bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Loss of ΔNp63α Promotes Invasion of Urothelial Carcinomas via N-Cadherin/Src Homology and Collagen/Extracellular Signal-Regulated Kinase Pathway
Authors: Fukushima Hiroshi , Koga Fumitaka , Kawakami Satoru , Fujii Yasuhisa , Yoshida Soichiro , Ratovitski Edward , Trink Barry , Kihara Kazunori
Published: March 2024

Abstract
p63 plays a critical role in normal development and maintenance of stratified epithelia, including the urothelium. In the normal urothelium, urothelial cells in the basal layers abundantly express the predominant p63 isoform ΔNp63α. We previously showed that (a) ΔNp63α expression at the similar level to the normal urothelium is retained in most low-grade papillary noninvasive (LPN) tumors, whereas frequently lost in high-grade invasive carcinomas, and that (b) loss of ΔNp63α is associated with poor prognosis of invasive bladder urothelial carcinoma patients. However, a functional role of ΔNp63α in progression of urothelial carcinomas remains to be elucidated. Here, we show that loss of ΔNp63α expression promotes invasion of urothelial carcinoma cells. In 5637 cells substantially expressing only ΔNp63α isoform at the protein level, knockdown of endogenous p63 upregulated N-cadherin, which recruited more Src homology and collagen to N-cadherin and activated extracellular signal-regulated kinase (ERK) signaling, and consequently potentiated cell motility, excretion of matrix metalloproteinase-9, and invasion. In T24 cells originally lacking endogenous ΔNp63α expression, exogenous expression of ΔNp63α attenuated invasion by downregulating N-cadherin expression and ERK activity, confirming an invasion-suppressive role of ΔNp63α in urothelial carcinoma cells. We further documented loss of ΔNp63 expression accompanied by N-cadherin upregulation during muscle-invasive recurrence in patients whose bladder cancer had progressed from LPN tumors to muscle-invasive disease. These results suggest that loss of ΔNp63α and subsequent upregulation of N-cadherin is one of the mechanisms underlying progression of bladder cancer. [Cancer Res 2009;69(24):9263–70]
Data from Tripeptidyl Peptidase II Plays a Role in the Radiation Response of Selected Primary Cell Types but not Based on Nuclear Translocation and p53 Stabilization
Authors: Firat Elke , Tsurumi Chizuko , Gaedicke Simone , Huai Jisen , Niedermann Gabriele
Published: March 2024

Abstract
The giant cytosolic protease tripeptidyl peptidase II (TPPII) was recently proposed to play a role in the DNA damage response. Shown were nuclear translocation of TPPII after γ-irradiation, lack of radiation-induced p53 stabilization in TPPII-siRNA–treated cells, and complete tumor regression in mice after γ-irradiation when combined with TPPII-siRNA silencing or a protease inhibitor reported to inhibit TPPII. This suggested that TPPII could be a novel target for tumor radiosensitization and prompted us to study radiation responses using TPPII-knockout mice. Neither the sensitivity to total body irradiation nor the radiosensitivity of resting lymphoid cells, which both strongly depend on p53, was altered in the absence of TPPII. Functional integrity of p53 in TPPII-knockout cells is further shown by a proper G1 arrest and by the accumulation of p53 and its transcriptional targets, p21, Bax, and Fas, on γ-irradiation. Furthermore, we could not confirm radiation-induced nuclear translocation of TPPII. Nevertheless, after γ-irradiation, we found slightly increased mitotic catastrophe of TPPII-deficient primary fibroblasts and increased apoptosis of TPPII-deficient activated CD8+ T cells. The latter was accompanied by delayed resolution of the DNA double-strand break marker γH2AX. This could, however, be due to increased apoptotic DNA damage rather than reduced DNA damage repair. Our data do not confirm a role for TPPII in the DNA damage response based on nuclear TPPII translocation and p53 stabilization but nevertheless do show increased radiation-induced cell death of selected nontransformed cell types in the absence of the TPPII protease. [Cancer Res 2009;69(8):3325–31]
Data from Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the α-Emitting Radionuclides Bismuth-213 or Astatine-211
Authors: Nakamae Hirohisa , Wilbur D. Scott , Hamlin Donald K. , Thakar Monica S. , Santos Erlinda B. , Fisher Darrell R. , Kenoyer Aimee L. , Pagel John M. , Press Oliver W. , Storb Rainer , Sandmaier Brenda M.
Published: March 2024

Abstract
We previously investigated the potential of targeted radiotherapy using a bismuth-213 (213Bi)–labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. Although this approach allowed sustained marrow engraftment, limited availability, high cost, and short half-life of 213Bi induced us to investigate an alternative α-emitting radionuclide, astatine-211 (211At), for the same application. Biodistribution and toxicity studies were conducted with conjugates of the anti-murine CD45 antibody 30F11 with either 213Bi or 211At. Mice were injected with 2 to 50 μCi on 10 μg or 20 μCi on 2 or 40 μg of 30F11 conjugate. Biodistribution studies showed that the spleen contained the highest concentration of radioactivity, ranging from 167 ± 23% to 417 ± 109% injected dose/gram (% ID/g) after injection of the 211At conjugate and 45 ± 9% to 166 ± 11% ID/g after injection of the 213Bi conjugate. The higher concentrations observed for 211At-labeled 30F11 were due to its longer half-life, which permitted better localization of isotope to the spleen before decay. 211At was more effective at producing myelosuppression for the same quantity of injected radioactivity. All mice injected with 20 or 50 μCi 211At, but none with the same quantities of 213Bi, had lethal myeloablation. Severe reversible acute hepatic toxicity occurred with 50 μCi 213Bi, but not with lower doses of 213Bi or with any dose of 211At. No renal toxicity occurred with either radionuclide. The data suggest that smaller quantities of 211At-labeled anti-CD45 antibody are sufficient to achieve myelosuppression and myeloablation with less nonhematologic toxicity compared with 213Bi-labeled antibody. [Cancer Res 2009;69(6):2408–15]
Data from DDB1 Targets Chk1 to the Cul4 E3 Ligase Complex in Normal Cycling Cells and in Cells Experiencing Replication Stress
Authors: Leung-Pineda Van , Huh Jiwon , Piwnica-Worms Helen
Published: March 2024

Abstract
The Chk1 protein kinase preserves genome integrity in normal proliferating cells and in cells experiencing replicative and genotoxic stress. Chk1 is currently being targeted in anticancer regimens. Here, we identify damaged DNA-binding protein 1 (DDB1) as a novel Chk1-interacting protein. DDB1 is part of an E3 ligase complex that includes the cullin proteins Cul4A and Cul4B. We report that Cul4A/DDB1 negatively regulates Chk1 stability in vivo. Chk1 associates with Cul4A/DDB1 during an unperturbed cell division cycle and both Chk1 phosphorylation and replication stress enhanced these interactions. Cul4A/DDB1 regulates Chk1 ubiquitination in vivo and Chk1 is directly ubiquitinated in vitro in a Cul4A/DDB1–dependent manner. Furthermore, Chk1 is stabilized in cells deficient for Cul4A/DDB1. This study shows that Chk1 abundance is regulated by the Cul4A/DDB1 ubiquitin ligase during an unperturbed cell division cycle, in response to replicative stress and on heat shock protein 90 inhibition, and that deregulation of the Chk1/Cul4A/DDB1 pathway perturbs the ionizing radiation–induced G2 checkpoint. [Cancer Res 2009;69(6):2630–7]
Data from Targeting Fibroblast Growth Factor Receptors Blocks PI3K/AKT Signaling, Induces Apoptosis, and Impairs Mammary Tumor Outgrowth and Metastasis
Authors: Dey Julien H. , Bianchi Fabrizio , Voshol Johannes , Bonenfant Debora , Oakeley Edward J. , Hynes Nancy E.
Published: March 2024

Abstract
Members of the fibroblast growth factor receptor (FGFR) family have essential roles in normal physiology and in cancer where they control diverse processes. FGFRs have been associated with breast cancer development. Thus, models to study the role of FGFR in breast cancer and their targeting potential are important. We present an in vitro and in vivo analysis of FGFRs in the breast cancer model cell lines 67NR and 4T1. We show that both tumor cell lines coexpress FGFRs and ligands and display autocrine FGFR signaling activity. Fibroblast growth factor receptor substrate 2 (FRS2), a downstream mediator of FGFR, is constitutively tyrosine phosphorylated and multiple signaling pathways are active. Treatment of 67NR and 4T1 cultures with TKI258, an FGFR tyrosine kinase inhibitor (TKI), caused a rapid decrease in FRS2 phosphorylation; decreased the activity of extracellular signal–regulated kinase 1/2 (ERK1/2), AKT, and phospholipase Cγ; and blocked proliferation of both tumor lines. Furthermore, TKI258 induced 4T1 apoptotic cell death via blockade of the phosphoinositide 3-kinase/AKT pathway. In vivo, one dose of TKI258 rapidly lowered FRS2 phosphorylation and ERK1/2 and AKT activity in mammary tumors. Long-term TKI258 treatment of 4T1 tumor– and 67NR tumor–bearing mice had a significant effect on primary tumor outgrowth and 4T1 tumor–induced lung metastases. A microarray analysis was carried out to identify targets with roles in TKI258 antitumor activity and potential prognostic markers in human breast tumors. Of interest are the downregulated matrix metalloproteases (MMP), in particular MMP9, which is essential for metastatic spread of 4T1 tumors. Cancer Res; 70(10); 4151–62. ©2010 AACR.
Data from Identification of a Stat3-Dependent Transcription Regulatory Network Involved in Metastatic Progression
Authors: Ranger Jill J. , Levy David E. , Shahalizadeh Solmaz , Hallett Michael , Muller William J.
Published: March 2024

Abstract
High levels of activated Stat3 are often found in human breast cancers and can correlate with poor patient outcome. We employed an activated ErbB2 mouse model of breast cancer to investigate the in vivo role of Stat3 in mammary tumor progression and found that Stat3 does not alter mammary tumor initiation but dramatically affects metastatic progression. Four-fold fewer animals exhibited lung metastases in the absence of Stat3 and a 12-fold reduction in the number of lung lesions was observed in animals bearing Stat3-null tumors when compared with the wild-type cohort. The decreased malignancy in Stat3-deficient tumors is attributed to a reduction in both angiogenic and inflammatory responses associated with a Stat3-dependent transcriptional cascade involving CCAAT/enhancer binding protein δ. [Cancer Res 2009;69(17):6823–30]
Data from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer
Authors: Hernandez Lídia , Hsu Sarah C. , Davidson Ben , Birrer Michael J. , Kohn Elise C. , Annunziata Christina M.
Published: March 2024

Abstract
The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-κB kinase β (IKKβ) and confirmed with RNA interference toward IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the nine-gene ovarian cancer–specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion, and adhesion. Downregulating IKKβ activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity. Cancer Res; 70(10); 4005–14. ©2010 AACR.
Data from The Absence of Pericytes Does Not Increase the Sensitivity of Tumor Vasculature to Vascular Endothelial Growth Factor-A Blockade
Authors: Nisancioglu Maya H. , Betsholtz Christer , Genové Guillem
Published: March 2024

Abstract
Recent progress with therapies targeting endothelial cells has drawn attention also to the pericytes as potential target cells for antiangiogenic therapy. Published data suggest that pericytes might confer resistance to vascular endothelial growth factor (VEGF) withdrawal in tumors. This hypothesis has been supported by experiments using tumors with reversible transgenic expression of VEGF-A as well as by individual pharmacologically targeting VEGF and platelet-derived growth factor receptor signaling in endothelial cells and pericytes using receptor tyrosine kinase (RTK) inhibitors with different specificities. However, the RTK inhibitors applied thus far are not entirely specific to the mentioned pathways, and therefore, the effects putatively attributed to pericyte targeting might reflect other antitumor effects. Here, we have reinvestigated the putative benefits of doubly targeting endothelial cells and pericytes in the treatment of experimental tumors. For this purpose, we used two highly specific tools, the pericyte-deficient pdgfbret/ret mouse and the recently developed specific anti–VEGF-A antibody G6-31, which neutralizes both murine and human VEGF-A. We generated B16, Lewis lung carcinoma, and T241 subcutaneous tumors in both pdgfbret/ret and control mice and treated these mice with G6-31. Our results fail to show any improved effect of VEGF inhibition, as measured by tumor growth or decrease in vascular density, in pericyte-deficient tumors compared with controls. Our observations suggest that additional targeting of pericytes does not increase the antitumor effect already generated by anti-VEGF drugs. Cancer Res; 70(12); 5109–15. ©2010 AACR.
Data from Tgfbr1 Haploinsufficiency Inhibits the Development of Murine Mutant Kras-Induced Pancreatic Precancer
Authors: Adrian Kevin , Strouch Matthew J. , Zeng Qinghua , Barron Morgan R. , Cheon Eric C. , Honasoge Akilesh , Xu Yanfei , Phukan Sharbani , Sadim Maureen , Bentrem David J. , Pasche Boris , Grippo Paul J.
Published: March 2024

Abstract
To dissect the role of constitutively altered Tgfbr1 signaling in pancreatic cancer development, we crossed Elastase-KrasG12D (EL-Kras) mice with Tgfbr1 haploinsufficient mice to generate EL-Kras/Tgfbr1+/− mice. Mice were euthanized at 6 to 9 months to compare the incidence, frequency, and size of precancerous lesions in the pancreas. Only 50% of all EL-Kras/Tgfbr1+/− mice developed preinvasive lesions compared with 100% of EL-Kras (wild-type Tgfbr1) mice. The frequency of precancerous lesions was 4-fold lower in haploinsufficient than in control mice. Paradoxically, the precancerous lesions of EL-Kras/Tgfbr1+/− mice were considerably larger than those in EL-Kras mice. Yet, the mitotic index of precancerous cells and the observable levels of fibrosis, lipoatrophy, and lymphocytic infiltration were reduced in EL-Kras/Tgfbr1+/− mice. We conclude that Tgfbr1 signaling promotes the development of precancerous lesions in mice. These findings suggest that individuals with constitutively decreased TGFBR1 expression may have a decreased risk of pancreatic cancer. [Cancer Res 2009;69(24):9169–74]
Data from EZH2 Is Essential for Glioblastoma Cancer Stem Cell Maintenance
Authors: Suvà Mario-Luca , Riggi Nicolò , Janiszewska Michalina , Radovanovic Ivan , Provero Paolo , Stehle Jean-Christophe , Baumer Karine , Le Bitoux Marie-Aude , Marino Denis , Cironi Luisa , Marquez Victor E. , Clément Virginie , Stamenkovic Ivan
Published: March 2024

Abstract
Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) occurs in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma multiforme (GBM). Based on its ability to modulate transcription of key genes implicated in cell cycle control, DNA repair, and cell differentiation, EZH2 is believed to play a crucial role in tissue-specific stem cell maintenance and tumor development. Here, we show that targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep), or its specific downregulation by short hairpin RNA (shRNA), strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. Using genome-wide expression analysis of DZNep-treated GBM CSCs, we found the expression of c-myc, recently reported to be essential for GBM CSCs, to be strongly repressed upon EZH2 depletion. Specific shRNA-mediated downregulation of EZH2 in combination with chromatin immunoprecipitation experiments revealed that c-myc is a direct target of EZH2 in GBM CSCs. Taken together, our observations provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a novel mechanism underlying GBM CSC maintenance and suggest that EZH2 may be a valuable new therapeutic target for GBM management. [Cancer Res 2009;69(24):9211–8]
Data from Blockade of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase and Murine Double Minute Synergistically Induces Apoptosis in Acute Myeloid Leukemia via BH3-Only Proteins Puma and Bim
Authors: Zhang Weiguo , Konopleva Marina , Burks Jared K. , Dywer Karen C. , Schober Wendy D. , Yang Jer-Yen , McQueen Teresa J. , Hung Mien-Chie , Andreeff Michael
Published: March 2024

Abstract
Molecular aberrations of the Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK and/or Murine double minute (MDM2)/p53 signaling pathways have been reported in 80% and 50% of primary acute myeloid leukemia (AML) samples and confer poor outcome. In this study, antileukemic effects of combined MEK inhibition by AZD6244 and nongenotoxic p53 activation by MDM2 antagonist Nutlin-3a were investigated. Simultaneous blockade of MEK and MDM2 signaling by AZD6244 and Nutlin-3a triggered synergistic proapoptotic responses in AML cell lines [combination index (CI) = 0.06 ± 0.03 and 0.43 ± 0.03 in OCI/AML3 and MOLM13 cells, respectively] and in primary AML cells (CI = 0.52 ± 0.01). Mechanistically, the combination upregulated levels of BH3-only proteins Puma and Bim, in part via transcriptional upregulation of the FOXO3a transcription factor. Suppression of Puma and Bim by short interfering RNA rescued OCI/AML3 cells from AZD/Nutlin-induced apoptosis. These results strongly indicate the therapeutic potential of combined MEK/MDM2 blockade in AML and implicate Puma and Bim as major regulators of AML cell survival. Cancer Res; 70(6); 2424–34
Data from DC-HIL/Glycoprotein Nmb Promotes Growth of Melanoma in Mice by Inhibiting the Activation of Tumor-Reactive T Cells
Authors: Tomihari Mizuki , Chung Jin-Sung , Akiyoshi Hideo , Cruz Ponciano D. , Ariizumi Kiyoshi
Published: March 2024

Abstract
DC-HIL/glycoprotein nmb (Gpnmb) expressed on antigen-presenting cells attenuates T-cell activation by binding to syndecan-4 (SD-4) on activated T cells. Because DC-HIL/Gpnmb is expressed abundantly by mouse and human melanoma lines, we posited that melanoma-associated DC-HIL/Gpnmb exerts similar inhibitory function on melanoma-reactive T cells. We generated small interfering RNA–transfected B16F10 melanoma cells to completely knock down DC-HIL/Gpnmb expression, with no alteration in cell morphology, melanin synthesis, or MHC class I expression. This knockdown had no effect on B16F10 proliferation in vitro or entry into the cell cycle following growth stimulation, but it markedly reduced the growth of these cells in vivo following their s.c. injection into syngeneic immunocompetent (but not immunodeficient) mice. This reduction in tumor growth was due most likely to an augmented capacity of DC-HIL–knocked down B16F10 cells (compared with controls) to activate melanoma-reactive T cells as documented in vitro and in mice. Whereas DC-HIL knockdown had no effect on susceptibility of melanoma to killing by cytotoxic T cells, blocking SD-4 function enhanced the reactivity of CD8+ T cells to melanoma-associated antigens on parental B16F10 cells. Using an assay examining the spread to the lung following i.v. injection, DC-HIL–knocked down cells produced lung foci at similar numbers compared with that produced by control cells, but the size of the former foci was significantly smaller than the latter. We conclude that DC-HIL/Gpnmb confers upon melanoma the ability to downregulate the activation of melanoma-reactive T cells, thereby allowing melanoma to evade immunologic recognition and destruction. As such, the DC-HIL/SD-4 pathway is a potentially useful target for antimelanoma immunotherapy. Cancer Res; 70(14); 5778–87. ©2010 AACR.
Data from Low Ascorbate Levels Are Associated with Increased Hypoxia-Inducible Factor-1 Activity and an Aggressive Tumor Phenotype in Endometrial Cancer
Authors: Kuiper Caroline , Molenaar Ilona G.M. , Dachs Gabi U. , Currie Margaret J. , Sykes Peter H. , Vissers Margreet C.M.
Published: March 2024

Abstract
Activation of the transcription factor hypoxia-inducible factor (HIF)-1 allows solid tumors to thrive under conditions of metabolic stress. Because HIF-1 is switched off by hydroxylation reactions that require ascorbate, inadequate intracellular ascorbate levels could contribute to HIF-1 overactivation. In this study, we investigated whether the ascorbate content of human endometrial tumors [known to be driven by HIF-1 and vascular endothelial growth factor (VEGF)] influenced HIF-1 activity and tumor pathology. We measured protein levels of HIF-1α and three downstream gene products [glucose transporter 1 (GLUT-1), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), and VEGF], as well as the ascorbate content of tumor and patient-matched normal endometrial tissue samples. HIF-1α and its downstream gene products were upregulated in tumor tissue, with the highest levels being present in high-grade tumors. High-grade tumors also had reduced capacity to accumulate ascorbate compared with normal tissue; however, all grades contained tumors with low ascorbate content. Tumors with the highest HIF-1α protein content were ascorbate deficient. Low ascorbate levels were also associated with elevated VEGF, GLUT-1, and BNIP3 protein levels and with increased tumor size, and there was a significant association between low tissue ascorbate levels and increased activation of the HIF-1 pathway (P = 0.007). In contrast, tumors with high ascorbate levels had lesser levels of HIF-1 activation. This study shows for the first time a likely in vivo relationship between ascorbate and HIF-1, with low tumor tissue ascorbate levels being associated with high HIF-1 activation and tumor growth. Cancer Res; 70(14); 5749–58. ©2010 AACR.
Data from Histone Methyltransferase Gene SETD2 Is a Novel Tumor Suppressor Gene in Clear Cell Renal Cell Carcinoma
Authors: Duns Gerben , van den Berg Eva , van Duivenbode Inge , Osinga Jan , Hollema Harry , Hofstra Robert M.W. , Kok Klaas
Published: March 2024

Abstract
Sporadic clear cell renal cell carcinoma (cRCC) is genetically characterized by the recurrent loss of the short arm of chromosome 3, with a hotspot for copy number loss in the 3p21 region. We applied a method called “gene identification by nonsense-mediated mRNA decay inhibition” to a panel of 10 cRCC cell lines with 3p21 copy number loss to identify biallelic inactivated genes located at 3p21. This revealed inactivation of the histone methyltransferase gene SETD2, located on 3p21.31, as a common event in cRCC cells. SETD2 is nonredundantly responsible for trimethylation of the histone mark H3K36. Consistent with this function, we observed loss or a decrease of H3K36me3 in 7 out of the 10 cRCC cell lines. Identification of missense mutations in 2 out of 10 primary cRCC tumor samples added support to the involvement of loss of SETD2 function in the development of cRCC tumors. Cancer Res; 70(11); 4287–91. ©2010 AACR.
Data from E2F3a Is Critically Involved in Epidermal Growth Factor Receptor–Directed Proliferation in Ovarian Cancer
Authors: Reimer Daniel , Hubalek Michael , Riedle Svenja , Skvortsov Sergej , Erdel Martin , Concin Nicole , Fiegl Heidi , Müller-Holzner Elisabeth , Marth Christian , Illmensee Karl , Altevogt Peter , Zeimet Alain G.
Published: March 2024

Abstract
We describe for the first time a new integral molecular pathway, linking transcription factor E2F3a to epidermal growth factor receptor (EGFR) activation in ovarian cancer cells. Investigations on the role of E2F family members in EGFR-mediated mitogenic signaling revealed that E2F3a was selectively upregulated following EGFR activation, whereas all other E2F family members remained unaffected. In contrast, EGF treatment of healthy ovarian surface epithelial and mesothelial cells yielded a selective upregulation of proliferation-promoting E2F1 and E2F2 without influencing E2F3a expression. In ovarian cancer cell lines, the extent of EGF-induced proliferative stimulus was closely related to the magnitude of E2F3a increase, and proliferation inhibition by E2F3a knockdown was not overcome by EGF exposure. Furthermore, the EGFR-E2F3a axis was found to be signal transducer and activator of transcription 1/3 dependent and the ratio of IFN-regulatory factor (IRF)-1 to IRF-2 was shown to be determinative for E2F3a control. In a pilot study on 32 primary ovarian cancer specimens, a highly significant correlation between activated EGFR and E2F3a expression was disclosed. This new integral pathway in the EGFR-driven mitogenic cell response, which through its key player E2F3a was found to be essential in triggering proliferation in ovarian cancer cells, provides new insights into EGFR signaling and could represent the basis for appealing new therapeutic approaches in ovarian cancer. Cancer Res; 70(11); 4613–23. ©2010 AACR.
Data from PML-RARα and Dnmt3a1 Cooperate in vivo to Promote Acute Promyelocytic Leukemia
Authors: Subramanyam Deepa , Belair Cassandra D. , Barry-Holson Keegan Q. , Lin Haijiang , Kogan Scott C. , Passegué Emmanuelle , Blelloch Robert
Published: March 2024

Abstract
The PML-RARα oncogene is the central effector of acute promyelocytic leukemia (APL). PML-RARα physically interacts with epigenetic-modifying enzymes including DNA methyltransferases (Dnmt) to suppress critical downstream targets. Here, we show that increased expression of Dnmt3a1 cooperates with PML-RARα in vivo to promote early lethality secondary to myeloid expansion and dysfunction in primary mice. Bone marrow cells from these mice cause leukemogenesis with a shortened latency and a higher penetrance on transplantation into irradiated recipients. Furthermore, leukemic cells overexpressing PML-RARα and Dnmt3a1 display increased methylation at a target promoter compared with PML-RARα or Dnmt3a1 controls. Our findings show a cooperation between the PML-RARα oncogene and the Dnmt3a1 enzyme in vivo and that Dnmt levels can be rate limiting in APL progression. Cancer Res; 70(21); 8792–801. ©2010 AACR.
Data from Optimized Peptide Vaccines Eliciting Extensive CD8 T-Cell Responses with Therapeutic Antitumor Effects
Authors: Cho Hyun-Il , Celis Esteban
Published: March 2024

Abstract
A major challenge for developing effective therapeutic vaccines against cancer is overcoming immunologic tolerance to tumor-associated antigens that are expressed on both malignant cells and normal tissues. Herein, we describe a novel vaccination approach, TriVax, that uses synthetic peptides representing CD8 T-cell epitopes, Toll-like receptor agonists that function as potent immunologic adjuvants and costimulatory anti-CD40 antibodies to generate large numbers of high-avidity antigen-reactive T cells capable of recognizing and killing tumor cells. Our results show that TriVax induced huge numbers of long-lasting antigen-specific CD8 T cells that displayed significant antitumor effects in vivo. The administration of a TriVax formulation containing a CD8 T-cell epitope derived from a melanosomal antigen (Trp2180-188) elicited antigen-specific CD8 T cells that induced systemic autoimmunity (vitiligo). More important, TriVax immunization was effective in eliciting potent protective antitumor immunity as well as remarkable therapeutic effects against established B16 melanoma. This therapeutic effect was mediated by CD8 T cells via perforin-mediated lysis and required the participation of type-I IFN but not IFNγ. These results suggest that similar strategies would be applicable for the design of effective vaccination for conducting clinical studies in cancer patients. [Cancer Res 2009;69(23):9012–9]
Data from Hypoxia-Regulated Delta-like 1 Homologue Enhances Cancer Cell Stemness and Tumorigenicity
Authors: Kim Yuri , Lin Qun , Zelterman Daniel , Yun Zhong
Published: March 2024

Abstract
Reduced oxygenation, or hypoxia, inhibits differentiation and facilitates stem cell maintenance. Hypoxia commonly occurs in solid tumors and promotes malignant progression. Hypoxic tumors are aggressive and exhibit stem cell–like characteristics. It remains unclear, however, whether and how hypoxia regulates cancer cell differentiation and maintains cancer cell stemness. Here, we show that hypoxia increases the expression of the stem cell gene DLK1, or delta-like 1 homologue (Drosophila), in neuronal tumor cells. Inhibition of DLK1 enhances spontaneous differentiation, decreases clonogenicity, and reduces in vivo tumor growth. Overexpression of DLK1 inhibits differentiation and enhances tumorigenic potentials. We further show that the DLK1 cytoplasmic domain, especially Tyrosine339 and Serine355, is required for maintaining both clonogenicity and tumorigenicity. Because elevated DLK1 expression is found in many tumor types, our observations suggest that hypoxia and DLK1 may constitute an important stem cell pathway for the regulation of cancer stem cell–like functionality and tumorigenicity. [Cancer Res 2009;69(24):9271–80]
Data from Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion
Authors: Heikkinen Pekka T. , Nummela Marika , Jokilehto Terhi , Grenman Reidar , Kähäri Veli-Matti , Jaakkola Panu M.
Published: March 2024

Abstract
Smad7 is an inhibitor of the transforming growth factor-β–activated signaling pathway. Under well-oxygenated conditions, Smad7 is a potent inhibitor of carcinoma cell invasion. Paradoxically, however, the expression of Smad7 is upregulated across several cancers and may promote cancer progression. Hypoxia, which is frequently met in solid tumors, is an enhancer of carcinoma cell invasion and cancer progression. Here, we report that hypoxia activates the expression of Smad7 in a hypoxia-inducible factor– and von Hippel-Lindau protein–dependent manner. As expected, in normoxia, the forced expression of Smad7 inhibited carcinoma cell invasion. In contrast with the normoxic condition, the inhibitory effect of Smad7 was lost under hypoxia. The block in carcinoma cell invasion by forced expression of Smad7 was released by hypoxia in two invasive carcinoma cell lines. Moreover, the noninvasive HaCaT keratinocytes become invasive upon simultaneous hypoxia and transforming growth factor-β stimulus. The hypoxia-activated invasion was attenuated by inhibiting Smad7 expression by short interfering RNA. Finally, the increased Smad7 expression in human carcinomas correlated with hypoxic gene expression. The data provide evidence that hypoxia could convert Smad7 function from an invasion inhibitor into an activator of invasion. Furthermore, they might shed light as to why increased Smad7 expression is detected in cancers. Cancer Res; 70(14); 5984–93. ©2010 AACR.
Data from Absence of Both Cytochrome P450 3A and P-glycoprotein Dramatically Increases Docetaxel Oral Bioavailability and Risk of Intestinal Toxicity
Authors: van Waterschoot Robert A.B. , Lagas Jurjen S. , Wagenaar Els , van der Kruijssen Cornelia M.M. , van Herwaarden Antonius E. , Song Ji-Ying , Rooswinkel Rogier W. , van Tellingen Olaf , Rosing Hilde , Beijnen Jos H. , Schinkel Alfred H.
Published: March 2024

Abstract
Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can therefore only be administered i.v. The drug-metabolizing enzyme cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp; MDR1) are considered to be major determinants of docetaxel pharmacokinetics. It has been hypothesized that CYP3A and P-gp work synergistically in limiting the systemic exposure to many orally ingested drugs. However, it has been difficult to examine this interplay in vivo. We therefore generated mice lacking all CYP3A and P-gp genes. Although missing two primary detoxification systems, Cyp3a/Mdr1a/1b−/− mice are viable, fertile, and without spontaneous abnormalities. When orally challenged with docetaxel, a disproportionate (>70-fold) increase in systemic exposure was observed compared with the increases in single Cyp3a−/− (12-fold) or Mdr1a/1b−/− (3-fold) mice. Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in lowering docetaxel exposure, their individual efficacy was not dependent on activity of the other protein. On reflection, this absence of functional synergism makes biological sense, as synergism would conflict with a robust detoxification defense. Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b−/− mice resulted in dramatically altered and lethal toxicity, with severe intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp might thus be a highly effective strategy to improve oral drug bioavailability but with serious risks when applied to drugs with narrow therapeutic windows. [Cancer Res 2009;69(23):8996–9002]
Data from Signatures of MicroRNAs and Selected MicroRNA Target Genes in Human Melanoma
Authors: Philippidou Demetra , Schmitt Martina , Moser Dirk , Margue Christiane , Nazarov Petr V. , Muller Arnaud , Vallar Laurent , Nashan Dorothee , Behrmann Iris , Kreis Stephanie
Published: March 2024

Abstract
Small noncoding microRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation or orchestrating their sequence-specific degradation. In this study, we investigated miRNA and miRNA target gene expression patterns in melanoma to identify candidate biomarkers for early and progressive disease. Because data presently available on miRNA expression in melanoma are inconsistent thus far, we applied several different miRNA detection and profiling techniques on a panel of 10 cell lines and 20 patient samples representing nevi and primary or metastatic melanoma. Expression of selected miRNAs was inconsistent when comparing cell line–derived and patient-derived data. Moreover, as expected, some discrepancies were also detected when miRNA microarray data were correlated with qPCR-measured expression levels. Nevertheless, we identified miRNA-200c to be consistently downregulated in melanocytes, melanoma cell lines, and patient samples, whereas miRNA-205 and miRNA-23b were markedly reduced only in patient samples. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Whole-genome microarrays were performed for analysis of selected melanoma cell lines to identify potential transcriptionally regulated miRNA target genes. Using Ingenuity pathway analysis, we identified a deregulated gene network centered around microphthalmia-associated transcription factor, a transcription factor known to play a key role in melanoma development. Our findings define miRNAs and miRNA target genes that offer candidate biomarkers in human melanoma. Cancer Res; 70(10); 4163–73. ©2010 AACR.
Data from Development of a Lung Cancer Therapeutic Based on the Tumor Suppressor MicroRNA-34
Authors: Wiggins Jason F. , Ruffino Lynnsie , Kelnar Kevin , Omotola Michael , Patrawala Lubna , Brown David , Bader Andreas G.
Published: March 2024

Abstract
Tumor suppressor microRNAs (miRNA) provide a new opportunity to treat cancer. This approach, “miRNA replacement therapy,” is based on the concept that the reintroduction of miRNAs depleted in cancer cells reactivates cellular pathways that drive a therapeutic response. Here, we describe the development of a therapeutic formulation using chemically synthesized miR-34a and a lipid-based delivery vehicle that blocks tumor growth in mouse models of non–small-cell lung cancer. This formulation is effective when administered locally or systemically. The antioncogenic effects are accompanied by an accumulation of miR-34a in the tumor tissue and downregulation of direct miR-34a targets. Intravenous delivery of formulated miR-34a does not induce an elevation of cytokines or liver and kidney enzymes in serum, suggesting that the formulation is well tolerated and does not induce an immune response. The data provide proof of concept for the systemic delivery of a synthetic tumor suppressor mimic, obviating obstacles associated with viral-based miRNA delivery and facilitating a rapid route for miRNA replacement therapy into the clinic. Cancer Res; 70(14); 5923–30. ©2010 AACR.
Data from High Aldehyde Dehydrogenase Activity Identifies Tumor-Initiating and Metastasis-Initiating Cells in Human Prostate Cancer
Authors: van den Hoogen Christel , van der Horst Geertje , Cheung Henry , Buijs Jeroen T. , Lippitt Jenny M. , Guzmán-Ramírez Natalia , Hamdy Freddie C. , Eaton Colby L. , Thalmann George N. , Cecchini Marco G. , Pelger Rob C.M. , van der Pluijm Gabri
Published: March 2024

Abstract
Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer. Cancer Res; 70(12); 5163–73. ©2010 AACR.
Data from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation
Authors: Wu Wenwen , Sato Ko , Koike Ayaka , Nishikawa Hiroyuki , Koizumi Hirotaka , Venkitaraman Ashok R. , Ohta Tomohiko
Published: March 2024

Abstract
The breast cancer suppressor BRCA1 forms a stable heterodimeric E3 ubiquitin ligase with BARD1. Each protein controls the abundance and stability of the other, and loss of the interaction leads to BRCA1 degradation. Here, we show that HERC2, a protein recently implicated in DNA damage repair, targets BARD1-uncoupled BRCA1 for degradation. HERC2 shuttles between the nucleus and the cytoplasm. Its COOH-terminal HECT-containing domain interacts with an NH2-terminal degron domain in BRCA1. HERC2 ubiquitinates BRCA1; this reaction depends on Cys4762 of HERC2, the catalytic ubiquitin binding site, and the degron of BRCA1. The HERC2-BRCA1 interaction is maximal during the S phase of the cell cycle and rapidly diminishes as cells enter G2-M, inversely correlated with the steady-state level of BRCA1. Significantly, HERC2 depletion antagonizes the effects of BARD1 depletion by restoring BRCA1 expression and G2-M checkpoint activity. Conversely, BARD1 protects BRCA1 from HERC2-mediated ubiquitination. Collectively, our findings identify a function for HERC2 in regulating BRCA1 stability in opposition to BARD1. The HERC2 expression in breast epithelial cells and breast carcinomas suggests that this mechanism may play a role in breast carcinogenesis. Cancer Res; 70(15); 6384–92. ©2010 AACR.
Data from Cancer Stem Cell Tumor Model Reveals Invasive Morphology and Increased Phenotypical Heterogeneity
Authors: Sottoriva Andrea , Verhoeff Joost J.C. , Borovski Tijana , McWeeney Shannon K. , Naumov Lev , Medema Jan Paul , Sloot Peter M.A. , Vermeulen Louis
Published: March 2024

Abstract
The recently developed concept of cancer stem cells (CSC) sheds new light on various aspects of tumor growth and progression. Here, we present a mathematical model of malignancies to investigate how a hierarchical organized cancer cell population affects the fundamental properties of solid malignancies. We establish that tumors modeled in a CSC context more faithfully resemble human malignancies and show invasive behavior, whereas tumors without a CSC hierarchy do not. These findings are corroborated by in vitro studies. In addition, we provide evidence that the CSC model is accompanied by highly altered evolutionary dynamics compared with the ones predicted to exist in a stochastic, nonhierarchical tumor model. Our main findings indicate that the CSC model allows for significantly higher tumor heterogeneity, which may affect therapy resistance. Moreover, we show that therapy which fails to target the CSC population is not only unsuccessful in curing the patient, but also promotes malignant features in the recurring tumor. These include rapid expansion, increased invasion, and enhanced heterogeneity. Cancer Res; 70(1); 46–56
Data from Genetically Engineered Salmonella typhimurium as an Imageable Therapeutic Probe for Cancer
Authors: Nguyen Vu H. , Kim Hyung-Seok , Ha Jung-Min , Hong Yeongjin , Choy Hyon E. , Min Jung-Joon
Published: March 2024

Abstract
Tumor-targeting bacteria have been studied in terms of their ability to visualize the infection pathway (through imaging probes) or to carry therapeutic molecules to tumors. To integrate these monitoring and therapeutic functions, we engineered attenuated Salmonella typhimurium defective in guanosine 5′-diphosphate-3′-diphosphate synthesis to carry cytotoxic proteins (cytolysin A) and express reporter genes. We successfully visualized the therapeutic process with these engineered bacteria in mice and found that they often mediated complete tumor (CT-26) eradication on cytotoxic gene induction. Furthermore, treatment with the engineered bacteria markedly suppressed metastatic tumor growth. Cancer Res; 70(1); 18–23
Data from The Skin Cancer Chemotherapeutic Agent Ingenol-3-Angelate (PEP005) Is a Substrate for the Epidermal Multidrug Transporter (ABCB1) and Targets Tumor Vasculature
Authors: Li Luowei , Shukla Suneet , Lee Andrew , Garfield Susan H. , Maloney David J. , Ambudkar Suresh V. , Yuspa Stuart H.
Published: March 2024

Abstract
Ingenol-3-angelate (Ing3A), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma, actinic keratosis, and squamous cell carcinoma (SCC) in situ by topical application. Although structurally related to phorbol esters and a protein kinase C activator, topical Ing3A, but not phorbol 12-myristate 13-acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). Ing3A and PMA both induced acute neutrophilic inflammation on mouse skin, but only Ing3A caused subcutaneous hemorrhage and vascular damage. Both Ing3A and PMA activated extracellular signal-regulated kinase 1/2 (ERK1/2) in epidermis, but Ing3A also activated ERK1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil, or XR9576, modulators of P-glycoprotein (P-gp), prevented Ing3A-induced hemorrhage but not neutrophil infiltration. CsA also impaired the anticancer activity of Ing3A, whereas the anti-inflammatory dexamethasone did not. Ing3A, but not PMA, blocked photoaffinity labeling of human P-gp with [125I]iodoaryazidoprazosin and inhibited P-gp–mediated drug resistance to HCT-15 cells. The intracellular levels of Ing3A were significantly lower in P-gp–expressing cells, and treatment with XR9576 increased the levels to those of cells that do not express P-gp, showing that Ing3A binds to and is transported by P-gp. Taken together, our results suggest that P-gp–mediated absorptive transport, dermal penetration, and vascular damage contribute to the anticancer activity of Ing3A in vivo. Cancer Res; 70(11); 4509–19. ©2010 AACR.
Data from Tetraspanin CD151 Regulates Growth of Mammary Epithelial Cells in Three-Dimensional Extracellular Matrix: Implication for Mammary Ductal Carcinoma In situ
Authors: Novitskaya Vera , Romanska Hanna , Dawoud Marwa , Jones J. Louise , Berditchevski Fedor
Published: March 2024

Abstract
Tetraspanin CD151 is associated with laminin-binding integrins (i.e., α3β1, α6β1, and α6β4) and regulates tumor cell migration and invasion. Here, we examined the role of CD151 in proliferation of mammary epithelial cells using in vitro and in vivo models. Depletion of CD151 suppressed growth of HB2 cells, a nontumorigenic breast epithelial cell line, in three-dimensional (3D) extracellular matrices (ECM) and in Matrigel-based xenografts. Whereas the presence of α3β1 (but not α6 integrins) was necessary to support growth of HB2 cells in 3D ECM, the pro-proliferative activity of CD151 did not require direct interaction with integrins. Furthermore, depletion of CD151 potentiated formation of the internal lumen and partial restoration of polarity when HB2 cells were cultured in 3D ECM. This correlated with a decrease in phosphorylation levels of extracellular signal-regulated kinase 1/2 and cAkt in CD151-negative cells and increase in activation of caspase-3. Accordingly, the number of CD151-positive colonies with internal lumen was increased by ∼5-fold when cells were cultured in the presence of MAP/ERK kinase (U0126) and phosphoinositide 3-kinase (LY29004) inhibitors. To establish the physiologic relevance of pro-proliferative and morphogenetic activities of CD151, we analyzed the expression of this tetraspanin in ductal carcinoma in situ (DCIS), which is characterized by neoplastic proliferation of mammary epithelial cells. Strong homogeneous membrane expression of CD151 was found to be associated with a high grade of DCIS (P = 0.004). Taken together, these results strongly suggest that CD151 complexes play a crucial role in the development of hyperproliferative diseases in the mammary gland. Cancer Res; 70(11); 4698–708. ©2010 AACR.