Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane.

Introduction Whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response is not known.

The cellular process of autophagy (literally “self-eating”) is important for maintaining the homeostasis and bioenergetics of mammalian cells. Two of the best-studied mechanisms of autophagy are macroautophagy and chaperone-mediated autophagy (CMA). Changes in macroautophagy activity have been described in cancer cells and in solid tumors, and inhibition of macroautophagy promotes tumorigenesis. Because normal cells respond to inhibition of macroautophagy by up-regulation of the CMA pathway, we aimed to characterize the CMA status in different cancer cells and to determine the contribution of changes in CMA to tumorigenesis. Here, we show consistent up-regulation of CMA in different types of cancer cells regardless of the status of macroautophagy. We also demonstrate an increase in CMA components in human cancers of different types and origins. CMA is required for cancer cell proliferation in vitro because it contributes to the maintenance of the metabolic alterations characteristic of malignant cells. Using human lung cancer xenografts in mice, we confirmed the CMA dependence of cancer cells in vivo. Inhibition of CMA delays xenograft tumor growth, reduces the number of cancer metastases, and induces regression of existing human lung cancer xenografts in mice. The fact that similar manipulations of CMA also reduce tumor growth of two different melanoma cell lines suggests that targeting this autophagic pathway may have broad antitumorigenic potential.