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Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage., The bacterial pathogen Helicobacter pylori is known for its ability to induce DNA double-strand breaks in the genome of its target cells. Here, we show that H. pylori-induced DNA damage and replication stress occurs in S-phase cells as a result of R-loop-mediated transcription/replication conflicts that are triggered by activation of the ALPK1/TIFA/NF-κB signaling axis.

Lymphoid neogenesis gives rise to tertiary lymphoid structures (TLS) in the periphery of multiple cancer types including muscle invasive bladder cancer (MIBC) where it has positive prognostic and predictive associations. Here, we explored molecular, clinical, and histological data of The Cancer Genome Atlas, as well as the IMvigor210 dataset to study factors associated with TLS development and function in the tumor microenvironment (TME) of MIBC. We also analyzed tumor immune composition including TLS in an independent, retrospective MIBC cohort. We found that the combination of TLS density and tumor mutational burden provides a novel independent prognostic biomarker in MIBC. Gene expression profiles obtained from intratumoral regions that rarely contain TLS in MIBC showed poor correlation with the prognostic TLS density measured in tumor periphery. Tumors with high TLS density showed increased gene signatures as well as infiltration of activated lymphocytes. Intratumoral B-cell and CD8+ T-cell co-infiltration was frequent in TLS-high samples, and such regions harbored the highest proportion of PD-1+TCF1+ progenitor-like T cells, naïve T cells, and activated B cells when compared to regions predominantly infiltrated by either B cells or CD8+ T cells alone. We found four TLS maturation subtypes; however, differences in TLS composition appeared to be dictated by the TME and not by the TLS maturation status. Finally, we identified one downregulated and three upregulated non-immune cell-related genes in TME with high TLS density, which may represent candidates for tumor-intrinsic regulation of lymphoid neogenesis. Our study provides novel insights into TLS-associated gene expression and immune contexture of MIBC and indicates towards the relevance of B-cell and CD8+ T-cell interactions in anti-tumor immunity within and outside TLS.

Summary: The two human pathogens Helicobacter pylori and Mycobacterium tuberculosis (Mtb) co-exist in many geographical areas of the world. Here, using a co-infection model of H. pylori and the Mtb relative M. bovis bacillus Calmette-Guérin (BCG), we show that both bacteria affect the colonization and immune control of the respective other pathogen. Co-occurring M. bovis boosts gastric Th1 responses and H. pylori control and aggravates gastric immunopathology. H. pylori in the stomach compromises immune control of M. bovis in the liver and spleen. Prior antibiotic H. pylori eradication or M. bovis-specific immunization reverses the effects of H. pylori. Mechanistically, the mutual effects can be attributed to the redirection of regulatory T cells (Treg cells) to sites of M. bovis infection. Reversal of Treg cell redirection by CXCR3 blockade restores M. bovis control. In conclusion, the simultaneous presence of both pathogens exacerbates the problems associated with each individual infection alone and should possibly be factored into treatment decisions.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer worldwide. Cancer-associated stroma (CAS) is central to tumor development and strongly influences therapy response. Perineural infiltration (PNI) represents a major risk factor for cSCC and likely influences CAS reprogramming. However, stromal reprogramming in cSCC remains poorly characterized, and it is unknown whether and how PNI influences CAS. To address these questions, we analyzed CAS and matched normal stroma from 20 cSCC cases (11 without PNI and 9 with PNI) by laser-capture microdissection using RNA sequencing. Our analysis reveals extensive stromal reprogramming strongly driven by changes in immune cells, as validated using immunohistochemistry. Furthermore, CAS of cSCC displays markers of immune exhaustion, and multiplex spatial analysis suggests that PD-L1 expression on NK T cells contributes to T-cell exhaustion and immunosuppression. Finally, PNI is characterized by increased IL-17A. In PNI-negative cases, IL-17A derives predominantly from CD3+ cells. However, with PNI, we observe an increased contribution of fibroblasts to high IL-17A, which coincides with a significant increase in FAP+ cells. Our analysis elucidates the molecular landscape of CAS in cSCC and identifies the presence of immunosuppressive mechanisms, supporting further research into immunotherapy and anti‒IL-17A in cSCC, especially for cases with PNI.

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a−/− melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism., Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes.

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BackgroundThe embryo of the crustacean Parhyale hawaiensis has a total, unequal and invariant early cleavage pattern. It specifies cell fates earlier than other arthropods, including Drosophila, as individual blastomeres of the 8-cell stage are allocated to the germ layers and the germline. Furthermore, the 8-cell stage is amenable to embryological manipulations. These unique features make Parhyale a suitable system for elucidating germ layer specification in arthropods. Since asymmetric localization of maternally provided RNA is a widespread mechanism to specify early cell fates, we asked whether this is also true for Parhyale. A candidate gene approach did not find RNAs that are asymmetrically distributed at the 8-cell stage. Therefore, we designed a high-density microarray from 9400 recently sequenced ESTs (1) to identify maternally provided RNAs and (2) to find RNAs that are differentially distributed among cells of the 8-cell stage.ResultsMaternal-zygotic transition takes place around the 32-cell stage, i.e. after the specification of germ layers. By comparing a pool of RNAs from early embryos without zygotic transcription to zygotic RNAs of the germband, we found that more than 10% of the targets on the array were enriched in the maternal transcript pool. A screen for asymmetrically distributed RNAs at the 8-cell stage revealed 129 transcripts, from which 50% are predominantly expressed in the early embryonic stages. Finally, we performed knockdown experiments for two of these genes and observed cell-fate-related defects of embryonic development.ConclusionsIn contrast to Drosophila, the four primary germ layer cell lineages in Parhyale are specified during the maternal control phase of the embryo. A key step in this process is the asymmetric distribution of a large number of maternal RNAs to the germ layer progenitor cells.

In addition to improving insulin sensitivity in type 2 diabetes, the thiazolidinedione family of compounds and the pharmacologic activation of their best-characterized target PPARγ have been proposed as a therapeutic option for cancer treatment. In this study, we reveal a new mode of action for the thiazolidinedione rosiglitazone that can contribute to tumorigenesis. Rosiglitazone activated a tumorigenic paracrine communication program in a subset of human melanoma cells that involves the secretion of cytokines, chemokines, and angiogenic factors. This complex blend of paracrine signals activated nonmalignant fibroblasts, endothelial cells, and macrophages in a tumor-friendly way. In agreement with these data, rosiglitazone promoted human melanoma development in xenografts, and tumors exposed to rosiglitazone exhibited enhanced angiogenesis and inflammation. Together, these findings establish an important tumorigenic action of rosiglitazone in a subset of melanoma cells. Although studies conducted on cohorts of diabetic patients report overall benefits of thiazolidinediones in cancer prevention, our data suggest that exposure of established tumors to rosiglitazone may be deleterious. These findings uncover a novel mechanism by which the thiazolidinedione compound rosiglitazone contributes to tumorigenesis, thus highlighting a potential risk associated with its use in patients with established tumors. .

Tumor lymphangiogenesis is accompanied by a higher incidence of sentinel lymph node metastasis and shorter overall survival in several types of cancer. We asked whether tumor lymphangiogenesis might also occur in distant organs with established metastases and whether it might promote further metastatic spread of those metastases to other organs. Using mouse metastasis models, we found that lymphangiogenesis occurred in distant lung metastases and that some metastatic tumor cells were located in lymphatic vessels and draining lymph nodes. In metastasis-bearing lungs of melanoma patients, a higher lymphatic density within and around metastases and lymphatic invasion correlated with poor outcome. Using a transgenic mouse model with inducible expression of vascular endothelial growth factor C (VEGF-C) in the lung, we found greater growth of lung metastases, with more abundant dissemination to other organs. Our findings reveal unexpected contributions of lymphatics in distant organs to the promotion of growth of metastases and their further spread to other organs, with potential clinical implications for adjuvant therapies in patients with metastatic cancer.

Real-time reverse transcription polymerase chain reaction (RT-PCR) remains a gold standard in detection of various viral diseases. In the COVID-19 pandemic, multiple RT-PCR based tests were developed to screen for viral infection. As an emergency response to growing testing demand, we established a SARS-CoV-2 PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial (CDC 2019-nCoV, Applied BiosystemsTM 2019-nCoV Assay Kit v1 TF-SinglePlex, 2019-nCoV Assay Kit v2 TF-MultiPlex, and EURORealTime SARS-CoV-2), one customized (Institute Pasteur), and one in-house RT-PCR protocols were evaluated with 92 SARS-CoV-2 positive and 92 SARS-CoV-2 negative samples. Furthermore, economical and practical characteristics of these protocols were compared. Additionally, a highly sensitive digital droplet PCR (ddPCR) method was developed and application of RT- and ddPCR methods on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/μL), high sensitivities (93.6-97.8%) and specificities (98.7-100%) for the tested RT-PCR protocols were found. Furthermore, the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity was demonstrated. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity, and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

MAPK inhibitors (MAPKi) show outstanding clinical response rates in melanoma patients harbouring BRAF mutations, but resistance is common. The ability of melanoma cells to switch from melanocytic to mesenchymal phenotypes appears to be associated with therapeutic resistance. High-throughput, subcellular proteome analyses and RNAseq on two panels of primary melanoma cells that were either sensitive or resistant to MAPKi revealed that only 15 proteins were sufficient to distinguish between these phenotypes. The two proteins with the highest discriminatory power were PTRF and IGFBP7, which were both highly upregulated in the mesenchymal-resistant cells. Proteomic analysis of CRISPR/Cas-derived PTRF knockouts revealed targets involved in lysosomal activation, endocytosis, pH regulation, EMT, TGFβ signalling and cell migration and adhesion, as well as a significantly reduced invasive index and ability to form spheres in 3D culture. Overexpression of PTRF led to MAPKi resistance, increased cell adhesion and sphere formation. In addition, immunohistochemistry of patient samples showed that PTRF expression levels were a significant biomarker of poor progression-free survival, and IGFBP7 levels in patient sera were shown to be higher after relapse.

The recent convergence of high-dimensional molecular datasets with small-molecule inhibitor pipelines allows for selective targeting of aberrantly regulated pathways in many cancer types. But intra-tumor heterogeneity, paradoxical activation, intrinsic or acquired therapeutic resistance, and non-linear pathway interactions confound most simple targeting strategies(Widmer et al., 2015). For instance, MAPK signaling is activated by hot-spot mutations in BRAF, which are found in about 40-50% of melanoma cases. This article is protected by copyright. All rights reserved.

Background Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression. Results Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients. Conclusions Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0594-4) contains supplementary material, which is available to authorized users.

We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.