MitoNews, Volume 9, Issue 4

Edited by James Murray, PhD.

Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how our MitoSciences range of research tools has contributed to this effort. Read the full list of 69 original research papers published this month.

Past issues are available for review in the archives.

This issue of MitoNews highlights 3 papers investigating different aspects of mitochondrial dysfunction in breast cancer.

NAD+/NADH ratio and complex I

Santidirian et al. utilized in vitro and in vivo assays to elucidate the role of complex I alterations in breast cancer tumorigenesis and metastasis. MDA-MB-435 and MDA-MB-231 are triple-negative human breast cancer cell lines; they lack estrogen receptor, progesterone receptor, and do not have HER-2/Neu amplification. The authors first showed that transduction and full integration of yeast NADH dehydrogenase Ndi1 resulted in increased complex I but did not affect OXPHOS expression. They then injected F-luc-tagged MDA-MB-435 and MDA-MB-231 cells expressing Ndi1 into SCID mice and monitored them with bioluminescence imaging. These mice experienced decreased tumor growth in the mammary fat pad and metastasis. Conversely, when they knocked down complex I subunit NDUFV1 in MDA-MB-435 cells, they observed a decrease in complex I activity and increased metastasis.

The depression in metastasis accompanying an increase in complex I activity was then shown to be an effect of increased autophagy. The authors measured p62 levels, a ubiquitin-binding scaffold protein, and mTORC1 and AKT activity by following phosphorylation of substrates by Western blot. Expression of Ndi1 resulted in decreased p62 and decreased mTORC1 activity in both cell lines and decreased AKT activity in MDA-MB-231, results consistent with increased autophagy. Conversely, increases in p62 and mTORC1 and AKT activity followed NDUFV1 knockdown. Furthermore, knockdown of ATG5, a protein necessary for induction of autophagy, by shRNA transduction in Ndi1-expressing cells inhibited autophagy and eliminated the Ndi1-induced inhibition of metastasis. Knockdown of nicotinamide phosphoribosyltransferase (NAMPT), which is required for biosynthesis of NAD+, by transduction of shRNA, reduced NAD+/NADH ratio and lung metastasis and was associated with decreased mTORC1 activity and increased autophagy. Neither expression of Ndi1 nor knockdown of NDUFV1 significantly affected production of ROS or NADPH. These results support the idea that an increase in complex I activity affects autophagy and metastasis through alterations in cellular NAD+/NADH ratios.

Next, they showed treatment of MDA-MB-231 and MDA-MB-435 cells with nicotinic acid (NIC) or NAM, NAD+ precursors, increased NAD+/NADH ratios in vitro. Interestingly, when mice were given 1% NIC or NAM in drinking water and i.v. injections of the cancer cell lines, metastasis was reduced. As expected, NIC and NAM treatment of MDA-MB-231 and MDA-MB-435 was associated with decreased p62 , mTORC1 and AKT activities. The effect on mTORC1 involved SIRT1, a NAD+-dependent deacetylase, in a different cell line, MDA-MG-453, with a predilection for brain metastasis, but not MDA-MB-231 or MDA-MB-435, suggesting multiple mechanisms. Finally, the authors used additional cell and mouse lines to confirm the inhibitory effects of treatment with NAD+ precursor on tumor growth and metastasis in vivo.

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression. J Clin Invest. 2013 Santidrian AF, Matsuno-Yagi A, Ritland M, Seo BB, LeBoeuf SE, Gay LJ, Yagi T, Felding-Habermann B.

Oxidative stress

Breast cancer is an estrogen-dependent malignancy, occurring in higher frequency in postmenopausal women. Presence of the estrogen receptor is considered a diagnostic and prognostic indicator and current treatments target binding of estrogen to the receptor. Sastre-Serra et al. categorized samples of invasive ductal carcinoma from 13 postmenopausal women according to the ratio of ER alpha and ER beta, two isoforms of the estrogen receptor. The ER alpha/ER beta ratio was classified as high (HER), medium (MER) and low (LER). They then measured markers of oxidative stress, mitochondrial function, and activation of signaling pathways using immunoblot and Western blots for HER, MER, and LER ratio breast tumors. LER ratio tumors were associated with increased oxidative stress (more carbonyl groups), increased antioxidant markers (Mn-SOD, CuZn-SOD, catalase, and GRd), and decreased OXPHOS complexes II, III, IV, and V. Levels of uncoupling protein 5 (UCP5) and sirtuin 3 (SIRT3) also increased with LER ratios as did phosphorylation of AKT and JNK/SAPK, signaling pathways associated with proliferation and oxidative stress. Interestingly, LER ratio tumors were also associated with increases in glutathione peroxidase and complex I and no change in UCP2.

Sastre-Setta et al. proposed that the decrease in complexes II, III, IV, and V, but not I in LER ratio breast tumors would result in an electron burst and increased in ROS. Antioxidant enzymes increase in response. They point out that ROS could play a role in proliferation during tumor initiation, but high ROS could instead lead to apoptosis. They conclude by suggesting inhibitors of UCPs and SIRT3 might be therapeutic as they could result in increased ROS and consequently, apoptosis.

The oxidative stress in breast tumors of postmenopausal women is ER alpha/ER beta ratio dependent. Free Radic Biol Med. 2013 Sastre-Serra J, Nadal-Serrano M, Pons DG, Valle A, Garau I, García-Bonafé M, Oliver J, Roca P.

Uncoupling proteins and mitochondrial dysfunction

Sanchez-Alvarez et al. sought to understand the effects of mitochondrial dysfunction in breast cancer tumorigenesis in both epithelial cancer cells and the surrounding stromal fibroblasts. This paper follows previous work showing that release of H₂O₂ from cancer cells results in oxidative stress in associated stromal fibroblasts, which induces catabolic processes and the production of fuels utilized by the cancer cells. The authors overexpressed uncoupling protein isoforms (UCP1, UCP2, UCP3) in hTERT-BJ1 human fibroblasts. They observed that when fibroblasts overexpressing UCP1 were co-injected with MDA-MB-231 breast cancer cells into mice, the resulting tumors were 2.4 to 3.6 times larger than controls. Increased tumor growth was not associated with neovascularization, as immunostaining of CD31 showed no difference in vascular density. Immunoblots of fibroblasts overexpressing UCP1 displayed induced oxidative stress (decreased Cav-1 and increased MCT4), autophagy (increased in BNIP3 and LC3), and specific lysosomal/autophagy markers (Lamp1 and cathepsin B). Overexpression of UCP2 and UCP3 had no effect on tumor growth when co-injected with MDA-MB-231 cells. These fibroblasts did show similar changes in oxidative stress and autophagy markers, but not the lysosomal/autophagy markers.

Increased levels of beta-hydroxybutyrate, a mitochondrial fuel, were measured in the conditioned media of UCP1, UCP2, and UCP3 fibroblasts, but an increase in fatty acid beta-oxidation rate was only detected in UCP1 fibroblasts. Quinicrine staining also revealed an increase in vesicles containing ATP in UCP1 fibroblasts. Exposure to chloroquine, an inhibitor or autophagy, decreased staining of vesicles, from which the authors conclude that autophagy is an important means of ATP release. Mitochondrial activity was decreased in UCP1 fibroblasts, but increased in MDA-MB-231 cells co-cultured with the fibroblasts. This effect was heightened in hypoxic conditions. Finally, results of overexpression of UCP1, UCP2, and UCP3 in MDA-MB-231 cancer cells were opposite of those in fibroblast mitochondrial fractions. Autophagy was induced, mitochondrial activity was depressed, and, when injected into mice, tumor growth was reduced.

The authors conclude that this two-compartment tumor dynamic and the importance of mitochondrial dysfunction could inform cancer treatments. Metformin, a complex I inhibitor, has been effective in clinical studies. When used in combination with tamoxifen, metformin initiates mitochondrial dysfunction in the cancer cells, predisposing them to oxidative stress and cell death induced by tamoxifen. As tamoxifen resistance can also develop as stromal fibroblasts “feed” cancer cells, metformin has been successfully used to reverse this resistance.

Mitochondrial dysfunction in breast cancer cells prevents tumor growth: understanding chemoprevention with metformin. Cell Cycle. 2013 Sanchez-Alvarez R, Martinez-Outschoorn UE, Lamb R, Hulit J, Howell A, Gandara R, Sartini M, Rubin E, Lisanti MP, Sotgia F.

The over-expression of ERbeta modifies estradiol effects on mitochondrial dynamics in breast cancer cell line. Int J Biochem Cell Biol. 2013 Sastre-Serra J, Nadal-Serrano M, Pons DG, Roca P, Oliver J.

Related papers using MitoSciences products this month:

 Unraveling the mechanisms behind the enhanced MTT conversion by irradiated breast cancer cells. Radiat Res. 2013 Blockhuys S, Vanhoecke B, Smet J, De Paepe B, Van Coster R, Bracke M, De Wagter C.

Creating a tumor-resistant microenvironment: cell-mediated delivery of TNFα completely prevents breast cancer tumor formation in vivo. Cell Cycle. 2013 Al-Zoubi M, Salem AF, Martinez-Outschoorn UE, Whitaker-Menezes D, Lamb R, Hulit J, Howell A, Gandara R, Sartini M, Arafat H, Bevilacqua G, Sotgia F, Lisanti MP.

The biarylpyrazole compound AM251 alters mitochondrial physiology via proteolytic degradation of ERR&alpha Mol Pharmacol. 2013 Krzysik-Walker SM, González-Mariscal I, Scheibye-Knudsen M, Indig FE, Bernier M.

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