ABSTRACT
BACKGROUND: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. METHODS: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. RESULTS: Using a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. CONCLUSIONS: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.
Subject(s)
Breast Neoplasms , Humans , Animals , Mice , Female , Trastuzumab/pharmacology , Trastuzumab/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Docetaxel/pharmacology , Apoptosis , Leukocytes, Mononuclear/metabolism , Receptor, ErbB-2/metabolism , Cell Line, Tumor , G2 Phase Cell Cycle Checkpoints , Mitosis , Drug Resistance, Neoplasm , Tumor Microenvironment , Receptors, CXCR4/geneticsABSTRACT
BACKGROUND: Prostate cancer (PCa) bone metastases have been shown to be more resistant to docetaxel than soft tissue metastases. The proinflammatory chemokine receptor CXCR4 has been shown to confer resistance to docetaxel (DOC) in PCa cells. Balixafortide (BLX) is a protein epitope mimetic inhibitor of CXCR4. Accordingly, we hypothesized that BLX would enhance DOC-mediated antitumor activity in PCa bone metastases. METHODS: PC-3 luciferase-labeled cells were injected into the tibia of mice to model bone metastases. Four treatment groups were created: vehicle, DOC (5 mg/kg), BLX (20 mg/kg), and combo (receiving both DOC and BLX). Mice were injected twice daily subcutaneously with either vehicle or BLX starting on Day 1 and weekly intraperitoneally with DOC starting on Day 1. Tumor burden was measured weekly via bioluminescent imaging. At end of study (29 days), radiographs were taken of the tibiae and blood was collected. Serum levels of TRAcP, IL-2, and IFNγ levels were measured using ELISA. Harvested tibiae were decalcified and stained for Ki67, cleaved caspase-3, and CD34 positive cells or microvessels were quantified. RESULTS: Tumor burden was lower in the combo group compared to the DOC alone group. Treatment with the combination had no impact on the number of mice with osteolytic lesions, however the area of osteolytic lesions was lower in the combo group compared to the vehicle and BLX groups, but not the DOC group. Serum TRAcP levels were lower in the combo compared to vehicle group, but not the other groups. No significant difference in Ki67 staining was found among the groups; whereas, cleaved caspase-3 staining was lowest in the Combo group and highest in the BLX group. The DOC and combo groups had more CD34+ microvessels than the control and BLX groups. There was no difference between the treatment groups for IL-2, but the combo group had increased levels of IFNγ compared to the DOC group. CONCLUSIONS: Our data demonstrate that a combination of BAL and DOC has greater antitumor activity in a model of PCa bone metastases than either drug alone. These data support further evaluation of this combination in metastatic PCa.
Subject(s)
Bone Neoplasms , Prostatic Neoplasms , Humans , Male , Animals , Mice , Docetaxel/pharmacology , Docetaxel/therapeutic use , Caspase 3 , Disease Models, Animal , Interleukin-2 , Ki-67 Antigen , Tartrate-Resistant Acid Phosphatase , Prostatic Neoplasms/pathology , Cell Line, Tumor , Bone Neoplasms/drug therapy , Bone Neoplasms/secondary , Receptors, CXCR4ABSTRACT
Background: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. Methods: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4expression. Three-dimensional co-culture (tumor cells/ breast cancer-associated fibroblasts / human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effect of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. Results: Using multiple cell lines and patient breast cancer samples we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated that the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using multiple trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Conclusions: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.
ABSTRACT
Polyphor's macrocycle platform led to the discovery of novel antibiotics addressing specifically Gramnegative bacteria by targeting outer membrane proteins. Furthermore, POL6014, an inhibitor of neutrophile elastase and balixafortide, a CXCR4 inhibitor have been discovered and developed from the platform. Currently a combination of balixafortide and eribulin is in Phase III clinical trial for the treatment of patients with advanced metastatic HER2-negative breast cancer.
Subject(s)
Breast Neoplasms , Ketones , Female , HumansABSTRACT
Signaling through chemokine receptor, C-X-C chemokine receptor type 4 (CXCR4) regulates essential processes in normal physiology, including embryogenesis, tissue repair, angiogenesis, and trafficking of immune cells. Tumors co-opt many of these fundamental processes to directly stimulate proliferation, invasion, and metastasis of cancer cells. CXCR4 signaling contributes to critical functions of stromal cells in cancer, including angiogenesis and multiple cell types in the tumor immune environment. Studies in animal models of several different types of cancers consistently demonstrate essential functions of CXCR4 in tumor initiation, local invasion, and metastasis to lymph nodes and distant organs. Data from animal models support clinical observations showing that integrated effects of CXCR4 on cancer and stromal cells correlate with metastasis and overall poor prognosis in >20 different human malignancies. Small molecules, Abs, and peptidic agents have shown anticancer efficacy in animal models, sparking ongoing efforts at clinical translation for cancer therapy. Investigators also are developing companion CXCR4-targeted imaging agents with potential to stratify patients for CXCR4-targeted therapy and monitor treatment efficacy. Here, pre-clinical studies demonstrating functions of CXCR4 in cancer are reviewed.
Subject(s)
Neoplasms/metabolism , Receptors, CXCR4/metabolism , Animals , Humans , Mutation/genetics , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Receptors, CXCR4/genetics , Signal Transduction , Tumor MicroenvironmentABSTRACT
BACKGROUND: The processes of prostate cancer (PCa) invasion and metastasis are facilitated by proteolytic cascade involving multiple proteases, such as matrix metalloproteinases, serine proteases and cysteine proteases including cathepsin K (CatK). CatK is predominantly secreted by osteoclasts and specifically degrades collagen I leading to bone destruction. PCa and breast cancer preferentially metastasize to the bone. Importantly, CatK expression level is greater in PCa bone metastatic sites compared to primary tumor and normal prostate tissues. However, the underlying mechanism of CatK during PCa metastases into the bone remains to be elucidated. We investigated the functional role of CatK during the PCa establishment and growth process in the murine bone. METHODS: CatK mRNA expression was validated by RT-PCR, protein expression by immunoblotting in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Its protein production was measured using ELISA assay. The effect of both knockdowns via siRNA and CatK inhibitor was compared in regard to PCa cell invasion. We further studied the dose-dependent CatK inhibitor effect on conditioned media-induced bone resorption. In setting up an animal model, C4-2B cells were injected into the tibiae of SCID mice. The animals treated with either vehicle or CatK inhibitor for 8 weeks at the time of tumor cell injection (tumor establishment model; protocol I) or 4 weeks after tumor cell injection (tumor progression model; protocol II) were applied to histological and histomorphometric analyses. RESULTS: We confirmed CatK expression in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Furthermore, we observed the inhibitory effects of a selective CatK inhibitor on PCa cell invasion. The CatK inhibitor dose-dependently inhibited PCa-conditioned media-induced bone resorption. Upon injection of C4-2B cells into the tibiae of SCID mice, the selective CatK inhibitor significantly prevented the tumor establishment in protocol I, and reduced the tumor growth in bone in protocol II. It also decreased serum PSA levels in both animal models. The inhibitory effects of the CatK inhibitor were enhanced in combination with zoledronic acid (ZA). CONCLUSION: The selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.
Subject(s)
Bone Neoplasms/prevention & control , Bone Neoplasms/secondary , Cathepsin K/antagonists & inhibitors , Molecular Targeted Therapy/methods , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Protease Inhibitors/therapeutic use , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Bone Neoplasms/genetics , Cathepsin K/genetics , Cell Proliferation/drug effects , Cells, Cultured , Humans , Male , Mice , Mice, SCID , PC-3 Cells , Prostatic Neoplasms/genetics , Protease Inhibitors/pharmacology , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Neutrophil elastase (NE) has been linked to lung neutrophil dysfunction in bronchiectasis and cystic fibrosis (CF), making NE inhibition a potential therapeutic target. NE inhibitor trials have given mixed result perhaps because not all patients have elevated airway NE activity. METHODS: We tested whether a single baseline sputum NE measurement or a combination of clinical parameters could enrich patient populations with elevated NE activity for "personalised medicine". Intra- and interindividual variations of total and active NE levels in induced sputum from patients with CF or bronchiectasis were monitored over 14â days. Patients with established CF and bronchiectasis (n=5 per group) were recruited. NE was measured using three different methods: one total and two active NE assays. Subsequently, we analysed the association between clinical parameters and NE from a large bronchiectasis cohort study (n=381). RESULTS: All three assays showed a high degree of day-to-day variability (0-233% over 14â days). There were strong correlations found between all assays (p<0.0001). Despite high day-to-day variability, patients could be stratified into "high" or "low" groups based on moderate cut-off levels. In the bronchiectasis cohort study, factors most associated with high sputum NE levels were: Pseudomonas aeruginosa infection (ß-estimate 11.5, 95% CI -6.0-29.0), sputum colour (ß-estimate 10.4, 95% CI 4.3-16.6), Medical Research Council dyspnoea score (ß-estimate 6.4, 95% CI 1.4-11.4) and exacerbation history (ß-estimate 3.4, 95% CI 1.4-5.3). Collectively, P. aeruginosa infection, sputum colour and exacerbation frequency provided the greatest specificity for "high" NE (98.7%, 95% CI 7.0-99.6%). CONCLUSION: These results show that patients with bronchiectasis and CF can be effectively divided into "high" or "low" groups, based on sputum NE assays or clinical inclusion criteria.
ABSTRACT
Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Geranyltranstransferase/antagonists & inhibitors , Quinolines/pharmacology , Salicylic Acid/pharmacology , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Geranyltranstransferase/metabolism , Humans , Molecular Structure , Quinolines/chemical synthesis , Quinolines/chemistry , Salicylic Acid/chemical synthesis , Salicylic Acid/chemistry , Structure-Activity RelationshipABSTRACT
The importance of the cell surface receptor CXCR4 and the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) is well-established in normal and malignant hematopoiesis. The Protein Epitope Mimetic POL5551 is a novel and potent antagonist of CXCR4. POL5551 efficiently mobilizes hematopoietic stem and progenitor cells, but its effects in acute lymphoblastic leukemia (ALL) have not been reported. Here, we demonstrate that POL5551 is a potent antagonist of CXCR4 in pre-B and T cell ALL cell lines and pediatric ALL primary samples. POL5551 has activity at nanomolar concentrations in decreasing CXCR4 antibody binding, blocking SDF-1α-mediated phosphorylation of ERK1/2, inhibiting SDF-1α-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy. POL5551 is significantly more effective at inhibiting CXCR4 antibody binding than the FDA-approved CXCR4 inhibitor plerixafor in ALL cell lines and primary samples. We also show that treatment with POL5551 in vitro and cytarabine +/- POL5551 in vivo modulates surface expression of adhesion molecules, findings that may guide the optimal clinical use of POL5551. Finally, we demonstrate that POL5551 increases sensitivity to cytarabine in a xenograft model of a high-risk pediatric ALL, infant MLL-rearranged (MLL-R) ALL. Therefore, disruption of the CXCR4/SDF-1 axis with POL5551 may improve outcomes in children with high-risk ALL.
Subject(s)
Cell Movement/drug effects , Drug Resistance, Neoplasm/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Proteins/pharmacology , Receptors, CXCR4/antagonists & inhibitors , Stromal Cells/drug effects , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Chemotaxis/drug effects , Child , Flow Cytometry , Humans , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Stromal Cells/metabolism , Stromal Cells/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Proteins/pharmacology , Receptors, CXCR4/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Animals , Biomimetic Materials/administration & dosage , Biomimetic Materials/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Chemokine CXCL12/metabolism , Epitopes/metabolism , Furans/administration & dosage , Furans/pharmacology , Humans , Ketones/administration & dosage , Ketones/pharmacology , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Neoplasm Metastasis , Protein Binding/drug effects , Proteins/administration & dosage , Receptors, CXCR4/metabolism , Signal Transduction/drug effects , Survival Analysis , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The CXCR4/stromal cell-derived factor-1 (SDF-1) axis is essential for cell trafficking and has been shown to regulate tumor progression and metastasis in many tumors including multiple myeloma (MM). A second chemokine receptor for SDF-1, CXCR7 was discovered recently and found on activated endothelial cells. We examined the role of CXCR7 in angiogenic mononuclear cells (AMCs) trafficking in MM. Our data demonstrate that AMCs are circulating in patients with MM and in vivo studies show that they specifically home to areas of MM tumor growth. CXCR7 expression is important for regulating trafficking and homing of AMCs into areas of MM tumor growth and neoangiogenesis. We demonstrate that the CXCR7 inhibitor, POL6926, abrogated trafficking of AMCs to areas of MM tumor progression leading to a significant inhibition of tumor progression. These effects were through regulation of endothelial cells and not through a direct tumor effect, indicating that targeting a bone marrow microenvironmental cell can lead to a delay in MM tumor progression. In conclusion, our studies demonstrate that CXCR7 may play an important role in the regulation of tumor progression in MM through an indirect effect on the recruitment of AMCs to areas of MM tumor growth in the bone marrow niche.
Subject(s)
Multiple Myeloma/etiology , Multiple Myeloma/immunology , Receptors, CXCR/metabolism , Animals , Biomimetic Materials/pharmacology , Cell Line, Tumor , Disease Progression , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Multiple Myeloma/pathology , Neoplastic Cells, Circulating/immunology , Neoplastic Cells, Circulating/pathology , Neovascularization, Pathologic , Plasma Cells/immunology , Plasma Cells/pathology , Receptors, CXCR/antagonists & inhibitors , Stem Cell Niche/immunology , Tumor Microenvironment/immunologyABSTRACT
The insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed and constitutively activated in pancreatic cancer, thus representing a promising target for therapy. We investigated the impact of a novel inhibitor of IGF-IR (NVP-AEW541) on signalling and growth of pancreatic cancer. Human pancreatic cancer cells and endothelial cells were employed, and effects of NVP-AEW541 on signalling pathways investigated by Western blotting. NVP-AEW541 diminished the activation of IGF-IR, IRS-1, Erk, Akt and STAT3. Furthermore, NVP-AEW541 reduced cancer cell proliferation and abrogated migratory effects of IGF-I. NVP-AEW541 elicited a direct effect on endothelial cells in terms of reducing endothelial cell migration. In vivo, treatment of mice with NVP-AEW541 significantly reduced orthotopic pancreatic tumour growth, vascularisation, and VEGF expression. Interestingly, NVP-AEW541 lowered serum levels of IGF-binding-protein-3 (IGFBP-3). In conclusion, the IGF-IR inhibitor NVP-AEW541 effectively disrupts IGF-I signalling and reduces pancreatic tumour growth. Hence, blocking IGF-IR could prove valuable for targeted therapy of pancreatic cancer.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Neovascularization, Pathologic/prevention & control , Pancreatic Neoplasms , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Animals , Cell Communication/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathologyABSTRACT
The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate-binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imaging-based assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids.
Subject(s)
Geranyltranstransferase/antagonists & inhibitors , Image Processing, Computer-Assisted/methods , Mevalonic Acid/metabolism , Protein Prenylation , Cell Line, Tumor , Cell Membrane/metabolism , Dimethylallyltranstransferase/metabolism , Diphosphonates/pharmacology , Enzyme Inhibitors/pharmacology , Genes, Reporter , Green Fluorescent Proteins/metabolism , Humans , Imidazoles/pharmacology , Luciferases/metabolism , Methionine/analogs & derivatives , Methionine/pharmacology , Mevalonic Acid/antagonists & inhibitors , RNA, Small Interfering/pharmacology , Reproducibility of Results , Transfection , Zoledronic Acid , ras Proteins/metabolismABSTRACT
Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of monoclonal plasma cells in the bone marrow (BM). Despite recent advances in the treatment, MM represents an incurable disease for which development of new therapies is required. We report the antimyeloma effect of NVP-AEW541, a small molecule that belongs to the pyrrolo[2,3-d]pyrimidine class, identified as a selective inhibitor of the insulin-like growth factor-I receptor (IGF-IR) in vitro kinase activity. NVP-AEW541 had a potent cytotoxic effect on fresh cells and in a murine MM model. NVP-AEW541 partially abrogated the proliferative advantage conferred by the coculture with BM stromal cells and the presence of growth factors produced by the BM microenvironment. In addition, NVP-AEW541 potentiated the action of drugs, such as bortezomib, lenalidomide, dexamethasone or melphalan. Moreover the triple combination of NVP-AEW541, dexamethasone and bortezomib resulted in a significant increase in growth inhibition. Mechanistic studies indicated that NVP-AEW541 provoked a marked cell cycle blockade accompanied by pRb downregulation. Interestingly, NVP-AEW541 increased the levels of p27 associated with a reduction in the CDK2 activity. Finally, NVP-AEW541 induced cell death through caspase-dependent and -independent mechanisms. All these data, suggest the potential effect of IGF-IR kinase inhibitors as therapeutic agents for MM patients.
Subject(s)
Antineoplastic Agents/pharmacology , Multiple Myeloma/pathology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Caspases/physiology , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cytokines/pharmacology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Insulin-Like Growth Factor I/metabolism , Mice , Mice, Nude , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Neoplasm Transplantation , Phosphorylation/drug effects , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Osteoclasts mediate bone destruction in breast cancer skeletal metastases. Cathepsin K is a proteinase that is secreted by osteoclasts and degrades bone. Here, immunohistochemistry revealed that cathepsin K was expressed not only by osteoclasts but also by breast cancer cells that metastasize to bone. Following intratibial injection with cathepsin K-expressing human BT474 breast cancer cells, tumor-bearing mice treated with a clinical dosing regimen of cathepsin K inhibitor (CKI; 50 mg/kg, twice daily) had osteolytic lesions that were 79% smaller than those of tumor-bearing mice treated with the vehicle. The effect of CKI was also studied in a mouse model in which the i.v. inoculation of human B02 breast cancer cells expressing cathepsin K leads to bone metastasis formation. Drug administration was started before (preventive protocol) or after (treatment protocol) the occurrence of osteolytic lesions. In treatment protocols, CKI (50 mg/kg, twice daily) or a single clinical dose of 100 microg/kg zoledronic acid (osteoclast inhibitor) reduced the progression of osteolytic lesions by 59% to 66%. CKI therapy also reduced skeletal tumor burden by 62% compared with vehicle, whereas zoledronic acid did not decrease the tumor burden. The efficacy of CKI at inhibiting skeletal tumor burden was similar in the treatment and preventive protocols. By contrast, CKI did not block the growth of s.c. B02 tumor xenografts in animals. Thus, CKI may render the bone a less favorable microenvironment for tumor growth by inhibiting bone resorption. These findings raise the possibility that cathepsin K could be a therapeutic target for the treatment of bone metastases.
Subject(s)
Bone Neoplasms/drug therapy , Bone Neoplasms/secondary , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Animals , Bone Neoplasms/enzymology , Bone Neoplasms/prevention & control , Breast Neoplasms/pathology , Cathepsin K , Cathepsins/biosynthesis , Cell Line, Tumor , Humans , Immunohistochemistry , Mice , Mice, Nude , Osteolysis/drug therapy , Osteolysis/enzymology , Osteolysis/prevention & control , Random Allocation , Xenograft Model Antitumor AssaysABSTRACT
Deubiquitinating enzymes can prevent the destruction of protein substrates prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) deubiquitinates the antiapoptotic proteins Fatty Acid Synthase and Mdm2. Here, we show that when USP2a is overexpressed in nontransformed cells, it exhibits oncogenic behavior both in vitro and in vivo and prevents apoptosis induced by chemotherapeutic agents. Notably, USP2a silencing in several human cancer cell lines results in apoptosis. Gene set enrichment analysis, which focuses on groups of genes sharing biological function or regulatory pathways, was done on microarray expression data from human prostate cancers. The cell death-related gene set, as well as a selected cluster of validated p53 target genes, were significantly enriched in the low USP2a expression group of tumors. Conversely, genes implicated in fatty acid metabolism were significantly associated with tumors expressing high USP2a (44%). The expression profile analysis is consistent with the effects of USP2a on its known targets, i.e., Fatty Acid Synthase and Mdm2, defining a subset of prostate tumors resistant to apoptosis. USP2a thus represents a therapeutic target in prostate cancer.
Subject(s)
Apoptosis/physiology , Carbon-Nitrogen Lyases/metabolism , Endopeptidases/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Cell Line, Tumor , Colony-Forming Units Assay , Endopeptidases/metabolism , Gene Silencing , Humans , Male , Prostatic Neoplasms/enzymology , RNA, Neoplasm/genetics , RNA, Small Interfering/genetics , Recombinant Proteins/metabolism , Transfection , Ubiquitin/metabolism , Ubiquitin ThiolesteraseABSTRACT
We have investigated the application of near-infrared (NIR) fiber-optic spectroscopy for the diagnosis of pancreatic cancer. Cluster analysis of the Fourier transformed near-infrared (FTNIR) fiber-optic spectra of surgically resected pancreatic tumor tissues allowed discrimination of tumor from normal tissue with high sensitivity and specificity. The sensitivity of the method using spectral information of the CH stretching first overtone region (5951-5608 cm(-1)) was 83.3% with a specificity of 83.3%. Based on the CH stretching second overtone region (8605-7938 cm(-1)) we could achieve a sensitivity of 88.9% and specificity of 72.2%. These findings suggest that NIR spectroscopy offers the potential for minimally invasive in-vivo diagnosis of pancreatic cancer.
Subject(s)
Artificial Intelligence , Biomarkers, Tumor/analysis , Diagnosis, Computer-Assisted/methods , Pancreatic Neoplasms/chemistry , Pancreatic Neoplasms/diagnosis , Spectrophotometry, Infrared/methods , Adult , Aged , Aged, 80 and over , Cluster Analysis , Female , Fiber Optic Technology , Humans , Male , Middle Aged , Optical Fibers , Pattern Recognition, Automated/methods , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Infrared/instrumentationABSTRACT
Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.
Subject(s)
Fluorescent Dyes/chemical synthesis , Ubiquitin/analogs & derivatives , Ubiquitin/chemical synthesis , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Endopeptidases/chemistry , Endopeptidases/metabolism , Fluorescent Dyes/chemistry , Kinetics , Lysine/chemistry , Lysine/metabolism , Rhodamines/chemistry , Rhodamines/metabolism , Time Factors , Ubiquitin/chemistry , Ubiquitin Thiolesterase/chemistry , Ubiquitin Thiolesterase/metabolismABSTRACT
Proteasome inhibition is a therapeutic concept of current interest in anticancer research. We report here the design, synthesis, and biological characterization of prototypes of a new class of noncovalent proteasome inhibitors showing high activity in biochemical and cellular assays.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Binding Sites , Cell Division/drug effects , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/metabolism , Drug Screening Assays, Antitumor/methods , Humans , Hydrogen Bonding , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Oligopeptides/chemistry , Oligopeptides/metabolism , Oligopeptides/pharmacology , Proteasome Endopeptidase Complex , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
IGF-IR-mediated signaling promotes survival, anchorage-independent growth, and oncogenic transformation, as well as tumor growth and metastasis formation in vivo. NVP-AEW541 is a pyrrolo[2,3-d]pyrimidine derivative small molecular weight kinase inhibitor of the IGF-IR, capable of distinguishing between the IGF-IR (IC50 = 0.086 microM) and the closely related InsR (IC50 = 2.3 microM) in cells. As expected for a specific IGF-IR kinase inhibitor, NVP-AEW541 abrogates IGF-I-mediated survival and colony formation in soft agar at concentrations that are consistent with inhibition of IGF-IR autophosphorylation. In vivo, this orally bioavailable compound inhibits IGF-IR signaling in tumor xenografts and significantly reduces the growth of IGF-IR-driven fibrosarcomas. Thus, NVP-AEW541 represents a class of selective, small molecule IGF-IR kinase inhibitors with proven in vivo antitumor activity and potential therapeutic application.