On the role of ROS and antioxidants in cancer progression

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1 On the role of ROS and antioxidants in cancer progression Martin O. Bergö Dept. Biosciences and Nutrition Karolinska Institutet Martin Bergö

2 Reactive oxygen species pools in cancer O 2 OH H 2 O 2 Pro-tumorigenic signaling Oxidative stress DNA damage Tumor initiation Tumor progression

3 The dogma on dietary antioxidants and cancer Free radicals (ROS) cause DNA damage that can lead to cancer......thus; antioxidants, which neutralize ROS, must protect against cancer UV O 2 O 2 DNA-damage CANCER > Billion $ industry Cancer patients heavy users

4 A research question Do antioxidants protect against cancer?

5 Clinical trials show inconsistent results Linxian General Population Nutrition Intervention Trial. J. Natl. Cancer Inst Alpha-Tocopherol/Beta-Carotene Cancer Prevention Study (ATBC) N. Engl. J. Med Carotene and Retinol Efficacy Trial (CARET) Cancer Res Physicians Health Study I (PHS I) N. Engl. J. Med Women s Health Study. J. Natl. Cancer Inst EUROSCAN study. J. Natl. Cancer Inst Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) Archives Int. Med Heart Outcomes Prevention Evaluation The Ongoing Outcomes (HOPE-TOO) Study J. Am. Med. Assoc Physicians Health Study II (PHS II) J. Am. Med. Assoc Selenium and Vitamin E Cancer Prevention Trial (SELECT) J. Am. Med. Assoc Three trials had to be stopped early due to more cancer in the antioxidant groups

6 Sharper questions Do antioxidants protect against cancer? Would antioxidants protect a tumor-free person from cancer in the future? Would antioxidants affect the progression of an existing tumor? Tumor initiation Tumor progression

7 Defining the impact of antioxidants on tumor progression: lung cancer Genetic switch models Kras2G12D/+ Kras2LSL/+ Cre-adenovirus or lentivirus Jackson et al. (2001) Genes & Dev.

8 Study design: antioxidant added after tumor initiation N-acetylcysteine (NAC) 1g/L in drinking water Vitamin E 0.1 och 0.5 g/kg in chow Start NAC 7 weeks Survival 0 weeks Birth 6 weeks AdCre 12 weeks End

9 Antioxidants increased KRAS- and BRAF-induced lung tumor progression Short-term: 12 weeks after Cre-adenovirus inhalation

10 Antioxidants reduced survival in mice with B-RAF V600E -induced lung tumors

11 Antioxidants reduced ROS levels in lung tumors DCF fluorescence NLT = Normal Lung Tissue Green = DCF fluorescence

12 Antioxidants reduced ROS and DNA damage and increased proliferation Antioxidants Control ROS DNA damage Proliferation + NAC IHC staining for BrdU

13 Antioxidants increased the proliferation of some human lung cancer cell lines TP53 WT TP53 MUT

14 Antioxidants reduced DNA damage signaling and p53 levels Human cell lysates Mouse tumor lysates

15 Antioxidants reduced DNA damage signaling and p53 levels Human cell lysates Mouse tumor lysates Is p53 required for the ability of antioxidants to accelerate tumor progression?

16 Knockout of p53 abolishes the antioxidant effect on tumor growth These experiments were done in the setting of tumor-specific p53 deficiency

17 NAC and vitamin E accelerate lung cancer progression in mice by suppressing a ROS-p53 axis The effect is cell autonomous, requires p53, and is similar in mouse and human tumor cells Antioxidants protect tumor cells from free radicals Sayin et al., Science Transl. Med. 2014

18 Is the antioxidant effect limited to lung tumors: What about malignant melanoma? 1. Increasing in incidence and lethality 2. Exposed to antioxidants from topical and dietary sources 3. Known to be sensitive to ROS Braf CA/+ Pten fl/fl Melanocyte-ER-Cre

19 NAC and vitamin E has no impact on the number and size of primary melanoma tumors Braf CA/+ Pten fl/fl Melanocyte-ER-Cre

20 NAC and vitamin E increases malignant melanoma metastasis

21 NAC and Trolox do not affect proliferation of human malignant melanoma cell lines...

22 ...but markedly and acutely increased migration and invasion The antioxidant effect requires new glutathione synthesis Analyzed by XCelligence real-time cell invasion assay

23 The increased migration and invasion depends on new glutathione synthesis BSO = Buthionine sulfoximine

24 Antioxidants increased RHOA activation in human melanoma cells

25 The increased migration and invasion depends on RHOA signaling ROCK inhibitor = Y27632, inhibits RHO associated kinase (ROCK)

Antioxidants increases metastasis in mice with malignant melanoma Increases invasive properties of cultured human melanoma cells Cell autonomous effect driven by glutathione synthesis and RHOA

26 Antioxidants increases metastasis in mice with malignant melanoma Increases invasive properties of cultured human melanoma cells Cell autonomous effect driven by glutathione synthesis and RHOA activation Suggest What that melanoma about lung cells tumors: exhibit why oxidative didn t we stress observe that increased limit their metastasis? ability to metastasize Le Gal et al., Science Transl. Med. 2015

27 Would long-term antioxidant administraion stimulate metastasis in Kras2 LSL/+ mice? N-acetylcysteine (NAC) 1g/L in drinking water Vitamin E 0.1 g/kg in chow Start NAC 7 weeks Survival Birth 6 weeks AdCre 10 5 pfu 60 weeks Survival exp.

28 Antioxidants increased metastasis in mice with K-RAS-induced lung cancer Similar effect in Trp53-deficient and Trp53-WT tumors

29 Antioxidants reduced the expression of the metastasis suppressor Nkx2.1 in some tumors Nkx2.1 **** 100 ** ** low staining % of mice 50 high staining 0 Ctrl NAC VitE Is the antioxidant effect tumor cell automonous rather than mediated by effects on the immune system or microenvironment?

30 We isolated lung tumor cells from antioxidanttreated and control mice... NAC-treated or Ctrl mouse Kras2 LSL/+

31 Lung tumor cells isolated from antioxidanttreated mice show increased invasion in vitro Tumor cells were cultured without antioxidants 0.4 Passage Late passages (>15) Invasion Index index Aox-TC Ctrl-TC Invasion Index index Ctrl-TC Aox-TC Hours Hours Antioxidants in vivo cause long-term but reversible reprogramming of lung tumor cells into an invasive phenotype

32 Acute antioxidant administration has no impact on human lung cancer cell invasion H838 H1975 A549 Invasion Index Ctrl NAC Invasion Index Ctrl Trolox Hours Hours

33 Chronic antioxidant administration increased human lung cancer cell invasion H838 H1975 A NAC Trolox Invasion index Ctrl Invasion Index Ctrl Hours Hours 7 days pre-incubation and subsequent invasion assay

34 Is the increased invasiveness and metastasis driven by epigenetic alterations? ChIP-seq medip-seq RNA-seq 1. Antioxidants de-methylated the promoter of a lesser-known chemokine and upregulate its expression by 2-fold 2. Antioxidants increased the transcription of a gene-package controlled by a redox-sensitive transcription factor

35 Defining the role of proteins-of-interest in antioxidant-induced metastasis Kras2G12D/+ Kras2LSL/+ Lentivirus-CreCas9-gRNA Tumor growth occurs in the absence of gene-of-interest

36 Summary A range of different antioxidants accelerate lung cancer and malignant melanoma metastasis, and increase invasiveness of cultured tumor cells Kinetics and mechanisms differ Tumor cells produce ROS that limit their ability to proliferate and metastasize Antioxidants supplementation helps them overcome those limitations and thereby stimulate tumor progression Martin Bergö

37 Recent findings by other groups Nature Oxidative stress inhibits distant metastasis by human melanoma cells S. Morrisson J. Natl. Cancer Inst. Reactivation of p53 by a Cytoskeletal Sensor to Control the Balance Between DNA Damage and Tumor Dissemination V. Sanz-Moreno Science Transl. Med. NRF2 activation by antioxidant antidiabetic agents accelerates tumor metastasis H. Zheng, H. Wang

38 Recommendation Cancer patients and survivors, and people with increased risk of cancer should avoid antioxidant supplements No evidence of beneficial effects Strong evidence of harmful effects Martin Bergö

39 Simplest intepretation of data ROS is harmful in healthy cells and tumor cells Antioxidants protect healthy cells and tumor cells from ROS This reasoning could explain the varying outcomes of clinical trials Martin Bergö

40 Potential explanation for the inconsistent results of clinical trials Linxian General Population Nutrition Intervention Trial. J. Natl. Cancer Inst Alpha-Tocopherol/Beta-Carotene Cancer Prevention Study (ATBC) N. Engl. J. Med Carotene and Retinol Efficacy Trial (CARET) Cancer Res Physicians Health Study I (PHS I) N. Engl. J. Med Women s Health Study. J. Natl. Cancer Inst EUROSCAN study. J. Natl. Cancer Inst Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) Archives Int. Med Heart Outcomes Prevention Evaluation The Ongoing Outcomes (HOPE-TOO) Study J. Am. Med. Assoc Physicians Health Study II (PHS II) J. Am. Med. Assoc Selenium and Vitamin E Cancer Prevention Trial (SELECT) J. Am. Med. Assoc What The trials clinical included trial would from show the start, that varying antioxidants degrees protect of tumor-free against cancer? people Potentially and one people where with people undiagnosed are cancer-free tumors from start!

41 Ongoing: questions to answer 1. Is there a specific form of ROS that limit metastasis? (Reduction of which ROS mediates the antioxidant effect?) 2. Would the marked sensitivity to changes in ROS expose any therapeutic opportunities? Martin Bergö

42 Probes: NAC and Trolox reduce basal and pro-oxidant-stimulated cytosolic oxidation

43 Antioxidant pretreatment might sensitize tumor cells (not healthy cells) to cytotoxic drugs medip-seq

) Tony Zou (PhD student) Dr. Xiufeng Xiu (senior scientist) Dr. Kashif Muhammad (postdoc) Dr. Xue Chen (PhD student) Dr.

44 Group members Mohamed Ibrahim (postdoc) Dr. Martin Dalin (MD, postdoc) Murali Akula (PhD student) Dr. Christin Karlsson (postdoc) Dr. Jaroslaw Cisowski (postdoc) Dr. Ella Äng (MD/PhD student) Emil Ivarsson (PhD student) Oskar Persson (animal tech.) Tony Zou (PhD student) Dr. Xiufeng Xiu (senior scientist) Dr. Kashif Muhammad (postdoc) Dr. Xue Chen (PhD student) Dr. Haidong Yao (postdoc) Elin Tüksammel (animal tech.) Volkan Sayin Kristell le Gal Clotilde Wiel Per Lindahl Jonas Nilsson Mike Murphy Tobias Dick Göran Gustafssonstiftelsen

45 MitoQ administration did not affect lung cancer and melanoma progression Kras2 LSL/+ Braf CA/+ Pten fl/fl Tyr-Cre

46 MitoQ administration did not appear to affect lung cancer invasiveness Ctrl dtpp MitoQ Ctrl MitoQ dtpp

47 Reactive oxygen species in cancer O 2 OH H 2 O 2 Pro-tumorigenic ROS signaling Anti-tumorigenic ROS DNA damage Tumor initiation Tumor progression

48 Tumor cells from NAC- and vitamin E treated mice exhibit reduced expression of endogenous antioxidants: expression is normalized over 15 passages Fold regulation ctrl NAC <15 NAC >15

49 Simplest intepretation of data ROS is harmful in healthy cells and tumor cells Antioxidants protect healthy cells and tumor cells from ROS Progression Antioxidants O 2 O 2 ROS CANCER Martin Bergö

50 People in blue zones, who eat antioxidant-rich diets, live long and have low incidence of cancer They ate the diet throughout life

51 Unanswered questions NAC, vitamin E, trolox, beta-carotene produce same phenotypic effects but what are they doing in the tumor cells? Which form(s) of free radicals limit tumor cell proliferation and metastasis? Which enzymes are involved? Similar effect in lung, melanoma, and liver; what about professional ROS-producing cells? Do antioxidants activate RHOA by deglutathionylation? Impact of mitochondria-targeted compounds? Martin Bergö

Group members Kristell Le Gal (PhD student) Clotilde Wiel (postdoc) Volkan Sayin (postdoc)

Christin Karlsson (postdoc) Dr. Jaroslaw Cisowski (postdoc) Dr.

Zou (PhD student) Dr. Xiufeng Xiu (senior scientist) Dr. Kashif Muhammad (postdoc) Dr.

52 Group members Kristell Le Gal (PhD student) Clotilde Wiel (postdoc) Volkan Sayin (postdoc) Mohamed Ibrahim (postdoc) Dr. Martin Dalin (MD, postdoc) Murali Akula (PhD student) Dr. Christin Karlsson (postdoc) Dr. Jaroslaw Cisowski (postdoc) Dr. Ella Äng (MD/PhD student) Emil Ivarsson (PhD student) Oskar Persson (animal technician) Tony Zou (PhD student) Dr. Xiufeng Xiu (senior scientist) Dr. Kashif Muhammad (postdoc) Dr. Xue Chen (PhD student) Dr. Haidong Yao (postdoc) Elin Tüksammel (animal tech) Prof. Per Lindahl Prof. Jonas Nilsson Göran Gustafssonstiftelsen

Mouse Models of K-RAS- and B-RAFinduced

Mouse Models of K-RAS- and B-RAFinduced Cancer Martin O. Bergö, Professor Sahlgrenska Cancer Center University of Gothenburg www.gu.se Why models? Why mice? Critical to understanding pathogenesis and identifying