Treatment of breast cancer with autophagy inhibitory microRNAs carried by AGO2-conjugated nanoparticles.

Nanoparticle based gene delivery systems holds great promise. Superparamagnetic iron oxide nanoparticles (SPIONs) are being heavily investigated due to good biocompatibility and added diagnostic potential, rendering such nanoparticles theranostic. Yet, commonly used cationic coatings for efficient delivery of such anionic cargos, results in significant toxicity limiting translation of the technology to the clinic. Here, we describe a highly biocompatible, small and non-cationic SPION-based theranostic nanoparticles as novel gene therapy agents. We propose for the first-time, the usage of the microRNA machinery RISC complex component Argonaute 2 (AGO2) protein as a microRNA stabilizing agent and a delivery vehicle. In this study, AGO2 protein-conjugated, anti-HER2 antibody-linked and fluorophore-tagged SPION nanoparticles were developed (SP-AH nanoparticles) and used as a carrier for an autophagy inhibitory microRNA, MIR376B. These functionalized nanoparticles selectively delivered an effective amount of the microRNA into HER2-positive breast cancer cell lines in vitro and in a xenograft nude mice model of breast cancer in vivo, and successfully blocked autophagy. Furthermore, combination of the chemotherapy agent cisplatin with MIR376B-loaded SP-AH nanoparticles increased the efficacy of the anti-cancer treatment both in vitro in cells and in vivo in the nude mice. Therefore, we propose that AGO2 protein conjugated SPIONs are a new class of theranostic nanoparticles and can be efficiently used as innovative, non-cationic, non-toxic gene therapy tools for targeted therapy of cancer.

Artificial intelligence assisted patient blood and urine droplet pattern analysis for non-invasive and accurate diagnosis of bladder cancer.

Bladder cancer is one of the most common cancer types in the urinary system. Yet, current bladder cancer diagnosis and follow-up techniques are time-consuming, expensive, and invasive. In the clinical practice, the gold standard for diagnosis remains invasive biopsy followed by histopathological analysis. In recent years, costly diagnostic tests involving the use of bladder cancer biomarkers have been developed, however these tests have high false-positive and false-negative rates limiting their reliability. Hence, there is an urgent need for the development of cost-effective, and non-invasive novel diagnosis methods. To address this gap, here we propose a quick, cheap, and reliable diagnostic method. Our approach relies on an artificial intelligence (AI) model to analyze droplet patterns of blood and urine samples obtained from patients and comparing them to cancer-free control subjects. The AI-assisted model in this study uses a deep neural network, a ResNet network, pre-trained on ImageNet datasets. Recognition and classification of complex patterns formed by dried urine or blood droplets under different conditions resulted in cancer diagnosis with a high specificity and sensitivity. Our approach can be systematically applied across droplets, enabling comparisons to reveal shared spatial behaviors and underlying morphological patterns. Our results support the fact that AI-based models have a great potential for non-invasive and accurate diagnosis of malignancies, including bladder cancer.

Role of autophagy in cancer-associated fibroblast activation, signaling and metabolic reprograming.

Tumors not only consist of cancerous cells, but they also harbor several normal-like cell types and non-cellular components. cancer-associated fibroblasts (CAFs) are one of these cellular components that are found predominantly in the tumor stroma. Autophagy is an intracellular degradation and quality control mechanism, and recent studies provided evidence that autophagy played a critical role in CAF formation, metabolic reprograming and tumor-stroma crosstalk. Therefore, shedding light on the autophagy and its role in CAF biology might help us better understand the roles of CAFs and the TME in cancer progression and may facilitate the exploitation of more efficient cancer diagnosis and treatment. Here, we provide an overview about the involvement of autophagy in CAF-related pathways, including transdifferentiation and activation of CAFs, and further discuss the implications of targeting tumor stroma as a treatment option.

Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis.

Macroautophagy/autophagy is an evolutionarily conserved cellular stress response mechanism. Autophagy induction in the tumor microenvironment (stroma) has been shown to support tumor metabolism. However, cancer cell-derived secreted factors that initiate communication with surrounding cells and stimulate autophagy in the tumor microenvironment are not fully documented. We identified CTF1/CT-1 (cardiotrophin 1) as an activator of autophagy in fibroblasts and breast cancer-derived carcinoma-associated fibroblasts (CAFs). We showed that CTF1 stimulated phosphorylation and nuclear translocation of STAT3, initiating transcriptional activation of key autophagy proteins. Additionally, following CTF1 treatment, AMPK and ULK1 activation was observed. We provided evidence that autophagy was important for CTF1-dependent ACTA2/α-SMA accumulation, stress fiber formation and fibroblast activation. Moreover, promotion of breast cancer cell migration and invasion by activated fibroblasts depended on CTF1 and autophagy. Analysis of the expression levels of CTF1 in patient-derived breast cancer samples led us to establish a correlation between CTF1 expression and autophagy in the tumor stroma. In line with our in vitro data on cancer migration and invasion, higher levels of CTF1 expression in breast tumors was significantly associated with lymph node metastasis in patients. Therefore, CTF1 is an important mediator of tumor-stroma interactions, fibroblast activation and cancer metastasis, and autophagy plays a key role in all these cancer-related events.Abbreviations: ACTA2/α-SMA: actin, alpha 2, smooth muscle CAFs: cancer- or carcinoma-associated fibroblasts CNT Ab.: control antibody CNTF: ciliary neurotrophic factor CTF1: cardiotrophin 1 CTF1 Neut. Ab.: CTF1-specific neutralizing antibody GFP-LC3 MEF: GFP-fused to MAP1LC3 protein transgenic MEF LIF: leukemia inhibitory factor IL6: interleukin 6 MEFs: mouse embryonic fibroblasts MEF-WT: wild-type MEFs OSM: oncostatin M TGFB/TGFß: transforming growth factor beta.

Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.

Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson's Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy.

A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress.

The maintenance of cellular homeostasis in living organisms requires a balance between anabolic and catabolic reactions. Macroautophagy (autophagy herein) is determined as one of the major catabolic reactions. Autophagy is an evolutionarily conserved stress response pathway that is activated by various insults including DNA damage. All sorts of damage to DNA potentially cause loss of genetic information and trigger genomic instability. Most of these lesions are repaired by the activation of DNA damage response following DNA repair mechanisms. Here we describe, a novel protein complex containing the autophagy protein ATG5 and the non-homologous end-joining repair system proteins. We discovered for the first time that ATG5 interacted with both Ku80 (XRCC5) and Ku70 (XRCC6). This novel interaction is facilitated mainly via Ku70. Our results suggest that this interaction is dynamic and enhanced upon genotoxic stresses. Strikingly, we identified that ATG5-Ku70 interaction is necessary for DNA repair and effective recovery from genotoxic stress. Therefore, our results are demonstrating a novel, direct, dynamic, and functional interaction between ATG5 and Ku70 proteins that plays a crucial role in DNA repair under genotoxic stress conditions.

Autophagy and Hepatic Tumor Microenvironment Associated Dormancy.

The goal of successful cancer treatment is targeting the eradication of cancer cells. Although surgical removal of the primary tumors and several rounds of chemo- and radiotherapy reduce the disease burden, in some cases, asymptomatic dormant cancer cells may still exist in the body. Dormant cells arise from the disseminated tumor cells (DTCs) from the primary lesion. DTCs escape from immune system and cancer therapy and reside at the secondary organ without showing no sign of proliferation. However, under some conditions. dormant cells can be re-activated and enter a proliferative state even after decades. As a stress response mechanism, autophagy may help the adaptation of DTCs at this futile foreign microenvironment and may control the survival and re-activation of dormant cells. Studies indicate that hepatic microenvironment serves a favorable condition for cancer cell dormancy. Although, no direct study was pointing out the role of autophagy in liver-assisted dormancy, involvement of autophagy in both liver microenvironment, health, and disease conditions has been indicated. Therefore, in this review article, we will summarize cancer dormancy and discuss the role and importance of autophagy and hepatic microenvironment in this context.

Autophagy and Cancer Dormancy.

Metastasis and relapse account for the great majority of cancer-related deaths. Most metastatic lesions are micro metastases that have the capacity to remain in a non-dividing state called "dormancy" for months or even years. Commonly used anticancer drugs generally target actively dividing cancer cells. Therefore, cancer cells that remain in a dormant state evade conventional therapies and contribute to cancer recurrence. Cellular and molecular mechanisms of cancer dormancy are not fully understood. Recent studies indicate that a major cellular stress response mechanism, autophagy, plays an important role in the adaptation, survival and reactivation of dormant cells. In this review article, we will summarize accumulating knowledge about cellular and molecular mechanisms of cancer dormancy, and discuss the role and importance of autophagy in this context.

Crosstalk between autophagy and DNA repair systems.

Autophagy and DNA repair are two essential biological mechanisms that maintain cellular homeostasis. Impairment of these mechanisms was associated with several pathologies such as premature aging, neurodegenerative diseases, and cancer. Intrinsic or extrinsic stress stimuli (e.g., reactive oxygen species or ionizing radiation) cause DNA damage. As a biological stress response, autophagy is activated following insults that threaten DNA integrity. Hence, in collaboration with DNA damage repair and response mechanisms, autophagy contributes to the maintenance of genomic stability and integrity. Yet, connections and interactions between these two systems are not fully understood. In this review article, current status of the associations and crosstalk between autophagy and DNA repair systems is documented and discussed.

Transcriptional landscape of cellular networks reveal interactions driving the dormancy mechanisms in cancer.

Primary cancer cells exert unique capacity to disseminate and nestle in distant organs. Once seeded in secondary sites, cancer cells may enter a dormant state, becoming resistant to current treatment approaches, and they remain silent until they reactivate and cause overt metastases. To illuminate the complex mechanisms of cancer dormancy, 10 transcriptomic datasets from the literature enabling 21 dormancy-cancer comparisons were mapped on protein-protein interaction networks and gene-regulatory networks to extract subnetworks that are enriched in significantly deregulated genes. The genes appearing in the subnetworks and significantly upregulated in dormancy with respect to proliferative state were scored and filtered across all comparisons, leading to a dormancy-interaction network for the first time in the literature, which includes 139 genes and 1974 interactions. The dormancy interaction network will contribute to the elucidation of cellular mechanisms orchestrating cancer dormancy, paving the way for improvements in the diagnosis and treatment of metastatic cancer.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

MicroRNAs as major regulators of the autophagy pathway.

Autophagy is a cellular stress response mechanism activation of which leads to degradation of cellular components, including proteins as well as damaged organelles in lysosomes. Defects in autophagy mechanisms were associated with several pathologies (e.g. cancer, neurodegenerative diseases, and rare genetic diseases). Therefore, autophagy regulation is under strict control. Transcriptional and post-translational mechanisms that control autophagy in cells and organisms studied in detail. Recent studies introduced non-coding small RNAs, and especially microRNAs (miRNAs) in the post-translational orchestration of the autophagic activity. In this review article, we analyzed in detail the current status of autophagy-miRNA connections. Comprehensive documentation of miRNAs that were directly involved in autophagy regulation resulted in the emergence of common themes and concepts governing these complex and intricate interactions. Hence, a better and systematic understanding of these interactions reveals a central role for miRNAs in the regulation of autophagy.

Autophagy as a molecular target for cancer treatment.

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed.

Bypassing pro-survival and resistance mechanisms of autophagy in EGFR-positive lung cancer cells by targeted delivery of 5FU using theranostic Ag2S quantum dots.

Targeted drug delivery systems that combine imaging and therapeutic functions in a single structure have become very popular in nanomedicine. Near-infrared (NIR) emitting Ag2S quantum dots (QDs) are excellent candidates for this task. Here, we have developed PEGylated Ag2S QDs functionalized with Cetuximab (Cet) antibody and loaded with an anticancer drug, 5-fluorouracil (5FU). These theranostic QDs were used for targeted NIR imaging and treatment of lung cancer using low (H1299) and high (A549) Epidermal Growth Factor Receptor (EGFR) overexpressing cell lines. The Cet conjugated QDs effectively and selectively delivered 5FU to A549 cells and provided significantly enhanced cell death associated with apoptosis. Interestingly, while treatment of cells with free 5FU activated autophagy, a cellular mechanism conferring resistance to cell death, these EGFR targeting multimodal QDs significantly overcame drug resistance compared to 5FU treatment alone. The improved therapeutic outcome of 5FU delivered to A549 cells by Cet conjugated Ag2S QDs is suggested as the synergistic outcome of enhanced receptor mediated uptake of nanoparticles, and hence the drug, coupled with suppressed autophagy even in the absence of addition of an autophagy suppressor.

Autophagy and liver cancer.

Autophagy is a key biological phenomenon conserved from yeast to mammals. Under basal conditions, activation of autophagy leads to the protein degradation as well as damaged organelles for maintaining cellular homeostasis. Deregulation of autophagy has been identified as a key mechanism contributing to the pathogenesis and progression of several liver diseases, including hepatocellular carcinoma (HCC), one of the most common and mortal types of cancer. Currently used treatment strategies in patients with HCC result in variable success rates. Therefore, novel early diagnosis and treatment techniques should be developed. Manipulation of autophagy may improve responses of cancer cell to treatments and provide novel targeted therapy options for HCC. In this review, we summarized how our understanding of autophagy-cell death connection may have an impact on HCC therapy.

Development of tailored SPION-PNIPAM nanoparticles by ATRP for dually responsive doxorubicin delivery and MR imaging.

Biocompatible, colloidally stable and ultra-small Fe3O4 nanoparticles (SPIONs) coated with poly(N-isopropylacrylamide) (PNIPAM) were synthesized via surface-initiated ATRP (atom transfer radical polymerization) to prevent excessive aggregation of magnetic cores and interparticle crosslinking, and to provide control over polymer content. These SPION-PNIPAM nanoparticles (NPs) have a hydrodynamic size between 8 and 60 nm depending on the PNIPAM content, and hence are ultrasmall in size and have an LCST around 38 °C. They had a high drug-loading capacity reaching 9.6 wt% doxorubicin in the final composition. The Dox release studies revealed pH and temperature-dependent release, which was not reported for PNIPAM before. Release of Dox under physiological conditions was below 20%, but around 90% at 42 °C and pH 5. This dually responsive nature is very advantageous to increase the drug efficacy and reduce side-effects, simultaneously. The cytocompatability of the SPION-PNIPAM NPs and the influence of Dox delivery to cells were investigated via in vitro cell viability, apoptosis, DNA-damage and confocal microscopy studies. The NPs were shown to be highly cytocompatible and induce significant cell death due to Dox when loaded with the drug. Besides, it was seen that the polymeric content can be used as an additional factor in tuning the release kinetics. Lastly, these nanoparticles reduced the signal intensity significantly in the T2 mode, acting as a potential SPION-based contrast agent. Overall, here, we demonstrate the design of small, smart theranostic nanoparticles with high drug-loading capacity and pH-dependent temperature-sensitive release characteristics with the ability to generate contrast in MRI.

Autophagy-Regulating microRNAs and Cancer.

Macroautophagy (autophagy herein) is a cellular stress response and a survival pathway that is responsible for the degradation of long-lived proteins, protein aggregates, as well as damaged organelles in order to maintain cellular homeostasis. Consequently, abnormalities of autophagy are associated with a number of diseases, including Alzheimers's disease, Parkinson's disease, and cancer. According to the current view, autophagy seems to serve as a tumor suppressor in the early phases of cancer formation, yet in later phases, autophagy may support and/or facilitate tumor growth, spread, and contribute to treatment resistance. Therefore, autophagy is considered as a stage-dependent dual player in cancer. microRNAs (miRNAs) are endogenous non-coding small RNAs that negatively regulate gene expression at a post-transcriptional level. miRNAs control several fundamental biological processes, and autophagy is no exception. Furthermore, accumulating data in the literature indicate that dysregulation of miRNA expression contribute to the mechanisms of cancer formation, invasion, metastasis, and affect responses to chemotherapy or radiotherapy. Therefore, considering the importance of autophagy for cancer biology, study of autophagy-regulating miRNA in cancer will allow a better understanding of malignancies and lead to the development of novel disease markers and therapeutic strategies. The potential to provide study of some of these cancer-related miRNAs were also implicated in autophagy regulation. In this review, we will focus on autophagy, miRNA, and cancer connection, and discuss its implications for cancer biology and cancer treatment.

Surface modifications for phase change cooling applications via crenarchaeon Sulfolobus solfataricus P2 bio-coatings.

Due to its high heat removal capability and exploitation of latent heat, boiling is considered to be one of the most effective cooling methods in industry. Surface structure and wettability are two factors imposing boiling phenomena. Here, we propose an effective and facile method for surface enhancement via crenarchaeon Sulfolobus Solfataricus P2 bio-coatings. The positive effects of such surfaces of bio-coatings were assessed, and enhancements in heat transfer and cooling were obtained. Visualization was also performed, and bubble dynamics of generated bubbles and vapor columns from the tested surfaces with bio-coatings are here presented. Superior performance in terms of boiling heat transfer and cooling was reached with the use of crenarchaeon Sulfolobus Solfataricus P2 coated surfaces. Thus, this study clearly demonstrates the potential of futuristic surfaces with bio-coatings to achieve substantial energy saving and efficiency.

MIR376 family and cancer.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level. They have been implicated in several fundamental biological processes including development, differentiation, apoptosis and stem cell maintenance. There is increasing evidence that microRNAs also play roles in cellular transformation and carcinogenesis by acting either as tumor suppressors or oncogenes. Recent studies introduced MIR376 as an important microRNA family for cancer formation and progression. The MIR376 family is located on human chromosome 14 and it has several members containing identical or similar seed sequences. Biological roles of family members were studied in different cancer settings, including gliomas, leukemia, breast and ovarian cancers. Furthermore, two MIR376 family members, namely MIR376A and MIR376B were implicated in the regulation of macroautophagy (autophagy herein). Since autophagy dysregulation underlies various diseases including cancer, it is essential to understand the role of the MIR376 family in this context. In this article, we summarize the miRNA-cancer connection, and review accumulating data about the involvement of the MIR376 family in cancer biology.

Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis.

Autophagy and apoptosis are two important molecular mechanisms that maintain cellular homeostasis under stress conditions. Autophagy represents an intracellular mechanism responsible for turnover of organelles and long-lived proteins through a lysosome-dependent degradation pathway. Cell death signals or sustained stress might trigger programmed cell death pathways, and among them, apoptosis is the most extensively studied one. Recent studies indicate the presence of a complex interplay between autophagy and apoptosis. Physiological relevance of autophagy-apoptosis crosstalk was mainly shown in vitro. However, in vivo consequences possibly exist both during health and disease. In this review, we will summarize the current knowledge about molecular mechanisms connecting autophagy and apoptosis, and about the significance of this crosstalk for human health.