bluuebunny/crossref_metadata_2025 huggingface dataset:
Supplementary Figure S2 from YAP1 Mediates Resistance to MEK1/2 Inhibition in Neuroblastomas with Hyperactivated RAS Signaling
Authors: Coggins Grace E. , Farrel Alvin , Rathi Komal S. , Hayes Colin M. , Scolaro Laura , Rokita Jo Lynne , Maris John M.
Published: March 2023

Figure S2 shows Sanger sequencing of NLF YAP1 knockout isogenic lines used to confirm mutational status.
Supplementary Table S3 from Activation of Canonical BMP4-SMAD7 Signaling Suppresses Breast Cancer Metastasis
Authors: Eckhardt Bedrich L. , Cao Yuan , Redfern Andrew D. , Chi Lap Hing , Burrows Allan D. , Roslan Suraya , Sloan Erica K. , Parker Belinda S. , Loi Sherene , Ueno Naoto T. , Lau Peter K. H. , Latham Bruce , Anderson Robin L.
Published: March 2023

Anoikis genes regulated by BMP4
Supplementary Data from Inhibition of IL1β by Canakinumab May Be Effective against Diverse Molecular Subtypes of Lung Cancer: An Exploratory Analysis of the CANTOS Trial
Authors: Wong Connie C. , Baum Jason , Silvestro Angela , Beste Michael T. , Bharani-Dharan Bharani , Xu Siyan , Wang Ying A. , Wang Xiaoshan , Prescott Margaret F. , Krajkovich Lynne , Dugan Margaret , Ridker Paul M. , Martin Anne-Marie , Svensson Eric C.
Published: March 2023

Supplementary Data
Supplementary Figure S4 from Activation of Canonical BMP4-SMAD7 Signaling Suppresses Breast Cancer Metastasis
Authors: Eckhardt Bedrich L. , Cao Yuan , Redfern Andrew D. , Chi Lap Hing , Burrows Allan D. , Roslan Suraya , Sloan Erica K. , Parker Belinda S. , Loi Sherene , Ueno Naoto T. , Lau Peter K. H. , Latham Bruce , Anderson Robin L.
Published: March 2023

Figure S4 demonstrates a role for SMAD7 in mediating the inhibition of metastasis by BMP4.
Supplementary Table 1 from Truncated ASPP2 Drives Initiation and Progression of Invasive Lobular Carcinoma via Distinct Mechanisms
Authors: Schipper Koen , Drenth Anne Paulien , van der Burg Eline , Cornelissen Samuel , Klarenbeek Sjoerd , Nethe Micha , Jonkers Jos
Published: March 2023

List of primers used for cloning and genotyping
Supplementary Figures from P2X7 Receptor Activity Limits Accumulation of T Cells within Tumors
Authors: Romagnani Andrea , Rottoli Elsa , Mazza Emilia Maria Cristina , Rezzonico-Jost Tanja , De Ponte Conti Benedetta , Proietti Michele , Perotti Michela , Civanelli Elisa , Perruzza Lisa , Catapano Alberico L. , Baragetti Andrea , Tenedini Elena , Tagliafico Enrico , Falzoni Simonetta , Di Virgilio Francesco , Norata Giuseppe Danilo , Bicciato Silvio , Grassi Fabio
Published: March 2023

Supplementary Figures
Tables 1-7 from Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia
Authors: Sachs Karen , Sarver Aaron L. , Noble-Orcutt Klara E. , LaRue Rebecca S. , Antony Marie Lue , Chang Daniel , Lee Yoonkyu , Navis Connor M. , Hillesheim Alexandria L. , Nykaza Ian R. , Ha Ngoc A. , Hansen Conner J. , Karadag Fatma K. , Bergerson Rachel J. , Verneris Michael R. , Meredith Matthew M. , Schomaker Matthew L. , Linden Michael A. , Myers Chad L. , Largaespada David A. , Sachs Zohar
Published: March 2023

Supplementary Tables: ST1: Differentially Expressed Genes; ST2: IPA, ST3: Clinical Data, ST4: Mutational Profiles, ST5: qPCR Gene Panel, ST6: qPCR Enriched, ST7: NGS Gene panel
Figure S5 from Modeling Acquired Resistance to the Second-Generation Androgen Receptor Antagonist Enzalutamide in the TRAMP Model of Prostate Cancer
Authors: Cerasuolo Marianna , Maccarinelli Federica , Coltrini Daniela , Mahmoud Ali Mokhtar , Marolda Viviana , Ghedini Gaia Cristina , Rezzola Sara , Giacomini Arianna , Triggiani Luca , Kostrzewa Magdalena , Verde Roberta , Paris Debora , Melck Dominique , Presta Marco , Ligresti Alessia , Ronca Roberto
Published: March 2023

Sensitivity analysis
Supplementary Data from Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia
Authors: Sachs Karen , Sarver Aaron L. , Noble-Orcutt Klara E. , LaRue Rebecca S. , Antony Marie Lue , Chang Daniel , Lee Yoonkyu , Navis Connor M. , Hillesheim Alexandria L. , Nykaza Ian R. , Ha Ngoc A. , Hansen Conner J. , Karadag Fatma K. , Bergerson Rachel J. , Verneris Michael R. , Meredith Matthew M. , Schomaker Matthew L. , Linden Michael A. , Myers Chad L. , Largaespada David A. , Sachs Zohar
Published: March 2023

Supplementary Materials and Methods and Figures. Suppl Figs: SF1. Workflow; SF2: Cell Surface Markers; SF3: HSPC Markers; SF4: Human AML, SF5: Sorting Strategy, SF6: Post-sort; SF7: Viability and Immunophenotype; SF8: CD36 and CD69 in Human AML
Data from ΔDNMT3B Variants Regulate DNA Methylation in a Promoter-Specific Manner
Authors: Wang Jie , Bhutani Manisha , Pathak Ashutosh K. , Lang Wenhua , Ren Hening , Jelinek Jaroslav , He Rong , Shen Lanlan , Issa Jean-Pierre , Mao Li
Published: March 2023

Abstract
DNA methyltransferase 3B (DNMT3B) is critical in de novo DNA methylation during development and tumorigenesis. We recently reported the identification of a DNMT3B subfamily, ΔDNMT3B, which contains at least seven variants, resulting from alternative pre-mRNA splicing. ΔDNMT3Bs are the predominant expression forms of DNMT3B in human lung cancer. A strong correlation was observed between the promoter methylation of RASSF1A gene but not p16 gene (both frequently inactivated by promoter methylation in lung cancer) and expression of ΔDNMT3B4 in primary lung cancer, suggesting a role of ΔDNMT3B in regulating promoter-specific methylation of common tumor suppressor genes in tumorigenesis. In this report, we provide first experimental evidence showing a direct involvement of ΔDNMT3B4 in regulating RASSF1A promoter methylation in human lung cancer cells. Knockdown of ΔDNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells. Conversely, normal bronchial epithelial cells with stably transfected ΔDNMT3B4 gained an increased DNA methylation in RASSF1A promoter but not p16 promoter. We conclude that promoter DNA methylation can be differentially regulated and ΔDNMT3Bs are involved in regulation of such promoter-specific de novo DNA methylation. [Cancer Res 2007;67(22):10647–52]
Data from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo
Authors: Huang Dan , Ding Yan , Luo Wang-Mei , Bender Stephanie , Qian Chao-Nan , Kort Eric , Zhang Zhong-Fa , VandenBeldt Kristin , Duesbery Nicholas S. , Resau James H. , Teh Bin Tean
Published: March 2023

Abstract
The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH2 kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature. [Cancer Res 2008;68(1):81–8]
Data from The Nucleolin Targeting Aptamer AS1411 Destabilizes Bcl-2 Messenger RNA in Human Breast Cancer Cells
Authors: Soundararajan Sridharan , Chen Weiwei , Spicer Eleanor K. , Courtenay-Luck Nigel , Fernandes Daniel J.
Published: March 2023

Abstract
We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 μmol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 μmol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 μmol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death. [Cancer Res 2008;68(7):2358–65]
Data from Oncogenic BARD1 Isoforms Expressed in Gynecological Cancers
Authors: Li Lin , Ryser Stephan , Dizin Eva , Pils Dietmar , Krainer Michael , Jefford Charles Edward , Bertoni Francesco , Zeillinger Robert , Irminger-Finger Irmgard
Published: March 2023

Abstract
BARD1 is required for protein stability and tumor suppressor functions of BRCA1, which depend on the ubiquitin ligase activity of the BRCA1-BARD1 heterodimer. The NH2-terminal RING domains of both proteins act as interaction modules and form a ubiquitin ligase, which has functions in DNA repair, cell cycle checkpoint regulation, and mitosis. Interestingly, up-regulated expression of truncated BARD1 isoforms was found to be associated with poor prognosis in breast and ovarian cancers and, in a hormonally regulated fashion, in the human cytotrophoblast, a cell type with properties reminiscent of cancer cells. We therefore performed reverse transcription-PCR to determine the structure of BARD1 isoforms in cell lines derived from hormone-dependent and hormone-independent cancers. We found a specific combination of isoforms, generated by differential splicing and alternative transcription initiation, mostly lacking the BRCA1 interaction domain, in gynecologic but not hematologic cancer cell lines. To investigate the prevalence of BARD1 isoforms in tumors, we applied immunohistochemistry to ovarian cancers, using antibodies distinguishing full-length BARD1 and isoforms. Expression of NH2 terminally truncated BARD1 was correlated with advanced stage of cancer, and expression of spliced isoforms was typical for clear cell carcinoma, the ovarian cancer with worst prognosis, suggesting a role of BARD1 isoforms in cancer progression. To challenge this hypothesis, we silenced BARD1 isoforms in ovarian cancer cells that lacked wild-type BARD1 by siRNA interference, which led to a complete proliferation arrest. Thus, BARD1 isoform expression is required for cancer cell proliferation, which is compatible with the notion that BARD1 isoforms act as cancer maintenance genes. [Cancer Res 2007;67(24):11876–85]
Data from Nuclear Factor-κB, an Unappreciated Tumor Suppressor
Authors: Chen Fei , Castranova Vince
Published: March 2023

Abstract
The notion that nuclear factor-κB (NF-κB) is a tumor-promoting transcription factor has become a widely accepted dogma in biology. However, recent findings suggest an inhibitory role for NF-κB in carcinogenesis and tumorigenesis. Although the tumor suppressor–like effect of NF-κB remains to be rigorously established by further studies using cellular and animal models, these latest findings warrant caution with respect to blockage of NF-κB activation as a broad strategy in treating cancers. [Cancer Res 2007;67(23):11093–8]
Data from A Smac Mimetic Rescue Screen Reveals Roles for Inhibitor of Apoptosis Proteins in Tumor Necrosis Factor-α Signaling
Authors: Gaither Alex , Porter Dale , Yao Yao , Borawski Jason , Yang Guang , Donovan Jerry , Sage David , Slisz Joanna , Tran Mary , Straub Christopher , Ramsey Tim , Iourgenko Vadim , Huang Alan , Chen Yan , Schlegel Robert , Labow Mark , Fawell Stephen , Sellers William R. , Zawel Leigh
Published: March 2023

Abstract
Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor α (TNFα) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFα signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFα induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFα-mediated apoptosis. [Cancer Res 2007;67(24):11493–8]
Data from Association of Matrix Metalloproteinase-8 Gene Variation with Breast Cancer Prognosis
Authors: Decock Julie , Long Ji-Rong , Laxton Ross C. , Shu Xiao-Ou , Hodgkinson Conrad , Hendrickx Wouter , Pearce Eve G. , Gao Yu-Tang , Pereira Andresa C. , Paridaens Robert , Zheng Wei , Ye Shu
Published: March 2023

Abstract
Animal and cell studies indicate an inhibitory effect of matrix metalloproteinase-8 (MMP8) on tumorigenesis and metastasis. We investigated whether MMP8 gene variation was associated with breast cancer metastasis and prognosis in humans. We first studied nine tagging single nucleotide polymorphisms (SNP) in the MMP8 gene in 140 clinically and pathologically well-characterized breast cancer patients. Four of the SNPs were found to be associated with lymph node metastasis, the most pronounced being a promoter SNP (rs11225395) with its minor allele (T) associating with reduced susceptibility to lymph node metastasis (P = 0.02). This SNP was further evaluated for association with cancer relapse and survival among a cohort of ∼1,100 breast cancer patients who had been followed for cancer recurrence and mortality for a median of 7.1 years. The T allele was associated with reduced cancer relapse and greater survival, particularly among patients with earlier stage cancer. Among patients of tumor-node-metastasis stage 0 to II, the adjusted hazard ratio of disease-free survival was 0.7 [95% confidence interval (95% CI), 0.5–0.9] for patients carrying T allele compared with those homozygous for the C allele (P = 0.02). In vitro experiments showed that the T allele had higher promoter activity than the C allele in breast cancer cells. Electrophoretic mobility shift assays showed binding of nuclear proteins to the DNA sequence at the SNP site of the T allele but not that of the C allele. The data suggest that MMP8 gene variation may influence breast cancer prognosis and support the notion that MMP8 has an inhibitory effect on cancer metastasis. [Cancer Res 2007;67(21):10214–21]
Data from Lapatinib (Tykerb, GW572016) Reverses Multidrug Resistance in Cancer Cells by Inhibiting the Activity of ATP-Binding Cassette Subfamily B Member 1 and G Member 2
Authors: Dai Chun-ling , Tiwari Amit K. , Wu Chung-Pu , Su Xiao-dong , Wang Si-Rong , Liu Dong-geng , Ashby Charles R. , Huang Yan , Robey Robert W. , Liang Yong-ju , Chen Li-ming , Shi Cheng-Jun , Ambudkar Suresh V. , Chen Zhe-Sheng , Fu Li-wu
Published: March 2023

Abstract
Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E217βG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [125I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic. [Cancer Res 2008;68(19):7905–14]
Data from An Actin-Binding Protein Girdin Regulates the Motility of Breast Cancer Cells
Authors: Jiang Ping , Enomoto Atsushi , Jijiwa Mayumi , Kato Takuya , Hasegawa Taisaku , Ishida Maki , Sato Tomoko , Asai Naoya , Murakumo Yoshiki , Takahashi Masahide
Published: March 2023

Abstract
Girdin (girders of actin filaments) is a novel actin-binding Akt substrate that plays an important role in actin organization and Akt-dependent cell motility in fibroblasts. Here, we find that Girdin is expressed in a variety of cancer cell lines, including the breast cancer cell line MDA-MB-231, and is phosphorylated by the stimulation of insulin-like growth factor (IGF-I). In vitro migration and invasion assays showed that Girdin is required for the IGF-I–dependent cell movement of MDA-MB-231 cells. Short hairpin interfering RNA directed against Girdin markedly inhibited the metastasis of s.c. transplanted MDA-MB-231 cells in nude mice. In addition, Girdin is highly expressed in a variety of human malignant tissues, including breast, colon, lung, and uterine cervical carcinomas. These findings highlight the important role of Girdin in tumor progression in which the Akt signaling pathway is aberrantly activated. [Cancer Res 2008;68(5):1310–8]
Data from Inactivation of gadd45a Sensitizes Epithelial Cancer Cells to Ionizing Radiation In vivo Resulting in Prolonged Survival
Authors: Lu Xiangdong , Yang Chunyu , Hill Reginald , Yin Chaoyin , Hollander M. Christine , Fornace Albert J. , Van Dyke Terry
Published: March 2023

Abstract
Ionizing radiation (IR) therapy is one of the most commonly used treatments for cancer patients. The responses of tumor cells to IR are often tissue specific and depend on pathway aberrations present in the tumor. Identifying molecules and mechanisms that sensitize tumor cells to IR provides new potential therapeutic strategies for cancer treatment. In this study, we used two genetically engineered mouse carcinoma models, brain choroid plexus carcinoma (CPC) and prostate, to test the effect of inactivating gadd45a, a DNA damage response p53 target gene, on tumor responses to IR. We show that gadd45a deficiency significantly increases tumor cell death after radiation. Effect on survival was assessed in the CPC model and was extended in IR-treated mice with gadd45a deficiency compared with those expressing wild-type gadd45a. These studies show a significant effect of gadd45a inactivation in sensitizing tumor cells to IR, implicating gadd45a as a potential drug target in radiotherapy management. [Cancer Res 2008;68(10):3579–83]
Data from Identification and Characterization of Ovarian Cancer-Initiating Cells from Primary Human Tumors
Authors: Zhang Shu , Balch Curt , Chan Michael W. , Lai Hung-Cheng , Matei Daniela , Schilder Jeanne M. , Yan Pearlly S. , Huang Tim H-M. , Nephew Kenneth P.
Published: March 2023

Abstract
The objective of this study was to identify and characterize a self-renewing subpopulation of human ovarian tumor cells (ovarian cancer-initiating cells, OCICs) fully capable of serial propagation of their original tumor phenotype in animals. Ovarian serous adenocarcinomas were disaggregated and subjected to growth conditions selective for self-renewing, nonadherent spheroids previously shown to derive from tissue stem cells. To affirm the existence of OCICs, xenoengraftment of as few as 100 dissociated spheroid cells allowed full recapitulation of the original tumor (grade 2/grade 3 serous adenocarcinoma), whereas >105 unselected cells remained nontumorigenic. Stemness properties of OCICs (under stem cell–selective conditions) were further established by cell proliferation assays and reverse transcription–PCR, demonstrating enhanced chemoresistance to the ovarian cancer chemotherapeutics cisplatin or paclitaxel and up-regulation of stem cell markers (Bmi-1, stem cell factor, Notch-1, Nanog, nestin, ABCG2, and Oct-4) compared with parental tumor cells or OCICs under differentiating conditions. To identify an OCIC cell surface phenotype, spheroid immunostaining showed significant up-regulation of the hyaluronate receptor CD44 and stem cell factor receptor CD117 (c-kit), a tyrosine kinase oncoprotein. Similar to sphere-forming OCICs, injection of only 100 CD44+CD117+ cells could also serially propagate their original tumors, whereas 105 CD44−CD117− cells remained nontumorigenic. Based on these findings, we assert that epithelial ovarian cancers derive from a subpopulation of CD44+CD117+ cells, thus representing a possible therapeutic target for this devastating disease. [Cancer Res 2008;68(11):4311–20]
Data from Cyclooxygenase-2–Derived Prostaglandin E2 Activates β-Catenin in Human Cholangiocarcinoma Cells: Evidence for Inhibition of These Signaling Pathways by ω3 Polyunsaturated Fatty Acids
Authors: Lim Kyu , Han Chang , Xu Lihong , Isse Kumiko , Demetris Anthony J. , Wu Tong
Published: March 2023

Abstract
Cholangiocarcinoma is a highly malignant neoplasm of the biliary tree. It has a high rate of mortality, and currently, there is no effective chemoprevention and treatment. This study was designed to investigate the potential effect of ω3 polyunsaturated fatty acids (ω3-PUFA) on human cholangiocarcinoma cell growth and to determine their mechanisms of actions. Treatment of three human cholangiocarcinoma cells (CCLP1, HuCCT1, SG231) with two ω3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for 12 to 72 h resulted in a dose- and time-dependent inhibition of cell growth; in contrast, arachidonic acid, a ω6-PUFA, had no significant effect. The ω3-PUFA effect is due to the induction of apoptosis, given that DHA induced the cleaved form of PARP, caspase-3, and caspase-9. DHA and EPA treatment caused dephosphorylation (and hence, the activation) of glycogen synthase kinase-3β (GSK-3β) with a decline of β-catenin protein. Accordingly, DHA treatment also decreased the β-catenin–mediated T cell factor/lymphoid enhancer factor (TCF/LEF) reporter activity, and inhibited the expression of c-Met, a β-catenin–controlled downstream gene implicated in cholangiocarcinogenesis. The GSK-3β inhibitor, SB216763, partially prevented DHA-induced reduction of β-catenin protein and TCF/LEF reporter activity, and restored cell growth, suggesting the involvement of GSK-3β dephosphorylation in ω3-PUFA–induced β-catenin degradation. In parallel, DHA treatment also induced the formation of the β-catenin/Axin/GSK-3β binding complex, further leading to β-catenin degradation. Moreover, DHA inhibited the expression of cyclooxygenase-2 (COX-2) and enhanced the expression of 15-hydroxyprostaglandin dehydrogenase, a physiologic COX-2 antagonist, in human cholangiocarcinoma cells. These findings suggest that ω3-PUFAs block cholangiocarcinoma cell growth at least in part through inhibition of Wnt/β-catenin and COX-2 signaling pathways. Thus, utilization of ω3-PUFAs may represent an effective and safe therapeutic approach for the chemoprevention and treatment of human cholangiocarcinoma. [Cancer Res 2008;68(2):553–60]
Data from Inhibition of the Ras-Net (Elk-3) Pathway by a Novel Pyrazole that Affects Microtubules
Authors: Wasylyk Christine , Zheng Hong , Castell Christelle , Debussche Laurent , Multon Marie-Christine , Wasylyk Bohdan
Published: March 2023

Abstract
Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras–extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)–induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment. [Cancer Res 2008;68(5):1275–83]
Data from Loss of E-Cadherin Promotes Metastasis via Multiple Downstream Transcriptional Pathways
Authors: Onder Tamer T. , Gupta Piyush B. , Mani Sendurai A. , Yang Jing , Lander Eric S. , Weinberg Robert A.
Published: March 2023

Abstract
Loss of the epithelial adhesion molecule E-cadherin is thought to enable metastasis by disrupting intercellular contacts—an early step in metastatic dissemination. To further investigate the molecular basis of this notion, we use two methods to inhibit E-cadherin function that distinguish between E-cadherin's cell-cell adhesion and intracellular signaling functions. Whereas the disruption of cell-cell contacts alone does not enable metastasis, the loss of E-cadherin protein does, through induction of an epithelial-to-mesenchymal transition, invasiveness, and anoikis resistance. We find the E-cadherin binding partner β-catenin to be necessary, but not sufficient, for induction of these phenotypes. In addition, gene expression analysis shows that E-cadherin loss results in the induction of multiple transcription factors, at least one of which, Twist, is necessary for E-cadherin loss–induced metastasis. These findings indicate that E-cadherin loss in tumors contributes to metastatic dissemination by inducing wide-ranging transcriptional and functional changes. [Cancer Res 2008;68(10):3645–53]
Data from Genome-Wide Analysis of the Homeobox C6 Transcriptional Network in Prostate Cancer
Authors: McCabe Colleen D. , Spyropoulos Demetri D. , Martin David , Moreno Carlos S.
Published: March 2023

Abstract
Homeobox transcription factors are developmentally regulated genes that play crucial roles in tissue patterning. Homeobox C6 (HOXC6) is overexpressed in prostate cancers and correlated with cancer progression, but the downstream targets of HOXC6 are largely unknown. We have performed genome-wide localization analysis to identify promoters bound by HOXC6 in prostate cancer cells. This analysis identified 468 reproducibly bound promoters whose associated genes are involved in functions such as cell proliferation and apoptosis. We have complemented these data with expression profiling of prostates from mice with homozygous disruption of the Hoxc6 gene to identify 31 direct regulatory target genes of HOXC6. We show that HOXC6 directly regulates expression of bone morphogenic protein 7, fibroblast growth factor receptor 2, insulin-like growth factor binding protein 3, and platelet-derived growth factor receptor α (PDGFRA) in prostate cells and indirectly influences the Notch and Wnt signaling pathways in vivo. We further show that inhibition of PDGFRA reduces proliferation of prostate cancer cells, and that overexpression of HOXC6 can overcome the effects of PDGFRA inhibition. HOXC6 regulates genes with both oncogenic and tumor suppressor activities as well as several genes such as CD44 that are important for prostate branching morphogenesis and metastasis to the bone microenvironment. [Cancer Res 2008;68(6):1988–96]
Data from RhoC Promotes Metastasis via Activation of the Pyk2 Pathway in Prostate Cancer
Authors: Iiizumi Megumi , Bandyopadhyay Sucharita , Pai Sudha K. , Watabe Misako , Hirota Shigeru , Hosobe Sadahiro , Tsukada Taisei , Miura Kunio , Saito Ken , Furuta Eiji , Liu Wen , Xing Fei , Okuda Hiroshi , Kobayashi Aya , Watabe Kounosuke
Published: March 2023

Abstract
RhoC is a member of the Ras-homologous family of genes which have been implicated in tumorigenesis and tumor progression. However, the exact role of RhoC is controversial and is yet to be clarified. We have examined the effect of RhoC on prostate tumor cells and found that RhoC had no effect on cell proliferation in vitro or on tumor growth in mice. However, RhoC significantly enhanced the metastatic ability of the tumor cells in these animals, suggesting that RhoC affects only the metastasis but not the growth of prostate tumor cells. The results of our immunohistochemical analyses on tumor specimens from 63 patients with prostate cancer indicate that RhoC expression had no significant correlation with Gleason grade. However, the expression of RhoC showed significant positive correlation with both lymph node and distant metastasis, and it was inversely correlated with patient survival. We also found that RhoC significantly augmented the invasion and motility of prostate tumor cells by activating matrix metalloproteinases 2 and 9 (MMP2 and MMP9) in vitro. The results of our antibody array analysis for signal molecules revealed that RhoC significantly activated kinases including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt, and Pyk2. Inhibition of Pyk2 kinase blocked the RhoC-dependent activation of FAK, MAPK, and Akt, followed by the suppression of MMP2 and MMP9. Inhibitors of both MAPK and Akt also significantly blocked the activities of these MMPs. Therefore, our results indicate that RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt followed by the up-regulation of MMP2 and MMP9, which results in the stimulation of invasiveness of tumor cells. [Cancer Res 2008;68(18):7613–20]
Data from The Kringle 1 Domain of Hepatocyte Growth Factor Has Antiangiogenic and Antitumor Cell Effects on Hepatocellular Carcinoma
Authors: Shen Zan , Yang Zhen Fan , Gao Yi , Li Ji Cheng , Chen Hai Xiao , Liu Ching Chiu , Poon Ronnie T.P. , Fan Sheung Tat , Luk John M. , Sze Kong Hung , Li Tsai Ping , Gan Ren Bao , He Ming Liang , Kung Hsiang Fu , Lin Marie C.M.
Published: March 2023

Abstract
The kringle 1 domain of human hepatocyte growth factor (HGFK1) was previously shown to inhibit bovine aortic endothelial cell proliferation, suggesting that it might be an antiangiogenic molecule. Here, we evaluated the in vivo efficacy of a recombinant adenoassociated virus carrying HGFK1 (rAAV-HGFK1) for the treatment of hepatocellular carcinoma (HCC) in a rat orthotopic HCC model and explored its molecular mechanisms in vitro in both endothelial and tumor cells. We first showed that rAAV-HGFK1 treatment significantly prolonged the survival time of rats transplanted with tumor cells. Treatment with rAAV-HGFK1 inhibited tumor growth, decreased tumor microvessel density, and completely prevented intrahepatic, lung, and peritoneal metastasis in this in vivo model. In vitro, rAAV-HGFK1 exhibited both antiangiogenic and antitumor cell effects, inhibiting the proliferation of both murine microvascular endothelial cells (MEC) and tumor cells, and inducing apoptosis and G0-G1 phase arrest in these cells. To our surprise, rAAV-HGFK1 did not act through the hepatocyte growth factor/hepatocyte growth factor receptor pathway. Instead, it worked mainly through epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling, with more minor contributions from vascular endothelial growth factor/vascular endothelial growth factor receptor and β fibroblast growth factor (bFGF)/β fibroblast growth factor receptor (bFGFR) signaling. In both MECs and tumor cells, rAAV-HGFK1 acted through two pathways downstream of EGFR, namely inhibition of extracellular signal-regulated kinase activation and stimulation of p38 mitogen-activated protein kinase/c-Jun-NH2-kinase activation. These results suggest for the first time that HGFK1 exerts both antiangiogenic and antitumor cell activities mainly through EGF/EGFR signaling, and may thus be considered as a novel therapeutic strategy for the treatment of HCC. [Cancer Res 2008;68(2):404–14]
Data from RETRACTED: Methylation Mediated Silencing of MicroRNA-1 Gene and Its Role in Hepatocellular Carcinogenesis
Authors: Datta Jharna , Kutay Huban , Nasser Mohd W. , Nuovo Gerard J. , Wang Bo , Majumder Sarmila , Liu Chang-Gong , Volinia Stefano , Croce Carlo M. , Schmittgen Thomas D. , Ghoshal Kalpana , Jacob Samson T.
Published: March 2023

Abstract
MicroRNAs (miR) are a class of small (∼21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1−/− HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3′-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC–treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049–58]
Data from 14-3-3ζ Down-regulates p53 in Mammary Epithelial Cells and Confers Luminal Filling
Authors: Danes Christopher G. , Wyszomierski Shannon L. , Lu Jing , Neal Christopher L. , Yang Wentao , Yu Dihua
Published: March 2023

Abstract
Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3ζ is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3ζ overexpression occurs, and we found that increased expression of 14-3-3ζ begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3ζ overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3ζ in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3ζ overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3ζ overexpression conferred resistance to anoikis. Additionally, 14-3-3ζ overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3ζ transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3ζ induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3ζ–overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3ζ overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3ζ alters MEC acini structure and increases the risk of breast cancer. [Cancer Res 2008;68(6):1760–7]
Data from Tumor-Induced Suppressor of Cytokine Signaling 3 Inhibits Toll-like Receptor 3 Signaling in Dendritic Cells via Binding to Tyrosine Kinase 2
Authors: Zeng Bin , Li Haijie , Liu Yu , Zhang Zhuohan , Zhang Yuan , Yang Rongcun
Published: March 2023

Abstract
The suppressor of cytokine signaling (SOCS) family of negative regulatory proteins is up-regulated in response to several cytokines and pathogen-associated molecular patterns (PAMP) and suppresses cellular signaling responses by binding receptor phosphotyrosine residues. Exposure of bone marrow–derived dendritic cells (BMDC) to 1D8 cells, a murine model of ovarian carcinoma, suppresses their ability to express CD40 and stimulate antigen-specific responses in response to PAMPs and, in particular, to polyinosinic acid:poly-CMP (polyI:C) with the up-regulated SOCS3 transcript and protein levels. The ectopic expression of SOCS3 in both the macrophage cell line RAW264.7 and BMDCs decreased signaling in response to both polyI:C and IFNα. Further, knockdown of SOCS3 transcripts significantly enhanced the responses of RAW264.7 and BMDCs to both polyI:C and IFNα. Immunoprecipitation and pull-down studies show that SOCS3 binds to the IFNα receptor tyrosine kinase 2 (TYK2). Because polyI:C triggers autocrine IFNα signaling, binding of SOCS3 to TYK2 may thereby suppress the activation of BMDCs by polyI:C and IFNα. Thus, elevated levels of SOCS3 in tumor-associated DCs may potentially resist the signals induced by Toll-like receptor 3 ligands and type I IFN to decrease DC activation via binding with IFNα receptor TYK2. [Cancer Res 2008;68(13):5397–404]
Data from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair
Authors: Chen Xi , Zhong Shijun , Zhu Xiao , Dziegielewska Barbara , Ellenberger Tom , Wilson Gerald M. , MacKerell Alexander D. , Tomkinson Alan E.
Published: March 2023

Abstract
Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents. [Cancer Res 2008;68(9):3169–77]
Data from A First-Generation Multiplex Biomarker Analysis of Urine for the Early Detection of Prostate Cancer
Authors: Laxman Bharathi , Morris David S. , Yu Jianjun , Siddiqui Javed , Cao Jie , Mehra Rohit , Lonigro Robert J. , Tsodikov Alex , Wei John T. , Tomlins Scott A. , Chinnaiyan Arul M.
Published: March 2023

Abstract
Although prostate-specific antigen (PSA) serum level is currently the standard of care for prostate cancer screening in the United States, it lacks ideal specificity and additional biomarkers are needed to supplement or potentially replace serum PSA testing. Emerging evidence suggests that monitoring the noncoding RNA transcript PCA3 in urine may be useful in detecting prostate cancer in patients with elevated PSA levels. Here, we show that a multiplex panel of urine transcripts outperforms PCA3 transcript alone for the detection of prostate cancer. We measured the expression of seven putative prostate cancer biomarkers, including PCA3, in sedimented urine using quantitative PCR on a cohort of 234 patients presenting for biopsy or radical prostatectomy. By univariate analysis, we found that increased GOLPH2, SPINK1, and PCA3 transcript expression and TMPRSS2:ERG fusion status were significant predictors of prostate cancer. Multivariate regression analysis showed that a multiplexed model, including these biomarkers, outperformed serum PSA or PCA3 alone in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.758 for the multiplexed model versus 0.662 for PCA3 alone (P = 0.003). The sensitivity and specificity for the multiplexed model were 65.9% and 76.0%, respectively, and the positive and negative predictive values were 79.8% and 60.8%, respectively. Taken together, these results provide the framework for the development of highly optimized, multiplex urine biomarker tests for more accurate detection of prostate cancer. [Cancer Res 2008;68(3):645–9]
Data from The Tumor Suppressor LKB1 Regulates Lung Cancer Cell Polarity by Mediating cdc42 Recruitment and Activity
Authors: Zhang Shumin , Schafer-Hales Katherine , Khuri Fadlo R. , Zhou Wei , Vertino Paula M. , Marcus Adam I.
Published: March 2023

Abstract
The tumor suppressor LKB1 is mutated in 30% of non–small cell lung cancer (NSCLC) tumors and cell lines and is proposed to be a key regulator of epithelial cell polarity; however, how LKB1 regulates cancer cell polarity is not known. The experiments described herein show for the first time that LKB1 is a dynamic, actin-associated protein that rapidly polarizes to the leading edge of motile cancer cells. LKB1 proves to be essential for NSCLC polarity, because LKB1 depletion results in classic cell polarity defects, such as aberrant Golgi positioning, reduced lamellipodia formation, and aberrant morphology. To probe how LKB1 regulates these events, we show that LKB1 colocalizes at the cellular leading edge with two key components of the polarity pathway — the small rho GTPase cdc42 and its downstream binding partner p21-activated kinase (PAK). Importantly, LKB1 functionality is required for cdc42 polarization to the leading edge, maintaining active cdc42 levels, and downstream PAK phosphorylation. To do this, LKB1 interacts only with active form of cdc42 and PAK, but not with inactive cdc42. Taken together, these results show that LKB1 is a critical mediator of the NSCLC polarity program in lung cancer cells through a novel LKB1-cdc42-PAK pathway. [Cancer Res 2008;68(3):740–8]
Data from Identification of a Novel Subgroup of Melanomas with KIT/Cyclin-Dependent Kinase-4 Overexpression
Authors: Smalley Keiran S.M. , Contractor Rooha , Nguyen Thiennga K. , Xiao Min , Edwards Robin , Muthusamy Viswanathan , King Alastair J. , Flaherty Keith T. , Bosenberg Marcus , Herlyn Meenhard , Nathanson Katherine L.
Published: March 2023

Abstract
Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G1 phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27KIP. Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy. [Cancer Res 2008;68(14):5743–52]