The pluripotency factor NANOG contributes to mesenchymal plasticity and is predictive for outcome in esophageal adenocarcinoma.

BACKGROUND: Despite the advent of neoadjuvant chemoradiotherapy (CRT), overall survival rates of esophageal adenocarcinoma (EAC) remain low. A readily induced mesenchymal transition of EAC cells contributes to resistance to CRT. METHODS: In this study, we aimed to chart the heterogeneity in cell state transition after CRT and to identify its underpinnings. A panel of 12 esophageal cultures were treated with CRT and ranked by their relative epithelial-mesenchymal plasticity. RNA-sequencing was performed on 100 pre-treatment biopsies. After RNA-sequencing, Ridge regression analysis was applied to correlate gene expression to ranked plasticity, and models were developed to predict mesenchymal transitions in patients. Plasticity score predictions of the three highest significant predictive models were projected on the pre-treatment biopsies and related to clinical outcome data. Motif enrichment analysis of the genes associated with all three models was performed. RESULTS: This study reveals NANOG as the key associated transcription factor predicting mesenchymal plasticity in EAC. Expression of NANOG in pre-treatment biopsies is highly associated with poor response to neoadjuvant chemoradiation, the occurrence of recurrences, and median overall survival difference in EAC patients (>48 months). Perturbation of NANOG reduces plasticity and resensitizes cell lines, organoid cultures, and patient-derived in vivo grafts. CONCLUSIONS: In conclusion, NANOG is a key transcription factor in mesenchymal plasticity in EAC and a promising predictive marker for outcome.

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Although chemotherapy combined with radiotherapy (chemoradiotherapy) followed by surgery has improved survival, tumor recurrence and metastatic disease (that has spread to other parts of the body) are often observed after several months. In this study, we assessed the effect of chemoradiotherapy on esophageal cells in the lab to predict the effect in patients with esophageal cancer. To investigate this, genes were assessed from 12 different cell lines and 100 patient tissues. We revealed that levels of one of the genes, NANOG, associates with poor response in patients. NANOG could be a promising marker to predict outcome in patients with esophageal cancer. This knowledge might help clinicians to treat patients with esophageal cancer appropriately, or may lead to new or optimized treatments.

Strategies to reprogram anti-inflammatory macrophages towards pro-inflammatory macrophages to support cancer immunotherapies.

Tumor-associated myeloid cells, including macrophages and myeloid-derived suppressor cells, can be highly prevalent in solid tumors and play a significant role in the development of the tumor. Therefore, myeloid cells are being considered potential targets for cancer immunotherapies. In this review, we focused on strategies aimed at targeting tumor-associated macrophages (TAMs). Most strategies were studied preclinically but we also included a limited number of clinical studies based on these strategies. We describe possible underlying mechanisms and discuss future challenges and prospects.

Prognostic value of Mandard score and nodal status for recurrence patterns and survival after multimodal treatment of oesophageal adenocarcinoma.

BACKGROUND: This study evaluated the association of pathological tumour response (tumour regression grade, TRG) and a novel scoring system, combining both TRG and nodal status (TRG-ypN score; TRG1-ypN0, TRG>1-ypN0, TRG1-ypN+ and TRG>1-ypN+), with recurrence patterns and survival after multimodal treatment of oesophageal adenocarcinoma. METHODS: This Dutch nationwide cohort study included patients treated with neoadjuvant chemoradiotherapy followed by oesophagectomy for distal oesophageal or gastro-oesophageal junctional adenocarcinoma between 2007 and 2016. The primary endpoint was the association of Mandard score and TRG-ypN score with recurrence patterns (rate, location, and time to recurrence). The secondary endpoint was overall survival. RESULTS: Among 2746 inclusions, recurrence rates increased with higher Mandard scores (TRG1 30.6%, TRG2 44.9%, TRG3 52.9%, TRG4 61.4%, TRG5 58.2%; P < 0.001). Among patients with recurrent disease, the distribution (locoregional versus distant) was the same for the different TRG groups. Patients with TRG1 developed more brain recurrences (17.7 versus 9.8%; P = 0.001) and had a longer mean overall survival (44 versus 35 months; P < 0.001) than those with TRG>1. The TRG>1-ypN+ group had the highest recurrence rate (64.9%) and worst overall survival (mean 27 months). Compared with the TRG>1-ypN0 group, patients with TRG1-ypN+ had a higher risk of recurrence (51.9 versus 39.6%; P < 0.001) and worse mean overall survival (33 versus 41 months; P < 0.001). CONCLUSION: Improved tumour response to neoadjuvant therapy was associated with lower recurrence rates and higher overall survival rates. Among patients with recurrent disease, TRG1 was associated with a higher incidence of brain recurrence than TRG>1. Residual nodal disease influenced prognosis more negatively than residual disease at the primary tumour site.

Estrogen-related receptor alpha drives mitochondrial biogenesis and resistance to neoadjuvant chemoradiation in esophageal cancer.

Neoadjuvant chemoradiotherapy (nCRT) improves outcomes in resectable esophageal adenocarcinoma (EAC), but acquired resistance precludes long-term efficacy. Here, we delineate these resistance mechanisms. RNA sequencing on matched patient samples obtained pre-and post-neoadjuvant treatment reveal that oxidative phosphorylation was the most upregulated of all biological programs following nCRT. Analysis of patient-derived models confirms that mitochondrial content and oxygen consumption strongly increase in response to nCRT and that ionizing radiation is the causative agent. Bioinformatics identifies estrogen-related receptor alpha (ESRRA) as the transcription factor responsible for reprogramming, and overexpression and silencing of ESRRA functionally confirm that its downstream metabolic rewiring contributes to resistance. Pharmacological inhibition of ESRRA successfully sensitizes EAC organoids and patient-derived xenografts to radiation. In conclusion, we report a profound metabolic rewiring following chemoradiation and demonstrate that its inhibition resensitizes EAC cells to radiation. These findings hold broader relevance for other cancer types treated with radiation as well.

Re-polarization of immunosuppressive macrophages to tumor-cytotoxic macrophages by repurposed metabolic drugs.

M2-like tumor-associated macrophages promote tumor progression by establishing an immunosuppressive tumor microenvironment. The phenotype and activity of immunosuppressive macrophages are related to their mitochondrial metabolism. Thus, we studied if drugs targeting mitochondrial metabolic pathways can repolarize macrophages from M2 into an M1-like phenotype or can prevent M0-to-M2 polarization. The drugs selected are clinically approved or in clinical trials and target M2-specific metabolic pathways: fatty acid oxidation (Perhexiline and Trimetazidine), glutaminolysis (CB-839), PPAR activation (HX531), and mitochondrial electron transport chain (VLX-600). Murine bone marrow-derived macrophages were either polarized to M2 using IL-4 in the presence of the drugs or polarized first into M2 and then treated with the drugs in presence of IFN-γ for re-polarization. Targeting both fatty acid oxidation with Perhexiline or the electron transport chain with VLX-600 in the presence of IFN-γ, impaired mitochondrial basal, and maximal respiration and resulted in M2 to M1-like re-polarization (increased iNOS expression, NO production, IL-23, IL-27, and TNF-α secretion), similar to LPS+IFN-γ re-polarization. Moreover, drug-induced macrophage re-polarization resulted in a strong tumor-cytotoxic activity. Furthermore, the polarization of M0- to M2-like macrophages was impaired by CB-839, Trimetazidine, HX531, and Perhexiline, while Hx531 and Perhexiline also reduced MCP-1 secretion. Our results show that by targeting cell metabolism, macrophages could be re-polarized from M2- into an anti-tumoral M1-like phenotype and that M0-to-M2 polarization could be prevented. Overall, this study provides rational for the use of clinically applicable drugs to change an immunosuppressive tumor environment into a pro-inflammatory tumor environment that could support cancer immunotherapies.

First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies.

A filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer.

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.

A short hairpin RNA-based adjuvant targeting NF-κB repressor IκBα promotes migration of dermal dendritic cells to draining lymph nodes and antitumor CTL responses induced by DNA vaccination.

DNA vaccination is an attractive approach to elicit tumor-specific cytotoxic CD8+ T lymphocytes (CTL), which can mediate protective immunity against tumors. To initiate CTL responses, antigen-encoding plasmids employed for DNA vaccination need to activate dendritic cells (DC) through the stimulation of DNA-sensing innate immune receptors that converge in the activation of the master transcription factor NF-κB. To this end, NF-κB repressor IκBα needs to be degraded, allowing NF-κB to translocate to the nucleus and transcribe proinflammatory target genes, as well as its repressor IκBα. Therefore, NF-κB activation is self-limited by de novo synthesis of IκBa, which sequesters NF-κB in the cytosol. Hence, we tested whether co-delivering a shRNA-based adjuvant able to silence IκBα expression would further promote DNA-induced NFκB activation, DC activation and tumor-protective CTL responses induced by DNA vaccination in a preclinical model. First, an IκBα-targeting shRNA plasmid (shIκBα) was shown to reduce IκBα expression and promote NFκB-driven transcription in vitro, as well as up-regulate inflammatory target genes in vivo. Then, we showed that intradermal DNA electroporation induced the migration of skin migratory dendritic cells to draining lymph nodes and maturation of dermal dendritic cells (dDC). Interestingly, shIκBα further promoted the migration of mature skin migratory dendritic cells, in particular dDC, which are specialized in antigen cross-presentation and activation of CD8+ T cells. Consistently, mice vaccinated with a plasmid encoding the melanoma-associated antigen tyrosinase-related protein 2 (TRP2) in combination with shIκBα enhanced TRP2-specific CTL responses and reduced the number of lung melanoma foci in mice challenged with intravenous injection of B16F10 cells. Moreover, therapeutic vaccination with pTRP2 and shIκBα delayed the growth of B16F10 melanoma subcutaneous tumors. Our data suggest that adjuvants promoting NF-κB activation represent an attractive strategy to boost DC activation and promote the generation of tumor-protective CTL responses elicited by DNA vaccines.

Inhibition of dopamine receptor D3 signaling in dendritic cells increases antigen cross-presentation to CD8+ T-cells favoring anti-tumor immunity.

Dendritic cells (DCs) display the unique ability for cross-presenting antigens to CD8+ T-cells, promoting their differentiation into cytotoxic T-lymphocytes (CTLs), which play a pivotal role in anti-tumor immunity. Emerging evidence points to dopamine receptor D3 (D3R) as a key regulator of immunity. Accordingly, we studied how D3R regulates DCs function in anti-tumor immunity. The results show that D3R-deficiency in DCs enhanced expansion of CTLs in vivo and induced stronger anti-tumor immunity. Co-culture experiments indicated that D3R-inhibition in DCs potentiated antigen cross-presentation and CTLs activation. Our findings suggest that D3R in DCs constitutes a new therapeutic target to strengthen anti-tumor immunity.

Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8+ T Cell Responses.

Dendritic cell (DC) trafficking from peripheral tissues to lymph nodes (LNs) is a key step required to initiate T cell responses against pathogens as well as tumors. In this context, cellular membrane protrusions and the actin cytoskeleton are essential to guide DC migration towards chemotactic signals. Caveolin-1 (CAV1) is a scaffolding protein that modulates signaling pathways leading to remodeling of the actin cytoskeleton and enhanced migration of cancer cells. However, whether CAV1 is relevant for DC function and specifically for DC migration to LNs is unknown. Here, we show that CAV1 expression is upregulated in DCs upon LPS- and TNF-α-induced maturation. CAV1 deficiency did not affect differentiation, maturation, or the ability of DCs to activate CD8+ T cells in vitro. However, CAV1-deficient (CAV1-/-) DCs displayed reduced in vivo trafficking to draining LNs in control and inflammatory conditions. In vitro, CAV1-/- DCs showed reduced directional migration in CCL21 gradients in transwell assays without affecting migration velocity in confined microchannels or three-dimensional collagen matrices. In addition, CAV1-/- DCs displayed reduced activation of the small GTPase Rac1, a regulator of actin cytoskeletal remodeling, and lower numbers of F-actin-forming protrusions. Furthermore, mice adoptively transferred with peptide-pulsed CAV1-/- DCs showed reduced CD8+ T cell responses and antitumor protection. Our results suggest that CAV1 promotes the activation of Rac1 and the formation of membrane protrusions that favor DC chemotactic trafficking toward LNs where they can initiate cytotoxic T cell responses.

GSK-3ß/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy.

Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT) is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3ß (GSK-3ß) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3ß signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3ß inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc) in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3ß activity as determined by increased GSK-3ß phosphorylation at Ser9 and ß-catenin protein accumulation, and also by reduction in ß-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3ß inhibition with 1-azakenpaullone or a GSK-3ß-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3ß mutant (GSK-3ßS9A) inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [3H]-leucine incorporation (as a measurement of cellular protein synthesis). Calcineurin-NFAT inhibition abolished and GSK-3ß inhibition promoted the hypertrophy stimulated by testosterone. GSK-3ß activation by GSK-3ßS9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results suggest that cardiac myocyte hypertrophy induced by testosterone involves a cooperative mechanism that links androgen signaling with the recruitment of NFAT through calcineurin activation and GSK-3ß inhibition.

Harnessing DNA-induced immune responses for improving cancer vaccines.

DNA vaccines have emerged as an attractive strategy to promote protective cellular and humoral immunity against the encoded antigen. DNA vaccines are easy to generate, inexpensive to produce and purify at large-scale, highly stable and safe. In addition, plasmids used for DNA vaccines act as powerful "danger signals" by stimulating several DNA-sensing innate immune receptors that promote the induction of protective adaptive immunity. The induction of tumor-specific immune responses represents a major challenge for DNA vaccines because most of tumor-associated antigens are normal non-mutated self-antigens. As a consequence, induction of potentially self-reactive T cell responses against such poorly immunogenic antigens is controlled by mechanisms of central and peripheral tolerance as well as tumor-induced immunosuppression. Although several DNA vaccines against cancer have reached clinical testing, disappointing results have been observed. Therefore, the development of new adjuvants that strongly stimulate the induction of antitumor T cell immunity and counteract immune-suppressive regulation is an attractive approach to enhance the potency of DNA vaccines and overcome tumor-associated tolerance. Understanding the DNA-sensing signaling pathways of innate immunity that mediate the induction of T cell responses elicited by DNA vaccines represents a unique opportunity to develop novel adjuvants that enhance vaccine potency. The advance of DNA adjuvants needs to be complemented with the development of potent delivery systems, in order to step toward successful clinical application. Here, we briefly discuss recent evidence showing how to harness DNA-induced immune response to improve the potency of cancer vaccines and counteract tumor-associated tolerance.

Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway.

Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of beta-myosin heavy chain, alpha-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP(3)/Ca(2+) and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.