Letícia F. Terra

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.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Aims/hypothesisTransplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process.MethodsHuman pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays.ResultsThe proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3.Conclusions/interpretationOur study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

Generation and characterization of human insulin-releasing cell lines

BackgroundThe in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.ResultsWe generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate.ConclusionWe have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

The non-canonical NF-κB pathway is induced by cytokines in pancreatic beta cells and contributes to cell death and proinflammatory responses in vitro.

Aims/hypothesisActivation of the transcription factor nuclear factor (NF)-κB by proinflammatory cytokines plays an important role in beta cell demise in type 1 diabetes. Two main signalling pathways are known to activate NF-κB, namely the canonical and the non-canonical pathways. Up to now, studies on the role of NF-κB activation in beta cells have focused on the canonical pathway. The aim of this study was to investigate whether cytokines activate the non-canonical pathway in beta cells, how this pathway is regulated and the consequences of its activation on beta cell fate.MethodsNF-κB signalling was analysed by immunoblotting, promoter reporter assays and real-time RT-PCR, after knockdown or overexpression of key genes/proteins. INS-1E cells, FACS-purified rat beta cells and the human beta cell line EndoC-βH1 exposed to cytokines were used as models.ResultsIL-1β plus IFN-γ induced stabilisation of NF-κB-inducing kinase and increased the expression and cleavage of p100 protein, culminating in the nuclear translocation of p52, the hallmark of the non-canonical signalling. This activation relied on different crosstalks between the canonical and non-canonical pathways, some of which were beta cell specific. Importantly, cytokine-mediated activation of the non-canonical pathway controlled the expression of ‘late’ NF-κB-dependent genes, regulating both pro-apoptotic and inflammatory responses, which are implicated in beta cell loss in early type 1 diabetes.Conclusions/interpretationThe atypical activation of the non-canonical NF-κB pathway by proinflammatory cytokines constitutes a novel ‘feed-forward’ mechanism that contributes to the particularly pro-apoptotic effect of NF-κB in beta cells.

Dysfunctional autophagy following exposure to pro-inflammatory cytokines contributes to pancreatic β-cell apoptosis.

Type 1 diabetes (T1D) results from β-cell destruction due to concerted action of both innate and adaptive immune responses. Pro-inflammatory cytokines, such as interleukin-1β and interferon-γ, secreted by the immune cells invading islets of Langerhans, contribute to pancreatic β-cell death in T1D. Cytokine-induced endoplasmic reticulum (ER) stress plays a central role in β-cell demise. ER stress can modulate autophagic response; however, no study addressed the regulation of autophagy during the pathophysiology of T1D. In this study, we document that cytokines activate the AMPK-ULK-1 pathway while inhibiting mTORC1, which stimulates autophagy activity in an ER stress-dependent manner. On the other hand, time-course analysis of LC3-II accumulation in autophagosomes revealed that cytokines block the autophagy flux in an ER stress independent manner, leading to the formation of large dysfunctional autophagosomes and worsening of ER stress. Cytokines rapidly impair lysosome function, leading to lysosome membrane permeabilization, Cathepsin B leakage and lysosomal cell death. Blocking cathepsin activity partially protects against cytokine-induced or torin1-induced apoptosis, whereas blocking autophagy aggravates cytokine-induced CHOP overexpression and β-cell apoptosis. In conclusion, cytokines stimulate the early steps of autophagy while blocking the autophagic flux, which aggravate ER stress and trigger lysosomal cell death. Restoration of autophagy/lysosomal function may represent a novel strategy to improve β-cell resistance in the context of T1D.

Proteins differentially expressed in human beta-cells-enriched pancreatic islet cultures and human insulinomas ☆

In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas.

Heat shock protein B1 is required for the prolactin-induced cytoprotective effects on pancreatic islets

The success of islet transplantation has improved lately. Unfortunately, it is still compromised by cell loss. We have shown that prolactin (PRL) inhibits beta-cell apoptosis and up-regulates the antiapoptotic Heat Shock Protein B1 (HSPB1) in human islets. Since its function in pancreatic islets has not been studied, we explored the role of HSPB1 in PRL-induced beta-cell survival. The significant PRL-induced cytoprotection in control cells was abrogated in HSPB1 silenced cells, overexpression of HSPB1 recovered survival. PRL-mediated inhibition of cytokine-induced caspase activities and cytokine-induced decrease of BCL-2/BAX ratio was significantly reverted in knocked-down cells. Kinetics of HSPB1 and HSF1 expression were studied in primary cultures of murine and human pancreatic islets. These findings are highly relevant for the improvement of clinical islet transplantation success rate since our results demonstrated a key role for HSPB1 pointing it as a promising target for beta-cell cytoprotection through the up-regulation of an endogenous protective pathway.

High‐fat diet and fish oil affect adipocyte metabolism in a depot‐specific manner

It is well known that the white adipose tissue plays a central role in the control of metabolism; therefore it can be related to several disorders, such as obesity, type-2 diabetes mellitus and metabolic syndrome, mainly by inflammation and hypertrophy. Chronic overfeeding with a high-fat (HF) diet causes an increase in body weight and promotes fat accumulation. The omega-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are dietary compounds that are intensively studied as potent anti-inflammatory products. The most important source of EPA and DHA is marine fish, and although fish oil (FO) has been shown in many studies to protect against metabolic diseases, not one has focused on adipocyte metabolism. More recently a study by de Sá et al. (2016), published in The Journal of Physiology, provided evidence to suggest that FO prevents the changes in adipocyte metabolism and adipokine secretion induced by the HF diet in a depot-specific manner. The well-conducted experiments, complemented by the relevant findings, make this work attractive. First of all the authors showed that the HF-diet-fed mice exhibited more adipose tissue, glucose and insulin intolerance, increased fasting blood glucose and insulin levels, augmented blood cholesterol and LDL-cholesterol concentrations, and elevated homeostatic model assessment of

Abstract 508: Development of a human model to study the role of Glypican-3 (GPC3) on tumor progression of the mammary gland.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC GPC3 was proposed as a negative regulator of cell proliferation in the mammary gland. Whereas normal breast tissues present high GPC3 levels, its expression is reduced in breast tumors. We have transfected LM3 murine mammary tumor cells with the GPC3 gene. We found that GPC3 reexpression inhibited metastasis in vivo. Although GPC3 signaling pathway is unclear, we reported that this glypican modulates Wnt, p38 and Akt pathways. However, regulation hierarchy has not been determined and we have no data in human cells. We believe it possible to generate an anti-metastasis therapy targeting GPC3 pathway. To test it, we developed a pre-clinical human model. First, we showed by qPCR that GPC3 expression levels are inversely proportional to the metastatic ability of a panel of human breast cancer cells. The metastatic Hs578T and MDA-MB231 cells expressed less GPC3 than the poorly-metastatic ZR-75-1 and MCF7 lines. We selected MDA-MB231 and MCF7 to be modified by genetic engineering. GPC3 expression was blocked by RNA interference technology. We transfected four shRNA-GPC3 and one shRNA scramble (shNC) into MCF7. After selection and cloning, we chose clone 2 that expressed about 75% less GPC3 for further characterization. On the other hand, GPC3 was reexpressed in MDA-MB231 cells by transduction of pLV-GPC3-GFP (empty vector was used as control). GPC3 mRNA increased up to 200-fold in pLV-GPC3-GFP transduced cells. Then we evaluated cell properties in the generated models. Surprisingly, MDA-MB231-GPC3 cells showed a higher proliferation rate than controls, that became significant from 48 h of exponential growth (Population doubling time= 45 h for control vs. 24 h for GPC3 reexpressing cells). On the other side, MCF7-shGPC3 bulk and clone 2 proliferated faster than -shNC. This suggests that, in this cell line, GPC3 is inhibiting proliferation. GPC3 reexpression induced a 30% inhibition on MDA-MB231 viability when cells were serum starved for 96 h, while parental and -GFP remained 100% viable. No differences were detected among MCF7 engineered cells. So, the effect of GPC3 on cell viability would depend on the cellular context. We performed a wound healing assay to evaluate migration, determining that MDA-MB231-GPC3 cells were significantly less migrant than their controls (Wound coverage: MDA-MB231-GPC3 10% vs. parental and -GFP 90%). In sum, we have developed a human cell model to study the role of GPC3 on tumor progression. Our preliminary in vitro results reveal the importance of GPC3 in the biology of breast cancer cells. Citation Format: Lilian F. Castillo, Rocio Tascon, Maria Amparo Lago Huvelle1, Ancely Ferreira dos Santos, Leticia Ferreira Terra, Rosangela A. m. Wailemann, Talita C. Oliveira, Mari Sogayar, Leticia Labriola, Elisa Bal de Kier Joffe, Maria Giselle Peters. Development of a human model to study the role of Glypican-3 (GPC3) on tumor progression of the mammary gland. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2013-508