BackgroundNutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype.MethodsTo investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells.ResultsER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts.ConclusionsOur work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0657-0) contains supplementary material, which is available to authorized users.

SUMMARY Primary hyperparathyroidism (PHPT) is a common cause of bone loss that is modeled by continuous PTH (cPTH) infusion. Here we show that the inflammatory cytokine IL-17A is upregulated by PHPT in humans and cPTH in mice. In humans IL-17A is normalized by parathyroidectomy. In mice treatment with anti-IL-17A antibody and silencing of IL-17A receptor IL-17RA prevent cPTH induced osteocytic and osteoblastic RANKL production and bone loss. Mechanistically, cPTH stimulates conventional T cell production of TNFα (TNF), which increases the differentiation of IL-17A producing Th17 cells via TNF receptor 1 (TNFR1) signaling in CD4+ cells. Moreover, cPTH enhances the sensitivity of naïve CD4+ cells to TNF via GαS/cAMP/Ca++ signaling. Accordingly, conditional deletion of GαS in CD4+ cells and treatment with the calcium channel blocker diltiazem prevents Th17 cell expansion and blocks cPTH induced bone loss. Neutralization of IL-17A and calcium channel blockers may thus represent novel therapeutic strategies for hyperparathyroidism.

Doxorubicin is one of the leading drugs for osteosarcoma standard chemotherapy. A total of 40% to 45% of high-grade osteosarcoma patients are unresponsive, or only partially responsive, to doxorubicin (Dox), due to the overexpression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). The aim of this work is to improve Dox-based regimens in resistant osteosarcomas. We used a chemically modified mitochondria-targeted Dox (mtDox) against Pgp-overexpressing osteosarcomas with increased resistance to Dox. Unlike Dox, mtDox accumulated at significant levels intracellularly, exerted cytotoxic activity, and induced necrotic and immunogenic cell death in Dox-resistant/Pgp-overexpressing cells, fully reproducing the activities exerted by anthracyclines in drug-sensitive tumors. mtDox reduced tumor growth and cell proliferation, increased apoptosis, primed tumor cells for recognition by the host immune system, and was less cardiotoxic than Dox in preclinical models of drug-resistant osteosarcoma. The increase in Dox resistance was paralleled by a progressive upregulation of mitochondrial metabolism. By widely modulating the expression of mitochondria-related genes, mtDox decreased mitochondrial biogenesis, the import of proteins and metabolites within mitochondria, mitochondrial metabolism, and the synthesis of ATP. These events were paralleled by increased reactive oxygen species production, mitochondrial depolarization, and mitochondria-dependent apoptosis in resistant osteosarcoma cells, where Dox was completely ineffective. We propose mtDox as a new effective agent with a safer toxicity profile compared with Dox that may be effective for the treatment of Dox-resistant/Pgp-positive osteosarcoma patients, who strongly need alternative and innovative treatment strategies. Mol Cancer Ther; 15(11); 2640-52. ©2016 AACR.

Doxorubicin is one of the most effective drugs for the first-line treatment of high-grade osteosarcoma. Several studies have demonstrated that the major cause for doxorubicin resistance in osteosarcoma is the increased expression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp).We recently identified a library of H 2 S-releasing doxorubicins (Sdox) that were more effective than doxorubicin against resistant osteosarcoma cells. Here we investigated the molecular mechanisms of the higher efficacy of Sdox in human osteosarcoma cells with increasing resistance to doxorubicin.Differently from doxorubicin, Sdox preferentially accumulated within the endoplasmic reticulum (ER), and its accumulation was only modestly reduced in Pgp-expressing osteosarcoma cells. The increase in doxorubicin resistance was paralleled by the progressive down-regulation of genes of ER-associated protein degradation/ER-quality control (ERAD/ERQC), two processes that remove misfolded proteins and protect cell from ER stress-triggered apoptosis. Sdox, that sulfhydrated ERassociated proteins and promoted their subsequent ubiquitination, up-regulated ERAD/ERQC genes. This up-regulation, however, was insufficient to protect cells, since Sdox activated ER stress-dependent apoptotic pathways, e.g. the C/EBP-β LIP/CHOP/PUMA/caspases 12-7-3 axis. Sdox also promoted the sulfhydration of Pgp, that was subsequently ubiquitinated: this process further enhanced Sdox retention and toxicity in resistant cells.Our work suggests that Sdox overcomes doxorubicin resistance in osteosarcoma cells by at least two mechanisms: it induces the degradation of Pgp following its sulfhydration and produces a huge misfolding of ER-associated proteins, triggering ERdependent apoptosis. Sdox may represent the prototype of innovative anthracyclines, effective against doxorubicinresistant/Pgp-expressing osteosarcoma cells by perturbing the ER functions.

Teriparatide is a bone anabolic treatment for osteoporosis, modeled in animals by intermittent PTH (iPTH) administration. iPTH-induced bone anabolism involves CD8+ T cell production of the osteogenic Wnt ligand Wnt10b, induced via yet unknown mechanisms. Here we show that Teriparatide and iPTH cause a ~2-3-fold increase in the number of regulatory T cells (Tregs) in humans and mice. Attesting in vivo relevance, blockade of the Treg increase in mice prevents iPTH-induced CD8+ T cell Wnt10b production and bone anabolism. Mechanistically, Treg action on CD8+ T cells promotes binding of NFAT1/2 and SMAD3 complexes to a NFAT/SMADactivated Wnt10b promoter region that stimualtes Wnt10b gene expression in CD8+ T cells.