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Background Metastasis 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. Methods The 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. Results In 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. Conclusion Altogether, 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.

Background Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear. Methods In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm 2 . We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing. Results We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures. Conclusions Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant’s outcome, several strategies are being studied. We have previously shown that prolactin induces protection against proinflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat-shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1 attenuated levels of pro-apoptotic proteins such as CHOP and BIM, as well as increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and, thus, enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aimed at the optimization of beta-cell replacement therapies.

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.

Aims/hypothesis Activation 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. Methods NF-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. Results IL-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/interpretation The 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.

Metastasis 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-[beta]1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. The mRNA expression levels of TGF-[beta] 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-[beta]1 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. In general, TGF-[beta]2, T[beta]RI and T[beta]RII are over-expressed in more aggressive cells, except for T[beta]RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-[beta]1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-[beta]1 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-[beta]1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-[beta]1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-[beta]1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-[beta]1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Altogether, our results support that TGF-[beta]1 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-[beta]1 still remains a promising target for breast cancer treatment.

Aims/hypothesis Activation of the transcription factor nuclear factor (NF)-[kappa]B by proinflammatory cytokines plays an important role in beta cell demise in type 1 diabetes. Two main signalling pathways are known to activate NF-[kappa]B, namely the canonical and the non-canonical pathways. Up to now, studies on the role of NF-[kappa]B 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. Methods NF-[kappa]B 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-[beta]H1 exposed to cytokines were used as models. Results IL-1[beta] plus IFN-γ induced stabilisation of NF-[kappa]B-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-[kappa]B-dependent genes, regulating both pro-apoptotic and inflammatory responses, which are implicated in beta cell loss in early type 1 diabetes. Conclusions/interpretation The atypical activation of the non-canonical NF-[kappa]B pathway by proinflammatory cytokines constitutes a novel 'feed-forward' mechanism that contributes to the particularly pro-apoptotic effect of NF-[kappa]B in beta cells.

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We previously reported that Photodynamic Therapy using Methylene Blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness. Through lipidomic and biochemical approaches we demonstrated that MB-PDT efficiency and specificity relies on polyunsaturated fatty acids-enriched membranes and on the better capacity to deal with photooxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results broadened the understanding of MB-PDT-induced photooxidation mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.