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Keratinocytes undergo significant structural remodeling during epidermal differentiation, including a broad transformation of the proteome coupled with a reduction in total cellular biomass. This suggests that intracellular digestion of proteins and organelles is necessary for keratinocyte differentiation. Here, we use both genetic and pharmacologic approaches to demonstrate that autophagy and lysosomal functions are required for keratinocyte differentiation in organotypic human skin. Lysosomal activity was required for mechanistic target of rapamycin signaling and mitochondrial oxidative metabolism. In turn, mitochondrial reactive oxygen species, produced as a natural byproduct of oxidative phosphorylation, were necessary for keratinocyte differentiation. Finally, treatment with exogenous reactive oxygen species rescued the differentiation defect in lysosome-inhibited keratinocytes. These findings highlight a reciprocal relationship between lysosomes and mitochondria, in which lysosomes support mitochondrial metabolism and the associated production of mitochondrial reactive oxygen species. The mitochondrial reactive oxygen species released to the cytoplasm in suprabasal keratinocytes triggers autophagy and lysosome-mediated degradation necessary for epidermal differentiation. As defective lysosome-dependent autophagy is associated with common skin diseases including psoriasis and atopic dermatitis, a better understanding of the role of lysosomes in epidermal homeostasis may guide future therapeutic strategies., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e., autophagy and macropinocytosis) while promoting mTORC1-dependent anabolism. Antimalarial compounds such as chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQ) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An in situ photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the in vivo tumor growth of melanoma, pancreatic cancer, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer. Significance: This study identifies chemical features of dimeric compounds that increase their lysosomal specificity, and a new molecular target for these compounds, reclassifying these compounds as targeted therapies. Targeting PPT1 blocks mTOR signaling in a manner distinct from catalytic inhibitors, while concurrently inhibiting autophagy, thereby providing a new strategy for cancer therapy. Cancer Discov; 7(11); 1266-83. ©2017 AACR. See related commentary by Towers and Thorburn, p. 1218 This article is highlighted in the In This Issue feature, p. 1201 ., (©2017 American Association for Cancer Research.)

Cells depend on the lysosome for sequestration and degradation of macromolecules in order to maintain metabolic homeostasis. These membrane-enclosed organelles can receive intracellular and extracellular cargo through endocytosis, phagocytosis, and autophagy. Lysosomes establish acidic environments to activate enzymes that are able to break down biomolecules engulfed through these various pathways. Recent advances in methods to study the lysosome have allowed the discovery of extended roles for the lysosome in various diseases, including cancer, making it an attractive and targetable node for therapeutic intervention. This review focuses on key aspects of lysosomal biology in the context of cancer and how these properties can be exploited for the development of new therapeutic strategies. This will provide a contextual framework for how advances in methodology could be applied in future translational research.

Lysosomal autophagy inhibitors (LAI) such as hydroxychloroquine (HCQ) have significant activity in a subset of cancer cell lines. LAIs are being evaluated in cancer clinical trials, but genetic determinants of sensitivity to LAIs are unknown, making it difficult to predict which tumors would be most susceptible. Here we characterize differentially expressed genes in HCQ-sensitive (-S) and -resistant (-R) cancer cells. Notably, expression of canonical macroautophagy/autophagy genes was not associated with sensitivity to HCQ. Expression patterns of ALDH1A1 (aldehyde dehydrogenase 1 family member A1) and HLTF (helicase like transcription factor) identified HCQ-S (ALDH1A1 high HLTF low ; ALDH1A1 low HLTF low ) and HCQ-R (ALDH1A1 low HLTF high ) cells. ALDH1A1 overexpression was found to enhance LAI cell entry and cytotoxicity without directly affecting lysosome function or autophagic flux. Expression of HLTF allows repair of DNA damage caused by LAI-induced reactive oxygen species, leading to HCQ resistance. Sensitivity to HCQ is increased in cells where HLTF is silenced by promoter methylation. HLTF overexpression blunted the antitumor efficacy of chloroquine derivatives in vitro and in vivo. Analysis of tumor RNA sequencing data from >700 patients in the Cancer Genome Atlas identified cancers including colon cancer, renal cell carcinoma, and gastric cancers, that were enriched for the HCQ-S or HCQ-R signature. These results provide mechanistic insights into LAI efficacy, and guidance for LAI clinical development.

Lysosomes are membrane-bound intracellular organelles that receive macromolecules delivered by endocytosis, phagocytosis, and autophagy for degradation and recycling. Over the last decade, advances in lysosome research have established a broad role for the lysosome in the pathophysiology of disease. In this review, we highlight the recent discoveries in lysosome biology, with an emphasis on their implications for cancer therapy. We focus on targeting the lysosome in cancer by exploring lysosomal biogenesis and its role in the crosstalk between apoptosis and autophagy. We also discuss how lysosomal inhibition could emerge as a new therapeutic strategy to overcome drug resistance in cancer., (© 2015 New York Academy of Sciences.)

Lys05 is a previously undescribed dimeric chloroquine which more potently accumulates in the lysosome and blocks autophagy compared with HCQ. Lys05 produced more potent antitumor activity as a single agent both in vitro and in vivo in multiple human cancer cell lines and xenograft models compared with HCQ. Initial structure-activity relationship studies demonstrated that the increased activity associated with Lys05 was due to the bivalent aminoquinoline rings, C7-Chlorine, and a short triamine linker. While lower doses of Lys05 were well tolerated and associated with antitumor activity, at the highest dose tested, Lys05 produced Paneth cell dysfunction and intestinal toxicity, similar to what can be observed in mice and humans with genetic defects in the autophagy gene ATG16L1. Lys05 is therefore a new lysosomal autophagy inhibitor that has potential to be developed further into a drug for cancer and other medical applications.

Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer.

Eukaryotes use autophagy as a mechanism for maintaining cellular homeostasis by degrading and recycling organelles and proteins. This process assists in the proliferation and survival of advanced cancers. There is mounting preclinical evidence that targeting autophagy can enhance the efficacy of many cancer therapies. Hydroxychloroquine (HCQ) is the only clinically-approved autophagy inhibitor, and this systematic review focuses on HCQ use in cancer clinical trials. Preclinical trials have shown that HCQ alone and in combination therapy leads to enhancement of tumor shrinkage. This has provided the base for multiple ongoing clinical trials involving HCQ alone and in combination with other treatments. However, due to its potency, there is still a need for more potent and specific autophagy inhibitors. There are multiple autophagy inhibitors in the pre-clinical stage at various stages of development. Additional studies on the mechanism of HCQ and other autophagy inhibitors are still required to answer questions surrounding how these agents will eventually be used in the clinic. [ABSTRACT FROM AUTHOR]

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER(TAM)/p53ER(TAM) mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ) can induce cell death in a subset of cancer cell lines, and some melanoma cell lines are quite susceptible. Although it is well known that CQ impairs lysosomal function and can serve as an autophagy inhibitor, the molecular target of CQ and the subsequent cascade of events that lead to cell death are not fully understood. Recent evidence indicates that in melanoma cell lines, CQ induces apoptosis by preventing degradation of the pro-apoptotic BH3-only protein p53-upregulated modulator of apoptosis. This finding adds to the unfolding story of CQ's mechanism of action as a cancer therapeutic agent. [ABSTRACT FROM AUTHOR]

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor—Lys05—with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells. [ABSTRACT FROM AUTHOR]