IRE1α regulates macrophage polarization, PD-L1 expression, and tumor survival.

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

Photopiperazines A-D, Photosensitive Interconverting Diketopiperazines with Significant and Selective Activity against U87 Glioblastoma Cells, from a Rare, Marine-Derived Actinomycete of the Family Streptomycetaceae.

Photopiperazines A-D (1-4), unsaturated diketopiperazine derivatives, were isolated from the culture broth of a rare, marine-derived actinomycete bacterium, strain AJS-327. This strain shows very poor 16S rRNA sequence similarity to other members of the actinomycete family Streptomycetaceae, indicating it is likely a new lineage within this group. The structures of the photopiperazines were defined by analysis of HR-ESI-TOF-MS spectra in conjunction with the interpretation of 1D and 2D NMR data. The photopiperazines are sensitive to light, causing interconversion among the four olefin geometrical isomers, which made purification of each isomer challenging. The photopiperazines are highly cytotoxic metabolites that show selective toxicity toward U87 glioblastoma and SKOV3 ovarian cancer cell lines.

Synthesis and delivery of short, noncoding RNA by B lymphocytes.

Evolutionarily conserved short (20-30 nucleotides) noncoding RNAs (microRNAs) are powerful regulators of gene expression in a variety of physiological and pathological processes. As such, means to efficiently modulate microRNA function constitute an important therapeutic opportunity. Here we demonstrate that primary B lymphocytes can be genetically programmed with nonviral plasmid DNA for the biogenesis and delivery of antisense sequences (anti-microRNA) against microRNA-150 (miR-150). Within 18 h of transfection with an anti-miR-150 construct, primary B lymphocytes secrete ∼3,000 copies of anti-miR-150 molecules per cell. Anti-miR-150 molecules released by B lymphocytes were internalized by CD8 T lymphocytes during cross-priming in vitro and in vivo, resulting in marked down-regulation of endogenous miR-150. However, such internalization was not observed in the absence of cross-priming. These results suggest that shuttling anti-miR-150 molecules from B lymphocytes to T cells requires the activation of receiver T cells via the antigen receptor. Finally, anti-miR-150 synthesized in B cells were secreted both as free and extracellular vesicle-associated fractions, but only extracellular vesicle-associated anti-miR-150 were apparently taken up by CD8 T cells. Collectively, these data indicate that primary B lymphocytes represent an efficient platform for the synthesis and delivery of short, noncoding RNA, paving the way for an approach to immunogenomic therapies.

IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

Insidious communication amongst cancer cells.

The tumor microenvironment is home to various types of cognate and non-cognate cell interactions. Here we comment on a newly discovered form of intercellular communication, which is based on endoplasmic reticulum stress signaling. Through this mechanism transmitter cancer cells impart receiver cancer cells with resistance to secondary metabolic, pharmacologic and genotoxic stress, providing survival advantage. The implications of this finding are briefly discussed.

Extracellular vesicles produced in B cells deliver tumor suppressor miR-335 to breast cancer cells disrupting oncogenic programming in vitro and in vivo.

The successful implementation of miRNA (miR) therapies in humans will ultimately rely on the use of vehicles with improved cellular delivery capability. Here we tested a new system that leverages extracellular vesicles (EVs) laden with a tumor suppressor miRNA (miR-335) produced in B cells by plasmid DNA induction (iEVs). We demonstrate that iEVs-335 efficiently and durably restored the endogenous miR-335 pool in human triple negative breast cancer cells, downregulated the expression of the miR-335 target gene SOX4 transcription factor, and markedly inhibited tumor growth in vivo. Remarkably, iEVs-335 mediated transcriptional effects that persisted in tumors after 60 days post orthotopic implantation. Genome-wide RNASeq analysis of cancer cells treated in vitro with iEVs-335 showed the regulation of a discrete number of genes only, without broad transcriptome perturbations. This new technology may be ideally suited for therapies aimed to restore tumor suppressor miRNAs in cancer cells, disrupting the oncogenic program established after escape from miRNA control.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Tumor microenvironment on the move and the Aselli connection.

The tumor microenvironment is involved in many activities that promote tumor cell growth, local spreading, and metastasis. In this issue of Science Signaling, Jung et al found that lymphangiogenesis may result from the cooperation of two molecules, sphingosine-1-phosphate (S1P) and lipocalin 2 (LCN2), produced by tumor cells and macrophages, respectively. The new S1P-LCN2 axis stresses the importance of innate immunity in remodeling the tumor microenvironment and in lymphangiogenesis.

Immune modulation by ER stress and inflammation in the tumor microenvironment.

It is now increasingly evident that the immune system represents a barrier to tumor emergence, growth, and recurrence. Although this idea was originally proposed almost 50 years ago as the "immune surveillance hypothesis", it is commonly recognized that, with few rare exceptions, tumor cells always prevail. Thus, one of the central unsolved paradoxes of tumor immunology is how a tumor escapes immune control, which is reflected in the lack of effective autochthonous or vaccine-induced anti-tumor T cell responses. In this review, we discuss the role of the endoplasmic reticulum (ER) stress response/unfolded protein response (UPR) in the immunomodulation of myeloid cells and T cells. Specifically, we will discuss how the tumor cell UPR polarizes myeloid cells in a cell-extrinsic manner, and how in turn, thus polarized myeloid cells negatively affect T cell activation and clonal expansion.

A Janus-faced role of the unfolded protein response in antitumor immunity.

The unfolded protein response (UPR) has been established as a cell-intrinsic mechanism of survival for malignant cells facing microenvironmental stressors. Recent evidence indicates that the UPR also modulates antitumor immunity. Here, we discuss the bi-faced role of the UPR as it both promotes and antagonizes antitumor T-cell immunity.

Lipocalin 2 in cancer: when good immunity goes bad.

The innate immune molecule Lipocalin 2 (LCN2) was initially shown to combat bacterial infection by binding bacterial siderophores, hence impairing microbial iron sequestration. In recent years, it has become apparent that LCN2 is over-expressed in cancers of diverse histological origin and that it facilitates tumorigenesis by promoting survival, growth, and metastasis. Herein, we discuss emerging evidence that substantiates two functional roles for LCN2 in cancer: promotion of the epithelial-to-mesenchymal transition (EMT) that facilitates an invasive phenotype and metastasis, and sequestration of iron that results in cell survival and tumorigenesis. Further, we present evidence that upregulated LCN2 expression in solid tumors is induced by hypoxia and pro-inflammation, microenvironmental noxae that converge to cause an endoplasmic reticulum (ER) stress response. Taken together, it appears that tumor cells exploit the beneficial innate immune function of LCN2 to support uncontrolled growth. This duplicity in function highlights LCN2 and its upstream driver, the ER stress response, as key targets for cancer therapy.

Cell-extrinsic effects of tumor ER stress imprint myeloid dendritic cells and impair CD8⁺ T cell priming.

Tumor-infiltrating myeloid cells, such as dendritic cells (BMDC), are key regulators of tumor growth. However, the tumor-derived signals polarizing BMDC to a phenotype that subverts cell-mediated anti-tumor immunity have yet to be fully elucidated. Addressing this unresolved problem we show that the tumor unfolded protein response (UPR) can function in a cell-extrinsic manner via the transmission of ER stress (TERS) to BMDC. TERS-imprinted BMDC upregulate the production of pro-inflammatory, tumorigenic cytokines but also the immunosuppressive enzyme arginase. Importantly, they downregulate cross-presentation of high-affinity antigen and fail to effectively cross-prime CD8(+) T cells, causing T cell activation without proliferation and similarly dominantly suppress cross-priming by bystander BMDC. Lastly, TERS-imprinted BMDC facilitate tumor growth in vivo with fewer tumor-infiltrating CD8(+) T cells. In sum, we demonstrate that tumor-borne ER stress imprints ab initio BMDC to a phenotype that recapitulates several of the inflammatory/suppressive characteristics ascribed to tumor-infiltrating myeloid cells, highlighting the tumor UPR as a critical controller of anti-tumor immunity and a new target for immune modulation in cancer.

Prostate cancer cells undergoing ER stress in vitro and in vivo activate transcription of pro-inflammatory cytokines.

BACKGROUND: Several micro-environmental and cell-intrinsic stimuli cause tumor cells to undergo endoplasmic reticulum (ER) stress in vivo. The occurrence of an ER stress response has been associated with tumor progression and angiogenesis. Recently, we found that pharmacological induction of ER stress in B lymphoma cells upregulates the transcription of several pro-inflammatory cytokines. RESULTS: Here, we show that transgenic adenocarcinoma of the mouse prostate (TRAMP) C1 murine prostate cancer cells induced to undergo ER stress in vitro activate the transcription of interleukin 6 (IL-6), interleukin 23p19 (IL-23p19), and tumor necrosis factor α (TNF-α). Furthermore we show that TRAMP C1 tumors growing in vivo spontaneously experience ER stress and that transcription of IL-6, IL-23p19, and TNF-α correlates with the in vivo ER stress response. CONCLUSIONS: These results suggest that an ER stress response in prostate cancer cells activates a program of pro-inflammatory cytokine transcription. A possible implication of this finding is that cancer cells may use the ER stress response to modify their microenvironment.