The endoplasmic reticulum (ER) is a site of protein biogenesis in eukaryotic cells. Perturbing ER homeostasis activates stress programs collectively called the unfolded protein response (UPR). The UPR enhances production of ER-resident chaperones and enzymes to reduce the burden of misfolded proteins. On resolution of ER stress, ill-defined, selective autophagic programs remove excess ER components. Here we identify Sec62, a constituent of the translocon complex regulating protein import in the mammalian ER, as an ER-resident autophagy receptor. Sec62 intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance in a series of events that we name recovER-phagy. Sec62 contains a conserved LC3-interacting region in the C-terminal cytosolic domain that is required for its function in recovER-phagy, but is dispensable for its function in the protein translocation machinery. Our results identify Sec62 as a critical molecular component in maintenance and recovery of ER homeostasis. DOI: https://doi.org/10.1038/ncb3423Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127515 Accepted Version Originally published at: Fumagalli, Fiorenza; Noak, Julia; Bergmann, Timothy J; Presmanes, Eduardo Cebollero; Pisoni, Giorgia Brambilla; Fasana, Elisa; Fregno, Ilaria; Galli, Carmela; Loi, Marisa; Solda, Tatiana; D'Antuono, Rocco; Raimondi, Andrea; Jung, Martin; Melnyk, Armin; Schorr, Stefan; Schreiber, Anne; Simonelli, Luca; Varani, Luca; Wilson-Zbinden, Caroline; Zerbe, Oliver; Hofmann, Kay; Peter, Matthias; Quadroni, Manfredo; Zimmermann, Richard; Molinari, Maurizio (2016 To define mechanisms that regulate the return of ER-resident chaperones and folding factors to their physiologic intracellular level after resolution of an ER stress, we established a protocol for reversible induction of UPR in cultured mammalian cells (Fig. 1a). Briefly, human embryonic kidney cells (HEK293) or mouse embryonic fibroblasts (MEF) were exposed for 12 h to non-toxic doses of cyclopiazonic acid (CPA), a reversible inhibitor of the sarco/endoplasmic reticulum calcium pump 6 . The return of ER-resident gene products at their pre-stress level was monitored during resolution of the UPR obtained upon CPA wash out ( CPA wash out initiated a recovery phase characterized by the rapid return of ER stress-induced transcripts at, or below, their pre-stress levels (Fig. 1b, recovery, T 1/2 average ≈ 1 h, blue line). The corresponding ER stress-induced proteins returned to their physiologic levels with much slower kinetics (Fig. 1c, d, T 1/2 average ≈ 10 h, blue). 3With the exception of Herp, which is rapidly turned over with intervention of proteasomes (Fig. 1c, d (Fig. 1g, 2a) and other membrane and luminal ER marker proteins such as Sec62 and Crt ( Fig. 2b and Extended data Fig. 3) in 0.5-1.5 µm diameter cytoplasmic puncta that rapidly disappeared upon BafA1 wash out (Extended data Fig. 4). Cytosolic puncta containing ER marker prot...

Aberrant activation of oncogenes or loss of tumour suppressor genes opposes malignant transformation by triggering a stable arrest in cell growth, which is termed cellular senescence. This process is finely tuned by both cell-autonomous and non-cell-autonomous mechanisms that regulate the entry of tumour cells to senescence. Whether tumour-infiltrating immune cells can oppose senescence is unknown. Here we show that at the onset of senescence, PTEN null prostate tumours in mice are massively infiltrated by a population of CD11b(+)Gr-1(+) myeloid cells that protect a fraction of proliferating tumour cells from senescence, thus sustaining tumour growth. Mechanistically, we found that Gr-1(+) cells antagonize senescence in a paracrine manner by interfering with the senescence-associated secretory phenotype of the tumour through the secretion of interleukin-1 receptor antagonist (IL-1RA). Strikingly, Pten-loss-induced cellular senescence was enhanced in vivo when Il1ra knockout myeloid cells were adoptively transferred to PTEN null mice. Therapeutically, docetaxel-induced senescence and efficacy were higher in PTEN null tumours when the percentage of tumour-infiltrating CD11b(+)Gr-1(+) myeloid cells was reduced using an antagonist of CXC chemokine receptor 2 (CXCR2). Taken together, our findings identify a novel non-cell-autonomous network, established by innate immunity, that controls senescence evasion and chemoresistance. Targeting this network provides novel opportunities for cancer therapy.

Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.

The mechanisms by which mitochondrial metabolism supports cancer anabolism are still unclear. Here, we unexpectedly find that genetic and pharmacological inactivation of Pyruvate Dehydrogenase A1 (PDHA1), a subunit of pyruvate dehydrogenase complex (PDC) inhibits prostate cancer development in different mouse and human xenograft tumour models by affecting lipid biosynthesis. Mechanistically, we show that in prostate cancer, PDC localizes in both mitochondria and nucleus. While nuclear PDC controls the expression of Sterol regulatory element-binding transcription factor (SREBF) target genes by mediating histone acetylation, mitochondrial PDC provides cytosolic citrate for lipid synthesis in a coordinated effort to sustain anabolism. In line with these evidence, we find that PDHA1 and the PDC activator, Pyruvate dehydrogenase phosphatase 1 (PDP1), are frequently amplified and overexpressed at both gene and protein level in prostate tumours. Taken together, these findings demonstrate that both mitochondrial and nuclear PDC sustain prostate tumourigenesis by controlling lipid biosynthesis thereby pointing at this complex as a novel target for cancer therapy.

Summary Tumor-associated macrophages (TAMs) represent a major component of the tumor microenvironment supporting tumorigenesis. TAMs re-education has been proposed as a strategy to promote tumor inhibition. However, whether this approach may work in prostate cancer is unknown. Here we find that Pten -null prostate tumors are strongly infiltrated by TAMs expressing C-X-C chemokine receptor type 2 (CXCR2), and activation of this receptor through CXCL2 polarizes macrophages toward an anti-inflammatory phenotype. Notably, pharmacological blockade of CXCR2 receptor by a selective antagonist promoted the re-education of TAMs toward a pro-inflammatory phenotype. Strikingly, CXCR2 knockout monocytes infused in Pten pc−/− ; Trp53 pc−/− mice differentiated in tumor necrosis factor alpha (TNF-α)-releasing pro-inflammatory macrophages, leading to senescence and tumor inhibition. Mechanistically, PTEN -deficient tumor cells are vulnerable to TNF-α-induced senescence, because of an increase of TNFR1 . Our results identify TAMs as targets in prostate cancer and describe a therapeutic strategy based on CXCR2 blockade to harness anti-tumorigenic potential of macrophages against this disease.