Tumor Stress Inside Out: Cell-Extrinsic Effects of the Unfolded Protein Response in Tumor Cells Modulate the Immunological Landscape of the Tumor Microenvironment

The unfolded protein response (UPR) is a eukaryotic cellular adaptive mechanism that functions to cope with stress of the endoplasmic reticulum (ER). Accumulating evidence demonstrates that the tumor microenvironment contains stressors that elicit a UPR, which has been demonstrated to be a cell-intrinsic mechanism crucial for tumorigenesis. In addition, the UPR is a source of proinflammatory signaling whose downstream mediators may hamper antitumor immunity. We discuss how the UPR may impair Ag presentation, which could result in defective T cell priming, also leading to tumor escape and growth. Further, we discuss the recent finding that ER stress and attendant proinflammation can be transmitted from ER-stressed tumor cells to myeloid cells. The ideas presented suggest that, in addition to being a cell-intrinsic mechanism of tumor survival, the tumor UPR can serve as a cell-extrinsic regulator of tumorigenesis by remodeling the immune response in the tumor microenvironment

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

Navin R Mahadevan, Antonio Fernandez, Jeffrey J Rodvold, Gonzalo Almanza, Maurizio ZanettiThe Laboratory of Immunology, Department of Medicine and Moores Cancer Center, University of California, San Diego, USABackground: 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 a (TNF-a). Furthermore we show that TRAMP C1 tumors growing in vivo spontaneously experience ER stress and that transcription of IL-6, IL-23p19, and TNF-a 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.Keywords: unfolded protein response, tumorigenesis, inflammatio

ER stress drives Lipocalin 2 upregulation in prostate cancer cells in an NF-κB-dependent manner

<p>Abstract</p> <p>Background</p> <p>Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.</p> <p>Methods</p> <p>We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.</p> <p>Results</p> <p>Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.</p> <p>Conclusions</p> <p>We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.</p

Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.</p

Cell-extrinsic effects of tumor endoplasmic reticulum stress on myeloid cells

The unfolded protein response (UPR) is an evolutionarily- conserved group of signaling pathways that eukaryotic cells use to adapt to periods of perturbed endoplasmic reticulum (ER) function caused by the accumulation of un/ misfolded proteins the ER lumen. Tumor cells undergo a constitutive UPR to survive the ER stress-inducing noxae within their microenvironment, such as hypoxia and nutrient deprivation. Elements of the tumor UPR have been shown to be key cell-intrinsic mechanisms of tumor survival but only few reports have considered the cell- extrinsic influence of the tumor UPR. Tumor-infiltrating myeloid cells, such as macrophages and dendritic cells, are key players in the cell-extrinsic regulation of tumor growth. Upon entering the tumor microenvironment, however, these cells are polarized to an inflammatory/suppressive phenotype that exacerbate the pro-inflammatory nature of the tumor microenvironment while concomitantly suppressing cell-mediated anti-tumor immune responses. However, the tumor-derived signals driving this mixed inflammatory/ suppressive phenotype have yet to be elucidated. Herein, we show that the tumor cell UPR can function in a cell- extrinsic manner by transmitting ER stress to myeloid cells that infiltrate the tumor microenvironment, a phenomenon we have termed TERS (transmissible ER stress). TERS-imprinted myeloid cells upregulate production of tumorigenic, inflammatory cytokines but also upregulate immunosuppressive markers, culminating in a pro- inflammatory/suppressive phenotype. In macrophages, TERS is sensed, in part, by TLR4. Furthermore, TERS-imprinted myeloid cells display a unique functional phenotype, upregulating costimulatory molecule expression, antigen- presentation machinery, while downregulating effective high-affinity antigen cross-presentation. We demonstrate that TERS-imprinted BMDC do not effectively prime CD8⁺ T cells, causing activation without proliferation, in part due to increased arginase activity. CD8⁺ T cells cross- primed by TERS-imprinted BMDC display a regulatory phenotype and abnormally highly splicing of Xbp-1. We also show that TERS-imprinted BMDC can also dominantly suppress the proper cross-priming function of normal bystander BMDC. Lastly, TERS-imprinted BMDC facilitate tumor growth in vivo, even promoting the transient escape of highly immunogenic tumor cells. Taken together, we demonstrate the ab initio generation of tumor-imprinted myeloid cells that have many of the inflammatory/suppressive characteristics of previously-described tumor-infiltrating myeloid cells and bring to the forefront the tumor UPR as a fundamental driver of myeloid cell polarizatio

ER stress drives Lipocalin 2 upregulation in prostate cancer cells in an NF-kappaB-dependent manner

Abstract Background Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production. Methods We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways. Results Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation. Conclusions We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression

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

A novel GIT2-BRAF fusion in pilocytic astrocytoma

Abstract Background KIAA1549-BRAF fusion is the most common genetic event in pilocytic astrocytoma (PA), and leads to activation of the mitogen activated protein kinase (MAPK) signaling pathway. Fusions of BRAF with other partner genes, as well as other genetic alterations not involving BRAF but also leading to MAPK pathway activation have been described rarely. Case presentation We present a new fusion partner in the low-grade glioma of a 10-year-old male, who presented with headaches and recent episodes of seizures. Magnetic resonance imaging (MRI) demonstrated a right temporal lobe tumor. Histological and immunohistochemical evaluation, and a next generation sequencing assay (Oncopanel, Illumina, 500 genes) including breaKmer analysis for chromosomal rearrangements were performed. Histology was remarkable for a low-grade glioma composed of mildly atypical astrocytes with piloid processes, in a focally microcystic background. Mitoses were not seen; unequivocal Rosenthal fibers or eosinophilic granular bodies were absent. The tumor was positive for OLIG2 and GFAP and negative for BRAF V600E and IDH1 R132H mutant protein immunostains. Oncopanel showed low SOX2 (3q26.33) copy number gain, and no gains at 7q34. There were no significant single nucleotide variants. BreaKmer detected a GIT2-BRAF fusion with loss of BRAF exons 1–8. The integrated diagnosis was low-grade glioma with piloid features, most consistent with pilocytic astrocytoma, WHO grade I. Conclusion GIT2-BRAF fusion has not been reported in the literature in any tumor. Given that the BRAF sequence deleted is identical to that seen in other fusion events in PA, it most likely acts as tumor driver by activation of the MAPK pathway

Figure S4

Metabolic, infectious and tumor cell-intrinsic noxae can all evoke the endoplasmic reticulum (ER) stress response in tumor cells, which is critical for tumor cell growth and cancer progression. Evidence exists that the ER stress response can drive a pro-inflammatory program in tumor cells and macrophages, but, to our knowledge, no one has suggested a role for the tumor ER stress response in influencing macrophages and inflammation in the tumor microenvironment. Here we show that macrophages cultured in conditioned medium from ER stressed tumor cells become activated, themselves undergo ER stress with the upregulation of Grp78, Gadd34, Chop and Xbp-1 splicing, suggesting a general activation of the ER stress signaling pathways. Furthermore, they recapitulate, amplify and expand the pro-inflammatory response of tumor cells. We term this phenomenon "transmissible" ER stress. While neither Toll-Like Receptor (TLR)2 nor interleukin 6 receptor (IL6R) signaling is involved, we noticed a reduction in the transmission of ER stress to TLR4 KO macrophages consistent with the fact that a second signal through TLR4 combined with exposure to tumor ER stress conditioned medium results in a faster ER stress response and an enhancement of pro-inflammatory cytokine production in macrophages. The injection of tumor ER stress conditioned medium into wild-type mice elicited a generalized ER stress response in the liver. We suggest that "transmissible" ER stress is a mechanism through which tumor cells can control myeloid cells by directing them towards a pro-inflammatory phenotype, thus facilitating tumor progression