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Tamoxifen resistance has remained a major setback in an otherwise successful treatment of estrogen receptor positive(ER+) breast cancer. Most breast cancers are estrogen-receptor positive (ER+). Approximately 70% of patients with ER+ cancer will respond to anti-estrogen therapy, such as tamoxifen . In addition, a significant number of tumors that initially
respond to tamoxifen treatment often progress into a resistant phenotype over time, in spite of sustained expression of ERÎ± [2, 3].
Over the years, several pathways and postulates have been proposed and examined as possible mechanisms of breast cancer resistance to tamoxifen. While some of these have translated into beneficial therapies to a large number of patients, tamoxifen resistance still remains a significant problem. In addition, though studies have examined the roles of ERÎ± and Î², and their co-regulatory proteins; Growth factor receptors and the activity of their downstream kinases; Cas/c-Src/BCAR3 and cell cycle regulators like cyclins D/E and p27, there are currently no studies investigating the effect of ROS on p27 in tamoxifen resistance.
Cancers have been shown to be under persistent oxidative stress . Oxidative stress which is triggered by an imbalance between the production and detoxification of ROS  has been implicated in the therapeutic resistance to tamoxifen . Tamoxifen, a selective estrogen receptor modulator (SERM) has been reported to possess both pro- and anti-oxidant properties. Among its antioxidant actions are: its cardio-protective effect in its prevention of atherosclerosis [7, 8]; its inhibition of lipid peroxidation  and its ability to protect human low density lipoproteins (LDL) against copper ion-mediated oxidative damage . Conversely its pro-oxidant effects include its ability to induce oxidative liver damage  and its explication as a hepato-carcinogen in rodents due to ROS over-production that occurs during its metabolism [12, 13]. Additionally, it has been suggested that oxidative stress might trigger the pathogenesis of tamoxifen-induced toxicity . Though it has not been ascertained whether the elevated oxidative state of tamoxifen resistant breast cancer is due to prolong tamoxifen exposure, the possibility that the pro-oxidant property of prolong tamoxifen exposure may play a role in breast cancer resistance cannot be ignored.
Reactive oxygen species consist of a number of partially reduced metabolites of oxygen such as superoxide anions (O2âˆ’ â€¢), hydrogen peroxide (H2O2), and hydroxyl radicals (OHÂ·), characterized by higher reactivity than molecular oxygen. These reactive species are known to play a vital role in cell growth mediated by 17Î² estradiol (E2) . Oxidative stress has been shown to instigate a number of varied responses including cell proliferation and transformation. Furthermore, an excessive and/or sustained increase in ROS production has been implicated in the pathogenesis of cancer . Redox balance is achieved by various enzymatic and non enzymatic antioxidant systems that counteract the harmful effect of ROS. For example Superoxide dismutase (SOD) catalyses the dismutation of the superoxide anion into H2O2, while Catalase and glutathione peroxidase (GPx) metabolize H2O2 into water and molecular oxygen [17, 18]. It is interesting to note that physiological ROS play a significant role in the regulation of signaling pathways such as kinase and phosphatase activation/inactivation.
Oxidative stress induced redox surges could bring about changes in the thiol status of signaling proteins with ensuing alterations in their Phosphorylation/dephosphorylations functions. These modifications could lead to changes in cellular signaling transduction pathways, DNA and RNA syntheses, protein syntheses, enzyme activation and cell cycle regulations [19-27]. Signaling proteins such as mitogen-activated protein kinases (MAPK) and protein tyrosine phosphatases (PTPs) like PTEN and CDC25 have been shown to be susceptible to oxidation as they contain essential cysteines which serve as possible targets for ROS in different pathways [30, 31].
Protein tyrosine phosphatases (PTPs) are important enzymes in cell cycle control and signal transduction. They act in conjunction with protein tyrosine kinases (PTKs) to regulate levels of protein tyrosine phosphorylation in response to cellular signals [32, 33]. Many studies have shown that PTKs may be directly activated through the inhibition of PTPs by ROS [34, 35]. For example PTEN is a redox sensitive phosphatase which has been shown to be inactivated by ROS. The inactivation of PTEN leads to the sustained phosporylation and activation of AKT. AKT has been shown to phosphorylate p27 on threonine 157 with a resultant cytoplasmic sequestration and a consequent inhibition of its nuclear function.
p27 is a cyclin-kinase inhibitor (CKI) which was originally discovered as a protein whose expression was induced by different growth inhibitory conditions/agents such as serum starvation, contact inhibition, transforming growth factor-Î± (TGF-Î±), or by lovastatin . Its expression is reduced by mitogens like epidermal growth factors and Estrogens. Mice with p27 knockout develop multiorgan hyperplasia and pituitary tumors, thus underscoring a role for p27 in both proliferation and differentiation . p27 is also well-known for its ability to inhibit G1 cyclin/CDK complexes. It is noteworthy that its activity is regulated by its post- translational modification [38, 39]. Hence one may infer that because of their capacity to oxidize and inactivate PTEN, ROS may be able to regulate p27 function by modulating its post-translational modification.
We therefore hypothesized that (i) In addition to its known action at the ER, tamoxifen also prevents estrogen-mediated progression of cell cycle by counteracting estrogen-induced reactive oxygen species signaling (ii) As a result of chronic oxidative stress, the conversion of estrogen-sensitive breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment. Therefore, an increase in ROS levels promotes the loss of p27 inhibitory function through the inactivation of protein tyrosine phosphates (PTPs) and a consequent change in p27 phosphorylation.
To test these hypotheses, we conducted experiments to i) Determine the relationship between tamoxifen and oxidative stress ii) Determine the effect of ROS on p27 expression and its phoshorylation in tamoxifen resistant LCC2 cells iii) Determine if pre-treatment of LCC2 cells with biological or chemical antioxidants can restore tamoxifen sensitivity by re-establishing redox balance within the cells and iv) Determine the effect of antioxidants on p27 stability and its inhibitory function.
In this study, we hypothesized that, (i) In addition to its known action at the ER, Tamoxifen also prevents estrogen-mediated progression of cell cycle by counteracting estrogen-induced reactive oxygen species signaling, and (ii) As a result of chronic oxidative stress, the conversion of estrogen-sensitive breast tumors to a Tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment. Therefore, an increase in ROS levels promotes the loss of p27 inhibitory function through the inactivation of protein tyrosine phosphates (PTPs) and a consequent change in p27 phosphorylation.
Tamoxifen, a known selective estrogen receptor modulator (SERM) has been widely reported to possess either pro- or anti-oxidant properties [7-13]. Our results show that Tamoxifen possesses the capacity to act as both an oxidant and an anti-oxidant. Exposure of LCC2 cells to TAM or E2 induced the formation of reactive oxidants (Fig.1A). These reactive oxidants were then inhibited by the co-treatment of E2 with TAM, much like the ROS inhibition observed in cells pre-treated with antioxidants (Figs.1B & C). We were able to further demonstrate that E2 and TAM induced ROS were capable of inducing cell proliferation which was then inhibited by pre-exposure of cell to biological or chemical antioxidant or the co-treatment of TAM with E2 (see Figs.2 & 3). These findings make a case for the dual role of Tamoxifen as both a pro- and an anti-oxidant. Based on these results, it appears that the antioxidant effect of Tamoxifen is observed in the presence of Estrogen.
Exposure of Tamoxifen resistant LCC2 cells to Tamoxifen resulted in a decrease in p27 expression in contrast to the increase in p27 expression observed when the same cells were treated with Fulvestrant (see Fig.4A). Fulvestrant is an anti-estrogen to which LCC2 cells are known to be sensitive. We also observed that the exposure of these cells to Tamoxifen increased p27 phosphorylation on T157 and T187 while exposure to Fulvestrant appeared to have the opposite effect (Fig5. Panels 1& IIA).
Conversely, following anti-oxidant pre-treatment/over expression, a Fulvestrant-like effect i.e. an increase in p27 expression (see Figs. 4B &C) and a decrease in p27 phosphorylation on T157 and T187 was observed in Tamoxifen treated cells compared to cells which were not pre-exposed to antioxidants (Figs. 5 Panels I & II B & C). It can thus be inferred that the over expression of antioxidant in LCC2 cells leads to an increase in p27 expression and a decrease in its phosphorylation on T157 and T187. This is in line with other previous studies [40 - 42], which showed that treatment with anti-estrogen drugs like Tamoxifen or Fulvestrant caused cell cycle arrest, with up-regulation of p21 and p27 levels, an increase in their binding to cyclin E-Cdk2, and kinase inhibition . Additionally, results from our comparison of p27 expression between Tamoxifen sensitive parental MCF7 and Tamoxifen resistant LCC2 cells showed a 40% decrease in p27 expression in LCC2 cells compared to MCF7 (Fig.11). This implies that the problem of Tamoxifen resistance could be associated with a decrease in p27 expression rather than its loss function. In which case, an increase in p27 expression following antioxidant pre-treatment would be a very significant finding.
Though the observed experimental outcomes imitate the effect of Fulvestrant on LCC2 cells, we cannot interpret this to denote either an increase in Tamoxifen sensitivity or the growth inhibitory function of p27. This is because previous studies have demonstrated the propensity for p27 to have a dual role in tumor - suppression and -promotion [43-46] depending on its subcellular redistribution and which cyclin /Cdk complex it is bound to. The binding of p27 to the cyclin E/Cdk2 complex in the nucleus promotes its function as a CKI while cytoplasmic sequestration takes it away from its nuclear cyclin/Cdk target to the growth promoting interaction with cyclin D/Cdk4/6 complex .
In line with previous studies , we also found an increase in the binding of cyclin E-Cdk2, and p27 in Tamoxifen treated cells over expressing anti-oxidants compared to cells not exposed to antioxidants (see Figs. 8, 9 & 10). This outcome, in addition to our finding of increased p27 stability in cells over expressing anti-oxidants (Fig. 12), when taken in context with our intial finding of increased p27 expression and decreased phosphorylation, are highly suggestive of an increase in the Tamoxifen sensitivity and p27 inhibitory function of LCC2 cells over expressing anti-oxidants.
In summary, we have been able to demonstrate that Tamoxifen has the capacity to induce ROS formation and act as an anti-oxidant in the presence of E2. Following prolonged exposure to Tamoxifen and an increasingly pro-oxidant environment, the oxidants formed are able to promote cell proliferation. This could be by decreasing p27 expression, and regulation of its activity by post-translational modification involving an increase in p27 phosphorylation on T157 and T187. This is accompanied by a resultant cytoplasmic sequestration, decreased binding to cyclin E/Cdk2 complex and loss of p27 stability resulting in loss of sensitivity to Tamoxifen. However, because these events are oxidant driven they can be reversed by over expression or anti-oxidant pre-treatment with a resultant growth inhibitory effect and increased Tamoxifen sensitivity.