Luciana R. Gomes

Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential

BackgroundThe metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. The role of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in the metastatic process has previously been established. However, in all published studies only a limited number of MMPs/MMP inhibitors was analyzed in a limited number of cell lines. Here, we propose a more comprehensive approach by analyzing the expression levels of several MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors (TIMP-1, TIMP-2 and RECK) in different models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues).MethodsWe analyzed the expression levels of MMP-2, MMP-9 and MMP-14 and their inhibitors (TIMP-1, TIMP-2 and RECK) by quantitative RT-PCR (qRT-PCR) in five human breast cancer cell lines presenting increased invasiveness and metastatic potential, 72 primary breast tumors and 30 adjacent normal tissues. Moreover, the role of cell-extracellular matrix elements interactions in the regulation of expression and activity of MMPs and their inhibitors was analyzed by culturing these cell lines on plastic or on artificial ECM (Matrigel).ResultsThe results demonstrated that MMPs mRNA expression levels displayed a positive and statistically significant correlation with the transcriptional expression levels of their inhibitors both in the cell line models and in the tumor tissue samples. Furthermore, the expression of all MMP inhibitors was modulated by cell-Matrigel contact only in highly invasive and metastatic cell lines. The enzyme/inhibitor balance at the transcriptional level significantly favors the enzyme which is more evident in tumor than in adjacent non-tumor tissue samples.ConclusionOur results suggest that the expression of MMPs and their inhibitors, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, the multi-factorial analysis of these molecules could provide new and independent prognostic information contributing to the determination of more adequate therapy strategies for each patient.

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

BackgroundMetastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models.MethodsThe mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays.ResultsIn general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors.ConclusionAltogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.

Epithelial-mesenchymal transition: implications in cancer progression and metastasis.

During the past few years, Epithelial-Mesenchymal Transition (EMT) has emerged as one of the most hot spots in clinical research. Its existence in human tumors can form the basis for explaining characteristics of cancer progression and metastasis, as well as certain cases of drug resistance and relapses after treatment. These cellular responses are tightly regulated by intracellular signaling pathways evoked by humoral factors that include growth factors, chemokines and cytokines. Indeed, several gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. A deeper understanding of the cellular and molecular basis of these different programs should aid in both the development of better diagnosis methods, as well as of specific treatments for invasive cancer. In this review we set out to summarize recent novel insights into the molecular players underlying EMT and its relation with cancer progression and metastasis.

Multivariate gene expression analysis reveals functional connectivity changes between normal/tumoral prostates.

BackgroundProstate cancer is a leading cause of death in the male population, therefore, a comprehensive study about the genes and the molecular networks involved in the tumoral prostate process becomes necessary. In order to understand the biological process behind potential biomarkers, we have analyzed a set of 57 cDNA microarrays containing ~25,000 genes.ResultsPrincipal Component Analysis (PCA) combined with the Maximum-entropy Linear Discriminant Analysis (MLDA) were applied in order to identify genes with the most discriminative information between normal and tumoral prostatic tissues. Data analysis was carried out using three different approaches, namely: (i) differences in gene expression levels between normal and tumoral conditions from an univariate point of view; (ii) in a multivariate fashion using MLDA; and (iii) with a dependence network approach. Our results show that malignant transformation in the prostatic tissue is more related to functional connectivity changes in their dependence networks than to differential gene expression. The MYLK, KLK2, KLK3, HAN11, LTF, CSRP1 and TGM4 genes presented significant changes in their functional connectivity between normal and tumoral conditions and were also classified as the top seven most informative genes for the prostate cancer genesis process by our discriminant analysis. Moreover, among the identified genes we found classically known biomarkers and genes which are closely related to tumoral prostate, such as KLK3 and KLK2 and several other potential ones.ConclusionWe have demonstrated that changes in functional connectivity may be implicit in the biological process which renders some genes more informative to discriminate between normal and tumoral conditions. Using the proposed method, namely, MLDA, in order to analyze the multivariate characteristic of genes, it was possible to capture the changes in dependence networks which are related to cell transformation.

Three-dimensional microenvironment confers enhanced sensitivity to doxorubicin by reducing p53-dependent induction of autophagy

Preclinical studies of anticancer drugs are typically performed using cancer cell lines maintained in two-dimensional (2D) cultures, ignoring the influences of the extracellular matrix (ECM) and three-dimensional (3D) microenvironment. In this study, we evaluated the microenvironmental control of human breast cancer cells responses to doxorubicin (DOXO) using the 3D laminin-rich ECM (3D lrECM) cell culture model. Under 3D culture conditions, MCF-7 cells displayed drastic morphological alterations, a decrease in proliferation and elevated sensitivity to DOXO. Interestingly, the chemotherapy-mediated activation of autophagy was compromised in the 3D matrix, suggesting an association between the increased cytotoxicity of DOXO and hindered autophagy induction. Indeed, while chloroquine or ATG5 knockdown potentiated DOXO-induced cell death under the 2D culture conditions, the autophagy inducer rapamycin improved the resistance of 3D-cultured cells to this drug. Moreover, in the monolayer-cultured cells, DOXO treatment led to increases in p53 and DRAM-1 expression, which is a p53-dependent activator of autophagy that functions in response to DNA damage. Conversely, p53 and DRAM-1 expression was impaired in 3D-cultured cells. The knockdown of p53 by shRNA blocked DRAM-1 activation, impaired autophagy induction and sensitized only those cells maintained under 2D conditions to DOXO. In addition, 2D-cultured MDA-MB-231 cells (a p53-mutated breast cancer cell line) not only showed increased sensitivity to DOXO compared with MCF-7 cells but also failed to induce DRAM-1 expression or autophagy. Similar to p53 silencing, DRAM-1 knockdown potentiated DOXO cytotoxicity only in 2D-cultured cells. These results suggest that the 3D tissue microenvironment controls tumor cell sensitivity to DOXO treatment by preventing p53-DRAM-autophagy axis activation.

Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

ABSTRACT Chaperone-mediated autophagy (CMA), a selective form of protein lysosomal degradation, is maximally activated in stress situations to ensure maintenance of cellular homeostasis. CMA activity decreases with age and in several human chronic disorders, but in contrast, in most cancer cells, CMA is upregulated and required for tumor growth. However, the role of CMA in malignant transformation remains unknown. In this study, we demonstrate that CMA inhibition in fibroblasts augments the efficiency of MYC/c-Myc-driven cellular transformation. CMA blockage contributes to the increase of total and nuclear MYC, leading to enhancement of cell proliferation and colony formation. Impaired CMA functionality accentuates tumorigenesis-related metabolic changes observed upon MYC-transformation. Although not a direct CMA substrate, we have found that CMA regulates cellular MYC levels by controlling its proteasomal degradation. CMA promotes MYC ubiquitination and degradation by regulating the degradation of C330027C09Rik/KIAA1524/CIP2A (referred to hereafter as CIP2A), responsible for MYC stabilization. Ubiquitination and proteasomal degradation of MYC requires dephosphorylation at Ser62, and CIP2A inhibits the phosphatase responsible for this dephosphorylation. Failure to degrade CIP2A upon CMA blockage leads to increased levels of phosphorylated MYC (Ser62) and to stabilization of this oncogene. We demonstrate that this phosphorylation is essential for the CMA-mediated effect, since specific mutation of this site (Ser62 to Ala62) is enough to normalize MYC levels in CMA-incompetent cells. Altogether these data demonstrate that CMA mitigates MYC oncogenic activity by promoting its proteasomal degradation and reveal a novel tumor suppressive role for CMA in nontumorigenic cells.

Microenvironment and autophagy cross-talk: Implications in cancer therapy

There are many ongoing clinical trials to validate tumour microenvironment or autophagic pathway components as targets for anticancer therapies. Different components of the tumour microenvironment play important roles in tumour cell responses, directly affecting malignant transformation, drug resistance and metastasis. Autophagy is also related to chemotherapy responses by inducing tumour cell death or survival. Thus, the autophagy pathway may act as oncosuppressor, in addition to protecting cells from chemotherapy. The cross-talk between the microenvironment and autophagy is very complex and poorly understood. In a recent study using a three-dimensional (3D) cell culture model, the well-documented chemotherapy-mediated activation of autophagy was impaired in breast cancer cells, suggesting a context-dependent outcome for autophagy modulators, under the control of the p53 protein. A deeper understanding of this microenvironment/autophagy interplay may provide important clues for identifying differences in the tumour cell signalling network from in vitro basic research studies to the actual clinical context. In this work, we summarize the role of the microenvironment and autophagy in physiological and tumourigenic conditions, their interactions, and the challenges related to the use of drugs that target these pathways in cancer treatment protocols, emphasizing the potential use of 3D cell culture models in preclinical studies.

RECK is not an independent prognostic marker for breast cancer

BackgroundThe REversion-inducing Cysteine-rich protein with Kazal motif (RECK) is a well-known inhibitor of matrix metalloproteinases (MMPs) and cellular invasion. Although high expression levels of RECK have already been correlated with a better clinical outcome for several tumor types, its main function, as well as its potential prognostic value for breast cancer patients, remain unclear.MethodsThe RECK expression profile was investigated in a panel of human breast cell lines with distinct aggressiveness potential. RECK functional analysis was undertaken using RNA interference methodology. RECK protein levels were also analyzed in 1040 cases of breast cancer using immunohistochemistry and tissue microarrays (TMAs). The association between RECK expression and different clinico-pathological parameters, as well as the overall (OS) and disease-free (DFS) survival rates, were evaluated.ResultsHigher RECK protein expression levels were detected in more aggressive breast cancer cell lines (T4-2, MDA-MB-231 and Hs578T) than in non-invasive (MCF-7 and T47D) and non-tumorigenic (S1) cell lines. Indeed, silencing RECK in MDA-MB-231 cells resulted in elevated levels of pro-MMP-9 and increased invasion compared with scrambled (control) cells, without any effect on cell proliferation. Surprisingly, by RECK immunoreactivity analysis on TMAs, we found no association between RECK positivity and survival (OS and DFS) in breast cancer patients. Even considering the different tumor subtypes (luminal A, luminal B, Her2 type and basal-like) or lymph node status, RECK remained ineffective for predicting the disease outcome. Moreover, by multivariate Cox regression analysis, we found that RECK has no prognostic impact for OS and DFS, relative to standard clinical variables.ConclusionsAlthough it continues to serve as an invasion and MMP inhibitor in breast cancer, RECK expression analysis is not useful for prognosis of these patients.

Autophagy Roles in the Modulation of DNA Repair Pathways

Autophagy and DNA repair are biological processes vital for cellular homeostasis maintenance and when dysfunctional, they lead to several human disorders including premature aging, neurodegenerative diseases, and cancer. The interchange between these pathways is complex and it may occur in both directions. Autophagy is activated in response to several DNA lesions types and it can regulate different mechanisms and molecules involved in DNA damage response (DDR), such as cell cycle checkpoints, cell death, and DNA repair. Thus, autophagy may modulate DNA repair pathways, the main focus of this review. In addition to the already well-documented autophagy positive effects on homologous recombination (HR), autophagy has also been implicated with other DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Given the relevance of these cellular processes, the clinical applications of drugs targeting this autophagy-DNA repair interface emerge as potential therapeutic strategies for many diseases, especially cancer.

DUOX1 Silencing in Mammary Cell Alters the Response to Genotoxic Stress

DUOX1 is an H2O2-generating enzyme related to a wide range of biological features, such as hormone synthesis, host defense, cellular proliferation, and fertilization. DUOX1 is frequently downregulated in lung and liver cancers, suggesting a tumor suppressor role for this enzyme. Here, we show that DUOX1 expression is decreased in breast cancer cell lines and also in breast cancers when compared to the nontumor counterpart. In order to address the role of DUOX1 in breast cells, we stably knocked down the expression of DUOX1 in nontumor mammary cells (MCF12A) with shRNA. This led to higher cell proliferation rates and decreased migration and adhesion properties, which are typical features for transformed cells. After genotoxic stress induced by doxorubicin, DUOX1-silenced cells showed reduced IL-6 and IL-8 secretion and increased apoptosis levels. Furthermore, the cell proliferation rate was higher in DUOX1-silenced cells after doxorubicin medication in comparison to control cells. In conclusion, we demonstrate here that DUOX1 is silenced in breast cancer, which seems to be involved in breast carcinogenesis.