bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting of Nuclear Factor κB Pathways by Dehydroxymethylepoxyquinomicin, a Novel Inhibitor of Breast Carcinomas: Antitumor and Antiangiogenic Potential In vivo
Authors: Matsumoto Gaku , Namekawa Jun-ichi , Muta Mariko , Nakamura Tadahiko , Bando Hiroko , Tohyama Kazumi , Toi Masakazu , Umezawa Kazuo
Published: June 2022

We previously designed and synthesized the new nuclear factor κB (NF-κB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) derived from the structure of the antibiotic epoxyquinomicin C. We looked into the effect of DHMEQ on cellular phenotypes and tumor growth in mice injected with human breast carcinoma cell line MDA-MB-231 or MCF-7. In estrogen-independent breast adenocarcinoma cell line MDA-MB-231, NF-κB is constitutively activated. The addition of DHMEQ (10 μg/mL) completely inhibited the activated NF-κB for at least 8 hours. On the other hand, NF-κB is not activated in estrogen-dependent MCF-7 cells. In this cell line, DHMEQ completely inhibited the tumor necrosis factor-α-induced activation of NF-κB. DHMEQ did not inhibit the degradation of IκB but inhibited the nuclear translocation of NF-κB by both p65/p50 and RelB/p52 pathways. MDA-MB-231 cells secrete interleukin (IL)-6 and IL-8 without stimulation, and DHMEQ decreased the secretion levels of both cytokines. When MDA-MB-231 or MCF-7 cells were stimulated by tumor necrosis factor-α, the inhibitory effects of DHMEQ were still maintained. I.p. administration of DHMEQ (thrice a week) significantly inhibited the tumor growth of MDA-MB-231 (12 mg/kg) or MCF-7 (4 mg/kg) in severe combined immunodeficiency mice. No toxicity was observed during the experiment, including the loss of body weight. An immunohistological study on resected MCF-7 tumors showed that DHMEQ inhibited angiogenesis and promoted apoptosis. Furthermore, in Adriamycin-resistant MCF-7 cells highly expressing multidrug resistance gene-1, DHMEQ also exhibited the above capability, including down-regulation of IL-8. Thus, DHMEQ might be a potent drug for the treatment of various breast carcinomas by inhibiting the NF-κB activity.
Loss of Reduced Folate Carrier Function and Folate Depletion Result in Enhanced Pemetrexed Inhibition of Purine Synthesis
Authors: Zhao Rongbao , Zhang Shubing , Hanscom Marie , Chattopadhyay Shrikanta , Goldman I. David
Published: June 2022

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 μmol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC50, in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.
Potentiation of a Dendritic Cell Vaccine for Murine Renal Cell Carcinoma by CpG Oligonucleotides
Authors: Chagnon Fanny , Tanguay Simon , Ozdal Ozdem Levent , Guan Meng , Ozen Zeynep Z. , Ripeau Jean-Sébastien , Chevrette Mario , Elhilali Mostafa M. , Thompson-Snipes Lu Ann
Published: June 2022

Purpose: An ideal vaccine therapy for tumors should activate both effector and memory immune responses against tumor-specific antigens. Here we investigated the effect of CpG oligodeoxynucleotides (CpG-ODN) for their ability to potentiate the activity of tumor antigen–pulsed bone marrow–derived dendritic cells (DC) in a vaccine model for the treatment of murine renal cell carcinoma (RENCA).

Experimental Design: First we evaluated the effects of a murine renal cell carcinoma (RENCA) on immune cell activity in a mouse model using in vitro assays for T-cell proliferation and natural killer cell activation. To overcome the immune suppression of the tumor, we s.c. injected groups of 10 mice with dendritic cells and tumor cells. We compared the effect of different conditioning regimens of the DCs with RENCA antigen and/or CpG-ODNs before injection by measuring tumor size twice a week.

Results: Tumor growth was shown to negatively affect spleen cell and T-cell proliferation, IFN-γ production, natural killer cell activity, and NF-κB activation in T cells. In this model, we have shown that RENCA-pulsed CpG-ODN-treated DCs were able not only to significantly reduce tumor growth but also to prevent tumor implantation in 60% of mice. Tumor-free mice were resistant to tumor challenge and the immunity conferred by the vaccine was transferable and tumor specific.

Conclusions: This data show that RENCA down-modulates the immune response, and DC vaccine therapy, in conjunction with CpG-ODN, can restore tumor-specific immunity.
Transcriptional Profiling Identifies Gene Expression Changes Associated with IFN-α Tolerance in Hepatitis C–Related Hepatocellular Carcinoma Cells
Authors: Wong Nathalie , Chan Kathy Y-Y. , Macgregor Pascale F. , Lai Paul B-S. , Squire Jeremy A. , Beheshti Ben , Albert Monique , Leung Thomas W-T.
Published: June 2022

Purpose: Treatment with IFN-α therapy has been shown to exhibit antitumor effects on patients with hepatocellular carcinoma (HCC). However, individual responses remained unpredictable because of the frequent presence of intrinsic or acquired IFN-α resistance. Hence, delineation of molecular targets implicated in the resistant pathway holds value in refining the therapeutic benefits of IFN-α.

Experimental Design: The current study analyzed the effect of IFN-α in human HCC cells. Three hepatitis C virus (HCV)–related, five hepatitis B virus (HBV)–related and two non-B non-C–related cell lines were subjected to IFN-α treatment and the cytotoxic effect on cell viability was measured. Further analysis by cDNA microarray and quantitative reverse transcription-PCR were conducted to examine the gene expression changes that mediated the IFN-α resistance observed.

Results: According to the IC50 values determined, HCV-related cell lines indicated distinct resistance (IC50, 389-1468 units/mL) compared with the HBV-related (IC50, 11-77 units/mL) and non-B non-C–related cell lines (IC50, 24-108 units/mL). Unsupervised hierarchical clustering on array data indicated three HCV-related cell lines to cluster independently from the sensitive cell lines, suggesting discrete features in association with IFN-α tolerance. Moreover, Significance Analysis of Microarrays analysis indicated the differential expression of 149 expressed sequence tags that represented 51 up-regulated and 98 down-regulated genes in the resistant cell lines. Comparing the temporal pattern of gene expression between 6- and 24-hour treatments, candidate genes that were considerably induced with time were further highlighted in the tolerant HCV-related cell lines. These candidates were verified by quantitative reverse transcription-PCR, which confirmed the down-regulation of UBA2, ZNF185, and FOXF1 and up-regulation of UBE4B in the drug-tolerant cells.

Conclusions: Our present study showed that the insensitivity to IFN-α therapy in HCC cells is associated with drug-inducible transcriptional alterations. Furthermore, our investigation highlighted potential candidate genes in conferring an anti-apoptotic effect toward IFN-α treatment.
Dual Inhibition of RET and FGFR4 Restrains Medullary Thyroid Cancer Cell Growth
Authors: Ezzat Shereen , Huang Ping , Dackiw Alan , Asa Sylvia L.
Published: June 2022

Medullary thyroid cancer is frequently an aggressive form of carcinoma for which there are currently no effective forms of systemic therapy. These carcinomas arise as a result of activating mutations in the RET proto-oncogene transmembrane tyrosine kinase receptor. We, therefore, examined the potential efficacy of the tyrosine kinase inhibitor STI571 on the growth of human TT medullary cancer cells in vitro and in xenografted severe combined immunodeficiency mice. Treatment with STI571 resulted in inhibition of RET phosphorylation, cell proliferation, tumor growth and invasiveness. Based on the profile of expression of fibroblast growth factor receptors (FGFR), we examined the effects of FGFR tyrosine kinase inhibition using the small molecule FGFR inhibitor PD173074. This inhibitor resulted in abrogation of fibroblast growth factor-1-mediated FGFR4 phosphorylation in TT cells, an effect that was accompanied by significant arrest of cell proliferation and tumor growth in vivo. Moreover, the combination of STI571 and PD173074 resulted in greater suppression of cell proliferation in vitro and tumor control in vivo than that achieved with either agent alone. These data highlight RET and FGFR4 as therapeutic targets and suggest a potential role for the combined use of tyrosine kinase inhibitors in the management of inoperable medullary thyroid cancers.
Recombinant Adeno-Associated Virus 2-Mediated Antiangiogenic Prevention in a Mouse Model of Intraperitoneal Ovarian Cancer
Authors: Isayeva Tatyana , Ren Changchun , Ponnazhagan Selvarangan
Published: June 2022

Purpose: In the present study, we sought to determine the potential of sustained transgene expression by a single i.m. administration of recombinant adeno-associated virus 2 (rAAV) encoding angiostatin and endostatin in inhibiting i.p. ovarian cancer growth and dissemination in a preclinical mouse model.

Experimental Design: Cohorts of female athymic nude mice received either no virus or 1.2 × 1011 particles of rAAV encoding green fluorescence protein or endostatin plus angiostatin, i.m. Three weeks later, the mice were i.p. injected with 106 human epithelial ovarian cancer cell line SKOV3.ip1. As a measure of effectiveness of the therapy, tumor weight, abdominal distension, ascites volume and vascular endothelial growth factor level, and tumor weight were determined. Immunohistochemistry was done to determine tumor cell apoptosis and endothelial cell proliferation following the therapy. Tumor-free survival was recorded as the end point.

Results: Results indicated a significant tumor-free survival (P < 0.003) following therapy with rAAV encoding endostatin and angiostatin compared with untreated or rAAV-green fluorescence protein–treated mice. Ascites volume in rAAV endostatin and angiostatin–treated mice was significantly lower than naive mice and contained less hemorrhage and tumor conglomerates. The level of vascular endothelial growth factor in the ascites of antiangiogenic vector treated mice was also significantly less compared with the untreated mice. Immunohistochemical analyses indicated increased tumor cell apoptosis and decreased blood vasculature following rAAV endostatin and angiostatin treatment.

Conclusion: The results indicate that antiangiogenic genetic prevention from stable systemic levels of angiostatin and endostatin by i.m. administration of rAAV can be used for the treatment of i.p. ovarian cancer growth and dissemination.
A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor Imaging and Therapy
Authors: Zitzmann Sabine , Mier Walter , Schad Arno , Kinscherf Ralf , Askoxylakis Vasileios , Krämer Susanne , Altmann Annette , Eisenhut Michael , Haberkorn Uwe
Published: June 2022

Purpose: Prostate carcinomas belong to the most widespread tumors, and their number is increasing. Imaging modalities used for diagnosis, such as ultrasound, computed tomography, and positron emission tomography, often produce poor results. Radiolabeled peptides with high sensitivity and specificity for prostate cancer would be a desirable tool for tumor diagnosis and treatment.

Experimental Design: We used phage display and the prostate-specific membrane antigen–negative cell line DU-145 to identify a peptide. The isolated DUP-1 was tested invitro for its binding specificity, kinetics, and affinity. Internalization of the peptide was evaluated with confocal microscopy. The tumor accumulation in a nude mouse model was analyzed with 131I-labeled DUP-1 in PC-3 and DU-145 prostate tumors as well as in the rat prostate tumor model AT-1.

Results: The synthesized peptide showed rapid binding kinetics peaking at 10 minutes. It shows specific binding to prostate carcinoma cells but low binding affinity to nontumor cells. Peptide binding is competed with unlabeled DUP-1, and a time-dependent internalization into DU-145 cells was shown. Biodistribution studies of DUP-1 in nude mice with s.c. transplanted DU-145 and PC-3 tumors showed a tumor accumulation of 5% and 7% injected dose per gram, and bound peptide could not be removed by perfusion. The rat prostate tumor model showed an increase of radioactivity in the prostate tumor up to 300% in comparison with normal prostate tissue.

Conclusions: DUP-1 holds promise as a lead peptide structure applicable in the development of new diagnostic tracers or anticancer agents that specifically target prostate carcinoma.
Prognostic Significance of Metastasis-Associated Protein S100A4 (Mts1) in Prostate Cancer Progression and Chemoprevention Regimens in an Autochthonous Mouse Model
Authors: Saleem Mohammad , Adhami Vaqar Mustafa , Ahmad Nihal , Gupta Sanjay , Mukhtar Hasan
Published: June 2022

Purpose: We recently showed that metastasis-promoting Mts1 gene (S100A4) and protein is overexpressed during progression of prostate cancer in humans. The purpose of this study was to assess the expression of S100A4 during autochthonous prostate cancer progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Because oral consumption of green tea polyphenols (GTP) has been shown to inhibit metastasis and prostate cancer in TRAMP, we further assessed the significance of S100A4 during chemoprevention regimen.

Experimental Design: Male TRAMP mice 8 weeks of age were equally divided into two groups. A freshly prepared 0.1% GTP solution in tap water was supplied thrice a week to experimental animals as the sole source of drinking fluid for 24 weeks, whereas the control group of animals received the same tap water throughout the experiment. The animals were sacrificed at 0, 8, 16, and 24 weeks of GTP feeding and were analyzed for S100A4 and E-cadherin. Additional untreated and treated nontransgenic controls were also included in the study.

Results: With the progression of age and prostate cancer growth in TRAMP mice, an increase in the expression of S100A4 at mRNA and protein level in dorsolateral prostate, but not in nontransgenic mice, occurred. GTP feeding to TRAMP mice resulted in marked inhibition of prostate cancer progression, which was associated with reduction of S100A4 and restoration of E-cadherin.

Conclusions: S100A4 represents a promising marker for prostate cancer progression and could be employed as a biomarker in chemoprevention regimens.
Expression of Receptor Activator of Nuclear Factor-κB Ligand Is Inversely Correlated with Metastatic Phenotype in Breast Carcinoma
Authors: Bhatia Pardeep , Sanders M. Melinda , Hansen Marc F.
Published: June 2022

During normal bone remodeling, the receptor activator of nuclear factor-κB (RANK) interacts with its ligand RANKL, which is present on pre-osteoclasts, resulting in bone resorption and initiation of new bone formation. When breast cancer metastasizes to bone, normal bone remodeling is disturbed by invasion of tumor cells, resulting in osteolytic lesions. We have studied the expression of both RANK and RANKL in 10 nonneoplastic breast samples, 58 infiltrating ductal carcinoma (IDC), and 43 breast cancer bony metastases (BTM). RANK seemed to be present in all samples tested. However, whereas RANKL expression was observed in 90% of nonneoplastic breast, RANKL expression was only observed in 62% of nonmetastatic IDC, 31% of metastatic IDC, and 2% of osteolytic BTM lesions. This decreased or absent expression of RANKL in the tumor cells may allow RANK, which is normally expressed as a receptor on the cell surface, to target RANKL present on the cell surface of normal osteoblasts and stromal cells of the bone. Stimulation of the normal osteoblasts and stromal cells by the tumor cells may then lead to secondary osteoclastogenesis, resulting in the osteolytic phenotype common to breast metastases.
Promoter Hypermethylation in Benign Breast Epithelium in Relation to Predicted Breast Cancer Risk
Authors: Lewis Cheryl M. , Cler Leslie R. , Bu Da-Wei , Zöchbauer-Müller Sabine , Milchgrub Sara , Naftalis Elizabeth Z. , Leitch A. Marilyn , Minna John D. , Euhus David M.
Published: June 2022

Introduction: The tumor suppressor genes RASSF1A, APC, H-cadherin, RARβ2, and cyclin D2 are methylated more frequently in breast cancer than in adjacent benign tissue. However, it is unclear whether promoter methylation of tumor suppressor genes in benign breast tissue is associated with an increased risk for breast cancer.

Methods: Promoter hypermethylation was measured in benign and malignant breast samples obtained by fine needle aspiration biopsy from 27 breast cancer patients and 55 unaffected women whose risk of breast cancer had been defined using the Gail, Claus, and BRCAPRO models.

Results: Cyclin D2 methylation occurred in 57% of tumor samples but not in corresponding benign breast samples and in only one sample from an unaffected patient (P < 0.0001). RARβ2 methylation occurred in 32% of benign breast samples from cancer patients but only 9% of similar samples from unaffected women (P = 0.002). Promoter methylation of RASSF1A and APC occurred more frequently (70% and 56%, respectively) in unaffected women at high-risk for breast cancer as defined by the Gail model than in low/intermediate risk women (29% and 20%, P = 0.04 and P = 0.03). Of the Gail model risk factors, only number of prior breast biopsies was highly correlated with APC and RASSF1A methylation (P = 0.0001 and 0.02, respectively).

Conclusions: Since cyclin D2 promoter methylation occurs almost exclusively in tumors, it may be possible to exploit it for the early detection of breast cancer. Promoter methylation of APC, RARβ2, and RASSF1A in benign breast epithelium is associated with epidemiologic markers of increased breast cancer risk.
ATBF1-A Messenger RNA Expression Is Correlated with Better Prognosis in Breast Cancer
Authors: Zhang Zhenhuan , Yamashita Hiroko , Toyama Tatsuya , Sugiura Hiroshi , Ando Yoshiaki , Mita Keiko , Hamaguchi Maho , Kawaguchi Makoto , Miura Yutaka , Iwase Hirotaka
Published: June 2022

Purpose: The AT motif-binding factor 1 (ATBF1) gene was first identified as a suppressor of the α-fetoprotein (AFP) gene through its binding to an AT-rich enhancer element of this gene. The gene is located at chromosome 16q22.3-q23.1 where loss of heterozygosity has been observed in various malignant tumors, especially in breast cancer. It was also found that in highly malignant AFP-producing gastric cancer cells the expression of AFP is inhibited by ATBF1-A. This led us to hypothesize that there was a link between levels of ATBF1 expression and the metastatic potential of breast cancer and also, therefore, the prognosis of these patients.

Experimental Design: In the present study, the level of ATBF1-A mRNA expression was analyzed using quantitative real-time reverse transcriptase-PCR, in 153 female patients with invasive carcinoma of the breast. ATBF1-A protein expression was also determined by immunohistochemistry from available 90 cases of paired tissues. An association was sought between ATBF1-A expression and various clinicopathologic factors.

Results: ATBF1-A mRNA was expressed at significantly higher levels in breast cancer patients with no axillary lymph node involvement, with small tumors measuring <2 cm and in estrogen receptor-α–positive tumors. By contrast, no relationship was found between ATBF1-A mRNA expression and ATBF1-A protein expression, and also no relationship was found between ATBF1-A protein expression and any of the other clinicopathologic factors. Patients expressing high levels of ATBF1-A mRNA tended to have a better prognosis than those expressing low levels. Univariate and multivariate prognostic analyses showed that ATBF1-A mRNA expression is an independent prognostic factor for disease-free survival.

Conclusions: In breast cancer, levels of ATBF1-A mRNA may serve as a predictive indicator of lymph node metastasis. The results of this study also imply that ATBF1-A gene expression may have potential both as a marker of endocrine responsiveness and also as a prognostic indicator for breast cancer progression.
Overexpression of h-prune in Breast Cancer is Correlated with Advanced Disease Status
Authors: Zollo Massimo , Andrè Alessandra , Cossu Antonio , Sini Maria C. , D'Angelo Anna , Marino Natascia , Budroni Mario , Tanda Francesco , Arrigoni Gianluigi , Palmieri Giuseppe
Published: June 2022

Purpose: The h-prune gene is involved in cellular motility and metastasis formation in breast cancer through interacting with the nm23-H1 protein. The aim of this study was to better define the clinical and pathologic role of h-prune in breast cancer patients.

Experimental Design: Using immunohistochemistry, we assessed h-prune and nm23-H1 protein expression in two series of breast cancer patients: (i) in 2,109 cases with pathologic reports on primary tumors and (ii) in 412 cases with detailed clinical information. To assess the role of DNA amplification in gene activation, the h-prune copy number was evaluated by fluorescence in situ hybridization analysis in 1,016 breast cancer cases.

Results: In the patients tested (n = 2,463), 1,340 (54%) had an increased level of h-prune expression; a positive immunostaining for nm23-H1 was observed in 615 of 2,061 (30%) cases. Overexpression of h-prune was associated with multiple gene copy number at chromosome 1q21.3 in a very limited fraction of cases (68 of 1,016; 6.7%), strongly indicating that alternative pathways induce h-prune activation in breast cancer. Multivariate Cox regression analysis showed that neither h-prune overexpression nor decreased nm23-H1 immunostaining is independent prognostic factors. However, a significant association of h-prune overexpression with either advanced lymph node status (P = 0.017) or presence of distant metastases (P = 0.029) was observed.

Conclusions: Although not significantly correlated with overall survival, positive h-prune immunostaining identifies subsets of breast cancer patients with higher tumor aggressiveness. Further investigations using larger collections of advanced breast cancer patients are required for assessing the predictive role of h-prune in breast cancer.
A Robust Assay for Alternative Lengthening of Telomeres in Tumors Shows the Significance of Alternative Lengthening of Telomeres in Sarcomas and Astrocytomas
Authors: Henson Jeremy D. , Hannay Jonathan A. , McCarthy Stanley W. , Royds Janice A. , Yeager Thomas R. , Robinson Robert A. , Wharton Stephen B. , Jellinek David A. , Arbuckle Susan M. , Yoo Jinyoung , Robinson Bruce G. , Learoyd Diana L. , Stalley Paul D. , Bonar S. Fiona , Yu Dihua , Pollock Raphael E. , Reddel Roger R.
Published: June 2022

Purpose and Experimental Design: Telomeres of tumor cells may be maintained by telomerase or by alternative lengthening of telomeres (ALT). The standard ALT assay requires Southern analysis of high molecular weight genomic DNA. We aimed to establish and validate an ALT assay suitable for archived paraffin-embedded tumors and to use it to examine the prevalence and clinical significance of ALT in various types of tumors that are often telomerase negative.

Results: To assay for ALT, we detected ALT-associated promyelocytic leukemia (PML) bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization. APBs are PML nuclear domains containing telomeric DNA and are a known hallmark of ALT in cell lines. The APB assay concurred with the standard ALT assay in 62 of 62 tumors and showed that 35% of 101 soft tissue sarcomas (STS), 47% of 58 osteosarcomas (especially younger patients), 34% of 50 astrocytomas, and 0% of 17 papillary thyroid carcinomas were ALT positive (ALT+). The prevalence of ALT varied greatly among different STS subtypes: malignant fibrous histiocytomas, 77%; leiomyosarcomas, 62%; liposarcomas, 33%; synovial sarcomas, 9%; and rhabdomyosarcomas, 6%. ALT correlated with survival in glioblastoma multiforme and occurred more often in lower-grade astrocytomas, but ALT+ and ALT− sarcomas were equally aggressive in terms of grade and clinical outcome.

Conclusion: The APB assay for ALT is suitable for paraffin-embedded tumors. It showed that a substantial proportion of STS, osteosarcomas, and astrocytomas, but not papillary thyroid carcinomas use ALT. APB positivity correlated strongly with survival of patients with astrocytomas.
Cyclin D3 Immunoreactivity Is an Independent Predictor of Survival in Laryngeal Squamous Cell Carcinoma
Authors: Pruneri Giancarlo , Pignataro Lorenzo , Valentini Stefano , Fabris Sonia , Maisonneuve Patrick , Carboni Nadia , Pece Salvatore , Capra Maria , Del Curto Barbara , Neri Antonino , Viale Giuseppe
Published: June 2022

Purpose: To analyze the prevalence and clinical relevance of cyclin D3 abnormalities in laryngeal squamous cell carcinoma (LSCC).

Experimental Design: Cyclin D3 immunoreactivity was evaluated in 223 formalin-fixed and paraffin-embedded samples of LSCC patients with a mean follow-up of 62.8 ± 43.2 months. The occurrence of cyclin D3 extra signals was analyzed by fluorescence in situ hybridization in 47 randomly selected cases collected in a tissue microarray. Cyclin D1 immunoreactivity had been previously investigated in 133 cases.

Results: Cyclin D3 immunoreactivity and gene extra signals were found in 39.5% and 42.6% of the cases, respectively, and the concordance between immunohistochemical and fluorescence in situ hybridization results was 70.2% (P = 0.0085). Cyclin D3 immunoreactivity was significantly associated with a high risk of death. Multivariate analysis showed that high tumor grade, exophytic/ulcerating tumor type, low performance status, and cyclin D3 immunoreactivity were the only independent predictors of poor overall survival. In the 133 cases analyzed for both cyclin D1 and cyclin D3, patients with cyclin D1+/cyclin D3+ tumors experienced the worst prognosis, patients with cyclin D1−/cyclin D3− exhibited the most prolonged survival, and with cyclin D1−/cyclin D3+ or cyclin D1+/cyclin D3− tumors an intermediate course was associated.

Conclusions: Our data suggest that cyclin D3 immunoreactivity, possibly due to the occurrence of gene extra copies, may represent an adjunct in LSCC patients' prognostication and contribute to identify D-type cyclins as potential targets of newly developed therapies.
Small Molecule Inhibitors of IκB Kinase Are Selectively Toxic for Subgroups of Diffuse Large B-Cell Lymphoma Defined by Gene Expression Profiling
Authors: Lam Lloyd T. , Davis R. Eric , Pierce Jackie , Hepperle Michael , Xu Yajun , Hottelet Maria , Nong Yuhua , Wen Danyi , Adams Julian , Dang Lenny , Staudt Louis M.
Published: June 2022

Constitutive activation of the NF-κB pathway is required for survival of the activated B cell–like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL). Here we show that a small molecule IκB kinase (IKK) inhibitor, PS-1145, and related compounds are toxic for ABC DLBCL cell lines but not for cell lines derived from the other prevalent form of DLBCL, germinal center B cell–like DLBCL. Treatment of ABC lines with these inhibitors rapidly induced a series of gene expression changes that were attributable to cessation of constitutive IKK activity, similar to changes induced by acute expression of genetic inhibitors of NF-κB, confirming the effectiveness and specificity of this compound. Before cell death, inhibition of IKK also induced features of apoptosis and an arrest in the G1 phase of the cell cycle. To test further the specificity of this toxicity, an inducible form of NF-κB was created by fusing the p65 NF-κB subunit with the ligand-binding domain of the estrogen receptor (p65-ERD). In the presence of tamoxifen, p65-ERD reversed the toxicity of IKK inhibition and restored expression of many NF-κB target genes. Another subgroup of DLBCL, primary mediastinal B-cell lymphoma (PMBL), also expresses NF-κB target genes, and treatment of a PMBL cell line with an IKK inhibitor was toxic and induced gene expression changes of a distinct group of NF-κB target genes. These studies validate the NF-κB pathway as a promising therapeutic target in ABC DLBCL, PMBL, and other lymphomas that depend on the activity of NF-κB for survival and proliferation.
Addition of Histamine to Interleukin 2 Treatment Augments Type 1 T-Cell Responses in Patients with Melanoma In vivo: Immunologic Results from a Randomized Clinical Trial of Interleukin 2 with or without Histamine (MP 104)
Authors: Asemissen Anne Marie , Scheibenbogen Carmen , Letsch Anne , Hellstrand Kristoffer , Thorén Fredrik , Gehlsen Kurt , Schmittel Alexander , Thiel Eckhard , Keilholz Ulrich
Published: June 2022

Purpose: Preclinical investigations suggest that histamine dihydrochloride (HDC) protects T cells and natural killer cells from inhibition by monocyte-derived reactive oxygen metabolites and synergizes with interleukin (IL) 2 in inducing T-cell activation. Here, we investigate whether this mechanism is operational in patients with melanoma treated with HDC as an adjunct to IL-2.

Experimental Design: Melanoma patients having liver metastases were treated with IL-2 with or without HDC within a randomized, multicenter, phase III trial. The effect of HDC on type 1 and type 2 T-cell cytokine production was investigated in peripheral blood samples from 19 patients with the use of intracellular cytokine flow cytometry. Melanoma-specific T-cell responses were analyzed in eight HLA-A2–positive patients.

Results: Frequencies of CD3+ T cells producing IFN-γ (type 1 T cells) in response to phorbol myristate acetate/ionomycin increased (median, 1.8-fold) in patients receiving IL-2 plus HDC but not in those receiving IL-2 alone (P < 0.01 for comparison between arms). In contrast, the number of IL-13-producing type 2 T cells that increased in patients after treatment with IL-2 was not modulated by HDC. Melanoma- and tyrosinase-specific IFN-γ and IL-13-producing T cells were detected in two of four HLA-A2–positive patients with melanoma following treatment with HDC + IL-2.

Conclusions: Treatment of patients with stage IV melanoma with HDC in combination with IL-2 increases type 1 T-cell responses and may promote induction of melanoma-specific T cells. These effects are of relevance for tumor immunotherapy and provide a potential mechanism for the clinical efficacy of HDC added to IL-2.
Class III β-Tubulin Overexpression Is a Prominent Mechanism of Paclitaxel Resistance in Ovarian Cancer Patients
Authors: Mozzetti Simona , Ferlini Cristiano , Concolino Paola , Filippetti Flavia , Raspaglio Giuseppina , Prislei Silvia , Gallo Daniela , Martinelli Enrica , Ranelletti Franco Oreste , Ferrandina Gabriella , Scambia Giovanni
Published: June 2022

The vast majority of women with advanced ovarian cancer will ultimately relapse and develop a drug-resistant disease with an overall 5-year survival of <50%. Unfortunately, the mechanisms of drug resistance actually operating in patients are still unknown. To address this issue, in 41 patients affected by advanced ovarian cancer the three main mechanisms of paclitaxel resistance were investigated: overexpression of MDR-1 gene, point mutations at prominently expressed α-tubulin and β-tubulin genes and selective alterations in the expression of β-tubulin isotypes. MDR-1 and the β-tubulin isotypes expression were evaluated by semiquantitative and real-time PCR. On the same specimens, quantitative immunohistochemistry was also done in the tumor area. No statistically significant changes of MDR-1 expression were noticed between the sensitive and resistant patients either at the mRNA or protein level. The tubulin mutations for the ubiquitous α-tubulin and β-tubulin genes were evaluated by automated DNA sequencing, and in all patients, no mutations were detected in both resistant and sensitive cases. With regard to the expression of tubulin isoforms, a statistically significant up-regulation of class III β-tubulin was found in the resistant subset. It is worth noting that this statistically significant increase of the expression of class III β-tubulin was detectable at the mRNA and protein level. By a direct comparison of the three main known mechanisms of paclitaxel resistance, this study indicates that overexpression of class III β-tubulin is the most prominent mechanism of paclitaxel resistance in ovarian cancer.
TAS-108, a Novel Oral Steroidal Antiestrogenic Agent, Is a Pure Antagonist on Estrogen Receptor α and a Partial Agonist on Estrogen Receptor β with Low Uterotrophic Effect
Authors: Yamamoto Yasuji , Shibata Jiro , Yonekura Kazuhiko , Sato Kouji , Hashimoto Akihiro , Aoyagi Yoshimi , Wierzba Konstanty , Yano Shingo , Asao Tetsuji , Buzdar Aman U. , Terada Tadafumi
Published: June 2022

Purpose: Investigators are currently conducting phase II trials on TAS-108, a novel oral steroidal antiestrogenic agent. The purpose of this study is to investigate the molecular and pharmacologic properties of TAS-108 comparedwith other antiestrogenic agents such as tamoxifen,raloxifene, and fulvestrant.

Experimental Design: The antagonistic or agonistic activities of these agents against both estrogen receptors (ER) α and β were compared in the reporter assay systems. Their effects on the uterus were evaluated in ovariectomized rat models. The antitumor activity of TAS-108 given p.o. was evaluated in both dimethylbenzanthracene-induced mammary tumor model and human breast cancer MCF-7 cell line xenografts.

Results: TAS-108 inhibited the transactivation of ERα under the presence of 17β-estradiol (E2) and did not induce the transactivation of ERα in the absence of E2, unlike the agonistic activity of tamoxifen. On the other hand, it exhibited the most agonistic activity on ERβ among the antiestrogenic agents tested. When given p.o. in the ovariectomized rat, TAS-108 showed a much weaker estrogenic effect on utterine weight compared to tamoxifen, or with similar levels of raloxifene, a selective estrogen receptor modulator. Also, TAS-108 strongly inhibited tumor growth in dimethylbenzanthracene-induced mammary carcinomain the rat, the endogenous E2 model, at a dosage of 1 to 3 mg/kg/day. It also inhibited high exogenous E2, inducing tumor growth against MCF-7 xenografts at a dosage of 1 mg/kg/day without any toxic manifestation.

Conclusions: Taken together, p.o. treatment with TAS-108 has a novel mode of action on ERs and inhibits E2-dependent tumor growth with little uterotrophic effect.
Clinical and Molecular Evidence for c-kit Receptor as a Therapeutic Target in Neuroblastic Tumors
Authors: Uccini Stefania , Mannarino Olga , McDowell Heather P. , Pauser Ursula , Vitali Roberta , Natali Pier Giorgio , Altavista Pierluigi , Andreano Tiziana , Coco Simona , Boldrini Renata , Bosco Sandro , Clerico Anna , Cozzi Denis , Donfrancesco Alberto , Inserra Alessandro , Kokai George , Losty Paul D. , Nicotra Maria R. , Raschellà Giuseppe , Tonini Gian Paolo , Dominici Carlo
Published: June 2022

Purpose: Clinicobiological characteristics of neuroblastic tumor (NT) expressing c-kit tyrosine kinase receptor and/or its ligand, stem cell factor (SCF), are debated. This study aimed at investigating the clinicobiological features of primary NTs expressing c-kit and/or SCF in order to define the clinical relevance of selective therapeutic targeting.

Experimental Design: c-Kit and SCF expression was studied in 168 NTs using immunohistochemistry and in 106 of 168 using Northern blot. Quantitative determination of c-kit expression in 54 additional NTs was also done using real-time reverse transcription-PCR. Correlations between c-kit and SCF expression and clinicobiological features were analyzed using χ2 test, univariate, and multivariate regression analyses.

Results: c-Kit protein was detected in 21 of 168 NTs (13%) and its mRNA in 23 of 106 NTs (22%). SCF protein was shown in 30 of 106 NTs (28%) and its mRNA in 33 of 106 NTs (31%). No mutations in exon 11 of c-kit gene were identified. By univariate analysis, c-kit and SCF expression correlated with advanced stage, MYCN amplification, and 1p36 allelic loss. Cox simple regression analysis showed that overall survival probability was 17% in the c-kit–positive subset versus 68% in the negative (P < 0.001), 43% in the SCF-positive subset versus 78% in the negative (P < 0.001). When using real-time reverse transcription-PCR, significant levels of c-kit mRNA were found in 35 of 54 NTs (65%), but the correlations with clinicobiological features were no longer documented.

Conclusions: c-Kit expression can be detected in the majority of primary NTs. High levels of expression are preferentially found in tumors with unfavorable clinicobiological variables. c-Kit may represent a useful therapeutic target in a subset of otherwise untreatable NTs.
Results of a Phase II Double-Blinded Randomized Clinical Trial of Difluoromethylornithine for Cervical Intraepithelial Neoplasia Grades 2 to 3
Authors: Vlastos Anne-Thérèse , West Loyd A. , Atkinson E. Neely , Boiko Iouri , Malpica Anais , Hong Waun K. , Follen Michele
Published: June 2022

Purpose: Our purpose was to conduct a double-blinded randomized trial of difluoromethylornithine (DFMO) at 0.125, 0.5 gm/m2, versus placebo in the treatment of cervical intraepithelial neoplasia (CIN) grades 2 to 3. A promising phase I study has shown histopathologic responses at these dose levels.

Experimental Design: Patients with histopathologically confirmed CIN 2-3 lesions were recruited from a colposcopy clinic and underwent Papanicolaou testing, human papillomavirus testing, and colpophotography. They took oral contraception and DFMO or placebo elixir for 28 days and filled out the National Cancer Institute common toxicity calendars. They returned for follow-up and a repeat Papanicolaou smear, colpophotograph, and loop excision of the cervix.

Results: There were no statistically significant differences among the arms in histopathologic response. This could no be explained by any biases in risk factors. The prominent toxicities were diarrhea, dizziness, nausea, and headaches. There were no differences in the toxicities among arms. The Papanicolaou smear was a poor biomarker of response and correlated poorly with the histopathology.

Conclusions: DFMO is no active at 0.125 and 0.5 gm/m2 for 28 days when given orally in CIN 2-3. Higher oral doses or longer administration is necessary, supporting data from breast trials. Alternatively, a trial of topical DFMO might merit attention as activity has been noed in trials of actinic keratoses.
Cyclooxygenase-2 Is a Target of KRASD12, Which Facilitates the Outgrowth of Murine C26 Colorectal Liver Metastases
Authors: Smakman Niels , Kranenburg Onno , Vogten Jan M. , Bloemendaal Alexander L.A. , van Diest Paul , Borel Rinkes Inne H.M.
Published: June 2022

Purpose: Mutational activation of the KRAS oncogene and overexpression of cyclooxygenase-2 (COX-2) contribute to colorectal carcinoma (CRC) development, but the relationship between these two events is unclear. This study was designed to clarify that relationship and to assess the contribution of KRAS-dependent COX-2 to the seeding of CRC cells in the liver and to their outgrowth as liver metastases in an experimental mouse model.

Experimental Design: The effect of RNA interference–mediated KRAS knockdown on COX-2 expression and activity was tested in murine C26 CRC cells. The contribution of KRAS-dependent COX-2 to early metastatic tumor cell seeding (by intravital microscopy) and outgrowth of metastases in the liver (by bioluminescence imaging) was studied by using parecoxib, a novel and highly selective liver-activated COX-2 inhibitor. Intratumoral cell proliferation, apoptosis, and tumor-associated angiogenesis were assessed by immunohistochemistry on liver tissue sections.

Results: Stable knockdown of mutant KRASD12 in murine C26 CRC cells by RNA interference lead to a dramatic reduction of COX-2 synthesis and prostaglandin E2 production. Inhibition of host or tumor cell COX-2 activity had no effect on early metastatic cell seeding in the liver but greatly reduced intrahepatic tumor cell proliferation and the rate of liver metastasis outgrowth. COX-2 inhibition had no effect on early tumor vascularization or on tumor cell apoptosis.

Conclusions: The high levels of COX-2 enzyme and prostaglandin production in C26 CRC cells are primarily caused by the presence of endogenous mutant KRASD12. Furthermore, COX-2 inhibition affects the tumoral rather than the vascular compartment during the early stages of C26 liver metastasis outgrowth.
Differentiation and Definition of Vascular-Targeted Therapies
Authors: Siemann Dietmar W. , Bibby Michael C. , Dark Graham G. , Dicker Adam P. , Eskens Ferry A.L.M. , Horsman Michael R. , Marmé Dieter , LoRusso Patricia M.
Published: June 2022

The therapeutic potential of targeting the tumor vascular supply is now widely recognized. Intense research and development activity has resulted in a variety of investigational agents, a number of which are currently in clinical development. As these novel agents are quite distinct from the cytotoxic drugs conventionally used in the treatment of solid tumors, it will be particularly important to ensure early differentiation of these vascular-targeted therapies in order to encourage widespread understanding of their potential benefits and application in the clinic. Two distinct groups of vascular-targeted therapies have evolved: antiangiogenic agents and vascular-disrupting approaches. These differ in three key respects: their physiologic target, the type or extent of disease that is likely to be susceptible, and the treatment scheduling. Inhibitors of angiogenesis interfere with new vessel formation and therefore have a preventative action, require chronic administration, and are likely to be of particular benefit in early-stage or asymptomatic metastatic disease. Vascular-disrupting agents target the established tumor blood vessels, resulting in tumor ischemia and necrosis. These agents are therefore given acutely, show more immediate effects, and may have particular efficacy against advanced disease. It is essential that these agents can be readily distinguished from conventional therapies and that an understanding of key differences between the two types of vascular-targeted therapies is fostered. Here, a simple taxonomy and nomenclature is proposed in anticipation that the therapeutic potential of this novel class can be realized as these approaches advance in clinical settings and a new anticancer strategy becomes available in the clinic.
Geranylgeranylated Proteins are Involved in the Regulation of Myeloma Cell Growth
Authors: van de Donk Niels W.C.J. , Lokhorst Henk M. , Nijhuis Evert H.J. , Kamphuis Marloes M.J. , Bloem Andries C.
Published: June 2022

Purpose: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. This study examined the role of farnesylated and geranylgeranylated proteins in the regulation of myeloma cell proliferation.

Experimental Design: Antiproliferative and apoptotic effects of various modulators of farnesylated and geranylgeranylated proteins were investigated in myeloma cells.

Results: Depletion of geranylgeranylpyrophosphate inhibited myeloma cell proliferation through accumulation of cells in G1 phase of the cell cycle and loss of cells in S phase. In contrast, depletion of farnesylpyrophosphate had no or only minor effects. Furthermore, inhibition of geranylgeranyl transferase I activity was more effective in reducing myeloma cell growth when compared with inhibition of farnesyl transferase activity. This indicates that protein geranylgeranylation is important for myeloma cell proliferation and cell cycle progression through G1. Geranylgeranylated target proteins involved in the control of proliferation include GTPases, such as Rac-1, Cdc42, and RhoA. Inhibition of Rho, Rac, and Cdc42 GTPases by toxin B reduced proliferation, without affecting cell viability, whereas specific inhibition of Rho GTPases by C3 exoenzyme was without effect. This suggests a role for Rac and/or Cdc42 GTPases in myeloma cell growth. Rac-1 activity was found in all myeloma cell lines and was suppressed by the depletion of intracellular pools of geranylgeranylpyrophosphate, whereas interleukin-6 rapidly induced Rac-1 activation. Furthermore, dominant-negative Tat-Rac-1 reduced myeloma cell proliferation, whereas constitutively active Tat-Rac-1 enhanced proliferation.

Conclusion: These results indicate that protein geranylgeranylation is essential for myeloma cell proliferation and suggest that Rac-1 is a regulator of myeloma cell growth.
Alternative mRNA Splicing of Liver Intestine-Cadherin in Hepatocellular Carcinoma
Authors: Wang Xiao Qi , Luk John M. , Leung Pauline P. , Wong Bonnie W. , Stanbridge Eric J. , Fan Sheung Tat
Published: June 2022

Purpose: To identify alternative splicing of the liver intestine-cadherin (LI-cadherin) gene in hepatocellular carcinoma (HCC) and correlate its aberrant expression with clinical outcomes.

Experimental Design: Reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR were used to examine alternative mRNA splicing and mRNA level of LI-cadherin in 50 paired tumor-peritumor tissues of 50 HCC and 8 normal liver specimens. The minigene exon-trapping strategy was employed to investigate the splicing mechanism introduced by nucleotide polymorphisms. Association of LI-cadherin splicing with tumor venous infiltration, first-year tumor recurrence, and overall survival after partial hepatectomy were determined.

Results: Alternative mRNA splicing of LI-cadherin was identified in half of the HCC specimens. Sequencing analysis indicated the loss of exon 7 in the spliced LI-cadherin gene. LI-cadherin mRNA was up-regulated from 2.58-fold to 800-fold in over 80% of HCC samples when compared with normal liver by quantitative PCR. Furthermore, nucleotide polymorphisms were identified in putative branch point at IVS6 + 35 (intron 6) as well as in coding sequence 651 (exon 6) in HCC tissues, which may affect alternative mRNA splicing. Clinically, those patients who harbored the alternative splicing of LI-cadherin were strongly associated with shorter overall survival time (P < 0.01) as well as higher incidences of tumor recurrences and venous infiltration (both P < 0.05) after hepatectomy.

Conclusions: Over-expression of LI-cadherin was frequently detected in liver cancer patients. Aberrant alternative splicing of LI-cadherin was detected in 50% of HCC specimens and its clinical significance hinted at early tumor recurrence and poor overall survival of HCC patients.