Necroptosis microenvironment directs lineage commitment in liver cancer

Primary liver cancer is a major health problem with markedly increasing incidence rates. It is mainly represented by hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) which are quite different regarding their morphology and treatment options. Although both share many risk factors as chronic liver damage or inflammation and cirrhosis, the molecular determinants resulting in either HCC or ICC lineage are largely unknown. Here, using two different transposon-based liver cancer mouse models we show that both HCC and ICC can be induced by overexpressing the same oncogenes. Taking advantage of a lineage tracing mouse model we prove that both tumors are derived from adult hepatocytes. Lineage commitment is independent of somatic mutations but depends on the hepatic microenvironment in the pre-tumorigenic phase. While in an apoptotic milieu there is an outgrowth of HCC a necroptotic milieu determines ICC development. This is independent of the composition of infiltrating immune cells, however, it is associated with a specific cytokine signature. The specific cytokine signature can be reversed when inhibiting necroptosis chemically or genetically and is followed by a switch from ICC to HCC development. Epigenetic and transcriptomic analyses of cell lines isolated from hepatocyte-derived HCC or ICC driven by the same oncogene showed specific signatures for each tumor type. Integrative analyses of epigenetic and transcriptomic data revealed Tbx3 and Prdm5 as differentially regulated transcription factors which could also be found in a large cohort of human patients. Functional validation further showed that the interplay of Tbx3 and Prdm5 is sufficient to switch tumor lineage commitment from HCC to ICC. Finally, we identified downstream targets for both transcription factors which revealed quite converse downstream pathways highlighting the importance for their simultaneous interaction in determining lineage commitment in primary liver cancer

Breast cancer prevention by short-term inhibition of TGFβ signaling.

peer reviewedCancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFβ signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFβ in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy

Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases

PTBP1-Mediated alternative splicing regulates the inflammatory secretome and the pro-tumorigenic effects of senescent cells

Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer. By performing a genetic screen for regulators of the senescence-associated secretory phenotype (SASP), Georgilis et al. identify PTBP1, which controls SASP by regulating alternative splicing of genes involved in intracellular trafficking such as EXOC7. PTBP1 knockdown blocks the tumor-promoting functions of SASP.</p

Platelet PD-L1 reflects collective intratumoral PD-L1 expression and predicts immunotherapy response in non-small cell lung cancer

Immune-checkpoint inhibitors (ICI) have transformed oncological therapy. Up to 20% of all non-small cell lung cancers (NSCLCs) show durable responses upon treatment with ICI, however, robust markers to predict therapy response are missing. Here we show that blood platelets interact with lung cancer cells and that PD-L1 protein is transferred from tumor cells to platelets in a fibronectin 1, integrin α5β1 and GPIbα-dependent manner. Platelets from NSCLC patients are found to express PD-L1 and platelet PD-L1 possess the ability to inhibit CD4 and CD8 T-cells. An algorithm is developed to calculate the activation independent adjusted PD-L1 payload of platelets (pPD-L1(Adj.)), which is found to be superior in predicting the response towards ICI as compared to standard histological PD-L1 quantification on tumor biopsies. Our data suggest that platelet PD-L1 reflects the collective tumor PD-L1 expression, plays important roles in tumor immune evasion and overcomes limitations of histological quantification of often heterogeneous intratumoral PD-L1 expression

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells

Altres ajuts: We are grateful to A.J. Innes and members of J.G.'s laboratory for reagents, comments, and other contributions to this project. We thank S. Vernia, J. Ule, and R. Faraway for advice and members of the Proteomics (P. Faull and A. Montoya) and Genomics (L. Game, K. Rekopoulou, and A. Ivan) LMS facilities for help with the proteomics and RNA-seq, respectively. We thank T.-W. Kang and C. Fellmeth for technical support and Life Science Editors for editorial assistance. J.A. is funded by the Breast Cancer Research Foundation (BCRF, grant BCRF-17-008) and Instituto de Salud Carlos III. N.L.B.-M.'s laboratory is supported by EMBO (Installation grant 3057) and Fundação para a Ciência e a Tecnologia, Portugal (FCT Investigator Starting grant IF/00595/2014). M.H. was supported by an ERC consolidator grant (HepatoMetabopath). Core support from MRC (grants MC-A652-5PZ00 and MC_U120085810) funded the research in J.G.'s laboratory.Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer. By performing a genetic screen for regulators of the senescence-associated secretory phenotype (SASP), Georgilis et al. identify PTBP1, which controls SASP by regulating alternative splicing of genes involved in intracellular trafficking such as EXOC7. PTBP1 knockdown blocks the tumor-promoting functions of SASP

microRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-associated Protein 1 to Suppress Hepatocarcinogenesis

Background & Aims: We performed an integrated analysis to identify microRNAs (miRNAs) and mRNAs with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. Methods: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta , or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in-silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched non-tumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 non-tumor liver samples using the Taqman assay. Results: Levels of the microRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with non-tumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of AKT1-MYC–induced tumors in mice. Conclusions: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans

Necroptosis microenvironment directs lineage commitment in liver cancer

Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC