bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1
Authors: Nilsson Jeanette , Helou Khalil , Kovács Anikó , Bendahl Pär-Ola , Bjursell Gunnar , Fernö Mårten , Carlsson Peter , Kannius-Janson Marie
Published: February 2010

Progression to metastasis is the proximal cause of most cancer-related mortality. Yet much remains to be understood about what determines the spread of tumor cells. This paper describes a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. We identify two transcription factors, nuclear factor 1-C2 (NF1-C2) and Forkhead box F1 (FoxF1), downstream of prolactin/nuclear Janus-activated kinase 2, with opposite effects on these processes. We show that NF1-C2 is lost during mammary tumor progression and is almost invariably absent from lymph node metastases. NF1-C2 levels in primary tumors correlate with better patient survival. Manipulation of NF1-C2 levels by expression of a stabilized version or using small interfering RNA showed that NF1-C2 counteracts EMT, motility, invasiveness, and tumor growth. FoxF1 was found to be a direct repressed target of NF1-C2. We provide the first evidence for a role of FoxF1 in cancer and in the regulation of EMT in cells of epithelial origin. Overexpression of FoxF1 was associated with a mesenchymal phenotype, increased invasiveness in vitro, and enhanced growth of breast carcinoma xenografts in nude mice. The relevance of these findings is strengthened by the correlation between FoxF1 expression and a mesenchymal phenoype in breast cancer cell isolates, consistent with the interpretation that FoxF1 promotes invasion and metastasis. Cancer Res; 70(5); 2020–9
Transcriptional Profiles of CD133+ and CD133− Glioblastoma-Derived Cancer Stem Cell Lines Suggest Different Cells of Origin
Authors: Lottaz Claudio , Beier Dagmar , Meyer Katharina , Kumar Praveen , Hermann Andreas , Schwarz Johannes , Junker Markus , Oefner Peter J. , Bogdahn Ulrich , Wischhusen Jörg , Spang Rainer , Storch Alexander , Beier Christoph P.
Published: February 2010

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. Growing evidence suggests that different types of CSC lead to the formation of GBM. This has prompted the present comparison of gene expression profiles between 17 GBM CSC lines and their different putative founder cells. Using a newly derived 24-gene signature, we can now distinguish two subgroups of GBM: Type I CSC lines display “proneural” signature genes and resemble fetal neural stem cell (fNSC) lines, whereas type II CSC lines show “mesenchymal” transcriptional profiles similar to adult NSC (aNSC) lines. Phenotypically, type I CSC lines are CD133 positive and grow as neurospheres. Type II CSC lines, in contrast, display (semi-)adherent growth and lack CD133 expression. Molecular differences between type I and type II CSC lines include the expression of extracellular matrix molecules and the transcriptional activity of the WNT and the transforming growth factor-β/bone morphogenetic protein signaling pathways. Importantly, these characteristics were not affected by induced adherence on laminin. Comparing CSC lines with their putative cells of origin, we observed greatly increased proliferation and impaired differentiation capacity in both types of CSC lines but no cancer-associated activation of otherwise silent signaling pathways. Thus, our data suggest that the heterogeneous tumor entity GBM may derive from cells that have preserved or acquired properties of either fNSC or aNSC but lost the corresponding differentiation potential. Moreover, we propose a gene signature that enables the subclassification of GBM according to their putative cells of origin. Cancer Res; 70(5); 2030–40
FOXQ1 Is Overexpressed in Colorectal Cancer and Enhances Tumorigenicity and Tumor Growth
Authors: Kaneda Hiroyasu , Arao Tokuzo , Tanaka Kaoru , Tamura Daisuke , Aomatsu Keiichi , Kudo Kanae , Sakai Kazuko , De Velasco Marco A. , Matsumoto Kazuko , Fujita Yoshihiko , Yamada Yasuhide , Tsurutani Junji , Okamoto Isamu , Nakagawa Kazuhiko , Nishio Kazuto
Published: February 2010

Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21CIP1/WAF1 expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 ± 301 versus 1735 ± 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family. Cancer Res; 70(5); 2053–63
Noninvasive Detection of Target Modulation following Phosphatidylinositol 3-Kinase Inhibition Using Hyperpolarized 13C Magnetic Resonance Spectroscopy
Authors: Ward Christopher S. , Venkatesh Humsa S. , Chaumeil Myriam M. , Brandes Alissa H. , VanCriekinge Mark , Dafni Hagit , Sukumar Subramaniam , Nelson Sarah J. , Vigneron Daniel B. , Kurhanewicz John , James C. David , Haas-Kogan Daphne A. , Ronen Sabrina M.
Published: February 2010

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol 3-kinase (PI3K) pathway are in clinical trials. However, it remains challenging to assess responses by traditional imaging methods. Here, we show for the first time the efficacy of hyperpolarized 13C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K inhibition by monitoring hyperpolarized [1-13C]lactate levels produced from hyperpolarized [1-13C]pyruvate through lactate dehydrogenase (LDH) activity. In GS-2 glioblastoma cells, PI3K inhibition by LY294002 or everolimus caused hyperpolarized lactate to drop to 42 ± 12% and to 76 ± 5%, respectively. In MDA-MB-231 breast cancer cells, hyperpolarized lactate dropped to 71 ± 15% after treatment with LY294002. These reductions were correlated with reductions in LDH activity to 48 ± 4%, 63 ± 4%, and 69 ± 12%, respectively, and were associated with a drop in levels of LDHA mRNA and LDHA and hypoxia-inducible factor-1α proteins. Supporting these findings, tumor growth inhibition achieved by everolimus in murine GS-2 xenografts was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio detected by in vivo MRS imaging, whereas an increase in this ratio occurred with tumor growth in control animals. Taken together, our findings illustrate the application of hyperpolarized 13C MRS of pyruvate to monitor alterations in LDHA activity and expression caused by PI3K pathway inhibition, showing the potential of this method for noninvasive imaging of drug target modulation. Cancer Res; 70(4); 1296–305
miR-31 Ablates Expression of the HIF Regulatory Factor FIH to Activate the HIF Pathway in Head and Neck Carcinoma
Authors: Liu Chung-Ji , Tsai Meng-Miao , Hung Pei-Shih , Kao Shou-Yen , Liu Tsung-Yun , Wu Kou-Juey , Chiou Shih-Hwa , Lin Shu-Chun , Chang Kuo-Wei
Published: February 2010

MicroRNAs (miRNA) are endogenously expressed noncoding RNAs with important biological and pathological functions that are yet to be fully defined. This study investigated alterations in miRNA expression in head and neck squamous cell carcinoma (HNSCC), the incidence of which is rising throughout the world. Initial screening and subsequent analysis identified a panel of aberrantly expressed miRNAs in HNSCC tissues, with miR-31 among the most markedly upregulated. Ectopic expression of miR-31 increased the oncogenic potential of HNSCC cells under normoxic conditions in cell culture or tumor xenografts. Conversely, blocking miR-31 expression reduced the growth of tumor xenografts. The in silico analysis suggested that miR-31 may target the 3′ untranslated region (UTR) of factor-inhibiting hypoxia-inducible factor (FIH), a hypoxia-inducible factor (HIF) regulatory factor that inhibits the ability of HIF to act as a transcriptional regulator under normoxic conditions. In support of this likelihood, miR-31 expression repressed FIH expression and mutations within the predictive miR-31 target site in the FIH 3′ UTR abrogated FIH repression. Furthermore, miR-31 expression increased HIF transactivation activity. We found that FIH suppressed oncogenic phenotypes under normoxic conditions and that this activity was abrogated by functional mutations. Lastly, increased miR-31 expression was correlated with decreased levels of FIH in tumor tissues. Our findings suggest that miR-31 contributes to the development of HNSCC by impeding FIH to activate HIF under normoxic conditions. Cancer Res; 70(4); 1635–44
Activation of the Integrin Effector Kinase Focal Adhesion Kinase in Cancer Cells Is Regulated by Crosstalk between Protein Kinase Cα and the PDZ Adapter Protein mda-9/Syntenin
Authors: Hwangbo Cheol , Kim Jaekyung , Lee Jung Joon , Lee Jeong-Hyung
Published: February 2010

Aberrant adhesion signaling pathways in cancer cells underlie their deadly invasive capabilities. The adhesion-related PDZ adapter protein mda-9/syntenin is a positive regulator of cancer cell progression in breast cancer, melanoma, and other human cancers. In this study, we report that mda-9/syntenin mediates adhesion-mediated activation of protein kinase Cα (PKCα) and focal adhesion kinase (FAK) by fibronectin (FN) in human breast cancer and melanoma cells. FN rapidly stimulated the expression of mda-9/syntenin and the activation of PKCα prior to activation of FAK. Inhibiting PKCα suppressed basal or FN-induced expression of mda-9/syntenin, as well as cell migration and invasion toward FN stimulated by mda-9/syntenin. Several lines of evidence suggested that activation of PKCα and expression of mda-9/syntenin were interdependent. First, mda-9/syntenin inhibition suppressed basal or FN-induced phosphorylation of PKCα at Thr638/641, whereas PKCα inhibition suppressed basal or FN-induced expression of mda-9/syntenin. Second, inhibiting either mda-9/syntenin or PKCα suppressed FN-induced formation of integrin-β1/FAK/c-Src signaling complexes. Third, inhibiting either mda-9/syntenin or PKCα suppressed FN-induced phosphorylation of FAK Tyr397 and c-Src Tyr416 and the induction of downstream effector signals to p38 and mitogen-activated protein kinase, Cdc42, and NF-κB. In summary, our findings offer evidence that mda-9/syntenin acts as a molecular adaptor linking PKCα and FAK activation in a pathway of FN adhesion by human breast cancer and melanoma cells. Cancer Res; 70(4); 1645–55
MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor
Authors: Pan Bo-Sheng , Chan Grace K.Y. , Chenard Melissa , Chi An , Davis Lenora J. , Deshmukh Sujal V. , Gibbs Jackson B. , Gil Susana , Hang Gaozhen , Hatch Harold , Jewell James P. , Kariv Ilona , Katz Jason D. , Kunii Kaiko , Lu Wei , Lutterbach Bart A. , Paweletz Cloud P. , Qu Xianlu , Reilly John F. , Szewczak Alexander A. , Zeng Qinwen , Kohl Nancy E. , Dinsmore Christopher J.
Published: February 2010

The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer. Here, we describe MK-2461, a novel ATP-competitive multitargeted inhibitor of activated c-Met. MK-2461 inhibited in vitro phosphorylation of a peptide substrate recognized by wild-type or oncogenic c-Met kinases (N1100Y, Y1230C, Y1230H, Y1235D, and M1250T) with IC50 values of 0.4 to 2.5 nmol/L. In contrast, MK-2461 was several hundredfold less potent as an inhibitor of c-Met autophosphorylation at the kinase activation loop. In tumor cells, MK-2461 effectively suppressed constitutive or ligand-induced phosphorylation of the juxtamembrane domain and COOH-terminal docking site of c-Met, and its downstream signaling to the phosphoinositide 3-kinase–AKT and Ras–extracellular signal-regulated kinase pathways, without inhibiting autophosphorylation of the c-Met activation loop. BIAcore studies indicated 6-fold tighter binding to c-Met when it was phosphorylated, suggesting that MK-2461 binds preferentially to activated c-Met. MK-2461 displayed significant inhibitory activities against fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor, and other receptor tyrosine kinases. In cell culture, MK-2461 inhibited hepatocyte growth factor/c-Met–dependent mitogenesis, migration, cell scatter, and tubulogenesis. Seven of 10 MK-2461–sensitive tumor cell lines identified from a large panel harbored genomic amplification of MET or FGFR2. In a murine xenograft model of c-Met–dependent gastric cancer, a well-tolerated oral regimen of MK-2461 administered at 100 mg/kg twice daily effectively suppressed c-Met signaling and tumor growth. Similarly, MK-2461 inhibited the growth of tumors formed by s.c. injection of mouse NIH-3T3 cells expressing oncogenic c-Met mutants. Taken together, our findings support further preclinical development of MK-2461 for cancer therapy. Cancer Res; 70(4); 1524–33
NOTCH Signaling Is Required for Formation and Self-Renewal of Tumor-Initiating Cells and for Repression of Secretory Cell Differentiation in Colon Cancer
Authors: Sikandar Shaheen S. , Pate Kira T. , Anderson Scott , Dizon Diana , Edwards Robert A. , Waterman Marian L. , Lipkin Steven M.
Published: February 2010

NOTCH signaling is critical for specifying the intestinal epithelial cell lineage and for initiating colorectal adenomas and colorectal cancers (CRC). Based on evidence that NOTCH is important for the maintenance and self-renewal of cancer-initiating cells in other malignancies, we studied the role of NOTCH signaling in colon cancer–initiating cells (CCIC). Tumors formed by CCICs maintain many properties of the primary CRCs from which they were derived, such as glandular organization, cell polarity, gap junctions, and expression of characteristic CRC molecular markers. Furthermore, CCICs have the property of self-renewal. In this study, we show that NOTCH signaling is 10- to 30-fold higher in CCIC compared with widely used colon cancer cell lines. Using small-molecule inhibition and short hairpin RNA knockdown, we show that NOTCH prevents CCIC apoptosis through repression of cell cycle kinase inhibitor p27 and transcription factor ATOH1. NOTCH is also critical to intrinsic maintenance of CCIC self-renewal and the repression of secretory cell lineage differentiation genes such as MUC2. Our findings describe a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest that inhibition of NOTCH signaling may improve CRC chemoprevention and chemotherapy. Cancer Res; 70(4); 1469–78
A Placental Growth Factor Variant Unable to Recognize Vascular Endothelial Growth Factor (VEGF) Receptor-1 Inhibits VEGF-Dependent Tumor Angiogenesis via Heterodimerization
Authors: Tarallo Valeria , Vesci Loredana , Capasso Onofrio , Esposito Maria Teresa , Riccioni Teresa , Pastore Lucio , Orlandi Augusto , Pisano Claudio , De Falco Sandro
Published: February 2010

Angiogenesis is one of the crucial events for cancer development and growth. Two members of the vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if coexpressed in the same cell, are both required for pathologic angiogenesis. We have generated a PlGF1 variant, named PlGF1-DE in which the residues Asp72 and Glu73 were substituted with Ala, which is unable to bind and activate VEGF receptor-1 but is still able to heterodimerize with VEGF. Here, we show that overexpression in tumor cells by adenoviral delivery or stable transfection of PlGF1-DE variant significantly reduces the production of VEGF homodimer via heterodimerization, determining a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel lumen and stabilization, and monocyte-macrophage infiltration. Conversely, the overexpression of PlGF1wt, also reducing the VEGF homodimer production comparably with PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, does not inhibit tumor growth and vessel density compared with controls but induces increase of vessel lumen, vessel stabilization, and monocyte-macrophage infiltration. The property of PlGF and VEGF-A to generate heterodimer represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a “dominant negative” of VEGF and is a new candidate for antiangiogenic gene therapy in cancer treatment. Cancer Res; 70(5); 1804–13
Regulation of p53 Family Member Isoform ΔNp63α by the Nuclear Factor-κB Targeting Kinase IκB Kinase β
Authors: Chatterjee Aditi , Chang Xiaofei , Sen Tanusree , Ravi Rajani , Bedi Atul , Sidransky David
Published: February 2010

The p53 family gene p63 plays an instrumental role in cellular stress responses including responses to DNA damage. In addition to encoding a full-length transcriptional activator, p63 also encodes several dominant inhibitory isoforms including the isoform ΔNp63α, the function of which is not fully understood. ΔNp63α is degraded in response to DNA damage, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying regulation of ΔNp63α expression in response to chemotherapeutic agents or tumor necrosis factor-α. We found that ΔNp63α interacts with IκB kinase (IKK), a multisubunit protein kinase that consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ. The IKKβ kinase promotes ubiquitin-mediated proteasomal degradation of ΔNp63α, whereas a kinase-deficient mutant IKKβ-K44A fails to do so. Cytokine- or chemotherapy-induced stimulation of IKKβ caused degradation of ΔNp63α and augmented transactivation of p53 family–induced genes involved in the cellular response to DNA damage. Conversely, IKKβ inhibition attenuated cytokine- or chemotherapy-induced degradation of ΔNp63α. Our findings show that IKKβ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. IKK activation represents one mechanism by which levels of ΔNp63α can be reduced, thereby rendering cells susceptible to cell death in the face of cellular stress or DNA damage. Cancer Res; 70(4); 1419–29
Identification of EP4 as a Potential Target for the Treatment of Castration-Resistant Prostate Cancer Using a Novel Xenograft Model
Authors: Terada Naoki , Shimizu Yosuke , Kamba Tomomi , Inoue Takahiro , Maeno Atsushi , Kobayashi Takashi , Nakamura Eijiro , Kamoto Toshiyuki , Kanaji Toshiya , Maruyama Takayuki , Mikami Yoshiki , Toda Yoshinobu , Matsuoka Toshiyuki , Okuno Yasushi , Tsujimoto Gozoh , Narumiya Shuh , Ogawa Osamu
Published: February 2010

More effective therapeutic approaches for castration-resistant prostate cancer (CRPC) are urgently needed, thus reinforcing the need to understand how prostate tumors progress to castration resistance. We have established a novel mouse xenograft model of prostate cancer, KUCaP-2, which expresses the wild-type androgen receptor (AR) and which produces the prostate-specific antigen (PSA). In this model, tumors regress soon after castration, but then reproducibly restore their ability to proliferate after 1 to 2 months without AR mutation, mimicking the clinical behavior of CRPC. In the present study, we used this model to identify novel therapeutic targets for CRPC. Evaluating tumor tissues at various stages by gene expression profiling, we discovered that the prostaglandin E receptor EP4 subtype (EP4) was significantly upregulated during progression to castration resistance. Immunohistochemical results of human prostate cancer tissues confirmed that EP4 expression was higher in CRPC compared with hormone-naïve prostate cancer. Ectopic overexpression of EP4 in LNCaP cells (LNCaP-EP4 cells) drove proliferation and PSA production in the absence of androgen supplementation in vitro and in vivo. Androgen-independent proliferation of LNCaP-EP4 cells was suppressed when AR expression was attenuated by RNA interference. Treatment of LNCaP-EP4 cells with a specific EP4 antagonist, ONO-AE3-208, decreased intracellular cyclic AMP levels, suppressed PSA production in vitro, and inhibited castration-resistant growth of LNCaP-EP4 or KUCaP-2 tumors in vivo. Our findings reveal that EP4 overexpression, via AR activation, supports an important mechanism for castration-resistant progression of prostate cancer. Furthermore, they prompt further evaluation of EP4 antagonists as a novel therapeutic modality to treat CRPC. Cancer Res; 70(4); 1606–15
R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer
Authors: Holland Sacha J. , Pan Alison , Franci Christian , Hu Yuanming , Chang Betty , Li Weiqun , Duan Matt , Torneros Allan , Yu Jiaxin , Heckrodt Thilo J. , Zhang Jing , Ding Pingyu , Apatira Ayodele , Chua Joanne , Brandt Ralf , Pine Polly , Goff Dane , Singh Rajinder , Payan Donald G. , Hitoshi Yasumichi
Published: February 2010

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. Cancer Res; 70(4); 1544–54
miR-181a and miR-630 Regulate Cisplatin-Induced Cancer Cell Death
Authors: Galluzzi Lorenzo , Morselli Eugenia , Vitale Ilio , Kepp Oliver , Senovilla Laura , Criollo Alfredo , Servant Nicolas , Paccard Caroline , Hupé Philippe , Robert Thomas , Ripoche Hugues , Lazar Vladimir , Harel-Bellan Annick , Dessen Philippe , Barillot Emmanuel , Kroemer Guido
Published: February 2010

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non–small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27Kip1 as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC. Cancer Res; 70(5); 1793–803
Reduced Levels of IGF-I Mediate Differential Protection of Normal and Cancer Cells in Response to Fasting and Improve Chemotherapeutic Index
Authors: Lee Changhan , Safdie Fernando M. , Raffaghello Lizzia , Wei Min , Madia Federica , Parrella Edoardo , Hwang David , Cohen Pinchas , Bianchi Giovanna , Longo Valter D.
Published: February 2010

Inhibitors of the insulin-like growth factor-I (IGF-I) receptor have been widely studied for their ability to enhance the killing of a variety of malignant cells, but whether IGF-I signaling differentially protects the host and cancer cells against chemotherapy is unknown. Starvation can protect mice, but not cancer cells, against high-dose chemotherapy [differential stress resistance (DSR)]. Here, we offer evidence that IGF-I reduction mediates part of the starvation-dependent DSR. A 72-hour fast in mice reduced circulating IGF-I by 70% and increased the level of the IGF-I inhibitor IGFBP-1 by 11-fold. LID mice, with a 70% to 80% reduction in circulating IGF-I levels, were protected against three of four chemotherapy drugs tested. Restoration of IGF-I was sufficient to reverse the protective effect of fasting. Sixty percent of melanoma-bearing LID mice treated with doxorubicin achieved long-term survival whereas all control mice died of either metastases or chemotherapy toxicity. Reducing IGF-I/IGF-I signaling protected primary glia, but not glioma cells, against cyclophosphamide and protected mouse embryonic fibroblasts against doxorubicin. Further, S. cerevisiae lacking homologs of IGF-I signaling proteins were protected against chemotherapy-dependent DNA damage in a manner that could be reversed by expressing a constitutively active form of Ras. We conclude that normal cells and mice can be protected against chemotherapy-dependent damage by reducing circulating IGF-I levels and by a mechanism that involves downregulation of proto-oncogene signals. Cancer Res; 70(4); 1564–72
The Bioreductive Prodrug PR-104A Is Activated under Aerobic Conditions by Human Aldo-Keto Reductase 1C3
Authors: Guise Christopher P. , Abbattista Maria R. , Singleton Rachelle S. , Holford Samuel D. , Connolly Joanna , Dachs Gabi U. , Fox Stephen B. , Pollock Robert , Harvey Justin , Guilford Parry , Doñate Fernando , Wilson William R. , Patterson Adam V.
Published: February 2010

PR-104, currently in phase II clinical trials, is a phosphate ester pre-prodrug which is converted in vivo to its cognate alcohol, PR-104A, a prodrug designed to exploit tumor hypoxia. Bioactivation occurs via one-electron reduction to DNA crosslinking metabolites in the absence of oxygen. However, certain tumor cell lines activate PR-104A in the presence of oxygen, suggesting the existence of an aerobic nitroreductase. Microarray analysis identified a cluster of five aldo-keto reductase (AKR) family members whose expressions correlated with aerobic metabolism of PR-104A. Plasmid-based expression of candidate genes identified aldo-keto reductase 1C3 as a novel nitroreductase. AKR1C3 protein was detected by Western blot in 7 of 23 cell lines and correlated with oxic PR-104A metabolism, an activity which could be partially suppressed by Nrf2 RNAi knockdown (or induced by Keap1 RNAi), indicating regulation by the ARE pathway. AKR1C3 was unable to sensitize cells to 10 other bioreductive prodrugs and was associated with single-agent PR-104 activity across a panel of 9 human tumor xenograft models. Overexpression in two AKR1C3-negative tumor xenograft models strongly enhanced PR-104 antitumor activity. A population level survey of AKR1C3 expression in 2,490 individual cases across 19 cancer types using tissue microarrays revealed marked upregulation of AKR1C3 in a subset including hepatocellular, bladder, renal, gastric, and non–small cell lung carcinoma. A survey of normal tissue AKR1C3 expression suggests the potential for tumor-selective PR-104A activation by this mechanism. These findings have significant implications for the clinical development of PR-104. Cancer Res; 70(4); 1573–84
DNA Methylation–Dependent Repression of PDZ-LIM Domain–Containing Protein 2 in Colon Cancer and Its Role as a Potential Therapeutic Target
Authors: Qu Zhaoxia , Yan Pengrong , Fu Jing , Jiang Jing , Grusby Michael J. , Smithgall Thomas E. , Xiao Gutian
Published: February 2010

Constitutive activation of the nuclear factor-κB (NF-κB) transcription factor plays a key role in chronic colonic inflammation and colon tumorigenesis. However, the mechanisms by which the tightly regulated NF-κB pathway becomes constitutively activated during colonic pathogenesis remain obscure. Here, we report that PDLIM2, an essential terminator of NF-κB activation, is repressed in various human colorectal cancer cell lines, suggesting one important mechanism for the constitutive activation of NF-κB. Indeed, expression of exogenous PDLIM2 inhibited constitutive NF-κB activation in these colorectal cancer cells. Importantly, the PDLIM2 expression was sufficient to suppress in vitro anchorage-independent growth and in vivo tumor formation of these malignant cells. We have further shown that the PDLIM2 repression involves promoter methylation. Accordingly, treatment of the colorectal tumor cell lines with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored PDLIM2 expression and resulted in growth arrest. These studies thus provide new mechanistic insights into colon tumorigenesis by identifying a novel tumor suppressor role for PDLIM2. Cancer Res; 70(5); 1766–72
Antiestrogens Induce Transforming Growth Factor β–Mediated Immunosuppression in Breast Cancer
Authors: Joffroy Christian M. , Buck Miriam B. , Stope Matthias B. , Popp Simone L. , Pfizenmaier Klaus , Knabbe Cornelius
Published: February 2010

Antiestrogens are universally used to treat estrogen receptor--positive breast cancer, but relapses occur commonly due to the development of drug resistance. The ability of antiestrogen to induce transforming growth factor β (TGFβ) in breast cancer cells may be relevant to the emergence of resistance, not only at the level of cell autonomous effects of TGFβ on cancer progression but also at the level of its effects on the host immune system. To evaluate the potential role of tumor-derived, antiestrogen-induced TGFβ as an immune suppressor, we established in vitro mixed lymphocyte tumor reactions (MLTR) using MCF-7 cells and peripheral blood mononuclear cells (PBMC), as well as tumor tissue and autologous tumor infiltrating lymphocytes (TIL) obtained from primary breast cancer biopsies. In allogeneic MLTR, antiestrogen-treated MCF-7 cells caused downregulation of the effector molecules granzyme B, perforin, and Fas ligand in CD8+ T cells, and suppressed the generation of cytotoxic effector cells in a TGFβ-dependent manner. Furthermore, we documented induction of regulatory T cells in CD4+ T cells, based on Foxp3 expression and T-cell activation in cocultures. In autologous MLTR, antiestrogen treatment gave rise to enhanced Foxp3 expression of TIL/PBMC and decreased the number of apoptotic tumor cells. These effects were reversed by addition of a TGFβ neutralizing antibody. Our findings offer evidence that antiestrogen induces immunosuppression in the tumor microenvironment, through a TGFβ-dependent mechanism that may contribute to the development of antiestrogen resistance in breast cancer. Cancer Res; 70(4); 1314–22
Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan
Authors: Zander Serge A.L. , Kersbergen Ariena , van der Burg Eline , de Water Niels , van Tellingen Olaf , Gunnarsdottir Sjöfn , Jaspers Janneke E. , Pajic Marina , Nygren Anders O.H. , Jonkers Jos , Borst Piet , Rottenberg Sven
Published: February 2010

There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This defect may serve as the tumor's Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2−/−;Brca1−/−;p53−/− tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans. Cancer Res; 70(4); 1700–10
Remote Control of Intestinal Tumorigenesis by Innate Immunity
Authors: Secher Thomas , Gaillot Olivier , Ryffel Bernhard , Chamaillard Mathias
Published: February 2010

Chronic intestinal inflammation has been linked to the development of colorectal cancer. Recent studies suggest that during chronic inflammation, the innate immune system may facilitate colon tumorigenesis in genetically predisposed individuals in response to certain indigenous microorganisms and/or cell remnants. In these individuals, therapeutic approaches that reroute the innate immune system's inflammatory and antimicrobial activities might help prevent colon tumorigenesis and metastasis. Cancer Res; 70(5); 1749–52
General Transcription Factor Binding at CpG Islands in Normal Cells Correlates with Resistance to De novo DNA Methylation in Cancer Cells
Authors: Gebhard Claudia , Benner Chris , Ehrich Mathias , Schwarzfischer Lucia , Schilling Elmar , Klug Maja , Dietmaier Wolfgang , Thiede Christian , Holler Ernst , Andreesen Reinhard , Rehli Michael
Published: February 2010

Aberrant DNA methylation at CpG islands is thought to contribute to cancer initiation and progression, but mechanisms that establish and maintain DNA methylation status during tumorigenesis or normal development remain poorly understood. In this study, we used methyl-CpG immunoprecipitation to generate comparative DNA methylation profiles of healthy and malignant cells (acute leukemia and colorectal carcinoma) for human CpG islands across the genome. While searching for sequence patterns that characterize DNA methylation states, we discovered several nonredundant sequences in CpG islands that were resistant to aberrant de novo methylation in cancer and that resembled consensus binding sites for general transcription factors (TF). Comparing methylation profiles with global CpG island binding data for specific protein 1, nuclear respiratory factor 1, and yin-yang 1 revealed that their DNA binding activity in normal blood cells correlated strictly with an absence of de novo methylation in cancer. In addition, global evidence showed that binding of any of these TFs to their consensus motif depended on their co-occurrence with neighboring consensus motifs. In summary, our results had two major implications. First, they pointed to a major role for cooperative binding of TFs in maintaining the unmethylated status of CpG islands in health and disease. Second, our results suggest that the majority of de novo methylated CpG islands are characterized by the lack of sequence motif combinations and the absence of activating TF binding. Cancer Res; 70(4); 1398–407
Estrogen Suppresses MLK3-Mediated Apoptosis Sensitivity in ER+ Breast Cancer Cells
Authors: Rangasamy Velusamy , Mishra Rajakishore , Mehrotra Suneet , Sondarva Gautam , Ray Rajarshi S. , Rao Arundhati , Chatterjee Malay , Rana Basabi , Rana Ajay
Published: February 2010

Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17β-Estradiol (E2) hinders cytotoxic drug–induced cell death in estrogen receptor–positive (ER+) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor–negative (ER−) breast tumors, suggesting that E2 might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH2-terminal kinase, were rapidly inhibited by E2 in ER+ but not in ER− cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E2, indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E2 involved phosphorylation of MLK3 Ser674 by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E2 accentuated this limitation. Taken together, our findings indicate that E2 inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug–induced death of ER+ breast cancer cells. Cancer Res; 70(4); 1731–40
Common Genetic Variants Associated with Breast Cancer and Mammographic Density Measures That Predict Disease
Authors: Odefrey Fabrice , Stone Jennifer , Gurrin Lyle C. , Byrnes Graham B. , Apicella Carmel , Dite Gillian S. , Cawson Jennifer N. , Giles Graham G. , Treloar Susan A. , English Dallas R. , Hopper John L. , Southey Melissa C. ,   for the Australian Twins and Sisters Mammographic Density Study
Published: February 2010

Mammographic density for age and body mass index (BMI) is a heritable risk factor for breast cancer. We aimed to determine if recently identified common variants associated with small gradients in breast cancer risk are associated with mammographic density. We genotyped 497 monozygotic and 330 dizygotic twin pairs and 634 of their sisters from 903 families for 12 independent variants. Mammographic dense area, percent dense area, and nondense area were measured by three observers using a computer-thresholding technique. Associations with mammographic density measures adjusted for age, BMI, and other determinants were estimated (a) cross-sectionally using a multivariate normal model for pedigree analysis (Px), (b) between sibships, and (c) within sibships using orthogonal transformations of outcomes and exposures. A combined test of association (Pc) was derived using the independent estimates from b and c. We tested if the distributions of P values across variants differed from the uniform distribution (Pu). For dense area and percent dense area, the distributions of Pc values were not uniform (both Pu <0.007). Consistent with their breast cancer associations, rs3817198 (LSP1) and rs13281615 (8q) were associated with dense area and percent dense area (all Px and Pc <0.05), and rs889312 (MAP3K1), rs2107425 (H19), and rs17468277 (CASP8) were marginally associated with dense area (some Px or Pc <0.05). All associations were independent of menopausal status. At least two common breast cancer susceptibility variants are associated with mammographic density measures that predict breast cancer. These findings could help elucidate how those variants and mammographic density measures are associated with breast cancer susceptibility. Cancer Res; 70(4); 1449–58
Interaction of Developmental Transcription Factor HOXC11 with Steroid Receptor Coactivator SRC-1 Mediates Resistance to Endocrine Therapy in Breast Cancer
Authors: McIlroy Marie , McCartan Damian , Early Sarah , Gaora Peadar Ó , Pennington Stephen , Hill Arnold D.K. , Young Leonie S.
Published: February 2010

Mechanisms of acquired resistance to endocrine therapy in breast cancer, a major clinical challenge, are poorly understood. We have used a mass spectrometry–based screen to identify proteins that are associated with the endocrine-resistant phenotype. In this study, we report the identification of a novel pathway of resistance to endocrine therapy involving interactions of the developmental transcription HOXC11 with the steroid receptor coactivator protein SRC-1, which is a strong predictor of reduced disease-free survival in breast cancer patients. HOXC11 and SRC-1 cooperate to regulate expression of the calcium-binding protein S100β in resistant breast cancer cells. Nuclear HOXC11 and S100β were found to strongly predict poor disease-free survival in breast cancer patients (n = 560; hazard ratios: 5.79 and 5.82, respectively; P < 0.0001). Elevated serum levels of S100β detected in patients also predicted reduced disease-free survival (n = 80; hazard ratio: 5.3; P = 0.004). Our findings define a biomolecular interaction network that drives an adaptive response to endocrine therapy with negative consequences for survival in breast cancer. Cancer Res; 70(4); 1585–94
Targeting the RB-pathway in Cancer Therapy
Authors: Knudsen Erik S. , Wang Jean Y. J.
Published: February 2010

The RB-pathway, consisting of inhibitors and activators of cyclin-dependent kinases, the retinoblastoma tumor suppressor (RB), and the E2F-family of transcription factors, plays critical roles in the regulation of cell cycle progression and cell death. Components of this pathway, particularly p16Ink4a, cyclin D1, and RB, are frequently altered in sporadic human cancers to promote deregulated cellular proliferation. The consistent disruption of the RB-pathway in human cancers raises the possibility of exploiting tumor-specific RB-pathway defects to improve the efficacy of current therapies and to develop new therapeutic strategies. This article discusses how the RB-pathway status impacts the cellular responses to cytotoxic, cytostatic, and hormone therapies, and how the components of the RB-pathway may be directly targeted to treat cancer. Clin Cancer Res; 16(4); 1094–9
A Phase I Safety and Pharmacokinetic Study of the Death Receptor 5 Agonistic Antibody PRO95780 in Patients with Advanced Malignancies
Authors: Camidge D. Ross , Herbst Roy S. , Gordon Michael S. , Eckhardt S. Gail , Kurzrock Razelle , Durbin Blythe , Ing Josephine , Tohnya Tanyifor M. , Sager Jason , Ashkenazi Avi , Bray Gordon , Mendelson David
Published: February 2010

Purpose: PRO95780 is a fully human IgG1 monoclonal antibody that triggers the extrinsic apoptosis pathway through death receptor 5. This first-in-human study assessed the safety, tolerability, pharmacokinetics, and any early evidence of efficacy of PRO95780 in patients with advanced malignancies.

Experimental Design: Target concentrations were predicted to occur at 10 mg/kg. Patients received up to eight cycles of PRO95780 i.v. using a 3+3 dose escalation design at 1 to 20 mg/kg every 14 days (every 28 days in cycle 1; stage 1), with cohort expansion at either the maximum tolerated dose or 10 mg/kg, whichever was lower (stage 2). Patients were evaluated for response every other cycle.

Results: The maximum tolerated dose was not reached within this study. Four (8%) of 50 patients reported adverse events of greater than grade 2 at least possibly related to PRO95780, including 2 patients with reversible grade 3 transaminase elevation. The mean terminal half-life was 8.8 to 19.3 days, with dose-dependent increases in exposure (peak plasma concentration and area under the concentration) across 1 to 15 mg/kg. Most patients treated with 10 mg/kg or above achieved trough concentration above the target efficacious concentration at day 15 with moderate accumulation after multiple doses. No objective responses occurred, although three minor responses were observed in patients with colorectal and granulosa cell ovarian cancers (each treated with 4 mg/kg) and chondrosarcoma (10 mg/kg).

Conclusions: PRO95780 is safe and well tolerated at doses up to 20 mg/kg. Evidence of activity was noted in several different tumor types at 4 and 10 mg/kg. Pharmacokinetic analysis supports a dosing regimen of 10 to 15 mg/kg every 2 to 3 weeks. Clin Cancer Res; 16(4); 1256–63
Phase I Clinical and Magnetic Resonance Imaging Study of the Vascular Agent NGR-hTNF in Patients with Advanced Cancers (European Organization for Research and Treatment of Cancer Study 16041)
Authors: van Laarhoven Hanneke W.M. , Fiedler Walter , Desar Ingrid M.E. , van Asten Jack J.A. , Marréaud Sandrine , Lacombe Denis , Govaerts Anne-Sophie , Bogaerts Jan , Lasch Peter , Timmer-Bonte Johanna N.H. , Lambiase Antonio , Bordignon Claudio , Punt Cornelis J.A. , Heerschap Arend , van Herpen Carla M.L.
Published: February 2010

Purpose: This phase I trial investigating the vascular targeting agent NGR-hTNF aimed to determine the (a) dose-limiting toxicities, (b) maximum tolerated dose (MTD), (c) pharmacokinetics and pharmacodynamics, (d) vascular response by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and (e) preliminary clinical activity in solid tumors.

Experimental Design: NGR-hTNF was administered once every 3 weeks by a 20- to 60-minute i.v. infusion to cohorts of three to six patients with solid tumors in escalating doses. Pharmacokinetic and pharmacodynamic analyses in blood were done during the first four cycles. DCE-MRI was done in cycle 1 at baseline and 2 hours after the start of the infusion.

Results: Sixty-nine patients received a total of 201 cycles of NGR-hTNF (0.2-60 μg/m2). Rigors and fever were the most frequently observed toxicities. Four dose-limiting toxicities were observed (at doses of 1.3, 8.1, and 60 μg/m2), of which three were infusion related. The MTD was 45 μg/m2. The mean apparent terminal half-life ranged from 0.963 to 2.08 hours. DCE-MRI results of tumors showed a vascular response to NGR-hTNF. No objective responses were observed, but 27 patients showed stable disease with a median duration of 12 weeks.

Conclusions: NGR-hTNF was well tolerated. The MTD was 45 μg/m2 administered in 1 hour once every 3 weeks. DCE-MRI results showed the antivascular effect of NGR-hTNF. These findings call for further research for defining the optimal biological dose and clinical activity of NGR-hTNF as a single agent or in combination with cytotoxic drugs. Clin Cancer Res; 16(4); 1315–23