Pharmacological Inhibition of MCT4 Reduces 4-Hydroxytamoxifen Sensitivity by Increasing HIF-1α Protein Expression in ER-Positive MCF-7 Breast Cancer Cells

Abstract

The rate of glycolysis in cancer cells is higher than that of normal cells owing to high energy demands, which results in the production of excess lactate. Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, play a critical role in maintaining an appropriate pH environment through lactate transport, and their high expression is associated with poor prognosis in breast cancer. Thus, we hypothesized that inhibition of MCTs is a promising therapeutic target for adjuvant breast cancer treatment. We investigated the effect of MCT inhibition in combination with 4-hydroxytamoxifen (4-OHT), an active metabolite of tamoxifen, using two estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. Lactate transport was investigated in cellular uptake studies. The cytotoxicity of 4-OHT was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In both cell lines evaluated, MCT1 and MCT4 were constitutively expressed at the mRNA and protein levels. [¹⁴C]-L-lactate uptake by both cells was significantly inhibited by bindarit, a selective MCT4 inhibitor, but weakly affected by 5-oxoploline (5-OP), a selective MCT1 inhibitor. The results of the MTT assay showed that combination with bindarit, but not 5-OP, decreased 4-OHT sensitivity. Bindarit significantly increased the levels of hypoxia-inducible factor-1α (HIF-1α) in MCF-7 cells. Moreover, HIF-1α knockdown significantly increased 4-OHT sensitivity, whereas induction of HIF-1α by hypoxia decreased 4-OHT sensitivity in MCF-7 cells. In conclusion, pharmacological MCT4 inhibition confers resistance to 4-OHT rather than sensitivity, by increasing HIF-1α protein levels. In addition, HIF-1α inhibition represents a potential therapeutic strategy for enhancing 4-OHT sensitivity.

INTRODUCTION

Breast cancer is one of the most common cancers in women. In 2018, the incidence and mortality associated with breast cancer were estimated the highest worldwide, accounting for approximately 20% of all women cancers.¹⁾ Breast cancer has five main breast cancer molecular subtypes: luminal A and luminal B, normal breast-like, human epidermal growth factor receptor positive, and triple-negative breast cancer.²⁾ Approximately 70% of breast cancers are diagnosed as estrogen receptor (ER)-positive breast cancer, like Luminal A and Luminal B. Tamoxifen (TAM), a selective ER antagonist, is the major anti-cancer drug for the ER positive breast cancer drug treatment, and used widely before or after operative treatment. TAM is metabolized by CYP 2D6 to metabolites, 4-hydroxytamoxifen (4-OHT) and 4-hydroxy-N-desmethyl-tamoxifen. Both of metabolites is exhibited the 100-fold high affinity to ER by TAM in vitro and central role for breast cancer treatment.^3,4) Although breast cancers demonstrating ER-positivity are treated with TAM, approximately 30% of treated patients acquire resistance to TAM therapy.^5,6)

Cancer tissues consume considerably larger amounts of glucose than normal tissues and predominantly generate ATP from glycolysis, followed by lactate fermentation (Otto–Warburg effect).⁷⁾ Additionally, it has been reported that lactate can be utilized by cancer cells, and is responsible for resistance to treatment.⁸⁾ Monocarboxylate transporters (MCTs) are comprised of 14 isoforms.^9,10) MCTs 1–4 show a broad tissue distribution and transport endogenous monocarboxylate compounds such as lactate. The expression of MCT1 and MCT4 has been characterized in multiple tumor types, and high expression has been associated with poor prognosis in cancer patients, including breast cancer.^11–13) In higher glycolytic cancer cells, MCT4 is expressed at a higher level than MCT1. Furthermore, Baenke et al. demonstrated that MCT4 is a critical regulator of cell survival in breast cancer.¹⁴⁾ Thus, MCT4 is considered a crucial therapeutic target and prognostic marker for breast cancer. Several nonspecific MCT inhibitors, including α-cyano-4-hydroxycinnamic acid (CHC) and lonidamine, are known to reportedly inhibit breast cancer cell viability in vitro.¹⁵⁾ Although our recent study identified a selective MCT4 inhibitor, bindarit, the anti-cancer potential of this compound in breast cancer cell lines has not been evaluated.¹⁶⁾

We hypothesized that inhibition of MCT4 can enhance the sensitivity of ER-positive breast cancer to TAM by inhibiting lactate transport. To test this hypothesis, in the present study, we aimed to demonstrate the contribution of MCT4 to lactate transport in ER-positive breast cancer cell lines and investigate the effect of 4-OHT on cell viability when MCT4 was inhibited, either pharmacologically or genetically with small interfering RNA (siRNA).

MATERIALS AND METHODS

Materials

[¹⁴C]-L-Lactate was purchased from PerkinElmer, Inc. (Waltham, MA, U.S.A.). 4-OHT, 5-oxoproline (5-OP), and cobalt (II) chloride hexahydrate (Co) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Bindarit was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.).

Cell Culture

ER-dependent human breast cancer MCF-7 cells and T47D cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) containing 10% fetal bovine serum at 37 °C in 5% CO₂. The medium was changed every 2 d, and both the cell lines were sub-cultured every 4 d after reaching subconfluence.

For hypoxia experiments, MCF-7 cells were cultured at 1% O₂ using a BIONIX-3 hypoxic culture kit (Sugiyamagen, Tokyo, Japan).

RT-PCR

Total RNA was extracted from MCF-7 and T47D cells using ISOGEN II (Nippon Gene, Tokyo, Japan). cDNA was prepared by reverse transcriptase reaction using ReverTra Ace^® (Toyobo Co., Ltd., Osaka, Japan). RT-PCR was performed using the KAPA Taq Extra kit (Kapa Biosystems, Wilmington, MA, U.S.A.). The PCR conditions were as follows: 25 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min. The sequences of specific primers for PCR reactions were as follows: hβ-actin sense, 5′-TGG CAC CCA GCA CAA TGA A-3′; hβ-actin antisense, 5′-CTA AGT CAT AGT CCG CCT AGA AGC A-3′; hMCT1 sense, 5′-CCA GCT CTG ACC ATG ATT GG-3′; hMCT1 antisense, 5′-GGC GCC AGA GTA CAG AGG AAC-3′; hMCT4 sense, 5′-GTT GGG TTT GGC ACT CAA CTT CC-3′; and hMCT4 antisense, 5′-CAG GAA GAC AGG GCT ACC TGC TG-3′. The estimated PCR product size was 186, 101, and 112 bp for hβ-actin, hMCT1, and hMCT4, respectively.

Western Blotting

Western blotting was performed as described in our previous study, with minor modifications.¹⁷⁾ The following antibodies were used for Western blotting: MCT1 (sc-365501, diluted 1 : 250; Santa Cruz Biotechnology, Dallas, TX, U.S.A.), MCT4 (22787-1-AP, diluted 1 : 1000; ProteinTech Group, Inc., Chicago, IL, U.S.A.), hypoxia-inducible factor 1α (HIF-1α) (sc-10790, diluted 1 : 200; Santa Cruz Biotechnology), β-actin (MAB1501, diluted 1 : 1000; Millipore, Bedford, MA, U.S.A.), anti-mouse secondary antibody (1070-05, diluted 1 : 4000; Southern Biotech, Birmingham, AL, U.S.A.), and anti-rabbit secondary antibody (sc-2357, diluted 1 : 4000; Santa Cruz Biotechnology).

Uptake Study

The MCF-7 and T47D cells were seeded on 24-well plates at a density of 5 × 10⁴ and 1.6 × 10⁵ cells/well, respectively, and used for the uptake experiments on day 3 after plating. The uptake of radiolabeled lactate by the MCF-7 and T47D cells was evaluated as previously described, with minor modifications.¹⁸⁾ Briefly, the cells were washed and pre-incubated with Hank’s balanced salt saline (HBSS) buffer, adjusted to pH 6.0 at 37 °C. The uptake was subsequently initiated by the addition of HBSS-containing [¹⁴C]-L-lactate in the presence or absence of each inhibitor, and the cell monolayers were incubated for the indicated time periods at 37 °C. Following incubation, each cell monolayer was rapidly washed twice with ice-cold HBSS. In order to quantify the radioactivity of [¹⁴C]-L-lactate, the cells were solubilized in 1% sodium dodecyl sulfate (SDS)/0.2 N NaOH. The resulting solution was mixed with a scintillation cocktail, and the radioactivity was measured. The uptake levels of [¹⁴C]-L-lactate were corrected for the protein content.

RNA Interference

The cells were transfected with siRNA using Lipofectamine RNAi MAX (Invitrogen, Waltham, MA, U.S.A.), according to the manufacturer’s instructions. After 24 h of siRNA transfection, the medium with or without the compounds was replaced and the cells were incubated for an additional 48 h, following which the cells were subjected to further experimentation. Silencer^® Validated siRNA against MCT4 gene (siRNA ID No. s17416), HIF-1α gene (siRNA ID No. 42840), and nontargeting siRNA as a Silencer^® negative control siRNA were purchased from Ambion (Austin, TX, U.S.A.).

Cell Viability and Cytotoxicity Assay

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.¹⁹⁾ MCF-7 and T47D cells were seeded on a 96-well plastic plate. Twenty-four hours after seeding, varying concentrations of 4-OHT were added for 48 h in the absence or presence of inhibitors. MTT solution was next added to the medium, and the cells were further incubated for 1 h. The MTT medium was then replaced with dimethyl sulfoxide (DMSO), and the absorbance was measured at 590 nm. Cell viability and cytotoxicity after treatment with vehicle (control) were set at 100%. For 4-OHT, the IC₅₀ value was calculated using SigmaPlot 12.5 (Systat Software, Inc.; San Jose, CA, U.S.A.). The cytotoxicity in MCF-7 cells was determined using the CytoTox-Glo™ cytotoxicity assay (Promega, Madison, WI, U.S.A.), according to the manufacturer’s instructions. Luminescence was measured with an Infinite 200 PRO multimode reader (Tecan, Männedorf, Switzerland).

Statistical Analysis

All the experiments were performed at least in triplicate and the results are presented as the mean ± standard error (S.E.). Statistical significance was calculated using unpaired Student’s t-test or Dunnett’s test. p-Values of <0.05 were considered statistically significant.

RESULTS

Effect of MCT Inhibitors on Lactate Uptake in ER-Positive Breast Cancer Cells

First, RT-PCR and Western blotting analysis confirmed the expression of MCT1 and MCT4 in both MCF-7 and T47D cells (Figs. 1a, b). Next, we examined the effect of MCT inhibitors on lactate transport activity in these cell lines (Table 1). CHC, a well-characterized nonspecific inhibitor of MCT, at a low concentration (0.1 mM), showed a weak inhibitory effect on lactate uptake by these cell lines, inhibiting uptake at a higher concentration (5 mM). Bindarit, a selective MCT4 inhibitor, significantly inhibited lactate uptake in both cell lines. Conversely, the inhibitory effects of 5-OP, an MCT1 inhibitor, but not MCT4, were relatively weak when compared with the inhibition mediated by bindarit.

Fig. 1. Expression of MCT1 and MCT4 in ER-Positive Breast Cancer Cell Lines

(a) Gene expression of MCT1 and MCT4 was assessed by RT-PCR. (b) Western blotting of MCF-7 and T47D cells for measuring MCT1 (40–48 kDa) and MCT4 (42–45 kDa) protein expression. β-Actin (43 kDa) was used as a housekeeping gene.

Table 1. The Inhibitory Effects of Various MCT Inhibitors on the Uptake of L-Lactate by ER-Positive Breast Cancer Cells

Cells	Treatment	% of control
MCF-7	CHC 5 mM	45.3 ± 2.2*
	CHC 0.1 mM	73.2 ± 6.1*
	5-OP 10 mM	76.5 ± 3.9*
	Bindarit 100 µM	45.0 ± 0.8*
T47D	CHC 5 mM	50.8 ± 1.8*
	CHC 0.1 mM	108.7 ± 5.3
	5-OP 10 mM	87.5 ± 2.4*
	Bindarit 100 µM	51.6 ± 1.1*

Effect of MCT inhibitors on the uptake of [¹⁴C]-L-lactate in ER-positive breast cancer cells. All uptake studies were performed at 37 °C, pH 6.0, for 5 min after preincubation in the absence or presence of each MCT inhibitors at 37 °C, pH 7.4, for 10 min. * p < 0.01 compared with control using Dunnett’s test. Each value represents the mean ± standard error (S.E.).

Suppression of 4-OHT-Induced Cytotoxicity by MCT Inhibitors

To determine whether the MCT inhibitor altered cell sensitivity to 4-OHT, we examined the effects of MCT inhibitors on the reduced MCF-7 and T47D cell viability by 4-OHT (Fig. 2). 4-OHT showed IC₅₀ values of 12 µM and 15 µM in MCF-7 and T47D cells, respectively, consistent with previous reports.²⁰⁾ Bindarit significantly reduced 4-OHT sensitivity in both cell lines. Furthermore, 5-OP did not alter 4-OHT sensitivity.

Fig. 2. Effect of 4-OHT Combined with MCT Inhibitors on ER-Positive Breast Cancer Cell Viability

The sigmoid curve and IC₅₀ values (µM) of 4-OHT cell growth inhibition were determined using the MTT assay. * p < 0.01, compared to the control using Dunnett’s test. Data are presented as the mean ± S.E. of three independent experiments.

Effect of MCT4 siRNA on Cell Viability and Cytotoxicity in 4-OHT-Treated MCF-7 Cells

In order to confirm the effect of MCT4 knockdown on cell sensitivity to 4-OHT, we examined the effect of 4-OHT on the viability and cytotoxicity of MCF-7 cells transfected with MCT4 siRNA. Western blot analysis revealed that MCT4 siRNA decreased MCT4 protein levels (Figs. 3a, b). However, MCT4 knockdown did not impact 4-OHT-induced cell viability inhibition and cytotoxicity in MCF-7 cells (Figs. 3c, d).

Fig. 3. Effect of MCT4 siRNA on Cell Viability and Cytotoxicity in MCF-7 Cells Treated with 4-OHT

(a), (b) Protein expression of MCT4 after siRNA transfection was analyzed by Western blot analysis in MCF-7 cells. (c), (d) Cell viability and cytotoxicity after siRNA transfection of MCT4 were determined by MTT assay and CytoTox-Glo™ cytotoxicity assay, respectively, in MCF-7 cells. * p < 0.01 for control versus each group by unpaired Student’s t-test. Data are presented as mean ± S.E. of three independent experiments.

Effect of MCT Inhibitors on HIF-1α Expression in MCF-7 Cells

We further determined whether the expression of HIF-1α, a key transcription factor that regulates MCT4 expression and promotes the Warburg effect, is modulated by MCT inhibitors. Following exposure of MCF-7 cells to bindarit, a significant increase in HIF-1α protein levels was observed; this effect was not observed with 5-OP (Fig. 4). Interestingly, bindarit significantly increased lactate accumulation; however, 5-OP did not affect it (Supplementary Fig. 1). Moreover, the impacts on cell sensitivity to 4-OHT of HIF-1α induction and knockdown were investigated. Hypoxia significantly increased HIF-1α protein levels compared with control (normoxia) (Fig. 5a). Induction of HIF-1α by hypoxia decreased 4-OHT sensitivity with qualitative similarity to the effect of bindalit (Fig. 5b). These results suggest that inhibition of MCT4 activity by bindarit may reduce 4-OHT sensitivity by increasing HIF-1α protein levels.

Fig. 4. Effect of Bindarit and 5-OP on HIF-1α Protein Levels in MCF-7 Cells

HIF-1α protein expression levels were analyzed by Western blot analysis in MCF-7 cells. * p < 0.05 compared with control versus each group using Dunnett’s test. Each data point represents the mean ± S.E. of three independent experiments.

Fig. 5. Effect of Hypoxia on Viability of MCF-7 Cells Treated with 4-OHT

(a) MCF-7 cells were exposed to hypoxic conditions at 1% O₂ for 72 h, and HIF-1α protein levels were measured by Western blotting. (b) The sigmoid curve and IC₅₀ values (µM) of 4-OHT cell growth inhibition under hypoxic conditions (1% O₂) for 48 h were determined by the MTT assay in MCF-7 cells. * p < 0.05, ** p < 0.01, compared to the control using unpaired Student’s t-test. Data are presented as the mean ± S.E. of three independent experiments.

SiRNA silencing HIF-1α inhibited the increased expression of HIF-1α induced by Co, a chemical hypoxia-mimicking agent, in addition to the basal HIF-1α protein level in MCF-7 cells (Fig. 6a). As shown in Fig. 6b, HIF-1α siRNA significantly increased 4-OHT sensitivity compared with cells transfected with a negative control siRNA, suggesting that HIF-1α protein inhibition might enhance sensitivity to 4-OHT.

Fig. 6. Effects of HIF-1α siRNA on Cell Sensitivity to 4-OHT in MCF-7 Cells

(a) Expression of HIF-1α protein levels after siRNA transfection was analyzed by Western blot analysis in MCF-7 cells. (b) The sigmoid curve and IC₅₀ values (µM) of 4-OHT cell growth inhibition by MTT assay in MCF-7 cells. * p < 0.01 compared with control by unpaired Student’s t-test. Data are presented as the mean ± S.E. of three independent experiments.

DISCUSSION

MCT1 and MCT4 mainly transport lactate, and their expression is related to cancer growth and the Warburg effect, which is considered a metabolic hallmark of cancer.²¹⁾ Several studies have suggested that high MCT expression is associated with poor prognosis in breast cancer.^12,13) However, the MCT isoform functionally expressed in breast cancer cells and contributing to cell viability and growth remains unknown. Recently, numerous MCT inhibitors have been developed and employed in cancer treatment.^22,23) However, the anti-cancer efficacy of MCT inhibitors is limited; therefore, its applicability in clinical cancer treatment is limited. The aim of this study was to clarify the contribution of MCT1 and MCT4 to lactate transport in different ER-positive breast cancer cell lines and to investigate the effect of 4-OHT on cell viability when MCTs were inhibited pharmacologically or genetically with siRNA.

As shown in Fig. 1, expression of MCT1 and MCT4 was detected in both MCF-7 and T47D cells, in line with a previous study.^24,25) Lactate transport via MCTs involves bidirectional facilitated diffusion dependent on the pH, and therefore low extracellular pH favors lactate uptake.²⁶⁾ Thus, we examined the contribution of each MCT isoform toward lactate transport using a [¹⁴C]-L-lactate uptake study at pH 6.0, by employing various MCT inhibitors. In the presence of 5 mM CHC, a nonspecific inhibitor of MCTs, lactate uptake was reduced by approximately 50% in both cell types. Furthermore, bindarit significantly inhibited the uptake of lactate, suggesting that MCT4 is functionally expressed for lactate transport in both cell lines. In contrast, lactate uptake was reduced by only 10–20% in the presence of 5-OP, an MCT1 specific inhibitor. Reportedly, the inhibition constant (K_i) of CHC for MCT1 and MCT4 are 0.17 and 1.0 mM, respectively.²⁷⁾ We observed a marginal inhibition of lactate uptake by 5-OP, or a lower concentration of CHC; hence, major involvement of MCT1 in lactate transport in these cells is unlikely.

Based on our data, we hypothesized that inhibition of MCT4 could increase 4-OHT-induced cytotoxicity in ER-positive breast cancer cells by inhibiting lactate transport. To evaluate this hypothesis, we examined the effects of MCT inhibitors on the viability of MCF-7 and T47D cells treated with 4-OHT. Unexpectedly, combination with MCT inhibitors failed to increase cytotoxicity when compared with 4-OHT treatment alone (Fig. 2). In contrast, bindarit reduced the sensitivity of both cell lines to 4-OHT treatment. However, 5-OP had little effect on 4-OHT-induced cytotoxicity. Moreover, bindarit (100 µM) alone did not affect cell viability in either breast cancer cell line investigated (Supplementary Fig. 2). These results suggest that inhibition of MCT4 may result in 4-OHT resistance in ER-positive breast cancer.

In order to determine whether the suppression of MCT4 expression is associated with resistance to 4-OHT, we investigated the effects of MCT4 knockdown on the sensitivity of MCF-7 cells to 4-OHT, as the expression of MCT4 in MCF-7 cells is higher than that in T47D cells. MCT4 siRNA decreased MCT4 protein levels but had little effect on 4-OHT-induced changes in cell viability and cytotoxicity in MCF-7 cells (Fig. 3). These results are inconsistent with our experimental results obtained using pharmacological inhibitors. Similarly, although pharmacological MCT4 inhibitors (i.e., bindarit) significantly inhibited lactate transport by cells, interestingly, the suppression of MCT4 expression by siRNA exhibited an increasing trend in lactate transport (Table 1, Supplementary Fig. 3). This inconsistency may be explained by the enhancement of MCT1 expression or other compensatory mechanisms when MCT4 is genetically inhibited.^28,29) Given that MCT4 is a proton-dependent lactate transporter that is essential for maintaining both intracellular and extracellular pH, suppression of lactate transport by pharmacological MCT4 inhibitors may contribute to changes in pH, which in turn could influence the uptake and intracellular accumulation of 4-OHT. TAM is a known substrate for drug transporters, including organic anion transporting polypeptides and P-glycoprotein.^30,31) However, for 4-OHT, data regarding transport properties are lacking. At present, we cannot exclude the possibility that intracellular accumulation of 4-OHT might be altered by inhibiting the function of MCT4 in ER-positive breast cancer cells. To clarify the sensitivity of ER-positive breast cancer to 4-OHT when MCT4 is pharmacologically inhibited by targeted therapies, further investigations regarding changes in pharmacokinetic properties such as cellular uptake are warranted.

Another possibility is that inhibition of lactate transport via MCT4 affects HIF-1α protein levels. Although MCT1 and MCT4 are known as predominant lactate influx and efflux pathways, respectively, MCT4 has lower lactate affinity when compared with MCT1 and is considered to be particularly well suited for the export of lactate from highly glycolytic cells.³²⁾ Hunt et al. reported that accumulation of lactate stabilizes HIF-1α expression.³³⁾ Moreover, HIF-1α reportedly contributes to TAM resistance.³⁴⁾ The findings of the present study indicated that bindarit increased HIF-1α protein levels and L-lactate accumulation, whereas it suppressed 4-OHT-induced decrease in cell viability in MCF-7 cells, which was not altered by 5-OP treatment (Figs. 2, 4, Supplementary Fig. 1). Furthermore, HIF-1α induction by hypoxia decreased 4-OHT sensitivity in MCF-7 cells, whereas HIF-1α knockdown increased these cells’ sensitivity (Figs. 5, 6). These results suggested that HIF-1α protein levels might be related to TAM resistance in ER-positive breast cancer. Although these findings were limited by the fact that the present study evaluated the transport activity of MCTs by measuring the uptake and accumulation of L-lactate, we found that bindarit induced HIF-1α accumulation by inhibiting lactate export via MCT4 inhibition, thereby reducing the sensitivity of MCF-7 cells to 4-OHT. Based on these findings, inhibition of HIF-1α activity might be an important approach to combination therapy strategies with MCT4 inhibitors. However, our study focused only on 4-OHT-treated ER-positive breast cancer. Therefore, whether this mechanism is applicable to other cancers or chemotherapies requires further investigation.

Our present study revealed that MCT4, but not MCT1, was functionally expressed for lactate transport in ER-positive breast cancer cell lines and that pharmacological inhibition of MCT4 decreased sensitization to 4-OHT. Moreover, the mechanism underlying the reduced sensitivity to 4-OHT may partially rely on the increased expression of HIF-1α. Thus, we concluded that inhibition of MCT4 may confer resistance, rather than sensitivity, to 4-OHT in breast cancer. HIF-1α inhibition increased 4-OHT sensitivity. Although our findings implicate a potential challenge for MCT inhibitors in targeting ER-positive breast cancer, HIF-1α inhibition could be a potential therapeutic strategy for improving sensitivity to 4-OHT.

Acknowledgments

This research was supported by JSPS KAKENHI (Grant No. JP 18K14416 to KN). We would like to thank the Nagai Memorial Research Scholarship (to TN) awarded by the Pharmaceutical Society of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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