Defining the roles of antioxidants and a chemokine ligand in cancer

<p dir="ltr">Reactive oxygen species (ROS) are involved in many cellular processes, from modulating intracellular signaling to mediating immune responses. Antioxidants neutralize ROS and maintain intracellular redox balance, because ROS in high concentrations can induce oxidative stress. While ROS can contribute to cancer development, oxidative stress has also been shown to limit tumor progression and metastasis. Previously, it has been discovered that antioxidant supplementation with N-acetylcysteine (NAC) and vitamin E accelerates tumor growth and metastasis in lung cancer mouse models. Similar effects have been observed in mice with melanoma, where NAC accelerated metastasis to the lymph nodes. Yet, it is unclear whether other compounds share the pro-metastatic properties of NAC and vitamin E, and whether antioxidants affect tumor angiogenesis— another critical characteristic of tumor progression.</p><p dir="ltr">Chemokines are another group of essential signaling molecules that orchestrate cell migration and mediate immune responses. Among those, C-C motif chemokine ligand 28 (CCL28) is expressed in mucosal tissues and has been found to be involved in tumor-associated processes, such as angiogenesis and immune evasion. However, little is known about its functional significance in melanoma and lung tumor progression.</p><p dir="ltr">The aim of this thesis is to investigate antioxidant effects and underlying molecular mechanisms in melanoma and lung tumor progression, and to explore the role of the chemokine CCL28 in both cancer types.</p><p dir="ltr">In <b>Study I</b>, we screened 104 redox-modulating compounds and identified 18 that concurrently reduced ROS levels and increased melanoma cell migration. Four compounds typically found in foods and dietary supplements were further tested in vivo: vitamin C, b-carotene, canthaxanthin, and retinyl palmitate. Supplementing the diet with these compounds increased the numbers of metastatic lymph nodes in a BRAF-driven melanoma mouse model. Transcriptomic profiling of human melanoma cells incubated with the four antioxidants indicated that BTB and CNC homolog 1 (BACH1) is involved in mediating antioxidant-induced effects in melanoma. In addition, <i>Bach1</i> knockout reduced experimental melanoma metastasis to the liver and lymph nodes in mice.</p><p dir="ltr">In <b>Study II</b>, we investigated the effects of antioxidants on lung tumor angiogenesis in 3D lung tumor cell and xenograft models. Vitamin C, vitamin E and NAC increased the expression of the transcription factor BACH1, which regulated angiogenesis gene expression. In addition, antioxidants induced hypoxia- inducible factor 1-alpha (HIF1a) expression, and antioxidant-induced BACH1 upregulation was found to be dependent on HIF1a under normoxia. However, the BACH1-mediated upregulation of angiogenesis genes was not dependent on HIF1a. BACH1 expression also increased under hypoxia and prolyl hydroxylase (PHD) inhibitors stabilized BACH1 protein levels in both HIF1a-expressing and <i>HIF1A</i>- knockout cells. In mice, antioxidant supplementation of the diet increased tumor vascularity in BACH1-expressing but not BACH1-deficient lung tumors. Moreover, BACH1 expression correlated with a wide range of angiogenesis genes, and both vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 2 (VEGFR2) in lung tumors from patients. In line with this, high BACH1 expression sensitized xenograft lung tumors to a VEGFR2-blocking agent.</p><p dir="ltr">In <b>Study III</b>, we explored the functional role of CCL28 in melanoma and lung cancer. Doxycycline-inducible CCL28 knockdown in human melanoma and lung cancer cells reduced colony formation and proliferation, while increasing apoptosis in a tumor cell-autonomous fashion. In immunodeficient mice, doxycycline-inducible CCL28 knockdown reduced subcutaneous tumor growth and inhibited experimental metastasis. Incubating melanoma and lung cancer cells with a recombinant CCL28 did not restore colony formation. In immunocytochemistry and cell fractionation experiments, CCL28 predominately localized to the cell nucleus. It was also enriched in DNA-binding protein fractions from melanoma and lung cancer cells. In addition, CCL28 expression was elevated in melanoma and lung tumors from patients compared to normal tissues, and high CCL28 expression was associated with poor overall survival in melanoma. In contrast to observations in human melanoma and lung cancer cells, <i>Ccl28</i> expression was nearly undetectable in lung tumor cells from mice with KRAS-driven lung cancer, and its loss did not affect survival in these mice.</p><p dir="ltr">In conclusion, these findings provide novel insights into antioxidant effects in tumor progression and metastasis, and the role of the chemokine ligand CCL28 in melanoma and lung cancer.</p><h3>List of scientific papers</h3><p dir="ltr">I. ROS-lowering doses of vitamin C and A accelerate malignant melanoma metastasis. Muhammad Kashif, Haidong Yao, <b>Sarah Schmidt</b>, Xue Chen, Michelle Truong, Elin Tüksammel, Yiran Liu, Martin O. Bergo (2023). Redox Biology, 60, 102619. <a href="https://doi.org/10.1016/j.redox.2023.102619">https://doi.org/10.1016/j.redox.2023.102619</a></p><p dir="ltr">II. Antioxidants stimulate BACH1-dependent tumor angiogenesis. Ting Wang, Yongqiang Dong, Zhiqiang Huang, Guoqing Zhang, Ying Zhao, Haidong Yao, Jianjiang Hu, Elin Tüksammel, Huan Cai, Ning Liang, Xiufeng Xu, Xijie Yang, <b>Sarah Schmidt</b>, Xi Qiao, Susanne Schlisio, Staffan Strömblad, Hong Qian, Changtao Jiang, Eckardt Treuter, and Martin O. Bergo (2023). The Journal of Clinical Investigation, 133(20), e169671. <a href="https://doi.org/10.1172/JCI169671">https://doi.org/10.1172/JCI169671</a></p><p dir="ltr">III. Nuclear CCL28 sustains tumor cell survival and metastasis in melanoma and lung cancer. <b>Sarah Schmidt</b>, Nina Spagone-Wolf, Mirja Simon, Jan Schlegel, Xiufeng Xu, Xi Qiao^#, and Martin O. Bergo^#. [Manuscript]</p><p dir="ltr"># Co-corresponding authors</p>