Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Methods in Mammalian Autophagy Research

The clinical use of anthracyclines like doxorubicin and daunorubicin can be viewed as a sort of double-edged sword. On the one hand, anthracyclines play an undisputed key role in the treatment of many neoplastic diseases; on the other hand, chronic administration of anthracyclines induces cardiomyopathy and congestive heart failure usually refractory to common medications. Second-generation analogs like epirubicin or idarubicin exhibit improvements in their therapeutic index, but the risk of inducing cardiomyopathy is not abated. It is because of their janus behavior (activity in tumors vis-a-vis toxicity in cardiomyocytes) that anthracyclines continue to attract the interest of preclinical and clinical investigations despite their longer-than-40-year record of longevity. Here we review recent progresses that may serve as a framework for reappraising the activity and toxicity of anthracyclines on basic and clinical pharmacology grounds. We review 1) new aspects of anthracycline-induced DNA damage in cancer cells; 2) the role of iron and free radicals as causative factors of apoptosis or other forms of cardiac damage; 3) molecular mechanisms of cardiotoxic synergism between anthracyclines and other anticancer agents; 4) the pharmacologic rationale and clinical recommendations for using cardioprotectants while not interfering with tumor response; 5) the development of tumor-targeted anthracycline formulations; and 6) the designing of third-generation analogs and their assessment in preclinical or clinical settings. An overview of these issues confirms that anthracyclines remain "evergreen" drugs with broad clinical indications but have still an improvable therapeutic index.

/pdf/anthracyclines-molecular-advances-and-pharmacologic-4vygbx1i0b.pdf

Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity

The survival benefits in prostate cancer (PCa) conferred by androgen deprivation therapies (ADT) and antiandrogens are often blunted by the development of castration resistant disease (CRPC). There is increasing evidence that the primary cellular response to ADT is senescence rather than apoptosis, and this may influence progression to CRPC. However, the majority of in vitro studies employ the charcoal stripped serum model, and very few studies use antiandrogens, despite the fact that antiandrogens are currently one of the primary modes of therapy for prostate cancer. In an effort to enhance our understanding of antiandrogen effects in PCa, we evaluated the cellular response to the clinically relevant antiandrogen, bicalutamide, in the human LNCaP and murine Myc-CaP cell lines. Acute treatment (48 hours) with bicalutamide resulted in a growth arrest followed by proliferative recovery in both cell lines. However, while the growth arrest was prolonged (lasting at least 6 days) in the LnCaP cells, growth arrest was exceedingly brief (lasting approximately 24 hours) in the Myc-CaP cells. Consistent with these observations, we were able to detect increased beta galactosidase staining (a hallmark of senescence) in the LnCaP treated cells but not the Myc-CaP treated cells. Despite the fact that profound morphological changes were evident in the Myc-Cap cells, including an enlarged and elongated cell body, the highly transient nature of the growth arrest suggests that the Myc-Cap cells undergo quiescence rather than senescence. The absence of senescence induction in the Myc-CaP cells may be a consequence of the androgen receptor copy number gain. However, androgen duplication is highly uncommon in human prostate cancers prior to development of CRPC. Thus, it is likely that antiandrogens can induce senescence in treatment-naive patients, as seen in the LNCaP cell line. The ability of bicalutamide to induce senescence, albeit perhaps conditionally, suggests that some prostate cancers treated with ADT and/or antiandrogens may be susceptible to drugs that target senescent cells (senolytics). We therefore propose that elimination of senescent cells prior to recovery could attenuate or prevent the development of CRPC. Citation Format: Valerie J. Carpenter, Tareq Saleh, David A. Gewirtz. The antiandrogen bicalutamide induces senescence in LNCaP cells and quiescence in Myc CaP cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 902.

Abstract 902: The antiandrogen bicalutamide induces senescence in LNCaP cells and quiescence in Myc CaP cells

Special issue: Autophagic molecules, mediators and modulators in cancer.

Cisplatin is a potent first-line therapy for many solid malignancies, such as breast, ovarian, lung, testicular, and head and neck cancer. However, acute kidney injury (AKI) is a major dose-limiting toxicity in cisplatin therapy, which often hampers the continuation of cisplatin treatment. The endocannabinoid system, consisting of anandamide (AEA) and 2-arachidonoylglycerol and cannabinoid receptors, participates in different kidney diseases. Inhibition of fatty acid amide hydrolase (FAAH), the primary enzyme for the degradation of AEA and AEA-related N-acylethanolamines, elicits anti-inflammatory effects; however, little is known about its role in cisplatin nephrotoxicity. The current study tested the hypothesis that genetic deletion of Faah mitigates cisplatin-induced AKI. Male wild-type C57BL6 (WT) and Faah−/− mice were administered a single dose of intraperitoneal injection of cisplatin (30 mg/kg) and euthanatized 72 hours later. Faah−/− mice showed a reduction of cisplatin-induced blood urea nitrogen, plasma creatinine levels, kidney injury markers, and tubular damage in comparison with WT mice. The renal protection from Faah deletion was associated with enhanced tone of AEA-related N-acylethanolamines (palmitoylethanolamide and oleoylethanolamide), attenuated nuclear factor-κB/p65 activity, DNA damage markers p53 and p21, and decreased expression of the inflammatory cytokine interleukin-1β, as well as infiltration of macrophages and leukocytes in the kidneys. Notably, a selective FAAH inhibitor (PF-04457845) did not interfere with or perturb the antitumor effects of cisplatin in two head and neck squamous cell carcinoma cell lines, HN30 and HN12. Our work highlights that FAAH inactivation prevents cisplatin-induced nephrotoxicity in mice and that targeting FAAH could provide a novel strategy to mitigate cisplatin-induced nephrotoxicity. SIGNIFICANCE STATEMENT Mice lacking the Faah gene are protected from cisplatin-induced inflammation, DNA damage response, tubular damage, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to mitigate cisplatin-induced nephrotoxicity.

Genetic Knockout of Fatty Acid Amide Hydrolase Ameliorates Cisplatin-Induced Nephropathy in Mice

Autophagy is a natural self-degradative process by which cells eliminate misfolded proteins and damaged organelles. Autophagy has been shown to have multiple functions in tumor cells that may be dependent on the tumor type and the treatment conditions. Autophagy can have a cytoprotective role and be thought of as a survival mechanism or be cytotoxic in nature and mediate cell death. Radiation, one of the primary treatments for many different types of cancer, almost uniformly promotes autophagy in tumor cells. While autophagy produced in response to radiation is often considered to be cytoprotective, radiation-induced autophagy has also been shown to mediate susceptibility to radiation. This review addresses the complexity of autophagy in response to radiation treatment in three different cancer models, specifically lung cancer, breast cancer and glioblastoma. A deeper understanding of the different roles played by autophagy in response to radiation should facilitate the development of approaches for enhancing the therapeutic utility of radiation by providing strategies for combination treatment with unique radiosensitizers as well as preventing the initiation of strategies which are likely to attenuate the effectiveness of radiation therapy.

Review article: AUTOPHAGY AND RADIOSENSITIZATION IN CANCER

Fatty acid amide hydrolase (FAAH) is the primary enzyme that degrades the endocannabinoid anandamide (AEA). Inhibition of FAAH by pharmacology or genetic approach has been shown to reduce inflammation in the brain, colon, heart and kidneys. Kidney medullary infusion of a FAAH inhibitor exerts diuretic and natriuretic effects. Faah knockout mice are protected from both post ischemia reperfusion injury and cisplatin-induced acute kidney injury (AKI) but through distinct mechanisms. The presented study tested the hypothesis that pharmacological inhibition of FAAH activity mitigates cisplatin-induced AKI and explored the potential renoprotective mechanism. Male wild type C57BL/6 (WT) were treated with oral gavage of a FAAH inhibitor (PF-04457845, PF, 5mg/kg) or vehicle (10% PEG200+5% Tween80+normal saline) at 72, 48, 24, 2-hr before and 24, 48-hr after a single dose of intraperitoneal injection of cisplatin (Cis, 25 mg/kg). Mice were euthanatized 72 hours after cisplatin treatment. PF-treated mice showed a decrease of cisplatin-induced plasma creatinine levels compared with vehicle (Veh)-treated mice (mean: 0.23±0.17, 1.98± 1.09, 0.31±0.16 and 1.11±0.56 mg/dL in Veh, Veh+Cis, PF and PF+Cis, respectively, p=0.02). Western blot analysis revealed that increased kidney injury molecule-1 (KIM-1) levels in kidney tissue of Veh-mice by cisplatin were reduced in PF-treated mice (2-fold increase vs. 1.1-fold in Veh+Cis vs. PF+Cis compared to the Veh-mice, respectively, p=0.04). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that PF+Cis mice displayed enhanced AEA tone compared with Veh+Cis mice (1.3-fold increase vs. 2.9-fold in Veh+Cis vs. PF+Cis compared to the Veh-mice, respectively, p=0.03). These results suggest that the renal protection from oral gavage of the FAAH inhibitor against cisplatin nephrotoxicity may be associated with enhanced tone of AEA and that pharmacological inactivation of FAAH could be a novel strategy to prevent cisplatin-induced AKI. NIH grant R01HL145163 and P30DA033934 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

David A. Gewirtz

Papers

Abstract 902: The antiandrogen bicalutamide induces senescence in LNCaP cells and quiescence in Myc CaP cells

Special issue: Autophagic molecules, mediators and modulators in cancer.

Genetic Knockout of Fatty Acid Amide Hydrolase Ameliorates Cisplatin-Induced Nephropathy in Mice

Review article: AUTOPHAGY AND RADIOSENSITIZATION IN CANCER

Pharmacological inhibition of the endocannabinoid anandamide metabolism alleviates cisplatin-induced acute kidney injury