bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Fibroblasts Isolated from Common Sites of Breast Cancer Metastasis Enhance Cancer Cell Growth Rates and Invasiveness in an Interleukin-6–Dependent Manner
Authors: Studebaker Adam W. , Storci Gianluca , Werbeck Jillian L. , Sansone Pasquale , Sasser A. Kate , Tavolari Simona , Huang Tim , Chan Michael W.Y. , Marini Frank C. , Rosol Thomas J. , Bonafé Massimiliano , Hall Brett M.
Published: March 2023

Abstract
Common sites of breast cancer metastasis include the lung, liver, and bone, and of these secondary metastatic sites, estrogen receptor α (ERα)–positive breast cancer often favors bone. Within secondary organs, cancer cells would predictably encounter tissue-specific fibroblasts or their soluble factors, yet our understanding of how tissue-specific fibroblasts directly affect cancer cell growth rates and survival remains largely unknown. Therefore, we tested the hypothesis that mesenchymal fibroblasts isolated from common sites of breast cancer metastasis provide a more favorable microenvironment with respect to tumor growth rates. We found a direct correlation between the ability of breast, lung, and bone fibroblasts to enhance ERα-positive breast cancer cell growth and the level of soluble interleukin-6 (IL-6) produced by each organ-specific fibroblast, and fibroblast-mediated growth enhancement was inhibited by the removal or inhibition of IL-6. Interestingly, mice coinjected with MCF-7 breast tumor cells and senescent skin fibroblasts, which secrete IL-6, developed tumors, whereas mice coinjected with presenescent skin fibroblasts that produce little to no IL-6 failed to form xenograft tumors. We subsequently determined that IL-6 promoted growth and invasion of breast cancer cells through signal transducer and activator of transcription 3–dependent up-regulation of Notch-3, Jagged-1, and carbonic anhydrase IX. These data suggest that tissue-specific fibroblasts and the factors they produce can promote breast cancer disease progression and may represent attractive targets for development of new therapeutics. [Cancer Res 2008;68(21):9087–95]
Data from Mechanisms of Antileukemic Activity of the Novel Bcl-2 Homology Domain-3 Mimetic GX15-070 (Obatoclax)
Authors: Konopleva Marina , Watt Julie , Contractor Rooha , Tsao Twee , Harris David , Estrov Zeev , Bornmann William , Kantarjian Hagop , Viallet Jean , Samudio Ismael , Andreeff Michael
Published: March 2023

Abstract
In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G2 cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC50 of 3.59 ± 1.23 μmol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics. [Cancer Res 2008;68(9):3413–20]
Data from Mutational Analysis of Thirty-two Double-Strand DNA Break Repair Genes in Breast and Pancreatic Cancers
Authors: Wang Xianshu , Szabo Csilla , Qian Chiping , Amadio Peter G. , Thibodeau Stephen N. , Cerhan James R. , Petersen Gloria M. , Liu Wanguo , Couch Fergus J.
Published: March 2023

Abstract
Inactivating mutations in several genes that encode components of the DNA repair machinery have been associated with an increased risk of breast cancer. To assess whether alterations in other DNA repair genes contribute to breast cancer and to further determine the relevance of these genes to pancreatic cancer, we performed mutational analysis of 32 DNA double-strand break repair genes in genomic DNA from 38 breast tumors, 48 pancreatic tumors, and 10 non-BRCA1/BRCA2 hereditary breast cancer patients. A total of 494 coding exons were screened by denatured high-performance liquid chromatography and direct DNA sequencing. Two inactivating mutations were identified in breast tumor samples, a germline single-nucleotide deletion in POLQ (c.3605delT) and a somatic nonsense change in PRKDC (c.2408C>A, p.Ser803X). Two germline-inactivating mutations in RAD50 (c.1875C>G, p.Tyr625X and IVS14+1G>A) were also detected in separate pancreatic tumor samples. In addition, 35 novel nonsynonymous amino acid substitutions, resulting from two in-frame deletions and 33 single nucleotide alterations, were identified. Seven of these were predicted to influence protein function. A separate analysis of the CLSPN c.3839C>T (rs35490896) variant that was observed more frequently in breast tumors than in pancreatic tumors or normal controls failed to detect a significant association with breast cancer risk in a Mayo Clinic breast cancer case-control study. In conclusion, this screen of DNA repair genes implicates PRKDC and POLQ as candidate tumor suppressor genes involved in breast cancer and suggests that inactivating mutations in RAD50 predispose to pancreatic cancer as well as breast cancer. [Cancer Res 2008;68(4):971–5]
Data from A Novel Lung Cancer Signature Mediates Metastatic Bone Colonization by a Dual Mechanism
Authors: Vicent Silvestre , Luis-Ravelo Diego , Antón Iker , García-Tuñón Ignacio , Borrás-Cuesta Francisco , Dotor Javier , De Las Rivas Javier , Lecanda Fernando
Published: March 2023

Abstract
Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage–related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-β (TGF-β) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-β blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-β–dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization. [Cancer Res 2008;68(7):2275–85]
Data from Rad6B Is a Positive Regulator of β-Catenin Stabilization
Authors: Shekhar Malathy P.V. , Gerard Brigitte , Pauley Robert J. , Williams Bart O. , Tait Larry
Published: March 2023

Abstract
Mutations in β-catenin or other Wnt pathway components that cause β-catenin accumulation occur rarely in breast cancer. However, there is some evidence of β-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of β-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces β-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, β-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus–Wnt-1 mammary tumor—derived cells, implicating Rad6B in physiologic regulation of β-catenin stability and activity. Ubiquitinated β-catenin was detectable in chromatin immunoprecipitations performed with β-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of β-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular β-catenin but with minimal effects on cell membrane–associated β-catenin. Measurement of β-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular β-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates β-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of β-catenin, reveal a positive regulatory feedback loop between Rad6B and β-catenin and a novel mechanism of β-catenin stabilization/activation in breast cancer cells. [Cancer Res 2008;68(6):1741–50]
Data from OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model
Authors: Sargeant Aaron M. , Rengel Robert C. , Kulp Samuel K. , Klein Russell D. , Clinton Steven K. , Wang Yu-Chieh , Chen Ching-Shih
Published: March 2023

Abstract
Histone deacetylase (HDAC) inhibitors suppress tumor cell growth via a broad spectrum of mechanisms, which should prove advantageous in the context of cancer prevention. Here, we examined the effect of dietary administration of OSU-HDAC42, a novel HDAC inhibitor, on prostate tumor progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Based on a series of pilot studies, an AIN-76A diet was formulated containing 208 ppm OSU-HDAC42, which was estimated to deliver ∼25 mg/kg of drug per day to each mouse and found to cause a suppression of PC-3 xenograft tumor growth equivalent to that achieved by gavage administration of a similar dose. At 6 weeks of age, TRAMP mice received this drug-containing or control diet for 4 or 18 weeks and were evaluated for prostatic intraepithelial neoplasia (PIN) and carcinoma development, respectively. OSU-HDAC42 not only decreased the severity of PIN and completely prevented its progression to poorly differentiated carcinoma (74% incidence in controls versus none in drug-treated mice), but also shifted tumorigenesis to a more differentiated phenotype, suppressing absolute and relative urogenital tract weights by 86% and 85%, respectively, at 24 weeks of age. This tumor suppression was associated with the modulation of intraprostatic biomarkers, including those indicative of HDAC inhibition, increased apoptosis and differentiation, and decreased proliferation. With the exception of completely reversible hematologic alterations and testicular degeneration, no significant changes in body weight or other indicators of general health were observed in drug-treated mice. These results suggest that OSU-HDAC42 has value in prostate cancer prevention. [Cancer Res 2008;68(10):3999–4009]
Data from Systematic High-Content Proteomic Analysis Reveals Substantial Immunologic Changes in Colorectal Cancer
Authors: Berndt Uta , Philipsen Lars , Bartsch Sebastian , Wiedenmann Bertram , Baumgart Daniel C. , Hämmerle Marcus , Sturm Andreas
Published: March 2023

Abstract
The immune system is a significant determinant of epithelial tumorigenesis, but its role in colorectal cancer pathogenesis is not well understood. The function of the immune system depends upon the integrity of the protein network environment, and thus, we performed MELC immunofluorescence microscopy focusing on the lamina propria. By analyzing structurally intact tissues from colorectal cancer, ulcerative colitis, and healthy colonic mucosa, we used this unique and novel highly multiplexed robotic-imaging technology, which allows visualizing dozens of proteins simultaneously, and explored the toponome in colorectal cancer mucosa for the first time. We identified 1,930 motifs that distinguish control from colorectal cancer tissue. In colorectal cancer, the number of activated T cells is increased, explained by a lack of bax, caspase-3, and caspase-8. Whereas CD4+CD25+ T cells are decreased and are, other than in ulcerative colitis, not activated, cytotoxic T cells are significantly increased in colorectal cancer. Furthermore, the number of activated human lymphocyte antigen (HLA)-DR+ T-cells is increased in colorectal cancer, pointing to an altered antigen presentation. In colorectal cancer, CD3+CD29+ expression and assembly of the LFA-1 and LFA-3 receptor are differentially changed, indicating a distinct regulation of T-cell adhesion in colorectal cancer. We also identified increased numbers of natural killer and CD44+ cells in the colorectal cancer mucosa and nuclear factor-κB as regulator of apoptosis in these cell populations. High-content proteomic analysis showed that colorectal cancer induces a tremendous modification of protein expression profiles in the lamina propria. Thus, topological proteomic analysis may help to unravel the role of the adaptive immune system in colorectal cancer and aid the development of new antitumor immunotherapy approaches. [Cancer Res 2008;68(3):880–8]
Data from Oxidative Stress Plays a Critical Role in Inactivating Mutant BRAF by Geldanamycin Derivatives
Authors: Fukuyo Yayoi , Inoue Masahiro , Nakajima Takuma , Higashikubo Ryuji , Horikoshi Nobuko T. , Hunt Clayton , Usheva Anny , Freeman Michael L. , Horikoshi Nobuo
Published: March 2023

Abstract
The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of mitogen-activated protein/extracellular signal-regulated kinase 1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells. Moreover, after treatment with 17-DMAG, the kinase activity of residual, undegraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine, partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the antitumor effect of geldanamycin and its derivatives is also mediated though the production of ROS, which may directly inactivate tumorigenic mutant BRAF(V600E). [Cancer Res 2008;68(15):6324–30]
Data from Statins Increase p21 through Inhibition of Histone Deacetylase Activity and Release of Promoter-Associated HDAC1/2
Authors: Lin Yi-Chu , Lin Jung-Hsin , Chou Chia-Wei , Chang Yu-Fan , Yeh Shu-Hao , Chen Ching-Chow
Published: March 2023

Abstract
Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors broadly used for the control of hypercholesterolemia. Recently, they are reported to have beneficial effects on certain cancers. In this study, we show that statins inhibited the histone deacetylase (HDAC) activity and increased the accumulation of acetylated histone-H3 and the expression of p21WAF/CIP in human cancer cells. Computational modeling showed the direct interaction of the carboxylic acid moiety of statins with the catalytic site of HDAC2. In the subsequent enzymatic assay, it was shown that lovastatin inhibited HDAC2 activity competitively with a Ki value of 31.6 μmol/L. Sp1 but not p53 sites were found to be the statins-responsive element shown by p21 luciferase-promoter assays. DNA affinity protein binding assay and chromatin immunoprecipitation assay showed the dissociation of HDAC1/2 and association of CBP, leading to the histone-H3 acetylation on the Sp1 sites of p21 promoter. In vitro cell proliferation and in vivo tumor growth were both inhibited by statins. These results suggest a novel mechanism for statins through abrogation of the HDAC activity and promoter histone-H3 acetylation to regulate p21 expression. Therefore, statins might serve as novel HDAC inhibitors for cancer therapy and chemoprevention. [Cancer Res 2008;68(7):2375–83]
Data from The Regulatory T Cell–Associated Transcription Factor FoxP3 Is Expressed by Tumor Cells
Authors: Ebert Lisa M. , Tan Bee Shin , Browning Judy , Svobodova Suzanne , Russell Sarah E. , Kirkpatrick Naomi , Gedye Craig , Moss Denis , Ng Sweet Ping , MacGregor Duncan , Davis Ian D. , Cebon Jonathan , Chen Weisan
Published: March 2023

Abstract
FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25+ regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo, suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines. [Cancer Res 2008;68(8):3001–9]
Data from A Human Breast Cell Model of Preinvasive to Invasive Transition
Authors: Rizki Aylin , Weaver Valerie M. , Lee Sun-Young , Rozenberg Gabriela I. , Chin Koei , Myers Connie A. , Bascom Jamie L. , Mott Joni D. , Semeiks Jeremy R. , Grate Leslie R. , Mian I. Saira , Borowsky Alexander D. , Jensen Roy A. , Idowu Michael O. , Chen Fanqing , Chen David J. , Petersen Ole W. , Gray Joe W. , Bissell Mina J.
Published: March 2023

Abstract
A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur “spontaneously” in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA–based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context. [Cancer Res 2008;68(5):1378–87]
Data from Ectopic Expression of P-Cadherin Correlates with Promoter Hypomethylation Early in Colorectal Carcinogenesis and Enhanced Intestinal Crypt Fission In vivo
Authors: Milicic Anita , Harrison Lea-Anne , Goodlad Robert A. , Hardy Robert G. , Nicholson Anna M. , Presz Michal , Sieber Oliver , Santander Sonia , Pringle James H. , Mandir Nikki , East Philip , Obszynska Jolanta , Sanders Scott , Piazuelo Elena , Shaw Jacqui , Harrison Rebecca , Tomlinson Ian P. , McDonald Stuart A.C. , Wright Nicholas A. , Jankowski Janusz A.Z.
Published: March 2023

Abstract
P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential “field effect” of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia. [Cancer Res 2008;68(19):7760–8]
Data from Regulation of Apoptosis and Caspase-8 Expression in Neuroblastoma Cells by Isoforms of the IG20 Gene
Authors: Li Liang Cheng , Sheng Jian Rong , Mulherkar Nirupama , Prabhakar Bellur S. , Meriggioli Matthew N.
Published: March 2023

Abstract
The IG20 gene undergoes alternative splicing resulting in the differential expression of six putative splice variants. Four of these (IG20pa, MADD, IG20-SV2, and DENN-SV) are expressed in virtually all human tissues. However, investigations examining alternative splicing of the IG20 gene to date have been largely limited to nonneural malignant and nonmalignant cells. In this study, we investigated the expression of alternative splice isoforms of the IG20 gene in human neuroblastoma cells. We found that six IG20 splice variants (IG20-SVs) were expressed in two human neuroblastoma cell lines (SK-N-SH and SH-SY5Y), highlighted by the expression of two unique splice isoforms (i.e., KIAA0358 and IG20-SV4). Similarly, we found enriched expression of these two IG20-SVs in human neural tissues derived from cerebral cortex, hippocampus, and, to a lesser extent, spinal cord. Using gain-of-function studies and siRNA technology, we determined that these “neural-enriched isoforms” exerted significant and contrasting effects on vulnerability to apoptosis in neuroblastoma cells. Specifically, expression of KIAA0358 exerted a potent antiapoptotic effect in both the SK-N-SH and SH-SY5Y neuroblastoma cell lines, whereas expression of IG20-SV4 had proapoptotic effects directly related to the activation of caspase-8 in these cells, which have minimal or absent constitutive caspase-8 expression. These data indicate that the pattern of expression of these neural-enriched IG20-SVs regulates the expression and activation of caspase-8 in certain neuroblastoma cells, and that manipulation of IG20-SV expression pattern may represent a potent therapeutic strategy in the therapy of neuroblastoma and perhaps other cancers. [Cancer Res 2008;68(18):7352–61]
Data from Phosphatidylinositol 3-Kinase Inhibition Broadly Sensitizes Glioblastoma Cells to Death Receptor– and Drug-Induced Apoptosis
Authors: Opel Daniela , Westhoff Mike-Andrew , Bender Ariane , Braun Veit , Debatin Klaus-Michael , Fulda Simone
Published: March 2023

Abstract
The aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway has been reported to correlate with adverse clinical outcome in human glioblastoma in vivo. However, the question of how this survival network can be successfully targeted to restore the sensitivity of glioblastoma to apoptosis induction has not yet been answered. Here, we report that inhibition of PI3K by LY294002 broadly sensitizes wild-type and mutant PTEN glioblastoma cells to both death receptor– and chemotherapy-induced apoptosis, whereas mammalian target of rapamycin (mTOR) inhibition is not sufficient to restore apoptosis sensitivity. LY294002 significantly enhances apoptosis triggered by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), agonistic anti-CD95 antibodies, or several anticancer drugs (i.e., doxorubicin, etoposide, and vincristine) in a highly synergistic manner. In addition, LY294002 cooperates with TRAIL or doxorubicin to suppress colony formation, thus also showing a strong effect on long-term survival. Similarly, genetic knockdown of PI3K subunits p110α and/or p110β by RNA interference (RNAi) primes glioblastoma cells for TRAIL- or doxorubicin-mediated apoptosis. In contrast to PI3K inhibition, pharmacologic or genetic blockade of mTOR by RAD001 (everolimus), rapamycin, or RNAi fails to enhance TRAIL- or doxorubicin-induced apoptosis. Analysis of apoptosis pathways reveals that PI3K inhibition acts in concert with TRAIL or doxorubicin to trigger mitochondrial membrane permeabilization, caspase activation, and caspase-dependent apoptosis, which are abolished by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Most importantly, PI3K inhibition by LY294002 sensitizes primary cultured glioblastoma cells obtained from surgical specimens to TRAIL- or chemotherapy-induced cell death. By showing that PI3K inhibition broadly primes glioblastoma cells for apoptosis, our findings provide the rationale for using PI3K inhibitors in combination regimens to enhance TRAIL- or chemotherapy-induced apoptosis in glioblastoma. [Cancer Res 2008;68(15):6271–80]
Data from Epigenetic Silencing of MicroRNA-34b/c and B-Cell Translocation Gene 4 Is Associated with CpG Island Methylation in Colorectal Cancer
Authors: Toyota Minoru , Suzuki Hiromu , Sasaki Yasushi , Maruyama Reo , Imai Kohzoh , Shinomura Yasuhisa , Tokino Takashi
Published: March 2023

Abstract
Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2′-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR-34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR-34b/c and BTG4, is a frequent target of epigenetic silencing in CRC. [Cancer Res 2008;68(11):4123–32]
Data from Gemcitabine and Cytosine Arabinoside Cytotoxicity: Association with Lymphoblastoid Cell Expression
Authors: Li Liang , Fridley Brooke , Kalari Krishna , Jenkins Gregory , Batzler Anthony , Safgren Stephanie , Hildebrandt Michelle , Ames Matthew , Schaid Daniel , Wang Liewei
Published: March 2023

Abstract
Two cytidine analogues, gemcitabine (dFdC) and 1-β-d-arabinofuranosylcytosine (AraC), show significant therapeutic effect in a variety of cancers. However, response to these drugs varies widely. Evidence from tumor biopsy samples shows that expression levels for genes involved in the cytidine transport, metabolism, and bioactivation pathway contribute to this variation in response. In the present study, we set out to test the hypothesis that variation in gene expression both within and outside of this “pathway” might influence sensitivity to gemcitabine and AraC. Specifically, Affymetrix U133 Plus 2.0 GeneChip and cytotoxicity assays were performed to obtain basal mRNA expression and IC50 values for both drugs in 197 ethnically defined Human Variation Panel lymphoblastoid cell lines. Genes with a high degree of association with IC50 values were involved mainly in cell death, cancer, cell cycle, and nucleic acid metabolism pathways. We validated selected significant genes by performing real-time quantitative reverse transcription-PCR and selected two representative candidates, NT5C3 (within the pathway) and FKBP5 (outside of the pathway), for functional validation. Those studies showed that down-regulation of NT5C3 and FKBP5 altered tumor cell sensitivity to both drugs. Our results suggest that cell-based model system studies, when combined with complementary functional characterization, may help to identify biomarkers for response to chemotherapy with these cytidine analogues. [Cancer Res 2008;68(17):7050–8]
Data from In vivo Switching of Human Melanoma Cells between Proliferative and Invasive States
Authors: Hoek Keith S. , Eichhoff Ossia M. , Schlegel Natalie C. , Döbbeling Udo , Kobert Nikita , Schaerer Leo , Hemmi Silvio , Dummer Reinhard
Published: March 2023

Abstract
Metastatic melanoma represents a complex and heterogeneous disease for which there are no therapies to improve patient survival. Recent expression profiling of melanoma cell lines identified two transcription signatures, respectively, corresponding with proliferative and invasive cellular phenotypes. A model derived from these findings predicts that in vivo melanoma cells may switch between these states. Here, DNA microarray–characterized cell lines were subjected to in vitro characterization before s.c. injection into immunocompromised mice. Tumor growth rates were measured and postexcision samples were assessed by immunohistochemistry to identify invasive and proliferative signature cells. In vitro tests showed that proliferative signature melanoma cells are faster growing but less motile than invasive signature cells. In vivo proliferative signature cells initiated tumor growth in 14 ± 3 days postinjection. By comparison, invasive signature cells required a significantly longer (P < 0.001) period of 59 ± 11 days. Immunohistochemistry showed that regardless of the seed cell signature, tumors showed evidence for both proliferative and invasive cell types. Furthermore, proliferative signature cell types were detected most frequently in the peripheral margin of growing tumors. These data indicate that melanoma cells undergo transcriptional signature switching in vivo likely regulated by local microenvironmental conditions. Our findings challenge previous models of melanoma progression that evoke one-way changes in gene expression. We present a new model for melanoma progression that accounts for transcription signature plasticity and provides a more rational context for explaining observed melanoma biology. [Cancer Res 2008;68(3):650–6]
Data from Targeted Delivery of Gemcitabine to Pancreatic Adenocarcinoma Using Cetuximab as a Targeting Agent
Authors: Patra Chitta Ranjan , Bhattacharya Resham , Wang Enfeng , Katarya Aaron , Lau Julie S. , Dutta Shamit , Muders Michael , Wang Shanfeng , Buhrow Sarah A. , Safgren Stephanie L. , Yaszemski Michael J. , Reid Joel M. , Ames Matthew M. , Mukherjee Priyabrata , Mukhopadhyay Debabrata
Published: March 2023

Abstract
One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR. [Cancer Res 2008;68(6):1970–8]
Data from Genetic Variants Contributing to Daunorubicin-Induced Cytotoxicity
Authors: Huang R. Stephanie , Duan Shiwei , Kistner Emily O. , Bleibel Wasim K. , Delaney Shannon M. , Fackenthal Donna L. , Das Soma , Dolan M. Eileen
Published: March 2023

Abstract
Identifying heritable genetic variants responsible for chemotherapeutic toxicities has been challenging due in part to its multigenic nature. To date, there is a paucity of data on genetic variants associated with patients experiencing severe myelosuppression or cardiac toxicity following treatment with daunorubicin. We present a genome-wide model using International HapMap cell lines that integrate genotype and gene expression to identify genetic variants that contribute to daunorubicin-induced cytotoxicity. A cell growth inhibition assay was used to measure variations in the cytotoxicity of daunorubicin. Gene expression was determined using the Affymetrix GeneChip Human Exon 1.0ST Array. Using sequential analysis, we evaluated the associations between genotype and cytotoxicity, those significant genotypes with gene expression and correlated gene expression of the identified candidates with cytotoxicity. A total of 26, 9, and 18 genetic variants were identified to contribute to daunorubicin-induced cytotoxicity through their effect on 16, 9, and 36 gene expressions in the combined, Centre d' Etude du Polymorphisme Humain (CEPH), and Yoruban populations, respectively. Using 50 non-HapMap CEPH cell lines, single nucleotide polymorphisms generated through our model predicted 29% of the overall variation in daunorubicin sensitivity and the expression of CYP1B1 was significantly correlated with sensitivity to daunorubicin. In the CEPH validation set, rs120525235 and rs3750518 were significant predictors of transformed daunorubicin IC50 (P = 0.005 and P = 0.0008, respectively), and rs1551315 trends toward significance (P = 0.089). This unbiased method can be used to elucidate genetic variants contributing to a wide range of cellular phenotypes. [Cancer Res 2008;68(9):3161–8]
Data from Defining the Cooperative Genetic Changes That Temporally Drive Alveolar Rhabdomyosarcoma
Authors: Naini Sarasija , Etheridge Katherine T. , Adam Stacey J. , Qualman Stephen J. , Bentley Rex C. , Counter Christopher M. , Linardic Corinne M.
Published: March 2023

Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of RMS known as alveolar (aRMS) have a 5-year survival rate of <30%. aRMS tissues exhibit a number of genetic changes, including loss-of-function of the p53 and Rb tumor suppressor pathways, amplification of MYCN, stabilization of telomeres, and most characteristically, reciprocal translocation of loci involving the PAX and FKHR genes, generating the PAX7-FKHR or PAX3-FKHR fusion proteins. We previously showed that PAX3-FKHR expression in primary human myoblasts, cells that can give rise to RMS, cooperated with loss of p16INK4A to promote extended proliferation. To better understand the genetic events required for aRMS formation, we then stepwise converted these cells to their transformed counterpart. PAX3-FKHR, the catalytic unit of telomerase hTERT, and MycN, in cooperation with down-regulation of p16INK4A/p14ARF expression, were necessary and sufficient to convert normal human myoblasts into tumorigenic cells that gave rise to aRMS tumors. However, the order of expression of these transgenes was critical, as only those cells expressing PAX3-FKHR early could form tumors. We therefore suggest that the translocation of PAX3 to FKHR drives proliferation of myoblasts, and a selection for loss of p16INK4A/p14ARF. These early steps, coupled with MycN amplification and telomere stabilization, then drive the cells to a fully tumorigenic state. [Cancer Res 2008;68(23):9583–8]
Data from An Epigenetic Genome-Wide Screen Identifies SPINT2 as a Novel Tumor Suppressor Gene in Pediatric Medulloblastoma
Authors: Kongkham Paul N. , Northcott Paul A. , Ra Young Shin , Nakahara Yukiko , Mainprize Todd G. , Croul Sidney E. , Smith Christian A. , Taylor Michael D. , Rutka James T.
Published: March 2023

Abstract
Medulloblastoma (MB) is a malignant cerebellar tumor that occurs primarily in children. The hepatocyte growth factor (HGF)/MET pathway has an established role in both normal cerebellar development as well as the development and progression of human brain tumors, including MB. To identify novel tumor suppressor genes involved in MB pathogenesis, we performed an epigenome-wide screen in MB cell lines, using 5-aza-2′deoxycytidine to identify genes aberrantly silenced by promoter hypermethylation. Using this technique, we identified an inhibitor of HGF/MET signaling, serine protease inhibitor kunitz-type 2 (SPINT2/HAI-2), as a putative tumor suppressor silenced by promoter methylation in MB. In addition, based on single nucleotide polymorphism array analysis in primary MB samples, we identified hemizygous deletions targeting the SPINT2 locus in addition to gains on chromosome 7 encompassing the HGF and MET loci. SPINT2 gene expression was down-regulated and MET expression was up-regulated in 73.2% and 45.5% of tumors, respectively, by quantitative real-time PCR. SPINT2 promoter methylation was detected in 34.3% of primary MBs examined by methylation-specific PCR. SPINT2 reexpression in MB cell lines reduced proliferative capacity, anchorage independent growth, cell motility in vitro, and increased overall survival times in vivo in a xenograft model (P < 0.0001). Taken together, these data support the role of SPINT2 as a putative tumor suppressor gene in MB, and further implicate dysregulation of the HGF/MET signaling pathway in the pathogenesis of MB. [Cancer Res 2008;68(23):9945–53]
Data from MicroRNA-7, a Homeobox D10 Target, Inhibits p21-Activated Kinase 1 and Regulates Its Functions
Authors: Reddy Sirigiri Divijendra Natha , Ohshiro Kazufumi , Rayala Suresh K. , Kumar Rakesh
Published: March 2023

Abstract
MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner and play crucial roles during oncogenesis. Here we show that microRNA-7 (miR-7) inhibits p21-activated kinase 1 (Pak1) expression, a widely up-regulated signaling kinase in multiple human cancers, by targeting the 3′-untranslated region (UTR) of Pak1 mRNA. We noticed an inverse correlation between the levels of endogenous miR-7 and Pak1 expression in human cancer cells. We discovered that endogenous miR-7 expression is positively regulated by a homeodomain transcription factor, HoxD10, the loss of which leads to an increased invasiveness. HoxD10 directly interacts with the miR-7 chromatin. Accordingly, the levels of Pak1 protein are progressively up-regulated whereas those of miR-7 and its upstream activator HoxD10 are progressively down-regulated in a cellular model of breast cancer progression from low to highly invasive phenotypes. Furthermore, HoxD10 expression in highly invasive breast cancer cells resulted in an increased miR-7 expression but reduced Pak1 3′-UTR-luciferase activity and reduced Pak1 protein. Finally, we show that miR-7 introduction inhibits the motility, invasiveness, anchorage-independent growth, and tumorigenic potential of highly invasive breast cancer cells. Collectively, these findings establish for the first time that Pak1 is a target of miR-7 and that HoxD10 plays a regulatory role in modifying the expression of miR-7 and, consequently, the functions of the miR-7-Pak1 pathway in human cancer cells. [Cancer Res 2008;68(20):8195–200]
Data from Genome-Wide Functional Synergy between Amplified and Mutated Genes in Human Breast Cancer
Authors: Nikolsky Yuri , Sviridov Evgeny , Yao Jun , Dosymbekov Damir , Ustyansky Vadim , Kaznacheev Valery , Dezso Zoltan , Mulvey Laura , Macconaill Laura E. , Winckler Wendy , Serebryiskaya Tatiana , Nikolskaya Tatiana , Polyak Kornelia
Published: March 2023

Abstract
A single cancer cell contains large numbers of genetic alterations that in combination create the malignant phenotype. However, whether amplified and mutated genes form functional and physical interaction networks that could explain the selection for cells with combined alterations is unknown. To investigate this issue, we characterized copy number alterations in 191 breast tumors using dense single nucleotide polymorphism arrays and identified 1,747 genes with copy number gain organized into 30 amplicons. Amplicons were distributed unequally throughout the genome. Each amplicon had distinct enrichment pattern in pathways, networks, and molecular functions, but genes within individual amplicons did not form coherent functional units. Genes in amplicons included all major tumorigenic pathways and were highly enriched in breast cancer–causative genes. In contrast, 1,188 genes with somatic mutations in breast cancer were distributed randomly over the genome, did not represent a functionally cohesive gene set, and were relatively less enriched in breast cancer marker genes. Mutated and gained genes did not show statistically significant overlap but were highly synergistic in populating key tumorigenic pathways including transforming growth factor β, WNT, fibroblast growth factor, and PIP3 signaling. In general, mutated genes were more frequently upstream of gained genes in transcription regulation signaling than vice versa, suggesting that mutated genes are mainly regulators, whereas gained genes are mostly regulated. ESR1 was the major transcription factor regulating amplified but not mutated genes. Our results support the hypothesis that multiple genetic events, including copy number gains and somatic mutations, are necessary for establishing the malignant cell phenotype. [Cancer Res 2008;68(22):9532–40]
Data from Resveratrol Suppresses Growth of Human Ovarian Cancer Cells in Culture and in a Murine Xenograft Model: Eukaryotic Elongation Factor 1A2 as a Potential Target
Authors: Lee Mee-Hyun , Choi Bu Young , Kundu Joydeb Kumar , Shin Young Kee , Na Hye-Kyung , Surh Young-Joon
Published: March 2023

Abstract
The eukaryotic elongation factor 1A2 (eEF1A2) is known to retain oncogenic potential and is recognized as a novel target for cancer prevention and therapy. Resveratrol (trans-3,4′,5-trihydroxystilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the growth-inhibitory effects of resveratrol in human ovarian cancer PA-1 cells, considering eEF1A2 as a potential molecular target. Pretreatment with resveratrol attenuated proliferation of serum-starved PA-1 cells stimulated with insulin or serum. Resveratrol also activated caspase-9, -7, and -3 and induced apoptosis in PA-1 cells in the presence of insulin or serum. Insulin or serum stimulation of PA-1 cells resulted in the marked induction of eEF1A2, which was suppressed by pretreatment with resveratrol. Moreover, resveratrol inhibited insulin- or serum-induced soft-agar colony formation in eEF1A2-transfected NIH3T3 cells. An antibody array directed to assess the phosphorylation of protein kinases revealed that treatment with insulin or serum induced the phosphorylation of Akt in PA-1 cells. Pharmacologic inhibition of Akt with LY294002 abrogated insulin- or serum-induced eEF1A2 expression and increased the caspase-3 activity. In another experiment, i.p. administration of resveratrol retarded the growth of PA-1 cell xenograft and the expression of eEF1A2 in athymic nude mice in association with decreased bromodeoxyuridine positivity, reduced expression of proliferating cell nuclear antigen, increased the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and caspase-3 staining, and diminished CD31 positivity. Taken together, eEF1A2 may be considered as a potential molecular target for the antiproliferative effects of resveratrol in PA-1 ovarian cancer cells. [Cancer Res 2009;69(18):7449–58]