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
Cancer is a disease of aging, and aged cancer patients have a poorer prognosis. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumor progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression1–4 we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. We find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signaling cascade in melanoma cells that results in a decrease in β-catenin and MITF, and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to ROS-induced DNA damage, rendering them more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumor progression, offering new paradigms for the design of therapy for the elderly.
An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the non-canonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Further, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In melanoma patients treated with the BRAF inhibitor, Vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance.
Edited by Noboru MizushimaKeywords: Nbr1 p62/SQSTM1 Ubiquitin Vesicular transport LC3 Autophagy Myopathy a b s t r a c t Nbr1, a ubiquitous kinase scaffold protein, contains a PB1, and a ubiquitin-associated (UBA) domain. We show here that the nbr1 UBA domain binds to lysine-48 and -63 linked polyubiquitin-B chains. Nbr1 also binds to the autophagic effector protein LC3-A via a novel binding site. Ubiquitin-binding, but not PB1-mediated p62/SQSTM1 interaction, is required to target nbr1 to LC3 and polyubiquitinpositive bodies. Nbr1 binds additionally to proteins implicated in ubiquitin-mediated protein turnover and vesicle trafficking: ubiquitin-specific peptidases USP8, and the endosomal transport regulator p14/Robld3. Nbr1 thus contributes to specific steps in protein turnover regulation disrupted in several hereditary human diseases. Structured summary:MINT-7034452: USP8 (uniprotkb:P40818) physically interacts (MI:0218) with NBR1 (uniprotkb:Q14596) by pull down (MI:0096) MINT-7034438: SQSTM1 (uniprotkb:Q13501) and LC3
We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAFV600E mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence-associated β-galactosidase (SA-β-gal), senescence associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence like SA-β-gal and SAHF, these Wnt5A-high cells are able to colonize the lungs in in vivo tail-vein colony forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.
The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, a key driver of melanoma invasiveness, induces Ca 2؉ signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive cell lines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.
Purpose Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment, are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein, whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. Experimental Design PPARγ increases klotho levels, and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/ Wnt5A crosstalk, in vitro and in vivo, and the implications of that for targeted therapy in young vs. aged animals. Results We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF-inhibitor sensitive, and BRAF inhibitor-resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. Conclusions Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types.
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