Maria Høyer-Hansen

Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium.

Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) either by unfolded protein response that leads to an increase in the capacity of the ER to fold its client proteins or by apoptosis when the function of ER cannot be restored. Emerging data now indicate that ER stress is also a potent inducer of macroautophagy, a process whereby eukaryotic cells recycle their macromolecules and organelles. Depending on the context, autophagy counterbalances ER stress-induced ER expansion, enhances cell survival or commits the cell to non-apoptotic death. Here, we discuss the signaling pathways linking ER stress to autophagy and possibilities for their clinical exploitation.

Heat Shock Protein 70 Promotes Cell Survival by Inhibiting Lysosomal Membrane Permeabilization

Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c–induced, apoptosome-dependent caspase activation in vitro and Fas ligand–induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.

TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells

The capacity of tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) to trigger apoptosis preferentially in cancer cells, although sparing normal cells, has motivated clinical development of TRAIL receptor agonists as anti‐cancer therapeutics. The molecular mechanisms responsible for the differential TRAIL sensitivity of normal and cancer cells are, however, poorly understood. Here, we show a novel signalling pathway that activates cytoprotective autophagy in untransformed human epithelial cells treated with TRAIL. TRAIL‐induced autophagy is mediated by the AMP‐activated protein kinase (AMPK) that inhibits mammalian target of rapamycin complex 1, a potent inhibitor of autophagy. Interestingly, the TRAIL‐induced AMPK activation is refractory to the depletion of the two known AMPK‐activating kinases, LKB1 and Ca(2+)/calmodulin‐dependent kinase kinase‐β, but depends on transforming growth factor‐β‐activating kinase 1 (TAK1) and TAK1‐binding subunit 2. As TAK1 and AMPK are ubiquitously expressed kinases activated by numerous cytokines and developmental cues, these data are most likely to have broad implications for our understanding of cellular control of energy homoeostasis as well as the resistance of untransformed cells against TRAIL‐induced apoptosis.

microRNA-101 is a potent inhibitor of autophagy

Autophagy is an evolutionarily conserved mechanism of cellular self‐digestion in which proteins and organelles are degraded through delivery to lysosomes. Defects in this process are implicated in numerous human diseases including cancer. To further elucidate regulatory mechanisms of autophagy, we performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR‐101, as a potent inhibitor of basal, etoposide‐ and rapamycin‐induced autophagy. Through transcriptome profiling, we identified three novel miR‐101 targets, STMN1, RAB5A and ATG4D. siRNA‐mediated depletion of these genes phenocopied the effect of miR‐101 overexpression, demonstrating their importance in autophagy regulation. Importantly, overexpression of STMN1 could partially rescue cells from miR‐101‐mediated inhibition of autophagy, indicating a functional importance for this target. Finally, we show that miR‐101‐mediated inhibition of autophagy can sensitize breast cancer cells to 4‐hydroxytamoxifen (4‐OHT)‐mediated cell death. Collectively, these data establish a novel link between two highly important and rapidly growing research fields and present a new role for miR‐101 as a key regulator of autophagy.

Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death.

A chemotherapeutic vitamin D analogue, EB1089, kills tumor cells via a caspase-independent pathway that results in chromatin condensation and DNA fragmentation. Employing transmission- and immunoelectronmicroscopy as well as detection of autophagosome-associated LC3-β protein in the vacuolar structures, we show here that EB1089 also induces massive autophagy in MCF-7 cells. Interestingly, inhibition of autophagy effectively hindered apoptosis-like nuclear changes and cell death in response to EB1089. Furthermore, restoration of normal levels of beclin 1, an autophagy-inducing tumor suppressor gene that is monoallelically deleted in MCF-7 cells, greatly enhanced the EB1089-induced nuclear changes and cell death. Thus, EB1089 triggers nuclear apoptosis via a pathway involving Beclin 1-dependent autophagy. Surprisingly, tumor cells depleted for Beclin 1 failed to proliferate suggesting that even though the monoallelic depletion of beclin 1 in human cancer cells suppresses EB1089-induced autophagic death, one intact beclin 1 allele is essential for tumor cell proliferation.

AMP-activated protein kinase: a universal regulator of autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests that AMPK inhibits mTORC1 and autophagy also in non-starved cells. Various Ca2+ mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate AMPK via activation of Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β), and this pathway is required for Ca2+-induced mTORC1 inhibition and autophagy. Thus, we propose that an increase in free cytosolic Ca2+ ([Ca2+]c) induces autophagy via the CaMKK/β-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected. Addendum to: Control of Macroautophagy by Calcium, Calmodulin-Dependent Kinase Kinase-β and Bcl-2 M. Høyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen and M. Jäättelä Mol Cell 2007; 25:193-205

Autophagy: An emerging target for cancer therapy

Macroautophagy (hereafter referred to as autophagy) is a lysosomal catabolic pathway whereby cells recycle macromolecules and organelles. The capacity of autophagy to maintain cellular metabolism under starvation conditions and to remove damaged organelles under stress conditions improves the survival of cells. Yet, autophagy appears to suppress tumorigenesis. In this review we discuss recent data that begin to elucidate the molecular basis for this apparent controversy. First, we summarize our current knowledge on the autophagy-mediated control of both cell survival and cell death in general. Then, we highlight the common cancer-associated changes in autophagy induction, regulation and execution. And finally we discuss the potential of pro- as well as anti-autophagic signaling pathways as targets for future cancer therapy.

Effective Tumor Cell Death by σ-2 Receptor Ligand Siramesine Involves Lysosomal Leakage and Oxidative Stress

Acquired resistance to classic caspase-mediated apoptosis is a common problem for the treatment of human cancer. Here, we show that siramesine, a novel sigma-2 receptor ligand, effectively induces caspase-independent programmed cell death in immortalized and transformed cells of various origins. Siramesine-treated tumor cells displayed increased levels of reactive oxygen species, lysosomal membrane permeabilization, chromatin condensation, and shrinkage and detachment of cells. Lipid antioxidants (alpha-tocopherol and gamma-tocopherol), but not other tested antioxidants (butylated hydroxyanisol or N-acetyl cysteine), effectively inhibited siramesine-induced morphologic changes and cell death. Cathepsin B inhibitors (CA-074-Me and R-2525) conferred similar, but less pronounced protection, whereas ectopic expression of antiapoptotic protein Bcl-2, lack of wild-type p53 as well as pharmacologic inhibitors of caspases (zVAD-fmk, DEVD-CHO, and LEHD-CHO), calpains (PD150606), and serine proteases (N-tosyl-L-phenylalanine chloromethyl ketone and pefabloc) failed to protect cells against siramesine-induced death. Importantly, transformation of murine embryonic fibroblasts with activated c-src or v-Ha-ras oncogenes greatly sensitized them to siramesine-induced cytotoxicity. Furthermore, p.o. administration of well-tolerated doses of siramesine had a significant antitumorigenic effect in orthotopic breast cancer and s.c. fibrosarcoma models in mice. These results present siramesine as a promising new drug for the treatment of tumors resistant to traditional therapies.

Ordered Organelle Degradation during Starvation-induced Autophagy

Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route. Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.

Vincristine induces dramatic lysosomal changes and sensitizes cancer cells to lysosome-destabilizing siramesine.

Vincristine is a microtubule-destabilizing antimitotic drug that has been used in cancer therapy for over 40 years. However, the knowledge on vincristine-induced cell death pathways is still sparse. Here, we show that vincristine induces dramatic changes in the lysosomal compartment and sensitizes cells to lysosomal membrane permeabilization. In HeLa cervix carcinoma cells, vincristine induced mitotic arrest and massive cell death associated with an early increase in the lysosomal volume and lysosomal leakage followed by the activation of the intrinsic apoptosis program. In contrast, the majority of vincristine-treated MCF-7 breast carcinoma cells resisted apoptosis. Instead, they adapted to the spindle assembly checkpoint and escaped the mitotic arrest as micronucleated and senescent cells with an increase in the volume and the activity of their lysosomal compartment. Consistent with its substantial effects on the lysosomes, vincristine greatly sensitized cultured cancer cells as well as orthotopic breast cancer xenografts in mice to the cytotoxicity induced by siramesine, a sigma-2 receptor ligand that kills cancer cells by destabilizing their lysosomes. Importantly, the combination of nontoxic concentrations of vincristine and siramesine resulted in massive cell death even in MCF-7 cells that were capable of escaping vincristine-induced spindle assembly checkpoint and cell death. Similar synergism was observed when siramesine was combined with a semisynthetic vincristine analogue, vinorelbine, or with microtubule-stabilizing paclitaxel. These data strongly suggest that combination therapies consisting of microtubule-disturbing and lysosome-destabilizing drugs may prove useful in the treatment of otherwise therapy-resistant human cancers.