Lactate Increases Renal Cell Carcinoma Aggressiveness through Sirtuin 1-Dependent Epithelial Mesenchymal Transition Axis Regulation

Abstract

Background: Renal cell carcinoma (RCC) displays a glycolytic phenotype (Warburg effect). Increased lactate production, impacting on tumor biology and microenvironment modulation, has been implicated in epigenetic mechanisms' regulation, leading to histone deacetylases inhibition. Thus, in-depth knowledge of lactate's impact on epigenome regulation of highly glycolytic tumors might allow for new therapeutic strategies. Herein, we investigated how extracellular lactate affected sirtuin 1 activity, a class III histone deacetylase (sirtuins, SIRTs) in RCC.

Methods: In vitro and in vivo interactions between lactate and SIRT1 in RCC were investigated in normal kidney and RCC cell lines. Finally, SIRT1 and N-cadherin immunoexpression was assessed in human RCC and normal renal tissues.

Results: Lactate inhibited SIRT1 expression in normal kidney and RCC cells, increasing global H3 and H3K9 acetylation. Cells exposed to lactate showed increased cell migration and invasion entailing a mesenchymal phenotype. Treatment with a SIRT1 inhibitor, nicotinamide (NAM), paralleled lactate effects, promoting cell aggressiveness. In contrast, alpha-cyano-4-hydroxycinnamate (CHC), a lactate transporter inhibitor, reversed them by blocking lactate transport. In vivo (chick chorioallantoic membrane (CAM) assay), lactate and NAM exposure were associated with increased tumor size and blood vessel recruitment, whereas CHC displayed the opposite effect. Moreover, primary RCC revealed N-cadherin upregulation whereas SIRT1 expression levels were downregulated compared to normal tissues.

Conclusions: In RCC, lactate enhanced aggressiveness and modulated normal kidney cell phenotype, in part through downregulation of SIRT1, unveiling tumor metabolism as a promising therapeutic target.

Keywords: Warburg effect; epigenetic regulation; lactate; renal cell carcinoma; sirtuin 1.

Figure 1

Lactate decreased sirtuin (SIRT)1’s expression and increased renal cell carcinoma (RCC) cell line aggressiveness. Characterization of SIRT1 expression in kidney tumor cell lines treated with 20 mM lactate by RT-qPCR (A), Western blot (B), and immunofluorescence (C). Characterization of global H3 acetylation and H3K9-specific mark in kidney tumor cell lines treated with 20 mM lactate by Western blot (D). Effect of 20 mM lactate treatment in kidney tumor cell lines at cell proliferation (5-bromo-2’-deoxyuridine (BrdU) assay) (E), cell migration (wound-healing assay), (F) and cell invasion (Matrigel Invasion Chambers) (G). Western blot and immunofluorescence quantification are represented as fold change of 20 mM lactate versus control condition; * p < 0.05, ** p < 0.01, *** p < 0.001, and ^ns p > 0.05 (non-significant).Abbreviations: C/CTR—control, L/LAC—20 mM lactate.

Figure 2

Lactate induced SIRT1 downregulation, contributing to normal kidney cell line pseudo-transformation. Characterization of SIRT1 expression in normal kidney cell line treated with 20 mM lactate by RT-qPCR (A), Western blot (B), and immunofluorescence (C). Characterization of global histone acetylation of histone H3 and specific histone marker H3K9 in normal kidney cell lines treated with 20 mM lactate by Western blot (D). Effect of 20 mM lactate treatment in cell proliferation (BrdU assay) (E), cell migration (wound-healing assay) (F), and cell invasion (Matrigel Invasion Chambers) (G). Western blot and immunofluorescence quantification are represented as fold change of 20 mM lactate versus control condition; * p < 0.05, **** p < 0.0001, and ^ns p > 0.05 (non-significant). Abbreviations: C/CTR—control, L/LAC—20 mM lactate.

Figure 3

Conditioned medium (CM) modulated SIRT1, increasing histone acetylation and cell invasion of normal kidney cell line. Characterization of SIRT1 expression in normal kidney cell line treated with conditioned medium from tumor cells, by RT-qPCR (A), Western blot (B), and immunofluorescence (C). Characterization of global histone acetylation of histone H3 and specific histone marker H3K9 (D) in normal kidney cell line treated with conditioned medium from tumor cells, by Western blot. Effect of conditioned medium from tumor cells in normal kidney cell line at cell proliferation (BrdU assay) (E), cell migration (wound-healing assay) (F), and cell invasion (Matrigel Invasion Chambers) (G). Western blot and immunofluorescence quantification are represented as fold change of 20 mM lactate versus control condition; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ^ns p > 0.05 (non-significant). Abbreviations: CM—conditioned medium, CTR—control.

Figure 4

Nicotinamide (NAM) mimicked lactate effect in kidney cell lines. Characterization of SIRT1 expression by RT-qPCR (A), Western blot (B), and immunofluorescence (C). Characterization of global histone acetylation of histone H3 and specific histone marker H3K9 (D) by Western blot. Effect at cell proliferation by BrdU assay (E), cell migration by wound-healing assay (F), and cell invasion by Matrigel invasion chambers (G). Western blot and immunofluorescence quantification are represented as fold change of 200 µM nicotinamide versus control condition; * p < 0.05, *** p < 0.001, **** p < 0.0001 and ^ns p > 0.05 (non-significant) Abbreviations: C/CTR—control, N/NAM—200 µM nicotinamide.

Figure 5

Lactate promoted epithelial–mesenchymal transition (EMT) phenotype through SIRT1-dependent SMAD4 axis in RCC cell lines. Characterization of EMT phenotype after 20 mM lactate, tumor cell-derived CM, and 200µM NAM treatment by Western blot (A). N-cadherin expression with 20 Mm lactate and 200µM NAM by immunofluorescence (B). Co-immunoprecipitation of acetylated lysine/SMAD4 (C) and SMAD4/SNAIL (D) in lactate and nicotinamide treatment conditions. Western blot and immunofluorescence quantification are represented as fold change of 20 mM lactate or 200µM nicotinamide versus control condition Abbreviations: C/CTR—control, CM—conditioned medium, L/LAC—20 mM lactate, N/NAM—200µM nicotinamide, NCAD—N-cadherin, VIM—vimentin.

Figure 6

Alpha-cyano-4-hydroxycinnamate (CHC) reverted the lactate effect in normal kidney cell lines. Characterization of SIRT1 expression by RT-qPCR (A,G) and Western blot (B,H). Characterization of global histone acetylation of histone H3 and specific histone marker H3K9 (C,I) by Western blot. Effect at cell proliferation (D,J) by BrdU assay and cell migration (E,K) by wound-healing assay. Measurement of lactate levels after CHC treatment in RCC cell lines (F); * p < 0.05, ** p < 0.01, *** p < 0.001, , **** p < 0.0001 and ^ns p > 0.05 (non-significant) Abbreviations: CTR—control, CHC—alpha-cyano-4-hydroxycinnamate, CM—conditioned medium, LAC—20 mM lactate.

Figure 7

Monocarboxylate transporter (MCT) inhibition reverted the lactate-derived EMT effect in RCC and normal kidney cell lines. Characterization of N-cadherin expression after CHC treatment in (A) RCC cell lines and (B) normal kidney cell line, by Western blot. N-cadherin, SMAD4, and SNAIL expression in HKC8 cell lines after CHC tumor cell line-derived CM treatment (C). Abbreviations: CTR—control, CHC—alpha-cyano-4-hydroxycinnamate, CM—conditioned medium, LAC—20 mM lactate, NCAD—N-cadherin.

Figure 8

In vivo effect of lactate, NAM, and CHC upon RCC microtumors. Representative in ovo and ex ovo pictures of chick chorioallantoic membrane (CAM) assay at final timepoint (A). Quantification of tumor growth (B) and total of blood vessels (C) in CAM tumors. Representative pictures (D) and graphics (E) of immunoexpression in CAM-developed tumors according to exposure to chemicals; ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ^ns p > 0.05 (non-significant) Abbreviations: CTR—control, CHC—alpha-cyano-4-hydroxycinnamate, LAC—20 mM lactate, NAM—200µM nicotinamide, NCAD—N-cadherin.

Figure 9

Characterization of SIRT1 and N-cadherin in kidney tissues. Expression of SIRT1 and NCAD in normal kidney and renal cell carcinoma tissues (clear cell RCC (ccRCC) and papillary RCC (pRCC) cases) by immunohistochemistry (A). Graphical representation of immunoexpression positive and negative cases (B). Abbreviations: ccRCC—clear cell renal cell carcinoma, pRCC—papillary renal cell carcinoma.