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Defining the Role of Antioxidants on Cancer progression and metastasis

Periodic Reporting for period 1 - RAC (Defining the Role of Antioxidants on Cancer progression and metastasis)

Reporting period: 2016-03-01 to 2018-02-28

Despite the striking discordance between the use of antioxidants and the lack of experimental support for their anti-cancer properties, no studies had examined their impact exclusively on the metastatic process of tumours in state of the art mouse models of cancer. In this project, I am approaching this issue using mouse models of lung cancer, a metastatic cancer that continues to be the cause of most cancer-related deaths worldwide.

Discoveries made in the project has a good chance of enhancing European excellence and competiveness in terms of public health. Indeed, the results I obtained during my postdoc will increase our understanding of the relationship between the use of antioxidant supplements and cancer progression. Antioxidant supplements fuels a billion dollar industry, and the belief that antioxidants will keep people cancer-free is wide-spread and deeply rooted in the general population. We know that the situation is not that simple. The project is addressing questions that are of public interest, as most people during his/her life will at one point or another have ingested an antioxidant supplement.
Well-being, health, and demographic changes are major societal challenges in Europe that are prioritized in Horizon 2020. Importantly, my results have a long-term impact and will likely be of high interest for translational research and clinicians. As the overall European population is growing older, it will become necessary to reduce age-related diseases, including cancer, for improving the well-being of people, in view of reducing spending in care systems. Economically speaking, European care systems will not be sustainable in the next decades. My research project show that antioxidant supplementation stimulates metastases of benign lung tumors. These findings could contribute significantly to the collective European effort of reducing health-related spending and to achieve a long-term sustainability of European finances.

The overall aim of the project is to answer this question: Can antioxidants increase metastasis of benign lung tumors?
During the reporting period I have focused my work on WP1, according to the gant chart, whose aim is to define the impact of antioxidant supplementation on lung cancer progression and metastasis. We hypothesized that antioxidant supplementation could stimulate or accelerate metastasis.
To answer our hypothesis we used 2 mouse models of lung cancer: one induced by KRASG12D alone where primary tumours do not/rarely metastasize, and another model where p53 expression is lost when KRASG12D is activated (KP mouse model). 1 week after tumor initiation, mice were fed with antioxidants in the drinking water (NAC) or in the chow pellet (VitaminE). Mice have been monitored over time, survival analysed, and organs harvested when mice reached the humane endpoint. During dissection, organs were thoroughly examined in order to detect macroscopic metastases. Organs have been fixed in formalin or snap-frozen. In some cases, small lung tumor pieces were collected in order to establish cell lines. Stainings and immunohistochemical analyses have been performed to score tumor burden, stage and metastasis. This in vivo work has been complemented with in vitro experiments on human lung cancer cells lines, and in-house cell lines established from the lung primary tumors. Long term exposition to antioxidants increase cells migration and invasion abilities. RNAseq data along with epigenomic analyses are currently being analysed in order to identify potential reprogramming induced by antioxidants that could explain this phenotype.
To determine whether antioxidants influence lung cancer metastasis, we administered NAC and vitE to Kras2LSL/+ mice that had previously received a low dose of Cre-adenovirus in their lungs (to induce only a few tumors) and followed them for one year. Tumors in Kras2LSL/+ mice rarely metastasize to lymph nodes, and never to distant sites unless Trp53 or another tumor suppressor gene is inactivated. Under these experimental conditions, NAC and vitE markedly increased lymph node metastasis and even triggered distant metastasis.
In a second metastasis-prone Kras2LSL/+Trp53fl/fl model, the antioxidants further increased lymph node metastasis and also induced metastasis to the inner wall of the rib cage—a site where we rarely observe metastasis. Thus, antioxidants increased lung cancer metastasis to both common and unique sites and of both Trp53-proficient and -deficient starting conditions. Although the antioxidants clearly reduce ROS levels in the tumor cells, we don’t yet know which type of ROS they reduce and where in the cell these ROS operate to inhibit tumor metastasis. Understanding cell intrinsic and dietary factors that regulate metastasis is important because it is metastases, rarely the primary tumor, that kill cancer patients.

Few scientific fields are fraught with more controversy than that of the role of reactive oxygen species (ROS) in cancer. This field has spawned a wide-spread dogma: Because ROS induces DNA damage and pro-tumorigenic signaling that can contribute to cancer, antioxidants, which neutralize ROS, must protect against cancer. But clinical trials with antioxidants do not support this dogma, and some even showed that antioxidants increase cancer risks. However, those trials have done little to change the belief that antioxidant supplements protect against cancer—a belief that fuels a billion-dollar industry that markets its products to healthy people and cancer patients alike.
Results from our group shed light on this issue. We discovered that supplementing the diet with pharmacological and dietary antioxidants such as N-acetylcysteine (NAC), vitamin E (vitE), and beta-carotene accelerates lung cancer progression in mice. We also found that antioxidants markedly accelerate metastasis in mouse models of malignant melanoma and lung cancer (this project). These results, which gain support from numerous subsequent studies, sparked a world-wide discussion about the potential dangers of antioxidant supplementation for cancer patients, and provide a plausible explanation for the failed clinical trials with these compounds.
The key to these results is that we focused exclusively on antioxidant effects on tumor progression, not initiation. The results suggest that oxidative stress potently suppresses tumor progression and metastasis; which explains why antioxidants—by eliminating oxidative stress—accelerate those processes. But interestingly, the underlying mechanisms seem to be different in melanoma and lung cancer cells: Antioxidants acutely increased invasion of mouse and human melanoma cells indicating that posttranslational mechanisms are involved. In contrast, we recently found that antioxidants in vivo cause long-term programming of lung cancer cells into a metastatic phenotype that is preserved for up to 15 passages when the tumor cells are introduced into cell culture. Also, preincubating human lung cancer cells with antioxidants for >7 days, but not acute administration, induces a long-lasting invasive phenotype, suggesting that epigenetic mechanisms are involved.

This project, along with previous results we obtained, markedly increased our understanding of how oxidative stress affects tumor progression and metastasis and will allow us to identify tumor-specific vulnerabilities that can be exploited for new therapies.
Antioxidants increased local and created distant metastasis in mice with KRAS-induced lung cancer