A. C. S. A. Herrera

Differential oxidative status and immune characterization of the early and advanced stages of human breast cancer

Breast cancer is the malignant neoplasia with the highest incidence in women worldwide. Chronic oxidative stress and inflammation have been indicated as major mediators during carcinogenesis and cancer progression. Human studies have not considered the complexity of tumor biology during the stages of cancer advance, limiting their clinical application. The purpose of this study was to characterize systemic oxidative stress and immune response parameters in early (ED; TNM I and II) and advanced disease (AD; TNM III and IV) of patients diagnosed with infiltrative ductal carcinoma breast cancer. Oxidative stress parameters were evaluated by plasmatic lipoperoxidation, carbonyl content, thiobarbituric reactive substances (TBARS), nitric oxide levels (NO), total radical antioxidant parameter (TRAP), superoxide dismutase, and catalase activities and GSH levels. Immune evaluation was determined by TNF-α, IL-1β, IL-12, and IL-10 levels and leukocytes oxidative burst evaluation by chemiluminescence. Tissue damage analysis included heart (total CK and CKMB), liver (AST, ALT, GGT), and renal (creatinine, urea, and uric acid) plasmatic markers. C-reactive protein (CRP) and iron metabolism were also evaluated. Analysis of the results verified different oxidative stress statuses occur at distinct cancer stages. ED was characterized by reduction in catalase, 8-isoprostanes, and GSH levels, with enhanced lipid peroxidation and TBARS levels. AD exhibited more pronounced oxidative status, with reduction in catalase activity and TRAP, intense lipid peroxidation and high levels of NO, TBARs, and carbonyl content. ED patients presented a Th2 immune pattern, while AD exhibited Th1 status. CRP levels and ferritin were increased in both stages of disease. Leukocytes burst impairment was observed in both the groups. Plasma iron levels were significantly elevated in AD. The data obtained indicated that oxidative stress enhancement and immune response impairment may be necessary to ensure cancer progression to advanced stages and may result from both host and tumor inflammatory mediators.

Putative circulating markers of the early and advanced stages of breast cancer identified by high-resolution label-free proteomics.

This study evaluated the plasmatic proteomic profile of breast cancer patients in the early (ED) and advanced (AD) stages, employing high-throughput proteomics. We identified 92 differentially expressed proteins in ED and 73 proteins in AD patients. Gelsolin, lumican, clusterin, SALL4 and PMS2, as well hTERT, TNF-α and GRHL3 were chosen for further investigation. ED presented augmented expression of GRHL3 and reduced circulating TNF-α with high expression of GRHL3 in tumors. AD displayed high TNF-α and a significant expression of PMS2 in tumors. These findings suggest processes enrolling stem cell division in ED, with TNF-α signaling and DNA mismatch repair in the advanced stage.

Molecular subtype is determinant on inflammatory status and immunological profile from invasive breast cancer patients

Breast cancer consists in a chronic inflammatory disease with multiple biological and clinical behaviors. Based on high throughput technologies data, this disease is currently classified according to the molecular expression of estrogen (ER), progesterone (PR) and human epidermal growth factor (HER-2) receptors. In this study, we defined the inflammatory profile of the main molecular subtypes of breast cancer patients: luminal (ER and PR positive, HER-2 negative), HER-2 enriched (HER-2 positive) and triple negative (ER, PR and HER-2 negative). Cytokines panel was assessed by measurement of TNF-α, TGF-β, IL-1, IL-10 and IL-12 plasmatic levels. Oxidative profile was assessed by determination of lipid peroxidation, total antioxidant capacity of plasma, malondialdehyde levels, carbonyl content and nitric oxide (NO). Clinical data were correlated with inflammatory findings. Our findings demonstrated that patients bearing the luminal subtype displayed high TNF-α, TGF-β and enhanced oxidative stress levels associated with reduced IL-12. HER-2-enriched group exhibited higher levels of TNF-α, IL-12 and TGF-β associated with enhanced oxidative stress. Triple-negative subtype exhibited the most aggressive profile of disease behavior, with reduction in both TNF-α and TGF-β, with high levels of lipid peroxidation and NO. The clinical importance of our findings lies in the fact that the inflammatory status varies in distinct ways due to molecular subtype of breast cancer, opening potential therapeutic targets to future therapies.

Immunological effects of Taxol and Adryamicin in breast cancer patients

Antineoplastic chemotherapy still consists in the major first-line therapeutics against cancer. Several reports have described the immunomodulatory effects of these drugs based on in vitro treatment, but no previous data are known about these effects in patients and its association with immunological-mediated toxicity. In this study, we first characterize the immunological profile of advanced breast cancer patients treated with doxorubicin and paclitaxel protocols, immediately after chemotherapy infusion. Our findings included an immediate plasmatic reduction in IL-1, IL-10, and TNF-α levels in doxorubicin-treated patients, as well as high levels of IL-10 in paclitaxel patients. Further, it was demonstrated that both drugs led to leukocytes oxidative burst impairment. In vitro analysis was performed exposing healthy blood to both chemotherapics in the same concentration and time of exposition of patients, resulting in low IL-10 and high IL-1β in doxorubicin exposition, as low TNF-α and high IL-1 in paclitaxel treatment. Nitric oxide levels were not altered in both in vivo and in vitro treatments. In conclusion, our data revealed for the first time that the immediate effects of chemotherapy could be mediated by cytokines signaling in patients and that the results observed in patients could be a resultant of host immune cells activation.

Label-Free Proteomic Analysis of Breast Cancer Molecular Subtypes

To better characterize the cellular pathways involved in breast cancer molecular subtypes, we performed a proteomic study using a label-free LC-MS strategy for determining the proteomic profile of Luminal A, Luminal-HER2, HER2-positive, and triple-negative (TN) breast tumors compared with healthy mammary tissue. This comparison aimed to identify the aberrant processes specific for each subtype and might help to refine our understanding regarding breast cancer biology. Our results address important molecular features (both specific and commonly shared) that explain the biological behavior of each subtype. Changes in proteins related to cytoskeletal organization were found in all tumor subtypes, indicating that breast tumors are under constant structural modifications to invade and metastasize. We also found changes in cell-adhesion processes in all molecular subtypes, corroborating that invasiveness is a common property of breast cancer cells. Luminal-HER2 and HER2 tumors also presented altered cell cycle regulation, as shown by the several DNA repair-related proteins. An altered immune response was also found as a common process in the Luminal A, Luminal-HER2, and TN subtypes, and complement was the most important pathway. Analysis of the TN subtype revealed blood coagulation as the most relevant biological process.

Breast cancer in Brazil: epidemiology and treatment challenges.

Notwithstanding the advances in tumor research, diagnosis, and treatment, breast cancer is still a challenge worldwide. This global burden of disease has been associated with population aging and the persistence of cancer-related behaviors. The number of women diagnosed with breast cancer has been estimated as increasing, especially in middle-income countries such as Brazil. Estimates from the Instituto Nacional de Câncer (INCA) point to breast cancer as the major malignant neoplasia in Brazilian women and the main cause of death from cancer in the country. This fact has been associated with increased life expectancy, urbanization, and cancer-related behaviors. Given this scenario, it is clear that there is a need for identifying and discussing which factors have substantially contributed to this growing number of cases in Brazil, including access to treatment, prevention and early diagnosis, weaknesses of the local health policy, and intrinsic genetic peculiarities of the Brazilian population. This review aims to address the role of such factors.

Trastuzumab-based chemotherapy modulates systemic redox homeostasis in women with HER2-positive breast cancer

Trastuzumab is an immunotargeting therapeutic against breast tumors with amplification of the human epithelial growth factor receptor 2 (HER2). HER2 patients naturally exhibit disruption in the pro-oxidant inflammatory profiling; however, the impact of trastuzumab-based chemotherapy in modulating this process is still unknown. Here we determined the systemic pro-inflammatory profile of women diagnosed with HER2-amplified tumors, undergoing trastuzumab-based chemotherapy (TZ), and compared the results with that of healthy controls (CTR) and untreated patients with HER2-amplified breast cancer (CA). The plasmatic inflammatory profile was assessed by evaluating pro-oxidant parameters such as lipid peroxidation, total antioxidant capacity (TRAP), levels of advanced oxidation protein products (AOPPs), nitric oxide (NO), C-reactive protein (CRP), and total thiol content. Markers of cardiac damage were also assessed. Our findings showed increased NO levels in TZ than that in either CA or CTR groups. Furthermore, TZ augmented TRAP and reduced total thiol than that of the CA group. Our data also revealed that AOPP levels were significantly higher in the TZ than the CA group. AOPP and the MB fraction of creatine-kinase (CKMB) levels were positively correlated in TZ patients. These findings suggest that trastuzumab-associated chemotherapy can modulate the pro-inflammatory markers of HER2-positive breast cancer patients to the levels found in healthy controls.

The positive is inside the negative: HER2-negative tumors can express the HER2 intracellular domain and present a HER2-positive phenotype

Overexpression of human epithelial growth factor receptor 2 (HER2) is a poor prognostic factor in breast cancer. HER2 is a transmembrane receptor comprising an extracellular domain (ECD), a single transmembrane domain, and an intracellular domain (ICD) with tyrosine-kinase activity. Receptor dimerization triggers pivotal effector pathways in cancer, such as phosphatidylinositol 3-kinase (PI3K) signaling. Currently, screening of HER2 in breast tumors for prognostic and therapeutic purposes involves immunohistochemical (IHC) phenotyping for the ECD, in which tumors with IHC scores below 2+ are reported as HER2-negative. We used a label-free liquid chromatography-mass spectrometry (LC-MS) proteomic approach to compare plasma samples from patients with HER2-positive breast tumors and patients with HER2-negative tumors. Patients with HER2-negative tumors expressed higher circulating levels of calpain-10 than patients with HER2-positive tumors. Calpains cleave HER2, releasing its ECD and transforming phenotypically positive tumors into phenotypically negative tumors. Therefore, we investigated the expression of the ICD in HER2-negative samples that overexpressed calpain-10. We found that 16% of HER2-negative tumors were positive for HER2-ICD, which was associated with circulating HER2-ECD. HER2 gene amplification was also observed in some HER2-negative tumors. Positive staining for the PI3K pathway was observed in the HER2-negative, ICD-positive tumors, similar to the HER2-positive cohort. Microarray analysis revealed that HER2-negative, ICD-positive samples clustered between HER2-positive tumors and triple-negative tumors. Survival analysis revealed that outcome in women with HER2-negative, ICD-positive tumors was better than in women bearing HER2-negative, ICD-negative (triple negative) tumors but was quite similar to HER2-positive tumors and worse than women with luminal A tumors. Moreover, in vitro analyses revealed that MDA-MB 231, a triple negative cell line, possesses calpain-10 and HER2-p-ICD up-regulation and blockage of calpain-10 activity promoted an increase in HER2-p-ICD and p-AKT levels, suggesting an increase in these pathways signaling. These data indicate that HER2-negative tumors with HER2-ICD positivity exhibit clinical behavior closer to that of HER2-positive tumors. This indicates a need for HER2-ICD screening when determining the molecular profile of breast tumors. These findings further indicate that lapatinib should be investigated as a target therapy for HER2-ICD-positive breast tumors.

Identifying potential markers in Breast Cancer subtypes using plasma label-free proteomics

Breast Cancer (BC) is the most common neoplasia among women and has a high mortality rate worldwide. Over the past several decades, increasing molecular knowledge of BC has resulted in its stratification into 4 major molecular subtypes according to hormonal receptor expression. Unfortunately, although the data accumulated thus far has improved BC prognosis and treatment, there have been few achievements in its diagnosis. In this study, we applied a Label-free Nano-LC/MSMS approach to reveal systemic molecular features and possible plasma markers for BC patients. Compared to healthy control plasma donors, we identified 191, 166, 182, and 186 differentially expressed proteins in the Luminal, Lumina-HER2, HER2, and TN subtypes. In silico analysis demonstrated an overall downregulation of cellular basal machinery and, more importantly, brought new focus to the known pathways and signaling molecules in BC that are related to immune system alterations. Moreover, using western blot analysis, we verified high levels of BCAS3, IRX1, IRX4 and IRX5 in BC plasma samples, thus highlighting the potential use of plasma proteomics in investigations into cancer biomarkers. SIGNIFICANCE The results of this study provide new insight into Breast Cancer (BC). We determined the plasma proteomic profile of BC subtypes. Furthermore, we report that the signaling pathways correlating with late processes in BC already exhibit plasma alterations in less aggressive subtypes. Additionally, we validated the high levels of particular proteins in patient samples, which suggests the use of these proteins as potential disease markers.

Can Breast Tumors Affect the Oxidative Status of the Surrounding Environment? A Comparative Analysis among Cancerous Breast, Mammary Adjacent Tissue, and Plasma

In this paper, we investigated the oxidative profile of breast tumors in comparison with their normal adjacent breast tissue. Our study indicates that breast tumors present enhanced oxidative/nitrosative stress, with concomitant augmented antioxidant capacity when compared to the adjacent normal breast. These data indicate that breast cancers may be responsible for the induction of a prooxidant environment in the mammary gland, in association with enhanced TNF-α and nitric oxide.