Targeting the Lactate Transporter MCT1 in Endothelial Cells Inhibits Lactate-Induced HIF-1 Activation and Tumor Angiogenesis

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities

Cancer : lorsque recycler devient une faiblesse

À l’origine du processus cancéreux, les descendants d’une cellule capable de proliférer de manière débridée envahissent le tissu d’origine pour former une masse tumorale de volume croissant (Figure 1). Or, à distance du vaisseau sanguin le plus proche, l’oxygène nécessaire à la chaîne respiratoire cellulaire (phosphorylations oxydatives) s’amenuise. Un équilibre dynamique s’installe donc entre la prolifération des cellules recevant suffisamment d’oxygène et la mort des cellules hypoxiques, ce qui favorise l’élimination naturelle des microlésions tumorales. Toutefois, l’hypoxie favorise aussi l’émergence de cellules cancéreuses capables de produire la majorité de leur énergie par la glycolyse plutôt que par la chaîne respiratoire. Cette adaptation et sa pérennisation permettent à la tumeur d’entrer en phase de croissance exponentielle

Lactate activates HIF-1 in normoxic oxidative tumor cells.

<p>(A) HIF-1 activity was quantified using a dual reporter luciferase assay in oxidative SiHa (left panel, <i>n</i> = 6–8), HeLa (middle panel, <i>n</i> = 3–4), and FaDu cancer cells (left panel, <i>n</i> = 3–4). All cells were cultured during 24-h in fresh medium containing 10 mM lactate or not (control). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005. (B) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of wild-type (WT, <i>n</i> = 3) or mitochondria-depleted (ρ0, <i>n</i> = 5–6) SiHa TCs cultured during 24-h in the presence of 10 mM lactate or not. The upper panels show representative experiments and the graphs HIF-1α protein expression normalized to β-actin levels. <i>ns</i>, <i>p</i>>0.05, **<i>p</i><0.01, ***p<0.005. (C–E) The level of <i>VEGF-A</i> transcript was determined using RT-qPCR in TC lysates. (C) SiHa TCs were exposed to 10 mM lactate or not during 24-h. **<i>p</i> = 0.009; <i>n</i> = 6. (D) As in (C) but with WiDr TCs. <i>ns</i>, <i>p</i>>0.05; <i>n</i> = 3. (E) SiHa TCs were cultured during 24-h in the presence of 10 mM lactate, 10 mM lactate+10 nM echinomycin (an inhibitor of the transcriptional activity of HIF-1), or none of these drugs (control). ***<i>p</i><0.005 <i>versus</i> control; ^###<i>p</i><0.005 <i>versus</i> lactate condition; <i>n</i> = 3–4.</p

MCT1 expression in tumor cells regulates lactate-induced angiogenesis and tumor growth.

<p>Two groups of BALB/c nude mice were injected s.c. with Matrigel plugs containing 30 mM lactate (right flank) or and equal volume of saline (left flank). The plugs also contained 10⁶ SiHa TCs infected with a control shRNA (shCTR, Group 1) or 10⁶ SiHa TCs infected with a specific shRNA against MCT1 (shMCT1-1, Group 2). (A) Tumor growth was tracked over time. The graph shows tumor growth curves and the pictures are representative of mice 21 days after tumor implantation. **<i>p</i><0.01; <i>ns</i>, <i>p</i>>0.05; <i>n</i> = 5 for Group 1; <i>n</i> = 4 for Group 2. (B) In a second set of experiments, plugs were microdissected 12 days after implantation. Pictures show CD31-positive endothelial cells (red) and α-smooth muscle actin-positive pericytes (green). Nuclei are stained in blue with DAPI. Bar = 50 µm. CD31 staining was quantified and is expressed as % of positive surface area in the graph. <i>n</i> = 3–4 for Group 1, <i>n</i> = 4–5 for Group 2. *<i>p</i><0.05.</p

Lactate inhibits PHD2 activity in oxidative tumor cells.

<p>(A) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of SiHa TCs incubated during 24-h with 10 mM lactate or not and increasing doses of 2-oxoglutarate. The upper panels show representative experiments and the graph HIF-1α protein expression normalized to β-actin levels. Data are expressed as % of lactate induction. **<i>p</i><0.01, ***<i>p</i><0.005 <i>versus</i> 10 mM lactate without 2-oxoglutarate; <i>n</i> = 8. (B) ODD-driven luciferase activity was measured in SiHa TCs treated during 24-h with 10 mM lactate or not. *<i>p</i> = 0.0206; <i>n</i> = 6. (C) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of SiHa TCs transfected with a specific siRNA against PHD2 (siPHD2) or with a control siRNA (siCTR) and incubated during 24-h with 10 mM lactate or not. The upper panels show representative experiments and the graph HIF-1α protein expression normalized to β-actin levels. <i>ns</i>, <i>p</i>>0.05, *<i>p</i><0.05; <i>n</i> = 3.</p

Lactate induces normoxic HIF-1α protein stabilization in oxidative tumor cells, not in Warburg tumor cells.

<p>(A) Lactate release in the supernatant of SiHa and WiDr TCs was measured using a CMA600 enzymatic analyzer after 24-h of culture in fresh medium. ***<i>p</i> = 0.0002; <i>n</i> = 4. (B–G) HIF-1α and β-actin protein expression was detected using Western blotting in the lysates of oxidative SiHa or Warburg WiDr TCs. The upper panels show representative experiments and the graphs HIF-1α protein expression normalized to β-actin levels. (B) SiHa and WiDr cells were untreated to detect basal HIF-1α protein expression. *<i>p</i> = 0.0414; <i>n</i> = 3–4. (C) SiHa TCs were cultured during 24-h under hypoxia (1% O₂) or not. *<i>p</i> = 0.011; <i>n</i> = 3. (D) As in (C) but with WiDr TCs. **<i>p</i> = 0.0057; <i>n</i> = 5. (E) SiHa TCs were cultured during 24-h in the presence of 10 mM lactate or not. **<i>p</i> = 0.0029; <i>n</i> = 4. (F) As in (E) but with WiDr TCs. <i>ns</i>, <i>p</i> = 0.1449; <i>n</i> = 8. (G) SiHa TCs were exposed to increasing doses of lactate during 24-h. *<i>p</i><0.05, **<i>p</i><0.01 <i>versus</i> 0 mM lactate condition; <i>n</i> = 9–11. (H) SiHa TCs were exposed to 10 mM lactate during increasing periods of time. *<i>p</i><0.05, **<i>p</i><0.01 <i>versus</i> time 0; <i>n</i> = 3.</p

MCT1 and CD147 interact at the plasma membrane of oxidative tumor cells.

<p>(A) The left graph shows basal <i>SLC16A1</i>/MCT1 mRNA expression normalized to <i>RPL19</i> mRNA expression detected in SiHa and WiDr TCs using RT-qPCR. **<i>p</i> = 0.0062; <i>n</i> = 5–8. On the right, MCT1 and β-actin were detected using Western blotting (WB) in cell lysates. The upper panels show a representative experiment and the graph HIF-1α protein expression normalized to β-actin levels. ***<i>p</i> = 0.0004; <i>n</i> = 5. (B) as in (A) but detecting CD147 instead of MCT1. RT-qPCR: ***<i>p</i><0.0001; <i>n</i> = 7. WB: *<i>p</i> = 0.0237; <i>n</i> = 6. (C) as in (A) but detecting <i>LDH-B</i> and LDH-H instead of MCT1. RT-qPCR: **<i>p</i> = 0.008; <i>n</i> = 3. WB: ***<i>p</i><0.001; <i>n</i> = 6. (D) MCT1 (red, left upper panel) and CD147 (green, left medium panel) were detected using immunocytofluorimetry in SiHa TCs. The lower left panel is a merged picture in which cell nuclei have been stained in blue (Hoechst 33342). Right panels show control experiments in which primary antibodies were omitted. Pictures are representative of <i>n</i> = 4. Bar = 20 µm. (E) The <i>in situ</i> interaction between MCT1 and CD147 was verified in SiHa TCs using a proximity ligation assay. MCT1-CD147 interaction is identified with a red staining and cell nuclei are in blue (Hoechst 33342) in the left panel. The right panel shows control experiments in which the primary antibody against CD147 was omitted. Pictures are representative of <i>n</i> = 6. Bars = 10 µm.</p