TG-interacting factor transcriptionally induced by AKT/FOXO3A is a negative regulator that antagonizes arsenic trioxide-induced cancer cell apoptosis

https://doi.org/10.1016/j.taap.2015.03.007Get rights and content

Highlights

  • ATO-induced biphasic survival responses of cancer cells depend on low- or high-concentrations.

  • TGIF mediates low-concentration ATO-induced cancer cell proliferation, migration, and invasion.

  • ATO transcriptionally regulates TGIF expression via c-Src/EGFR/AKT/FOXO3A signalings.

  • Increased TGIF or AKT activation attenuates high-concentration ATO-induced CDKN1A expression and cellular apoptosis.

  • Suppression of these negative regulators might be a promising therapeutic strategy to improve therapeutic efficacy of ATO.

Abstract

Arsenic trioxide (ATO) is a multi-target drug approved by the Food and Drug Administration as the first-line chemotherapeutic agent for the treatment of acute promyelocytic leukemia. In addition, several clinical trials are being conducted with arsenic-based drugs for the treatment of other hematological malignancies and solid tumors. However, ATO's modest clinical efficacy on some cancers, and potential toxic effects on humans have been reported. Determining how best to reduce these adverse effects while increasing its therapeutic efficacy is obviously a critical issue. Previously, we demonstrated that the JNK-induced complex formation of phosphorylated c-Jun and TG-interacting factor (TGIF) antagonizes ERK-induced cyclin-dependent kinase inhibitor CDKN1A (p21WAF1/CIP1) expression and resultant apoptosis in response to ATO in A431 cells. Surprisingly, at low-concentrations (0.1–0.2 μM), ATO increased cellular proliferation, migration and invasion, involving TGIF expression, however, at high-concentrations (5–20 μM), ATO induced cell apoptosis. Using a promoter analysis, TGIF was transcriptionally regulated by ATO at the FOXO3A binding site (− 1486 to − 1479 bp) via the c-Src/EGFR/AKT pathway. Stable overexpression of TGIF promoted advancing the cell cycle into the S phase, and attenuated 20 μM ATO-induced apoptosis. Furthermore, blockage of the AKT pathway enhanced ATO-induced CDKN1A expression and resultant apoptosis in cancer cells, but overexpression of AKT1 inhibited CDKN1A expression. Therefore, we suggest that TGIF is transcriptionally regulated by the c-Src/EGFR/AKT pathway, which plays a role as a negative regulator in antagonizing ATO-induced CDKN1A expression and resultant apoptosis. Suppression of these antagonistic effects might be a promising therapeutic strategy toward improving clinical efficacy of ATO.

Introduction

Arsenic has been used in traditional Chinese medicine (TCM) for the treatment of syphilis, ulcers and psoriasis for over two millennia. In 2000, arsenic trioxide (As2O3, ATO) was approved by the Food and Drug Administration (FDA) as a first-line chemotherapeutic agent for the treatment of both de novo and relapsed acute promyelocytic leukemia (APL) (Dilda and Hogg, 2007, Hughes et al., 2011). Furthermore, clinical trials of other arsenical-based cancer drugs are being conducted for the treatment of various forms of cancer, including: multiple myeloma (MM), myelodysplasia (MDS) and some solid tumors (Dilda and Hogg, 2007).

However, patients with hepatocellular carcinoma (HCC) who were treated with ATO, used as a single-agent in a phase II clinical trial, showed resistance to the treatment (Lin et al., 2007). In addition, chronic exposure to low-concentrations of (< 1 mg/kg) ATO in a mouse model of HCC can enhance tumor growth, angiogenesis and metastasis, but cannot induce tumor cell apoptosis (Liu et al., 2006). Furthermore, dual effects of ATO on APL cells were also found: inducing differentiation at low concentrations (0.1–0.5 μM) and apoptosis at high concentrations (0.5–2 μM) (Chen et al., 1997). Some dermatological diseases have been attributed to the long-term use of arsenic-based Fowler's solution (1% potassium arsenite) which was used successfully in the treatment of psoriasis, an inflammatory skin disease characterized by excessive proliferation of keratinocytes (Cuzick et al., 1982). Due to its toxic effects on patients, the medication was eventually prohibited from medical use (Hughes et al., 2011). Therefore, we suggest that ATO is a multi-target drug that has remarkable efficacy in the treatment of some diseases; however, its adverse effects should be particularly monitored, and fully elucidated so as to enhance its therapeutic utility.

In our previous studies, 20 μM ATO induced the ERK pathway to enhance cyclin-dependent kinase inhibitor CDKN1A expression and cellular apoptosis (Liu and Huang, 2008a). In contrast, ATO also activated the JNK pathway to phosphorylate c-Jun to recruit TG-interacting factor (TGIF)/HDAC1 complex to inhibit the CDKN1A promoter and result in apoptosis (Huang et al., 2006, Liu and Huang, 2008b, Huang et al., 2010). TGIF belongs to the TALE (three-amino-acid loop extension) subfamily of atypical homeodomain proteins (Bertolino et al., 1995), which plays a transcriptional repressor/co-repressor to modulate TGF-β (Wotton et al., 2001) and retinoic acid signaling (Bartholin et al., 2006). It has been implicated in diverse biological and pathological functions, such as: brain development (Gripp et al., 2000), hematopoietic stem cell function (Yan et al., 2013), regulating vascularization of the embryonic placenta (Bartholin et al., 2008), differentiation of preadipocytes (Horie et al., 2008) and various types of human cancer, such as: leukemia, ovarian cancer, liver cancer and gastric carcinoma (Imoto et al., 2000, Borlak et al., 2005, Hu et al., 2005, Hamid and Brandt, 2009). Recently, we also demonstrated that increased TGIF expression in upper-tract urothelial carcinoma (UTUC) patients after radical nephroureterectomy is associated with progression of the disease and worsened prognosis (Huang et al., 2012, Yeh et al., 2012). However, to date, evidence for the regulation of TGIF is limited.

In the present studies, ATO was found to have biphasic effects on various cancer cells, depending low or high concentrations. In particular, TGIF was involved in low-concentration (0.1–0.2 μM) ATO-induced cellular proliferation, migration, and invasion. Furthermore, the antagonistic effects of TGIF on high-concentration ATO-induced cell apoptosis were explored.

Section snippets

Reagents and antibodies

ATO, actinomycin D, LY294002, protein A-agarose beads and streptavidin-agarose were purchased from Sigma-Aldrich. The Wizard Plus MiniPrep DNA purification system, luciferase plasmid pGL-3 and the luciferase assay system were obtained from Promega. PD153035 and PP1 were purchased from Calbiochem. Lipofectamine™ 2000 reagent, the TRIzol® RNA extraction kit, SuperScript™ III, Dulbecco's modified Eagle's medium (DMEM) and Opti-MEM were obtained from Invitrogen. Antibodies against TGIF, CDKN1A,

TGIF mediates low-concentration ATO-induced cancer cell proliferation

According to our previous findings, there are opposing effects of ATO on A431 cells (Huang et al., 2006, Liu and Huang, 2008b, Huang et al., 2010), which might depend on the concentrations of ATO. To evaluate the effects of different concentrations of ATO on cellular responses, A431 cells were treated with various concentrations of ATO to measure their viability. Treatment with low-concentration (0.1 and 0.5 μM) ATO for 48 h increases cellular growth by about 10%, but with high-concentration (> 5 

Discussion

As described above, ATO has been approved by the FDA as an effective chemotherapeutic agent for the treatment of patients with both de novo and relapsed APL (Dilda and Hogg, 2007, Hughes et al., 2011). Furthermore, several arsenical-based drugs are undergoing trials for the treatment of patients with other hematological malignancies and solid tumors (Dilda and Hogg, 2007); however, using ATO as a single-agent in a phase II clinical trial of patients with HCC indicates resistance to this

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grants from the National Science Council (Taipei, Taiwan; NSC98-2320-B-006-008-MY3; NSC101-2320-B-006-034; NSC102-2320-B-006-017), and by a grant from Health and Welfare surcharge of tobacco products, Ministry of Health and Welfare (MOHW103-TD-B-111-05).

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