Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma

Abstract

Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter plays a key role in coordinating metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation, and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial pathways has remained unexplored. Here, we investigate metabolic consequences of uniporter loss- and gain-of-function using uniporter knockout cells and the liver cancer fibrolamellar carcinoma (FLC), which we demonstrate to have elevated mitochondrial calcium levels. Our results reveal that branched-chain amino acid (BCAA) catabolism and the urea cycle are uniporter-regulated metabolic pathways. Reduced uniporter function boosts expression of BCAA catabolism genes, and the urea cycle enzyme ornithine transcarbamylase (OTC). In contrast, high uniporter activity in FLC suppresses their expression. This suppression is mediated by reduced expression of the transcription factor KLF15, a master regulator of liver metabolism. Thus, uniporter responsive calcium signaling plays a central role in FLC-associated metabolic changes, including hyperammonemia. Our study identifies an important role for mitochondrial calcium signaling in metabolic adaptation through transcriptional regulation of metabolism and elucidates its importance for BCAA and ammonia metabolism in FLC.

Figure 1:. MCU KO cells exhibit growth defects and altered mitochondrial proteome

(A) WT, MCU KO, and MCU rescue cells were counted on days 2, 3, and 5 after plating; n=4–6. (B) HeLa cell doubling times were calculated from cell counts on days 2 and 5 in (A); statistical significance was determined by Dunnett’s multiple comparisons test following one-way ANOVA; expression of MCU and MCU-FLAG was confirmed by Western blot. (C) Volcano plot shows relative abundance of mitochondrial proteins in MCU KO cells compared to WT cells. Red points indicate proteins in the valine, leucine, and isoleucine degradation KEGG pathway; n=5. (D) Volcano plot shows relative abundance of mRNAs encoding mitochondrial proteins in MCU KO cells compared to WT cells. Red points indicate genes in the valine, leucine and isoleucine degradation KEGG pathway; MCU is marked in light blue; n=3. (E, F) Fold change (FC) of valine, leucine, and isoleucine degradation-associated proteins (E) and genes (F) enriched in MCU KO cells in (C, D); proteins and genes are listed in order of ascending p-value. All error bars represent standard deviation; ns indicates non-significant, * indicates a p-value < 0.05, ** indicates a p-value < 0.01, *** indicates a p-value < 0.001, and **** indicates a p-value < 0.0001.

Figure 2.. Mitochondrial calcium uniporter regulates BCAA catabolism pathway

(A) Representative immunoblots of select BCAA catabolism proteins and uniporter components and their quantification in HeLa WT and MCU KO cells; n=3. (B) Representative immunoblots of select BCAA catabolism pathway proteins and their quantification in HeLa WT and EMRE KO cells; n=3. (C) Representative immunoblots of select BCAA pathway proteins and their quantification in AML12 cells following shRNA-mediated MCU knockdown; n=3. (D) Schematic of BCAA catabolism pathway. The committed step in the pathway is catalyzed by BCKDH complex which is active in the dephosphorylated state in (A-C) was determined by one-sample t-test. All error bars represent standard deviation; * indicates a p-value < 0.05 and ** indicates a p-value < 0.01.

Figure 3:. BCAA catabolism maintains NADH/NAD⁺ balance in MCU KO cells

(A) Cell numbers of WT and MCU KO cells with and without pharmacological activation of BCAA catabolism by BT2. Cells were counted three days after plating and treatment; n=4–5. Immunoblots show phosphorylated and total BCKD-E1α after vehicle DMSO or BT2 treatment. Statistical significance was determined by Dunnett’s multiple comparisons test following one-way ANOVA. (B) Schematic of BCAA catabolism and TCA cycle enzymes and metabolites; BCAA catabolism produces acetyl-CoA and succinyl-CoA, which can enter the TCA cycle; Ca²⁺-regulated enzymes and NAD^+/NADH coupled reactions are shown in red. Labelled leucine carbons (indicated as blue circles) and their incorporation into the TCA cycle are also shown. (C) Relative abundance of indicated metabolites and their fraction generated from labeled leucine in WT and MCU KO HeLa cells. (D) Relative NADH/NAD⁺ ratios in WT and MCU KO cells with and without 3hr BCAA starvation are shown. Statistical significance was determined by the Tukey-Kramer test following one-way ANOVA; n=3. All error bars indicate standard deviation; ns indicates non-significant, * indicates a p-value < 0.05. ** indicates a p-value < 0.01, *** indicates a p-value < 0.001, **** indicates a p-value < 0.0001.

Figure 4.. FLC is characterized by increased mitochondrial Ca²⁺ levels and uniporter expression

(A) Schematic of FLC liver tumor with heterozygous deletion in chromosome 19 producing the DNAJ-PKAc (DP) fusion protein. (B) Immunoblots of lysates from non-tumor (N) and tumor (T) liver from FLC patients show DP fusion protein expression in the tumor. (C) Electron micrographs at 10,000x magnification of non-tumor (NTL) and tumor (FLC) sections from patient 9; nuclei are labeled n; scale bars = 1 μm. (D) Micrographs of samples shown in (C) at 25,000x magnification; white arrowheads mark representative Ca²⁺ deposits in the tumor; scale bars = 600 nm. (E) Percentage of mitochondria from FLC patient 9 with Ca²⁺ deposits; the mean is reported from manual counting of >500 mitochondria per sample by two independent, blinded analysts. (F) Immunoblots of uniporter components and control mitochondrial proteins from paired non-tumor (N) and FLC tumor (T) samples. (G) Pooled quantification of immunoblots in (F) normalized to TOM20 levels; statistical significance was determined by one-sample t-test. (H) H&E and MCU IHC staining of non-tumor (N) and tumor (T) regions of liver from FLC patient 9; 40x and 100x image scale bars are 500 μm and 200 μm, respectively. (I) qPCR analysis of MCU RNA expression in paired non-tumor liver (NTL) and tumors from FLC patients 29, 42.1, 47, and 59. (J) MCU transcript expression in indicated datasets in tumor (FLC) and normal liver (NML). All error bars indicate standard deviation; numbers above error bars indicate p-values; * indicates a p-value < 0.05.

Figure 5.. Cellular models of FLC show DP-dependent increase in mitochondrial Ca²⁺ levels

(A) Immunoblot of lysates from WT and FLC clones c14 and c4 using an antibody against PKAc. (B) Proliferation curves of cellular models of FLC compared to WT AML12 cells; cells were counted on days 2, 3, and 4 after plating; n=3. (C) Representative traces and quantification of mitochondrial Ca²⁺ release assays in AML12 cells; cells were treated with the uncoupler CCCP, and the relative amount of Ca²⁺ released was quantified using a Ca²⁺ indicator dye; statistical significance was determined by one-sample t-test; n=10. (D) Representative trace of mitochondrial free Ca²⁺ levels of AML12 WT and c14 cells quantified using matrix-targeted Ca²⁺ reporter G-GECO (mito-G-GECO). (E) Baseline mito-G-GECO fluorescence normalized to minimum and maximum signals in AML12 WT and c14 or c4 cells; statistical significance was determined by Mann Whitney test; n >12 (F, G) Representative traces (F) and mitochondrial Ca²⁺ uptake rates in AML12 cells (G); mitochondrial Ca²⁺ uptake rates were calculated by monitoring Ca²⁺ clearance in the presence of a Ca²⁺ indicator dye; statistical significance was determined by one-sample t-test; n=9. Immunoblot of MCU shows comparable MCU expression in FLC clones compared to WT controls. (H) Mitochondrial Ca²⁺ release was assayed as in (C) after cells were treated with 5 μM PKA inhibitor BLU2864 or DMSO for 4 days. Ca²⁺ release was normalized to total protein levels; fold change in released Ca²⁺ is shown relative to DMSO control for each cell line; statistical significance was determined by paired t-test, n=7–9. (I) Seahorse extracellular flux analysis of oxygen consumption rates (OCR) in FLC clones compared to WT AML12 cells at baseline and after indicated treatments; n=10–16. All error bars indicate standard deviation; ns indicates non-significant, * indicates a p-value < 0.05, ** indicates a p-value < 0.01, *** indicates a p-value < 0.001, and **** indicates a p-value < 0.0001.

Figure 6:. FLC is characterized by uniporter-mediated suppression of BCAA catabolism

(A) Analysis of publicly available gene expression datasets of FLC tumor and non-tumor liver (NTL) show decreased in BCAA catabolism gene expression in FLC (B) Immunoblots of select BCAA catabolism enzymes from paired non-tumor (N) and FLC tumor (T) lysates and pooled quantification of protein levels normalized to TOM20 levels; statistical significance was determined by one-sample t-test. (C) Immunoblots and quantification of select BCAA catabolism proteins and uniporter components in AML12 cells; statistical significance was determined by one-sample t-test; n=4. (D) MCU knockdown increases expression of BCAA catabolism pathway proteins in FLC clones; immunoblots of select pathway proteins and uniporter components are shown. All error bars indicate standard deviation; * indicates a p-value < 0.05, ** indicates a p-value < 0.01, and *** indicates a p-value < 0.001.

Figure 7:. KLF15 and OTC expression are regulated by the uniporter in FLC

(A) Immunoblots of KLF15 in paired non-tumor (N) and FLC tumor (T) lysates. (B) IHC of MCU or KLF15 on non-tumor (N) and tumor (T) regions from FLC patients 9 and 58; 40x and 100x image scale bars are 500 μm and 200 μm, respectively. (C) Immunoblot of KLF15 from WT AML12, c14, and c4 lysates. (D) Immunoblots of KLF15 from WT AML12 and c14 lysates following MCU knockdown. (E) Schematic of the urea cycle, metabolites, and OTC, a mitochondrial protein with reduced levels in FLC. (F) qPCR analysis of OTC mRNA expression in paired non-tumor liver and tumors from FLC patients 29, 42.1, 47, and 59; statistical significance was determined by one sample t-test; n=4 (G) OTC mRNA expression in normal liver (NML) and FLC tumors in the indicated gene expression datasets. (H) Immunoblot of OTC and quantification of OTC levels relative to β-actin from paired non-tumor (N) and FLC tumor (T) lysates; statistical significance was determined by one sample t-test; n=5. (I) Immunoblots of OTC from WT AML12 and c14 lysates following MCU knockdown. All error bars indicate standard deviation; * indicates a p-value < 0.05 and ** indicates a p-value < 0.01.

Figure 8:. Model for regulation of KLF15, BCAAs, and the urea cycle by mitochondrial Ca²⁺ signaling.

Our data suggest that under conditions of low uniporter function in the liver cells, high KLF15 levels stimulate expression of BCAA catabolism pathway genes and OTC. Activation of this pathway helps maintain NADH/NAD⁺ balance. Increased uniporter activity, as observed in FLC, inhibits KLF15, leading to decreased BCAA catabolism enzyme and OTC expression. This inhibition conserves BCAAs for translation and cell growth. MCU inhibition also causes urea cycle impairment, which can lead to hyperammonemia. How the uniporter regulates KLF15 expression is not known.