bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Gene Expression Profiling Identifies BAX-δ as a Novel Tumor Antigen in Acute Lymphoblastic Leukemia
Authors: Maia Sara , Haining W. Nicholas , Ansén Sascha , Xia Zhinan , Armstrong Scott A. , Seth Nilufer P. , Ghia Paolo , den Boer Monique L. , Pieters Rob , Sallan Stephen E. , Nadler Lee M. , Cardoso Angelo A.
Published: March 2023

Abstract
The identification of new tumor-associated antigens (TAA) is critical for the development of effective immunotherapeutic strategies, particularly in diseases like B-cell acute lymphoblastic leukemia (B-ALL), where few target epitopes are known. To accelerate the identification of novel TAA in B-ALL, we used a combination of expression profiling and reverse immunology. We compared gene expression profiles of primary B-ALL cells with their normal counterparts, B-cell precursors. Genes differentially expressed by B-ALL cells included many previously identified as TAA in other malignancies. Within this set of overexpressed genes, we focused on those that may be functionally important to the cancer cell. The apoptosis-related molecule, BAX, was highly correlated with the ALL class distinction. Therefore, we evaluated BAX and its isoforms as potential TAA. Peptides from the isoform BAX-δ bound with high affinity to HLA-A*0201 and HLA-DR1. CD8+ CTLs specific for BAX-δ epitopes or their heteroclitic peptides could be expanded from normal donors. BAX-δ–specific T cells lysed peptide-pulsed targets and BAX-δ–expressing leukemia cells in a MHC-restricted fashion. Moreover, primary B-ALL cells were recognized by BAX-δ–specific CTL, indicating that this antigen is naturally processed and presented by tumor cells. This study suggests that (a) BAX-δ may serve as a widely expressed TAA in B-ALL and (b) gene expression profiling can be a generalizable tool to identify immunologic targets for cancer immunotherapy.
Data from Tumor Paint: A Chlorotoxin:Cy5.5 Bioconjugate for Intraoperative Visualization of Cancer Foci
Authors: Veiseh Mandana , Gabikian Patrik , Bahrami S-Bahram , Veiseh Omid , Zhang Miqin , Hackman Robert C. , Ravanpay Ali C. , Stroud Mark R. , Kusuma Yumiko , Hansen Stacey J. , Kwok Deborah , Munoz Nina M. , Sze Raymond W. , Grady William M. , Greenberg Norman M. , Ellenbogen Richard G. , Olson James M.
Published: March 2023

Abstract
Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies. [Cancer Res 2007;67(14):6882–8]
Data from Tumor αvβ3 Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases
Authors: Zhao Yingshe , Bachelier Richard , Treilleux Isabelle , Pujuguet Philippe , Peyruchaud Olivier , Baron Roland , Clément-Lacroix Philippe , Clézardin Philippe
Published: March 2023

Abstract
In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The αvβ3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor αvβ3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of αvβ3-overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of αvβ3-overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that αvβ3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of αvβ3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, αvβ3-expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor αvβ3 integrin stands as a therapeutic target for the prevention of skeletal metastases. [Cancer Res 2007;67(12):5821–30]
Data from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung
Authors: Chen Yuanbin , De Marco Melissa A. , Graziani Irene , Gazdar Adi F. , Strack Peter R. , Miele Lucio , Bocchetta Maurizio
Published: March 2023

Abstract
NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell fate during development and postnatal life. It has been increasingly linked to carcinogenesis, although its role in cancer seems to be highly context and tissue specific. Although NOTCH signaling is required for lung development, little is known about its role in lung cancer. In this study, we show that NOTCH signaling, as measured by the γ-secretase cleavage product NIC-1, is active in both normal human and lung tumor samples; however, downstream NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung tumors. Levels of NOTCH signaling components in primary human lung cells reflect observations in tissue samples, yet lung tumor cell lines showed little NOTCH signaling. Because oxygen concentrations are important in normal lung physiology and lung tumors are hypoxic, the effect of low oxygen on these lung tumor cell lines was evaluated. We found that hypoxia dramatically elevates NOTCH signaling (especially NOTCH-1) in lung tumor cell lines and concomitantly sensitizes them to inhibition via small-molecule γ-secretase inhibitors or NOTCH-1 RNA interference. γ-Secretase inhibitor–induced apoptosis of lung tumor cells grown under hypoxic conditions could be rescued by reintroduction of active NOTCH-1. Our data strengthen the role of NOTCH in lung cancer and as a therapeutic target for the treatment of lung and other hypoxic tumor types. [Cancer Res 2007;67(17):7954–9]
Data from Endoplasmic Reticulum Stress in the Proapoptotic Action of Edelfosine in Solid Tumor Cells
Authors: Nieto-Miguel Teresa , Fonteriz Rosalba I. , Vay Laura , Gajate Consuelo , López-Hernández Silvia , Mollinedo Faustino
Published: March 2023

Abstract
The endoplasmic reticulum (ER) has been posited as a potential anticancer target. The synthetic antitumor alkyl-lysophospholipid analogue edelfosine accumulates in the ER of solid tumor cells. This ER accumulation of the drug leads to the inhibition of phosphatidylcholine and protein synthesis, G2-M arrest, depletion of ER-stored Ca2+, Bax up-regulation and activation, transcriptional factor growth arrest and DNA damage–inducible gene 153 up-regulation, caspase-4 and caspase-8 activation, and eventually to apoptosis. Edelfosine prompted ER stress apoptotic signaling, but not the survival unfolded protein response. Edelfosine also induced persistent c-Jun NH2-terminal kinase (JNK) activation. Gene transfer–mediated overexpression of apoptosis signal–regulating kinase 1, which plays a crucial role in ER stress, enhanced edelfosine-induced JNK activation and apoptosis. Inhibition of JNK, caspase-4, or caspase-8 activation diminished edelfosine-induced apoptosis. Edelfosine treatment led to the generation of the p20 caspase-8 cleavage fragment of BAP31, directing proapoptotic signals between the ER and the mitochondria. bax−/−bak−/− double-knockout cells fail to undergo edelfosine-induced ER-stored Ca2+ release and apoptosis. Wild-type and bax−/−bak−/− cells showed similar patterns of phosphatidylcholine and protein synthesis inhibition, despite their differences in drug sensitivity. Thus, edelfosine-induced apoptosis is dependent on Bax/Bak-mediated ER-stored Ca2+ release, but phosphatidylcholine and protein synthesis inhibition is not critical. Transfection-enforced expression of Bcl-XL, which localizes specifically in mitochondria, prevented apoptosis without inhibiting ER-stored Ca2+ release. These data reveal that edelfosine induces an ER stress response in solid tumor cells, providing novel insights into the edelfosine-mediated antitumor activity. Our data also indicate that mitochondria are indispensable for this edelfosine-induced cell death initiated by ER stress. [Cancer Res 2007;67(21):10368–78]
Data from Haploinsufficiency of Krüppel-Like Factor 4 Promotes Adenomatous Polyposis Coli–Dependent Intestinal Tumorigenesis
Authors: Ghaleb Amr M. , McConnell Beth B. , Nandan Mandayam O. , Katz Jonathan P. , Kaestner Klaus H. , Yang Vincent W.
Published: March 2023

Abstract
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that the expression of KLF4 was activated by the colorectal cancer tumor suppressor adenomatous polyposis coli (APC) and that KLF4 repressed the Wnt/β-catenin pathway. Here, we examined whether KLF4 plays a role in modulating intestinal tumorigenesis by comparing the tumor burdens in mice heterozygous for the ApcMin allele (ApcMin/+) and those heterozygous for both the ApcMin and Klf4 alleles (Klf4+/−/ApcMin/+). Between 10 and 20 weeks of age, Klf4+/−/ApcMin/+ mice developed, on average, 59% more intestinal adenomas than ApcMin/+ mice (P < 0.0001). Immunohistochemical staining showed that Klf4 protein levels were lower in the normal-appearing intestinal tissues of Klf4+/−/ApcMin/+ mice compared with wild-type, Klf4+/−, or ApcMin/+ mice. In contrast, the levels of β-catenin and cyclin D1 were higher in the normal-appearing intestinal tissues of Klf4+/−/ApcMin/+ mice compared with the other three genotypes. Klf4 levels were further decreased in adenomas from both ApcMin/+ and Klf4+/−/ApcMin/+ mice compared with their corresponding normal-appearing tissues. Reverse transcription-PCR showed an inverse correlation between adenoma size and Klf4 mRNA levels in both Klf4+/−/ApcMin/+ and ApcMin/+ mice. There was also a progressive loss of heterozygosity of the wild-type Apc allele in adenomas with increasing size from Klf4+/−/ApcMin/+ and ApcMin/+ mice. Results from this study show that KLF4 plays an important role in promoting the development of intestinal adenomas in the presence of ApcMin mutation. [Cancer Res 2007;67(15):7147–54]
Data from BEX2 Is Overexpressed in a Subset of Primary Breast Cancers and Mediates Nerve Growth Factor/Nuclear Factor-κB Inhibition of Apoptosis in Breast Cancer Cell Lines
Authors: Naderi Ali , Teschendorff Andrew E. , Beigel Juergen , Cariati Massimiliano , Ellis Ian O. , Brenton James D. , Caldas Carlos
Published: March 2023

Abstract
We have identified a novel subtype of estrogen receptor (ER)-positive breast cancers with improved outcome after tamoxifen treatment and characterized by overexpression of the gene BEX2. BEX2 and its homologue BEX1 have highly correlated expression and are part of a cluster enriched for ER response and apoptosis genes. BEX2 expression is induced after estradiol (E2) treatment with a peak at 3 h, suggesting BEX2 is an estrogen-regulated gene. BEX2 belongs to a family of genes, including BEX1, NGFRAP1 (alias BEX3), BEXL1 (alias BEX4), and NGFRAP1L1 (alias BEX5). Both BEX1 and NGFRAP1 interact with p75NTR and modulate nerve growth factor (NGF) signaling through nuclear factor-κB (NF-κB) to regulate cell cycle, apoptosis, and differentiation in neural tissues. In breast cancer cells, NGF inhibits C2-induced apoptosis through binding of p75NTR and NF-κB activation. Here, we show that BEX2 expression is necessary and sufficient for the NGF-mediated inhibition (through NF-κB activation) of C2-induced apoptosis. We also show that BEX2 modulates apoptosis of breast cancer cells in response to E2 (50 nmol/L) and tamoxifen (5 and 10 μmol/L). Furthermore, BEX2 overexpression enhances the antiproliferative effect of tamoxifen at pharmacologic dose (1 μmol/L). These data suggest that a NGF/BEX2/NF-κB pathway is involved in regulating apoptosis in breast ancer cells and in modulating response to tamoxifen in primary tumors. [Cancer es 2007;67(14):6725–36]
Data from Selective Down-Regulation of Glioma-Associated Oncogene 2 Inhibits the Proliferation of Hepatocellular Carcinoma Cells
Authors: Kim Youngsoo , Yoon Joon Won , Xiao Xiaokun , Dean Nicholas M. , Monia Brett P. , Marcusson Eric G.
Published: March 2023

Abstract
The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC. [Cancer Res 2007;67(8):3583–93]
Data from Focal Adhesion Kinase Promotes the Aggressive Melanoma Phenotype
Authors: Hess Angela R. , Postovit Lynne-Marie , Margaryan Naira V. , Seftor Elisabeth A. , Schneider Galen B. , Seftor Richard E.B. , Nickoloff Brian J. , Hendrix Mary J.C.
Published: March 2023

Abstract
Malignant melanoma continues to remain a significant health threat, with death often occurring as a result of metastasis. The metastatic phenotype typically is characterized by augmented tumor cell invasion and migration in addition to tumor cell plasticity as shown by vasculogenic mimicry. Therefore, understanding the molecular mechanisms that promote an aggressive phenotype is essential to predicting the likelihood of metastasis at a stage when intervention may be possible. This study focuses on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, including cell survival, invasion, and migration. We found FAK to be phosphorylated on its key tyrosine residues, Tyr397 and Tyr576, in only aggressive uveal and cutaneous melanoma cells, which correlates with their increased invasion, migration, and vasculogenic mimicry plasticity. Additionally, we confirmed the presence of FAK phosphorylated on Tyr397 and Tyr576 in both cutaneous and uveal melanoma tumors in situ. Examination of a functional role for FAK in aggressive melanoma revealed that disruption of FAK-mediated signal transduction pathways, through the expression of FAK-related nonkinase (FRNK), results in a decrease in melanoma cell invasion, migration, and inhibition of vasculogenic mimicry. Moreover, we found that FRNK expression resulted in a down-regulation of Erk1/2 phosphorylation resulting in a decrease in urokinase activity. Collectively, these data suggest a new mechanism involved in promoting the aggressive melanoma phenotype through FAK-mediated signal transduction pathways, thus providing new insights into possible therapeutic intervention strategies.
Data from Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation
Authors: Sirianni Rosa , Chimento Adele , Malivindi Rocco , Mazzitelli Ignazio , Andò Sebastiano , Pezzi Vincenzo
Published: March 2023

Abstract
The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor α (ERα). Treatment with exogenous 17β-estradiol (E2) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E2/ERα–dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP–responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I–dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1–dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors. [Cancer Res 2007;67(17):8368–77]
Data from Novel Estrogen Receptor-α Binding Sites and Estradiol Target Genes Identified by Chromatin Immunoprecipitation Cloning in Breast Cancer
Authors: Lin Zhihong , Reierstad Scott , Huang Chiang-Ching , Bulun Serdar E.
Published: March 2023

Abstract
Estrogen receptor-α (ERα) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ERα was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ERα target loci in breast cancer cells. The ERα-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ERα-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ERα responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ERα that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer. [Cancer Res 2007;67(10):5017–8]
Data from Cells with Defective p53-p21-pRb Pathway Are Susceptible to Apoptosis Induced by p84N5 via Caspase-6
Authors: Garner Elizabeth , Martinon Fabio , Tschopp Jurg , Beard Peter , Raj Kenneth
Published: March 2023

Abstract
Adeno-associated virus (AAV) infection triggers a DNA damage response in the cell. This response is not induced by viral proteins but by virtue of the structure of AAV ssDNA being recognized by the cell as damaged DNA. The consequence of this is the killing of cells lacking p53 activity. We have observed that cells that lack p21 or pRb activity are also sensitive to AAV-induced cell death. We report that cells respond to AAV infection by activating two DNA damage signaling cascades. The first activates the p84N5 protein, which in turn activates caspase-6, leading to cell death. The second cascade activates the p53-21-pRb pathway, which inhibits activation of the p84N5 protein and thus prevents cell death. The result of the antagonistic interaction between these two pathways is that cells that do not exhibit functional p53-p21-pRb signaling undergo apoptosis as a consequence of AAV infection. Cells with a functional p53-21-pRb pathway are refractory to AAV-induced cell death. These results show that p53, although a proapoptotic protein, together with pRb and p21 proteins, is a member of an antiapoptotic cellular mechanism. As such, these experiments reveal features that may be exploited to specifically kill cells that lack the p53-p21-pRb pathway, such as cancer cells. The use of AAV to expose these subtle characteristics of intracellular signaling further highlights the advantages of using viruses as precision tools with which to address questions of cell biology. [Cancer Res 2007;67(16):7631–7]
Data from Epidermal Growth Factor Receptor Tyrosine Phosphorylation and Signaling Controlled by a Nuclear Receptor Coactivator, Amplified in Breast Cancer 1
Authors: Lahusen Tyler , Fereshteh Mark , Oh Annabell , Wellstein Anton , Riegel Anna T.
Published: March 2023

Abstract
The steroid receptor coactivator amplified in breast cancer 1 (AIB1) as well as epidermal growth factor receptor (EGFR) family members are frequently overexpressed in epithelial tumors, and their expression is associated with poor prognosis. However, a direct role of AIB1 in EGF signaling has not been determined. To address this, we reduced endogenous AIB1 levels using RNA interference in lung, breast, and pancreatic cancer cell lines. We found that a knockdown of AIB1 levels resulted in a loss of the growth response of these cell lines to EGF. Further analysis revealed that the depletion of AIB1 reduced tyrosine phosphorylation of EGFR at multiple residues both at autophosphorylation and Src kinase phosphorylation sites. AIB1 knockdown did not affect tyrosine phosphorylation of the receptor tyrosine kinases, platelet-derived growth factor receptor and HER3, or overall tyrosine phosphorylation of cellular proteins. However, EGF-dependent phosphorylation of HER2 was decreased. EGFR levels and membrane trafficking were not changed by AIB1 depletion, but there was less recruitment of Src homology 2 domain-containing proteins to the EGFR. This led to a substantial reduction in EGF-induced phosphorylation of signal transducers and activators of transcription 5 and c-Jun NH2-terminal kinase but no significant change in the activation of AKT. Vanadate treatment of cells revealed that the reduction in EGFR tyrosine phosphorylation is dependent in part on changes in cellular phosphatase activity. We propose that a portion of the oncogenic effect of AIB1 could be through control of EGFR and HER2 activity and subsequent modulation of cellular signaling pathways. [Cancer Res 2007;67(15):7256–65]
Data from Down-regulation of PU.1 by Methylation of Distal Regulatory Elements and the Promoter Is Required for Myeloma Cell Growth
Authors: Tatetsu Hiro , Ueno Shikiko , Hata Hiroyuki , Yamada Yasuhiro , Takeya Motohiro , Mitsuya Hiroaki , Tenen Daniel G. , Okuno Yutaka
Published: March 2023

Abstract
The transcription factor PU.1 is essential for myeloid and B-cell development. Down-regulation of PU.1 by disruption of its 14-kb 5′ upstream regulatory element induced acute myeloid leukemia, T-cell lymphoma, and chronic lymphocytic leukemia–like disease in murine models. In the present study, we found that PU.1 was down-regulated in the majority of human myeloma cell lines and a subset of freshly isolated myeloma cells, in contrast to relatively high expression of PU.1 in normal plasma cells. Patients in this low PU.1 expression subset may have a poor prognosis. In human myeloma cell lines, the 17-kb 5′ upstream enhancer and the promoter region of the PU.1 gene were highly methylated, and this is consistent with disappearance of DNase I–hypersensitive sites in these regions. To elucidate the significance of down-regulation of PU.1, we generated stable myeloma cell lines with an inducible PU.1 expression system. Exogenous expression of PU.1 in PU.1 null myeloma cell lines, U266 and KMS12PE, induced complete growth arrest and cell death. Up-regulation of PU.1 by 5-aza-2′-deoxycytidine also induced growth arrest of KMS12PE and KHM11 myeloma cells. These data suggest that down-regulation of PU.1 is an essential step for the survival of a subset of myeloma cells and that up-regulation of PU.1 by demethylation agents or other types of agents may represent a new therapeutic strategy for treatment of multiple myeloma patients. [Cancer Res 2007;67(11):5328–36]
Data from Tumor Suppressor Functions of ARLTS1 in Lung Cancers
Authors: Yendamuri Sai , Trapasso Francesco , Ferracin Manuela , Cesari Rossano , Sevignani Cinzia , Shimizu Masayoshi , Rattan Shashi , Kuroki Tamotsu , Dumon Kristoffel R. , Bullrich Florencia , Liu Chang-gong , Negrini Massimo , Williams Noel N. , Kaiser Larry R. , Croce Carlo M. , Calin George A.
Published: March 2023

Abstract
ARLTS1 is a newly characterized tumor suppressor gene located at chromosome 13q14.3 and involved in the pathogenesis of various types of tumors: two single-nucleotide polymorphisms, one of them responsible for protein truncation, were found statistically associated with familial malignancies, whereas DNA hypermethylation and genomic deletions have been identified as a mechanism of ARLTS1 down-regulation in sporadic cancers. We found that in a large portion of lung carcinomas (37%) and in all analyzed lung cancer cell lines, ARLTS1 is strongly down-regulated due to DNA methylation in its promoter region. After its restoration by adenoviral transduction, ARLTS1-negative A549 and H1299 cells underwent apoptosis and inhibition of cell growth. Furthermore, ARLTS1 reexpression significantly reduced the ability of A549 and H1299 to form tumors in nude mice. Finally, we identified ∼650 transcripts differentially expressed after restoration of ARLTS1 expression in A549 cells, suggesting that various pathways involved in cell survival, proliferation, signaling, and development mediate the effects of wild-type ARLTS1 in a lung cancer system. [Cancer Res 2007;67(16):7738–45]
Data from Zonal Heterogeneity for Gene Expression in Human Pancreatic Carcinoma
Authors: Nakamura Toru , Kuwai Toshio , Kitadai Yasuhiko , Sasaki Takamitsu , Fan Dominic , Coombes Kevin R. , Kim Sun-Jin , Fidler Isaiah J.
Published: March 2023

Abstract
Using Affymetrix HG-U133 Plus 2.0 array and laser capture microdissection techniques, we determined whether different zones of the same pancreatic tumor exhibited differential expression of genes. Human L3.6pl pancreatic cancer cells were implanted into the pancreas of nude mice. Three weeks later when tumors were 7 to 9 mm in diameter, gene expression patterns in tumor cells within the central and peripheral zones were compared, and 1,222 genes showed statistically significant differences. Bioinformatic functional analysis revealed that 346 up-regulated genes in the peripheral zone were related to cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrin-mediated signaling pathway, development, morphogenesis, and IκB kinase/nuclear factor-κB cascade; 876 up-regulated genes in the central zone were related to regulation of cell proliferation, regulation of transcription, transmembrane receptor protein tyrosine kinase signaling pathways, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, carbohydrate metabolism, and response to wounding. The reliability of the microarray results were confirmed by in situ hybridization analysis of the expression of two genes. Collectively, the data showed zonal heterogeneity for gene expression profiles in tumors and suggest that characterization of zonal gene expression profiles is essential if microarray analyses of genetic profiles are to produce reproducible data, predict disease prognosis, and allow design of specific therapeutics. [Cancer Res 2007;67(16):7597–604]
Data from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype
Authors: van Staveren Wilma C.G. , Solís David Weiss , Delys Laurent , Duprez Laurence , Andry Guy , Franc Brigitte , Thomas Gerry , Libert Frédérick , Dumont Jacques E. , Detours Vincent , Maenhaut Carine
Published: March 2023

Abstract
Cell lines are crucial to elucidate mechanisms of tumorigenesis and serve as tools for cancer treatment screenings. Therefore, careful validation of whether these models have conserved properties of in vivo tumors is highly important. Thyrocyte-derived tumors are very interesting for cancer biology studies because from one cell type, at least five histologically characterized different benign and malignant tumor types can arise. To investigate whether thyroid tumor–derived cell lines are representative in vitro models, characteristics of eight of those cell lines were investigated with microarrays, differentiation markers, and karyotyping. Our results indicate that these cell lines derived from differentiated and undifferentiated tumor types have evolved in vitro into similar phenotypes with gene expression profiles the closest to in vivo undifferentiated tumors. Accordingly, the absence of expression of most thyrocyte-specific genes, the nonresponsiveness to thyrotropin, as well as their large number of chromosomal abnormalities, suggest that these cell lines have acquired characteristics of fully dedifferentiated cells. They represent the outcome of an adaptation and evolution in vitro, which questions the reliability of these cell lines as models for differentiated tumors. However, they may represent useful models for undifferentiated cancers, and by their comparison with differentiated cells, can help to define the genes involved in the differentiation/dedifferentiation process. The use of any cell line as a model for a cancer therefore requires prior careful and thorough validation for the investigated property. [Cancer Res 2007;67(17):8113–20]
Data from Oncogenic Activity of Epidermal Growth Factor Receptor Kinase Mutant Alleles Is Enhanced by the T790M Drug Resistance Mutation
Authors: Godin-Heymann Nadia , Bryant Ianthe , Rivera Miguel N. , Ulkus Lindsey , Bell Daphne W. , Riese David J. , Settleman Jeffrey , Haber Daniel A.
Published: March 2023

Abstract
Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non–small cell lung cancers (NSCLC) with extraordinary sensitivity to targeted tyrosine kinase inhibitors (TKI). A single secondary EGFR mutation, T790M, arising in cis with the primary activating mutation, confers acquired resistance to these drugs. However, the T790M mutation is also detected in the absence of drug selection, suggesting that it may provide a growth advantage. We show here that although T790M alone has only a modest effect on EGFR function, when combined with the characteristic activating mutations L858R or del746–750, it results in a dramatic enhancement of EGFR activity. The double mutants show potent ligand-independent receptor autophosphorylation associated with altered cellular phenotypes, soft agar colony formation, and tumorigenesis in nude mice. The significant gain-of-function properties of these double mutants may explain their initial presence before drug selection and their rapid selection as the single drug resistance mutation during therapy with gefitinib/erlotinib, and suggests that they may contribute to the adverse clinical course of TKI-resistant NSCLC. [Cancer Res 2007;67(15):7319–26]
Data from Expression of L1-CAM and ADAM10 in Human Colon Cancer Cells Induces Metastasis
Authors: Gavert Nancy , Sheffer Michal , Raveh Shani , Spaderna Simone , Shtutman Michael , Brabletz Thomas , Barany Francis , Paty Phillip , Notterman Daniel , Domany Eytan , Ben-Ze'ev Avri
Published: March 2023

Abstract
L1-CAM, a neuronal cell adhesion receptor, is also expressed in a variety of cancer cells. Recent studies identified L1-CAM as a target gene of β-catenin-T-cell factor (TCF) signaling expressed at the invasive front of human colon cancer tissue. We found that L1-CAM expression in colon cancer cells lacking L1-CAM confers metastatic capacity, and mice injected in their spleen with such cells form liver metastases. We identified ADAM10, a metalloproteinase that cleaves the L1-CAM extracellular domain, as a novel target gene of β-catenin-TCF signaling. ADAM10 overexpression in colon cancer cells displaying endogenous L1-CAM enhanced L1-CAM cleavage and induced liver metastasis, and ADAM10 also enhanced metastasis in colon cancer cells stably transfected with L1-CAM. DNA microarray analysis of genes induced by L1-CAM in colon cancer cells identified a cluster of genes also elevated in a large set of human colon carcinoma tissue samples. Expression of these genes in normal colon epithelium was low. These results indicate that there is a gene program induced by L1-CAM in colon cancer cells that is also present in colorectal cancer tissue and suggest that L1-CAM can serve as target for colon cancer therapy. [Cancer Res 2007;67(16):7703–12]
Data from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia
Authors: Kojima Kyoko , Vickers Selwyn M. , Adsay N. Volkan , Jhala Nirag C. , Kim Hyung-Gyoon , Schoeb Trenton R. , Grizzle William E. , Klug Christopher A.
Published: March 2023

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of <5%. Genetic analysis of PDAC patient samples has shown that specific disease-associated mutations are correlated with histologically defined stages of neoplastic progression in the ductal epithelium. Activating mutations in KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16INK4A, p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating KrasG12D mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate KrasG12D and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;KrasG12D or Pdx1-Cre;Smad4lox/lox mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16Ink4A expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated KrasG12D allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4lox/lox animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple KrasG12D-dependent disease-associated phenotypes. [Cancer Res 2007;67(17):8121–30]
Data from Retinoblastoma Tumor Suppressor Status Is a Critical Determinant of Therapeutic Response in Prostate Cancer Cells
Authors: Sharma Ankur , Comstock Clay E.S. , Knudsen Erik S. , Cao Khanh H. , Hess-Wilson Janet K. , Morey Lisa M. , Barrera Jason , Knudsen Karen E.
Published: March 2023

Abstract
The retinoblastoma tumor suppressor protein (RB), a critical mediator of cell cycle progression, is functionally inactivated in the majority of human cancers, including prostatic adenocarcinoma. The importance of RB tumor suppressor function in this disease is evident because 25% to 50% of prostatic adenocarcinomas harbor aberrations in RB pathway. However, no previous studies challenged the consequence of RB inactivation on tumor cell proliferation or therapeutic response. Here, we show that RB depletion facilitates deregulation of specific E2F target genes, but does not confer a significant proliferative advantage in the presence of androgen. However, RB-deficient cells failed to elicit a cytostatic response (compared with RB proficient isogenic controls) when challenged with androgen ablation, AR antagonist, or combined androgen blockade. These data indicate that RB deficiency can facilitate bypass of first-line hormonal therapies used to treat prostate cancer. Given the established effect of RB on DNA damage checkpoints, these studies were then extended to determine the impact of RB depletion on the response to cytotoxic agents used to treat advanced disease. In this context, RB-deficient prostate cancer cells showed enhanced susceptibility to cell death induced by only a selected subset of cytotoxic agents (antimicrotubule agents and a topoisomerase inhibitor). Combined, these data indicate that RB depletion dramatically alters the cellular response to therapeutic intervention in prostate cancer cells and suggest that RB status could potentially be developed as a marker for effectively directing therapy. [Cancer Res 2007;67(13):6192–203]
Data from Inhibition of Nuclear Factor-κB Activity by Temozolomide Involves O6-Methylguanine–Induced Inhibition of p65 DNA Binding
Authors: Yamini Bakhtiar , Yu Xiaohong , Dolan M. Eileen , Wu Michael H. , Kufe Donald W. , Weichselbaum Ralph R.
Published: March 2023

Abstract
The alkylating agent temozolomide, commonly used in the treatment of malignant glioma, causes cellular cytotoxicity by forming O6-methylguanine adducts. In this report, we investigated whether temozolomide alters the activity of the transcription factor nuclear factor-κB (NF-κB). Temozolomide inhibits basal and tumor necrosis factor α (TNFα)–induced NF-κB transcriptional activity without altering phosphorylation or degradation of inhibitor of κB-α. Inhibition of NF-κB is secondary to attenuation of p65 DNA binding, not nuclear translocation. Inhibition of DNA binding is shown both in vitro, with gel shift studies and DNA binding assays, and in vivo at κB sites. Consistent with inhibition of NF-κB activity, temozolomide reduces basal and TNFα-induced κB-dependent gene expression. Temozolomide also inhibits NF-κB activated by inducers other than TNFα, including lipopolysaccharide, doxorubicin, and phorbol 12-myristate 13-acetate. The inhibitory action of temozolomide on NF-κB is observed to be maximal following pretreatment of cells with temozolomide for 16 h and is also seen with the SN1-type methylating agent methylnitrosourea. The ability of temozolomide to form O6-methylguanine adducts is important for inhibition of NF-κB as is the presence of a functioning mismatch repair system. Activation of NF-κB with TNFα before administration of temozolomide reduces the cytotoxicity of temozolomide, whereas 16-h pretreatment with temozolomide resensitizes cells to killing. This work shows a mechanism whereby O6-methylguanine adducts formed by temozolomide lead to inhibition of NF-κB activity and illustrates a link between mismatch repair processing of alkylator-induced DNA damage and cell death. [Cancer Res 2007;67(14):6889–98]
Data from Structural Analysis Identifies Imidazo[1,2-b]Pyridazines as PIM Kinase Inhibitors with In vitro Antileukemic Activity
Authors: Pogacic Vanda , Bullock Alex N. , Fedorov Oleg , Filippakopoulos Panagis , Gasser Christelle , Biondi Andrea , Meyer-Monard Sandrine , Knapp Stefan , Schwaller Juerg
Published: March 2023

Abstract
Much attention has recently been focused on PIM kinases as potential targets for the treatment of hematopoietic malignancies and some solid cancers. Using protein stability shift assays, we identified a family of imidazo[1,2-b]pyridazines to specifically interact with and inhibit PIM kinases with low nanomolar potency. The high-resolution crystal structure of a PIM1 inhibitor complex revealed that imidazo[1,2-b]pyridazines surprisingly interact with the NH2-terminal lobe helix αC rather than with the kinase hinge region. Thus, the identified inhibitors are ATP competitive but not ATP mimetic compounds, explaining their enhanced selectivity with respect to conventional type I kinase inhibitors. One of the identified imidazo[1,2-b]pyridazines (K00135) was further tested in several hematopoietic cellular systems. First, K00135 dose-dependently impaired survival of murine Ba/F3 cells that have been rendered cytokine independent by overexpression of human PIMs. Second, K00135 impaired survival and clonogenic growth of a panel of human acute leukemia cells. Third, exposure of K00135 significantly suppressed in vitro growth of leukemic blasts from five acute myelogenous leukemia patients but not of normal umbilical cord blood mononuclear cells. In vitro kinase assays and immunoblotting using lysates from human MV4;11 leukemic cells showed inhibition of phosphorylation of known PIM downstream targets, such as BAD and eukaryotic translation initiation factor 4E–binding protein 1, by K00135. Taken together, we report a family of small molecules that selectively interact and block PIM kinases and could serve as a lead to develop new targeted antileukemic therapeutics. [Cancer Res 2007;67(14):6916–24]
Data from Effect of a Single Nucleotide Polymorphism in the Murine Double Minute 2 Promoter (SNP309) on the Sensitivity to Topoisomerase II–Targeting Drugs
Authors: Nayak Mamatha S. , Yang Jin-Ming , Hait William N.
Published: March 2023

Abstract
A single nucleotide polymorphism (SNP) SNP309 (T→G) in the murine double minute 2 (MDM2) promoter creates a high-affinity Sp1 binding site and increases the expression of MDM2 mRNA and protein. Approximately 40% of the populations harbor at least one variant allele and 12% to 17% are homozygous G/G at codon 309. This MDM2 SNP increases susceptibility to cancer and decreases the response of cancer cells to certain forms of treatment, such as radiation therapy and DNA-damaging drugs. Topoisomerase II (TopoII)–targeting agents are commonly used chemotherapeutic drugs with a broad spectrum of activity. However, resistance to TopoII poisons limits their effectiveness. We show that MDM2 SNP309 rendered a panel of cancer cell lines that are homozygous for SNP309 selectively resistant (∼10-fold) to certain TopoII-targeting chemotherapeutic drugs (etoposide, mitoxantrone, amsacrine, and ellipticine). The mechanism underlying this observation was Mdm2-mediated down-regulation of TopoII; on drug exposure, MDM2 bound to TopoII and resulted in decreased cellular enzyme content. Knockdown of MDM2 by RNA interference stabilized TopoIIα and decreased resistance to TopoII-targeting drugs. Thus, MDM2 SNP309 (T→G) may represent a relatively common, previously unappreciated determinant of drug sensitivity. Given the frequency of SNP309 in the general population (40% in heterozygous T/G and 12% in homozygous G/G condition), our observation may have important implications for the individualization of cancer chemotherapy. [Cancer Res 2007;67(12):5831–9]
Data from Hemizygous Disruption of Cdc25A Inhibits Cellular Transformation and Mammary Tumorigenesis in Mice
Authors: Ray Dipankar , Terao Yasuhisa , Nimbalkar Dipali , Hirai Hiroyuki , Osmundson Evan C. , Zou Xianghong , Franks Roberta , Christov Konstantin , Kiyokawa Hiroaki
Published: March 2023

Abstract
CDC25A phosphatase activates multiple cyclin-dependent kinases (CDK) during cell cycle progression. Inactivation of CDC25A by ubiquitin-mediated degradation is a major mechanism of DNA damage-induced S-G2 checkpoint. Although increased CDC25A expression has been reported in various human cancer tissues, it remains unclear whether CDC25A activation is a critical rate-limiting step of carcinogenesis. To assess the role for CDC25A in cell cycle control and carcinogenesis, we used a Cdc25A-null mouse strain we recently generated. Whereas Cdc25A−/− mice exhibit early embryonic lethality, Cdc25A+/− mice show no appreciable developmental defect. Cdc25A+/− mouse embryonic fibroblasts (MEF) exhibit normal kinetics of cell cycle progression at early passages, modestly enhanced G2 checkpoint response to DNA damage, and shortened proliferative life span, compared with wild-type MEFs. Importantly, Cdc25A+/− MEFs are significantly resistant to malignant transformation induced by coexpression of H-rasV12 and a dominant negative p53 mutant. The rate-limiting role for CDC25A in transformation is further supported by decreased transformation efficiency in MCF-10A human mammary epithelial cells stably expressing CDC25A small interfering RNA. Consistently, Cdc25A+/− mice show substantially prolonged latency in mammary tumorigenesis induced by MMTV-H-ras or MMTV-neu transgene, whereas MMTV-myc–induced tumorigenesis is not significantly affected by Cdc25A heterozygosity. Mammary tissues of Cdc25A+/−;MMTV-neu mice before tumor development display less proliferative response to the oncogene with increased tyrosine phosphorylation of CDK1/2, but show no significant change in apoptosis. These results suggest that Cdc25A plays a rate-limiting role in transformation and tumor initiation mediated by ras activation. [Cancer Res 2007;67(14):6605–11]