bluuebunny/crossref_metadata_2025 huggingface dataset:
Matrix Metalloproteinase-2 Contributes to Cancer Cell Migration on Collagen
Authors: Xu Xiaoping , Wang Yao , Chen Zhihua , Sternlicht Mark D. , Hidalgo Manuel , Steffensen Bjorn
Published: June 2022

Matrix metalloproteinases (MMP) are central to tissue penetration by cancer cells, as tumors expand and form metastases, but the mechanism by which MMP-2 contributes to cancer cell migration is not well understood. In the present experiments, both a broad-spectrum MMP inhibitor and the isolated collagen binding domain (CBD) from MMP-2 inhibited cell migration on native type I collagen. These results verified the involvement of MMPs in general and showed that MMP-2, specifically, contributes to cell migration by a mechanism involving MMP-2 interaction with collagen. To exclude potential overlapping effects of MMP-9, additional experiments showed that MMP-2 also contributed to migration of MMP-9−/− cells. To investigate whether the homologous CBD from human fibronectin also inhibited cell migration, we first showed that fragmentation of fibronectin is a feature of breast cancer tumors and that several fragments contained the CBD. However, the recombinant fibronectin domain did not alter cell migration on collagen. This lack of effect on cell migration was explored in competitive protein-protein binding assays, which showed that the affinity of MMP-2 for collagen exceeds that of fibronectin. Furthermore, whereas the isolated MMP-2 CBD inhibited the gelatinolytic activities of MMP-2 and tumor extracts, such an inhibition was not characteristic of the corresponding fibronectin domain. Together, our results provide evidence that MMP-2 is an important determinant of cancer cell behavior but is not inhibited by the collagen binding segment of fibronectin.
Transcriptional Profiles of Intestinal Tumors in Apc
Min Mice are Unique from those of Embryonic Intestine and Identify Novel Gene Targets Dysregulated in Human Colorectal Tumors
Authors: Reichling Tim , Goss Kathleen Heppner , Carson Daniel J. , Holdcraft Robert W. , Ley-Ebert Cathy , Witte Dave , Aronow Bruce J. , Groden Joanna
Published: June 2022

The adenomatous polyposis coli (APC) tumor suppressor is a major regulator of the Wnt signaling pathway in normal intestinal epithelium. APC, in conjunction with AXIN and GSK-3β, forms a complex necessary for the degradation of β-catenin, thereby preventing β-catenin/T-cell factor interaction and alteration of growth-controlling genes such as c-MYC and cyclin D1. Inappropriate activation of the Wnt pathway, via Apc/APC mutation, leads to gastrointestinal tumor formation in both the mouse and human. In order to discover novel genes that may contribute to tumor progression in the gastrointestinal tract, we used cDNA microarrays to identify 114 genes with altered levels of expression in ApcMin mouse adenomas from the duodenum, jejunum, and colon. Changes in the expression of 24 of these 114 genes were not observed during mouse development at embryonic day 16.5, postnatal day 1, or postnatal day 14 (relative to normal adult intestine). These 24 genes are not previously known Wnt targets. Seven genes were validated by real-time reverse transcription-PCR analysis, whereas four genes were validated by in situ hybridization to mouse adenomas. Real-time reverse transcription-PCR analysis of human colorectal cancer cell lines and adenocarcinomas revealed that altered expression levels were also observed for six of the genes Igfbp5, Lcn2, Ly6d, N4wbp4 (PMEPA1), S100c, and Sox4.
cIAP1 Localizes to the Nuclear Compartment and Modulates the Cell Cycle
Authors: Samuel Temesgen , Okada Kazuya , Hyer Marc , Welsh Kate , Zapata Juan M. , Reed John C.
Published: June 2022

We explored the location and function of the human cIAP1 protein, a member of the inhibitor of apoptosis protein (IAP) family. Unlike family member X-linked IAP (XIAP), which was predominantly cytoplasmic, the cIAP1 protein localized almost exclusively to nuclei in cells, as determined by immunofluorescence microscopy and subcellular fractionation methods. Interestingly, apoptotic stimuli induced nuclear export of cIAP1, which was blocked by a chemical caspase inhibitor. In dividing cells, cIAP1 was released into the cytosol early in mitosis, then reaccumulated in nuclei in late anaphase and in telophase, with the exception of a pool of cIAP1 that associated with the midbody. Survivin, another IAP family member, and cIAP1 were both localized on midbody microtubules at telophase, and also interacted with each other during mitosis. Cells stably overexpressing cIAP1 accumulated in G2-M phase and grew slower than control-transfected cells. These cIAP1-overexpressing cells also exhibited cytokinesis defects over 10 times more often than control cells and displayed a mitotic checkpoint abnormality with production of polyploid cells when exposed to microtubule-targeting drugs nocodazole and paclitaxel (Taxol). Our findings demonstrate a role for overexpressed cIAP1 in genetic instability, possibly by interfering with mitotic functions of Survivin. These findings may have important implications for cancers in which cIAP1 overexpression occurs.
Identification of Brain-Derived Neurotrophic Factor as a Novel Functional Protein in Hepatocellular Carcinoma
Authors: Yang Zhen Fan , Ho David W. , Lam Chi Tat , Luk John M. , Lum Ching Tung , Yu Wan Ching , Poon Ronnie T. , Fan Sheung Tat
Published: June 2022

This study aims to identify a novel molecule that may contribute to hepatocarcinogenesis in a rat orthotopic hepatocellular carcinoma model. The hepatocellular carcinoma model was generated by injection of tumor cells into the left lobe of the liver. Proteomic approaches, including ProteinChip and two-dimensional electrophoresis, were used to identify proteins from serially collected rat serum samples. By both ProteinChip and two-dimensional electrophoresis techniques, the level of a 27-kDa protein was found to be augmented in serum samples during tumor development, decreased after left lobectomy, and reincreased at the time of tumor recurrence. The protein was identified to be brain-derived neurotrophic factor (BDNF). By using specific primers and monoclonal antibody, the expression pattern of BDNF was confirmed in tumor tissue but not in the adjacent nontumorous liver tissue. In addition, the truncated isoform of BDNF receptor-tyrosine protein kinase receptor B was only found in tumor tissue. An in vitro study showed that exogenous BDNF could induce tumor cell proliferation predominantly in relatively small numbers of inoculated cells. Administration of BDNF to tumor cell lines induced significantly increased expression of heat shock protein 90 (Hsp90) and cyclin D1, and blocking the activity of Hsp90 could reverse the up-regulation of cyclin D1 induced by BDNF. The present study revealed that BDNF and its receptor were uniquely expressed in tumor tissue and cell lines of hepatocellular carcimona but not in nontumorous liver tissue and normal cell line. BDNF could stimulate tumor cell proliferation in a Hsp90-dependent manner.
A Genome-Wide View of the In vitro Response to l-Asparaginase in Acute Lymphoblastic Leukemia
Authors: Fine Bernard M. , Kaspers Gertjan J.L. , Ho Minh , Loonen Anne H. , Boxer Linda M.
Published: June 2022

To investigate the effect of l-asparaginase on acute lymphoblastic leukemia (ALL), we used cDNA microarrays to obtain a genome-wide view of gene expression both at baseline and after in vitro exposure to l-asparaginase in cell lines and pediatric ALL samples. In 16 cell lines, a baseline gene expression pattern distinguished l-asparaginase sensitivity from resistance. However, for 28 pediatric ALL samples, no consistent baseline expression pattern was associated with sensitivity to l-asparaginase. In particular, baseline expression of asparagine synthetase (ASNS) was not predictive of response to l-asparaginase. After exposure to l-asparaginase, 5 cell lines and 10 clinical samples exhibited very similar changes in the expression of a large number of genes. However, the gene expression changes occurred more slowly in the clinical samples. These changes included a consistent increase in expression of tRNA synthetases and solute transporters and activating transcription factor and CCAAT/enhancer binding protein family members, a response similar to that observed with amino acid starvation. There was also a consistent decrease in many genes associated with proliferation. Taken together, the changes seem to reflect a consistent coordinated response to asparagine starvation in both cell lines and clinical samples. Importantly, in the clinical samples, increased expression of ASNS after l-asparaginase exposure was not associated with in vitro resistance to l-asparaginase, indicating that ASNS-independent mechanisms of in vitro l-asparaginase resistance are common in ALL. These results suggest that targeting particular genes involved in the response to amino acid starvation in ALL cells may provide a novel way to overcome l-asparaginase resistance.
Androgens Repress the Expression of the Angiogenesis Inhibitor Thrombospondin-1 in Normal and Neoplastic Prostate
Authors: Colombel Marc , Filleur Stéphanie , Fournier Pierick , Merle Carole , Guglielmi Julien , Courtin Aurélie , Degeorges Armelle , Serre Claire Marie , Bouvier Raymonde , Clézardin Philippe , Cabon Florence
Published: June 2022

In order to understand why the angiogenesis inhibitor thrombospondin-1 (TSP1) is often, although not always, associated with prostatic tumors, we have investigated its relationship with the testosterone and the vasculature on which both normal and tumorigenic prostatic epithelia depend. In vivo, androgen withdrawal led to increased TSP1 production and decreased vascularization in the normal rat prostate which was reversed by androgen replacement. Androgen repression of TSP1 production occurred at the transcriptional level and was dependent on the presence of the first intron of the TSP1 gene. In an experimental model of prostate tumorigenesis, TSP1, when delivered by admixed stromal fibroblasts, markedly delayed LNCaP tumor growth and limited tumor vascularization. However, prolonged exposure to TSP1 resulted in the growth of tumors secreting high levels of vascular endothelial growth factor in the bloodstream of tumor-bearing animals and tumor growth was no longer sensitive to TSP1 inhibitory effects. Clinical evidence also suggested that prostate carcinomas are able to adapt to escape the antiangiogenic effects of TSP1. In human androgen–dependent localized prostate carcinomas, TSP1 expression was inversely correlated with blood vessel density. Androgen deprivation in patients with hormone-responsive tumors led to increased TSP1 expression and vascular regression. In contrast, despite a sustained expression in the tumor bed, TSP1 was no longer associated with decreased vascularization in hormone-refractory prostate tumors. Overall, these results suggest that the high in situ TSP1 exposure triggered by androgen deprivation in patients with prostate cancer could lead to early tumor resistance. Such patients could benefit from a combination of androgen deprivation and antiangiogenic therapy in order to minimize the induction of such tumor escape.
Global Analysis of Altered Gene Expressions during the Process of Esophageal Squamous Cell Carcinogenesis in the Rat: A Study Combined with a Laser Microdissection and a cDNA Microarray
Authors: Nishida Koujiro , Mine Shinji , Utsunomiya Tohru , Inoue Hiroshi , Okamoto Masahiro , Udagawa Harushi , Hanai Taizo , Mori Masaki
Published: June 2022

The genetic alterations that occur during esophageal tumorigenesis have yet to be determined. We previously established a Wister rat carcinogenesis model of esophageal squamous cell carcinoma. To understand more about the molecular mechanisms during carcinogenesis, we produced esophageal neoplastic lesions by administering N-amyl-N-methylnitrosamine and 12-O-tetradecanoylphorbol-13-acetate to rats. We used laser microdissection to specifically isolate the cells from the normal epithelium, papilloma, dysplasia, and invasive carcinoma. Using a cDNA microarray representing 14,815 clones, we then analyzed the gene expression profiles for each esophageal lesion. The number of differentially expressed genes compared with the normal control dramatically increased in a step-by-step fashion from normal epithelium (1,151 ± 119 genes) to papilloma (1,899 ± 543 genes), dysplasia (1,991 ± 193 genes), and invasive carcinoma (2,756 ± 87 genes). A hierarchical clustering analysis showed that the three stages of normal epithelium, dysplasia (papilloma), and invasive carcinoma could be clearly classified, whereas the gene expression patterns of papilloma and dysplasia were indistinguishable. Using the Fisher criterion, we also identified 50 genes whose expression level had either significantly increased or decreased in a step-by-step manner from the normal epithelium to dysplasia and then finally to invasive carcinoma. Many of these genes were not previously known to be associated with esophageal carcinogenesis. The present findings in our rat model thus seem to provide us with a better understanding of the molecular alterations that occur during esophageal carcinogenesis and hopefully will also help lead to the development of novel diagnostic and therapeutic targets.
Mutations in Apc and p53 Synergize to Promote Mammary Neoplasia
Authors: Méniel Valérie , Hay Trevor , Douglas-Jones Anthony , Sansom Owen J. , Clarke Alan R.
Published: June 2022

Mutations of Apc and p53 have both been implicated in human and murine mammary neoplasia. To investigate potential interactions between Apc and p53, we conditionally inactivated Apc in both the presence and the absence of functional p53. Apc deficiency on its own leads to the development of metaplasia but not neoplasia. We show here that these areas of metaplasia are characterized by elevated levels of both p53 and p21. In the additional absence of p53,there is rapid progression to neoplasia, with 44.4% of lymphoma-free mice developing a mammary tumor with earliest observed onset at pregnancy. To investigate the mechanism by which p53 deficiency accelerates neoplasia, we used the Rosa26R reporter strain as a marker of Cre-mediated recombination and show a role for p53 in the loss of Apc-deficient cells. This role seems limited to pregnancy and subsequent time points. We therefore show clear synergy between these two mutations in mammary gland neoplasia and present data to suggest that at least one mechanism for this acceleration is the p53-dependent loss of Apc-deficient cells.
Candidate Genes in Breast Cancer Revealed by Microarray-Based Comparative Genomic Hybridization of Archived Tissue
Authors: Nessling Michelle , Richter Karsten , Schwaenen Carsten , Roerig Peter , Wrobel Gunnar , Wessendorf Swen , Fritz Björn , Bentz Martin , Sinn Hans-Peter , Radlwimmer Bernhard , Lichter Peter
Published: June 2022

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.
Decreased Accessibility and Lack of Activation of ErbB2 in JIMT-1, a Herceptin-Resistant, MUC4-Expressing Breast Cancer Cell Line
Authors: Nagy Peter , Friedländer Elza , Tanner Minna , Kapanen Anita I. , Carraway Kermit L. , Isola Jorma , Jovin Thomas M.
Published: June 2022

Overexpression of erbB2 in breast tumors is associated with poor prognosis and is a target of receptor-oriented cancer therapy. Trastuzumab (Herceptin), a monoclonal antibody against a membrane-proximal epitope in the extracellular region of erbB2, shows a therapeutic effect against a fraction of erbB2-amplified breast tumors. Unfortunately, resistance to Herceptin is common, and its cause is as yet unclear. Here we investigated the properties of erbB2 in a Herceptin-resistant cell line, JIMT-1, established from a breast cancer patient showing erbB2 gene amplification and primary resistance to Herceptin. The expression profile of erbB proteins, Herceptin-induced erbB2 internalization, and down-regulation in JIMT-1 were similar to those in Herceptin-sensitive lines. However, the mean number of Herceptin Mab binding sites in JIMT-1 was 1/5 that of the expressed erbB2 molecules, although 5% to 10% of the cells showed a ∼10-fold higher Herceptin binding than the main population. Herceptin Fab and Mab 2C4, an antibody binding to an epitope in the ectodomain further removed from the membrane, bound more efficiently to JIMT-1 cells than Herceptin Mab, implying that erbB2 was partly masked. The expression of MUC4, a membrane-associated mucin that according to reports contributes to the masking of membrane proteins, was higher in JIMT-1 than in Herceptin-sensitive lines, and its level was inversely correlated with the Herceptin binding capacity of single cells. Knockdown of MUC4 expression by RNA interference increased the binding of Herceptin. Western blotting showed a low level of proteolytic processing, shedding, and tyrosine phosphorylation of erbB2 in JIMT-1. The latter finding may explain its Herceptin-resistant phenotype characterizing both the low and high Herceptin binding subpopulations. We conclude that masking of erbB2 in JIMT-1 leads to diminished Herceptin binding and isolation of erbB2 from its normal interaction and activation partners.
The COOH-terminal End of R-Ras Alters the Motility and Morphology of Breast Epithelial Cells through Rho/Rho-Kinase
Authors: Jeong Ha-Won , Nam Ju-Ock , Kim In-San
Published: June 2022

R-Ras has a high degree of sequence homology with Ras and other members of the Ras subfamily, including Rap, TC21, and M-Ras. Although R-Ras has been suggested to regulate cell adhesion, migration, and invasion, the biological mechanism has not been well assessed. In this report, we show that constitutively active R-Ras (38V) induces a more rounded cell shape and redistribution of focal adhesion, and enhances the phosphorylation of focal adhesion kinase and paxillin. Active R-Ras (38V) induces cell adhesion to type I collagen, but inhibits cell motility. In active R-Ras (38V) cells, the activity of RhoA is increased and accompanied with translocation to plasma membrane, but not that of Rac1 or Cdc42. In parallel, dominant-negative RhoA (N19RhoA) and Y27632, a specific inhibitor of Rho-associated kinase, dramatically reverse the rounded cell morphology to a spread cell shape and enhance motility. Furthermore, coincident with the formation of cortical actin filaments in active R-Ras (38V) cells, myosin light chain and Ser-19-phosphorylated myosin light chain mainly accumulate at the peripheral region, which is inhibited by the treatment of Y27632. Using H-Ras/R-Ras and R-Ras/H-Ras hybrid constructs, we show that the COOH-terminal region of R-Ras contains the specific signal for inducing changes in motility and morphology. Our results suggest that R-Ras in breast epithelial cells disrupts cell polarity and motility through the Rho/Rho–associated kinase pathway triggered by a signal from the COOH-terminal end of R-Ras.
Estrogen-Mediated Post transcriptional Down-regulation of Breast Cancer Resistance Protein/ABCG2
Authors: Imai Yasuo , Ishikawa Etsuko , Asada Sakiyo , Sugimoto Yoshikazu
Published: June 2022

Breast cancer resistance protein (BCRP)/ABCG2 mediates concurrent resistance to chemotherapeutic agents, such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan, by pumping them out of cells. We previously reported that BCRP transports sulfated estrogens. In the present study, we show that at physiologic levels, estrogens markedly decrease endogenous BCRP expression in the estrogen-responsive and estrogen receptor α (ERα)–positive human breast cancer MCF-7 cells, but not in estrogen-nonresponsive human cancer cells. 17 β-Estradiol (E2) also significantly reduces exogenous BCRP expression, driven by a constitutive promoter, in BCRP-transduced estrogen-responsive and ERα-positive MCF-7 (MCF-7/BCRP) and T-47D cells, but not in BCRP-transduced estrogen-nonresponsive MDA-MB-231 and SKOV-3 cells. E2 potentiates the cytotoxicity of SN-38, but not vincristine, in MCF-7/BCRP cells significantly, and increases cellular topotecan uptake in MCF-7 and MCF-7/BCRP cells. Antiestrogen tamoxifen partially reverses E2-mediated BCRP down-regulation in MCF-7 and MCF-7/BCRP cells and treatment of MCF-7/BCRP cells with an ERα small interfering RNA abolished E2-mediated BCRP down-regulation, suggesting that interaction of E2 and ERα is necessary for BCRP down-regulation. E2 does not affect endogenous BCRP mRNA levels in MCF-7 cells or exogenous BCRP mRNA levels in MCF-7/BCRP cells. The results from pulse-chase labeling experiments with MCF-7/BCRP cells suggest that decreased protein biosynthesis and maturation, but not alterations in protein turnover, might underlie E2-mediated BCRP down-regulation. These data indicate that estrogen down-regulates BCRP expression by novel posttranscriptional mechanisms. This is the first report of small molecules that can affect BCRP protein expression in cells and may therefore assist in establishing new strategies for regulating BCRP expression.
Identification of a Novel Small-Molecule Inhibitor of the Hypoxia-Inducible Factor 1 Pathway
Authors: Tan Chalet , de Noronha Rita G. , Roecker Anthony J. , Pyrzynska Beata , Khwaja Fatima , Zhang Zhaobin , Zhang Huanchun , Teng Quincy , Nicholson Ainsley C. , Giannakakou Paraskevi , Zhou Wei , Olson Jeffrey J. , Pereira M. Manuela , Nicolaou K.C. , Van Meir Erwin G.
Published: June 2022

Hypoxia-inducible factor 1 (HIF-1) is the central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. Inhibition of HIF-1 is expected to result in the attenuation of hypoxia-inducible genes, which are vital to many aspects of tumor biology, including adaptative responses for survival under anaerobic conditions. To identify small molecules inhibiting the HIF-1 pathway, we did a biological screen on a 10,000-membered natural product-like combinatorial library. The compounds of the library, which share a 2,2-dimethylbenzopyran structural motif, were tested for their ability to inhibit the hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxia-responsive elements in human glioma cells. This effort led to the discovery of 103D5R, a novel small-molecule inhibitor of HIF-1α. 103D5R markedly decreased HIF-1α protein levels induced by hypoxia or cobaltous ions in a dose- and time-dependent manner, whereas minimally affecting global cellular protein expression levels, including that of control proteins such as HIF-1β, IκBα, and β-actin. The inhibitory activity of 103D5R against HIF-1α was clearly shown under normoxia and hypoxia in cells derived from different cancer types, including glioma, prostate, and breast cancers. This inhibition prevented the activation of HIF-1 target genes under hypoxia such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). Investigations into the molecular mechanism showed that 103D5R strongly reduced HIF-1α protein synthesis, whereas HIF-1α mRNA levels and HIF-1α degradation were not affected. 103D5R inhibited the phosphorylation of Akt, Erk1/2, and stress-activated protein kinase/c-jun-NH2-kinase, without changing the total levels of these proteins. Further studies on the mechanism of action of 103D5R will likely provide new insights into its validity/applicability for the pharmacologic targeting of HIF-1α for therapeutic purposes.
Immunogenicity without Immunoselection: A Mutant but Functional Antioxidant Enzyme Retained in a Human Metastatic Melanoma and Targeted by CD8+ T Cells with a Memory Phenotype
Authors: Sensi Marialuisa , Nicolini Gabriella , Zanon Marina , Colombo Chiara , Molla Alessandra , Bersani Ilaria , Lupetti Raffaella , Parmiani Giorgio , Anichini Andrea
Published: June 2022

Human melanomas can express unique tumor antigens, resulting from mutated proteins, and shared epitopes encoded for by normal genes, but these two classes of antigens have not been previously compared for immunogenicity and retention in metastatic cells. Here, we identified a new unique antigen generated by a point mutation in the peroxiredoxin 5 (Prdx5) gene in an HLA-A*0201+ human metastatic melanoma lacking the wild-type allele. An antioxidant assay, with recombinant Prdx5 proteins, and evaluation of peroxide accumulation in transiently transfected cells, indicated that the mutant protein retained its enzymatic activity. The mutation in the Prdx5 protein did not generate a new HLA agretope but yielded an HLA-A*0201–restricted T cell epitope (Prdx5110-119). By HLA-tetramer analysis, in a tumor-invaded lymph node, >50% of mutant Prdx5-specific CD8+ T cells (frequency 0.37%/CD8+) showed a CCR7+/− CD45RA− “TCM” or “TEM” phenotype, as found in Melan-A/MART-1–specific T cells (frequency 0.68%/CD8+) in the same tissue. In agreement with their memory phenotype, the Prdx5-specific T cells readily expanded in vitro in mixed lymphocyte-tumor culture, as did the Melan-/MART-1–specific T cells. By immunohistochemistry of the invaded lymph node, the mutant Prdx5 protein was expressed in all neoplastic cells, in contrast with the heterogeneous expression of shared antigens as Melan-A/MART-1, gp100 and tyrosinase. Thus, a unique tumor antigen can be as immunogenic as the melanoma differentiation antigens but, in contrast to the latter, may be retained in all metastatic cells possibly as result of the relevant cellular function exerted by the mutated protein.
Endogenous Anti-HER2 Antibodies Block HER2 Phosphorylation and Signaling through Extracellular Signal-Regulated Kinase
Authors: Montgomery R. Bruce , Makary Ekram , Schiffman Kathy , Goodell Vivian , Disis Mary L.
Published: June 2022

Immunologic targeting of the oncoprotein HER2/neu with monoclonal antibodies is an important component of current therapeutic strategies for patients with locally and systemically advanced breast cancer. Engineered antibodies targeting HER2 may have agonist or antagonist effects on HER2, but little is known about whether endogenous antibodies modulate HER2 activity. Vaccination of patients with HER2 peptides successfully induced antibodies in a minority of patients with HER2-expressing malignancy. A subset of antibodies specifically suppressed phosphorylation of HER2 on tyrosine Y1248, a residue critical for HER2 signaling through extracellular signal-regulated kinase. These antibodies also suppressed extracellular signal-regulated kinase phosphorylation and inhibited colony formation in soft agar. The majority of the antibodies that suppressed HER2 phosphorylation displayed specificity for amino acids 328 to 345 and 369 to 384. The isotype of anti-HER2 antibodies was predominantly IgG3 of low avidity, suggesting a Th1 response to peptide vaccine. Endogenous anti-HER2 antibodies can effectively suppress HER2 kinase activity and downstream signaling to inhibit the transformed phenotype of HER2-expressing tumor cells.
Levels of Cyclooxygenase-2 Are Increased in the Oral Mucosa of Smokers: Evidence for the Role of Epidermal Growth Factor Receptor and Its Ligands
Authors: Moraitis Dimitrios , Du Baoheng , De Lorenzo Mariana S. , Boyle Jay O. , Weksler Babette B. , Cohen Erik G. , Carew John F. , Altorki Nasser K. , Kopelovich Levy , Subbaramaiah Kotha , Dannenberg Andrew J.
Published: June 2022

Cyclooxygenase-2 (COX-2) is a promising pharmacologic target for preventing aerodigestive malignancies. In this study, we investigated the effects of tobacco smoke on the expression of COX-2 in oral mucosa. An ~4-fold increase in amount of COX-2 mRNA was observed in the oral mucosa of active smokers versus never smokers. Thus, a series of in vitro studies were carried out to elucidate the mechanism by which tobacco smoke induced COX-2. Treatment of a nontumorigenic oral epithelial cell line (MSK-Leuk1) with a saline extract of tobacco smoke (TS) stimulated COX-2 transcription, resulting in increased amounts of COX-2 mRNA, COX-2 protein, and prostaglandin E2 (PGE2) synthesis. Exposure of cells to TS also caused an increase in epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both an inhibitor of EGFR tyrosine kinase activity and a neutralizing anti-EGFR antibody blocked TS-mediated induction of COX-2. To define the mechanism by which TS activated EGFR, the release of amphiregulin and transforming growth factor α, two ligands of the EGFR, was measured. Exposure to TS caused a rapid increase in the release of both ligands. TS also markedly induced the expression of mRNAs for amphiregulin and transforming growth factor α. Importantly, increased expression of both ligands was also detected in the oral mucosa of active smokers. Taken together, these results suggest that activation of EGFR signaling contributes to the elevated levels of COX-2 found in the oral mucosa of smokers. Moreover, these findings strengthen the rationale for determining whether inhibitors of COX-2 or EGFR tyrosine kinase activity can reduce the risk of tobacco smoke–related malignancies of the aerodigestive tract.
N-Ras–Induced Growth Suppression of Myeloid Cells Is Mediated byIRF-1
Authors: Passioura Toby , Dolnikov Alla , Shen Sylvie , Symonds Geoff
Published: June 2022

Activating mutations in ras oncogenes occur at high frequency in human malignancies and expression of activated ras in immortalized cells lines is generally transforming. However, somewhat paradoxically, ectopic expression of ras in some myeloid cell lines has been shown to induce growth suppression associated with up-regulation of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 in a p16INK4a, p15INK4b, and p53 independent fashion. We have used cDNA array technology to compare the expression profile induced by activated N-ras (N-rasG13R) in growth-suppressed myeloid cells with that induced in myeloid cells, which are transformed by N-rasG13R. The expression profile induced in growth suppressed cells was consistent with differentiation and included the up-regulation of the transcription factor IFN regulatory factor-1 (IRF-1), a known transcriptional activator of p21CIP/WAF1 expression and a target of oncogenic mutations associated with myeloid leukemia. Antisense suppression of IRF-1 prevented N-rasG13R–associated growth arrest and up-regulation of p21CIP1/WAF1. These results define a novel tumor suppressive response to oncogenic signaling and provide a mechanistic link between growth suppression and differentiation in myeloid cells.
CASPASE-8 Gene Is Inactivated by Somatic Mutations in Gastric Carcinomas
Authors: Soung Young Hwa , Lee Jong Woo , Kim Su Young , Jang Jin , Park Yong Gyu , Park Won Sang , Nam Suk Woo , Lee Jung Young , Yoo Nam Jin , Lee Sug Hyung
Published: June 2022

Several lines of evidence indicate that deregulation of apoptosis is involved in the mechanisms of cancer development. Caspase-8 activation plays a central role in the initiation phase of apoptosis. The aim of this study was to explore the possibility that genetic alteration of CASPASE-8 gene is involved in the development of human cancers, including gastric cancers. We have analyzed the entire coding region of human CASPASE-8 gene for the detection of somatic mutations in 162 gastric carcinomas (40 early and 122 advanced cancers), 185 non–small cell lung cancers, 93 breast carcinomas, and 88 acute leukemias by PCR-single-strand conformation polymorphism. Of the cancers analyzed, 13 cancers harbored CASPASE-8 somatic mutations. Interestingly, all of the mutations were detected in the advanced gastric cancers (10.7% of the 122 samples). We expressed the tumor-derived caspase-8 mutants in 293T, 293, and HT1080 cells and found that most of the mutants (9 of the 10 mutations tested) markedly decreased the cell death activity of caspase-8. In addition, in the cells with the inactivating caspase-8 mutants, cleavage of poly(ADP-ribose)polymerase was markedly reduced compared with that of wild-type caspase-8. The occurrence of CASPASE-8 mutation and the inactivation of cell death activity by the mutants suggest that CASPASE-8 gene mutation may affect the pathogenesis of gastric cancers, especially at the late stage of gastric carcinogenesis.
Diminished Expression of C/EBPα in Skin Carcinomas Is Linked to Oncogenic Ras and Reexpression of C/EBPα in Carcinoma Cells Inhibits Proliferation
Authors: Shim Minsub , Powers Kristina L. , Ewing Sarah J. , Zhu Songyun , Smart Robert C.
Published: June 2022

The basic leucine zipper transcription factor, CCAAT/enhancer binding protein α (C/EBPα) is involved in mitotic growth arrest and has been implicated as a human tumor suppressor in acute myeloid leukemia. We have previously shown that C/EBPα is abundantly expressed in mouse epidermal keratinocytes. In the current study, the expression of C/EBPα was evaluated in seven mouse skin squamous cell carcinoma (SCC) cell lines that contain oncogenic Ha-Ras. C/EBPα mRNA and protein levels were greatly diminished in all seven SCC cell lines compared with normal primary keratinocytes, whereas C/EBPβ levels were not dramatically changed. Reexpression of C/EBPα in these SCC cell lines resulted in the inhibition in SCC cell proliferation. To determine whether the decrease in C/EBPα expression observed in the SCC cell lines also occurred in the carcinoma itself, immunohistochemical staining for C/EBPα in mouse skin SCCs was conducted. All 14 SCCs evaluated displayed negligible C/EBPα protein expression and normal C/EBPβ levels compared with the epidermis and all 14 carcinomas contained mutant Ras. To determine whether oncogenic Ras is involved in the down-regulation of C/EBPα, BALB/MK2 keratinocytes were infected with a retrovirus containing Ras12V, and C/EBPα protein, mRNA and DNA binding levels were determined. Keratinocytes infected with the retrovirus containing oncogenic Ras12V displayed greatly diminished C/EBPα protein, mRNA and DNA binding levels. In addition, BALB/MK2 cells containing endogenous mutant Ras displayed diminished C/EBPα expression and the ectopic expression of a dominant-negative RasN17 partially restored C/EBPα levels in these cells. These results indicate that oncogenic Ras negatively regulates C/EBPα expression and the loss of C/EBPα expression may contribute to the development of skin SCCs.
Testisin, a Glycosyl-Phosphatidylinositol–Linked Serine Protease, Promotes Malignant Transformation In vitro and In vivo
Authors: Tang Tenny , Kmet Muriel , Corral Laura , Vartanian Steffan , Tobler Andreas , Papkoff Jackie
Published: June 2022

Human testisin, a serine protease, is highly expressed in ovarian cancer and premeiotic spermatocytes with relatively little expression in other normal tissues. We first showed that testisin was localized on the surface of cultured tumor cells as a glycosyl-phosphatidylinositol–linked protein. We next explored the biological function of testisin in malignant transformation through manipulation of testisin expression in cell culture model systems. Small interfering RNA–mediated knockdown of endogenous testisin mRNA and protein expression in tumor cell lines led to increased apoptosis and diminished growth in soft agar. Conversely, overexpression of testisin in an epithelial cell line induced colony formation in soft agar as well as s.c. tumor growth in severe combined immunodeficient mice. A catalytic domain mutant was unable to induce soft-agar growth indicating that testisin protease activity is required for transformation. Ectopic expression of testisin in a human ovarian cancer cell line without endogenous testisin expression, led to the formation of larger tumors in severe combined immunodeficient mice. Data presented here provide the first demonstration that testisin can promote cellular processes that drive malignant transformation. Our functional data coupled with the restricted normal tissue distribution of testisin and its overexpression in a majority of ovarian cancers validates this cell surface protein as a target for therapeutic intervention.
Identification of OTX2 as a Medulloblastoma Oncogene Whose Product can be Targeted by All-Trans Retinoic Acid
Authors: Di Chunhui , Liao Shaoxi , Adamson David C. , Parrett Timothy J. , Broderick Daniel K. , Shi Qun , Lengauer Christoph , Cummins Jordan M. , Velculescu Victor E. , Fults Daniel W. , McLendon Roger E. , Bigner Darell D. , Yan Hai
Published: June 2022

Through digital karyotyping of permanent medulloblastoma cell lines, we found that the homeobox gene OTX2 was amplified more than 10-fold in three cell lines. Gene expression analyses showed that OTX2 transcripts were present at high levels in 14 of 15 (93%) medulloblastomas with anaplastic histopathologic features. Knockdown of OTX2 expression by siRNAs inhibited medulloblastoma cell growth in vitro, whereas pharmacologic doses of all-trans retinoic acid repressed OTX2 expression and induced apoptosis only in medulloblastoma cell lines that expressed OTX2. These observations suggest that OTX2 is essential for the pathogenesis of anaplastic medulloblastomas and that these tumors may be amenable to therapy with all-trans-retinoic acid.
Combined Haploinsufficiency for ATM and RAD9 as a Factor in Cell Transformation, Apoptosis, and DNA Lesion Repair Dynamics
Authors: Smilenov Lubomir B. , Lieberman Howard B. , Mitchell Stephen A. , Baker Ronald A. , Hopkins Kevin M. , Hall Eric J.
Published: June 2022

Loss of function of oncogenes, tumor suppressor genes and DNA damage processing genes has been implicated in the development of many types of cancer, but for the vast majority of cases, there is no link to specific germ line mutations. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair pathways was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced. Because the effect of haploinsufficiency for one protein is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes, critical for pathways that control DNA damage signaling, repair or apoptosis. To address this issue, primary mouse cells, haploinsufficient for one or two proteins, ATM and RAD9, related to the cellular response to DNA damage were examined. The results show that cells having low levels of both ATM and RAD9 proteins are more sensitive to transformation by radiation, have different DNA double-strand break repair dynamics and are less apoptotic when compared with wild-type controls or those cells haploinsufficient for only one of these proteins. Our conclusions are that under stress conditions, the efficiency and capacity for DNA repair mediated by the ATM/RAD9 cell signaling network depend on the abundance of both proteins and that, in general, DNA repair network efficiencies are genotype-dependent and can vary within a specific range.
Targeting Tumor Angiogenesis with Adenovirus-Delivered Anti-Tie-2 Intrabody
Authors: Popkov Mikhail , Jendreyko Nina , McGavern Dorian B. , Rader Christoph , Barbas Carlos F.
Published: June 2022

Inhibition of tumor angiogenesis is a promising approach for cancer therapy. As an endothelial cell–specific receptor kinase expressed almost exclusively on the surface of vascular endothelium, Tie-2 has an important role in tumor angiogenesis. To explore the therapeutic potential of blocking Tie-2 receptor-interaction pathway, an adenoviral vector was used to deliver a recombinant single-chain antibody fragment rabbit intrabody (pAd-2S03) capable of inhibition of both mouse and human Tie-2 surface expression. pAd-2S03 was given to mice with well-established primary tumors, either a human Kaposi's sarcoma (SLK) or a human colon carcinoma (SW1222). The intrabody significantly inhibited growth of both tumors (75% and 63%, respectively) when compared with pAd-GFP control-treated tumors (P < 0.01). Histopathologic analysis of cryosections taken from mice treated with pAd-2S03 revealed a marked decrease in vessel density, which was reduced by >87% in both tumor models when compared with control-treated tumors (P < 0.01). In contrast, human Tie-2-monospecific pAd-1S05 intrabody did not affect the growth of tumors, indicating that the antitumor effect of pAd-2S03 was due to the inhibition of tumor angiogenesis in these murine models. Our results show that the Tie-2 receptor pathway is essential for both SLK sarcoma and SW1222 colon carcinoma xenograft growth. The present study shows the potential utility of antiangiogenic agents that target the endothelium-specific receptor Tie-2 for down-regulation or genetic deletion.
Abstract 6284: Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells
Authors: Kimura Yuki , Ohzawa Hideyuki , Kaneko Yuki , Tamura Kohei , Futoh Yurie , Takahashi Kazuya , Saito Akira , Tojo Mineyuki , Miyato Hideyo , Kitayama Joji
Published: June 2022

Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). We reported miR-29b suppressed MMT of human peritoneal mesothelial cell (HPMC).

Methods: MiR-29b mimics and negative control miRNA (NC) were transfected to human bone marrow derived MSC by lipofection method. Exosomes were purified from the culture supernatants of the MSC with ultracentrifugation, and added to HPMC. Proliferation and migration of HPMC were examined with MTT assay and transwell assay, respectively. MMT of HPMC induced by 10 ng/ml TGF-β, and evaluated with immunofluorescence staining.

Results: MSC transfected with miR-29b mimics produced significantly increased amounts of miR-29b-containing exosomes (p<0.0001, n=3) as compared with negative control. After 24h-culture with PKH-26 stained exosomes, HPMC actively took the exosomes. After 48 hr-culture with TGF-β, HPMC apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMC was decreased while vimentin and Fibronectin tended to be upregulated by TGF-β. However, exosomes from miR-29b transfected MSC significantly suppressed the morphological changes and reduced the expression levels of vimentin and Fibronectin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMC. The exosome inhibited the proliferation and migration of HPMC by 31±14% (p<0.01, n=5) and 67±9% (p<0.01, n=3), and inhibited the adhesion of NUGC by 90±3% (p<0.001, n=5) as compared with NC exosome.

Conclusion: Exosomes from MSC transfected with miR-29b mimic efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell.

Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Joji Kitayama. Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6284.
Abstract 629: Benefit of using a spectral flow analyzer for the analysis of immune cell populations in tumors
Authors: Metzger Philipp , Malaisé Muriel , Obodozie Cynthia , Weber Holger
Published: June 2022

Flow cytometry is a widely applied approach for exploratory immune profiling and enables the identification of individual cells in a cell population, such as a tumor cell suspension, by staining cell markers with antibodies conjugated with a fluorophore. The markers allow for the differentiation of a large variety of immune cell populations, their change in frequency, and activation status upon treatment. Conventional flow cytometers, which use bandpass filters and need conventional compensation matrices, are limited by the number of parameters that can be simultaneously analyzed, restricting their utility. Spectral analysis technology promises a greater flexibility for panel design, a higher number of analyzed parameters, and more accurate visualization of results.

Our standard all-in-one flow cytometry panel uses all possible fluorescent detectors of the flow analyzer BD Fortessa, which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), B and NK cells, macrophages (M1/M2), MDSCs (granulocytic and monocytic), and dendritic cells. However, in addition to immune cell frequency, it would also be interesting to investigate cell surface activation and memory markers like PD-1, CD40, CD44, or CD62L or intracellular activation markers like IFN-gamma, perforin, granzyme B, or Ki67. The 32-channel spectral flow analyzer SP6800 ZE from Sony allows the combination of both.

Flow cytometry data of different tumor samples will be presented, as well as the comparison of the results and resolution of both flow analyzer systems. Auto-fluorescence of cells, which causes fluorescent signal contamination of other fluorescent markers in the conventional flow cytometer, can be subtracted using spectral technology to see the true fluorescent population, thereby allowing dim signal detection in rare populations. A comprehensive overview will be given regarding the pros and cons of the spectral analyzer system and difficulties switching from the one system to the other.

Citation Format: Philipp Metzger, Muriel Malaisé, Cynthia Obodozie, Holger Weber. Benefit of using a spectral flow analyzer for the analysis of immune cell populations in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 629.