Hexose‐6‐phosphate dehydrogenase controls cancer cell proliferation and migration through pleiotropic effects on the unfolded‐protein response, calcium homeostasis, and redox balance

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.

Impact of 17β-HSD12, the 3-ketoacyl-CoA reductase of long-chain fatty acid synthesis, on breast cancer cell proliferation and migration

“…Evaluation of mRNA abundance was performed by qPCR as described previously [26]. The sequences of the primers used are shown in Table 1.…”

“…17β-HSD12 0.05 μg/ml (SIGMA-Aldrich HPA016427), α-tubulin 0.062 μg/ml (GeneTex, Irvine, CA, USA, #GTX628802), actin 0.67 μg/ml (Santa Cruz Biotechnology, sc-1616), PPIA 0.05 μg/ml (Abcam, Ab41684), PERK 0.1 μg/ml (Cell signaling Technology, Danvers, MA, USA, #3192), eIF2α 0.1 μg/ml (Cell Signaling Technology, #9722S), peIF2α 0.3 μg/ml (Cell Signaling Technology, #119A11), ATF4 0.1 μg/ml (Cell Signaling Technology, #11815S), ATF6 0.25 μg/ml (Cell Signaling Technology, #65880S), CHOP 1.2 μg/ml (Cell Signaling Technology, #2895S), sXBP1 0.1 μg/ml (Cell Signaling Technology, #12782S), GRP78 0.62 μg/ml (BD Bioscience, San Jose, CA, USA, #610978), ERp44 1:1000 dilution [26], PDI 0.1 μg/ml (Abcam, Cambridge, UK, Ab2792), ERp72 1:1000 dilution (Stressgen), GRP94 1:1000 dilution [28], FADS1 0.5 μg/ml (Santa Cruz Biotechnology, sc-134337), FADS2 0.5 μg/ml (Abcam, ab72189), G6PD 1 μg/ml (Bethyl Laboratories, TX, USA, A300-404A), PGD 1:1000 (Abcam, ab129199) and RXRα 0.1 μg/ml (Santa Cruz Biotechnology, sc-515929), Akt 0.1 μg/ml (Abcam, ab126811), pAkt Ser473 0.1 μg/ml (Cell Signaling Technology, #9271), Caspase-9 (Cas-9)/cleaved Cas-9 0.1 μg/ml (Cell Signaling Technology, #9508), Caspase-3 (Cas-3) 0.1 μg/ml (Cell Signaling Technology, #9665), cleaved Cas-3 0.1 μg/ml (Cell Signaling Technology, #9664), Bcl-2 0.1 μg/ml (Cell Signaling Technology, #2870), Bax 0.1 μg/ml (Cell Signaling Technology, #5023), and ELOVL5 1 μg/ml (Origene, #TA315700). Samples were not boiled for detection of ELOVL5, due to the protein aggregation occurring otherwise.…”

“…Indirect immunofluorescence staining has been described earlier [26]. Visualization of the fluorescence signal and image acquisition was performed utilizing the 20 × objective Figure 7a).…”

Induction of the nicotinamide riboside kinase NAD+ salvage pathway in skeletal myopathy of H6PDH KO mice

“…In contrast to H6KO conditional and global murine models that show a profound upregulation of the ER stress response human cancer cell lines of breast origin transient knockdown of H6PD produced a down regulation in ER stress 30 . Also reported was a reduction in migration and enhanced cell adhesion, these data suggest not only that cancer cells show reliance upon H6PD mediated NADPH generation for their proliferation but also that H6PD maybe a novel target.…”

Lipase maturation factor 1 affects redox homeostasis in the endoplasmic reticulum

“…One such reductase, 11b-HSD1, acts on cortisone, but an enzyme that directly reduces PDIs in the ER is currently only hypothetical (Odermatt & Klusonova, 2015;Ellgaard et al, 2018). Loss of LMF1 resembles loss of H6PD in that the ER is more oxidized (Tsachaki et al, 2018). However, there is no evidence suggesting that LMF1 itself binds NADPH, but it could cooperate with such an enzyme.…”