In Vitro and In Vivo Efficacy of AZD3965 and Alpha-Cyano-4-Hydroxycinnamic Acid in the Murine 4T1 Breast Tumor Model

Abstract

Monocarboxylate transporter 1 (MCT1) represents a potential therapeutic target in cancer. The objective of this study was to determine the efficacy of AZD3965 (a specific inhibitor of MCT1) and α-cyano-4-hydroxycinnamic acid (CHC, a nonspecific inhibitor of MCTs) in the murine 4T1 tumor model of triple-negative breast cancer (TNBC). Expression of MCT1 and MCT4 in 4T1 and mouse mammary epithelial cells were determined by Western blot. Inhibition of MCT1-mediated L-lactate uptake and cellular proliferation by AZD3965 and CHC was determined. Mice bearing 4T1 breast tumors were treated with AZD3965 100 mg/kg i.p. twice-daily or CHC 200 mg/kg i.p. once-daily. Tumor growth, metastasis, intra-tumor lactate concentration, immune function, tumor MCT expression, and concentration-effect relationships were determined. AZD3965 and CHC inhibited cell growth and L-lactate uptake in 4T1 cells. AZD3965 treatment resulted in trough plasma and tumor concentrations of 29.1 ± 13.9 and 1670 ± 946 nM, respectively. AZD3965 decreased the tumor proliferation biomarker Ki67 expression, increased intra-tumor lactate concentration, and decreased tumor volume, although tumor weight was not different from untreated controls. CHC had no effect on tumor volume and weight, or intra-tumor lactate concentration. AZD3965 treatment reduced the blood leukocyte count and spleen weight and increased lung metastasis, while CHC did not. These findings indicate AZD3965 is a potent MCT1 inhibitor that accumulates to high concentrations in 4T1 xenograft tumors, where it increases tumor lactate concentrations and produces beneficial effects on markers of TNBC; however, overall effects on tumor growth were minimal and lung metastases increased.

INTRODUCTION

Triple-negative breast cancer (TNBC) represents a highly metastatic and aggressive form of breast cancer with no proven targeted therapy available. One metabolic alteration in cancer is the enhancement in the rate of glycolysis under both aerobic, termed as the “Warburg effect” (1) and anaerobic conditions. This is manifested by the high demand in glucose consumption and the large production of lactic acid along with a shift in intracellular pH. Among the different forms of breast cancer, TNBC has been reported to be the most glycolytic, as demonstrated by increased protein expression of a number of glycolytic markers including glucose transporter 1, GLUT1 (2). Lactic acid produced from glycolysis is transported across the cellular membrane by a family of transport proteins, known as monocarboxylate transporters (MCTs), which are encoded by the SLC16 gene family (3,4). MCT1, MCT4, and CD147 (a chaperone protein required for trafficking and function of MCTs to the plasma membrane) are overexpressed in a number of cancers (5–7); facilitation of the transport of lactate along with protons by MCTs out of cancer cells helps maintain the high glycolytic rate and prevents acidosis and apoptosis.

Elevated protein expression of MCT1 and CD147 has been shown to correlate with poor survival and metastases in TNBC (5,7), which suggests that inhibition of MCT1 transport could provide a potential therapeutic approach for the treatment of TNBC. Inhibitors of MCTs and silencing RNAs have been demonstrated to inhibit tumor growth through their ability to block lactate efflux in different human cancer xenograft models (8–18). One of these inhibitors includes α-cyano-4-hydroxycinnamic acid (CHC), which is a classical nonspecific inhibitor of MCTs with low millimolar potency (19) and has been shown to have activity against the pyruvate carrier in isolated mitochondria (20).

AZD3965, a pyrrole pyrimidine derivative, represents a first-in-class potent inhibitor of MCT1 that is currently being investigated in first-in-human study in the UK for advanced solid tumors and lymphomas (NCT01791595). AZD3965 was initially developed by AstraZeneca as part of a group of novel immunosuppressive agents that can potently inhibit MCT1-mediated lactate efflux in proliferating T cells at low nanomolar concentrations (21). AZD3965 is a potent inhibitor of MCT1 (binding affinity of 1.6 nM) and it also inhibits MCT2, but to a 6-fold lesser extent (11,22). AZD3965 does not inhibit MCT3/MCT4 at a concentration of 10 μM (11). AZD3965 has been demonstrated to be effective in various preclinical tumor models overexpressing MCT1 (9–11,23,24), and in a human TNBC breast cancer cell line–derived xenograft model expressing both MCT1 and MCT4 (25). The suppression of tumor growth has also been demonstrated with MCT1 knockdown in a human non-TNBC cell line–derived xenograft model that expresses both MCT1 and MCT4 (12). In addition to targeting MCT1-mediated lactate transport, several studies have reported involvement of AZD3965 in enhancing pyruvate/mitochondrial metabolism (25,26) and that mitochondrial complex I inhibitor, metformin and the mitochondrial pyruvate carrier inhibitor, UK5099 were able to increase sensitivity to AZD3965 (26).

The murine 4T1 breast tumor animal model mimics the metastatic phenotype of human breast cancer with similar TNBC characteristics (27,28), and overexpresses MCT1 (29). The objective of current research was to evaluate the effects of AZD3965 and CHC on tumor growth and metastasis in the murine 4T1 breast tumor xenografts with treatment started after the primary tumor reached a palpable size.

MATERIALS AND METHODS

Chemicals and Reagents

L-lactate (as calcium salt) and α-cyano-4-hydroxycinnamic acid (CHC) were purchased from Sigma-Aldrich (St. Louis, MO). AZD3965 was obtained from AstraZeneca (for the in vitro studies) and MedKoo Biosciences (Chapel Hill, NC) with > 98% purity for the in vivo studies.

Cell Culture

Mouse mammary tumor, 4T1 and mouse mammary epithelial, NMuMG cells were kindly provided by Dr. Elizabeth A. Repasky (Roswell Park Cancer Institute, Buffalo, NY) and Dr. Karen J.L. Burg (University of Clemson, SC), respectively. Cells were cultured, following the recommendations of American Type Culture Collection.

Western Blotting Analysis

Total plasma membrane protein was harvested using ultracentrifugation according to Zhang et al. (30). For tumor samples, tumors were thawed on ice and homogenized in lysis buffer (29), supplemented with protease inhibitors (20 mL/g tumor). Tumor homogenates were centrifuged at 12,000 rpm for 10 min at 4°C and the resulting supernatants were collected for Western blot analysis. The Western blotting (20 μg total protein per lane) was performed as previously described (29) using the following antibodies: MCT1 (1:5000; ab3538P, EMD Millipore), MCT4 (1:500; sc50329, Santa Cruz), CD147 (1:100; sc9757, 1:1000; sc9753, Santa Cruz), GLUT1 (1:500; ab40084), the proliferation biomarker Ki67 (1:500; sc23900, Santa Cruz), and GAPDH (1:5000; sc25778, Santa Cruz).

Cellular Uptake Studies

The concentration-dependent inhibition of radiolabeled L-lactate uptake by AZD3965 and CHC was investigated as previously described (29). The radioactivity was determined by liquid scintillation counting (1900 CA, Tri-carb liquid scintillation analyzer, Packard Instrument Co. Downers Grove, IL). All the results were normalized to total protein content and expressed as pmol mg protein⁻¹ min⁻¹.

Cell Proliferation Studies

Cell proliferation studies were carried out as previously described (29), using cell proliferation reagent WST-1 (Roche Applied Science). Cells were treated with various concentrations of AZD3965 or CHC for 24 or 48 h, respectively. Relative cell growth was normalized to the vehicle control (< 0.1% DMSO) and expressed as percentage of cell growth.

Intra and Extracellular l-Lactate Accumulation Studies

For the intra and extracellular L-lactate accumulation studies, 4T1 cells were plated in 6-well plates at a cell density of 2.0 × 10⁵ cells/mL 2 days before the study. On the day of the experiment, culture medium was removed and cells were washed with 1× PBS. Cells were treated with AZD3965 (50 and 250 nM) in 1 mL of serum free medium for 24 h. At the end of the incubation, the medium was removed and saved for quantifying extracellular lactate concentration. Cells then were washed with ice-cold 1× PBS for three times. Cells were lysed in 1 mL of 1× PBS and frozen at − 80°C for 30 min followed by thawing and shaking on a shaker for 1 h at 4°C. Cell lysates and media were centrifuged at 10,000×g for 10 min at 4°C. The resulting supernatants were stored in − 80°C until analysis.

Animals

Female, BALB/c mice, 5 to 6 weeks of age (18–20 g) were purchased from Envigo (Indianapolis, IN). Mice were housed in a filtered laminar airflow room in standard vinyl cages with air filter tops. Water and food were provided ad libitum. Animals were maintained under the standard 12-h light/dark cycle at 22–24°C. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University at Buffalo.

In Vivo Tumor Growth Experiments

After 2 weeks of acclimatization, mice were lightly anesthetized and injected with 100 μL of cell suspension subcutaneously into the fourth inguinal mammary fat pad at a cell density of 2.5 × 10⁵ cells/mL in 1× PBS as previously reported (31). When the tumor volume reached 100 mm³, animals were randomly divided into four treatment groups (8–11 mice per group): [i] vehicle control of AZD3965, [ii] vehicle control of CHC, [iii] AZD3965 (100 mg/kg, twice daily), and [iv]CHC (200 mg/kg or 42.6 μmol/animal, once daily). AZD3965 stock solution (10 mg/mL) was prepared in 20% (w/v) cyclodextrin in normal saline. A modified CHC stock solution (37 mg/mL) was prepared by dissolving 37 mg of CHC in 0.74 mL of normal saline and 0.26 mL of 1 N NaOH (32). The vehicle controls of AZD3965 and CHC consisted of 20% (w/v) cyclodextrin in normal saline and normal saline, respectively. AZD3965 and CHC were administered at volumes of 0.2 and 0.1 mL, respectively, by intraperitoneal (i.p.) injection for 17 days. Throughout the study, body weights were monitored weekly and tumor volumes were measured every 2–3 days using a digital caliper. Tumor volumes were calculated with Eq. 1.

$$\text{ Tumor Volume }0\frac{\text{ Length }\times {\text{ Width}}^2}{2}$$ (1)

At the end of the study, blood was obtained from the aortic artery and collected in lithium-heparinized tubes. Primary tumor and spleen were excised, weighed and snap frozen in liquid nitrogen. All samples were stored at − 80°C until analysis.

Quantification of Metastatic Nodules in the Lungs

To study the effect of AZD3965 and CHC on metastasis, lungs were freshly harvested at the end of the study and fixed in Z-FIX solution (Anatech LTD, Battle Creek, MI) (31) for 24 h at room temperature. Individual lobes were separated, and the numbers of surface-visible metastases were determined by using a cell counter (33,34). Tumor nodules are seen as white spots on the brownish lung tissue background. The numbers of lung metastases were counted three times on three different days in a blinded fashion. Micrometastases in other organs have been reported for this tumor model (27).

Total Leukocyte Count

To study the effect of AZD3965 and CHC on immune function, part of the freshly isolated blood was diluted in 3% (v/v) acetic acid with 0.001% (w/v) methylene blue to lyse all the non-nucleated red blood cells. Total leukocytes were counted twice using a hemocytometer and expressed in thousands of cells per microliter of blood (K/μL).

l-Lactate Assay

Plasma and intra-tumor lactate concentrations were measured by a lactate assay kit (Eton Biosciences, Inc., Union, NJ), as described previously (29). Intra-tumor and cellular lactate concentrations were normalized to gram of tumor tissue and milligram of total protein content, respectively.

Plasma and Tumor Sample Preparation and LC/MS/MS Analysis

AZD3965 plasma and tumor concentrations were measured using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), with an assay we previously developed and validated for mouse plasma and 4T1 tumor tissue (35). The detailed sample preparation, chromatograph conditions, mass spectrometer parameters, linear calibration ranges, accuracy/precision and recovery have been published (35).

Data and Statistical Analysis

All the data are presented as mean ± SD. Data analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Depending on the number of groups and variances, data were compared using a Student’s t test, one-way ANOVA followed by Dunnett’s post hoc test, or two-way ANOVA followed by Bonferroni’s post hoc test. Significant differences were based on the criterion P < 0.05. The inhibition of relative cell growth and L-lactate uptake by AZD3965 and CHC (IC₅₀) were calculated using Eq. 2.

$$R0R_0\left(1-\frac{I_{\text{max}}\times C^{\gamma }}{{\text{IC}}_{\text{50}}{}^{\gamma }+C^{\gamma }}\right)$$ (2)

where R and R₀ are the percentage of cell growth or L-lactate uptake in the presence and absence of the inhibitor, respectively, C is the concentration of the inhibitor, I_max is the maximal inhibition, γ is the Hill coefficient and IC₅₀ is the concentration of the inhibitor producing half maximal inhibition. IC₅₀ values were determined using weighted nonlinear regression analysis (ADAPT 5 Biomedical Simulations Resource, University of South California, Los Angeles, CA).

RESULTS

In Vitro Effect of AZD3965 and CHC in 4T1 Cells

The 4T1 breast tumor cell line was previously characterized by this laboratory and exhibited overexpression of MCT1 and CD147, no expression of MCT4 on the plasma membrane, and MCT1-mediated L-lactate uptake (29). The inhibition of L-lactate uptake by AZD3965 and CHC demonstrated IC₅₀ values of 17.0 ± 3.6 nM and 7.57 ± 15.2 mM, respectively (Fig. 1 a and b). The concentration-dependent inhibition of cell growth by AZD3965 (Fig. 1c) and CHC (Fig. 1d) demonstrated similar potency (IC₅₀ values of 22.2 ± 4.6 nM and 6.16 ± 0.19 mM for AZD3965 and CHC, respectively), as in the L-lactate uptake studies, indicating the anti-proliferative activity of the MCT inhibitors. Treatment with AZD3965 (50 and 250 nM) for 24 h significantly increased intracellular (Fig. 1e) and decreased extracellular (Fig. 1f) lactate concentrations. These results suggest that the net effect of MCT1 inhibition by AZD3965 results in the inhibition of MCT1-mediated L-lactate efflux in 4T1 cells.

Fig. 1.

Effect of AZD3965 and CHC on L-lactate uptake (n = 3) and cell growth (n = 4–6) in 4T1 cells. Inhibition of radiolabeled L-lactate (0.5 mM) uptake at pH 6.0 by AZD3965 (a) and CHC (b). Percentage of cell growth after 24 and 48 h of AZD3965 (c) and CHC (d) treatment, respectively, with results normalized to vehicle control–treated cells. To assess the inhibition of L-lactate uptake by AZD3965 or CHC, cells were pre-incubated with various concentrations of inhibitors for 30 min followed by L-lactate uptake (0.5 mM) for 1 min. Buffer at pH 6.0 was used to drive the transport of MCT1, which is dependent on the proton gradient. The intracellular (e) and extracellular (f) L-lactate accumulation after AZD3965 treatment (50 and 250 nM) for 24 h at physiological pH (n = 4). All the data are presented as mean ± SD. Symbols depict the observed mean data and the lines represent model fitted results. **P < 0.01, ***P < 0.001, compared with the vehicle control–treated group (one-way ANOVA followed by Dunnett’s post hoc test)

In Vivo Efficacy of AZD3965 and CHC in the 4T1 Breast Tumor Xenograft

Groups of eleven mice bearing 4T1 tumors (100 mm³) were treated with 200 mg/kg i.p. of CHC or vehicle control daily (Fig. 2a). The treatment with CHC did not alter the body weight (Fig. 2b) and did not decrease tumor volume (Fig. 2c) and weight (Fig. 2d) throughout the study. The effect of CHC on lung metastasis was quantitated and a representative metastatic lung is shown in Fig. 2e. CHC treatment demonstrated no effect on the number of lung macrometastases (Fig. 2f). Further analysis revealed that CHC treatment did not alter the plasma lactate (Fig. 2g) and intra-tumor lactate concentration (Fig. 2h).

Fig. 2.

In vivo efficacy of CHC in the 4T1 breast tumor xenograft. The schematic representation of the experimental timeline and dosing schedule for CHC following orthotropic inoculation of 4T1 cells subcutaneously into the mammary fat pad of female BALB/c mice. Mice were given i.p. injection of CHC (200 mg/kg) or vehicle control once a day (a). Body weight (b) and tumor volume (c) were monitored throughout the study. Tumor weight (d) was measured at the end of the treatment. The effect of CHC on the lung metastasis was quantified. The representative metastatic lungs are shown (e), where the number of surface-visible metastases as indicated by the arrows were counted. Quantification of the number of surface lung metastases in the vehicle control–treated and CHC-treated animals (f). The effect of CHC treatment on plasma (g) and intra-tumor (h) L-lactate concentration. All the data are presented as mean ± SD, n = 11 in each group. Statistical significant differences were based on the criterion P < 0.05

For AZD3965, groups of eight mice bearing 4T1 tumors (100 mm³) were treated with 100 mg/kg i.p. of AZD3965 or vehicle control twice daily (Fig. 3a). Similar to CHC, chronic administration of AZD3965 throughout the study produced no change in body weight (Fig. 3b). AZD3965 treatment decreased tumor volume (Fig. 3c); however, the primary tumor weight (Fig. 3d) did not change. AZD3965 treatment also decreased total Ki67 (a cellular proliferation biomarker used clinically for evaluating a number of cancers including breast cancer) expression in the tumor (Fig. 3e). Interestingly, although there were reductions in tumor volume and Ki67 expression, we saw a significant increase in the number of lung macrometastases (Fig. 3f) in AZD3965-treated mice compared with the vehicle control-treated mice. AZD3965 treatment had no effect on the plasma lactate concentration (Fig. 3g); however, there was a significant increase in the intra-tumor lactate concentration (Fig. 3h), indicating inhibition of MCT1-mediated efflux.

Fig. 3.

In vivo efficacy of AZD3965 in the 4T1 breast tumor xenograft. The schematic representation of the experimental timeline and dosing schedule for AZD3965 following orthotropic inoculation of 4T1 cells subcutaneously into the mammary fat pad of female BALB/c mice. Mice were given i.p. injection of AZD3965 (100 mg/kg) or vehicle control twice a day (a). Body weight (b) and tumor volume (c) were monitored throughout the study. Tumor weight (d), total tumor Ki67 expression (e) and lung metastases (f) at the end of the treatment (12 h after the last dose) were measured. The plasma (g) and intra-tumor (h) lactate concentration in vehicle control–treated and AZD3965-treated animals. All the data are presented as mean ± SD, n = 8 in each group. ***P < 0.001, compared with the vehicle control–treated group (two-way ANOVA followed by Bonferroni’s post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001, compared with the vehicle control–treated group (nonparametric Student’s t test)

Effect of AZD3965 and CHC on Immune Function

Since AZD3965 was initially developed as a novel immunosuppressive agent, we sought to evaluate the effect of AZD3965 on blood leukocyte count and spleen weight in our tumor model (Fig. 4). CHC treatment had no effect on blood leukocyte count and spleen weight (Fig. 4 a and b). However, there was a significant reduction in the leukocyte count and spleen weight in AZD3965-treated mice, compared with the vehicle control–treated mice (Fig. 4 c and d) indicating AZD3965 exerts immunosuppressive effects in these animals.

Fig. 4.

Effect of AZD3965 and CHC on immune function in the murine 4T1 breast tumor model. Blood leukocyte count in CHC-treated (n = 10–11) (a) and AZD3965-treated (n = 5–6) (c) animals. Spleen weight in CHC-treated (n = 8) (b) and AZD3965-treated (n = 8) (d) animals. All the data are presented as mean ± SD. **P < 0.01, ***P < 0.001, compared with the vehicle control–treated group (nonparametric Student’s t test)

Effect of AZD3965 on 4T1 Tumor MCT1, MCT4, GLUT1, and CD147 Expression

We characterized the potential effect of AZD3965 on tumor protein expression of MCTs, GLUT1, and CD147. Our results showed that there were no significant differences in the tumor protein expression of MCT1 and 4 (Fig. 5 a and b), CD147 (Fig. 5c), and GLUT1 (Fig. 5d) between the vehicle control- and AZD3965-treated groups, as determined at the end of the study (day 29). These results indicate that AZD3965 treatment has no effect on the protein expression of MCTs, GLUT1, and CD147. Interestingly, both vehicle control–treated and AZD3965-treated tumors showed expression of MCT4 (Fig. 5b), which was undetectable on the plasma membrane of 4T1 cells during tumor implantation.

Fig. 5.

Total 4T1 tumor expression of MCT1 (a), MCT4 (b), CD147 (c), and GLUT1 (d) in vehicle control–treated and AZD3965-treated mice. Plasma membrane protein from 4T1 and NMuMG cells were used as positive controls. GAPDH was used as loading control. Histograms show corresponding densitometry analysis of the Western blot membranes. All the data are presented as mean ± SD, n = 3–4. *P < 0.05, compared with vehicle control–treated tumor (nonparametric Student’s t test)

Plasma and Tumor AZD3965 Quantification and LCMS/MS Assay

The plasma and total tumor AZD3965 concentrations were investigated when the animals were sacrificed on day 29 (12 h after the last AZD3965 dose). The average total tumor AZD3965 concentration (1670 ± 946 nM) was found to be significantly higher (57.4-fold higher) than that in the plasma (29.1 ± 13.9 nM), indicating extensive tumor uptake of AZD3965 (Fig. 6). Average unbound (free) trough AZD3965 concentration in plasma was estimated as 6.31 ± 3.02 nM, based on a predicted fraction unbound in plasma (f_up) of 21.7% in the rat (MedChem Designer version 4.5.0.12) (36).

Fig. 6.

Plasma and total tumor AZD3965 concentrations measured at the end of the treatment (12 h after the last AZD3965 dose). Assuming tumor density of 1 g/cm³, tumor AZD3965 concentrations were normalized to gram of tumor tissue and expressed in nanomolars. All the data are presented as mean ± SD, n = 7–8. ***P < 0.001, compared with the plasma AZD3965 concentration (nonparametric Student’s t test)

DISCUSSION

The rationale for targeting MCT1 in TNBC came from two publications by Pinheiro et al. which reported that MCT1 protein expression is significantly elevated in human breast cancer tumors, as compared with normal breast tissue (7) and that the elevated expression of MCT1 and CD147 were significantly associated with poor prognosis in breast tumors characterized as TNBC (37). While the pharmacological inhibition of MCTs by AZD3965 and CHC has been demonstrated to be effective in various tumor xenograft models (8–11,15–18), few studies have examined the effect of MCT inhibition in breast cancer, particularly in TNBC. There are limited preclinical TNBC models (including patient-derived models) available that can fully mimic the metastatic phenotype and the underlying disease biology and heterogeneity. We chose to study the 4T1 tumor model because: [1] 4T1 breast tumor model has been extensively studied to exhibit similar characteristics as human TNBC (38,39). [2] Importantly, this tumor model enables us to study MCT1 inhibition in the absence of MCT4 (29), in contrast to other human TNBC cell lines that we have characterized and found to express both MCT1 and MCT4 (unpublished data). To our knowledge, the in vivo anticancer effects of AZD3965 and CHC have not been studied in breast tumor xenograft model of immunocompetent mice. The current study sought to evaluate the therapeutic potential of specific and nonspecific MCT1 inhibitors (AZD3965 and CHC) in the murine 4T1 syngeneic breast tumor model, which could also have some potential importance since AZD3965 was developed as an immunomodulatory agent.

CHC represents a widely used nonspecific inhibitor of MCTs 1, 2, and 4, with a reported K_i value in the low millimolar range (3,19), although it also has activity against mitochondrial pyruvate carrier (20). CHC has been shown to exhibit anticancer activity via inhibiting MCT-mediated lactate transport (16,17). Consistent with previous studies (8,40), CHC inhibited both L-lactate uptake and cell growth with low millimolar potency in the 4T1 cells indicating that inhibition of MCT1 has an anti-proliferative effect. In contrast to previous tumor xenograft studies where CHC was given either at a lower or similar dose and shown to inhibit tumor growth (8,16–18,41), CHC demonstrated a lack of in vivo efficacy in the 4T1 breast tumor xenograft.

For AZD3965, our in vitro experiments demonstrated potent inhibition of cell growth by AZD3965 (IC₅₀ value of 22.2 ± 4.57 nM), which is similar to the IC₅₀ determined from our cellular lactate uptake studies and consistent with previous findings (9–11,22). As expected, the overall effect of AZD3965 in 4T1 cells is blocking MCT1-mediated efflux of L-lactate out of the cells since the intracellular pH will be decreased and will function as the driving force for the proton-mediated MCT1 transport of lactate. Here the incubation with AZD3965 at 50 and 250 nM concentrations did not result in a dose-dependent accumulation of intracellular or reduction of extracellular L-lactate concentrations which is likely due to the low K_i value of AZD3965 resulting in maximal effects at 50 nM, consistent with a previous report (26). In vivo, AZD3965 treatment reduced tumor volume and Ki67 protein expression in mice bearing the 4T1 tumors, compared with the vehicle control. However, AZD3965 treatment had no effect on the tumor weight. The mechanism underlying this discrepancy is unclear, but one possible reason may be due to the considerable tumor necrosis observed in our study, as fluid retention in the tumor or tumor swelling may be possible (42). Since inhibiting lactate efflux would be expected to lead to lactate accumulation and thereby limiting the survival of cancer cells, we sought to evaluate the effect of AZD3965 on intra-tumor lactate concentration. Indeed, AZD3965 treatment resulted in a significant increase in intra-tumor lactate concentration, a 1.54-fold increase, indicating inhibition of MCT1-mediated lactate efflux. Although the fold increase was small, the change is similar to previous tumor xenograft studies, where they saw a 1.7-fold increase at similar pharmacokinetic exposure and showed AZD3965 was effective (10,26). Even though the fold increase in the intra-tumor lactate was very similar, it is not known to what extent tumor lactate accumulation would limit the ability of the tumor cells to survive, as the effects of lactate may vary across different cancer types.

Since AZD3965 was part of a series of pyrrole pyrimidine analogues originally developed by AstraZeneca as immunosuppressive agents, we evaluated the effect of AZD3965 on immune function in mice bearing 4T1 tumors. The development of splenomegaly and elevation of circulating leukocytes have been shown to increase progressively in murine 4T1 xenograft mice (27,43). AZD3965 treatment, but not CHC treatment, resulted in significantly decreased blood leukocyte counts and spleen weights. Furthermore, a recent study in a tumor xenograft of severely immunocompromised NSG mice also reported reduced spleen weight in animals treated with AZD3965 (9). These data suggest that the immunosuppressive effect of AZD3965 may be specific to the pyrrole pyrimidine derivatives class of MCT inhibitors or it could be due to low potency of CHC resulting in lack of immunosuppression; thus, further studies are needed.

An unexpected finding was the increased number of lung metastases, compared with controls. Here the induction in lung metastasis appears to be specific to AZD3965, as CHC and vehicle control treatments in animals bearing 4T1 tumors did not affect the extent of lung metastasis. It is not clear by what mechanism AZD3965 enhances lung metastasis but complex change in epithelial-mesenchymal transition (EMT) following MCT1 inhibition may be possible, as altered tumor metabolism has been recognized to play a role in governing EMT process (44). Our results conflict with those of Tasdogan et al. who reported that MCT1 inhibition by AZD3965 decreased the metastatic disease burden in both patient-derived xenograft and mouse syngenic melanoma models (45). The differences in findings may relate to AZD3965-mediated immunomodulation and the immuno-competence of subjects, and may be highly dependent on tumor type and the dosing regimens and duration of treatment. Further studies are needed to elucidate how the immunomodulatory property of AZD3965 may impact the chemotherapeutic efficacy of AZD3965 in breast cancer patients.

In the present study, AZD3965 was administered at a dose of 100 mg/kg i.p. twice-daily. We chose to use this dose because it has been previously reported to be effective in other tumor xenograft models expressing MCT1 (9–11). Our results demonstrated extensive accumulation in tumor tissue (57.4 times higher than the total plasma concentration), and much higher than the in vitro IC₅₀ value. This extensive accumulation of AZD3965 in the tumor tissue has not been reported previously. For CHC (administered at 42.6 μmol/animal in current study), we did not quantitate plasma or tissue concentrations of CHC. However, at a lower dose of 25 μmol/animal administered five times per week i.p. for 28 days, CHC was effective in reducing breast tumor growth in a xenograft model (8). Although the lack of in vivo efficacy with CHC was unanticipated, previous work from our laboratory has shown that even with another potent inhibitor of MCT1, AR-155858 (analogue of AZD3965 with similar potency), there was no in vivo activity in the same tumor model despite high tumor concentrations (29).

In addition to the effect of AZD3965 on the immune function, other factors may contribute to the observed in vivo results. Although the current study was limited by the lack of antibodies available for detecting MCTs on tumor cells via immunohistochemical analysis, we demonstrated that AZD3965 treatment had no effect on the total tumor protein expression of MCTs, GLUT1 and CD147. Here the total tumor includes fibroblasts, stem cells, as well as immune cells present in tumor tissue. Interestingly, while MCT4 expression was not present prior to 4T1 tumor injection, we observed detectable MCT4 expression regardless of treatment. This is consistent with previous studies showing that the existence of MCT4 could likely impact the sensitivity of MCT1 inhibition (10,13,46). Thus, the presence of MCT4 could represent a possible reason for the poor efficacy of AZD3965 in our in vivo study. Furthermore, we demonstrated lack of efficacy even with CHC, an inhibitor that can inhibit both MCT1 and MCT4. This finding is supported by a recent study that profiled 246 tumor cell lines and demonstrated that MCT4 alone may not be sufficient for predicting sensitivity to MCT1 inhibition (46). While many of the resistant cells expressed MCT4 to varying extents, the authors also identified biomarkers associated with metabolic adaptation that may play a role in resistance to MCT1 inhibition (46).

In this study, we observed a trend toward lower total tumor protein expression levels (at sacrifice) of MCT1, GLUT1 and CD147, relative to that in the implanted 4T1 cells. The reduced GLUT1 expression in the 4T1 tumors agrees with previous findings (47). Decreased tumor protein expression of MCT1 and CD147 in both vehicle control–treated and AZD3965-treated animals could have con-founded the in vivo efficacy of AZD3965. This is supported by a recent finding that statin-induced overexpression of MCT1, observed in both clinical tumor biopsies and xenograft tumors, enhanced the sensitivity to AZD3965 (48).

In summary, the findings presented here represent the first study examining the in vivo efficacy of AZD3965 and CHC and their effect on immune function in the murine 4T1 breast tumor animal model in immunocompetent mice; 4T1 cells overexpress MCT1, but not MCT4. While in vitro cell studies demonstrated potential efficacy, in vivo CHC and AZD3965 treatments exhibited minimal efficacy in reducing tumor volume and weight. AZD3965 treatment was also associated with increased lung metastasis. Previous work from our laboratory has shown that high tumor ARC-155858 (an analogue of AZD3965) accumulation also exhibited no in vivo activity in the syngeneic 4T1 breast tumor xenograft model (29). While the choice of the tumor model likely dictates the findings presented here, the current work highlights the need to consider the effect of AZD3965 on immune function and the changes in tumor MCT1 and MCT4 expression over time as potential mechanisms responsible for developing resistance toward AZD3965 treatment. Further studies are necessary to evaluate the therapeutic potential of MCT1 inhibition by AZD3965 in other tumor models of TNBC.

Abbreviations:

MCTs

Monocarboxylate transporters

TNBC

Triple-negative breast cancer

CD147

Basigin

GLUT1

Glucose transporter 1

CHC

Alpha-cyano-4-hydroxycinnamic acid