Heterogeneous Expression and Subcellular Localization of Pyruvate Dehydrogenase Complex in Prostate Cancer

Abstract

Background: Pyruvate dehydrogenase (PDH) complex converts pyruvate into acetyl-CoA by pyruvate decarboxylation, which drives energy metabolism during cell growth, including prostate cancer (PCa) cell growth. The major catalytic subunit of PDH, PDHA1, is regulated by phosphorylation/dephosphorylation by pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). There are four kinases, PDK1, PDK2, PDK3 and PDK4, which can phosphorylate and inactivate PDH; and two phosphatases, PDP1 and PDP2, that dephosphorylate and activate PDH.

Methods: We have analyzed by immunohistochemistry the expression and clinicopathological correlations of PDHA1, PDP1, PDP2, PDK1, PDK2, PDK3, and PDK4, as well as of androgen receptor (AR), in a retrospective PCa cohort of patients. A total of 120 PCa samples of representative tumor areas from all patients were included in tissue microarray (TMA) blocks for analysis. In addition, we studied the subcellular localization of PDK2 and PDK3, and the effects of the PDK inhibitor dichloroacetate (DCA) in the growth, proliferation, and mitochondrial respiration of PCa cells.

Results: We found heterogeneous expression of the PDH complex components in PCa tumors. PDHA1, PDP1, PDK1, PDK2, and PDK4 expression correlated positively with AR expression. A significant correlation of PDK2 immunostaining with biochemical recurrence and disease-free survival was revealed. In PCa tissue specimens, PDK2 displayed cytoplasmic and nuclear immunostaining, whereas PDK1, PDK3 and PDK4 showed mostly cytoplasmic staining. In cells, ectopically expressed PDK2 and PDK3 were mainly localized in mitochondria compartments. An increase in maximal mitochondrial respiration was observed in PCa cells upon PDK inhibition by DCA, in parallel with less proliferative capacity.

Conclusion: Our findings support the notion that expression of specific PDH complex components is related with AR signaling in PCa tumors. Furthermore, PDK2 expression associated with poor PCa prognosis. This highlights a potential for PDH complex components as targets for intervention in PCa.

Keywords: androgen receptor (AR); dichloroacetate (DCA); prostate cancer (PCa); pyruvate dehydrogenase (PDH); pyruvate dehydrogenase kinase (PDK).

Figure 1

Viability, proliferation and mitochondrial function of PCa cells treated with DCA. (A) Cell viability is shown for LNCaP and DU-145 PCa cells, as determined by MTS analysis, after 72 h in the presence of DCA (5 mM and 20 mM). (B) Cell viability is shown for LNCaP cells, as determined by MTS analysis, after 72 h in the presence of DCA (1 mM and 5 mM). (C) Cell growth is shown for LNCaP cells, as determined by Incucyte live-cell analysis, after 72 h in the presence of DCA (1 mM and 5 mM). (D) Mitochondrial respiration is shown for LNCaP cells, as determined by Seahorse extracellular flux analysis, after 48 h in the presence of DCA (1 mM and 5 mM). p value below 0.05 are indicated with *.

Figure 2

Expression of PDKs in PCa specimens. Immunohistochemical staining of expression of PDKs in four representative prostate carcinoma patient samples (1-4). Hematoxylin and eosin (H&E) staining (1A, 2A, 3A, 4A). High expression of all PDKs (case 3: 3B, 3C, 3D, 3E). Low expression of all PDKs (case 2: 2B, 2C, 2D, 2E). High expression of PDK2 and PDK4 (case 1: 1C, 1E), and low expression of PDK1 and PDK3 (case 1: 1B, 1D). High expression of PDK2 and PDK3 (case 4: 4C, 4D), and low expression of PDK1 and PDK4 (case 4: 4B, 4E). Magnification: X100.

Figure 3

Immunohistochemical profile of a prostate adenocarcinoma specimen, showing positive staining for androgen receptor (AR, nuclear), PDHA1 and PDP1 (cytoplasmic), and negative for PDP2. Magnification: X400.

Figure 4

Expression and subcellular localization of PDK2 and PDK3 in PCa cells. (A) Immunoblot of ectopically expressed PDK2-Flag, PDK3-Flag, and Flag-PTEN (as a control) in COS-7 and LNCaP cells using anti-Flag antibody. (B) Immunofluorescence of PDK2-Flag, PDK3-Flag, and Flag-PTEN in COS-7 cells, using anti-Flag antibody (green). (C) Immunofluorescence of PDK2-Flag and PDK3-Flag (green) as in B, with Mitotracker as a mitochondria marker (red). (D) Immunofluorescence of PDK2-Flag and PDK3-Flag (green) in LNCaP cells, with Mitotracker as a mitochondria marker (red). In (B–D) nuclei were stained with DAPI (blue). Note the punctuated and mitochondrial localization of PDK2 and PDK3, as compared to the cytoplasmic PTEN localization.