Tissue ACE inhibition and sodium status in left ventricular dysfunction

Left ventricular (LV) dysfunction is characterized by a progressive loss of cardiac pump function, which eventually leads to the clinical syndrome of chronic heart failure (CHF). Heart failure is associated with high morbidity and mortality. The incidence of heart failure has increased during the last decades, mainly because improved survival after acute myocardial infarction. Renin angiotensin aldosterone system (RAAS) activation plays a major role in the progression of left ventricular dysfunction towards chronic heart failure. Angiotensinconverting enzyme (ACE) inhibition therapy improves cardiac function and reduces morbidity and mortality. Although ACE-inhibition slows the gradual progression of myocardial dysfunction towards overt CHF, it does not prevent it. In order to reach maximal therapy response to ACE inhibition a low sodium intake may be required. The expected underlyingmechanism was that an activated RAAS – i.e. elevated renin production – induced by sodium restriction is required for an optimal therapeutic response to ACE inhibition. Hence, the first main subject of this thesis was to study intervention with the sodium balance in order to optimize ACE-I therapy

Mechanisms used by cancer cells to tolerate drug-induced replication stress

Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells

Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRAS(G12V) dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRAS(G12V) sensitized cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and TGF-beta pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRAS(G12V) exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 target genes is the prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase checkpoint inhibition in cancers with intact P53

Inferring single-cell protein levels and cell cycle behavior in heterogeneous cell populations

Individual cells in a genetically identical population can show highly variable behavior. Single-cell measurements allow us to study this variability, but the available measurement techniques have limitations: live-cell microscopy is typically restricted to one or a few molecular markers, while techniques that simultaneously measure large numbers of molecular markers are destructive and cannot be used to follow cells over time. To help overcome these limitations, we present here scMeMo (single cell Mechanistic Modeler): a mechanistic modeling framework that can leverage diverse sets of measurements in order to infer unobserved variables in heterogeneous single cells. We used this framework to construct a model describing cell cycle progression in human cells, and show that it can predict the levels of several proteins in individual cells, based on live-cell microscopy measurements of only one marker and information learned from other experiments. The framework incorporates an uncertainty calibration step that makes the posterior distributions robust against partial model misspecification. Our modeling framework can be used to integrate information from separate experiments with diverse readouts, and to infer single cell variables that may be difficult to measure directly

Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using the 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g. Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1 h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g. Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages

Atypical E2Fs either Counteract or Cooperate with RB during Tumorigenesis Depending on Tissue Context

Simple Summary In virtually all human malignancies, the CDK-RB-E2F pathway is dysregulated resulting in the activation of the E2F transcriptional network. Rb and atypical E2Fs are the most important negative regulators of E2F-dependent transcription during tumorigenesis. However, it is unknown whether they cooporate or act independently in tumor development. Here we show that combined loss of RB and atypical E2Fs in mice enhances tumorigenesis in the liver, while in the pituitary gland, we observe inhibition of tumorigenesis. These findings suggest that the interaction between RB and atypical E2Fs in controlling tumorigenesis occurs in a tissue cell-type specific manner. E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and -8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo. Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb+/- mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and -8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency

CDC6:A novel canine tumour biomarker detected in circulating extracellular vesicles

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non-invasive nature of these so-called liquid biopsies provides an attractive alternative to tissue biopsy-based cancer diagnostics. This study aimed to investigate if circulating cell cycle-related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real-time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non-invasive biomarker to diagnose canine tumours