Nrf2 S Interdependent Role in Multiple Forms of Cancer

Leschinsky1

Nrf2’s Interdependent Role in Multiple Forms of Cancer

Nicholas Leschinsky

Dr. Bert Ely

HNRS BIOL 303

1 November 2014

Due to recent advances in the fields of medicine and genetics, uncovering the underlying causes behind diseases hasbecome much easier to accomplish. With these data concerning the sources of mutations, specialized treatments and precautionary measuresare able to be put into place to either lessen or completely eliminate the harmful effects of these conditions. In addition, comparisons have beendrawn between different afflictions based on what factorslie at their sources. The conditions that result in either the facilitation or inhibition of certain genes are alsotraceable back to the presence or absence of certain factors within the cell. Wu et al. (2014) stated that “Nrf2 has become the subject of widespread interest due to its impact on the development and growth of many cancers”. Its role within several different regulatory feedback loops and protein expression pathways makes understanding the impact of Nrf2’s expression an important step toward treating cancer. Nrf2 expression contributes to the progression of many cancers, including liver cancer, lung cancer, breast cancer, and several other types of malignancies (Figure 1). Since this element is so crucial to the development of cancerous diseases, it is essential that we find out as much as possible about the effects of Nrf2 with regards to tumorgenesis and fibrosis.

Nrf2 was found to be a transcription factor within the cells of many different tissue systems. It acted as a regulatory agent within the immune systemand controlled the expression of antioxidants and cytoprotective genes (Thimmulappa et al. 2006). Figure 2 shows many of the different organ systems and genes which Nrf2 affects. Thimmulappa et al. (2006) went on to state that, in normal cases, nuclear levels of Nrf2 were low. This was due in part to the inactivation of Nrf2 through its binding to Kelch-like ECH-associated protein 1, otherwise known as Keap1 (Wu et al. 2014). Keap1 acted as a maintenance factor for the process of redox homeostasis. This binding and subsequent repression of Nrf2 caused both the pro-oncogenic and anti-oncogenic properties of the nuclear factor to be repressed. However, in times of stress or stimuli, Nrf2 has the potential to become both dissociated and up regulated, which has the chance to lead to tumor progression through the Antioxidant Response Element (Du et al. 2013). The way in which the problems surfaced varied in which type of cells were affected.

According to Ni et al. (2014), persistent activation of Nrf2 contributed to the pathogenesis of cell death, inflammation, fibrosis, and eventual liver tumorigenesis. However, the ways in which these symptoms were produced within the liver differed from cases of breast or lung cancer. In the case of liver cancer, Ni et al. (2014) demonstrated that Nrf2 becomes activated when the presence of p62, a receptor protein, increases. The increased presence of p62 caused the binding of Nrf2 to Keap1 to become disrupted, and the p62 began to take the place of Nrf2 within Keap1 (Ni et al. 2014). This allowed the Nrf2 to act independentofits binding complex. Ni et al. (2014) discovered this phenomenon through their comparison of the cultures of the deficient test mice with the wild type mice that did not show any expression of autophagy deficiency. They went on to show that as one factor increased, the other factor also increased. Thus, the interaction between Nrf2 and p62 in liver tissueswas shown to be located within a positive feedback loop (Fujjikawa et al. 2012). In samples that contained this feedback issue, higher levels of caspase activation was linked to the dissociation of Nrf2, and this led to the problems of pathogenesis previously mentioned (Figure 3). Caspase enzymes are enzymes which cut proteins in specific areas, resulting in the creation of smaller protein subunits.The mice that had the deficiency had significantly increased levels of caspase-8, -9, and -3(figure 3a, 3b, and 3c), that lead to compensatory activation of some anti-apoptotic and inflammation factors was observed in response to necrosis and other forms of cell death (Ni et al. 2014). As a result of this cell death, fibrosis factors such as collagen type1, connective tissue growth factor (CTGF), transforming growth factor β1 (TGF-β1), and α-SMA increased to act as a recovery agent within the cell (Ni et al. 2014). The unregulated presence of these factors in conjunction with inflammation led to higher levels of tumorgenesis and fibrosis within the mice livers.

In a similar sense, Wu et al. (2014) demonstrated that an increase in estrogen or E2 expression within a breast cell also acted through a positive feedback loop to increase the induction of Nrf2. However, the way in which Nrf2 was positively induced wasn’t as simple as having a competing binding agent present which allowed the activation of the nuclear factor. Instead, an increased presence of estrogen allowed the estrogen-receptor ERα to stimulate the expression of Nrf2 in these cells (Wu et al. 2014). While ERα did not directlystimulate the levels of Nrf2, it controlled the phosphorylation of different signaling pathways inhibiting the function of kinases that reduced the actions of Nrf2 (Chowdhry et al. 2012 and Ansell et al. 2005). In order to confirm that ERα and not ERβ was essential to E2 mediated increases in Nrf2, Wu et al. (2014) introduced both ERα and ERβ antagonists MMP and PHTPP respectively. However, only ERα and MMP blocked any E2 induced Nrf2 activity. The ERα pathway was only one factor within the breast cells which controlled the expression of Nrf2. Wu et al. (2014) also hypothesized that the activation of PI3K/Akt signaling by estrogen and later on the phosphorylation of GSK3β may have acted in assistance to the increased expression of Nrf2. Donahue et al. (2007) and Franke et al. (1995) established that Akt was a target factor for PI3K and in the instance of breast cells, the expression of Akt was rapidly increased in E2 treated cells (Wu et al. 2014). Akt isthe only mediatory factor in the GSK3β pathways (Das et al 2013 and Franke et al. 1995). Thus the increased expression of Akt by E2 led to the increased phosphorylation of the GSK3β pathways as shown in figure 4, and, in turn, that provided E2 with another pathway to increase Nrf2’s activity(Wu et al. 2014). As a result, Nrf2 was able to promote tumorgenesis through stress protection problems and loss of function mutations (Wu et al. 2014).

Another instance of the effects of Nrf2 in conjunction with the appearance of cancer and tumorgenesis occurred within the feedback loop containing p66shc. P66shc acted within breast cells to restrain Ras from overexpression and it also activated pro-apoptosis when certain oxidative stresses were present (Micliaccio et al. 1997). However, when p66shc was lost or silenced, metastasis of cancer within lung cells became especially aggressive (Ma et al. 2010). Du et al. (2013) stated that Nrf2 played a role in binding to the p66shc promoter and promoted the transcription of p66shc. This promotion can be seen in figure 5a where the normal processes were carried out. In this case, Nrf2 acted not as a competing factor for binding but instead as an inducer for another factor. However, the presence of Keap1 still resulted in the ubiquitination and dissociation of Nrf2. Their research continued on to state that methylation of the binding site of Nrf2 resulted in the silencing of p66shc and the upregulation of Nrf2 (Du et al. 2013). This competition may be seen in figure 5b where Nrf2 is rejected from the binding site. Since p66shc controlled apoptotic processes and Nrf2 controlled cellular drug resistance and the expression of cytoprotective genes, Du et al. (2013) stated thatthe feedback loop between these two factors in conjunction with each other led to the promotion of lung cancer. They were able to reinforce this idea through the induction of an oxidative stress environment by depriving a culture of serum (Du et al. 2013). As a result of this deprivation, Du et al. (2013) showed through chromatin immunoprecipitation that both Nrf2 and p66shc activity levels were both increased, lending to the idea that the two factors were indeed in an interconnected feedback loop. Following that, Du et al. (2014) tested samples of lung cells which expressed p66shc using a NucBuster protein extraction kit and found that out of all the tested cells, the cancer cells contained much higher levels of Nrf2 than surrounding tissues. With the decreased levels of p66shc as seen in figure 6a and 6d, the metastasis of cancer cells was increased and the subsequent increased presence of Nrf2 led to drug resistance and additional cancer progression (Du et al. 2013).

In these completely independent instances of Nrf2 expression, many similarities arosewhich enabled parallels to be drawn between the different genetic pathways. To reiterate, Nrf2 in its normal state acted a protective factor through its role in the Antioxidant Response Element (Du et al. 2013). Oxidative stress was the maincause of the initial problems and since Nrf2 acted as a protective factor against these stressors, it became upregulated. The feedback loops that were coded within the cells acted as another instance through which Nrf2 was expressed. However, additional problems ranged from an increased level of inhibiting proteins or methylation of certain coding or binding areasmay have caused Nrf2 to become overexpressed. These problems allowed Nrf2 to express genes that actually wound up damaging the cell even more. Ni et al. (2014) stated that the absence of certain cellular functions--such as autophagy-- may result in conditions which turned the normally positive effects that Nrf2 expressed into harmful ones. In the cases of lung, liver, and breast cancer, the regulation and supression of Nrf2 in response to these conditions decreased the drug-resistance of the cancerous cell (Du et al. 2013). Furthermore, regulation of Nrf2 might even reverse the determental effects of these conditions (Ni et al. 2014). With this information, the hope is that issues can be discovered and treated before their expression becomes an issue within the patient.

The genetic pathways found while researching the effets of Nrf2 hold many opportunities for the possible treatment of both existing and new conditions. The ability to attribute certain symptoms to specific factors—such as Nrf2—gives us insight into what role these factors may have in promoting, facilitating, and advancing disease. With the knowledge that we currently have, scientists and doctors may soon be able to pinpoint the exact location of problems or diseases within the cell and take the correct precautions to minimize damge. The discoveies of factors like Nrf2 are just stepping stones on the path towards curing the abomination known as cancer.

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