Metabolic adaption of cancer cells toward autophagy: Is there a role for ER-phagy?

Autophagy is an evolutionary conserved catabolic pathway that uses a unique double-membrane vesicle, called autophagosome, to sequester cytosolic components, deliver them to lysosomes and recycle amino-acids. Essentially, autophagy acts as a cellular cleaning system that maintains metabolic balance under basal conditions and helps to ensure nutrient viability under stress conditions. It is also an important quality control mechanism that removes misfolded or aggregated proteins and mediates the turnover of damaged and obsolete organelles. In this regard, the idea that autophagy is a non-selective bulk process is outdated. It is now widely accepted that forms of selective autophagy are responsible for metabolic rewiring in response to cellular demand. Given its importance, autophagy plays an essential role during tumorigenesis as it sustains malignant cellular growth by acting as a coping-mechanisms for intracellular and environmental stress that occurs during malignant transformation. Cancer development is accompanied by the formation of a peculiar tumor microenvironment that is mainly characterized by hypoxia (oxygen < 2%) and low nutrient availability. Such conditions challenge cancer cells that must adapt their metabolism to survive. Here we review the regulation of autophagy and selective autophagy by hypoxia and the crosstalk with other stress response mechanisms, such as UPR. Finally, we discuss the emerging role of ER-phagy in sustaining cellular remodeling and quality control during stress conditions that drive tumorigenesis.

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress.

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.

Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and Collagen quality control.

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.

Challenging Safety and Efficacy of Retinal Gene Therapies by Retinogenesis.

Gene-expression programs modulated by transcription factors (TFs) mediate key developmental events. Here, we show that the synthetic transcriptional repressor (TR; ZF6-DB), designed to treat Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP), does not perturb murine retinal development, while maintaining its ability to block Rho expression transcriptionally. To express ZF6-DB into the developing retina, we pursued two approaches, (i) the retinal delivery (somatic expression) of ZF6-DB by Adeno-associated virus (AAV) vector (AAV-ZF6-DB) gene transfer during retinogenesis and (ii) the generation of a transgenic mouse (germ-line transmission, TR-ZF6-DB). Somatic and transgenic expression of ZF6-DB during retinogenesis does not affect retinal function of wild-type mice. The P347S mouse model of RHO-adRP, subretinally injected with AAV-ZF6-DB, or crossed with TR-ZF6-DB or shows retinal morphological and functional recovery. We propose the use of developmental transitions as an effective mode to challenge the safety of retinal gene therapies operating at genome, transcriptional, and transcript levels.

Erector spine plane block as postoperative rescue analgesia in thoracic surgery.

OBJECTIVE: Erector spine plane block (ESPB) is a newly defined regional anesthesia technique performed by injection of local anesthetic beneath the erector spine muscle. We tested ESPB as a regional rescue analgesia bedside technique to be performed in the thoracic surgical ward, reporting a 7-patient case series. METHODS: We report our experience in rescue analgesia after thoracic surgery. During the postoperative stay, numeric rating scale (NRS) score >3 and inability to perform physiotherapy or effective cough due to postoperative pain represented the criteria for proposing rescue analgesia with ESPB. NRS at rest and during movements was recorded; blood gas analysis and spirometry were performed to evaluate PaO2/FiO2 (P/F), forced vital capacity (FVC), and forced expiratory volume in 1 second (FEV1) before ESPB execution. After performing the ESPB, static and dynamic NRS, P/F, and FVC and FEV1 were recorded at 40 minutes and 80 minutes. RESULTS: NRS had a reduction at rest and in dynamic assessment. The P/F did not improve but spirometric measures improved. FVC had a relevant improvement only after 80 minutes; FEV1 was increased after 40 minutes. CONCLUSION: The use of ESPB as postoperative rescue analgesia can offer several advantages due to effective rescue analgesia and safety that makes it easy to perform in the thoracic surgical ward or in an outpatient clinic setting.

Effectiveness of home-based preoperative pulmonary rehabilitation in COPD patients undergoing lung cancer resection.

OBJECTIVE: To investigate the effectiveness of a home-based preoperative rehabilitation program for improving preoperative lung function and surgical outcome of patients with chronic obstructive pulmonary disease (COPD) undergoing lobectomy for cancer. METHODS: This was a prospective, observational, single-center study including 59 patients with mild COPD who underwent lobectomy for lung cancer. All patients attended a home-based preoperative rehabilitation program including a minimum of 3 sessions each week for 4 weeks. Each session included aerobic and anaerobic exercises. Participants recorded the frequency and the duration of exercise performed in a diary. The primary end point was to evaluate changes in lung function including predicted postoperative (PPO) forced expiratory volume in 1 second (FEV1), 6-minute walking distance test (6MWD), PPO diffusing capacity for carbon monoxide (DLCO) %, and blood gas analysis values before and after the rehabilitation program. Postoperative pulmonary complications were recorded and multivariable analysis was used to identify independent prognostic factors (secondary end point). RESULTS: All patients completed the 4-week rehabilitation program. Thirteen of 59 (22%) patients (Group A) performed <3 sessions per week (mean sessions per week: 2.3±1.3); 46 of 59 (78%) patients (Group B) performed ⩾3 sessions per week (mean sessions per week: 3.5±1.6). The comparison of PPO FEV1% and 6MWD before and after rehabilitation showed a significant improvement only in Group B. No significant changes in PPO DLCO% or in blood gas analysis values were seen. Nine patients presented postoperative pulmonary complications, including atelectasis (n = 6), pneumonia (n = 1), respiratory failure (n = 1), and pulmonary embolism (n = 1). Group A presented higher number of postoperative pulmonary complications than Group B (6 vs 3; p = 0.0005). Multivariate analysis showed that the number of weekly rehabilitation sessions was the only independent predictive factor (p = 0.001). CONCLUSIONS: Our simple and low-cost rehabilitation program could improve preoperative clinical function in patients with mild to moderate COPD undergoing lobectomy and reduce postoperative pulmonary complications. All patients should be motivated to complete at least 3 rehabilitation sessions per week in order to obtain significant clinical benefits. Our preliminary results should be confirmed by larger prospective studies.

Preoperative optimization with levosimendan in heart failure patient undergoing thoracic surgery.

INTRODUCTION: We present the case of a patient with dilatative cardiomyopathy waiting for heart transplantation with pleural effusion to be subjected to pleural biopsy, treated with preoperative infusion of levosimendan to improve heart performances. PRESENTATION OF CASE: A 56-year-old man (BMI 22,49) with dilatative cardiomyopathy (EF 18%) presented right pleural effusion. The levosimendan treatment protocol consisted of 24h continuous infusion (0,1ug/kg/min), without bolus. The patient was under continuous hemodynamic monitoring prior, during and after levosimendan administration. The surgery for pleural biopsy was performed with uniportal Video Assisted Thoracoscopic approach (VATS). DISCUSSION: A significant increase of Cardiac Index (CI) and Stroke Volume Index (SVI) were observed at 4h after infusion initiation and was sustained during the next 24h after the end of infusion. Levosimendan administration was safe. CONCLUSION: In this case the prophylactic preoperative levosimendan administration is safe and effective in cardiac failure patient undergoing thoracic surgery, but prophylactic preoperative levosimendan treatment in these patients merits further study.

Intraoperative Extracorporeal Carbon Dioxide Removal During Apneic Oxygenation with an EZ-Blocker in Tracheal Surgery.

Tracheal surgery requires continued innovation to manage the anesthetic during an open airway phase. A common approach is apneic oxygenation with continuous oxygen flow, but the lack of effective ventilation causes hypercapnia, with respiratory acidosis. We used extracorporeal carbon dioxide removal for intraoperative decapneization during apneic oxygenation in a 64-year-old woman who was scheduled for tracheal surgery because of tracheal stenosis caused by long-term intubation. Our findings demonstrate that even after 40 minutes of total apnea, using an EZ-blocker for oxygenation and external decapneization, hemodynamic and gas exchange variables never demonstrated any dangerous alterations.

Tracheal ring fracture secondary to percutaneous tracheostomy: is tracheal flaccidity a risk factor?

OBJECTIVE: To evaluate the risk factors of tracheal ring fracture and whether previous tracheal ring flaccidity predisposes to it in consecutive, mechanically ventilated, intensive care unit patients undergoing different percutaneous dilatational tracheostomy procedures (Ciaglia Blue Rhino, PercuTwist, and Ciaglia BlueDolphin). DESIGN: Single-center retrospective study performed between November 2006 and July 2013. SETTING: Single-center university hospital. PARTICIPANTS: Two hundred nineteen consecutive intensive care unit patients. INTERVENTIONS: Video bronchoscopic percutaneous dilatational tracheostomies using different techniques, including Ciaglia Blue Rhino, PercuTwist, and Ciaglia BlueDolphin, were performed consecutively. During the procedure, the tracheal wall response to the routine external palpation was evaluated endoscopically to find the interanular space. An abnormal change in the tracheal ring shape (fingerprint) with anterior airway wall collapse was diagnosed as tracheal flaccidity. MEASUREMENTS AND MAIN RESULTS: Tracheal ring fracture occurred in 21 patients (9.6%). The proportion of tracheal ruptures was 16 (76.2%) after PercuTwist, 3 (14.3%) after Ciaglia Blue Dolphin, and 2 (9.5%) after the Blue Rhino technique. Significant risk factors for tracheal rupture were PercuTwist procedure (p = 0.02), tracheal flaccidity (p = 0.0001), and a period of intubation before a percutaneous dilatational tracheostomy procedure>14 days (p = 0.01). CONCLUSIONS: In addition to the PercuTwist technique and intubation>14 days before tracheostomy, tracheal flaccidity was a significant risk factor for tracheal ring fracture. In the presence of this finding, a less traumatic tracheostomy procedure should be applied.

Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4(+)CD25(+)FOXP3(+) T regulatory cell subpopulations.

Adoptive transfer of ex vivo expanded CD4(+)CD25(+)FOXP3(+) regulatory T cells is a successful therapy for autoimmune diseases and transplant rejection in experimental models. In man, equivalent manipulations in bone marrow transplant recipients appear safe, but questions regarding the stability of the transferred regulatory T cells during inflammation remain unresolved. In this study, protocols for the expansion of clinically useful numbers of functionally suppressive and stable human regulatory T cells were investigated. Regulatory T cells were expanded in vitro with rapamycin and/or all-trans retinoic acid and then characterized under inflammatory conditions in vitro and in vivo in a humanized mouse model of graft-versus-host disease. Addition of rapamycin to regulatory T-cell cultures confirms the generation of high numbers of suppressive regulatory T cells. Their stability was demonstrated in vitro and substantiated in vivo. In contrast, all-trans retinoic acid treatment generates regulatory T cells that retain the capacity to secrete IL-17. However, combined use of rapamycin and all-trans retinoic acid abolishes IL-17 production and confers a specific chemokine receptor homing profile upon regulatory T cells. The use of purified regulatory T-cell subpopulations provided direct evidence that rapamycin can confer an early selective advantage to CD45RA(+) regulatory T cells, while all-trans retinoic acid favors CD45RA(-) regulatory T-cell subset. Expansion of regulatory T cells using rapamycin and all-trans retinoic acid drug combinations provides a new and refined approach for large-scale generation of functionally potent and phenotypically stable human regulatory T cells, rendering them safe for clinical use in settings associated with inflammation.

The therapeutic effect of mesenchymal stem cell transplantation in experimental autoimmune encephalomyelitis is mediated by peripheral and central mechanisms.

Stem cells are currently seen as a treatment for tissue regeneration in neurological diseases such as multiple sclerosis, anticipating that they integrate and differentiate into neural cells. Mesenchymal stem cells (MSCs), a subset of adult progenitor cells, differentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their clinical development, however, has been significantly boosted by the demonstration that MSCs display significant therapeutic plasticity mainly occurring through bystander mechanisms. These features have been exploited in the effective treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a significant amelioration of disease and decrease of demyelination, immune infiltrates and axonal loss. Surprisingly, these effects do not require MSCs to engraft in the central nervous system but depend on the cells' ability to inhibit pathogenic immune responses both in the periphery and inside the central nervous system and to release neuroprotective and pro-oligodendrogenic molecules favoring tissue repair. These results paved the road for the utilization of MSCs for the treatment of multiple sclerosis.

IL-17- and IFN-γ-secreting Foxp3+ T cells infiltrate the target tissue in experimental autoimmunity.

CD4(+)Foxp3(+) regulatory T cells (Tregs) have been considered crucial in controlling immune system homeostasis, and their derangement is often associated to autoimmunity. Tregs identification is, however, difficult because most markers, including CD25 and Foxp3, are shared by recently activated T cells. We show in this paper that CD4(+)Foxp3(+) T cells are generated in peripheral lymphoid organs on immunization and readily accumulate in the target organ of an autoimmune reaction, together with classical inflammatory cells, constituting up to 50% of infiltrating CD4(+) T cells. Most CD4(+)Foxp3(+) T cells are, however, CD25(-) and express proinflammatory cytokines such as IL-17 and IFN-γ, questioning their suppressive nature. Moreover, in vitro CD4(+) T lymphocytes from naive and autoimmune mice, stimulated to differentiate into Th1, Th2, Th17, and induced Tregs, display early mixed expression of lineage-specific markers. These results clearly point to an unprecedented plasticity of naive CD4(+) T cells, that integrating inflammatory signals may change their fate from the initial lineage commitment to a different functional phenotype.

Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation.

Rapamycin is an oral immunosuppressant drug previously reported to efficiently induce naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T ((n)T(reg)) cells re-establishing long-term immune self-tolerance in autoimmune diseases. We investigated the effect of rapamycin administration to SJL/j mice affected by PLP(139-151)-induced relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. Rapamycin induced suppression of IFN-gamma, and IL-17 release from antigen-specific T cells in peripheral lymphoid organs. While CD4(+)FoxP3(+) cells were unaffected, we observed disappearance of CD4(+)CD45RB(high) effector T (T(eff)) cells and selective expansion of T(reg) cells bearing the CD4(+)CD45RB(low)FoxP3(+)CD25(+)CD103(+) extended phenotype. Finally, the dual action of rapamycin on both T(eff) and T(reg) cells resulted in modulation of their ratio that closely paralleled disease course. Our data show that rapamycin inhibits RR-EAE, provide evidence for the immunological mechanisms, and indicate this compound as a potential candidate for the treatment of multiple sclerosis.

Prevalence and long-term course of macro-aspartate aminotransferase in children.

OBJECTIVES: To investigate the prevalence, association with clinical conditions, and long-term course of macro-aspartate aminotransferase (macro-AST). STUDY DESIGN: Forty-four children with an isolated elevation of serum AST were screened for macro-AST with electrophoresis and % polyethylene glycol (PEG) precipitable activity (PPA). RESULTS: All children were healthy, except they had elevated AST values. Seventeen children (38.6%) were macro-AST-positive. They had higher AST values than the 27 children who were macro-AST-negative (P = .001). Values <67.1% PPA and >82.2% PPA were associated with a very low probability of being macro-AST-positive and macro-AST-negative, respectively. Thirty-eight children underwent clinical and laboratory follow-up (mean, 4.7 +/- 3.8; range, 1-16 years). All remained symptom-free. AST levels decreased significantly only in children who were macro-AST-negative (P = .006). Macroenzyme persisted in 6 of the 9 children who were macro-AST-positive after 6.0 +/- 4.1 years. CONCLUSIONS: Macro-AST was present in more than one-third of children with an isolated increase of AST levels. The lack of pathological correlates in a long period argues for the benign nature of this phenomenon in childhood. We suggest that our %PPA thresholds can be used as a screening test and that electrophoresis be reserved for confirming positive screen test results and cases in which %PPA levels are of intermediate discriminant accuracy.

Immunogenicity and therapeutic efficacy of phage-displayed beta-amyloid epitopes.

In vitro and in vivo studies indicate that Alzheimer's Disease (AD) could be prevented or treated by active immunization against self-peptide beta-amyloid. In this study, we compared the immunogenicity of different regions of beta-amyloid, displayed on filamentous phages. We established that a filamentous phage displaying epitope 2-6 (AEFRH) of beta-amyloid at the N-terminus of Major Capside Protein (phage fdAD(2-6)) is more immunogenic than a phage displaying epitope 1-7 (DAEFRHD) that differs only in flanking residues. Monthly injections of fdAD(2-6) trigger a robust anti-beta-amyloid antibody response, and afford a significant reduction of plaque pathology in a mouse model of AD, whereas the same treatment, performed with phage fdAD(1-7), induces a lower anti-beta-amyloid titer and does not protect from amyloid deposition. "Memory" anti-amyloid antibodies induced by a single prime-boost cycle with vaccine fdAD(2-6), that have a lower titer compared to antibodies induced by monthly restimulations, do not prevent plaque pathology. Our data show that optimization of epitope display is essential in vaccine design, and suggest that the titer of the anti-amyloid response is the crucial parameter to obtain therapeutic efficacy in vivo.