Discovery of synthetic lethal interactions from large-scale pan-cancer perturbation screens.

The development of cancer therapies is limited by the availability of suitable drug targets. Potential candidate drug targets can be identified based on the concept of synthetic lethality (SL), which refers to pairs of genes for which an aberration in either gene alone is non-lethal, but co-occurrence of the aberrations is lethal to the cell. Here, we present SLIdR (Synthetic Lethal Identification in R), a statistical framework for identifying SL pairs from large-scale perturbation screens. SLIdR successfully predicts SL pairs even with small sample sizes while minimizing the number of false positive targets. We apply SLIdR to Project DRIVE data and find both established and potential pan-cancer and cancer type-specific SL pairs consistent with findings from literature and drug response screening data. We experimentally validate two predicted SL interactions (ARID1A-TEAD1 and AXIN1-URI1) in hepatocellular carcinoma, thus corroborating the ability of SLIdR to identify potential drug targets.

GATA3 and MDM2 are synthetic lethal in estrogen receptor-positive breast cancers.

Synthetic lethal interactions, where the simultaneous but not individual inactivation of two genes is lethal to the cell, have been successfully exploited to treat cancer. GATA3 is frequently mutated in estrogen receptor (ER)-positive breast cancers and its deficiency defines a subset of patients with poor response to hormonal therapy and poor prognosis. However, GATA3 is not yet targetable. Here we show that GATA3 and MDM2 are synthetically lethal in ER-positive breast cancer. Depletion and pharmacological inhibition of MDM2 significantly impaired tumor growth in GATA3-deficient models in vitro, in vivo and in patient-derived organoids/xenograft (PDOs/PDX) harboring GATA3 somatic mutations. The synthetic lethality requires p53 and acts via the PI3K/Akt/mTOR pathway. Our results present MDM2 as a therapeutic target in the substantial cohort of ER-positive, GATA3-mutant breast cancer patients. With MDM2 inhibitors widely available, our findings can be rapidly translated into clinical trials to evaluate in-patient efficacy.

Epigenetic priming in chronic liver disease impacts the transcriptional and genetic landscapes of hepatocellular carcinoma.

Hepatocellular carcinomas (HCCs) usually arise from chronic liver disease (CLD). Precancerous cells in chronically inflamed environments may be 'epigenetically primed', sensitising them to oncogenic transformation. We investigated whether epigenetic priming in CLD may affect HCC outcomes by influencing the genomic and transcriptomic landscapes of HCC. Analysis of DNA methylation arrays from 10 paired CLD-HCC identified 339 shared dysregulated CpG sites and 18 shared differentially methylated regions compared with healthy livers. These regions were associated with dysregulated expression of genes with relevance in HCC, including ubiquitin D (UBD), cytochrome P450 family 2 subfamily C member 19 (CYP2C19) and O-6-methylguanine-DNA methyltransferase (MGMT). Methylation changes were recapitulated in an independent cohort of nine paired CLD-HCC. High CLD methylation score, defined using the 124 dysregulated CpGs in CLD and HCC in both cohorts, was associated with poor survival, increased somatic genetic alterations and TP53 mutations in two independent HCC cohorts. Oncogenic transcriptional and methylation dysregulation is evident in CLD and compounded in HCC. Epigenetic priming in CLD sculpts the transcriptional landscape of HCC and creates an environment favouring the acquisition of genetic alterations, suggesting that the extent of epigenetic priming in CLD could influence disease outcome.

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens.

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/ß-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.

Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo-Independent Regulation of Heat Shock Protein 70 Family Members.

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up-regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo. Additionally, RNA sequencing analysis of TEAD4-overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up-regulation of genes involved in epithelial-to-mesenchymal transition, proliferation, and protein-folding pathways. Among the most up-regulated genes following TEAD4 overexpression were the 70-kDa heat shock protein (HSP70) family members HSPA6 and HSPA1A. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4-overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ)-knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo-independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members.

Adenylosuccinate lyase is oncogenic in colorectal cancer by causing mitochondrial dysfunction and independent activation of NRF2 and mTOR-MYC-axis.

Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro, ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting.

Standardizing Patient-Derived Organoid Generation Workflow to Avoid Microbial Contamination From Colorectal Cancer Tissues.

The use of patient-derived organoids (PDO) as a valuable alternative to in vivo models significantly increased over the last years in cancer research. The ability of PDOs to genetically resemble tumor heterogeneity makes them a powerful tool for personalized drug screening. Despite the extensive optimization of protocols for the generation of PDOs from colorectal tissue, there is still a lack of standardization of tissue handling prior to processing, leading to microbial contamination of the organoid culture. Here, using a cohort of 16 patients diagnosed with colorectal carcinoma (CRC), we aimed to test the efficacy of phosphate-buffered saline (PBS), penicillin/streptomycin (P/S), and Primocin, alone or in combination, in preventing organoid cultures contamination when used in washing steps prior to tissue processing. Each CRC tissue was divided into 5 tissue pieces, and treated with each different washing solution, or none. After the washing steps, all samples were processed for organoid generation following the same standard protocol. We detected contamination in 62.5% of the non-washed samples, while the use of PBS or P/S-containing PBS reduced the contamination rate to 50% and 25%, respectively. Notably, none of the organoid cultures washed with PBS/Primocin-containing solution were contaminated. Interestingly, addition of P/S to the washing solution reduced the percentage of living cells compared to Primocin. Taken together, our results demonstrate that, prior to tissue processing, adding Primocin to the tissue washing solution is able to eliminate the risk of microbial contamination in PDO cultures, and that the use of P/S negatively impacts organoids growth. We believe that our easy-to-apply protocol might help increase the success rate of organoid generation from CRC patients.

Single cell polarity in liquid phase facilitates tumour metastasis.

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.

The Role of Long Non-Coding RNAs in Hepatocarcinogenesis.

Whole-transcriptome analyses have revealed that a large proportion of the human genome is transcribed in non-protein-coding transcripts, designated as long non-coding RNAs (lncRNAs). Rather than being "transcriptional noise", increasing evidence indicates that lncRNAs are key players in the regulation of many biological processes, including transcription, post-translational modification and inhibition and chromatin remodeling. Indeed, lncRNAs are widely dysregulated in human cancers, including hepatocellular carcinoma (HCC). Functional studies are beginning to provide insights into the role of oncogenic and tumor suppressive lncRNAs in the regulation of cell proliferation and motility, as well as oncogenic and metastatic potential in HCC. A better understanding of the molecular mechanisms and the complex network of interactions in which lncRNAs are involved could reveal novel diagnostic and prognostic biomarkers. Crucially, it may provide novel therapeutic opportunities to add to the currently limited number of therapeutic options for HCC patients. In this review, we summarize the current status of the field, with a focus on the best characterized dysregulated lncRNAs in HCC.

High mobility group A1 protein modulates autophagy in cancer cells.

High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasias with a causal role in cancer initiation and progression. HMGA1 promotes tumor growth by several mechanisms, including increase of cell proliferation and survival, impairment of DNA repair and induction of chromosome instability. Autophagy is a self-degradative process that, by providing energy sources and removing damaged organelles and misfolded proteins, allows cell survival under stress conditions. On the other hand, hyper-activated autophagy can lead to non-apoptotic programmed cell death. Autophagy deregulation is a common feature of cancer cells in which has a complex role, showing either an oncogenic or tumor suppressor activity, depending on cellular context and tumor stage. Here, we report that depletion of HMGA1 perturbs autophagy by different mechanisms. HMGA1-knockdown increases autophagosome formation by constraining the activity of the mTOR pathway, a major regulator of autophagy, and transcriptionally upregulating the autophagy-initiating kinase Unc-51-like kinase 1 (ULK1). Consistently, functional experiments demonstrate that HMGA1 binds ULK1 promoter region and negatively regulates its transcription. On the other hand, the increase in autophagosomes is not associated to a proportionate increase in their maturation. Overall, the effects of HMGA1 depletion on autophagy are associated to a decrease in cell proliferation and ultimately impact on cancer cells viability. Importantly, silencing of ULK1 prevents the effects of HMGA1-knockdown on cellular proliferation, viability and autophagic activity, highlighting how these effects are, at least in part, mediated by ULK1. Interestingly, this phenomenon is not restricted to skin cancer cells, as similar results have been observed also in HeLa cells silenced for HMGA1. Taken together, these results clearly indicate HMGA1 as a key regulator of the autophagic pathway in cancer cells, thus suggesting a novel mechanism through which HMGA1 can contribute to cancer progression.