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Several studies demonstrated that mitochondrial dynamics and metabolic pathways control T cell fate in the periphery. However, little is known about their implication in thymocyte development. Our results showed that thymic progenitors (CD3-CD4-CD8-triple negative, TN), in active division, have essentially a fused mitochondrial morphology and rely on high glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). As TN cells differentiate to double positive (DP, CD4+CD8+) and single positive (SP, CD4+ and CD8+) stages, they became more quiescent, their mitochondria fragment and they downregulate glycolysis and OXPHOS. Accordingly, in vitro inhibition of the mitochondrial fission during progenitor differentiation on OP9-DL4 stroma, affected the TN to DP thymocyte transition by enhancing the percentage of TN and reducing that of DP, leading to a decrease in the total number of thymic cells including SP T cells. We demonstrated that the stage 3 triple negative pre-T (TN3) and the stage 4 triple negative pre-T (TN4) have different metabolic and functional behaviors. While their mitochondrial morphologies are both essentially fused, the LC-MS based analysis of their metabolome showed that they are distinct: TN3 rely more on OXPHOS whereas TN4 are more glycolytic. In line with this, TN4 display an increased Hexokinase II expression in comparison to TN3, associated with high proliferation and glycolysis. The in vivo inhibition of glycolysis using 2-deoxyglucose (2-DG) and the absence of IL-7 signaling, led to a decline in glucose metabolism and mitochondrial membrane potential. In addition, the glucose/IL-7R connection affects the TN3 to TN4 transition (also called b-selection transition), by enhancing the percentage of TN3, leading to a decrease in the total number of thymocytes. Thus, we identified additional components, essential during b-selection transition and playing a major role in thymic development. [ABSTRACT FROM AUTHOR]

Simple Summary: Triple-negative breast cancer (TNBC) is characterized by a quick and high rate of recurrence. The benefits from neo-adjuvant chemotherapy associated with anti-PDL1 have been shown to be efficient in this aggressive form of breast cancer. Ferroptosis inducers (erastin/RSL3) induced the upregulation of CD274 in TNBC cells. Basal and TNBC subtypes of breast cancers overexpressed CD274 conjointly with three ferroptosis drivers: TNFAIP3, IFNG and IDO1 (IDO1: inhibitory immune checkpoint). These tumors present higher levels of recurrence. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested in recurrent TNBC. (1) Background: Triple-negative breast cancer (TNBC) is a distinct subgroup of breast cancer presenting a high level of recurrence, and neo-adjuvant chemotherapy is beneficial in its therapy management. Anti-PD-L1 immunotherapy improves the effect of neo-adjuvant therapy in TNBC. (2) Methods: Immune-modulation and ferroptosis-related R-packages were developed for integrative omics analyses under ferroptosis-inducer treatments: TNBC cells stimulated with ferroptosis inducers (GSE173905, GSE154425), single cell data (GSE191246) and mass spectrometry on breast cancer stem cells. Clinical association analyses were carried out with breast tumors (TCGA and METABRIC cohorts). Protein-level validation was investigated through protein atlas proteome experiments. (3) Results: Erastin/RSL3 ferroptosis inducers upregulate CD274 in TNBC cells (MDA-MB-231 and HCC38). In breast cancer, CD274 expression is associated with overall survival. Breast tumors presenting high expression of CD274 upregulated some ferroptosis drivers associated with prognosis: IDO1, IFNG and TNFAIP3. At the protein level, the induction of Cd274 and Tnfaip3 was confirmed in breast cancer stem cells under salinomycin treatment. In a 4T1 tumor treated with cyclophosphamide, the single cell expression of Cd274 was found to increase both in myeloid- and lymphoid-infiltrated cells, independently of its receptor Pdcd1. The CD274 ferroptosis-driver score computed on a breast tumor transcriptome stratified patients on their prognosis: low score was observed in the basal subgroup, with a higher level of recurrent risk scores (oncotypeDx, ggi and gene70 scores). In the METABRIC cohort, CD274, IDO1, IFNG and TNFAIP3 were found to be overexpressed in the TNBC subgroup. The CD274 ferroptosis-driver score was found to be associated with overall survival, independently of TNM classification and age diagnosis. The tumor expression of CD274, TNFAIP3, IFNG and IDO1, in a biopsy of breast ductal carcinoma, was confirmed at the protein level (4) Conclusions: Ferroptosis inducers upregulate PD-L1 in TNBC cells, known to be an effective target of immunotherapy in high-risk early TNBC patients who received neo-adjuvant therapy. Basal and TNBC tumors highly expressed CD274 and ferroptosis drivers: IFNG, TNFAIP3 and IDO1. The CD274 ferroptosis-driver score is associated with prognosis and to the risk of recurrence in breast cancer. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested for recurrent TNBC. [ABSTRACT FROM AUTHOR]

Background The association between different phenotypes and genotypes of circulating tumor cells (CTCs) and efficacy of neoadjuvant chemotherapy (NAC) remains uncertain. This study was conducted to evaluate the relationship of FTH1 gene-associated CTCs (F-CTC) with/without epithelial-mesenchymal transition (EMT) markers, or their dynamic changes with the efficacy of NAC in patients with non-metastatic breast cancer. Patients and Methods This study enrolled 120 patients with non-metastatic breast cancer who planned to undergo NAC. The FTH1 gene and EMT markers in CTCs were detected before NAC (T0), after 2 cycles of chemotherapy (T1), and before surgery (T2). The associations of these different types of CTCs with rates of pathological complete response (pCR) and breast-conserving surgery (BCS) were evaluated using the binary logistic regression analysis. Results F-CTC in peripheral blood ≥1 at T0 was an independent factor for pCR rate in patients with HER2-positive (odds ratio [OR]=0.08, 95% confidence interval [CI], 0.01-0.98, P =.048). The reduction in the number of F-CTC at T2 was an independent factor for BCS rate (OR = 4.54, 95% CI, 1.14-18.08, P =.03). Conclusions The number of F-CTC prior to NAC was related to poor response to NAC. Monitoring of F-CTC may help clinicians formulate personalized NAC regimens and implement BCS for patients with non-metastatic breast cancer. [ABSTRACT FROM AUTHOR]

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in women and causes a high number of mortality among this population due to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy, and inhibit distant metastases, potentially impacting the tumor microenvironment. (2) Methods: Through data mining, the ferroptosis/extracellular matrix remodeling literature text-mining results were integrated into the breast cancer transcriptome cohort, taking into account patients with distant relapse-free survival (DRFS) under adjuvant therapy (anthracyclin + taxanes) with validation in an independent METABRIC cohort, along with the MDA-MB-231 and HCC338 transcriptome functional experiments with ferroptosis activations (GSE173905). (3) Results: Ferroptosis/extracellular matrix remodeling text-mining identified 910 associated genes. Univariate Cox analyses focused on breast cancer (GSE25066) selected 252 individual significant genes, of which 170 were found to have an adverse expression. Functional enrichment of these 170 adverse genes predicted basal breast cancer signatures. Through text-mining, some ferroptosis-significant adverse-selected genes shared citations in the domain of ECM remodeling, such as TNF, IL6, SET, CDKN2A, EGFR, HMGB1, KRAS, MET, LCN2, HIF1A, and TLR4. A molecular score based on the expression of the eleven genes was found predictive of the worst prognosis breast cancer at the univariate level: basal subtype, short DRFS, high-grade values 3 and 4, and estrogen and progesterone receptor negative and nodal stages 2 and 3. This eleven-gene signature was validated as regulated by ferroptosis inductors (erastin and RSL3) in the triple-negative breast cancer cellular model MDA-MB-231. (4) Conclusions: The crosstalk between ECM remodeling-ferroptosis functionalities allowed for defining a molecular score, which has been characterized as an independent adverse parameter in the prognosis of breast cancer patients. The gene signature of this molecular score has been validated to be regulated by erastin/RSL3 ferroptosis activators. This molecular score could be promising to evaluate the ECM-related impact of ferroptosis target therapies in breast cancer. [ABSTRACT FROM AUTHOR]

Autophagy, a lysosome-mediated cellular degradation pathway, recycles intracellular components to maintain metabolic balance and survival. Autophagy plays an important role in tumor immunotherapy as a "double-edged sword" that can both promote and inhibit tumor progression. Autophagy acts on innate and adaptive immunity and interacts with immune cells to modulate tumor immunotherapy. The discovery of autophagy inducers and autophagy inhibitors also provides new insights for clinical anti-tumor therapy. However, there are also difficulties in the application of autophagy-related regulators, such as low bioavailability and the lack of efficient selectivity. This review focuses on autophagy-related immunogenic regulation and its application in cancer therapy. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment. Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone. [ABSTRACT FROM AUTHOR]

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito‐C. Mito‐C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito‐C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF‐1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER–mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito‐C counteracts dengue virus‐induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito‐C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti‐viral research. Synopsis: This study identifies the small molecule Mito‐C as an activator of mitochondrial fragmentation. Mito‐C suppresses dengue virus replication by counteracting virus induced mitochondrial fusion. Mito‐C targets the NEET family proteins and stabilizes ER‐mitochondria contact sites.NAF‐1, a NEET family protein, promotes Drp1 recruitment to ER‐mitochondria contact sites and induces mitochondrial fragmentation.Mito‐C promotes DRP1 local recruitment to mitochondria and mitochondrial fragmentation.Mito‐C inhibits dengue virus induced mitochondrial fusion and suppresses viral replication. [ABSTRACT FROM AUTHOR]

Abstract: The modulation of macroautophagy is now recognized as one of the hallmarks of cancer cells. There is accumulating evidence that autophagy plays a role in the various stages of tumorigenesis. Depending on the type of cancer and the context, macroautophagy can be tumor suppressor or it can help cancer cells to overcome metabolic stress and the cytotoxicity of chemotherapy. Recent studies have shed light on the role of macroautophagy in tumor-initiating cells, in tumor immune response cross-talk with the microenvironment. This review is intended to provide an up-date on these aspects, and to discuss them with regard to the role of the major signaling sub-networks involved in tumor progression (Beclin 1, MTOR, p53 and RAS) and in regulating autophagy. [Copyright &y& Elsevier]

The role of TH17 cells in cancer is being investigated, but the existence of tumor antigen-specific TH17 cells has yet to be ascertained. Here, we report the first description of a spontaneous TH17 (IL-17+) response to the important tumor antigen MAGE-A3, which occurred concurrently with a TH1 (IFN-γ+) response in a lung cancer patient. MAGE-A3-specific interleukin (IL)-17+) T cells were mainly CCR7+) central memory T cells, whereas IFN-γ+) cells were enriched for CCR7-) effector memory T cells. An assessment of the fine specificity of antigen recognition by these T cells indicated that the CCR6+) CCR4+) and CCR6+)CXCR3+) fractions contained the same TH17/TH1 population at early and late differentiation stages, respectively, whereas the CCR6-) CXCR3+) fraction contained a distinct TH1 population. These findings are important because they suggest a differentiation model in which tumor antigen-specific CD4+) T cells that are primed under TH17 polarizing conditions will progressively convert into IFN-γ-secreting cells in vivo as they differentiate into effector T cells that can effectively attack tumors. [ABSTRACT FROM AUTHOR]

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)- invasive ductal carcinomas of high grade, including both HER2- and HER2+ tumors. In line with these results, we detected ESO expression in 20% of primary HR- BC, including both ESO Ab+ and Ab- patients, but not in HR+ BC. Interestingly, whereas expression levels in ESO+ BC were not significantly different between ESO Ab+ and Ab- patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR- (HER2- or HER2+) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are a distinct subpopulation of tumor cells with stem cell-like features. Able to initiate and sustain tumor growth and mostly resistant to anti-cancer therapies, they are thought responsible for tumor recurrence and metastasis. Recent accumulated evidence supports that iron metabolism with the recent discovery of ferroptosis constitutes a promising new lead in the field of anti-CSC therapeutic strategies. Indeed, iron uptake, efflux, storage and regulation pathways are all over-engaged in the tumor microenvironment suggesting that the reprogramming of iron metabolism is a crucial occurrence in tumor cell survival. In particular, recent studies have highlighted the importance of iron metabolism in the maintenance of CSCs. Furthermore, the high concentration of iron found in CSCs, as compared to non-CSCs, underlines their iron addiction. In line with this, if iron is an essential macronutrient that is nevertheless highly reactive, it represents their Achilles' heel by inducing ferroptosis cell death and therefore providing opportunities to target CSCs. In this review, we first summarize our current understanding of iron metabolism and its regulation in CSCs. Then, we provide an overview of the current knowledge of ferroptosis and discuss the role of autophagy in the (regulation of) ferroptotic pathways. Finally, we discuss the potential therapeutic strategies that could be used for inducing ferroptosis in CSCs to treat cancer. [ABSTRACT FROM AUTHOR]

Abstract Iron is an essential nutrient that facilitates cell proliferation and growth. Iron can be detrimental, however. The ability of iron to cycle between oxidized and reduced forms contributes to the formation of free radicals. An excess of free radicals leads to lipid peroxidation, more reactive oxygen species and oxidative stress, damage to DNA and other biomolecules, and, if potentially, tumorigenesis. Iron also has a role in the maintenance of the tumor microenvironment and in metastasis. Pathways of iron acquisition, efflux, storage, and regulation are all perturbed in cancer, suggesting that reprogramming of iron metabolism is a central aspect of tumor cell survival. Recent studies have shed light on the role of iron metabolism in cancer stem cells (CSC) and suggest that specific targeting of iron metabolism in CSCs may improve the efficacy of cancer therapy. In this review, we first summarize briefly our current understanding of the intracellular processes involving iron, the effect of dietary iron, and its relation to cancer. We emphasize the importance of modifier "iron genes" in cancer and the possibility that these genes may encode biomarkers that may be used clinically. We then provide an update on the role of iron in metabolic reprogramming, the epithelial-mesenchymal transition, and the regulation of epigenetic marks essential for CSC maintenance and plasticity. Finally, we discuss the potential of targeting a recently discovered form of iron-regulated cell death, ferroptosis, in CSCs for treatment of cancer. [ABSTRACT FROM AUTHOR]

Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress. [ABSTRACT FROM AUTHOR]

(1) Background: Triple-negative breast cancer (TNBC) is a distinct subgroup of breast cancer presenting a high level of recurrence, and neo-adjuvant chemotherapy is beneficial in its therapy management. Anti-PD-L1 immunotherapy improves the effect of neo-adjuvant therapy in TNBC. (2) Methods: Immune-modulation and ferroptosis-related R-packages were developed for integrative omics analyses under ferroptosis-inducer treatments: TNBC cells stimulated with ferroptosis inducers (GSE173905, GSE154425), single cell data (GSE191246) and mass spectrometry on breast cancer stem cells. Clinical association analyses were carried out with breast tumors (TCGA and METABRIC cohorts). Protein-level validation was investigated through protein atlas proteome experiments. (3) Results: Erastin/RSL3 ferroptosis inducers upregulate CD274 in TNBC cells (MDA-MB-231 and HCC38). In breast cancer, CD274 expression is associated with overall survival. Breast tumors presenting high expression of CD274 upregulated some ferroptosis drivers associated with prognosis: IDO1, IFNG and TNFAIP3. At the protein level, the induction of Cd274 and Tnfaip3 was confirmed in breast cancer stem cells under salinomycin treatment. In a 4T1 tumor treated with cyclophosphamide, the single cell expression of Cd274 was found to increase both in myeloid- and lymphoid-infiltrated cells, independently of its receptor Pdcd1. The CD274 ferroptosis-driver score computed on a breast tumor transcriptome stratified patients on their prognosis: low score was observed in the basal subgroup, with a higher level of recurrent risk scores (oncotypeDx, ggi and gene70 scores). In the METABRIC cohort, CD274, IDO1, IFNG and TNFAIP3 were found to be overexpressed in the TNBC subgroup. The CD274 ferroptosis-driver score was found to be associated with overall survival, independently of TNM classification and age diagnosis. The tumor expression of CD274, TNFAIP3, IFNG and IDO1, in a biopsy of breast ductal carcinoma, was confirmed at the protein level (4) Conclusions: Ferroptosis inducers upregulate PD-L1 in TNBC cells, known to be an effective target of immunotherapy in high-risk early TNBC patients who received neo-adjuvant therapy. Basal and TNBC tumors highly expressed CD274 and ferroptosis drivers: IFNG, TNFAIP3 and IDO1. The CD274 ferroptosis-driver score is associated with prognosis and to the risk of recurrence in breast cancer. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested for recurrent TNBC.

Iron is an essential nutrient to life. However, the ability of iron to cycle between the oxidized and reduced forms contributes to the formation of reactive oxygen species. The generation of free radicals leads to oxidative stress and the initiation of signaling pathways involved in cell survival or cell death. The iron homeostasis is very carefully regulated and dysregulation of iron metabolism contributes to various human pathologies. The work carried out in recent years has revealed new cellular processes and mechanisms like ferritinophagy, that deepen our understanding of iron homeostasis. Ferritinophagy is a form of selective macroautophagy whereby ferritin, an iron storage protein, is degraded in the lysosome. Here, we describe iron homeostasis and review recent discoveries regarding the mechanism of ferritinophagy and its relationship to a new form of cell-death iron-dependent, the ferroptosis., (© 2017 médecine/sciences – Inserm.)

Macroautophagy (hereafter called autophagy) is a vacuolar, lysosomal pathway for catabolism of intracellular material that is conserved among eukaryotic cells. Autophagy plays a crucial role in tissue homeostasis, adaptation to stress situations, immune responses, and the regulation of the inflammatory response. Blockade or uncontrolled activation of autophagy is associated with cancer, diabetes, obesity, cardiovascular disease, neurodegenerative disease, autoimmune disease, infection, and chronic inflammatory disease. During the past decade, researchers have made major progress in understanding the three levels of regulation of autophagy in mammalian cells: signaling, autophagosome formation, and autophagosome maturation and lysosomal degradation. As we discuss in this review, each of these levels is potentially druggable, and, depending on the indication, may be able to stimulate or inhibit autophagy. We also summarize the different modulators of autophagy and their potential and limitations in the treatment of life-threatening diseases.

The cancer testis antigen (CTA), melanoma-associated antigen A3/6 (MAGE-A3/6), is expressed in human cancers of different histologic types, to variable extents, and is an important target for immunotherapy. In this study, to address the potential of MAGE-A3/6 as an immunotherapeutic target in breast cancer (BC), we assessed MAGE-A3/6 expression by PCR in a cohort of 362 primary BC tumors and analyzed the correlation between MAGE-A3/6 expression, tumors hormone receptor (HR) status, and other clinicopathologic features. We found expression of MAGE-A3/6 in 10% of primary BC tumors. MAGE-A3/6 expression was significantly correlated with estrogen receptor (ER) and progesterone receptor (PR) negative status and was frequent in ER (29%) and in PR (24%) tumors. MAGE-A3/6 expression was also significantly associated with high histologic grade but not with patients age, tumor size, tumor type, lymph-node invasion, and human epidermal growth factor receptor 2 (HER2) overexpression. Consistent with the associated poor clinicopathologic features, patients with MAGE-A3/6-expressing tumors had a worse disease-specific survival as compared with patients with MAGE-A3/6 tumors. The frequent expression of MAGE-A3/6 in tumors of patients with primary HR BC, who have, for a large part, limited therapeutic options, encourages the selection of BC patients bearing MAGE-A3/6-expressing tumors for targeted immunotherapy.

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K (+) /H (+) ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH (+) population in HMLER cells than in the ALDH (-) population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH (+) population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH (+) population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.

Macroautophagy is an evolutionarily conserved homeostatic process that mediates the degradation of long-lived cytoplasmic components in eukaryotes, which allows cells to survive stresses such as inflammation, hypoxia, and deprivation of nutrients or growth factors. At least 30 members of the Atg (autophagy-related) protein family orchestrate this degradative process. Additional complexity resides in the signaling networks controlling the autophagic process, which include various posttranslational modifications of key components. Evidence is accumulating that protein acetylation represents an evolutionarily conserved mechanism tightly regulating macroautophagy.

The role of T(H)17 cells in cancer is being investigated, but the existence of tumor antigen-specific T(H)17 cells has yet to be ascertained. Here, we report the first description of a spontaneous T(H)17 (IL-17(+)) response to the important tumor antigen MAGE-A3, which occurred concurrently with a T(H)1 (IFN-γ(+)) response in a lung cancer patient. MAGE-A3-specific interleukin (IL)-17(+) T cells were mainly CCR7(+) central memory T cells, whereas IFN-γ(+) cells were enriched for CCR7(-) effector memory T cells. An assessment of the fine specificity of antigen recognition by these T cells indicated that the CCR6(+)CCR4(+) and CCR6(+)CXCR3(+) fractions contained the same T(H)17/T(H)1 population at early and late differentiation stages, respectively, whereas the CCR6(-)CXCR3(+) fraction contained a distinct T(H)1 population. These findings are important because they suggest a differentiation model in which tumor antigen-specific CD4(+) T cells that are primed under T(H)17 polarizing conditions will progressively convert into IFN-γ-secreting cells in vivo as they differentiate into effector T cells that can effectively attack tumors.

Human primary melanoma cells (T1) were found to be more susceptible to lysis by a Melan-A/MART-1-specific CTL clone (LT12) than their metastatic derivative (G1). We show that this differential susceptibility does not involve antigen presentation by target cells, synapse formation between the metastatic target and CTL clone, or subsequent granzyme B (GrB) polarization. Although PI-9, an inhibitor of GrB, was found to be overexpressed in metastatic G1 cells, knockdown of the PI-9 gene did not result in the attenuation of G1 resistance to CTL-induced killing. Interestingly, we show that whereas T1 cells express high levels of intercellular adhesion molecule-1 (ICAM-1), a dramatically reduced expression was noted on G1 cells. We also showed that sorted ICAM-1+ G1 cells were highly sensitive to CTL-induced lysis compared with ICAM-1- G1 cells. Furthermore, incubation of metastatic G1 cells with IFN-gamma resulted in the induction of ICAM-1 and the potentiation of their susceptibility to lysis by LT12. More importantly, we found that the level of ICAM-1 expression by melanoma cells correlated with decreased PTEN activity. ICAM-1 knockdown in T1 cells resulted in increased phosphorylation of PTEN and the subsequent activation of AKT. We have additionally shown that inhibition of the phosphatidylinositol (3,4,5)-triphosphate kinase (PI3K)/AKT pathway by the specific inhibitor wortmannin induced a significant potentiation of susceptibility of G1 and ICAM-1 small interfering RNA-treated T1 cells to CTL-induced lysis. The present study shows that a shift in ICAM-1 expression, which was associated with an activation of the PI3K/AKT pathway, can be used by metastatic melanoma cells to escape CTL-mediated killing.

We investigated the relationship between the resistance to the proapoptotic action of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and cellular prion protein (PrPc) function, using the TRAIL-sensitive MCF-7 human breast adenocarcinoma cell line and two TRAIL-resistant sublines: 2101 and MCF-7/ADR. All of the cell lines tested expressed TRAIL-R1 and TRAIL-R2. TRAIL decoy receptors were not detected, suggesting that the resistance of 2101 and MCF-7/ADR cells, strongly expressing PrPc, to TRAIL-mediated cell death was independent from the expression of TRAIL receptors and death-inducing signaling complex formation. Down-regulation of PrPc by small interfering RNA increased the sensitivity of Adriamycin- and TRAIL-resistant cells to TRAIL, but not to epirubicin/Adriamycin. TRAIL-mediated apoptosis in PrPc knocked-down cells was associated with caspase processing, Bid cleavage, and Mcl-1 degradation. In addition, an increased sensitivity of apoptosis-resistant cells to TRAIL after PrPc silencing was not associated with the increased recruitment of receptors and intracellular signaling molecule to the death-inducing signaling complex. Bcl-2 expression was substantially decreased after PrPc knock-down but the levels of Bcl-X(L) and Mcl-1 were not affected. The down-regulation of Bcl-2 was concomitant with Bax delocalization. Our findings support the notion that silencing of PrPc facilitates the activation of proapoptotic Bax by down-regulation of Bcl-2 expression, thereby abolishing the resistance of breast cancer cells to TRAIL-induced apoptosis.