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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.

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.

Adult mesenchymal stromal cell‐based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage‐differentiating stress‐enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse‐AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse‐AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse‐AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse‐AT cells evidenced high stage‐specific embryonic antigen‐3 (SSEA‐3) expression (∼60% of cells) after 7–10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA‐3+‐Muse‐AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide‐stimulated macrophages and antigen‐challenged T‐cell assays, we have shown that Muse‐AT cells have anti‐inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon‐γ and tumor necrosis factor‐α. Muse‐AT cells spontaneously gained transforming growth factor‐β1 expression that, in a phosphorylated SMAD2‐dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T‐box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse‐AT cells that can potentially be harnessed as immunoregulators to treat immune‐related disorders. Stem Cells Translational Medicine 2017;6:161–173

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.

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.

The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the effect of the simultaneous block of these receptors on the MPA-induced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not affect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the first time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I. Oncogene (2001) 20, 34 - 47.

A global online survey was administered to 69 islet transplantation programs, covering 84 centers and 5 networks. The survey addressed questions on program organization and activity in the 2000–2020 period, including impact on activity of national health care coverage policies. We obtained full data from 55 institutions or networks worldwide and basic activity data from 6 centers. Additional data were obtained from alternative sources. A total of 94 institutions and 5 networks was identified as having performed islet allotransplantation. 4,365 islet allotransplants (2,608 in Europe, 1,475 in North America, 135 in Asia, 119 in Oceania, 28 in South America) were reported in 2,170 patients in the survey period. From 15 centers active at the start of the study period, the number of simultaneously active islet centers peaked at 54, to progressively decrease to 26 having performed islet allotransplants in 2020. Notably, only 16 centers/networks have done >100 islet allotransplants in the survey period. Types of transplants performed differed notably between North America and the rest of the world, in particular with respect to the near-absence of simultaneous islet-kidney transplantation. Absence of heath care coverage has significantly hampered transplant activity in the past years and the COVID-19 pandemic in 2020.

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.