Furthermore, using a mouse model of ovarian cancer metastasis, treatment with VCAM-1 function-blocking antibodies decreased tumor burden and increased survival. Together, these observations implicate VCAM-1-α4β1 integrin interactions in the regulation of ovarian cancer cell mesothelial invasion and metastatic progression and offer the possibility of novel therapeutic targets. [Cancer Res 2009;69(4):1469–76]
Data from Concordant Gene Expression Signatures Predict Clinical Outcomes of Cancer Patients Undergoing Systemic Therapy
Authors: Williams Paul D. , Cheon Sooyoung , Havaleshko Dmytro M. , Jeong Hyeon , Cheng Feng , Theodorescu Dan , Lee Jae K.
Published: March 2023

Abstract
Conventional development of multivariate gene expression models (GEM) predicting therapeutic response of cancer patients is based on analysis of patients treated with specific regimens, which limits generalization to different or novel drug combinations. We overcome this limitation by developing GEMs based on in vitro drug sensitivities and microarray analyses of the NCI-60 cancer cell line panel. These GEMs were evaluated in blind fashion as predictors of tumor response and/or patient survival in seven independent cohorts of patients with breast (n = 275), bladder (n = 59), and ovarian (n= 143) cancer treated with multiagent chemotherapy, of which 233 patients were from prospectively enrolled clinical trials. In all studies, GEMs effectively stratified tumor response and patient survival independent of established clinical and pathologic tumor variables. In bladder cancer patients treated with neoadjuvant methotrexate, vinblastine, Adriamycin (doxorubicin), and cisplatin, the 3-year overall survival for those with favorable GEM scores was 81% versus 33% for those with less favorable scores (P = 0.002). GEMs for breast cancer patients treated with 5-fluorouracil, Adriamycin (doxorubicin), and cyclophosphamide and ovarian cancer patients treated with platinum-containing regimens also stratified patient survival [5-year overall survival 100% versus 74% (P = 0.05) and 3-year overall survival 68% versus 43% (P = 0.008), respectively]. Importantly, clinical prediction using our in vitro GEM was superior to that of conventionally derived GEMs. We show a facile yet effective approach to GEM derivation that identifies patients most likely to benefit from selected multiagent therapy. Use of such in vitro–based GEMs may provide a robust and generalizable approach to personalized cancer therapy. [Cancer Res 2009;69(21):8302–9]
Data from A Key Regulatory Role of the Transcription Factor NFATc2 in Bronchial Adenocarcinoma via CD8+ T Lymphocytes
Authors: Maxeiner Joachim H. , Karwot Roman , Sauer Kerstin , Scholtes Petra , Boross Ildiko , Koslowski Michael , Türeci Özlem , Wiewrodt Rainer , Neurath Markus F. , Lehr Hans A. , Finotto Susetta
Published: March 2023

Abstract
The Ca2+-regulated calcineurin/nuclear factor of activated T cells (NFAT) cascade controls alternative pathways of T-cell activation and peripheral tolerance. Here, we describe reduction of NFATc2 mRNA expression in the lungs of patients with bronchial adenocarcinoma. In a murine model of bronchoalveolar adenocarcinoma, mice lacking NFATc2 developed more and larger solid tumors than wild-type littermates. The extent of central tumor necrosis was decreased in the tumors in NFATc2(−/−) mice, and this finding was associated with reduced tumor necrosis factor-α and interleukin-2 (IL-2) production by CD8+ T cells. Adoptive transfer of CD8+ T cells of NFATc2(−/−) mice induced transforming growth factor-β1 in the airways of recipient mice, thus supporting CD4+CD25+Foxp-3+glucocorticoid-induced tumor necrosis factor receptor (GITR)+ regulatory T (Treg) cell survival. Finally, engagement of GITR in NFATc2(−/−) mice induced IFN-γ levels in the airways, reversed the suppression by Treg cells, and costimulated effector CD4+CD25+ (IL-2Rα) and memory CD4+CD127+ (IL-7Rα) T cells, resulting in abrogation of carcinoma progression. Agonistic signaling through GITR, in the absence of NFATc2, thus emerges as a novel possible strategy for the treatment of human bronchial adenocarcinoma in the absence of NFATc2 by enhancing IL-2Rα+ effector and IL-7Rα+ memory-expressing T cells. [Cancer Res 2009;69(7):3069–76]
Data from p73 and p63 Sustain Cellular Growth by Transcriptional Activation of Cell Cycle Progression Genes
Authors: Lefkimmiatis Konstantinos , Caratozzolo Mariano Francesco , Merlo Paola , D'Erchia Anna Maria , Navarro Beatriz , Levrero Massimo , Sbisa' Elisabetta , Tullo Apollonia
Published: March 2023

Abstract
Despite extensive studies on the role of tumor suppressor p53 protein and its homologues, p73 and p63, following their overexpression or cellular stress, very little is known about the regulation of the three proteins in cells during physiologic cell cycle progression. We report a role for p73 and p63 in supporting cellular proliferation through the transcriptional activation of the genes involved in G1-S and G2-M progression. We found that in MCF-7 cells, p73 and p63, but not p53, are modulated during the cell cycle with a peak in S phase, and their silencing determines a significant suppression of proliferation compared with the control. Chromatin immunoprecipitation analysis shows that in cycling cells, p73 and p63 are bound to the p53-responsive elements (RE) present in the regulatory region of cell cycle progression genes. On the contrary, when the cells are arrested in G0-G1, p73 detaches from the REs and it is replaced by p53, which represses the expression of these genes. When the cells move in S phase, p73 is recruited again and p53 is displaced or is weakly bound to the REs. These data open new possibilities for understanding the involvement of p73 and p63 in cancer. The elevated concentrations of p73 and p63 found in many cancers could cause the aberrant activation of cell growth progression genes and therefore contribute to cancer initiation or progression under certain conditions. [Cancer Res 2009;69(22):8563–71]
Data from Equally Potent Inhibition of c-Src and Abl by Compounds that Recognize Inactive Kinase Conformations
Authors: Seeliger Markus A. , Ranjitkar Pratistha , Kasap Corynn , Shan Yibing , Shaw David E. , Shah Neil P. , Kuriyan John , Maly Dustin J.
Published: March 2023

Abstract
Imatinib is an inhibitor of the Abl tyrosine kinase domain that is effective in the treatment of chronic myelogenic leukemia. Although imatinib binds tightly to the Abl kinase domain, its affinity for the closely related kinase domain of c-Src is at least 2,000-fold lower. Imatinib recognition requires a specific inactive conformation of the kinase domain, in which a conserved Asp-Phe-Gly (DFG) motif is flipped with respect to the active conformation. The inability of c-Src to readily adopt this flipped DFG conformation was thought to underlie the selectivity of imatinib for Abl over c-Src. Here, we present a series of inhibitors (DSA compounds) that are based on the core scaffold of imatinib but which bind with equally high potency to c-Src and Abl. The DSA compounds bind to c-Src in the DFG-flipped conformation, as confirmed by crystal structures and kinetic analysis. The origin of the high affinity of these compounds for c-Src is suggested by the fact that they also inhibit clinically relevant Abl variants bearing mutations in a structural element, the P-loop, that normally interacts with the phosphate groups of ATP but is folded over a substructure of imatinib in Abl. Importantly, several of the DSA compounds block the growth of Ba/F3 cells harboring imatinib-resistant BCR-ABL mutants, including the Thr315Ile “gatekeeper” mutation, but do not suppress the growth of parental Ba/F3 cells. [Cancer Res 2009;69(6):2384–92
Data from Identification of a Novel Pathway That Selectively Modulates Apoptosis of Breast Cancer Cells
Authors: Tinnikov Alexander A. , Yeung Kay T. , Das Sharmistha , Samuels Herbert H.
Published: March 2023

Abstract
Expression of the nuclear receptor interacting factor 3 (NRIF3) coregulator in a wide variety of breast cancer cells selectively leads to rapid caspase-2–dependent apoptotic cell death. A novel death domain (DD1) was mapped to a 30–amino acid region of NRIF3. Because the cytotoxicity of NRIF3 and DD1 seems to be cell type–specific, these studies suggest that breast cancer cells contain a novel “death switch” that can be specifically modulated by NRIF3 or DD1. Using an MCF-7 cell cDNA library in a yeast two-hybrid screen, we cloned a factor that mediates apoptosis by DD1 and refer to this factor as DD1-interacting factor-1 (DIF-1). DIF-1 is a transcriptional repressor that mediates its effect through SirT1, and this repression is attenuated by the binding of NRIF3/DD1. DIF-1 expression rescues breast cancer cells from NRIF3/DD1-induced apoptosis. Small interfering RNA (siRNA) knockdown of DIF-1 selectively leads to apoptosis of breast cancer cells, further suggesting that DIF-1 plays a key role in NRIF3/DD1-mediated apoptosis. A protein kinase A inhibitor (H89) also elicits apoptosis of breast cancer cells but not of the other cell types examined, and DIF-1 also protects these cells from H89-mediated apoptosis. In addition, H89 incubation results in a rapid increase in NRIF3 levels and siRNA knockdown of NRIF3 protects breast cancer cells from H89-mediated apoptosis. Our results indicate that DIF-1 plays a key role in breast cancer cell survival and further characterizing this pathway may provide important insights into developing novel therapies to selectively target breast cancer cells for apoptosis. [Cancer Res 2009;69(4):1375–82]
Data from Senescent Stromal-Derived Osteopontin Promotes Preneoplastic Cell Growth
Authors: Pazolli Ermira , Luo Xianmin , Brehm Sarah , Carbery Kelly , Chung Jun-Jae , Prior Julie L. , Doherty Jason , Demehri Shadmehr , Salavaggione Lorena , Piwnica-Worms David , Stewart Sheila A.
Published: March 2023

Abstract
Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression. [Cancer Res 2009;69(3):1230–9]
Data from Human Postmeiotic Segregation 2 Exhibits Biased Repair at Tetranucleotide Microsatellite Sequences
Authors: Shah Sandeep N. , Eckert Kristin A.
Published: March 2023

Abstract
The mismatch repair (MMR) system plays a major role in removing DNA polymerization errors, and loss of this pathway results in hereditary cancers characterized by microsatellite instability. We investigated microsatellite stability during DNA replication within human postmeiotic segregation 2 (hPMS2)–deficient and proficient human lymphoblastoid cell lines. Using a shuttle vector assay, we measured mutation rates at reporter cassettes containing defined mononucleotide, dinucleotide, and tetranucleotide microsatellite sequences. A mutator phenotype was observed in the hPMS2-deficient cell line. The mutation rate of vectors containing [G/C]10 or [GT/CA]10 alleles was elevated 20-fold to 40-fold in hPMS2-deficient cells, relative to an hPMS2-expressing cell line. We observed a 6-fold and 12-fold relative increase in mutation rate of [TTTC/AAAG]9 and [TTCC/AAGG]9 sequences, respectively, in hPMS2-deficient cells. Mutational specificity analyses suggested that repair by hPMS2 is biased. In the absence of hPMS2, a greater number of microsatellite expansion versus deletion mutations was observed, and expansion rates of the tetranucleotide alleles were similar. In the presence of hPMS2, we observed a 29-fold decrease in the [TTCC/AAGG]9 expansion rate but only a 6-fold decrease for the [TTTC/AAAG]9 allele. Our data indicate that hPMS2 is more protective of tetranucleotide expansions than deletions and that hPMS2 displays a sequence bias, wherein [TTCC/AAGG] sequences are stabilized to a greater extent than [TTTC/AAAG]. Our results allow for greater accuracy during identification of MMR defects by providing a mutational signature characteristic of hPMS2 defect. This study also provides clues to possible mechanisms of repair by hPMS2 in the context of the MMR system. [Cancer Res 2009;69(3):1143–9]
Data from Sunitinib Inhibition of Stat3 Induces Renal Cell Carcinoma Tumor Cell Apoptosis and Reduces Immunosuppressive Cells
Authors: Xin Hong , Zhang Chunyan , Herrmann Andreas , Du Yan , Figlin Robert , Yu Hua
Published: March 2023

Abstract
The novel multitargeted tyrosine kinase inhibitor sunitinib is used as an antiangiogenic agent for the treatment of several types of cancer, including metastatic renal cell carcinoma (RCC). Sunitinib was shown to positively change the immunosuppressive phenotype in RCC patients. To improve its antitumor efficacy, and offer strategies for its combination with other approaches, it is critical to fully elucidate its mechanisms of action. We show that sunitinib induces tumor cell apoptosis and growth arrest in RCC tumor cells, which correlates with signal transducer and activator of transcription 3 (Stat3) activity inhibition. Sunitinib-mediated direct effects on tumor cells occur regardless of von Hippel-Lindau tumor suppressor gene status and hypoxia-inducible transcription factor-2α levels. Reduction of Stat3 activity enhances the antitumor effects of sunitinib, whereas expression of a constitutively activated Stat3 mutant rescues tumor cell death. Intravital multiphoton microscopy data show that sunitinib induces mouse Renca tumor cell apoptosis in vivo before tumor vasculature collapse. Sunitinib also inhibits Stat3 in Renca tumor–associated myeloid-derived suppressor cells (MDSC), down-regulates angiogenic gene expression, and reduces MDSCs and tumor T regulatory cells. These results suggest that Stat3 activity is important for RCC response to sunitinib, and Stat3 inhibition permits the direct proapoptotic activity of sunitinib on tumor cells and positive effects on tumor immunologic microenvironment. [Cancer Res 2009;69(6):2506–13]
Data from Recapitulation of Pancreatic Neuroendocrine Tumors in Human Multiple Endocrine Neoplasia Type I Syndrome via Pdx1-Directed Inactivation of Men1
Authors: Shen H.-C. Jennifer , He Mei , Powell Anathea , Adem Asha , Lorang Dominique , Heller Charles , Grover Amelia C. , Ylaya Kris , Hewitt Stephen M. , Marx Stephen J. , Spiegel Allen M. , Libutti Steven K.
Published: March 2023

Abstract
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal syndrome caused by mutations in the MEN1 tumor suppressor gene. Whereas the protein product of MEN1, menin, is ubiquitously expressed, somatic loss of the remaining wild-type MEN1 allele results in tumors primarily in parathyroid, pituitary, and endocrine pancreas. To understand the endocrine specificity of the MEN1 syndrome, we evaluated biallelic loss of Men1 by inactivating Men1 in pancreatic progenitor cells using the Cre-lox system. Men1 deletion in progenitor cells that differentiate into exocrine and endocrine pancreas did not affect normal pancreas morphogenesis and development. However, mice having homozygous inactivation of the Men1 in pancreas developed endocrine tumors with no exocrine tumor manifestation, recapitulating phenotypes seen in the MEN1 patients. In the absence of menin, the endocrine pancreas showed increase in cell proliferation, vascularity, and abnormal vascular structures; such changes were lacking in exocrine pancreas. Further analysis revealed that these endocrine manifestations were associated with up-regulation in vascular endothelial growth factor expression in both human and mouse MEN1 pancreatic endocrine tumors. Together, these data suggest the presence of cell-specific factors for menin and a permissive endocrine environment for MEN1 tumorigenesis in endocrine pancreas. Based on our analysis, we propose that menin's ability to maintain cellular and microenvironment integrity might explain the endocrine- restrictive nature of the MEN1 syndrome. [Cancer Res 2009;69(5):1858–66]