UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma.

Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.

Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes.

The development and functions of oenocytes.

Oenocytes have intrigued insect physiologists since the nineteenth century. Many years of careful but mostly descriptive research on these cells highlights their diverse sizes, numbers, and anatomical distributions across Insecta. Contemporary molecular genetic studies in Drosophila melanogaster and Tribolium castaneum support the hypothesis that oenocytes are of ectodermal origin. They also suggest that, in both short and long germ-band species, oenocytes are induced from a Spalt major/Engrailed ectodermal zone by MAPK signaling. Recent glimpses into some of the physiological functions of oenocytes indicate that they involve fatty acid and hydrocarbon metabolism. Genetic studies in D. melanogaster have shown that larval oenocytes synthesize very-long-chain fatty acids required for tracheal waterproofing and that adult oenocytes produce cuticular hydrocarbons required for desiccation resistance and pheromonal communication. Exciting areas of future research include the evolution of oenocytes and their cross talk with other tissues involved in lipid metabolism such as the fat body.

Aberrant corin and PCSK6 in placentas of the maternal hyperinsulinemia IUGR rat model.

OBJECTIVES: Corin is a protease that converts pro-atrial natriuretic peptide (pro-ANP) to ANP. While the involvement of ANP in the cardiovascular regulation is well established, there is increasing evidence that the pregnant uterus produces ANP, which promotes spiral artery remodeling. The present study examines the alterations in corin and PCSK6, a key enzyme in the conversion of pro-corin to corin, in the placenta of hyperinsulinemic dams (HD) featuring pregnancy-induced hypertension (PIH). MATERIALS AND METHODS: The study was conducted on female Wistar rats. Rats were rendered hyperinsulinemic by subcutaneous insulin pellet, mated and followed to the twenty-first day of pregnancy. Normal pregnant dams (NPD) served as controls. Both groups were sacrificed on day 21 of gestation and their placentas were dissected along with the mesometrial triangle (MT). The tissue was then sectioned from the maternal surface to the base of the MT, and processed for histological and molecular biology analysis of Corin, PCSK6 and ANP expression/immunoreactivity. RESULTS: Hyperinsulinemic dams developed PIH, along lower placental and fetal weights. Corin expression and immunoreactivity were significantly decreased in the placenta by ~40-50%, but not in the MT. Similarly, placental but not MT PCSK6 immunoreactivity was lower in HD. Concomitantly with the downregulation of corin/PCSK6, proANP levels increased in the placenta of HD. CONCLUSIONS: Corin and PCSK6 are expressed in the placenta and MT. The decline in these two enzymes in the placenta of HD suggests a role of corin/PCSK6 machinery in the development of PIH and intrauterine growth restriction characterizing hyperinsulinemia.

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Phosphorylation of Groucho mediates RTK feedback inhibition and prolonged pathway target gene expression.

BACKGROUND: Signaling by receptor tyrosine kinase (RTK) pathways plays fundamental roles in processes of cell-fate determination, often through the induction of specific transcriptional responses. Yet it is not fully understood how continuous target gene expression, required for irreversible cell-fate specification, is preserved after RTK signaling has ended. Here we address this question using the Drosophila embryo, a model system that has been instrumental in elucidating the developmental functions of RTK signal transduction. RESULTS: The Groucho corepressor is phosphorylated and downregulated in response to RTK signaling. Here we show that RTK pathways use Groucho phosphorylation as a general mechanism for inducing expression of pathway target genes encoding cell-fate determinants as well as feedback antagonists, indicating that relief of Groucho-dependent repression is an integral element of RTK signaling networks. We further demonstrate that after mitogen-activated protein kinase (MAPK) has been deactivated, sustained phosphorylation of Groucho is essential for persistent RTK-induced target gene expression and cell-fate determination in several developmental contexts. CONCLUSIONS: Phosphorylation of Groucho by MAPK plays a dual role in the regulation of RTK responses: (1) it mediates rapid feedback inhibition, and (2) it provides a stable memory mechanism of past MAPK activity. We propose that, in this manner, phosphorylation of Groucho enables transiently active RTK pathways to fix the spatiotemporal expression profiles of downstream targets over time.

Context-dependent regulation of Groucho/TLE-mediated repression.

Groucho/TLE proteins are global corepressors that are recruited to target promoters by different families of DNA-binding repressors. As these corepressors are widely expressed, the long-standing view had been that Groucho/TLE-mediated repression is regulated solely by the spatial and temporal distribution of partner repressors. It has recently emerged, however, that Groucho/TLE repressor activity is itself regulated, in a signal induced, context-dependent manner. Here we review the essential roles played by Groucho/TLE factors in different cell-signalling processes that illustrate different modes for regulating Groucho/TLE-mediated repression: (i) via the expression of partner repressors; (ii) by competition with coactivators and (iii) through post-translational modifications of Groucho/TLE. We also discuss how the intrinsic properties of repressors can result in differential responses to Groucho/TLE regulation.

Multiple RTK pathways downregulate Groucho-mediated repression in Drosophila embryogenesis.

RTK pathways establish cell fates in a wide range of developmental processes. However, how the pathway effector MAPK coordinately regulates the expression of multiple target genes is not fully understood. We have previously shown that the EGFR RTK pathway causes phosphorylation and downregulation of Groucho, a global co-repressor that is widely used by many developmentally important repressors for silencing their various targets. Here, we use specific antibodies that reveal the dynamics of Groucho phosphorylation by MAPK, and show that Groucho is phosphorylated in response to several RTK pathways during Drosophila embryogenesis. Focusing on the regulation of terminal patterning by the Torso RTK pathway, we demonstrate that attenuation of Groucho's repressor function via phosphorylation is essential for the transcriptional output of the pathway and for terminal cell specification. Importantly, Groucho is phosphorylated by an efficient mechanism that does not alter its subcellular localisation or decrease its stability; rather, modified Groucho endures long after MAPK activation has terminated. We propose that phosphorylation of Groucho provides a widespread, long-term mechanism by which RTK signals control target gene expression.

Capicua integrates input from two maternal systems in Drosophila terminal patterning.

In Drosophila, the maternal terminal system specifies cell fates at the embryonic poles via the localised stimulation of the Torso receptor tyrosine kinase (RTK). Signalling by the Torso pathway relieves repression mediated by the Capicua and Groucho repressors, allowing the restricted expression of the zygotic terminal gap genes tailless and huckebein. Here we report a novel positive input into tailless and huckebein transcription by maternal posterior group genes, previously implicated in abdomen and pole cell formation. We show that absence of a subset of posterior group genes, or their overactivation, leads to the spatial reduction or expansion of the tailless and huckebein posterior expression domains, respectively. We demonstrate that the terminal and posterior systems converge, and that exclusion of Capicua from the termini of posterior group mutants is ineffective, accounting for reduced terminal gap gene expression in these embryos. We propose that the terminal and posterior systems function coordinately to alleviate transcriptional silencing by Capicua, and that the posterior system fine-tunes Torso RTK signalling output, ensuring precise spatial domains of tailless and huckebein expression.