Cinthya Sternberg

Abstract 2571: Metformin inhibits proliferation and acts synergistically with paclitaxel and doxorubicin in triple negative breast cancer cell lines

Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BC) characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis and unavailability of effective target therapy. Indeed, despite good initial response to chemotherapy, even platinum- or anthracyclines-based, TNBC relapse and mortality rates are high even in early-stage disease. Metformin, one of the most commonly used medications for treatment of type 2 diabetes mellitus, has emerged as a potential anticancer agent. This study aimed to evaluate the combination treatment of metformin, at lower concentrations (10uM), with conventional chemotherapeutic agents on TNBC cell lines. We investigated the combination effect of metformin with doxorubicin (DOX), cisplatin (CDDP) and paclitaxel (PTX). Methods: Three TNBC cell lines (MDAMB-231, HCC-70, HCC-1937) and one luminal BC cell line (MCF-7) were used. Cell proliferation was determined by MTT assay. Clonogenic assay was conducted and apoptosis was analyzed by Annexin V-FITC assay. Western immunoblotting was performed to determine the expression of the downstream targets of PI3K, MAPK and AMPK pathways. Findings: Metformin potently inhibited the proliferation of all BC cell lines in a dose-dependent manner. Likewise, metformin caused a significant dose-dependent reduction in clonogenicity of TNBC cell lines (P Citation Format: Isabella S. Guimaraes, Nayara G. Tessarollo, Laura FRL Oliveira, Roger C. Zampier, Ian V. Silva, Cinthya Sternberg, Leticia BA Rangel. Metformin inhibits proliferation and acts synergistically with paclitaxel and doxorubicin in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2571. doi:10.1158/1538-7445.AM2015-2571

Functional analysis of polymorphisms in the COX‑2 gene and risk of lung cancer

The enzyme cyclooxygenase 2 (COX-2) is known to be involved in tumorigenesis and metastasis in certain types of cancer. Nevertheless, the prognostic value of COX-2 overexpression and its polymorphisms in patients with non-small cell lung cancer (NSCLC) have yet to be fully elucidated. The aim of the present study was to investigate the association between the three most commonly studied COX-2 gene polymorphisms (−1195 G/A, −765 G/C and 8473 T/C) with COX-2 expression and lung cancer risk in a Brazilian cohort. In the present hospital based, case-control retrospective study, 104 patients with NSCLC and 202 cancer free control subjects were genotyped for −1195 G/A, −765 G/C and 8473 T/C polymorphisms using allelic discrimination with a reverse transcription quantitative polymerase chain reaction method. COX-2 mRNA expression was analyzed in surgically resected tumors from 34 patients with NSCLC. The results revealed that COX-2 expression levels were higher in tumor tissue compared with normal lung tissue. However, this overexpression of COX-2 was not associated with the patient outcome, and furthermore, none of the analyzed polymorphisms were associated with the risk of developing lung cancer, COX-2 overexpression, or the overall survival of the patients with NSCLC. Taken together, the findings described in the present study do not support a major role for COX-2 polymorphisms and COX-2 overexpression in lung carcinogenesis within the Brazilian population.

An alert to Latin America: Current human papillomavirus vaccination trends highlight key barriers to successful implementation

&NA; Human papillomavirus vaccine programs run the risk of repeating the problems associated with Papanicolaou testing programs in low‐income and middle‐income countries: an efficient, life‐saving tool that unfortunately is underused for cancer prevention. There is a great need for vigilance in the ongoing implementation of the human papillomavirus vaccine in Latin America.

Positive crosstalk between EGFR and the TF-PAR2 pathway mediates resistance to cisplatin and poor survival in cervical cancer

Cisplatin-based chemoradiation is the standard treatment for cervical cancer, but chemosensitizing strategies are needed to improve patient survival. EGFR (Epidermal Growth Factor Receptor) is an oncogene overexpressed in cervical cancer that is involved in chemoresistance. Recent studies showed that EGFR upregulates multiple elements of the coagulation cascade, including tissue factor (TF) and the protease-activated receptors (PAR) 1 and 2. Moreover, many G protein-coupled receptors, including PARs, have been implicated in EGFR transactivation. However, the role of coagulation proteins in the progression of cervical cancer has been poorly investigated. Herein we employed cervical cancer cell lines and The Cancer Genome Atlas (TCGA) database to evaluate the role of EGFR, TF and PAR2 in chemoresistance. The SLIGKL-NH2 peptide (PAR2-AP) and coagulation factor VIIa (FVIIa) were used as PAR2 agonists, while cetuximab was used to inhibit EGFR. The more aggressive cell line CASKI showed higher expression levels of EGFR, TF and PAR2 than that of C33A. PAR2 transactivated EGFR, which further upregulated cyclooxygenase-2 (COX2) expression. PAR2-AP decreased cisplatin-induced apoptosis through an EGFR- and COX2-dependent mechanism. Furthermore, treatment of CASKI cells with EGF upregulated TF expression, while treatment with cetuximab decreased the TF protein levels. The RNA-seq data from 309 TCGA samples showed a strong positive correlation between EGFR and TF expression (P = 0.0003). In addition, the increased expression of EGFR, PAR2 or COX2 in cervical cancer patients was significantly correlated with poor overall survival. Taken together, our results suggest that EGFR and COX2 are effectors of the TF/FVIIa/PAR2 signaling pathway, promoting chemoresistance.

Chemosensitizing effects of metformin on cisplatin- and paclitaxel-resistant ovarian cancer cell lines

BACKGROUND Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Primary cytoreductive surgery with adjuvant taxane-platinum chemotherapy is the standard treatment to fight ovarian cancer, however, their side effects are severe, and chemoresistance emerges at high rates. Therefore, EOC clinic urges for novel treatment strategies to reverse chemoresistance and to improve the survival rates. Metformin has been shown to act in synergy with certain anti-cancer agents, overcoming chemoresistance in various types of tumors. This paper aims to investigate the use of metformin as a new treatment option for cisplatin- and paclitaxel-resistant ovarian cancer. METHODS The effects of metformin alone or in combination with conventional drugs on resistant EOC cell lines were investigated using the MTT assay for cell proliferation; Flow Cytometry analysis for cell cycle and the mRNA expression was analyzed using the real-time PCR technique. RESULTS We found that metformin exhibited antiproliferative effects in paclitaxel-resistant A2780-PR, and in cisplatin-resistant ACRP cell lines. The combined therapy containing conventional drugs and metformin improved the effect of the treatment in cell proliferation rate, especially in the resistant cells. We found that metformin, in clinical relevant doses, could significantly reduce the mRNA expression of inflammatory cytokines and NF-κB signaling pathway. CONCLUSIONS Taken together, our observations suggest that metformin inhibits the inflammatory pathway induced by paclitaxel and cisplatin treatment. Furthermore, metformin in combination with paclitaxel or cisplatin improved the sensitivity in drug-resistant ovarian cancer cells. Therefore, metformin may be beneficial treatment strategy, particularly in patients with tumors refractory to platinum and taxanes.

Exploring the link between anabolic-androgenic steroids abuse for performance improvement and hepatocellular carcinoma: a literature review

Introduction: Hepatocellular carcinoma (HCC) corresponds to 90% of primary malignant liver cell carcinomas and is a leading cause of cancer-related death worldwide. Objective: This compilation of cases aimed to identify evidence of correlation between anabolic androgen steroids (AAS) abuse for performance improvement by healthy subjects and HCC. Methods: We performed a literature review and identifi ed 935, 1148, 12 and 3 articles in Pubmed, Embase, Scopus and Lilacs, respectively. Only studies, reviews and case reports evaluating the association between androgens and hepatocarcinoma were included with no restrictions in time span or language. Further on, we excluded studies and case reports which patients were receiving therapeutic androgens and collected data only of those reporting androgen intake to improve physical performance. Results: Six articles fulfi lled the inclusion criteria, excluding the duplicates. HCC onset after AAS abuse seems to occur at earlier ages than those related to chronic hepatitis B/C, chronic alcohol consumption and nonalcoholic steatohepatitis (NASH). Timeframe ranged from two to seven years in the cases reports presented here. Many AAS subtypes were used by patients depicted in the case reports, so it is diffi cult to conclude if a specifi c AAS is safer or more harmful than the other. Carcinogenic mechanisms are poorly understood, but pre-clinical evidence shows that androgen receptors and oxidative stress may play a pivotal role in its development. Conclusion: The evidence that HCC has been linked to long term AAS abuse for performance improvement is scant but some association is suggested. AAS must be taken only under specialized supervision and the putative correlation with HCC calls for public policies to make high risk populations aware of the risks of misuse and self-administration.

Abstract 413: Targeting IL-6 as a preventive and therapeutic strategy for K-ras mutant lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Worldwide, lung cancer (LC) leads in cancer mortality and cigarette smoking (CS) is its principal cause. Activating mutations of K-ras are one of the most common alterations in LC and are associated with CS exposure. However, several studies have shown that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have a higher risk of developing LC when compared to smokers without COPD. These facts suggest a strong link between COPD-type inflammatory lung microenvironment and K-ras mutant lung cancer. Unfortunately, attempts to directly target K-ras have thus far failed, clearly stating the need for new approaches to bring clinical benefits to patients with this undrugabble profile. Using a conditional K-ras induced lung cancer mouse model (CC-LR) we showed that K-ras mutant lung tumors have intrinsic inflammatory characteristics, and a bacterial lysate-induced COPD-like airway inflammation promotes lung cancer in this model. This was associated with release of the cytokine IL-6 and activation of the IL-6-responsive transcription factor STAT3. We showed that genetic ablation of IL-6 in this model results in a significant tumor reduction indicating an essential role for IL-6/STAT3 pathway in lung cancer promotion and introducing IL-6 as a druggable target for K-ras mutant lung tumor. Therefore, we blocked IL-6 using a monoclonal anti-IL-6 immunotherapy in our K-ras mutant model. Six week old CC-LR mice were injected intraperitoneally with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody twice a week for eight weeks, while been exposed to the aerosolized bacterial lysate once weekly for 8 weeks. Anti-IL-6 therapy not only inhibited intrinsic lung cancer development by ∼78%, but also inhibited the tumor promoting effect of the COPD-like airway inflammation. It also decreased the expression of P-STAT3, proliferation marker, Ki67, and pro-angiogenic factors: VEGF, MMP-9 and CD31 in lung tissue. qPCR analysis of lung tissues from anti-IL-6 treated mice showed a decrease in Arginase1, FIZZ1, FOXP3, and IL-17 expression, with increased expression of Th1 markers, IFNγ and TBx21. Flow cytometry analysis of total lung inflammatory cells in CC-LR mouse showed a significant increase in the population of tumor associated macrophages (TAMs) during tumor progression from age 6 to 14 weeks, and COPD-like inflammation induced an increase in myeloid derived suppressor cell (MDSCs) population. Interestingly, we found a significant decrease in TAMs and MDSCs population after anti-IL-6 treatment. Taken together, we conclude that IL-6 blockade not only has cell intrinsic effect in K-ras mutant tumors but also edit the lung microenvironment toward an anti-tumor phenotype. Therefore, we propose pharmacological targeting of IL-6 alone or in combination with conventional cytotoxic therapy, or other targeted therapies for prevention and treatment of lung cancer patients with K-ras mutation. Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Seon H. Chang, Huiyuan Zhang, Humam N. Kadara, Cinthya Sternberg, Carlos G. Ferreira, Stephanie S. Watowich, Seyed J. Moghaddam. Targeting IL-6 as a preventive and therapeutic strategy for K-ras mutant lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 413. doi:10.1158/1538-7445.AM2015-413