ABSTRACT
BACKGROUND AND AIMS: We compared the safety and efficacy of bintrafusp alfa (BA) in combination with gemcitabine+cisplatin (GemCis), to those of GemCis alone, in patients with biliary tract cancer. APPROACH AND RESULTS: This randomized, double-blind, placebo-controlled, adaptive design phase 2/3 trial (NCT04066491) included adults who are treatment-naive with locally advanced/metastatic biliary tract cancer. Patients (N = 297) were randomized to receive an IV infusion of BA (2400 mg once/3 wk) plus GemCis (gemcitabine 1000 mg/m 2 +cisplatin 25 mg/m 2 on days 1 and 8/3 wk; 8 cycles) (BA group, n = 148) or placebo+GemCis (placebo group, n = 149). The primary end point was overall survival (OS). For adaptation analysis (phase 2-phase 3; data cutoff: May 20, 2021), efficacy was assessed in the first 150 patients who were antibiotic-naive when 80 progression-free survival events had occurred and ≥ 19 weeks of follow-up had been completed (BA, n = 73; placebo, n = 77). Median OS (95% CI) for the BA (11.5 mo [9.3-not estimable]) and placebo (11.5 mo [10.0-not estimable]) groups was comparable (hazard ration 1.23 [95% CI 0.66-2.28]; p = 0.7394); OS data maturity was 27.2% (41 events/151 patients). The most common grade ≥3 treatment-related adverse event was anemia (BA, 26.0%; placebo, 22.8%). Bleeding adverse events were reported more frequently in the BA group (28.8%) versus the placebo group (7.4%). Deaths within 60 days of the first dose were reported in 7.5% and 1.3% of patients in the BA and placebo groups, respectively. CONCLUSIONS: BA+GemCis did not provide a clinically meaningful benefit compared with GemCis alone as first-line treatment for biliary tract cancer, and the study was discontinued early (terminated: August 20, 2021).
ABSTRACT
We performed a comprehensive survey of commonly inactivated tumor suppressor genes in esophageal squamous cell carcinoma (ESCC) based on functional reactivation of epigenetically silenced tumor suppressor genes by 5-aza-2'-deoxycytidine and trichostatin A using microarrays containing 12599 genes. Among 58 genes identified by this approach, 44 (76%) harbored dense CpG islands in the promoter regions. Thirteen of twenty-two tested gene promoters were methylated in cell lines, and ten in primary ESCC accompanied by silencing at the mRNA level. Potent growth suppressive activity of three genes including CRIP-1, Apolipoprotein D, and Neuromedin U in ESCC cells was demonstrated by colony focus assays. Pharmacologic reversal of epigenetic silencing is a powerful approach for comprehensive identification of tumor suppressor genes in human cancers.
Subject(s)
Azacitidine/analogs & derivatives , Carcinoma, Squamous Cell/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , Gene Silencing , Genes, Tumor Suppressor , Azacitidine/pharmacology , Base Sequence , CpG Islands/genetics , Decitabine , Down-Regulation , Enzyme Inhibitors/pharmacology , Esophageal Neoplasms/genetics , Gene Silencing/drug effects , Genes, Tumor Suppressor/drug effects , Humans , Hydroxamic Acids/pharmacology , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
PURPOSE: Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domain of the transforming growth factor-ß (TGF-ß) receptor II fused to a human immunoglobulin G1 antibody blocking programmed death ligand 1 (PD-L1). The recommended phase 2 dose (RP2D) was selected based on phase 1 efficacy, safety, and pharmacokinetic (PK)-pharmacodynamic data, assuming continuous inhibition of PD-L1 and TGF-ß is required. Here, we describe a model-informed dose modification approach for risk management of BA-associated bleeding adverse events (AEs). METHODS: The PK and AE data from studies NCT02517398, NCT02699515, NCT03840915, and NCT04246489 (n = 936) were used. Logistic regression analyses were conducted to evaluate potential relationships between bleeding AEs and BA time-averaged concentration (Cavg), derived using a population PK model. The percentage of patients with trough concentrations associated with PD-L1 or TGF-ß inhibition across various dosing regimens was derived. RESULTS: The probability of bleeding AEs increased with increasing Cavg; 50% dose reduction was chosen based on the integration of modeling and clinical considerations. The resulting AE management guidance to investigators regarding temporary or permanent treatment discontinuation was further refined with recommendations on restarting at RP2D or at 50% dose, depending on the grade and type of bleeding (tumoral versus nontumoral) and investigator assessment of risk of additional bleeding. CONCLUSION: A pragmatic model-informed approach for management of bleeding AEs was implemented in ongoing clinical trials of BA. This approach is expected to improve benefit-risk profile; however, its effectiveness will need to be evaluated based on safety data generated after implementation.
Subject(s)
Hemorrhage , Immunologic Factors , Neoplasms , B7-H1 Antigen , Clinical Studies as Topic , Hemorrhage/chemically induced , Hemorrhage/prevention & control , Humans , Immunologic Factors/toxicity , Neoplasms/drug therapy , Risk Management , Transforming Growth Factor betaABSTRACT
BACKGROUND: Patients with esophageal squamous cell carcinoma (SCC) have limited treatment options. Blocking transforming growth factor-ß (TGFß), which can be overexpressed in these tumors, may enhance responses to programmed cell death protein 1/programmed death-ligand 1 [PD-(L)1] inhibitors. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGFß receptor II (TGFßRII) (a TGFß "trap") fused to a human IgG1 monoclonal antibody blocking PD-L1. OBJECTIVE: The objective of this study was to investigate the safety and efficacy of bintrafusp alfa in Asian patients with pretreated, PD-L1-unselected esophageal SCC. PATIENTS AND METHODS: In a phase 1 study, Asian patients with pretreated esophageal SCC received bintrafusp alfa 1200 mg every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint was safety/tolerability with a goal of exploring clinical activity. RESULTS: By the database cutoff of August 24, 2018, 30 patients (76.7% had two or more prior anticancer regimens) received bintrafusp alfa for a median of 6.1 weeks; two remained on treatment. Nineteen patients (63.3%) had treatment-related adverse events, seven (23.3%) with grade 3/4 events, and there were no treatment-related deaths. The confirmed objective response rate (ORR) per independent review was 10.0% (95% confidence interval [CI] 2.1-26.5); responses lasted 2.8-8.3 + months. All responses occurred in immune-excluded tumors. Investigator-assessed confirmed ORR was 20.0% (95% CI 7.7-38.6). Median overall survival was 11.9 months (95% CI 5.7-not reached). CONCLUSIONS: Bintrafusp alfa demonstrated a manageable safety profile and efficacy in Asian patients with pretreated esophageal SCC. CLINICAL TRIALS REGISTRATION: NCT02699515.
Subject(s)
B7-H1 Antigen/antagonists & inhibitors , Esophageal Neoplasms/drug therapy , Esophageal Squamous Cell Carcinoma/drug therapy , Transforming Growth Factor beta/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Asia , Female , Humans , Male , Middle AgedABSTRACT
PURPOSE: Patients with advanced gastric/gastroesophageal junction cancer (GC/GEJC) have limited treatment options after first-line therapy. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGFßRII receptor (a TGFß "trap") fused to a human IgG1 antibody against programmed death ligand 1 (PD-L1), potentially offering a new treatment approach for these patients. We report results for bintrafusp alfa in GC/GEJC. PATIENTS AND METHODS: Asian patients with recurrent GC/GEJC for whom standard therapy does not exist or for whom standard therapy has failed enrolled in this expansion cohort of an ongoing phase I trial and received bintrafusp alfa 1,200 mg once every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary objective was to assess safety/tolerability. RESULTS: By July 23, 2018, 31 heavily pretreated patients received bintrafusp alfa for a median of 10.1 weeks; 3 patients remained on treatment. Six patients (19%) experienced grade 3 treatment-related adverse events (AE); no grade 4 events occurred. One on-treatment death occurred (sudden death); rupture of a preexisting thoracic aortic aneurysm was the suspected cause. Ten patients (32%) had immune-related AEs. The confirmed objective response rate per independent review committee was 16%; disease control rate was 26%. Median duration of response was 8.7 months (range, 2.4-12.4+). Responses occurred irrespective of PD-L1 expression or microsatellite instability status and appeared to correlate with high tumor TGFB1 levels. CONCLUSIONS: In this first evaluation in Asian patients with heavily pretreated advanced GC/GEJC, bintrafusp alfa demonstrated a manageable safety profile and clinical activity.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , Aged , Aged, 80 and over , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/adverse effects , Drug Resistance, Neoplasm , Female , Humans , Male , Middle Aged , Prognosis , Recurrence , Retreatment , Stomach Neoplasms/pathology , Treatment OutcomeABSTRACT
BACKGROUND: Patients with biliary tract cancer (BTC) have poor prognosis with few treatment options. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor (TGF)-ßRII receptor (a TGF-ß 'trap') fused to a human IgG1 antibody blocking programmed death ligand 1 (PD-L1), has shown clinical efficacy in multiple solid tumors. METHODS: In this phase I, open-label trial expansion cohort, Asian patients with BTC whose disease progressed after first-line chemotherapy received bintrafusp alfa 1200 mg every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint is safety/tolerability, while the secondary endpoints include best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: As of August 24, 2018, 30 patients have received bintrafusp alfa for a median of 8.9 (IQR 5.7-32.1) weeks; 3 patients remained on treatment for >59.7 weeks. Nineteen (63%) patients experienced treatment-related adverse events (TRAEs), most commonly rash (17%), maculopapular rash and fever (13% each), and increased lipase (10%). Eleven (37%) patients had grade ≥3 TRAEs; three patients had grade 5 events (septic shock due to bacteremia, n=1; interstitial lung disease (reported term: interstitial pneumonitis), n=2). The objective response rate was 20% (95% CI 8 to 39) per independent review committee (IRC), with five of six responses ongoing (12.5+ to 14.5+ months) at data cut-off. Two additional patients with durable stable disease had a partial response per investigator. Median progression-free survival assessed by IRC and overall survival were 2.5 months (95% CI 1.3 to 5.6) and 12.7 months (95% CI 6.7 to 15.7), respectively. Clinical activity was observed irrespective of PD-L1 expression and microsatellite instability-high status. CONCLUSIONS: Bintrafusp alfa had clinical activity in Asian patients with pretreated BTC, with durable responses. Based on these results, bintrafusp alfa is under further investigation in patients with BTC (NCT03833661 and NCT04066491). TRIAL REGISTRATION NUMBER: NCT02699515.
Subject(s)
Antineoplastic Agents, Immunological/adverse effects , Biliary Tract Neoplasms/drug therapy , Immunoconjugates/adverse effects , Recombinant Fusion Proteins/adverse effects , Adult , Aged , Antineoplastic Agents, Immunological/administration & dosage , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , Biliary Tract Neoplasms/immunology , Biliary Tract Neoplasms/mortality , Female , Humans , Immunoconjugates/administration & dosage , Immunoconjugates/genetics , Male , Middle Aged , Progression-Free Survival , Receptor, Transforming Growth Factor-beta Type II/genetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Signal Transduction/drug effects , Signal Transduction/immunology , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/metabolism , Young AdultABSTRACT
Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2'-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.
Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , DNA Methylation , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Homeodomain Proteins/genetics , Promoter Regions, Genetic/genetics , Tumor Suppressor Proteins/genetics , Base Sequence , Cell Line, Tumor , CpG Islands/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, Neoplasm , Homeodomain Proteins/metabolism , Humans , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Prognosis , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Tumor Stem Cell Assay , Tumor Suppressor Proteins/metabolismABSTRACT
BACKGROUND: Fibroblast growth factor receptor (FGFR) 2 is overexpressed in several tumor types, including triple-negative breast cancer and gastric cancer, both of which have a high unmet medical need. Aprutumab ixadotin (BAY 1187982) is the first antibody-drug conjugate (ADC) to target FGFR2 and the first to use a novel auristatin-based payload. OBJECTIVE: This first-in-human trial was conducted to determine the safety, tolerability, and maximum tolerated dose (MTD) of aprutumab ixadotin in patients with advanced solid tumors from cancer indications known to be FGFR2-positive. PATIENTS AND METHODS: In this open-label, multicenter, phase I dose-escalation trial (NCT02368951), patients with advanced solid tumors received escalating doses of aprutumab ixadotin (starting at 0.1 mg/kg body weight), administered intravenously on day 1 of every 21-day cycle. Primary endpoints included safety, tolerability, and the MTD of aprutumab ixadotin; secondary endpoints were pharmacokinetic evaluation and tumor response to aprutumab ixadotin. RESULTS: Twenty patients received aprutumab ixadotin across five cohorts, at doses of 0.1-1.3 mg/kg. The most common grade ≥ 3 drug-related adverse events were anemia, aspartate aminotransferase increase, proteinuria, and thrombocytopenia. Dose-limiting toxicities were thrombocytopenia, proteinuria, and corneal epithelial microcysts, and were only seen in the two highest dosing cohorts. The MTD was determined to be 0.2 mg/kg due to lack of quantitative data following discontinuations at 0.4 and 0.8 mg/kg doses. One patient had stable disease; no responses were reported. CONCLUSIONS: Aprutumab ixadotin was poorly tolerated, with an MTD found to be below the therapeutic threshold estimated preclinically; therefore, the trial was terminated early. CLINICALTRIALS. GOV IDENTIFIER: NCT02368951.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Cholangiocarcinoma/drug therapy , Colorectal Neoplasms/drug therapy , Immunoconjugates/therapeutic use , Oligopeptides/therapeutic use , Adult , Aged , Early Termination of Clinical Trials , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Neoplasm Staging , Receptor, Fibroblast Growth Factor, Type 2/immunology , Treatment Failure , Young AdultABSTRACT
PGP9.5 is a controversial molecule from an oncologic point of view. We recently identified frequent methylation of PGP9.5 gene exclusively in primary head and neck squamous cell carcinoma (HNSCC), suggesting that it could be a tumor suppressor gene. On the other hand, PGP9.5 was reported to be overexpressed in a subset of human cancers presumably due to intrinsic oncogenic properties or as a result of transformation. To demonstrate that PGP9.5 possesses tumor suppressive activity, we examined forced expression by stable transfection of PGP9.5 in 4 HNSCC cell lines. Although all 4 cell lines demonstrated reduced log growth rates in culture after transfection, only 2 cell lines with wild type p53 (011, 022) demonstrated decreased growth in soft agar. In 2 cell lines with mutant p53 (013, 019), we observed no altered growth in soft agar and increased sensitivity to UV irradiation. We then tested for and found a high frequency of promoter methylation in a larger panel of primary tumors including HNSCC, esophageal SCC, gastric, lung, prostate and hepatocellular carcinoma. Our data support the notion that PGP9.5 is a tumor suppressor gene that is inactivated by promoter methylation or gene deletion in several types of human cancers.
Subject(s)
Genes, Tumor Suppressor , Neoplasms/genetics , Ubiquitin Thiolesterase/genetics , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , DNA Methylation , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/genetics , Humans , Neoplasms/enzymology , Transfection , Ubiquitin Thiolesterase/biosynthesisABSTRACT
Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA Methylation , Esophageal Neoplasms/genetics , Genes, DCC , Genes, Tumor Suppressor , Promoter Regions, Genetic , Aged , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Early Diagnosis , Esophageal Neoplasms/pathology , Female , Humans , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Invasiveness , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63+/+ mouse, it is undetectable in these tissues in the p63-/- mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.
Subject(s)
Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Phosphoproteins/metabolism , Protein Precursors/genetics , Response Elements/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Animals , Base Sequence , Bone Development/genetics , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins , Genes, Reporter , Genes, Tumor Suppressor , Humans , Membrane Proteins/analysis , Mice , Molecular Sequence Data , Mutation , Oligonucleotide Array Sequence Analysis , Pharynx/growth & development , Pharynx/immunology , Phosphoproteins/genetics , Promoter Regions, Genetic/genetics , Protein Precursors/analysis , Skin/growth & development , Skin/immunology , Trans-Activators/genetics , Transcriptional Activation , Tumor Suppressor Protein p53/genetics , Tumor Suppressor ProteinsABSTRACT
PURPOSE: To investigate whether the promoter methylation pattern in N-methyl-d-aspartate receptor 2B (NMDAR2B) is correlated with clinical features of human esophageal squamous cell carcinoma (ESCC), the methylation status of the gene was examined at three different sites (P1, P2, and P3) where two CpG islands reside within 1 kb upstream of the transcription start site. EXPERIMENTAL DESIGN: Three independent modalities for methylation analysis (bisulfite sequencing, combined bisulfite restriction analysis, and TaqMan methylation-specific PCR) were done to analyze total 67 ESCC tissues that included 43 primary tumors with well-characterized clinicopathologic variables including patient outcome. RESULTS: Using an optimized cutoff value based on quantitative methylation-specific PCR, we found that patients with higher NMDAR2B methylation ratio in the proximal region (P1) showed a worse 5-year disease-specific survival rate than those without NMDAR2B methylation (P < 0.006). A significant correlation was also seen between NMDAR2B promoter methylation and the presence of vascular permeation (P = 0.03). CONCLUSION: NMDAR2B promoter methylation could be a clinically applicable marker in ESCC.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/diagnosis , DNA Methylation , Esophageal Neoplasms/diagnosis , Receptors, N-Methyl-D-Aspartate/genetics , Adult , Aged , Carcinoma, Squamous Cell/mortality , Esophageal Neoplasms/mortality , Female , Humans , Male , Middle Aged , Promoter Regions, Genetic , Sequence Analysis, DNA , Survival AnalysisABSTRACT
Diffuse-type gastric cancer (DGC) is the most deadly form of gastric cancer and is frequently accompanied by peritoneal dissemination and metastasis. The specific molecular events involved in DGC pathogenesis remain elusive. Accumulating evidence of epigenetic inactivation in tumor suppressor genes led us to conduct a comprehensive screen to identify novel methylated genes in human cancers using pharmacologic unmasking and subsequent microarray analysis. We compared differential RNA expression profiles of DGC and intestinal-type gastric cancer (IGC) cell lines treated with 5-aza-2'-deoxycytidine using microarrays containing 22,284 genes. We identified 16 methylated genes, including many novel genes, in DGC cell lines and studied PGP9.5 with particular interest. In primary gastric cancers, PGP9.5 was found to be more frequently methylated in DGCs (78%) than in IGCs (36%; DGC versus IGC, P < 0.05). Furthermore, real-time methylation-specific PCR analysis of PGP9.5 showed relatively higher methylation levels in DGC than in IGC. Our data thus implicate a molecular event common in the DGC phenotype compared with IGC.
Subject(s)
DNA Methylation , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Ubiquitin Thiolesterase/genetics , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Base Sequence , Cell Line, Tumor , Decitabine , Genes, Tumor Suppressor , Humans , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Stomach Neoplasms/drug therapyABSTRACT
Promoter hypermethylation accompanied by gene silencing is a common feature of human cancers. We identified previously several new tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of reexpression on microarray analysis. In this study, we modified the selection of candidates from our previous microarray data by excluding genes that showed basal expression in cancer cell lines. With the new method, we found novel methylated genes with 90% accuracy. Among these 33 novel methylated genes that we identified in esophageal squamous cell carcinoma (ESCC) cell lines, N-methyl-D-aspartate receptor type 2B (NMDAR2B) was of particular interest. NMDAR2B was methylated in 95% of primary human ESCC tissue specimens and 12 ESCC cell lines by sequence analysis. NMDAR2B expression was silenced in all 12 ESCC cell lines and was reactivated by the demethylating agent 5-aza-2'-deoxycytidine. Moreover, reintroduction of the gene was accompanied by marked Ca(2+)-independent apoptosis in ESCC cell lines, suggesting that NMDAR2B can suppress tumor growth. Thus, NMDAR2B promoter methylation is common in ESCC, abrogating gene transcription and leading to cellular resistance to apoptosis.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA Methylation , Esophageal Neoplasms/genetics , Genes, Tumor Suppressor , Receptors, N-Methyl-D-Aspartate/genetics , Apoptosis/genetics , Base Sequence , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Epigenesis, Genetic , Esophageal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer.
Subject(s)
Acyltransferases/biosynthesis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Glycosylphosphatidylinositols/biosynthesis , Membrane Glycoproteins/biosynthesis , Oncogenes , Acyltransferases/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cloning, Molecular , Gene Amplification , Gene Dosage , Glycosylphosphatidylinositols/genetics , Humans , Membrane Glycoproteins/genetics , Neoplasm Invasiveness , Paxillin/metabolism , Phosphorylation , Protein SubunitsABSTRACT
Recent evidence indicates that in vitro p53 augments base excision repair (BER) activities in mammalian cells. To understand the role of p53 in BER, we analyzed the repair activity of hOgg1 in isogenic cell lines HCT116p53+/+ and HCT116p53-/-. We found that hOgg1 activity was significantly decreased in HCT116p53-/- cells as compared with HCT116p53+/+ cells, indicating a functional role for p53 in the regulation of hOGG1. Using gel-shift assays, we showed that p53 binds to its putative cis-elements within the hOGG1 promoter. In addition we demonstrated that supplementing p53 in HCT116p53-/- cells enhanced the transcription of hOGG1. To further strengthen our findings, we used p53-RNAi to study the effects of decreased p53 levels on hOgg1 activity. We observed that p53-RNAi resulted in decreased hOGG1 expression both at the mRNA and protein levels. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage and reduced hOgg1 activity as evidenced by the 8-oxoG incision assay. Taken together, our results indicate that loss of p53 function can lead to decreased hOgg1 repair activity.
Subject(s)
DNA Glycosylases/metabolism , Gene Deletion , RNA Interference , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , DNA Damage , DNA Glycosylases/genetics , DNA Repair , Down-Regulation , Gene Expression Regulation, Enzymologic , HCT116 Cells , Humans , Promoter Regions, Genetic , Protein Binding , Tumor Suppressor Protein p53/metabolismABSTRACT
HSP70, a stress response protein, is known to be a determinant of cell death and cell transformation. We show that different isoforms of p63 have different transcriptional activities on hsp70 genes. DeltaNp63alpha, an abundantly expressed isoform of p63, activates (in vitro and in vivo), whereas TAp63gamma down-regulates the expression of hsp70. We further show that the transactivation domain at the NH(2) terminus of p63 represses, whereas the COOH terminus activates hsp70 transcription. In addition, DeltaNp63alpha regulates transcription of the hsp70 gene through its interaction with the CCAAT binding factor and NF-Y transcription factors which are known to form a complex with the CCAAT box located in the hsp70 promoter. Moreover, DeltaNp63alpha expression correlates with HSP70 expression in all head and neck cancer cell lines. Finally, we show colocalization of DeltaNp63alpha and HSP70 in the epithelium and coexpression of both proteins in 41 primary head and neck cancers. Our study provides strong evidence for the physiologic association between DeltaNp63alpha and hsp70 in human cancer, thus further supporting the oncogenic potential of DeltaNp63alpha.
Subject(s)
HSP70 Heat-Shock Proteins/genetics , Neoplasms/genetics , Phosphoproteins/physiology , Trans-Activators/physiology , Animals , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , CCAAT-Binding Factor/metabolism , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , DNA-Binding Proteins , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , HSP70 Heat-Shock Proteins/biosynthesis , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Neoplasms/metabolism , Oligonucleotide Array Sequence Analysis , Osteosarcoma/genetics , Osteosarcoma/metabolism , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Structure, Tertiary , Trans-Activators/biosynthesis , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors , Transcriptional Activation , Tumor Suppressor Proteins , Up-RegulationABSTRACT
PGP9.5/UCHL1 is a member of the carboxyl-terminal ubiquitin hydrolase family with a potential role in carcinogenesis. We previously identified PGP9.5 as a putative tumor-suppressor gene and methylation of the promoter as a cancer-specific event in primary cancer tissues. In this current study, we analyzed PGP9.5 methylation in 50 esophageal squamous cell carcinoma (ESCC) primary tumors with well characterized clinicopathologic variables including patient outcome. Two independent modalities for methylation analysis (TaqMan methylation-specific PCR and combined bisulfite restriction analysis) were used to analyze these samples. The two data sets were consistent with each other, as the 21 patients (42%) with highest methylation levels by TaqMan analysis all showed visible combined bisulfite restriction analysis bands on acrylamide gels. Using an optimized cutoff value by TaqMan quantitation, we found that patients with higher PGP9.5 methylation ratios in the primary tumor showed poorer 5-year survival rates than those without PGP9.5 methylation (P = 0.01). A significant correlation was also seen between PGP9.5 promoter methylation and the presence of regional lymph node metastases (P = 0.03). Multivariate analysis subsequently revealed that PGP9.5 methylation was an independent prognostic factor for ESCC survival (P = 0.03). These results suggest that PGP9.5 promoter methylation could be a clinically applicable marker for ESCC progression.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA Methylation , Esophageal Neoplasms/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Esophageal Neoplasms/pathology , Female , Gene Silencing , Humans , Lymphatic Metastasis , Male , Middle Aged , Multivariate Analysis , Prognosis , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
PURPOSE: To evaluate the safety, tolerability, pharmacokinetics, and efficacy of the intravenously administered pan-PI3K inhibitor copanlisib in Japanese patients with advanced or refractory solid tumors. METHODS: A Phase I open-label study in Japanese patients with advanced or refractory solid tumors was carried out. Patients received a single intravenous dose of either copanlisib 0.4 mg/kg or copanlisib 0.8 mg/kg, dosed intermittently on days 1, 8, and 15 of a 28-day cycle. Safety was monitored throughout the study. Plasma copanlisib levels were measured for pharmacokinetic analysis. RESULTS: Ten patients were enrolled and treated; three received copanlisib 0.4 mg/kg and seven received copanlisib 0.8 mg/kg. Overall, median duration of treatment was 6.2 weeks. No patients treated at 0.4 mg/kg experienced a dose-limiting toxicity, and the maximum tolerated dose in Japanese patients was determined to be 0.8 mg/kg. Adverse events were recorded in all ten patients; the most common were hyperglycemia, hypertension, and constipation. Copanlisib pharmacokinetic exposures displayed near dose-proportionality, with no accumulation. No patients achieved a complete or partial response, and disease control rate was 40.0%. CONCLUSIONS: Copanlisib was well tolerated in Japanese patients with advanced or refractory solid tumors, and the maximum tolerated dose was determined to be 0.8 mg/kg. Copanlisib demonstrated near dose-proportional pharmacokinetics and preliminary disease control, warranting further investigation. CLINICAL TRIAL REGISTRATION NUMBER: NCT01404390.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Pyrimidines/therapeutic use , Quinazolines/therapeutic use , Aged , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Pyrimidines/adverse effects , Pyrimidines/pharmacokinetics , Quinazolines/adverse effects , Quinazolines/pharmacokineticsABSTRACT
Aberrant promoter hypermethylation of tumor suppressor genes is proposed to be a common feature of primary cancer cells. We recently developed a pharmacological unmasking microarray approach to screen unknown tumor suppressor gene candidates epigenetically silenced in human cancers. In this study, we applied this method to identify such genes in head and neck squamous cell carcinoma (HNSCC). We identified 12 novel methylated genes in HNSCC cell lines, including PGP9.5, cyclin A1, G0S2, bone-morphogenetic protein 2A, MT1G, and neuromedin U, which showed frequent promoter hypermethylation in primary HNSCC (60%, 45%, 35%, 25%, 25%, and 20%, respectively). Moreover, we discovered that cyclin A1 methylation was inversely related to p53 mutational status in primary tumors (P = 0.015), and forced expression of cyclin A1 resulted in robust induction of wild-type p53 in HNSCC cell lines. Pharmacological unmasking followed by microarray analysis is a powerful tool to identify key methylated tumor suppressor genes and relevant pathways.