ABSTRACT
A series of bifunctional compounds have been discovered for their dual functionality as MER/AXL inhibitors and immune modulators. The furanopyrimidine scaffold, renowned for its suitability in kinase inhibitor discovery, offers at least three distinct pharmacophore access points. Insights from molecular modeling studies guided hit-to-lead optimization, which revealed that the 1,3-diketone side chain hybridized with furanopyrimidine scaffold that respectively combined amino-type substituent and 1H-pyrazol-4-yl substituent on the top and bottom of the aryl regions to produce 22 and 33, exhibiting potent antitumor activities in various syngeneic and xenograft models. More importantly, 33 demonstrated remarkable immune-modulating activity by upregulating the expression of total T-cells, cytotoxic CD8+ T-cells, and helper CD4+ T-cells in the spleen. These findings underscored the bifunctional capabilities of 33 (BPR5K230) with excellent oral bioavailability (F = 54.6%), inhibiting both MER and AXL while modulating the tumor microenvironment and highlighting its diverse applicability for further studies to advance its therapeutic potential.
Subject(s)
Antineoplastic Agents , Axl Receptor Tyrosine Kinase , Protein Kinase Inhibitors , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases , Tumor Microenvironment , c-Mer Tyrosine Kinase , Animals , Tumor Microenvironment/drug effects , Humans , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/chemical synthesis , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/therapeutic use , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , c-Mer Tyrosine Kinase/antagonists & inhibitors , c-Mer Tyrosine Kinase/metabolism , Mice , Cell Line, Tumor , Structure-Activity Relationship , Drug Discovery , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/chemical synthesis , Female , Xenograft Model Antitumor Assays , Mice, Inbred BALB C , Cell Proliferation/drug effectsABSTRACT
BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common type of cancer and has a high mortality rate worldwide. Sorafenib is the only systemic treatment demonstrating a statistically significant but modest overall survival benefit. We previously have identified the aurora kinases (AURKs) and FMS-like tyrosine kinase 3 (FLT3) multikinase inhibitor DBPR114 exhibiting broad spectrum anti-tumor effects in both leukemia and solid tumors. The purpose of this study was to evaluate the therapeutic potential of DBPR114 in the treatment of advanced HCC. METHODS: Human HCC cell lines with histopathology/genetic background similar to human HCC tumors were used for in vitro and in vivo studies. Human umbilical vein endothelial cells (HUVEC) were used to evaluate the drug effect on endothelial tube formation. Western blotting, immunohistochemical staining, and mRNA sequencing were employed to investigate the mechanisms of drug action. Xenograft models of sorafenib-refractory and sorafenib-acquired resistant HCC were used to evaluate the tumor response to DBPR114. RESULTS: DBPR114 was active against HCC tumor cell proliferation independent of p53 alteration status and tumor grade in vitro. DBPR114-mediated growth inhibition in HCC cells was associated with apoptosis induction, cell cycle arrest, and polyploidy formation. Further analysis indicated that DBPR114 reduced the phosphorylation levels of AURKs and its substrate histone H3. Moreover, the levels of several active-state receptor tyrosine kinases were downregulated by DBPR114, verifying the mechanisms of DBPR114 action as a multikinase inhibitor in HCC cells. DBPR114 also exhibited anti-angiogenic effect, as demonstrated by inhibiting tumor formation in HUVEC cells. In vivo, DBPR114 induced statistically significant tumor growth inhibition compared with the vehicle control in multiple HCC tumor xenograft models. Histologic analysis revealed that the DBPR114 treatment reduced cell proliferation, and induced apoptotic cell death and multinucleated cell formation. Consistent with the histological findings, gene expression analysis revealed that DBPR114-modulated genes were mostly related to the G2/M checkpoint and mitotic spindle assembly. DBPR114 was efficacious against sorafenib-intrinsic and -acquired resistant HCC tumors. Notably, DBPR114 significantly delayed posttreatment tumor regrowth and prolonged survival compared with the regorafenib group. CONCLUSION: Our results indicated that targeting AURK signaling could be a new effective molecular-targeted agent in the treatment of patients with HCC.
Subject(s)
Antineoplastic Agents , Carcinoma, Hepatocellular , Liver Neoplasms , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm/genetics , Endothelial Cells , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Sorafenib/pharmacology , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Colony-stimulating factor-1 receptor (CSF1R) is implicated in tumor-associated macrophage (TAM) repolarization and has emerged as a promising target for cancer immunotherapy. Herein, we describe the discovery of orally active and selective CSF1R inhibitors by property-driven optimization of BPR1K871 (9), our clinical multitargeting kinase inhibitor. Molecular docking revealed an additional nonclassical hydrogen-bonding (NCHB) interaction between the unique 7-aminoquinazoline scaffold and the CSF1R hinge region, contributing to CSF1R potency enhancement. Structural studies of CSF1R and Aurora kinase B (AURB) demonstrated the differences in their back pockets, which inspired the use of a chain extension strategy to diminish the AURA/B activities. A lead compound BPR1R024 (12) exhibited potent CSF1R activity (IC50 = 0.53 nM) and specifically inhibited protumor M2-like macrophage survival with a minimal effect on antitumor M1-like macrophage growth. In vivo, oral administration of 12 mesylate delayed the MC38 murine colon tumor growth and reversed the immunosuppressive tumor microenvironment with the increased M1/M2 ratio.
Subject(s)
Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Drug Discovery , Immunomodulating Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Receptor, Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Line, Tumor , Colonic Neoplasms/pathology , Immunomodulating Agents/administration & dosage , Immunomodulating Agents/chemistry , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, ß-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.
Subject(s)
Bridged-Ring Compounds/pharmacology , Colorectal Neoplasms , Mutation , Neoplasm Proteins , Paclitaxel/pharmacology , Taxoids/pharmacology , Thrombospondins , Tumor Microenvironment/drug effects , Wnt Signaling Pathway , Animals , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Thrombospondins/antagonists & inhibitors , Thrombospondins/genetics , Thrombospondins/metabolism , Tumor Microenvironment/genetics , Wnt Signaling Pathway/drug effects , Wnt Signaling Pathway/genetics , Xenograft Model Antitumor AssaysABSTRACT
The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/ß-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.
Subject(s)
Antineoplastic Agents/pharmacology , Mitosis/drug effects , Taxoids/pharmacology , Wnt Proteins/antagonists & inhibitors , Cell Death/drug effects , Drug Resistance, Neoplasm/drug effects , Humans , Paclitaxel/pharmacology , Wnt Signaling Pathway/drug effects , beta Catenin/antagonists & inhibitorsABSTRACT
Deregulation of the ß-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and ß-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate ß-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate ß-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited ß-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of ß-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis.
Subject(s)
Antibodies, Monoclonal/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , Thrombospondins/metabolism , beta Catenin/metabolism , Animals , Carcinogenesis , Cell Line, Tumor , HEK293 Cells , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/pathology , Signal Transduction , Thrombospondins/biosynthesis , Thrombospondins/genetics , Thrombospondins/immunology , Wnt Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling. EXPERIMENTAL DESIGN: We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. RESULTS: We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature. CONCLUSIONS: These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Neoplasms, Experimental/drug therapy , Neoplastic Stem Cells/drug effects , Receptor, Notch2/antagonists & inhibitors , Receptors, Notch/antagonists & inhibitors , Animals , Humans , Immunohistochemistry , Mice , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Receptor, Notch3 , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy. EXPERIMENTAL DESIGN: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays. RESULTS: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition. CONCLUSIONS: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling.
Subject(s)
Antibodies, Anti-Idiotypic/administration & dosage , Intercellular Signaling Peptides and Proteins , Neoplastic Stem Cells , Pancreatic Neoplasms , Receptors, Notch/metabolism , Adaptor Proteins, Signal Transducing , Animals , Calcium-Binding Proteins , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/immunology , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/immunology , Neovascularization, Pathologic/drug therapy , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Receptors, Notch/immunology , Signal Transduction/drug effects , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/immunology , Vascular Endothelial Growth Factor A/metabolism , GemcitabineABSTRACT
The Wnt/ß-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Frizzled Receptors/metabolism , Neoplasms/drug therapy , Wnt Signaling Pathway/drug effects , Animals , Antibodies, Monoclonal/metabolism , Antineoplastic Agents/metabolism , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , Drug Synergism , Genetic Vectors/genetics , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Immunohistochemistry , Lentivirus , Luciferases , Neoplasms/metabolism , Peptide Library , Wnt Signaling Pathway/physiologyABSTRACT
KRAS mutations are frequent in colorectal cancer (CRC) and are associated with clinical resistance to treatment with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway and mediates stem cell self-renewal and vascular development. DLL4 inhibition in colon tumor cells reduces tumor growth and stem cell frequency. Considering the need for new drugs to treat colon cancers with oncogenic KRAS mutations, we examined in this study the efficacy of anti-DLL4 antibodies in KRAS mutant tumors in a panel of early passage colon tumor xenograft models derived from patients. Consistent with clinical findings, mutant KRAS colorectal xenograft tumors were insensitive to the EGFR therapeutic antibody cetuximab, whereas KRAS wild-type tumors responded to cetuximab. In contrast, anti-DLL4 was efficacious against both wild-type and mutant KRAS colon tumors as a single agent and in combination with irinotecan. Further analysis of mutant KRAS tumors indicated that the anti-DLL4/irinotecan combination produced a significant decrease in colon cancer stem cell frequency while promoting apoptosis in tumor cells. Our findings provide a rationale for targeting DLL4-Notch signaling for improved treatment of CRC patients with activating KRAS mutations.
Subject(s)
Antineoplastic Agents/therapeutic use , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Mutation , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Adaptor Proteins, Signal Transducing , Animals , Antibodies, Monoclonal/therapeutic use , Apoptosis/drug effects , Calcium-Binding Proteins , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Cell Separation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Irinotecan , Mice , Mice, SCID , Neoplastic Stem Cells/drug effects , Proto-Oncogene Proteins p21(ras) , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor AssaysABSTRACT
Previous studies have shown that blocking DLL4 signaling reduced tumor growth by disrupting productive angiogenesis. We developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. We found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes and reduced proliferation of tumor cells. Furthermore, we found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Intercellular Signaling Peptides and Proteins/immunology , Neoplasms/therapy , Neoplastic Stem Cells/immunology , Receptors, Notch/metabolism , Adaptor Proteins, Signal Transducing , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Apoptosis Regulatory Proteins/metabolism , Calcium-Binding Proteins , Camptothecin/analogs & derivatives , Camptothecin/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Chaperonin 60/agonists , Chaperonin 60/metabolism , Drug Synergism , Humans , Inhibitor of Apoptosis Proteins/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Irinotecan , Membrane Proteins/antagonists & inhibitors , Mice , Neoplasms/pathology , Neoplastic Stem Cells/drug effects , Neovascularization, Pathologic/metabolism , Secondary Prevention , Xenograft Model Antitumor AssaysABSTRACT
Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.
Subject(s)
Anti-Inflammatory Agents/adverse effects , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/drug therapy , Benzopyrans/adverse effects , Benzopyrans/therapeutic use , Benzylidene Compounds/adverse effects , Benzylidene Compounds/therapeutic use , Receptors, Glucocorticoid/agonists , Animals , Arthritis, Rheumatoid/drug therapy , Cells, Cultured , Drug Evaluation, Preclinical , Humans , Ligands , Male , Models, Biological , Prednisolone/adverse effects , Prednisolone/therapeutic use , Rats , Rats, Inbred Lew , Rats, Inbred WKY , Rats, Sprague-Dawley , Spodoptera , Substrate SpecificityABSTRACT
Acquired drug resistance is a major obstacle in cancer therapy. As for many other drugs, this is also the case for gemcitabine, a nucleoside analogue with activity against non-small cell lung cancer (NSCLC). Here, we evaluate the ability of bexarotene to modulate the acquisition and maintenance of gemcitabine resistance in Calu3 NSCLC models. In the prevention model, Calu3 cells treated repeatedly with gemcitabine alone gradually developed resistance. However, with inclusion of bexarotene, the cells remained chemosensitive. RNA analysis showed a strong increase of rrm1 (ribonucleotide reductase M1) expression in the resistant cells (Calu3-GemR), a gene known to be involved in gemcitabine resistance. In addition, the expression of genes surrounding the chromosomal location of rrm1 was increased, suggesting that resistance was due to gene amplification at the chr11 p15.5 locus. Analysis of genomic DNA confirmed that the rrm1 gene copy number was increased over 10-fold. Correspondingly, fluorescence in situ hybridization analysis of metaphase chromosomes showed an intrachromosomal amplification of the rrm1 locus. In the therapeutic model, bexarotene gradually resensitized Calu3-GemR cells to gemcitabine, reaching parental drug sensitivity after 10 treatment cycles. This was associated with a loss in rrm1 amplification. Corresponding with the in vitro data, xenograft tumors generated from the resistant cells did not respond to gemcitabine but were growth inhibited when bexarotene was added to the cytotoxic agent. The data indicate that bexarotene can resensitize gemcitabine-resistant tumor cells by reversing gene amplification. This suggests that bexarotene may have clinical utility in cancers where drug resistance by gene amplification is a major obstacle to successful therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Deoxycytidine/analogs & derivatives , Lung Neoplasms/drug therapy , Retinoid X Receptors/agonists , Tetrahydronaphthalenes/pharmacology , Animals , Bexarotene , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Growth Processes/drug effects , Cell Line, Tumor , Deoxycytidine/administration & dosage , Deoxycytidine/pharmacology , Drug Resistance, Neoplasm , Drug Synergism , Female , Gene Amplification/drug effects , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Nude , Tetrahydronaphthalenes/administration & dosage , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
BACKGROUND: We previously reported that a retinoid X receptor agonist bexarotene prevented and overcame acquired drug resistance in advanced breast cancer and non-small cell lung cancer. The present study was to evaluate the effect of bexarotene on the development of multidrug resistance in advanced prostate cancer. METHODS: Human prostate cancer cells PC3 were repeatedly treated in culture with paclitaxel, doxorubicin, or cisplatin with or without bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity. RESULTS: Compared to parental cells, cells treated with a single therapeutic agent was resistant to the therapeutic agent, whereas cells treated with the combination remained chemosensitive. Cells with acquired drug resistance showed increased sensitivity to the cytotoxic agent when treated with the combination. Fluctuation analysis demonstrated that treatment with bexarotene decreased the rate of spontaneous development of drug resistance. These in vitro findings were further confirmed in the PC3 xenograft model. CONCLUSION: Our results suggest a role of bexarotene in combination with chemotherapeutic agents in prevention and overcoming acquired drug resistance in advanced prostate cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Prostatic Neoplasms/drug therapy , Retinoid X Receptors/agonists , Tetrahydronaphthalenes/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Bexarotene , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/administration & dosage , Doxorubicin/administration & dosage , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Male , Mice , Mice, Nude , Paclitaxel/administration & dosage , Prostatic Neoplasms/genetics , RNA, Neoplasm/chemistry , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tetrahydronaphthalenes/administration & dosageABSTRACT
This study evaluated the anti-tumor efficacy of combining the RXR agonist, bexarotene, with the PPARgamma agonist, rosiglitazone, in colon cancer. Moser, a human colon cancer cell line, was treated with bexarotene and rosiglitazone alone or in combination and the effect on growth and differentiation were examined. The data demonstrated that the bexarotene/rosiglitazone combination produced greater efficacy in growth inhibition than either single agent. Furthermore, combination treatment acted cooperatively to decrease COX-2 expression and PGE2 synthesis while increasing expression of the differentiation marker, CEA. These findings were confirmed in vivo in a Moser xenograft tumor model. Collectively, our data suggest a potential role for utilizing a combination regimen of a RXR and PPARgamma agonist in the treatment of colon cancer.
Subject(s)
Cell Differentiation , Cell Proliferation , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , PPAR gamma/metabolism , Retinoid X Receptors/metabolism , Animals , Anticarcinogenic Agents/pharmacology , Bexarotene , Colonic Neoplasms/drug therapy , Cyclooxygenase 2/metabolism , Fibrinolytic Agents/pharmacology , Humans , Male , Membrane Proteins/metabolism , Mice , Mice, Nude , PPAR gamma/agonists , Prostaglandin D2/metabolism , Retinoid X Receptors/agonists , Rosiglitazone , Tetrahydronaphthalenes/pharmacology , Thiazolidinediones/pharmacology , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
This study was designed to evaluate, using preclinical models of non-small cell lung cancer (NSCLC), the growth inhibitory effects of the retinoid X receptor (RXR) agonist bexarotene (LGD1069, Targretin) in combination with cytotoxic agents currently used as standard first-line therapy in advanced disease. Although single-agent bexarotene had modest growth inhibitory effects in several cell lines, efficacy was observed only in the micromolar range (>1muM), which approximates the plasma C(max) measured in pharmacokinetic studies in patients. However, when combined with paclitaxel or vinorelbine, bexarotene produced a concentration-dependent enhancement of the growth inhibitory activities of paclitaxel and vinorelbine. Formal synergy analysis using the Calu3 cell line demonstrated that the combination of bexarotene with either cytotoxic agent produced synergistic activity (combination index, CI<1). The in vitro observations were confirmed in vivo in a NSCLC xenograft tumor model (Calu3), where both bexarotene/paclitaxel and bexarotene/vinorelbine combinations produced significantly greater antitumor effects than the single agents. These results demonstrate that bexarotene can cooperate with widely used cytotoxic agents to decrease the growth of NSCLC tumor cells both in vitro and in vivo, and suggest the potential benefit of adding a RXR-selective agonist in combination with chemotherapy for NSCLC treatment. Furthermore, the data support the clinical observation from phase I/IIa trials suggesting that bexarotene has beneficial effects on survival when used in combination with cytotoxic agents in advanced NSCLC.
Subject(s)
Anticarcinogenic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Lung Neoplasms/pathology , Tetrahydronaphthalenes/pharmacology , Animals , Bexarotene , Cell Line, Tumor , Drug Interactions , Humans , Male , Mice , Mice, Nude , Paclitaxel/pharmacology , Transplantation, Heterologous , Vinblastine/analogs & derivatives , Vinblastine/pharmacology , VinorelbineABSTRACT
Acquired drug resistance represents a major challenge in the therapeutic management of breast cancer patients. We reported previously that the retinoid X receptor-selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating animal models of tamoxifen-resistant breast cancer. The goal of this study was to evaluate the effect of bexarotene on development of acquired drug resistance and its role in overcoming acquired drug resistance in advanced breast cancer. Paclitaxel, doxorubicin, and cisplatin were chosen as model compounds to determine the effect of bexarotene on the development of acquired drug resistance. Human breast cancer cells MDA-MB-231 were repeatedly treated in culture with a given therapeutic agent with or without bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity. Compared with parental cells, cells treated with a single therapeutic agent became resistant to the therapeutic agent, whereas cells treated with the bexarotene combination remained chemosensitive. Cells with acquired drug resistance, when treated with the combination, showed increased sensitivity to the cytotoxic agent. Furthermore, cells treated with the combination regimen had reduced invasiveness and angiogenic potential than their resistant counterparts. These in vitro findings were further confirmed in an in vivo MDA-MB-231 xenograft model. Our results suggest a role for bexarotene in combination with chemotherapeutic agents in prevention and overcoming acquired drug resistance in advanced breast carcinoma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Neoplasm Invasiveness/prevention & control , Neovascularization, Pathologic/prevention & control , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Bexarotene , Breast Neoplasms/blood supply , Breast Neoplasms/secondary , Cisplatin/administration & dosage , Doxorubicin/administration & dosage , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Humans , Mice , Mice, Nude , Paclitaxel/administration & dosage , Retinoid X Receptors/agonists , Survival Rate , Tetrahydronaphthalenes/administration & dosage , Umbilical Veins/cytology , Umbilical Veins/drug effects , Umbilical Veins/metabolismABSTRACT
PURPOSE: Paclitaxel is an important anticancer agent for the treatment of non-small cell lung cancer (NSCLC). However, its use in cancer therapy is limited by development of acquired drug resistance. The goal of this study was to determine the effect of bexarotene on development of acquired paclitaxel resistance in NSCLC. EXPERIMENTAL DESIGN: Human NSCLC Calu3 cells were repeatedly treated in culture with intermittent paclitaxel alone or in combination with continuous bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity in vitro and in vivo. RESULTS: Repeat exposure to paclitaxel alone resulted in development of paclitaxel resistance with cross-resistance to multidrug resistance P-glycoprotein substrates, whereas the bexarotene/paclitaxel combination prevented the development of drug resistance and the cells remained chemosensitive. Furthermore, paclitaxel resistance could be overcome when the resistant cells were treated with the combination regimen. Fluctuation analysis showed that treatment with bexarotene decreased the rate of spontaneous development of paclitaxel resistance. In vivo, the bexarotene/paclitaxel combination regimen produced a statistically significant decrease in tumor growth in a Calu3 NSCLC xenograft model compared with the single agents (two-tailed, P < 0.05). In addition, paclitaxel-resistant Calu3 tumors treated with the bexarotene/paclitaxel combination showed greater delay in tumor growth compared with those treated with paclitaxel alone. CONCLUSIONS: Our results suggest that bexarotene may offer a novel approach to prevent and overcome paclitaxel resistance in patients with NSCLC.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/prevention & control , Drug Resistance, Neoplasm , Retinoid X Receptors/agonists , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Bexarotene , Carcinoma, Non-Small-Cell Lung/pathology , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/prevention & control , Male , Mice , Mice, Nude , Mutation , Paclitaxel/administration & dosage , Survival Rate , Tetrahydronaphthalenes/administration & dosage , Transplantation, Heterologous , Tumor Cells, Cultured/transplantationABSTRACT
We have previously shown that the retinoid X receptor (RXR) ligand bexarotene (LGD1069, Targretin) is efficacious as a chemopreventive and chemotherapeutic agent in rat N-nitroso-N-methylurea (NMU)-induced mammary carcinomas (Cancer Res 58: 479-484, 1998). To determine additional role for bexarotene in breast cancer treatment, we evaluated the effect of bexarotene on the efficacy of paclitaxel (Taxol) treatment in a rat NMU-derived mammary tumor cell line, NMU-417, in vitro and in rat NMU-induced mammary tumors in vivo. Our growth inhibition results showed that the bexarotene/paclitaxel combination produced a concentration-dependent synergy in NMU-417 tumor cell line. Synergistic growth inhibition by the combination was associated with an increase in cell death induced by both agents. In rat NMU-induced mammary tumor model in vivo, the benefit of combination therapy was observed as early as 1 week after treatment and increased as treatment continued. At the end of 6 weeks of treatment, the bexarotene/paclitaxel combination produced an overall objective response rate of 94% compared with a rate of 12% in paclitaxel-treated and 58% in bexarotene-treated animals, an effect that was more than the additive effects produced by single agents. Although both bexarotene alone and the bexarotene/paclitaxel combination reduced tumor multiplicity to similar extent, the combination regimen produced a statistically significant decrease in total tumor burden compared to single agents and untreated controls (two-tailed, p < 0.05). Combination therapy did not further alter body weight nor increase toxicity when compared to single agents. In summary, our results demonstrated the potential of using a RXR selective ligand in combination with chemotherapy for the treatment of breast cancer.