Protocol for the characterization of the pancreatic tumor microenvironment using organoid-derived mouse models and single-nuclei RNA sequencing.

Single-nuclei RNA sequencing (snRNA-seq) allows for obtaining gene expression profiles from frozen or hard-to-dissociate tissues at the single-nuclei level. Here, we describe a protocol to obtain snRNA-seq data of pancreatic tumors from orthotopically grafted organoid-derived mouse models. We provide details on the establishment of these mouse models, the isolation of single nuclei from pancreatic tumors, and the analysis of the snRNA-seq datasets. For complete details on the use and execution of this protocol, please refer to Mucciolo et al.1.

ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

EGFR-activated myofibroblasts promote metastasis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Cancer-associated fibroblasts (CAFs) are recognized potential therapeutic targets, but poor understanding of these heterogeneous cell populations has limited the development of effective treatment strategies. We previously identified transforming growth factor beta (TGF-ß) as a main driver of myofibroblastic CAFs (myCAFs). Here, we show that epidermal growth factor receptor/Erb-B2 receptor (EGFR/ERBB2) signaling is induced by TGF-ß in myCAFs through an autocrine process mediated by amphiregulin. Inhibition of this EGFR/ERBB2-signaling network in PDAC organoid-derived cultures and mouse models differentially impacts distinct CAF subtypes, providing insights into mechanisms underpinning their heterogeneity. Remarkably, EGFR-activated myCAFs promote PDAC metastasis in mice, unmasking functional significance in myCAF heterogeneity. Finally, analyses of other cancer datasets suggest that these processes might operate in other malignancies. These data provide functional relevance to myCAF heterogeneity and identify a candidate target for preventing tumor invasion in PDAC.

Molecular landscape and functional characterization of centrosome amplification in ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC) is characterised by poor outcome and extreme chromosome instability (CIN). Therapies targeting centrosome amplification (CA), a key mediator of chromosome missegregation, may have significant clinical utility in HGSOC. However, the prevalence of CA in HGSOC, its relationship to genomic biomarkers of CIN and its potential impact on therapeutic response have not been defined. Using high-throughput multi-regional microscopy on 287 clinical HGSOC tissues and 73 cell lines models, here we show that CA through centriole overduplication is a highly recurrent and heterogeneous feature of HGSOC and strongly associated with CIN and genome subclonality. Cell-based studies showed that high-prevalence CA is phenocopied in ovarian cancer cell lines, and that high CA is associated with increased multi-treatment resistance; most notably to paclitaxel, the commonest treatment used in HGSOC. CA in HGSOC may therefore present a potential driver of tumour evolution and a powerful biomarker for response to standard-of-care treatment.

The role of branched-chain aminotransferase 1 in driving glioblastoma cell proliferation and invasion varies with tumor subtype.

Background: Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase (IDH) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible. Methods: The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype. Results: In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux. Conclusions: There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype.

Identification of ChIP-seq and RIME grade antibodies for Estrogen Receptor alpha.

Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.

BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Whole blood gene expression profiling predicts therapeutic response at six months in patients with polyarticular juvenile idiopathic arthritis.

OBJECTIVE: To determine whether gene expression profiles identified in peripheral whole blood samples could be used to determine therapeutic outcome in a cohort of children with newly diagnosed polyarticular juvenile idiopathic arthritis (JIA). METHODS: Whole blood samples from the Trial of Early Aggressive Therapy (TREAT) in JIA patients were analyzed on Illumina microarrays, and differential gene expression was compared to expression in healthy controls. Microarray results were validated by real-time quantitative polymerase chain reaction in an independent cohort of samples. Pathway analysis software was used to characterize gene expression profiles. Support vector machines were used to develop predictive models for different patient classes. RESULTS: Differential gene expression profiles for rheumatoid factor (RF)-positive and RF-negative patients were remarkably similar. Pathway analysis revealed a broad range of affected pathways, consistent with current mechanistic theories. Modeling showed that the prognosis at 6 months was strongly linked to gene expression at presentation, irrespective of treatment. CONCLUSION: Gene expression is linked to therapeutic outcome, and gene expression in the peripheral blood may be a suitable target for a prognostic test.