The human high-affinity copper transporter 1 (hCtr1) transports both Cu(I) and cisplatin (cDDP). Because Cu deficiency is lethal yet Cu overload is poisonous, hCtr1 expression is transcriptionally ...upregulated in response to Cu deficiency but is downregulated under Cu replete conditions in controlling Cu homeostasis. The up- and down-regulation of hCtr1 is regulated by Specific protein 1 (Sp1), which itself is also correspondingly regulated under these Cu conditions. hCtr1 expression is also upregulated by cDDP via upregulation of Sp1. The underlying mechanisms of these regulations are unknown. Using gel-electrophoretic mobility shift assays, we demonstrated here that Sp1-DNA binding affinity is reduced under Cu replete conditions but increased under reduced Cu conditions. Similarly, Sp1-DNA binding affinity is increased by cDDP treatment. This in vitro system demonstrated, for the first time, that regulation of Sp1/hCtr1 expression by these agents is modulated by the stability of Sp1-DNA binding, the first step in the Sp1-mediated transcriptional regulation process.
Proposed models depicting Specific protein 1 (Sp1)-mediated transcriptional regulation of Sp1 itself and the human high-affinity copper transporter 1 (hCtr1) expression by bathocuproine sulfonate (BCS, a copper chelator) and cisplatin (cDDP) A and by copper B. Display omitted
•The human high-affinity copper transporter (hCtr1) transports both Cu and cisplatin.•hCtr1 is regulated by Cu availability and cisplatin via Specific protein 1 (Sp1) factor.•Cu(II) de-stabilizes Sp1-DNA binding.•Cu chelator and cisplatin increase Sp1-DNA binding.•Effects of Sp1-DNA binding by Cu(II)/cDDP mirror their effects on hCtr1/Sp1 expression.
Argininosuccinate synthetase 1 (ASS1) is the key enzyme that controls biosynthesis of arginine (Arg). ASS1 is silenced in many human malignancies therefore, these tumors require extracellular Arg for ...growth. The Arg-degrading recombinant protein, pegylated arginine deiminase (ADI-PEG20), has been in clinical trials for targeting Arg auxotrophic tumors by Arg starvation therapy. Resistance to Arg starvation is often developed through reactivation of ASS1 expression. We previously demonstrated that ASS1 silencing is controlled by HIF-1α and Arg starvation-reactivated ASS1 is associated with HIF-1α downregulation. However, mechanisms underlying ASS1 repression and HIF-1α turnover are not known. Here, we demonstrate that interplay of p300-HDAC2-Sin3A in the chromatin remodeling system is involved in HIF-1α degradation at the ASS1 promoter. The histone acetyltransferase p300 is normally associated with the ASS1 promoter to maintain acetylated H3K14ac and H3K27ac for ASS1 silencing. Arg starvation induces p300 dissociation, allowing histone HDAC2 and cofactor Sin3A to deacetylate these histones at the ASS1 promoter, thereby facilitating HIF-1α-proteasomal complex, driven by PHD2, to degrade HIF-1α in situ. Arg starvation induces PHD2 and HDAC2 interaction which is sensitive to antioxidants. This is the first report describing epigenetic regulation of chromosomal HIF-1α turnover in gene activation that bears important implication in cancer therapy.
Arginine deiminase (ADI)–based arginine depletion is a novel strategy under clinical trials for the treatment of malignant
melanoma with promising results. The sensitivity of melanoma to ADI ...treatment is based on its auxotrophy for arginine due
to a lack of argininosuccinate synthetase (AS) expression, the rate-limiting enzyme for the de novo biosynthesis of arginine. We show here that AS expression can be transcriptionally induced by ADI in melanoma cell lines
A2058 and SK-MEL-2 but not in A375 cells, and this inducibility was correlated with resistance to ADI treatment. The proximal
region of the AS promoter contains an E-box that is recognized by c-Myc and HIF-1α and a GC-box by Sp4. Through ChIP assays, we showed that
under noninduced conditions, the E-box was bound by HIF-1α in all the three melanoma cell lines. Under arginine depletion
conditions, HIF-1α was replaced by c-Myc in A2058 and SK-MEL-2 cells but not in A375 cells. Sp4 was constitutively bound to
the GC-box regardless of arginine availability in all three cell lines. Overexpressing c-Myc by transfection upregulated AS
expression in A2058 and SK-MEL-2 cells, whereas cotransfection with HIF-1α suppressed c-Myc–induced AS expression. These results
suggest that regulation of AS expression involves interplay among positive transcriptional regulators c-Myc and Sp4, and negative
regulator HIF-1α that confers resistance to ADI treatment in A2058 and SK-MEL-2 cells. Inability of AS induction in A375 cells
under arginine depletion conditions was correlated by the failure of c-Myc to interact with the AS promoter. Mol Cancer Ther 2009;8(12):3223–33
Abstract Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme that catalyzes the biosynthesis of arginine (Arg). Many malignant human tumors are auxotrophic for Arg because ASS1 is ...silenced. ASS1 has been established as a sensor of Arg auxotrophic response and a chemosensitivity marker for Arg starvation therapy. Here, we report that ASS1 is also a sensor for glutamine (Gln)-deprivation response, and that upregulation of ASS1 expression is associated with resistance to Gln-starvation treatments. Knockdown of ASS1 expression resulted in increased sensitivity to both Arg- and Gln-starvation, whereas increased ASS1 expression by ectopic transfection is associated with resistance to both Arg- and Gln-starvation. The addition of permeable fumarate, a metabolite that bridges the tricarboxylic acid and urea cycles, resulted in downregulation of ASS1 expression and increased sensitivity to both Arg- and Gln-deprivation treatments. Mechanistically, the Gln-deprivation response, like the arginine-auxotrophic response, downregulates HIF-1α resulting in de-silencing of ASS1. Our results demonstrate that ASS1 is a common biosensor for Arg and Gln deprivation response and a shared target for Arg- and Gln-starvation therapies which have been in several current clinical trials.
Three mechanisms have been proposed for the role of glutathione (GSH) in regulating cisplatin (CDDP) sensitivities that affects its ultimate cell-killing ability: (i) GSH may serve as a cofactor in ...facilitating multidrug resistance protein 2- (MRP2-) mediated CDDP efflux in mammalian cells, since MRP2-transfected cells were shown to confer CDDP resistance; (ii) GSH may serve as a redox-regulating cytoprotector based on the observations that many CDDP-resistant cells overexpress GSH and γ-glutamylcysteine synthesis (γ-GCS), the rate-limiting enzyme for GSH biosynthesis; (iii) GSH may function as a copper (Cu) chelator. Elevated GSH expression depletes the cellular bioavailable Cu pool, resulting in upregulation of the high-affinity Cu transporter (hCtr1) which is also a CDDP transporter. This has been demonstrated that overexpression of GSH by transfection with γ-GCS conferred sensitization to CDDP toxicity. This review describes how these three models were developed and critically reviews their importance to overall CDDP cytotoxicity in cancer cell treatments.
The platinum (Pt)-containing antitumor drugs including cisplatin (cis-diamminedichloroplatinum II, cDDP), carboplatin, and oxaliplatin, have been the mainstay of cancer chemotherapy. These drugs are ...effective in treating many human malignancies. The major cell-killing target of Pt drugs is DNA. Recent findings underscored the important roles of Pt drug transport system in cancer therapy. While many mechanisms have been proposed for Pt-drug transport, the high-affinity copper transporter (hCtr1), Cu chaperone (Atox1), and Cu exporters (ATP7A and ATP7B) are also involved in cDDP transport, highlighting Cu homeostasis regulation in Pt-based cancer therapy. It was demonstrated that by reducing cellular Cu bioavailable levels by Cu chelators, hCtr1 is transcriptionally upregulated by transcription factor Sp1, which binds the promoters of Sp1 and hCtr1. In contrast, elevated Cu poisons Sp1, resulting in suppression of hCtr1 and Sp1, constituting the Cu-Sp1-hCtr1 mutually regulatory loop. Clinical investigations using copper chelator (trientine) in carboplatin treatment have been conducted for overcoming Pt drug resistance due in part to defective transport. While results are encouraging, future development may include targeting multiple steps in Cu transport system for improving the efficacies of Pt-based cancer chemotherapy. The focus of this review is to delineate the mechanistic interrelationships between Cu homeostasis regulation and antitumor efficacy of Pt drugs.
Platinum-based antitumor agents are widely used in cancer chemotherapy. Drug resistance is a major obstacle to the successful use of these agents because once drug resistance develops, other ...effective treatment options are limited. Recently, we conducted a clinical trial using a copper-lowering agent to overcome platinum drug resistance in ovarian cancer patients and the preliminary results are encouraging. In supporting this clinical study, using three pairs of cisplatin (cDDP)-resistant cell lines and two ovarian cancer cell lines derived from patients who had failed in platinum-based chemotherapy, we showed that cDDP resistance associated with reduced expression of the high-affinity copper transporter (hCtr1), which is also a cDDP transporter, can be preferentially resensitized by copper-lowering agents because of enhanced hCtr1 expression, as compared with their drug-sensitive counterparts. Such a preferential induction of hCtr1 expression in cDDP-resistant variants by copper chelation can be explained by the mammalian copper homeostasis regulatory mechanism. Enhanced cell-killing efficacy by a copper-lowering agent was also observed in animal xenografts bearing cDDP-resistant cells. Finally, by analyzing a public gene expression dataset, we found that ovarian cancer patients with elevated levels of hCtr1 in their tumors, but not ATP7A and ATP7B, had more favorable outcomes after platinum drug treatment than those expressing low hCtr1 levels. This study reveals the mechanistic basis for using copper chelation to overcome cDDP resistance in clinical investigations.
The aim of this study was to comprehensively evaluate via a meta‐analysis the association between p27 expression and clinical outcome in breast cancer patients. We conducted a meta‐analysis of 20 ...studies (n= 6463 patients) that evaluated the correlation between p27 expression and indicators of breast cancer clinical outcome, including overall survival (OS), disease‐free survival (DFS) and relapse‐free survival (RFS). Data pooling was performed by RevMan 4.2. A total of 60% (9 of 15) of the studies showed a significant association between p27 high expression and OS, whereas 25% (2 of 8) and 60% (3 of 5) studies demonstrated a correlation between p27 high expression and DFS and RFS, respectively. The relative risks (RRs) were 1.34 (1.26–1.42) for OS (P < 0.00001), 1.27 (1.10–1.47) for DFS (P= 0.001) and 1.49 (0.92–2.42) for RFS (P= 0.10). In lymph node‐negative breast cancer patients, the RRs for OS and RFS were 1.84 (1.30–2.59; P= 0.0005) and 1.30 (0.20–8.50; P= 0.78), respectively. In lymph node‐positive breast cancer patients, the RRs for OS and RFS were 2.99 (1.77–5.07; P < 0.0001) and 1.49 (0.80–2.77; P= 0.21), respectively. This meta‐analysis indicates that reduced p27 is an independent prognostic factor for poor overall and disease‐free cancer survival.
Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer-related deaths worldwide. Preclinical studies found that copper-lowering agents could re-sensitize platinum-resistant ...cancer cells by enhancing the human copper transporter 1 (hCtr1)-mediated uptake of platinum. In the clinic, re-sensitization of platinum-resistance in relapsed EOC has been discovered by the application of trientine plus platinum (NCT01178112). However, no pharmacokinetic data of trientine has been reported in cancer patients.
Our study aimed to explore the safety and activity of trientine combined with carboplatin and pegylated liposomal doxorubicin (PLD) in patients with EOC, tubal, and peritoneal cancer who experienced disease progression during platinum-based chemotherapy or showed relapse <12 months after completing first-line chemotherapy. Also, we aimed to demonstrate pharmacokinetic parameters and to discover potential biomarkers in our EOC patients.
In this dose escalation study, 18 Asian patients in six dosing cohorts received fixed doses of carboplatin (AUC 4) and PLD (LipoDox®, TTY Biopharm Co. Ltd., Taipei, Taiwan) (40 mg/m
, day 1 per 4-week cycle), and escalated daily trientine doses (range: 300-1800 mg; initiated 7 days before the 1st combination cycle) according to a 3 + 3 design.
No dose-limiting toxicity or treatment-related death was observed. Four patients (22.2%) developed grade 3 drug-related adverse events (AEs), whereas no grade 4 AEs were encountered. Anemia and grade 2 dizziness were the most common hematological toxicity and neurotoxicity, respectively. In a pharmacokinetics comparison with healthy volunteers in the literature, our patients achieved greater absorption after oral trientinem, and more rapid elimination of triethylenetetramine dihydrochloride at high doses. The clinical benefit rate was 33.3 and 50.0% in the platinum-resistant and the partially platinum-sensitive group, respectively. A high baseline serum iron level and low serum copper level might help differentiate subgroups of patients with different clinical responses. Nevertheless, no associations of the clinical response with the levels of serum hCtr1, ceruloplasmin, or copper were observed.
Combination therapy with carboplatin, trientine, and PLD was well-tolerated and safe. Our results encourage the development of a future phase II trial.
ClinicalTrials.gov # NCT03480750.
Abstract Background Recent studies have shown that human copper transporter 1 (hCtr1), the major copper influx transporter, is involved in the transport of platinum-based antitumor agents. We ...investigated the predictive and prognostic values of hCtr1, and copper efflux transporters ATP7A and ATP7B, in patients with locally advanced non-small cell lung cancer (NSCLC) receiving first-line platinum-based chemotherapy. Methods From 2004 to 2009, we identified 54 consecutive stage III NSCLC patients who underwent first-line platinum-based doublet chemotherapy. Immunohistochemical studies of hCtr1, ATP7A and ATP7B on the paraffin-embedded pre-treatment tumor samples were performed and correlated with chemotherapy response and survival. Results Overexpression of hCtr1, ATP7A and ATP7B were observed in 68%, 48% and 74% of the participants, respectively. hCtr1 overexpression was associated with better chemotherapy responses ( P < 0.01); whereas ATP7A and ATP7B were not. Patients with hCtr1 overexpressing tumors had better progression-free survival (PFS) and overall survival (OS) ( P = 0.01 and 0.047, respectively). In multivariate analyses for chemotherapy response and PFS, only hCtr1 overexpression emerged as a favorable independent predictive and prognostic factor (all P < 0.01). Conclusion This is the first report to state that hCtr1 is not only an independent predictor of platinum-based chemotherapy response but also a prognostic factor in stage III NSCLC.
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