Caroline M. Woolston

Redox Environment, Free Radical, and Oxidative DNA Damage

SIGNIFICANCE Effective redox homeostasis is critical, and disruption of this process can have important cellular consequences. An array of systems protect the cell from potentially damaging reactive oxygen species (ROS), however if these systems are overwhelmed, for example, in aberrantly functioning cells, ROS can have a number of detrimental consequences, including DNA damage. Oxidative DNA damage can be repaired by a number of DNA repair pathways, such as base excision repair (BER). RECENT ADVANCES The role of ROS in oxidative DNA damage is well established, however, there is an emerging role for ROS and the redox environment in modulating the efficiency of DNA repair pathways targeting oxidative DNA lesions. CRITICAL ISSUES Oxidative DNA damage and modulation of DNA damage and repair by the redox environment are implicated in a number of diseases. Understanding how the redox environment plays such a critical role in DNA damage and repair will allow us to further understand the far reaching cellular consequence of ROS. FUTURE DIRECTIONS In this review, we discuss the detrimental effects of ROS, oxidative DNA damage repair, and the redox systems that exist to control redox homeostasis. We also describe how DNA pathways can be modulated by the redox environment and how the redox environment and oxidative DNA damage plays a role in disease.

Calpain system protein expression in basal-like and triple-negative invasive breast cancer

BACKGROUND Basal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required. MATERIALS AND METHODS We investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients. RESULTS The calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P=0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease. CONCLUSIONS Expression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.Background Basal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required. Materials and methods We investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients. Results The calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P = 0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease. Conclusions Expression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.

Expression of thioredoxin system and related peroxiredoxin proteins is associated with clinical outcome in radiotherapy treated early stage breast cancer.

BACKGROUND AND PURPOSE Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Expression of the thioredoxin system proteins (thioredoxin, thioredoxin reductase and thioredoxin interacting protein) and downstream peroxiredoxins (I-VI), was examined in tumor specimens from early stage breast cancer patients, subsequently treated by breast conserving surgery and locoregional radiotherapy, to determine if redox protein expression is associated with clinical outcome. MATERIAL AND METHODS Nuclear and cytoplasmic expression was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. RESULTS High expression of cytoplasmic peroxiredoxin-I correlated with a greater risk of local recurrence (p=0.009). When nuclear and cytoplasmic expression patterns were combined, patients with low nuclear but high cytoplasmic expression of peroxiredoxin-I increased significance (p=0.005). Both were independent factors (p=0.006 and 0.003) from multivariate analysis. Associations were obtained between tumor grade and nuclear thioredoxin interacting protein (p=0.01) and with cytoplasmic expression of peroxiredoxin-V (p=0.007) but not with peroxiredoxin-I suggesting that the latter may exert influence via regulation of oxidative stress rather than via altering the tumor phenotype. CONCLUSIONS Results highlight the potential of using redox protein expression, namely peroxiredoxin-I, to predict clinical outcome and support further studies to validate its usefulness as an independent prognostic, and potentially predictive, marker.

Calpain-1 expression is associated with relapse-free survival in breast cancer patients treated with trastuzumab following adjuvant chemotherapy.

The calpain family, and their endogenous inhibitor calpastatin, has been implicated in cancer progression, and recent in vitro data have indicated a role in trastuzumab resistance. The aims of our study were to examine expression levels of calpastatin, calpain‐1 and calpain‐2 in breast tumours from patients treated with trastuzumab following adjuvant chemotherapy to determine their potential as biomarkers to predict therapeutic response. The expression of calpastatin, calpain‐1 and calpain‐2 was determined, using immunohistochemistry (IHC), in tumours from a series of 93 patients with primary breast cancer treated with surgery and adjuvant chemotherapy with or without trastuzumab followed by trastuzumab to complete 1 year of therapy. IHC was performed using tissue microarrays constructed from cores taken from intratumour and peripheral tumour areas. Expression was correlated with clinicopathologic variables and patient outcome. Calpastatin expression was correlated with Nottingham prognostic index (p = 0.003) and lymph node status (p = 0.007). Trastuzumab resistance was defined as disease relapse during therapy. Calpain‐1 expression is associated with relapse‐free survival (p = 0.001) and remained significant in multivariate analysis accounting for confounding pathological and treatment variables (hazard ratio 4.60, 95% confidence interval 1.05–20.25; p = 0.043). Calpain‐1 may be a useful biomarker to predict relapse‐free survival in breast cancer patients treated with adjuvant trastuzumab and chemotherapy. A larger verification study is warranted.

Redox protein expression predicts progression-free and overall survival in ovarian cancer patients treated with platinum-based chemotherapy

Ovarian cancer is primarily treated with platinum-based chemotherapy, with ROS generation implicated in cytotoxicity. We examined redox protein expression in ovarian tumors, focusing on the thioredoxin system, to determine the role it might play in mediating response to therapy. Nuclear and cytoplasmic expression of thioredoxin, thioredoxin reductase, thioredoxin-interacting protein, metallothionein, and glutathione S-transferase Pi was assessed, using standard immunohistochemical techniques, on a tissue microarray of 154 primary ovarian carcinomas obtained from patients subsequently treated with adjuvant platinum-based chemotherapy. Low cytoplasmic expression of thioredoxin (p=0.032) and negative nuclear expression of metallothionein (p=0.04) significantly correlated with better progression-free survival. When nuclear and cytoplasmic expression patterns were combined those patients with tumors with low cytoplasmic but high nuclear expression of thioredoxin exhibited better progression-free (p=0.003) and overall survival (p=0.004). This combination was, using multivariate analysis, an independent predictive factor for overall survival (p=0.034). Improved progression-free survival was also seen with negative expression of metallothionein, cytoplasmic and nuclear (p=0.038), and was independent of other clinical parameters (p=0.048). Such results support the suitability of using redox protein expression to predict response and, potentially, to alter treatment options accordingly.

Calpastatin is associated with lymphovascular invasion in breast cancer

Metastasis of breast cancer is a major contributor to mortality. Histological assessment of vascular invasion (VI) provides important prognostic information and demonstrates that VI occurs predominantly via lymphatics in breast cancer. We sought to examine genes and proteins involved in lymphovascular invasion (LVI) to understand the mechanisms of this key disease process. A gene expression array of 91 breast cancer patients was analysed by an Artificial Neural Network (ANN) approach using LVI to supervise the analysis. 89 transcripts were significantly associated (p<0.001) with the presence of LVI. Calpastatin, a specific calpain inhibitor, had the second lowest selection error and was investigated in breast cancer specimens using real-time PCR (n=56) and immunohistochemistry (n=53). Both calpastatin mRNA and protein levels were significantly associated with the presence of LVI (p=0.014 and p=0.025 respectively). The data supports the hypothesis that calpastatin may play a role in regulating the initial metastatic dissemination of breast cancer.

REDOX PROTEIN EXPRESSION PREDICTS RADIOTHERAPEUTIC RESPONSE IN EARLY-STAGE INVASIVE BREAST CANCER PATIENTS

PURPOSE Early-stage invasive breast cancer patients have commonly undergone breast-conserving surgery and radiotherapy. In a large majority of these patients, the treatment is effective; however, a proportion will develop local recurrence. Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Therefore, the expression of redox proteins was examined in tumor specimens from this defined cohort to determine whether such expression could predict response. METHODS AND MATERIALS The nuclear and cytoplasmic expression of nine redox proteins (glutathione, glutathione reductase, glutaredoxin, glutathione peroxidase 1, 3, and 4, and glutathione S-transferase-θ, -π, and -α) was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. RESULTS A high cytoplasmic expression of glutathione S-transferase-θ significantly correlated with a greater risk of local recurrence (p = .008) and, when combined with a low nuclear expression (p = .009), became an independent predictive factor (p = .002) for local recurrence. High cytoplasmic expression of glutathione S-transferase-θ also correlated with a worse overall survival (p = .009). Low nuclear and cytoplasmic expression of glutathione peroxidase 3 (p = .002) correlated with a greater risk of local recurrence and was an independent predictive factor (p = .005). These proteins did not correlate with tumor grade, suggesting their function might be specific to the regulation of oxidative stress rather than alterations of tumor phenotype. Only nuclear (p = .005) and cytoplasmic (p = .001) expression of glutathione peroxidase 4 correlated with the tumor grade. CONCLUSIONS Our results support the use of redox protein expression, namely glutathione S-transferase-θ and glutathione peroxidase 3, to predict the response to radiotherapy in early-stage breast cancer patients. If incorporated into routine diagnostic tests, they have the potential to aid clinicians in their stratification of patients into more tailored treatment regimens. Future targeted therapies to these systems might improve the efficacy of reactive oxygen species-inducing therapies, such as radiotherapy.

The prognostic and predictive power of redox protein expression for anthracycline-based chemotherapy response in locally advanced breast cancer

Neoadjuvant chemotherapy has become the standard of care for locally advanced primary breast cancer. Anthracycline-based regimens have proven to be one of the most effective treatments in this setting. As certain cytotoxic antineoplastic agents, such as anthracyclines, generate reactive oxygen species as a by-product of their mechanism of action, we examined whether redox protein expression was involved in the response to anthracycline-based chemotherapy and with clinical outcome. Pre-treatment needle core biopsy and post-anthracycline treatment tumour sections were analysed from 98 cases. In all, 32 individuals had a complete clinical response and 17 had a complete pathological response. Immunohistochemical staining was performed for eight redox proteins: thioredoxin, thioredoxin reductase, thioredoxin interacting protein (TxNIP), glutathione S-transferase (GST) π, θ and α, catalase and manganese superoxide dismutase. GST π (P=0.05) and catalase (P=0.045) were associated with pathological complete response in pre-chemotherapy samples. TxNIP (P=0.017) and thioredoxin reductase (P=0.022) were independent prognostic factors for distant metastasis-free survival and TxNIP for overall survival (P=0.014). In oestrogen receptor negative patients that are known to have a poor overall survival, a considerably worse prognosis was seen in cases that exhibited low expression of TxNIP (P=0.000003), stratifying patients into more defined groups. This study indicates the importance of redox regulation in determining breast cancer response to anthracycline-based chemotherapy and provides ways of further stratifying pre-chemotherapy patients to potentially allow more tailored treatments.

Calpain-2 expression is associated with response to platinum based chemotherapy, progression-free and overall survival in ovarian cancer

Ovarian cancer is routinely treated with surgery and platinum‐based chemotherapy. Resistance is a major obstacle in the efficacy of this chemotherapy regimen and the ability to identify those patients at risk of developing resistance is of considerable clinical importance. The expression of calpain‐1, calpain‐2 and calpastatin were determined using standard immunohistochemistry on a tissue microarray of 154 primary ovarian carcinomas from patients subsequently treated with platinum‐based adjuvant chemotherapy. High levels of calpain‐2 expression was significantly associated with platinum resistant tumours (P = 0.031). Furthermore, high expression of calpain‐2 was significantly associated with progression‐free (P = 0.049) and overall survival (P = 0.006) in this cohort. The association between calpain‐2 expression and overall survival remained significant in multivariate analysis accounting for tumour grade, stage, optimal debulking and platinum sensitivity (hazard ratio = 2.174; 95% confidence interval = 1.144–4.130; P = 0.018). The results suggest that determining calpain‐2 expression in ovarian carcinomas may allow prognostic stratification of patients treated with surgery and platinum‐based chemotherapy. The findings of this study warrant validation in a larger clinical cohort.

Base excision repair, the redox environment and therapeutic implications.

Control of redox homeostasis is crucial for a number of cellular processes with deregulation leading to a number of serious consequences including oxidative damage such induction of DNA base lesions. The DNA lesions caused by oxidative damage are principally repaired by the base excision repair (BER) pathway. Pharmacological inhibition of BER is becoming an increasingly active area of research with the emergence of PARP inhibitors in cancer therapy. The redox status of the cell is modulated by a number of systems, including a large number of anti-oxidant enzymes who function in the control of superoxide and hydrogen peroxide, and ultimately in the release of the damaging hydroxyl radical. Here we provide an overview of reactive oxygen species (ROS) production and its modulation by antioxidant enzymes. The review also discusses the effect of ROS on the BER pathway, particularly in relation to cancer. Finally, as the modulation of the redox environment is of interest in cancer therapy, with certain agents having the potential to reverse chemo- and radiotherapy resistance or treat therapy related toxicity, we discuss redox modulating agents currently under development.