How being anti- can pro-mote metastasis

The popular belief that anti-oxidant dietary supplements could protect against cancer was overturned a few years ago when studies feeding them to mice showed an accelerated development of cancer. A new study uncovers an underlying mechanism of how anti-oxidants can promote lung cancer metastasis.

Isabel Hofman

Our cells produce their necessary energy using oxygen which leads to unwanted byproducts in the form of reactive oxygen species (ROS). Too much of these oxygen radicals is known to cause DNA damage, and a popular belief that ROS can cause cancer drove the trend for people, healthy or not, to supplement with anti-oxidants to neutralize the dangerous oxygen derivatives. Large randomized clinical trials could however not confirm any benefit of such supplements.1,2

In 2014, the group led by Martin Bergö at Karolinska Institutet demonstrated for the first time that anti-oxidants can actually accelerate cancer. This study showed faster lung cancer progression in mice whose feed was supplemented with the anti-oxidants N-acetylcysteine (NAC) and vitamin E.The compounds reduced DNA damage and subsequent TP53 activation in the tumors, thereby offering protection from apoptotic pathways that would otherwise limit their proliferation.3 A follow-up paper in 2015 showed that antioxidants promote metastasis in a mouse melanoma model.4

Anti-oxidants can be taken in the form of dietary supplements and are popular among athletes and health gurus. A healthy diet should however be sufficient in most circumstances.
Pictures from pexel.com

Recent studies show that lung tumors can intrinsically use similar mechanisms by stimulating endogenous production of anti-oxidants by activating the transcription factor NRF2 or inactivating its inhibitory chaperone KEAP1.5 The new paper of Wiel, Bergö and colleagues published in Cell is the first study to investigate the effect of anti-oxidants on lung cancer metastasis, a major factor in the high mortality rates of the disease.6

They used two mouse models for lung cancer, K and KP, reflecting their KRAS and TP53 mutations. These models have been described to develop KRAS-dependent lung tumors that can also form distant metastases under the influence of TP53 mutations. The new study shows that NAC and vitamin E can increase the formation of these secondary tumors in both mouse models. To make subsequent molecular analysis more accessible, cell lines were derived from lung tumors from K mice: three control lines (mTC) from mice not treated with anti-oxidants, and three treated lines (mTN) from animals that had been supplemented with NAC. In line with the in vivo experiments, the mTN lines had more invasive characteristics in vitro (as measured by migration and invasion assays) and caused more metastases when re-injected into wild type mice.

To get a deeper understanding of the cellular changes caused by anti-oxidant treatment, RNA sequencing was performed on the two sets of cell lines. This transcriptome analysis revealed that targets of NRF2 were downregulated in the mTC lines, and 1/5 of the genes differentially expressed contained a previously described NRF2 binding motif. Almost all of those were shown to also possess the known binding site for another transcription factor – BACH1. This factor is involved in a musical chair dance with NRF2, displacing it from its DNA binding sites and repressing its targets. qPCR analysis confirmed an elevated expression of specific BACH1 target genes, consistent with elevated levels of the protein itself. This could be a direct cause of lowered ROS levels through a known feedback mechanism linking free radicals with free heme molecules which promote the degradation of the BACH1 protein. Confirmation of this connection was obtained in several ways. Lowering ROS decreased free heme levels and could stabilize BACH1. On the other hand, addition of heme lowered BACH1 levels, while blocking the proteasome could inhibit the heme-induced degradation of the protein.

BACH1 was thus identified as a main actor of anti-oxidant induced transcriptional changes in lung cancer cells. The next step was to investigate the link between cellular BACH1 levels and the metastasis phenotype seen in vivo. First of all, KO of BACH1 in vitro could normalize the migration phenotype of the mTN cell lines without affecting proliferation. Secondly, these mTN-BACH1KO cells also had a reduced metastasis capacity in vivo when re-injected in mice. Finally, in a reverse experiment, increased expression of BACH1 in mTC cells was sufficient to raise their in vivo metastatic abilities to a level comparable to the mTN cells.

The last mystery was to uncover how BACH1 can promote metastasis. To identify the signature of BACH1 transcriptional regulation, chromatin bound to BACH1 was isolated and the captured fragments sequenced in a ChIPseq experiment. This confirmed the known BACH1 target sequence and known BACH1 target genes. Additionally, a pathway analysis uncovered the cellular metabolism as one of three top pathways regulated by the transcription factor. Genes regulated by BACH1 in this pathway include Hexokinase 2 (Hk2) and Gapdh, both coding for important enzymes in glucose metabolism. Glycolysis was indeed elevated in mTN cells, and reanalysis of RNAseq data showed that in addition to Hk2 and Gapdh, other glycolysis associated genes were overexpressed in these cells. A direct link between the metabolic and migratory phenotypes was confirmed through overexpression of HK2, which increased glycolysis and migration rates of mTC cells without affecting BACH1. These cells now also produced more metastasis when injected in mice, while reducing glycolysis rates had the opposite effect.

BACH1 is thus identified as a down-stream effector of anti-oxidant supplements, leading to an elevated glycolytic metabolism which can strengthen migration and metastasis in a lung cancer model. Importantly, the anti-oxidant treated cells were shown to have a higher dependency on glycolysis, making them more vulnerable to anti-glycolytic drugs. Indeed, these compounds could also inhibit the pro-metastatic effect of the anti-oxidants in vivo. BACH1 or glycolysis inhibitors could thus be viable options for lung tumors with the reported NRF2 or KEAP1 mutations, or after radiation of primary lung tumors to pre-emptively reduce the risk for metastasis.

References

1. van Zandwijk, N., Dalesio, O., Pastorino, U., de Vries, N., and van Tinteren, H. (2000). EUROSCAN, a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer. For the EUropean Organization for Research and Treatment of Cancer Head and Neck and Lung Cancer Cooperative Groups. J. Natl. Cancer Inst. 92, 977–986.

2. Klein, E.A., Thompson, I.M., Jr., Tangen, C.M., Crowley, J.J., Lucia, M.S., Goodman, P.J., Minasian, L.M., Ford, L.G., Parnes, H.L., Gaziano, J.M., et al. (2011). Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 306, 1549–1556.

3. Sayin V. I., Ibrahim M. X., Larsson E., Nilsson J. A., Lindahl P., Bergo M. O. (2014) Antioxidants accelerate lung cancer progression in mice. Sci Transl Med. Jan 29;6(221):221ra15.

4. Le Gal K., Ibrahim M. X., Wiel C., Sayin V. I., Akula M. K., Karlsson C., Dalin M. G., Akyürek L. M., Lindahl P., Nilsson J., Bergo M. O. (2015) Antioxidants can increase melanoma metastasis in mice. Sci Transl Med. Oct 7;7(308):308re8.

5. Berger, A.H., Brooks, A.N., Wu, X., Shrestha, Y., Chouinard, C., Piccioni, F., Bagul, M., Kamburov, A., Imielinski, M., Hogstrom, L., et al. (2016). Highthroughput Phenotyping of Lung Cancer Somatic Mutations. Cancer Cell 30, 214–228.

6. Wiel, C., Le Gal, K., Ibrahim, M.X., Jahangir, C.A., Kashif, M., Yao, H., Ziegler, D. V., Xu, X., Ghosh, T., Mondal, T., et al. (2019). BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis. Cell 178, 330-345.e22.

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