Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba, amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans-splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive ‘cross-talk’ between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba.

BackgroundThe ability of solid tumor cells to resist anoikis, apoptosis triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. ErbB2/Her2 oncoprotein is often overproduced by breast tumor cells and blocks their anoikis by partially understood mechanisms. In our effort to understand them better, we observed that detachment of nonmalignant human breast epithelial cells from the ECM upregulates the transcription factor Irf6. Irf6 is thought to play an important role in mammary gland homeostasis and causes apoptosis by unknown mechanisms. We noticed that ErbB2, when overproduced by detached breast epithelial cells, downregulates Irf6.MethodsTo test whether ErbB2 downregulates Irf6 in human ErbB2-positive breast cancer cells, we examined the effect of ErbB2 inhibitors, such as the anti-ErbB2 antibody trastuzumab or the ErbB2/epidermal growth factor receptor small-molecule inhibitor lapatinib, on Irf6 in these cells. Moreover, we performed Irf6 IHC analysis of tumor samples derived from the locally advanced ErbB2-positive breast cancers before and after neoadjuvant trastuzumab-based therapies. To examine the role of Irf6 in anoikis of nonmalignant and ErbB2-overproducing breast epithelial cells, we studied anoikis after knocking down Irf6 in the former cells by RNA interference and after overproducing Irf6 in the latter cells. To examine the mechanisms by which cell detachment and ErbB2 control Irf6 expression in breast epithelial cells, we tested the effects of genetic and pharmacological inhibitors of the known ErbB2-dependent signaling pathways on Irf6 in these cells.ResultsWe observed that trastuzumab and lapatinib upregulate Irf6 in ErbB2-positive human breast tumor cells and that neoadjuvant trastuzumab-based therapies tend to upregulate Irf6 in human breast tumors. We found that detachment-induced Irf6 upregulation in nonmalignant breast epithelial cells requires the presence of the transcription factor ∆Np63α and that Irf6 mediates their anoikis. We showed that ErbB2 blocks Irf6 upregulation in ErbB2-overproducing cells by activating the mitogen-activated protein kinases that inhibit ∆Np63α-dependent signals required for Irf6 upregulation. Finally, we demonstrated that ErbB2-driven Irf6 downregulation in ErbB2-overproducing breast epithelial cells blocks their anoikis and promotes their anchorage-independent growth.ConclusionsWe have demonstrated that ErbB2 blocks anoikis of breast epithelial cells by downregulating Irf6.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1080-1) contains supplementary material, which is available to authorized users.

The nucleus harbours numerous protein subdomains and condensates that regulate chromatin organization, gene expression and genomic stress. A novel nuclear subdomain that is formed following exposure of cells to excess fatty acids is the nuclear lipid droplet (nLD), which is composed of a neutral lipid core surrounded by a phospholipid monolayer and associated regulatory and lipid biosynthetic enzymes. While structurally resembling cytoplasmic LDs, nLDs are formed by distinct but poorly understood mechanisms that involve the emergence of lipid droplets from the lumen of the nucleoplasmic reticulum and de novo lipid synthesis. Luminal lipid droplets that emerge into the nucleoplasm do so at regions of the inner nuclear membrane that become enriched in promyelocytic leukemia (PML) protein. The resulting nLDs that retain PML on their surface are termed lipid-associated PML structures (LAPS), and are distinct from canonical PML nuclear bodies (NB) as they lack key proteins and modifications associated with these NBs. PML is a key regulator of nuclear signaling events and PML NBs are sites of gene regulation and post-translational modification of transcription factors. Therefore, the subfraction of nLDs that form LAPS could regulate lipid stress responses through their recruitment and retention of the PML protein. Both nLDs and LAPS have lipid biosynthetic enzymes on their surface suggesting they are active sites for nuclear phospholipid and triacylglycerol synthesis as well as global lipid regulation. In this review we have summarized the current understanding of nLD and LAPS biogenesis in different cell types, their structure and composition relative to other PML-associated cellular structures, and their role in coordinating a nuclear response to cellular overload of fatty acids.

The translocation of CCTα, the rate-limiting enzyme for phosphatidylcholine synthesis, to nuclear membranes and nuclear lipid droplets results in reversible dephosphorylation of S319 and sustained phosphorylation of Y359+S362. Independent regulation of these phosphosites in the P-domain of CCTα is required for activation on nuclear membranes.

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.