Abstract
Hexose-6-phosphate dehydrogenase (H6PD) produces reduced NADPH in the endoplasmic reticulum (ER) lumen. NADPH constitutes a cofactor for many reducing enzymes, and its inability to traverse biologic membranes makes in situ synthesis of NADPH in the ER lumen indispensable. The H6PD gene is amplified in several types of malignancies, and earlier work pointed toward a potential involvement of the enzyme in cancer cell growth. In the present study, we demonstrated a pivotal role of H6PD in proliferation and migratory potential of 3 human breast cancer cell lines. Knockdown of H6PD decreased proliferation and migration in SUM159, MCF7, and MDA-MB-453 cells. To understand the mechanism through which H6PD exerts its effects, we investigated the cellular changes after H6PD silencing in SUM159 cells. Knockdown of H6PD resulted in an increase in ER lumen oxidation, and down-regulation of many components of the unfolded protein response, including the transcription factors activating transcription factor-4, activating transcription factor-6, split X-box binding protein-1, and CCAAT/enhancer binding protein homologous protein. This effect was accompanied by an increase in sarco/endoplasmic reticulum Ca2+-ATPase-2 pump expression and an decrease in inositol trisphosphate receptor-III, which led to augmented levels of calcium in the ER. Further characterization of the molecular pathways involving H6PD could greatly broaden our understanding of how the ER microenvironment sustains malignant cell growth.—Tsachaki, M., Mladenovic, N., Štambergová, H., Birk, J., Odermatt, A. Hexose-6-phosphate dehydrogenase controls cancer cell proliferation and migration through pleiotropic effects on the unfolded protein response, calcium homeostasis, and redox balance.
Highlights
Hexose-6-phosphate dehydrogenase (H6PD) produces reduced NADPH in the endoplasmic reticulum (ER) lumen
To examine the impact of H6PD on cancer cell proliferation, we first silenced its expression by using specific short interfering RNA (siRNA) in the SUM159, MCF7, and MDA-MB-453 cell lines
To investigate whether components present in the serum would further influence the effect of H6PD down-regulation on cell proliferation, we performed the same experiments in SUM159 and MCF7 cells using one-fourth of the amount of serum present in their normal medium (Fig. 1B; low serum)
Summary
Hexose-6-phosphate dehydrogenase (H6PD) produces reduced NADPH in the endoplasmic reticulum (ER) lumen. ABBREVIATIONS: 11b-HSD1, 11b-hydroxysteroid dehydrogenase type 1; ATF, activating transcription factor; Cch, carbachol; CCN, cyclin or G1/S-specific cyclin; CFP, cyan fluorescent protein; CHOP, CCAAT/enhancer binding protein homologous protein; ECAR, extracellular acidification rate; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FRET, fluorescence resonance energy transfer; G6PD, glucose 6phosphate dehydrogenase; Grp, glucose-regulated protein; H6PD, hexose-6-phosphate dehydrogenase; HEPES, hydroxyethyl piperazineethanesulfonic acid; Her, human epidermal growth factor receptor; HHBSS, HEPES-HBSS; IP3R, inositol trisphosphate receptor; MMP, matrix metalloproteinase; OCR, oxygen consumption rate; OxD, oxidation; PARP, poly(ADP-ribose) polymerase; PDI, protein disulfide isomerase; PERK, protein kinase R-like ER kinase; qPCR, quantitative PCR; roGFP, reduction–oxidation-sensitive green fluorescent protein; ROS, reactive oxygen species; RTCA, real-time cell analyzer; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; siRNA, short interfering RNA; sXBP1, split X-box binding protein 1; UPR, unfolded protein response Muscles from H6PD2/2 mice showed decreased expression of the sarco/ER Ca2+-ATPase (SERCA), which is responsible for pumping calcium from the cytosol into the ER at the expense of ATP
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