Probes for INS

We have profiled, for the first time, an evolving human metastatic microenvironment, measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, ECM organization and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer (HGSOC) metastases that ranged from minimal to extensive disease, we show how non-malignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, whilst also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and twelve other primary solid cancers, suggesting that there may be a common matrix response to human cancer.

The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.

Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer, but there are no useful zebrafish pre-B ALL models. We describe the first highly- penetrant zebrafish pre-B ALL, driven by human MYC. Leukemias express B lymphoblast-specific genes and are distinct from T cell ALL (T-ALL)-which these fish also develop. Zebrafish pre-B ALL shares in vivo features and expression profiles with human pre-B ALL, and these profiles differ from zebrafish T-ALL or normal B and T cells. These animals also exhibit aberrant lymphocyte development. As the only robust zebrafish pre-B ALL model and only example where T-ALL also develops, this model can reveal differences between MYC-driven pre-B vs. T-ALL and be exploited to discover novel pre-B ALL therapies.

The contributions of the innate immune system to the development of pancreatic cancer are still ill defined. Inflammatory macrophages can initiate metaplasia of pancreatic acinar cells to a duct-like phenotype (acinar-to-ductal metaplasia [ADM]), which then gives rise to pancreatic intraepithelial neoplasia (PanIN) when oncogenic KRas is present. However, it remains unclear when and how this inflammatory macrophage population is replaced by tumor-promoting macrophages. Here, we demonstrate the presence of interleukin-13 (IL-13), which can convert inflammatory into Ym1+ alternatively activated macrophages, at ADM/PanIN lesions. We further show that Ym1+ macrophages release factors, such as IL-1ra and CCL2, to drive pancreatic fibrogenesis and tumorigenesis. Treatment of mice expressing oncogenic KRas under an acinar cell-specific promoter with a neutralizing antibody for IL-13 significantly decreased the accumulation of alternatively activated macrophages at these lesions, resulting in decreased fibrosis and lesion growth.

Understanding the roles of splicing factors and splicing events during tumorigenesis would open new avenues for targeted therapies. Here we identify an oncofetal splicing factor, MBNL3, which promotes tumorigenesis and indicates poor prognosis of hepatocellular carcinoma patients. MBNL3 knockdown almost completely abolishes hepatocellular carcinoma tumorigenesis. Transcriptomic analysis revealed that MBNL3 induces lncRNA-PXN-AS1 exon 4 inclusion. The transcript lacking exon 4 binds to coding sequences of PXN mRNA, causes dissociation of translation elongation factors from PXN mRNA, and thereby inhibits PXN mRNA translation. In contrast, the transcript containing exon 4 preferentially binds to the 3' untranslated region of PXN mRNA, protects PXN mRNA from microRNA-24-AGO2 complex-induced degradation, and thereby increases PXN expression. Through inducing exon 4 inclusion, MBNL3 upregulates PXN, which mediates the pro-tumorigenic roles of MBNL3. Collectively, these data demonstrate detailed mechanistic links between an oncofetal splicing factor, a splicing event and tumorigenesis, and establish splicing factors and splicing events as potential therapeutic targets.

Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation.

Genetic alterations that potentiate PI3K signalling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor prostate cancer patient survival. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110α-dependent invasive prostate carcinoma in-vivo. Furthermore, we report PIK3CA mutation and PTEN loss co-exist in prostate cancer patients, and can cooperate in-vivo to accelerate disease progressionvia AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de-novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients.