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Thioredoxin-1 (Trx-1) redox signaling regulates multiple aspects of cell growth and survival, and elevated tumor levels of Trx-1 have been associated with decreased patient survival. PX-12, an inhibitor of Trx-1 currently in clinical development, has been found to decrease tumor levels of the HIF-1α transcription factor. SSAT1 has been reported to bind to HIF-1α and RACK1, resulting in oxygen-independent HIF-1 ubiquitination and degradation. SSAT2, a related protein, stabilizes the interaction of the VHL protein and elongin C with HIF-1 leading to oxygen-dependent HIF-1α ubiquitination and degradation. We investigated the effects of PX-12 and Trx-1 on SSAT1, SSAT2, and inhibition of HIF-1α.
A panel of cell lines was treated with PX-12 to investigate its effects on SSAT1 and SSAT2 expression, and on HIF-1α protein levels. We also evaluated the regulation of SSAT1 through the Nrf2 and PMF-1, two trans-acting transcription factors.
We found that PX-12 increased nuclear Nrf2 activity and antioxidant response element binding. PX-12 also increased the expression of SSAT1 but not SSAT2 in a PMF-1-dependent manner that was independent of Trx-1. Inhibition of Nrf2 or PMF-1 prevented the increase in SSAT1 caused by PX-12.
The results show that PX-12, acting independently of Trx-1, increases nuclear Nrf2, which interacts with PMF-1 to increase the expression of SSAT1. The degradation of HIF-1α that results from binding with SSAT1 may explain the decrease in HIF-1α caused by PX-12 and could contribute to the antitumor activity of PX-12.
Thioredoxin-1 (Trx-1) is a small (12 kDa) redox protein that is an important regulator of many aspects of cell proliferation and survival . Thioredoxin reductase, a homodimeric flavoprotein, transfers reducing equivalents from NADPH to Trx-1, which then acts as a redox cofactor and a cellular antioxidant through the transfer of reducing equivalents to peroxiredoxins . Trx-1 also increases the DNA binding of redox-sensitive transcription factors such as activator protein-1 (AP-1) and nuclear factor kappa-B (NF-κB) , and binds to and inhibits proapoptotic proteins including apoptosis signal–regulating kinase-1 (Ask-1)  and the tumor suppressor PTEN (phosphatase and tensin homolog) , which attenuates the activity of the phosphatidylinositol-3-kinase/Akt cell survival pathway in cancer cells. Trx-1 is overexpressed in many human cancers including lung, colon, cervix, stomach, and pancreatic cancer , where it is associated with aggressive tumor growth, decreased patient survival [6, 7], and resistance to chemotherapy [8, 9]. We have previously reported that Trx-1 increases the levels of the hypoxia-inducible transcription factor-1alpha (HIF-1α) under both normoxic and hypoxic conditions and increases the expression of HIF-1α downstream targets such as vascular endothelial growth factor (VEGF) leading to increased tumor angiogenesis . We have also suggested that Trx-1 may play a role in the redox regulation of spermidine/spermine N(1)-acetyl transferase 1 (SSAT1) and polyamine homeostasis, which could contribute to the biological effects of Trx-1 . SSAT1 is a polyamine catabolic enzyme that acetylates spermidine and spermine, thus reducing their potency. SSAT1 acts in concert with spermine oxidase and N(1)-acetylpolyamine oxidase to regulate polyamine metabolism . SSAT1 is transcriptionally regulated through the interaction of two trans-acting transcription factors, nuclear factor erythroid-derived 2 (NF-E2)–related factor 2 (Nrf2) and polyamine modulated factor-1 (PMF-1) . SSAT1 binding has been reported to stabilize the interaction between hypoxia-induced HIF-1α, RACK-1 (receptor for activated C kinase-1), and elongin C, thus promoting oxygen-independent HIF-1α ubiquitination and degradation . SSAT2 has 64% similarity and 46% homology to SSAT1  but has a much lower level of acetyltransferase activity toward polyamines and a lower level of expression than SSAT1 [16–18]. SSAT2 has been reported to stabilize the interaction of the von Hippel-Lindau protein (pVHL) and elongin C with HIF-1α and lead to oxygen-dependent HIF-1α ubiquitination and degradation .
PX-12, 1-methylpropyl 2-imidazolyl disulfide, is a Trx-1 inhibitor that shows antitumor activity in both in vitro and in vivo  and that is currently in clinical development . We have previously reported that PX-12 lowers tumor HIF-1α protein levels and inhibits HIF-1α transactivating activity and the expression of the downstream targets of HIF-1α, including VEGF [21–24]. In the present study, we investigated the regulation of HIF-1α by PX-12 and the associated roles of Trx-1, SSAT1, and SSAT2. We found that PX-12 increases SSAT1 expression and decreases HIF-1α levels independently of Trx-1 and that SSAT2 does not play a role in HIF-1α degradation by PX-12.
Human MiaPaCa-2 pancreatic cancer cells, MCF-7 breast cancer cells, LN-229 glioma cancer cells, and HEK-293 transformed embryonic kidney cells were obtained from American Type Culture Collection (Manassas, VA). Monoclonal antibodies to human HIF-1α, RACK1, and elongin C were purchased from BD Transduction Laboratories (San Diego, CA), and antibodies to Trx-1, Trx-2, Trx-reductase-1 and -2, lamin A/C, β-actin, NAD(P)H dehydrogenase, quinone 1 (NQO1), and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PX-12 was synthesized , and MG-132 was purchased from Sigma–Aldrich (St. Louis, MO).
MiaPaCa-2, HEK-293, and LN-229 cells were cultured in Dulbecco’s minimum essential medium (Cellgro, Media-tech, Manassas, VA) and 10% fetal calf serum (Hyclone Laboratories, Logan, UT) at 37°C in 5% CO2. MCF-7 cells were cultured in minimum essential medium (Cellgro) and 10% fetal calf serum. All cell lines were genetically verified by the M. D. Anderson Cell Line Characterization Service. For assay, cells were incubated for various times at 37°C in humidified air, 5% CO2 (normoxia), or 1% O2, 5% CO2, 94% N2 (hypoxia) using an Invivo Hypoxia Workstation 400 with Ruskinn hypoxic gas mixer (Ruskinn Technology, Pencoed, UK). Cells (2.5 × 105)) were seeded and grown under normoxic conditions to 70% confluence and then incubated under hypoxic conditions for 16–24 h in the presence or absence of PX-12. Cells cultured under hypoxic conditions were collected under hypoxic conditions.
Cells were grown under normoxic or hypoxic conditions in the presence or absence of 25 μM PX-12. The cells were washed twice in phosphate-buffered saline, and Western blotting was performed as previously described . Blots were quantified using ImageQuant software (Molecular Dynamics/GE Healthcare Biosciences, Pittsburgh, PA). Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce/Thermo Fisher Scientific, Rockford, IL) in accordance with the manufacturer’s protocols.
Total RNA was isolated from cell lysates using the PARIS kit (Ambion/Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. TaqMan quantitative reverse transcription (RT)–PCR was performed on the ABI 7300 system using the TaqMan one-step reverse transcription–PCR Master Mix kit and predesigned primer/probe pairs for SSAT1, SSAT2, Nrf2, PMF-1, and β2-microglobulin (Applied Biosystems, Foster City, CA). Normalization and analyses were carried out with β2-microglobulin (B2M) using the 2(-delta-delta C(T)) method as the internal reference  and with Applied Biosystems GeneAmp 5700 SDS software. All measurements were performed in triplicate.
Small interfering RNA (siRNA) SMARTpool sequences were obtained from Dharmacon/Thermo Fisher Scientific (Waltham, MA), and the cells were transfected with 25 nM siRNA-Trx-1, siRNA-EIF2AK3 (PERK), siRNA-Nrf-2, siRNA-SSAT1, siRNA-SSAT2, siRNA-PMF-1, siRNA-HIF-1α, or a siRNA nontargeting control using Dharma-FECT-1 lipid transfection reagent. The transfection medium was removed after 24 h and replaced with fresh medium, and the cells were grown in 5% CO2 at 37°C for an additional 48–72 h. Western blot analyses were performed to confirm target knockdown by siRNA. Transfected cells were treated with PX-12 and cultured under normoxic or hypoxic conditions for an additional 16–18 h.
Nuclear extracts were obtained using the Nuclear Extract Kit (Active Motif, Carlsbad, CA) in accordance with the manufacturer’s protocol. The Nrf2/antioxidant response element (ARE)–binding assay was conducted using the TransAM Transcription Factor DNA-binding ELISA kit (Active Motif). Nuclear extract DNA was quantified using NanoDrop (Thermo Fisher Scientific).
Significance was determined using student’s t test to compare data points with control data. Results at p < 0.05 were considered significant.
PX-12 decreased hypoxia-induced HIF-1α protein levels in a panel of four cell lines (MiaPaCa-2, MCF-7, HEK-293, and LN-299) confirming earlier observations  (Fig. 1a). The proteosomal inhibitor MG-132 was used to determine whether the PX-12–mediated decrease in HIF-1α was the result of increased proteosomal degradation (Fig. 1b). The results show that PX-12 was still able to decrease HIF-1α protein level in the presence of MG-132. PX-12 also increased the expression of SSAT1 under both normoxic and hypoxic conditions in all 4 cell lines (Fig. 1c). SSAT2 expression was not increased by PX-12 under either normoxic or hypoxic conditions.
Trx-1 knockdown with siRNA did not decrease hypoxia-induced HIF-1α protein levels although Trx-1 protein levels were markedly decreased (Fig. 2a). We previously reported that transfection of cells with dominant-negative redox-inactive Trx-1 inhibited hypoxia-induced HIF-1α protein levels . The difference with the present results using siRNA-Trx-1 may be due to off target effects of the redox-inactive Trx-1. Furthermore, siRNA knockdown of Trx-1 did not inhibit the increase in SSAT1 caused by PX-12 in either normoxia or hypoxia (Fig. 2b). It should be noted that for unknown reasons, knockdown of Trx-1 actually increased SSAT1 expression. There was no significant effect of Trx-1 knockdown on SSAT2 expression.
PX-12 was found to increase nuclear Nrf2 protein levels in MiaPaCa-2 cells, but not to affect the levels of Nrf2 protein in the cytoplasm (Fig. 3a). At the same time, Nrf2 binding to the ARE was increased by PX-12 at 10 and 25 μM; however, at 50 μM, PX-12 inhibited ARE binding possibly due to a decrease in nuclear Nrf2 at this high concentration of PX-12 (Fig. 3b). The endoplasmic reticulum stress response acting through PERK can activate Nrf2 . However, PX-12, 25 μM, was able to increase Nrf2 expression under hypoxic conditions even when PERK was inhibited (Fig. 3c). PX-12 significantly increased the binding of Nrf2 to the ARE in the panel of 4 cell lines (Fig. 3d) but failed to increase the binding in siRNA-Nrf2-treated cells, while siRNA-PMF1, siRNA-SSAT1, and siRNA-Trx-1 had no effect on ARE binding (Fig. 3e). PX-12 increased total levels of SSAT1 in hypoxic MiaPaCa-2 and MCF-7 cells but only increased Nrf2 under hypoxic conditions and when Trx-1 was knocked down with siRNA–Trx-1 (Fig. 4a, b).
Although PX-12 increased hypoxia-induced Nrf2 levels in the absence of Trx-1, and increased Nrf2 binding to the ARE in hypoxic MiaPaca-2 cells, it had no effect on the levels of typical ARE downstream targets suggesting that ARE-bound Nrf2 is nonfunctional possibly (Fig. 4c). This could be as a result of modification of Nrf2 by PX-12. However, in hypoxic MCF-7 breast cancer cells, PX-12 caused a decrease in ARE downstream targets, including thioredoxin reductase 1, RACK1, and elongin C (Fig. 4d). As expected, PX-12 decreased HIF-1α and Trx-1 levels in both the MiaPaCa-2 and MCF-7 cell lines. The results suggest a complex effect on antioxidant activity that is not mediated by the ARE or SSAT1 and which is perhaps dependent on the intrinsic oxidant stress state of the cells. Hypoxia is known to increase the production of reactive oxygen species through a process mediated by the mitochondria , which could vary between cell lines.
Both Nrf2 and PMF-1 appear to be necessary for hypoxia-induced SSAT1 mRNA expression, and PX-12 does not induce SSAT1 mRNA expression when either Nrf2 or PMF-1 is knocked down (Fig. 5a). SSAT2 mRNA expression was not affected by a loss of Nrf2 or PMF-1 (Fig. 5b). Trx-1 siRNA treatment of MiaPaCa-2 cells showed an increase in PMF-1 in hypoxic conditions, with and without the addition of PX-12 (Fig. 5c).
In a previous study, we suggested that Trx-1 might regulate polyamine levels in breast cancer cells through a decrease in SSAT1 expression and this might contribute to the growth-stimulating and antiapoptotic effects of Trx-1 . SSAT1 expression is transcriptionally regulated through the interaction of two trans-acting transcription factors, Nrf2 and PMF-1 . SSAT1 functions in polyamine catabolism and contributes to the regulation of intracellular pools of the polyamines putresceine, spermidine, and spermine. The closely related SSAT2 cannot acetylate polyamines and its likely physiological substrate is thialysine, which is formed by the reaction of L-serine and cysteamine catalyzed by cystathionine-β-synthase . SSAT1 and SSAT2 both promote HIF-1α degradation but by different mechanisms. SSAT1 stabilizes the binding of RACK1 and elongin C with HIF-1α, thus promoting the oxygen-independent ubiquitination and degradation of HIF-1α ; SSAT2 stabilizes the binding of pVHL and elongin C with HIF-1α and because pVHL requires oxygen-dependent proline hydroxylation of HIF-1α, this leads to oxygen-dependent HIF-1α ubiquitination and degradation . We anticipated that PX-12 would counteract the effects of Trx-1, resulting in an increase in SSAT1, and thus a decrease in HIF-1α levels. We were therefore surprised to find that PX-12 increased SSAT1 expression and decreased HIF-1α levels independently of Trx-1. We were also interested to see if there might be an increase in SSAT2 finding that its expression level was not increased by PX-12 and that SSAT2 did not play a role in HIF-1α degradation by PX-12.
Nrf2 activates ARE-mediated gene expression and mediates the primary antioxidant response to an oxidative challenge in cells [27, 28]. Interaction of the leucine zipper region of Nrf2 with the c-terminal coiled-coil region of PMF-1 increases the expression of SSAT1 and its response to polyamines [13, 29]. Nrf2 is kept in the cytoplasm in a complex with Kelch-like ECH-associated protein 1 (Keap1), from which it is released by the oxidization of critical cysteine residues. This leads to the nuclear translocation and accumulation of Nrf2 . We found increased levels of nuclear Nrf2 and Nrf2 binding to the ARE in cells treated with PX-12. Nrf2 is also activated by PERK-dependent phosphorylation that leads to dissociation of the Nrf2/Keap1 complex ; however, we found that PX-12 increased Nrf2 independently of PERK. We found that PX-12 indirectly increases Nrf2 through its inhibitory effect on Trx-1, but also directly increases Nrf2 expression under hypoxic conditions by a process that is independent of Trx-1. The latter mechanism appears to be the primary means by which PX-12 increases Nrf2. It is noteworthy that the PX-12-induced Nrf2 increase we found in one of the cell lines was not accompanied by increased expression of ARE downstream targets. This suggests a possible cell context–dependent effect for the antioxidant response.
The Sat1 gene encoding SSAT1 contains a positive regulatory element (PRE)–binding site. Along with increases in the levels of Nrf2 and SSAT1 mRNA caused by PX-12, we also found evidence of increased PRE binding suggesting that a Nrf2/PMF-1 mechanism is responsible for the increase in SSAT1 by PX-12. Inhibiting either Nrf2 or PMF-1 prevented the increase in SSAT1 by PX-12. Baek et al.  have shown that SSAT1 plays an essential role in the RACK1-mediated ubiquitination of HIF-1α and suggested that this leads to 26S proteasome-dependent HIF-1α degradation. However, we found that the decrease in HIF-1α by PX-12 was insensitive to proteasome inhibition with MG-132, which suggests that another degradation mechanism may be involved.
Thus, the effect of PX-12 in decreasing HIF-1α may occur through multiple mechanisms (Fig. 6). The results of our study show that PX-12 decreases HIF-1α protein levels by an oxygen-independent pathway through an increase in SSAT1 expression mediated by Nrf2 activation and increased ARE binding. Furthermore, we found that this activity of PX-12 is independent of its ability to inhibit Trx-1. Although SSAT2 is essential to the stabilization of pVHL in the oxygen-dependent degradation pathway, PX-12 did not affect SSAT2 expression levels. In conclusion, we have shown that PX-12 is an inhibitor of HIF-1α through Nrf2/PMF-1 complex formation and the induction of SSAT1.
The authors thank LeeAnn Chastain for editorial assistance. The research was supported by National Institutes of Health grants CA129616, CA109552, and CA077204.
Conflict of interest GP is a stockholder in Oncothyreon the company that owns PX-12.
Yon Hui Kim, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Amy Coon, Arizona Cancer Center, University of Arizona, Tucson, AZ, USA.
Amanda F. Baker, Arizona Cancer Center, University of Arizona, Tucson, AZ, USA.
Garth Powis, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.