Modulating Chemosensitivity of Tumors to Platinum-Based Antitumor Drugs by Transcriptional Regulation of Copper Homeostasis.

Platinum (Pt)-based antitumor agents have been effective in treating many human malignancies. Drug importing, intracellular shuffling, and exporting-carried out by the high-affinity copper (Cu) transporter (hCtr1), Cu chaperone (Ato x1), and Cu exporters (ATP7A and ATP7B), respectively-cumulatively contribute to the chemosensitivity of Pt drugs including cisplatin and carboplatin, but not oxaliplatin. This entire system can also handle Pt drugs via interactions between Pt and the thiol-containing amino acid residues in these proteins; the interactions are strongly influenced by cellular redox regulators such as glutathione. hCtr1 expression is induced by acute Cu deprivation, and the induction is regulated by the transcription factor specific protein 1 (Sp1) which by itself is also regulated by Cu concentration variations. Copper displaces zinc (Zn) coordination at the zinc finger (ZF) domains of Sp1 and inactivates its DNA binding, whereas Cu deprivation enhances Sp1-DNA interactions and increases Sp1 expression, which in turn upregulates hCtr1. Because of the shared transport system, chemosensitivity of Pt drugs can be modulated by targeting Cu transporters. A Cu-lowering agent (trientine) in combination with a Pt drug (carboplatin) has been used in clinical studies for overcoming Pt-resistance. Future research should aim at further developing effective Pt drug retention strategies for improving the treatment efficacy.

C-Jun N-terminal kinase signalling pathway in response to cisplatin.

Cisplatin (cis diamminedichloroplatinum II, cDDP) is one of the most effective cancer chemotherapeutic agents and is used in the treatment of many types of human malignancies. However, inherent tumour resistance is a major barrier to effective cisplatin therapy. So far, the mechanism of cDDP resistance has not been well defined. In general, cisplatin is considered to be a cytotoxic drug, for damaging DNA and inhibiting DNA synthesis, resulting in apoptosis via the mitochondrial death pathway or plasma membrane disruption. cDDP-induced DNA damage triggers signalling pathways that will eventually decide between cell life and death. As a member of the mitogen-activated protein kinases family, c-Jun N-terminal kinase (JNK) is a signalling pathway in response to extracellular stimuli, especially drug treatment, to modify the activity of numerous proteins locating in the mitochondria or the nucleus. Recent studies suggest that JNK signalling pathway plays a major role in deciding the fate of the cell and inducing resistance to cDDP-induced apoptosis in human tumours. c-Jun N-terminal kinase regulates several important cellular functions including cell proliferation, differentiation, survival and apoptosis while activating and inhibiting substrates for phosphorylation transcription factors (c-Jun, ATF2: Activating transcription factor 2, p53 and so on), which subsequently induce pro-apoptosis and pro-survival factors expression. Therefore, it is suggested that JNK signal pathway is a double-edged sword in cDDP treatment, simultaneously being a significant pro-apoptosis factor but also being associated with increased resistance to cisplatin-based chemotherapy. This review focuses on current knowledge concerning the role of JNK in cell response to cDDP, as well as their role in cisplatin resistance.

Arginine deprivation in cancer therapy.

PURPOSE OF REVIEW: There has been an increased and renewed interest in metabolic therapy for cancer, particularly Arg deprivation. The purpose of this review is to highlight recent studies that focus on Arg-dependent malignancies with Arginine (Arg)-degrading enzymes, including arginase and Arg deiminase. RECENT FINDINGS: New developments in this area include understanding of the role of most significantly downregulated gene regulating amino acid metabolism, argininosuccinate synthetase and its expression and therapeutic relevance in different tumors. Recent studies have also shed light on the mechanism of tumor cell death with Arg deprivation, with arginase and pegylated Arg deiminase. Particularly important is understanding the mechanism of resistance that cancers develop after such drug exposure. Finally, recent clinical trials have been performed or are ongoing to use Arg deprivation as treatment for advanced malignancies. SUMMARY: Arg deprivation is a promising approach for the treatment of various malignancies.

Exploratory study of carboplatin plus the copper-lowering agent trientine in patients with advanced malignancies.

PURPOSE: Preclinical data showed that trientine, a copper-lowering agent, re-sensitized cancer cells to carboplatin through enhanced human copper transporter 1 (hCtr1) -mediated platinum uptake. EXPERIMENTAL DESIGN: We studied carboplatin and trientine in patients (n = 55; 45 who had failed platinum) with advanced malignancies (Phase I, modified 3 + 3 design). RESULTS: The most common cancers were head and neck (n = 13), non-small cell lung (n = 10) and epithelial ovarian (n = 8). The median number of prior regimens was four. No dose-limiting toxicity or treatment-related deaths were observed at doses up to carboplatin AUC 6 given with trientine. Eight patients achieved stable disease (SD) ≥ 6 months (six platinum failures) and one patient with platinum-resistant ovarian cancer, partial response (PR) (total SD ≥ 6 months/PR = 9, 16.4 %). The mean nadir serum copper level in the nine patients with SD ≥ 6 months/PR was 0.55 µg/mL (95 % CI, 0.34-0.75) versus 1.22 µg/mL (95 % CI, 1.02-1.42) (p < 0.001) in 38 tested patients with progression. In patients who maintained their ceruloplasmin (major copper-carrying protein) levels at 5-15 mg/dL (n = 9), the median progression-free and overall survivals were 9.2 and 15.2 months versus 1.9 (p = 0.001) and 5.7 months (p = 0.033) in patients who did not (n = 38), respectively. CONCLUSIONS: The combination of a copper-lowering agent with carboplatin was well tolerated and associated with antitumor activity, especially in patients in whom copper and/or ceruloplasmin levels were lowered. Further investigation of this strategy for reversing platinum resistance is warranted.

Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

Specificity protein 1 (sp1) oscillation is involved in copper homeostasis maintenance by regulating human high-affinity copper transporter 1 expression.

Copper is an essential micronutrient for cell growth but is toxic in excess. Copper transporter (Ctr1) plays an important role in regulating adequate copper levels in mammalian cells. We have shown previously that expression of the human high-affinity copper transporter (hCtr1) was transcriptionally up-regulated under copper-depleted conditions and down-regulated under replete conditions; moreover, elevated hCtr1 levels suppress hCtr1 expression. Specificity protein 1 (Sp1) regulates expression of hCtr1 under copper-stressed conditions. In this study, we made the following important observations: 1) Sp1 expression is down-regulated under copper-replete conditions but up-regulated under copper-depleted conditions. These up- and down-regulations of Sp1 in turn regulate hCtr1 expression to control copper homeostasis. 2) Copper-regulated Sp1 expression involved Sp1 binding to its own promoter as demonstrated by the chromatin immunoprecipitation assay; therefore, Sp1 is also transcriptionally self-regulated via hCtr1/copper intermediation. 3) Both zinc finger and glutamine-rich transactivation domains of Sp1 are involved in the Sp1-mediated hCtr1 and Sp1 regulation by copper stresses. 4) Although Sp3 expression is also regulated by copper availability, Sp3 does not regulate hCtr1 homeostasis. Collectively, our results demonstrated that mammalian cells use Sp1 oscillation in response to copper availability to regulate copper homeostasis through hCtr1 expression in a tripartite inter-regulatory relationship. These findings have important implications in mammalian copper physiology regulation.

Targeting the Proline-Glutamine-Asparagine-Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy.

Proline, glutamine, asparagine, and arginine are conditionally non-essential amino acids that can be produced in our body. However, they are essential for the growth of highly proliferative cells such as cancers. Many cancers express reduced levels of these amino acids and thus require import from the environment. Meanwhile, the biosynthesis of these amino acids is inter-connected but can be intervened individually through the inhibition of key enzymes of the biosynthesis of these amino acids, resulting in amino acid starvation and cell death. Amino acid starvation strategies have been in various stages of clinical applications. Targeting asparagine using asparaginase has been approved for treating acute lymphoblastic leukemia. Targeting glutamine and arginine starvations are in various stages of clinical trials, and targeting proline starvation is in preclinical development. The most important obstacle of these therapies is drug resistance, which is mostly due to reactivation of the key enzymes involved in biosynthesis of the targeted amino acids and reprogramming of compensatory survival pathways via transcriptional, epigenetic, and post-translational mechanisms. Here, we review the interactive regulatory mechanisms that control cellular levels of these amino acids for amino acid starvation therapy and how drug resistance is evolved underlying treatment failure.

Targeting the Copper Transport System to Improve Treatment Efficacies of Platinum-Containing Drugs in Cancer Chemotherapy.

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.

p27(Kip1) as a prognostic factor in breast cancer: a systematic review and meta-analysis.

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.

Collaboration Between RSK-EphA2 and Gas6-Axl RTK Signaling in Arginine Starvation Response That Confers Resistance to EGFR Inhibitors.

Many human malignancies require extracellular arginine (Arg) for survival because the key enzyme for de novo Arg biosynthesis, argininosuccinate synthetase 1 (ASS1), is silenced. Recombinant arginine deiminase (ADI-PEG20), which digests extracellular Arg, has been in clinical trials for treating ASS1-negative tumors. Reactivation of ASS1 is responsible for the treatment failure. We previously demonstrated that ASS1 reactivation is transcriptionally regulated by c-Myc via the upstream Gas6-Axl tyrosine kinase (RTK) signal. Here, we report that another RTK EphA2 is coactivated via PI3K-ERK/RSK1 pathway in a ligand-independent mechanism. EphA2 is also regulated by c-Myc. Moreover, we found that knockdown Axl upregulates EphA2 expression, demonstrating cross-talk between these RTKs. ADIR cell lines exhibits enhanced sensitivities to nutrient deprivation such as charcoal-stripped FBS and multiple RTK inhibitor foretinib but resistance to EGFR inhibitors. Knockdown EphA2, and to lesser extent, Axl, overcomes EGFRi resistance. c-Myc inhibitor JQ1 can also sensitize ADIR cells to ADI-PEG20. This study elucidates molecular interactions of multiple RTKs in Arg-stress response and offers approaches for developing strategies of overcoming ADI-PEG20 resistance.

A Dose Escalation Study of Trientine Plus Carboplatin and Pegylated Liposomal Doxorubicin in Women With a First Relapse of Epithelial Ovarian, Tubal, and Peritoneal Cancer Within 12 Months After Platinum-Based Chemotherapy.

Background: 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. Purpose: 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. Methods: 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/m2, 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. Results: 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. Conclusion: Combination therapy with carboplatin, trientine, and PLD was well-tolerated and safe. Our results encourage the development of a future phase II trial. Clinical trial registration: ClinicalTrials.gov # NCT03480750.

Elevated GSH level increases cadmium resistance through down-regulation of Sp1-dependent expression of the cadmium transporter ZIP8.

Cadmium is a nonessential toxic metal in mammals. Its toxicity is mainly caused by interactions with cellular proteins that result in protein dysfunction and then disturb normal cellular functions. Glutathione (GSH) has been reported to play a role in cadmium resistance by serving as a cofactor for multidrug resistance protein 1/GS-X pump-mediated cadmium elimination. To further investigate the role of GSH in cadmium toxicity, we carried out a comparative study using small-cell lung cancer-derived cell lines, SR3A, and those that were stably transfected with glutamate cysteine ligase catalytic subunit (GCLC), a rate-limiting enzyme in GSH biosynthesis. These GCLC stably transfected cell lines produced higher levels of GSH and were more resistant to cadmium toxicity than the parental cell line was. The rates of cadmium uptake were reduced in these GCLC-transfected cell lines, which were associated with down-regulation of the cadmium transporter ZIP8/SLC39A8. Further analyses demonstrated that Sp1 binding site at the proximal promoter region of ZIP8 was sensitive to the GSH level and that the expression level of transcription factor Sp1 was reduced by increased GSH levels. We also demonstrated that low concentrations of cadmium exposure down-regulated ZIP8 expression with concomitant reduction of Sp1 expression. Taken together, these results demonstrate the importance of Sp1 in the regulation of ZIP8 expression. More important, our results reveal a new mechanism by which elevated GSH levels confer cadmium resistance by down-regulation of ZIP8 expression through the suppression of Sp1.

Transcription factor Sp1 plays an important role in the regulation of copper homeostasis in mammalian cells.

Copper is an essential metal nutrient, yet copper overload is toxic. Here, we report that human copper transporter (hCtr) 1 plays an important role in the maintenance of copper homeostasis by demonstrating that expression of hCtr1 mRNA was up-regulated under copper-depleted conditions and down-regulated under copper-replete conditions. Overexpression of full-length hCtr1 by transfection with a recombinant hCtr1 cDNA clone reduced endogenous hCtr1 mRNA levels, whereas overexpression of N terminus-deleted hCtr1 did not change endogenous hCtr1 mRNA levels, suggesting that increased functional hCtr1 transporter, which leads to increased intracellular copper content, down-regulates the endogenous hCtr1 mRNA. A luciferase assay using reporter constructs containing the hCtr1 promoter sequences revealed that three Sp1 binding sites are involved in the basal and copper concentration-dependent regulation of hCtr1 expression. Modulation of Sp1 levels affected the expression of hCtr1. We further demonstrated that the zinc-finger domain of Sp1 functions as a sensor of copper that regulates hCtr1 up and down in response to copper concentration variations. Our results demonstrate that mammalian copper homeostasis is maintained at the hCtr1 mRNA level, which is regulated by the Sp1 transcription factor.

Elevated glutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of copper transporter hCtr1.

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to up-regulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.

Nrf2-dependent and -independent induction of ABC transporters ABCC1, ABCC2, and ABCG2 in HepG2 cells under oxidative stress.

Nrf2, an NF-E2-related transcription factor, plays a critical role in transcriptional upregulation of many target genes, including those for metabolizing enzymes and transporters essential for cellular defense in response to oxidative and/or electrophilic stress. In the present study, we have studied the potential involvement of Nrf2 in induction of human ABC transporter genes under oxidative stress. We created a real-time PCR primer set to quantitatively investigate the induction of human ABC transporters by a redox-active compound tert-butylhydroquinone (tBHQ) in HepG2 cells. We found that mRNA levels of ABCC1, ABCC2, ABCC3, and ABCG2 were significantly elevated in dose- and time-dependent manners. Translocation of Nrf2 into the nuclei occurred concomitantly with the induction of ABCC1 and ABCC2 as well as both heavy and light chains of gamma-glutamylcysteine synthetase (gamma-GCSh and gamma-GCSI) during tBHQ treatments. To examine the potential involvement of Nrf2 in upregulation of the ABC transporters, we treated cells with siRNA to knockdown the expression of Nrf2. Under such Nrf2-knockdown conditions, tBHQ-induced mRNA levels of ABCC2 and ABCG2 were significantly suppressed as were mRNA levels of gamma-GCSh and gamma-GCSI. Interestingly, however, the elevated mRNA level of ABCC1 was little affected by Nrf2 siRNA treatment. We also addressed the involvement of Keap1, which is a negative regulator of Nrf2 by retrieving it in the cytoplasm. When HepG2 cells were treated with Keap1-specifc siRNA, a significant increase was observed in mRNA levels of ABCC1, ABCC2, and ABCG2 as well as gamma-GCSI, suggesting that induction of ABCC2 and ABCG2 by tBHQ is mediated by the Nrf2/Keap1 system, whereas the induction of ABCC1 may involve a Keap1-dependent but Nrf2-independent mechanism.

Improving radiotherapy in cancer treatment: Promises and challenges.

Effective radiotherapy for cancer has relied on the promise of maximally eradicating tumor cells while minimally killing normal cells. Technological advancement has provided state-of-the-art instrumentation that enables delivery of radiotherapy with great precision to tumor lesions with substantial reduced injury to normal tissues. Moreover, better understanding of radiobiology, particularly the mechanisms of radiation sensitivity and resistance in tumor lesions and toxicity in normal tissues, has improved the treatment efficacy of radiotherapy. Previous mechanism-based studies have identified many cellular targets that can affect radiation sensitivity, notably reactive oxygen species, DNA-damaging response signals, and tumor microenvironments. Several radiation sensitizers and protectors have been developed and clinically evaluated; however, many of these results are inconclusive, indicating that improvement remains needed. In this era of personalized medicine in which patients' genetic variations, transcriptome and proteomics, tumor metabolism and microenvironment, and tumor immunity are available. These new developments have provided opportunity for new target discovery. Several radiotherapy sensitivity-associated "gene signatures" have been reported although clinical validations are needed. Recently, several immune modifiers have been shown to associate with improved radiotherapy in preclinical models and in early clinical trials. Combination of radiotherapy and immunocheckpoint blockade has shown promising results especially in targeting metastatic tumors through abscopal response. In this article, we succinctly review recent advancements in the areas of mechanism-driven targets and exploitation of new targets from current radio-oncogenomic and radiation-immunotherapeutic approaches that bear clinical implications for improving the treatment efficacy of radiotherapy.

Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression.

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.

Argininosuccinate synthetase 1 (ASS1) is a common metabolic marker of chemosensitivity for targeted arginine- and glutamine-starvation therapy.

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.

Effects of Cu(II) and cisplatin on the stability of Specific protein 1 (Sp1)-DNA binding: Insights into the regulation of copper homeostasis and platinum drug transport.

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.

Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1α, and c-Myc transcription network and is independent of ASS1 promoter DNA methylation.

Many human tumors require extracellular arginine (Arg) for growth because the key enzyme for de novo biosynthesis of Arg, argininosuccinate synthetase 1 (ASS1), is silenced. These tumors are sensitive to Arg-starvation therapy using pegylated arginine deiminase (ADI-PEG20) which digests extracellular Arg. Many previous studies reported that ASS1 silencing is due to epigenetic inactivation of ASS1 expression by DNA methylation, and that the demethylation agent 5-aza-deoxycytidine (Aza-dC) can induce ASS1 expression. Moreover, it was reported that cisplatin suppresses ASS1 expression through ASS1 promoter methylation, leading to synthetic lethality to ADI-PEG20 treatment. We report here that cisplatin supppresses ASS1 expression is due to upregulation of HIF-1α and downregulation of c-Myc, which function as negative and positive regulators of ASS1 expression, respectively, by reciprocal bindings to the ASS1 promoter. In contrast, we found that Aza-dC induces ASS1 expression by downregulation of HIF-1α but upregulation of c-Myc. We further demonstrated that the clock protein DEC1 is the master regulator of HIF-1α and c-Myc that regulate ASS1. cDDP upregulates DEC1, whereas Aza-dC suppresses its expression. Using two proteasomal inhibitors bortezomib and carfilzomib which induce HIF-1α accumulation, we further demonstrated that HIF-1α is involved in ASS1 silencing for the maintenance of Arg auxotrophy for targeted Arg-starvation therapy.