The transition from unicellular to multicellular life, which occurred several times during evolution, requires tight interaction and communication of neighboring cells. The multicellular cyanobacterium Nostoc punctiforme ATCC 29133 forms filaments of hundreds of interacting cells exchanging metabolites and signal molecules and is able to differentiate specialized cells in response to environmental stimuli. Mutation of cell wall amidase AmiC2 leads to a severe phenotype with formation of aberrant septa in the distorted filaments, which completely lack cell communication and potential for cell differentiation. Here we demonstrate the function of the amidase AmiC2 in formation of cell-joining structures. The AmiC2 protein localizes to the young septum between cells and shows bona fide amidase activity in vivo and in vitro. Vancomycin staining identified the overall septum morphology in living cells. By electron microscopy of isolated peptidoglycan sacculi, the submicroscopic structure of the cell junctions could be visualized, revealing a novel function for a cell wall amidase: AmiC2 drills holes into the cross-walls, forming an array of ~155 nanopores with a diameter of ~20 nm each. These nanopores seem to constitute a framework for cell-joining proteins, penetrating the cell wall. The entire array of junctional nanopores appears as a novel bacterial organelle, establishing multicellularity in a filamentous prokaryote.

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Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1 mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1 animals were then crossed with -methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1/MGMT double knockout (DKO) mice developed more, but much smaller tumors than MGMT animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar -methylguanine levels. Studies with PARP-1 cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to -methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

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Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.