bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from A Genome-Wide Screen in Saccharomyces cerevisiae Reveals Altered Transport As a Mechanism of Resistance to the Anticancer Drug Bleomycin
Authors: Aouida Mustapha , Pagé Nicolas , Leduc Anick , Peter Matthias , Ramotar Dindial
Published: March 2023

Abstract
The potent DNA damaging agent bleomycin (BLM) is highly effective for treating various cancers, although, in certain individuals, the development of cellular resistance to the drug can severely diminish its antineoplastic properties. We performed two independent genome-wide screens using a Saccharomyces cerevisiae mutant collection to isolate variants exhibiting either sensitivity or resistance to BLM. This procedure reproducibly identified a relatively large collection of 231 BLM-hypersensitive mutants, representing genes belonging to diverse functional groups. In contrast, only five BLM-resistant mutants could be recovered by our screens. Among these latter mutants, three were deleted for genes involved in plasma membrane transport, including the l-carnitine transporter Agp2, as well as the kinases Ptk2 and Sky1, which are involved in regulating polyamine transport. We further showed that Agp2 acts as a transporter of BLM and that overexpression of this transporter significantly enhances BLM-induced cell killing. Our data strongly implicate membrane transport as a key determinant in BLM resistance in yeast. This finding is critical, given that very little is known about BLM transport in human cells. Indeed, characterization of analogous mechanisms in humans may ultimately lead to enhancement of the antitumor properties of BLM.
Data from Potentiation of Cytotoxicity of Topoisomerase I Poison by Concurrent and Sequential Treatment with the Checkpoint Inhibitor UCN-01 Involves Disparate Mechanisms Resulting in Either p53-Independent Clonogenic Suppression or p53-Dependent Mitotic Catastrophe
Authors: Tse Archie N. , Schwartz Gary K.
Published: March 2023

Abstract
UCN-01 is a potent inhibitor of the S- and G2-M-phase cell cycle checkpoints by targeting chk1 and possibly chk2 kinases. It has been shown in some, but not all, instances that UCN-01 potentiates the cytotoxicity of DNA-damaging agents selectively in p53-defective cells. We have investigated this concept in HCT116 colon cancer cells treated with the topoisomerase I poison SN-38. SN-38 alone induced a senescence-like sustained G2 arrest without apoptosis. Sequential treatment with SN-38 followed by UCN-01 resulted in enhancement of cytotoxicity by apoptosis assay, whereas the reverse sequence or concurrent treatment did not potentiate apoptosis. Real-time visualization of HCT116 cells labeled with green fluorescent protein-histone 2B or green fluorescent protein-α-tubulin revealed that sequential treatment resulted in G2 checkpoint abrogation, and cells entered an aberrant mitosis despite normal assembly of bipolar spindles, resulting in either apoptosis or formation of micronucleated cells. Although p53-null cells were clearly more sensitive than parental HCT116 to undergoing checkpoint abrogation and mitotic death after sequential treatment, this was not accompanied by an increased inhibition of clonogenicity over that induced by SN-38 alone. Conversely, concurrent treatment with SN-38 and UCN-01 resulted in S-phase checkpoint override, an amplified DNA damage response including increased phosphorylation of the DNA double-strand breakage marker H2AX and augmentation of clonogenic inhibition, which was independent of p53. Thus, reported discrepancies in the pharmacology of UCN-01 and the influence of p53 status on treatment outcome appears to stem, in part, from the different schedules used, the specific checkpoints examined, and the assays used to assess cytotoxicity. Moreover, checkpoint abrogation and subsequent apoptosis induced by UCN-01 do not necessarily correlate with reproductive cell death.
Data from Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets
Authors: Andersen Claus Lindbjerg , Jensen Jens Ledet , Ørntoft Torben Falck
Published: March 2023

Abstract
Accurate normalization is an absolute prerequisite for correct measurement of gene expression. For quantitative real-time reverse transcription-PCR (RT-PCR), the most commonly used normalization strategy involves standardization to a single constitutively expressed control gene. However, in recent years, it has become clear that no single gene is constitutively expressed in all cell types and under all experimental conditions, implying that the expression stability of the intended control gene has to be verified before each experiment. We outline a novel, innovative, and robust strategy to identify stably expressed genes among a set of candidate normalization genes. The strategy is rooted in a mathematical model of gene expression that enables estimation not only of the overall variation of the candidate normalization genes but also of the variation between sample subgroups of the sample set. Notably, the strategy provides a direct measure for the estimated expression variation, enabling the user to evaluate the systematic error introduced when using the gene. In a side-by-side comparison with a previously published strategy, our model-based approach performed in a more robust manner and showed less sensitivity toward coregulation of the candidate normalization genes. We used the model-based strategy to identify genes suited to normalize quantitative RT-PCR data from colon cancer and bladder cancer. These genes are UBC, GAPD, and TPT1 for the colon and HSPCB, TEGT, and ATP5B for the bladder. The presented strategy can be applied to evaluate the suitability of any normalization gene candidate in any kind of experimental design and should allow more reliable normalization of RT-PCR data.
Data from The Retinoic Acid Synthesis Gene ALDH1a2 Is a Candidate Tumor Suppressor in Prostate Cancer
Authors: Kim Hanna , Lapointe Jacques , Kaygusuz Gulsah , Ong David E. , Li Chunde , van de Rijn Matt , Brooks James D. , Pollack Jonathan R.
Published: March 2023

Abstract
Prostate cancer is the most common cancer among men in the United States, and aberrant DNA methylation is known to be an early molecular event in its development. Here, we have used expression profiling to identify novel hypermethylated genes whose expression is induced by treatment of prostate cancer cell lines with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-aza-dC). Of the 271 genes that were induced by 5-aza-dC treatment, 25 also displayed reduced expression in primary prostate tumors compared with normal prostate tissue, and the decreased expression of only one gene, aldehyde dehydrogenase 1 family, member A2 (ALDH1a2), was also associated with shorter recurrence-free survival. ALDH1a2 encodes an enzyme responsible for synthesis of retinoic acid (RA), a compound with prodifferentiation properties. By immunohistochemistry, we observed that ALDH1a2 was expressed in epithelia from normal prostate but not prostate cancer. Using bisulfite sequencing, we determined that the ALDH1a2 promoter region was significantly hypermethylated in primary prostate tumors compared with normal prostate specimens (P = 0.01). Finally, transfection-mediated reexpression of wild-type ALDH1a2 (but not a presumptive catalytically dead mutant) in the prostate cancer cell line DU145 resulted in decreased colony growth (P < 0.0001), comparable with treatment with either 5-aza-dC or RA. Taken together, our findings implicate ALDH1a2 as a candidate tumor suppressor gene in prostate cancer and further support a role of retinoids in the prevention or treatment of prostate cancer.
Data from Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53
Authors: Li Chun-Qi , Robles Ana I. , Hanigan Christin L. , Hofseth Lorne J. , Trudel Laura J. , Harris Curtis C. , Wogan Gerald N.
Published: March 2023

Abstract
Loss of p53 function by inactivating mutations results in abrogation of NO·-induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO· in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO· was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO· also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO· treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO· exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.
Data from Antibody against Junctional Adhesion Molecule-C Inhibits Angiogenesis and Tumor Growth
Authors: Lamagna Chrystelle , Hodivala-Dilke Kairbaan M. , Imhof Beat A. , Aurrand-Lions Michel
Published: March 2023

Abstract
The junctional adhesion molecule-C (JAM-C) was recently described as an adhesion molecule localized at interendothelial contacts and involved in leukocyte transendothelial migration. The protein JAM-C interacts with polarity complex molecules and regulates the activity of the small GTPase Cdc42. The angiogenesis process involves rearrangement of endothelial junctions and implicates modulation of cell polarity. We tested whether JAM-C plays a role in angiogenesis using tumor grafts and hypoxia-induced retinal neovascularization. Treatment with a monoclonal antibody directed against JAM-C reduces tumor growth and infiltration of macrophages into tumors. The antibody decreases angiogenesis in the model of hypoxia-induced retinal neovascularization in vivo and vessel outgrowth from aortic rings in vitro. Importantly, the antibody does not induce pathologic side effects in vivo. These findings show for the first time a role for JAM-C in angiogenesis and define JAM-C as a valuable target for antitumor therapies.
Data from Chromatin Assembly Factor-1, a Marker of Clinical Value to Distinguish Quiescent from Proliferating Cells
Authors: Polo Sophie E. , Theocharis Stamatios E. , Klijanienko Jerzy , Savignoni Alexia , Asselain Bernard , Vielh Philippe , Almouzni Geneviève
Published: March 2023

Abstract
Histone synthesis and chromatin assembly are mainly associated with DNA replication and are thus intimately involved in cell cycle regulation. The expression of key components involved in these events in human cells was studied in relation to cell-proliferative status. Among several chromatin assembly factors, chromatin assembly factor (CAF)-1 stood out as the most discriminating marker of the proliferative state. We show, using both immunofluorescence and Western blot analysis, that the expression of both CAF-1 large subunits, p150 and p60, is massively down-regulated during quiescence in several cell lines. Upon exit from the quiescent state, the CAF-1 subunits are re-expressed early, before DNA replication. The amounts of either total or chromatin-associated pools of CAF-1 proteins correlate directly with cell proliferation. Regulation of CAF-1 expression is partly controlled at the RNA level, as shown by quantitative reverse transcription-PCR and Northern blot experiments. Biological material from benign and malignant human breast tumors analyzed by immunocytochemistry and immunohistochemistry exhibits a strong positive correlation between CAF-1 p60 expression and the following proliferation markers: S-phase fraction (r = 0.84, P < 0.0001); Ki-67 (r = 0.94, P < 0.0001); and proliferating cell nuclear antigen (r = 0.95, P = 0.0001). We discuss the advantages of using CAF-1 to assess cell proliferation. High CAF-1 p60 levels are also shown to be associated with various prognostic factors. Our data highlight the precise association of CAF-1 expression with the proliferative state and validate the use of this factor as a useful proliferation marker and prognostic indicator in malignant and benign breast lesions.
Data from Differential Gene Expression Analysis Reveals Activation of Growth Promoting Signaling Pathways by Tenascin-C
Authors: Ruiz Christian , Huang Wentao , Hegi Monika E. , Lange Katrin , Hamou Marie-France , Fluri Erika , Oakeley Edward J. , Chiquet-Ehrismann Ruth , Orend Gertraud
Published: March 2023

Abstract
Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of β-catenin and stimulated the expression of the β-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.
Data from Tumor Suppressor Activity and Epigenetic Inactivation of Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 in Papillary and Clear Cell Renal Cell Carcinoma
Authors: Morris Mark R. , Gentle Dean , Abdulrahman Mahera , Maina Esther N. , Gupta Kunal , Banks Rosamonde E. , Wiesener Michael S. , Kishida Takeshi , Yao Masahiro , Teh Bin , Latif Farida , Maher Eamonn R.
Published: March 2023

Abstract
Following treatment with a demethylating agent, 5 of 11 renal cell carcinoma (RCC) cell lines showed increased expression of hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI-2/SPINT2/Bikunin), a Kunitz-type protease inhibitor that regulates HGF activity. As activating mutations in the MET proto-oncogene (the HGF receptor) cause familial RCC, we investigated whether HAI-2/SPINT2 might act as a RCC tumor suppressor gene. We found that transcriptional silencing of HAI-2 in RCC cell lines was associated with promoter region methylation and HAI-2/SPINT2 protein expression was down-regulated in 30% of sporadic RCC. Furthermore, methylation-specific PCR analysis revealed promoter region methylation in 30% (19 of 64) of clear cell RCC and 40% (15 of 38) of papillary RCC, whereas mutation analysis (in 39 RCC cell lines and primary tumors) revealed a missense substitution (P111S) in one RCC cell line. Restoration of HAI-2/SPINT2 expression in a RCC cell line reduced in vitro colony formation, but the P111S mutant had no significant effect. Increased cell motility associated with HAI-2/SPINT2 inactivation was abrogated by treatment with extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phospholipase C-γ inhibitors, but not by an inhibitor of atypical protein kinase C. These findings are consistent with frequent epigenetic inactivation of HAI-2/SPINT2, causing loss of RCC tumor suppressor activity and implicate abnormalities of the MET pathway in clear cell and papillary sporadic RCC. This information provides opportunities to develop novel targeted approaches to the treatment of RCC.
Data from ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation
Authors: Stiff Tom , O’Driscoll Mark , Rief Nicole , Iwabuchi Kuniyoshi , Löbrich Markus , Jeggo Penny A.
Published: March 2023

Abstract
H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.
Data from An Msh2 Point Mutation Uncouples DNA Mismatch Repair and Apoptosis
Authors: Lin Diana P. , Wang Yuxun , Scherer Stefan J. , Clark Alan B. , Yang Kan , Avdievich Elena , Jin Bo , Werling Uwe , Parris Tchaiko , Kurihara Naoto , Umar Asad , Kucherlapati Raju , Lipkin Martin , Kunkel Thomas A. , Edelmann Winfried
Published: March 2023

Abstract
Mutations in the human DNA mismatch repair gene MSH2 are associated with hereditary nonpolyposis colorectal cancer as well as a significant proportion of sporadic colorectal cancer. The inactivation of MSH2 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of chemotherapeutic agents. Here we show that the DNA repair and DNA damage-induced apoptosis functions of Msh2 can be uncoupled using mice that carry the G674A missense mutation in the conserved ATPase domain. As a consequence, although Msh2G674A homozygous mutant mice are highly tumor prone, the onset of tumorigenesis is delayed as compared with Msh2-null mice. In addition, tumors that carry the mutant allele remain responsive to treatment with a chemotherapeutic agent. Our results indicate that Msh2-mediated apoptosis is an important component of tumor suppression and that certain MSH2 missense mutations can cause mismatch repair deficiency while retaining the signaling functions that confer sensitivity to chemotherapeutic agents.
Data from Mechanistic and Predictive Profiling of 5-Fluorouracil Resistance in Human Cancer Cells
Authors: Wang Weiguang , Cassidy James , O’Brien Vincent , Ryan Kevin M. , Collie-Duguid Elaina
Published: March 2023

Abstract
Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor κB (NFκB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFκB DNA-binding activity. Cotransfection of NFκB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFκB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G1-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G1, and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.
Data from BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells
Authors: Shellenberger Thomas D. , Wang Mary , Gujrati Manu , Jayakumar Arumugam , Strieter Robert M. , Burdick Marie D. , Ioannides Constantin G. , Efferson Clayton L. , El-Naggar Adel K. , Roberts Dianna , Clayman Gary L. , Frederick Mitchell J.
Published: March 2023

Abstract
BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.
Data from Simultaneous Suppression of Epidermal Growth Factor Receptor and c-erbB-2 Reverses Aneuploidy and Malignant Phenotype of a Human Ovarian Carcinoma Cell Line
Authors: Pack Svetlana D. , Alper Özgül M. , Stromberg Kurt , Augustus Meena , Özdemirli Metin , Miermont Anne M. , Klus Greg , Rusin Marek , Slack Rebecca , Hacker Neville F. , Ried Thomas , Szallasi Zoltan , Alper Özge
Published: March 2023

Abstract
Coexpression of epidermal growth factor receptor (EGFR) and c-erbB-2 in 47–68% of ovarian cancer cells indicate their strong association with tumor formation. We examined the effects of simultaneous antisense- or immunosuppression of EGFR and c-erbB-2 expression on the invasive phenotype, aneuploidy, and genotype of cultured human ovarian carcinoma cells (NIH:OVCAR-8). We report here that suppression of both EGFR and c-erbB-2 results in regression of aneuploidy and genomic imbalances in NIH:OVCAR-8 cells, restores a more normal phenotype, and results in a more normal gene expression profile. Combined with cytogenetic analysis, our data demonstrate that the regression of aneuploidy is due to the selective apoptosis of double antisense transfected cells with highly abnormal karyotype.
Data from Activated Signal Transducer and Activator of Transcription (STAT) 3
Authors: Silver Debra L. , Naora Honami , Liu Jinsong , Cheng Wenjun , Montell Denise J.
Published: March 2023

Abstract
Constitutive activation of the Janus-activated kinase/signal transducer and activator of transcription (STAT) pathway promotes the proliferation and survival of cancer cells in culture and is associated with various cancers, including those of the ovary. We found that constitutively activated STAT3 levels correlated with aggressive clinical behavior of ovarian carcinoma specimens. Furthermore, inhibition of STAT3 reduced the motility of ovarian cancer cells in vitro. Surprisingly, we found that activated STAT3 localized not only to nuclei but also to focal adhesions in these cells. Activated STAT3 coimmunoprecipitated with phosphorylated paxillin and focal adhesion kinase and required paxillin and Src for its localization to focal adhesions. These results suggest that Janus-activated kinase/STAT signaling may contribute to ovarian cancer cell invasiveness.
Data from JAGGED1 Expression Is Associated with Prostate Cancer Metastasis and Recurrence
Authors: Santagata Sandro , Demichelis Francesca , Riva Alberto , Varambally Sooryanarayana , Hofer Matthias D. , Kutok Jeffery L. , Kim Robert , Tang Jeffery , Montie James E. , Chinnaiyan Arul M. , Rubin Mark A. , Aster Jon C.
Published: March 2023

Abstract
Recent studies suggest that NOTCH signaling can promote epithelial-mesenchymal transitions and augment signaling through AKT, an important growth and survival pathway in epithelial cells and prostate cancer in particular. Here we show that JAGGED1, a NOTCH receptor ligand, is significantly more highly expressed in metastatic prostate cancer as compared with localized prostate cancer or benign prostatic tissues, based on immunohistochemical analysis of JAGGED1 expression in human tumor samples from 154 men. Furthermore, high JAGGED1 expression in a subset of clinically localized tumors was significantly associated with recurrence, independent of other clinical parameters. These findings support a model in which dysregulation of JAGGED1 protein levels plays a role in prostate cancer progression and metastasis and suggest that JAGGED1 may be a useful marker in distinguishing indolent and aggressive prostate cancers.
Data from Inhibition of Chk1 by CEP-3891 Accelerates Mitotic Nuclear Fragmentation in Response to Ionizing Radiation
Authors: Syljuåsen Randi G. , Sørensen Claus Storgaard , Nylandsted Jesper , Lukas Claudia , Lukas Jiri , Bartek Jiri
Published: March 2023

Abstract
The human checkpoint kinase Chk1 has been suggested as a target for cancer treatment. Here, we show that a new inhibitor of Chk1 kinase, CEP-3891, efficiently abrogates both the ionizing radiation (IR)-induced S and G2 checkpoints. When the checkpoints were abrogated by CEP-3891, the majority (64%) of cells showed fragmented nuclei at 24 hours after IR (6 Gy). The formation of nuclear fragmentation in IR-treated human cancer cells was directly visualized by time-lapse video microscopy of U2-OS cells expressing a green fluorescent protein-tagged histone H2B protein. Nuclear fragmentation occurred as a result of defective chromosome segregation when irradiated cells entered their first mitosis, either prematurely without S and G2 checkpoint arrest in the presence of CEP-3891 or after a prolonged S and G2 checkpoint arrest in the absence of CEP-3891. The nuclear fragmentation was clearly distinguishable from apoptosis because caspase activity and nuclear condensation were not induced. Finally, CEP-3891 not only accelerated IR-induced nuclear fragmentation, it also increased the overall cell killing after IR as measured in clonogenic survival assays. These results demonstrate that transient Chk1 inhibition by CEP-3891 allows premature mitotic entry of irradiated cells, thereby leading to accelerated onset of mitotic nuclear fragmentation and increased cell death.
Data from A Secreted Form of ADAM9 Promotes Carcinoma Invasion through Tumor-Stromal Interactions
Authors: Mazzocca Antonio , Coppari Roberto , De Franco Raffaella , Cho Je-Yoel , Libermann Towia A. , Pinzani Massimo , Toker Alex
Published: March 2023

Abstract
Tumor cell invasion is a process regulated by integrins, matrix-degrading enzymes, and interactions with host tissue stromal cells. The ADAM family of proteins plays an important role in modulating various cellular responses. Here, we show that an alternatively spliced variant of ADAM9 is secreted by hepatic stellate cells and promotes carcinoma invasion. ADAM9-S induced a highly invasive phenotype in several human tumor cell lines in Matrigel assays, and the protease activity of ADAM9-S was required for invasion. ADAM9-S binds directly to α6β4 and α2β1 integrins on the surface of colon carcinoma cells through the disintegrin domain. ADAM9-S was also able to cleave laminin and promote invasion. Analysis of human liver metastases revealed that ADAM9 is expressed by stromal liver myofibroblasts, particularly those that are localized within the tumor stroma at the invasive front. These results emphasize the importance of tumor-stromal interactions in invasion and suggest that ADAM9-S can be an important determinant in the ability of cancer cells to invade and colonize the liver.
Data from Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway by Inositol Pentakisphosphate Results in Antiangiogenic and Antitumor Effects
Authors: Maffucci Tania , Piccolo Enza , Cumashi Albana , Iezzi Manuela , Riley Andrew M. , Saiardi Adolfo , Godage H. Yasmin , Rossi Cosmo , Broggini Massimo , Iacobelli Stefano , Potter Barry V.L. , Innocenti Paolo , Falasca Marco
Published: March 2023

Abstract
The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)–induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.
Data from A Hypoxia-Driven Vascular Endothelial Growth Factor/Flt1 Autocrine Loop Interacts with Hypoxia-Inducible Factor-1α through Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase 1/2 Pathway in Neuroblastoma
Authors: Das Bikul , Yeger Herman , Tsuchida Rika , Torkin Risa , Gee Matthew F.W. , Thorner Paul S. , Shibuya Masabumi , Malkin David , Baruchel Sylvain
Published: March 2023

Abstract
Flt1, an “fms-like tyrosine kinase” receptor, has been suggested to play an active role in vascular endothelial growth factor (VEGF)–mediated autocrine signaling of tumor growth and angiogenesis. Here, we used a neuroblastoma model to investigate the role of VEGF/Flt1 signaling in hypoxia-mediated tumor cell survival, drug resistance, and in vivo angiogenesis. SK-N-BE(2), a highly malignant neuroblastoma cell line resistant to hypoxia-induced apoptosis expresses active Flt1 but lacks VEGFR2 expression. We found that 24-hour hypoxia (<0.1% O2) alone (no serum deprivation) showed sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) associated with bcl-2 up-regulation and resistance to etoposide-induced (5 μmol/L) apoptosis. Treatment with anti-VEGF and anti-Flt1 antibodies inhibited ERK1/2 activation, down-regulated bcl-2, and reversed the hypoxia-mediated drug resistance to etoposide. Similar results were obtained with U0126 and ursolic acid, specific and nonspecific inhibitors of ERK1/2, respectively. We confirmed the protective role of Flt1 receptor by small interfering RNA knockout and Flt1 overexpression studies. Subsequently, we found that inhibition of VEGF/Flt1 autocrine signaling led to reduced hypoxia-inducible factor-1α (HIF-1α) phosphorylation. Furthermore, the reduced phosphorylation was associated with down-regulation of basic fibroblast growth factor, a downstream target of the HIF-1α and VEGF pathways. Our findings suggested an expanded autocrine loop between VEGF/Flt1 signaling and HIF-1α. We investigated the angiogenic activity of the loop in an in vivo Matrigel plug assay. The hypoxia-treated conditioned medium induced a strong angiogenic response, as well as the cooption of surrounding vessels into the plugs; ursolic acid inhibited the angiogenesis process. We also found that three other Flt1-expressing neuroblastoma cell lines show hypoxia-mediated drug resistance to etoposide, melphalan, doxorubicin, and cyclophosphamide. Taken together, we conclude that a hypoxia-driven VEGF/Flt1 autocrine loop interacts with HIF-1α through a mitogen-activated protein kinase/ERK1/2 pathway in neuroblastoma. The interaction, in the form of an autocrine loop, is required for the hypoxia-driven cell survival, drug resistance, and angiogenesis in neuroblastoma.
Data from p300 Regulates Androgen Receptor–Independent Expression of Prostate-Specific Antigen in Prostate Cancer Cells Treated Chronically with Interleukin-6
Authors: Debes Jose D. , Comuzzi Barbara , Schmidt Lucy J. , Dehm Scott M. , Culig Zoran , Tindall Donald J.
Published: March 2023

Abstract
Prostate cancer is the most frequent non–skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor–dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6–treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.
Data from Identification of Aberrantly Methylated Genes in Association with Adult T-Cell Leukemia
Authors: Yasunaga Jun-ichirou , Taniguchi Yuko , Nosaka Kisato , Yoshida Mika , Satou Yorifumi , Sakai Tatsunori , Mitsuya Hiroaki , Matsuoka Masao
Published: March 2023

Abstract
In this study, we identified 53 aberrantly hypermethylated DNA sequences in adult T-cell leukemia (ATL) cells using methylated CpG island amplification/representational difference analysis method. We also observed a proportionate increase in the methylation density of these regions with disease progression. Seven genes, which were expressed in normal T cells, but suppressed in ATL cells, were identified near the hypermethylated regions. Among these silenced genes, Kruppel-like factor 4 (KLF4) gene is a cell cycle regulator and early growth response 3 (EGR3) gene is a critical transcriptional factor for induction of Fas ligand (FasL) expression. Treatment with 5-aza-2′-deoxycytidine resulted in the recovery of their transcription, indicating that their silencing might be associated with DNA hypermethylation. To study their functions in ATL cells, we transfected recombinant adenovirus vectors expressing KLF4 and EGR3 genes. Expression of KLF4 induced apoptosis of ATL cells whereas enforced expression of EGR3 induced the expression of FasL gene, resulting in apoptosis. Thus, suppressed expression of EGR3 enabled ATL cells to escape from activation-induced cell death mediated by FasL. Our results showed that the methylated CpG island amplification/representational difference analysis method allowed the isolation of hypermethylated DNA regions specific to leukemic cells and thus shed light on the roles of DNA methylation in leukemogenesis.
Data from Characterization of a Carcinogenesis Rat Model of Ovarian Preneoplasia and Neoplasia
Authors: Stewart Sherri L. , Querec Troy D. , Ochman Alexander R. , Gruver Briana N. , Bao Rudi , Babb James S. , Wong Thang S. , Koutroukides Theodoros , Pinnola Aaron D. , Klein-Szanto Andres , Hamilton Thomas C. , Patriotis Christos
Published: March 2023

Abstract
Animal models of ovarian cancer are crucial for understanding the pathogenesis of the disease and for testing new treatment strategies. A model of ovarian carcinogenesis in the rat was modified and improved to yield ovarian preneoplastic and neoplastic lesions that pathogenetically resemble human ovarian cancer. A significantly lower dose (2 to 5 μg per ovary) of 7,12-dimethylbenz(a)anthracene (DMBA) was applied to the one ovary to maximally preserve its structural integrity. DMBA-induced mutagenesis was additionally combined with repetitive gonadotropin hormone stimulation to induce multiple cycles of active proliferation of the ovarian surface epithelium. Animals were treated in three arms of different doses of DMBA alone or followed by hormone administration. Comparison of the DMBA-treated ovaries with the contralateral control organs revealed the presence of epithelial cell origin lesions at morphologically distinct stages of preneoplasia and neoplasia. Their histopathology and path of dissemination to other organs are very similar to human ovarian cancer. Hormone cotreatment led to an increased lesion severity, indicating that gonadotropins may promote ovarian cancer progression. Point mutations in the Tp53 and Ki-Ras genes were detected that are also characteristic of human ovarian carcinomas. Additionally, an overexpression of estrogen and progesterone receptors was observed in preneoplastic and early neoplastic lesions, suggesting a role of these receptors in ovarian cancer development. These data indicate that this DMBA animal model gives rise to ovarian lesions that closely resemble human ovarian cancer and it is adequate for additional studies on the mechanisms of the disease and its clinical management.
Data from Proteasome Inhibitors Trigger NOXA-Mediated Apoptosis in Melanoma and Myeloma Cells
Authors: Qin Jian-Zhong , Ziffra Jeffrey , Stennett Lawrence , Bodner Barbara , Bonish Brian K. , Chaturvedi Vijaya , Bennett Frank , Pollock Pamela M. , Trent Jeffrey M. , Hendrix Mary J.C. , Rizzo Paola , Miele Lucio , Nickoloff Brian J.
Published: March 2023

Abstract
Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration–approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.
Data from A Prostate-Specific Membrane Antigen-Targeted Monoclonal Antibody–Chemotherapeutic Conjugate Designed for the Treatment of Prostate Cancer
Authors: Henry Michael D. , Wen Shenghua , Silva Matthew D. , Chandra Sudeep , Milton Mark , Worland Peter J.
Published: March 2023

Abstract
MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane antigen (PSMA). PSMA is a transmembrane receptor whose expression is largely restricted to prostatic epithelium and prostate cancer cells with its expression level increasing during the progression of malignancy. MLN2704 consists of a de-immunized, monoclonal antibody that is specific for PSMA conjugated to drug maytansinoid 1 (DM1), a microtubule-depolymerizing compound. After antibody binding to PSMA and the subsequent cellular internalization of this complex, DM1 is released leading to cell death. MLN2704 has an approximate half-life of 39 hours in scid mice bearing CWR22 tumor tissue, and the antibody effectively penetrates xenograft tumor tissue. Optimization of dosage and schedule of MLN2704 administration defined interdependency between these conditions that maximized efficacy with no apparent toxicity. Tumor growth delays of ∼100 days could be achieved on the optimized schedule of one dose of 60 mg/kg MLN2704 every 14 days for five doses (q14d×5). The unconjugated antibody (MLN591) demonstrated essentially no antitumor activity and DM1 alone or a non-PSMA targeted antibody–DM1 conjugate was only weakly active. Furthermore, we show that MLN2704 is active in a novel model of osteoblastic prostate cancer metastasis.
Data from BRCA1 Induces Antioxidant Gene Expression and Resistance to Oxidative Stress
Authors: Bae Insoo , Fan Saijun , Meng Qinghui , Rih Jeong Keun , Kim Hee Jong , Kang Hyo Jin , Xu Jingwen , Goldberg Itzhak D. , Jaiswal Anil K. , Rosen Eliot M.
Published: March 2023

Abstract
Mutations of the breast cancer susceptibility gene 1 (BRCA1), a tumor suppressor, confer an increased risk for breast, ovarian, and prostate cancers. To investigate the function of the BRCA1 gene, we performed DNA microarray and confirmatory reverse transcription-PCR analyses to identify BRCA1-regulated gene expression changes. We found that BRCA1 up-regulates the expression of multiple genes involved in the cytoprotective antioxidant response, including glutathione S-transferases, oxidoreductases, and other antioxidant genes. Consistent with these findings, BRCA1 overexpression conferred resistance while BRCA1 deficiency conferred sensitivity to several different oxidizing agents (hydrogen peroxide and paraquat). In addition, in the setting of oxidative stress (due to hydrogen peroxide), BRCA1 shifted the cellular redox balance to a higher ratio of reduced to oxidized glutathione. Finally, BRCA1 stimulated antioxidant response element-driven transcriptional activity and enhanced the activity of the antioxidant response transcription factor nuclear factor erythroid-derived 2 like 2 [also called NRF2 (NFE2L2)]. The ability of BRCA1 to stimulate antioxidant response element-dependent transcription and to protect cells against oxidative stress was attenuated by inhibition of nuclear factor erythroid-derived 2 like 2. These findings suggest a novel function for BRCA1, i.e., to protect cells against oxidative stress. This function would be consistent with the postulated role of BRCA1 as a caretaker gene in preserving genomic integrity.