Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer.

BACKGROUND & AIMS: Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. METHODS: Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. RESULTS: We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. CONCLUSIONS: Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

Neutrophil-induced genomic instability impedes resolution of inflammation and wound healing.

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBDs). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified what we believe is a new, ROS-independent mechanism whereby activated tissue-infiltrating PMNs release microparticles armed with proinflammatory microRNAs (miR-23a and miR-155). Using IBD clinical samples, and in vitro and in vivo injury models, we show that PMN-derived miR-23a and miR-155 promote accumulation of double-strand breaks (DSBs) by inducing lamin B1-dependent replication fork collapse and inhibition of homologous recombination (HR) by targeting HR-regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR-23a and miR-155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, in wound healing, and potentially to prevent neoplasia.

In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines.

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of gastrointestinal inflammation, with significant implications for host defense, injury and repair. However, phenotypic and mechanistic aspects of PMN recruitment in inflamed intestines have not been explored in vivo. Using novel epithelial/PMN fluorescence reporter mice, advanced intravital imaging and 3D reconstruction analysis, we mapped the microvasculature architecture across the intestinal layers and determined that in response to Salmonella/endotoxin-induced inflammation, PMN transendothelial migration (TEM) was restricted to submucosal vessels. PMN TEM was not observed in villus or crypt vessels, proximal to the epithelium that underlies the intestinal lumen, and was partially dependent on (C-X-C motif) ligands 1 (CXCL1) and 2 (CXCL2) expression, which was found to be elevated in the submucosa layer. Restricted PMN extravasation at the submucosa and subsequent PMN interstitial migration may serve as a novel regulatory step of PMN effector function and recruitment to the luminal space in inflamed intestines.

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies.

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50-1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

Neutrophil Microparticles Deliver Active Myeloperoxidase to Injured Mucosa To Inhibit Epithelial Wound Healing.

Neutrophil (PMN) infiltration of the intestinal mucosa often leads to severe epithelial injury; however, how this process occurs is unclear. This article describes a novel mechanism whereby membrane-derived microparticles released by tissue infiltrating PMNs (PMN-MPs) serve as shuttles to protect and deliver active mediators to locally modulate cellular function during inflammation. Specifically, myeloperoxidase (MPO), which is abundantly expressed in PMN azurophilic granules and is used for microbial killing, was found to be mobilized to the PMN surface and subsequently released in association with PMN-MPs upon PMN activation and binding to intestinal epithelial cells (IECs). The enzymatic activity of PMN-MP-associated MPO was enhanced compared with soluble protein, leading to potent inhibition of wound closure following PMN-MP binding to IECs. Importantly, localized microinjection of PMN-MPs into wounded colonic mucosa was sufficient to impair epithelial wound healing in vivo. PMN-MP/MPO-dependent inhibition of IEC wound healing was due to impaired IEC migration and proliferation, resulting from impeded actin dynamics, cell spreading, and cell cycle arrest. Thus, our findings provide new insight into mechanisms governing PMN-induced tissue injury and implicate PMN-MPs and MPO as important regulators of cellular function.

Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration.

Neutrophil [polymorphonuclear leukocyte (PMN)] transepithelial migration (TEM) is a hallmark of inflammatory mucosal disorders. PMN TEM is associated with epithelial injury; however, mechanisms involved in this process are not well defined. The current work describes a new mechanism whereby deposition of PMN membrane-derived microparticles (PMN-MPs) onto intestinal epithelial cells (IECs) during TEM leads to loss of epithelial cadherins, thus promoting epithelial injury and increased PMN recruitment. PMN-MPs secreted by activated PMNs during TEM displayed a high level of enzymatically active matrix metalloproteinase 9 (MMP-9), and were capable of mediating potent effects on IEC integrity. Isolated PMN-MPs efficiently bound to IEC monolayers and induced cleavage of desmoglein-2 (DSG-2) but not E-cadherin, leading to disruption of IEC intercellular adhesions. Furthermore, PMN-MP binding to intestinal epithelium in vitro in transwell assays and in vivo in ligated intestinal loop preparations facilitated increases in PMN TEM. These effects were MMP-9 dependent and were reversed in the presence of specific pharmacological inhibitors. Finally, we demonstrated that IEC Dsg-2 serves as a barrier for migrating PMNs, and its removal by PMN-MP-associated MMP-9 facilitates PMN trafficking across epithelial layers. Our findings thus implicate PMN-MPs in PMN-mediated inflammation and epithelial damage, as observed in inflammatory disorders of mucosal surfaces.-Butin-Israeli, V., Houser, M. C., Feng, M., Thorp, E. B., Nusrat, A., Parkos, C. A, Sumagin, R. Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration.

p53 elevation in human cells halt SV40 infection by inhibiting T-ag expression.

SV40 large T-antigen (T-ag) has been known for decades to inactivate the tumor suppressor p53 by sequestration and additional mechanisms. Our present study revealed that the struggle between p53 and T-ag begins very early in the infection cycle. We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection. These findings shed new light on the long studied struggle between SV40 T-ag and p53, as developed during virus-host coevolution. Our studies indicate that the fate of SV40 infection is determined as soon as the viral DNA enters the nucleus, before the onset of viral gene expression.

Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells.

More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies.

Role of lamin b1 in chromatin instability.

Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions.

Regulation of nucleotide excision repair by nuclear lamin b1.

The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

Disruption of lamin B1 and lamin B2 processing and localization by farnesyltransferase inhibitors.

Lamin A and the B-type lamins, lamin B1 and lamin B2, are translated as pre-proteins that are modified at a carboxyl terminal CAAX motif by farnesylation, proteolysis and carboxymethylation. Lamin A is further processed by proteolysis to remove the farnesyl, but B-type lamins remain permanently farnesylated. Two childhood diseases, Hutchinson Gilford Progeria Syndrome and restrictive dermopathy are caused by defects in the processing of lamin A, resulting in permanent farnesylation of the protein. Farnesyltransferase inhibitors, originally developed to target oncogenic Ras, have recently been used in clinical trials to treat children with Hutchinson Gilford Progeria Syndrome. Lamin B1 and lamin B2 play important roles in cell proliferation and organ development, but little is known about the role of farnesylation in their functions. Treating normal human fibroblasts with farnesyltransferase inhibitors causes the accumulation of unprocessed lamin B2 and lamin A and a decrease in mature lamin B1. Normally, lamins are concentrated at the nuclear envelope/lamina, but when farnesylation is inhibited, the peripheral localization of lamin B2 decreases as its nucleoplasmic levels increase. Unprocessed prelamin A distributes into both the nuclear envelope/lamina and nucleoplasm. Farnesyltransferase inhibitors also cause a rapid cell cycle arrest leading to cellular senescence. This study suggests that the long-term inhibition of protein farnesylation could have unforeseen consequences on nuclear functions.

Virus strategies for passing the nuclear envelope barrier.

Viruses that replicate in the nucleus need to pass the nuclear envelope barrier during infection. Research in recent years indicates that the nuclear envelope is a major hurdle for many viruses. This review describes strategies to overcome this obstacle developed by seven virus families: herpesviridae, adenoviridae, orthomyxoviridae, lentiviruses (which are part of retroviridae), Hepadnaviridae, parvoviridae and polyomaviridae. Most viruses use the canonical nuclear pore complex (NPC) in order to get their genome into the nucleus. Viral capsids that are larger than the nuclear pore disassemble before or during passing through the NPC, thus allowing genome nuclear entry. Surprisingly, increasing evidence suggest that parvoviruses and polyomaviruses may bypass the nuclear pore by trafficking directly through the nuclear membrane. Additional studies are required for better understanding these processes. Since nuclear entry emerges as the limiting step in infection for many viruses, it may serve as an ideal target for antiviral drug development.

Nuclear lamin functions and disease.

Recent studies have shown that premature cellular senescence and normal organ development and function depend on the type V intermediate filament proteins, the lamins, which are major structural proteins of the nucleus. This review presents an up-to-date summary of the literature describing new findings on lamin functions in various cellular processes and emphasizes the relationship between the lamins and devastating diseases ranging from premature aging to cancer. Recent insights into the structure and function of the A- and B- type lamins in normal cells and their dysfunctions in diseased cells are providing novel targets for the development of new diagnostic procedures and disease intervention. We summarize these recent findings, focusing on data from mice and humans, and highlight the expanding knowledge of these proteins in both healthy and diseased cells.

The functions of the nuclear envelope in mediating the molecular crosstalk between the nucleus and the cytoplasm.

Recent studies of the nuclear envelope (NE) have emphasized its role in linking the nuclear and cytoplasmic compartments of mammalian cells. The inner face of the NE is bound to chromatin and this interaction is involved in regulating DNA replication and transcription. The outer face of the NE binds to different components of the cytoskeleton, and these interactions are involved in nuclear positioning. Many disease causing mutations in genes encoding NE proteins cause significant changes in nuclear architecture and cytoskeletal interactions with the NE. These mutations are also providing important new insights into nuclear-cytoplasmic interactions.

The role of nuclear lamin B1 in cell proliferation and senescence.

Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence of WI-38 cells. This overexpression eventually leads to cell cycle arrest at the G1/S boundary. These results demonstrate the importance of LB1 in regulating the proliferation and senescence of human diploid cells through a ROS signaling pathway.

Simian virus 40 induces lamin A/C fluctuations and nuclear envelope deformation during cell entry.

The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuclear pore, embedded within the nuclear envelope. However, we found that for SV40, the nuclear envelope poses a major hurdle to infection: FISH analysis revealed that the majority of viral DNA remains trapped in the ER; silencing of Lamin A/C rendered the cells more susceptible to infection; and proliferating cells are more susceptible to infection than quiescent cells. Surprisingly, we observed that following SV40 infection the nuclear envelope, including lamins A/C, B1, B2 and the nuclear pore complex, was dramatically deformed, as seen by immunohistochemistry. The infection induced fluctuations in the level of lamin A/C, dephosphorylation of an unknown epitope and leakage to the cytoplasm just prior to and during nuclear entry. Deformations were transient, and the spherical structure of the nuclear envelope was restored subsequent to nuclear entry. Nuclear envelope deformations and lamin A/C dephosphorylation depended on caspase-6 cleavage of lamin A/C. Notably, we have previously reported that inhibition of caspase-6 abolishes SV40 infection. Taken together the results suggest that alterations of the nuclear lamina, induced by the infecting virus, are involved in the nuclear entry of the SV40 genome. We propose that SV40 utilize this unique, previously unknown mechanism for direct trafficking of its genome from the ER to the nucleus. As SV40 serves as a paradigm for the pathogenic human BK, JC and Merkel cell polyomavirus, this study suggests nuclear entry as a novel drug target for these infections.

Simian virus 40 infection triggers a balanced network that includes apoptotic, survival, and stress pathways.

The infection process by simian virus 40 (SV40) and entry of its genome into nondividing cells are only partly understood. Infection begins by binding to GM1 receptors at the cell surface, cellular entry via caveolar invaginations, and trafficking to the endoplasmic reticulum, where the virus disassembles. To gain a deeper insight into the contribution of host functions to this process, we studied cellular signaling elicited by the infecting virus. Signaling proteins were detected by Western blotting and immunofluorescence staining. The study was assisted by a preliminary proteomic screen. The contribution of signaling proteins to the infection process was evaluated using specific inhibitors. We found that CV-1 cells respond to SV40 infection by activating poly(ADP-ribose) polymerase 1 (PARP-1)-mediated apoptotic signaling, which is arrested by the Akt-1 survival pathway and stress response. A single key regulator orchestrating the three pathways is phospholipase C-gamma (PLCgamma). The counteracting apoptotic and survival pathways are robustly balanced as the infected cells neither undergo apoptosis nor proliferate. Surprisingly, we have found that the apoptotic pathway, including activation of PARP-1 and caspases, is absolutely required for the infection to proceed. Thus, SV40 hijacks the host defense to promote its infection. Activities of PLCgamma and Akt-1 are also required, and their inhibition abrogates the infection. Notably, this signaling network is activated hours before T antigen is expressed. Experiments with recombinant empty capsids, devoid of DNA, indicated that the major capsid protein VP1 alone triggers this early signaling network. The emerging robust signaling network reflects a delicate evolutionary balance between attack and defense in the host-virus relationship.

DNA-free recombinant SV40 capsids protect mice from acute renal failure by inducing stress response, survival pathway and apoptotic arrest.

Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses.

An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses.

The checkpoint proteins Rad9, Rad1 and Hus1 form a clamp-like complex which plays a central role in the DNA-damage-induced checkpoint response. Here we address the function of the 9-1-1 complex in Drosophila. We decided to focus our analysis on the meiotic and somatic requirements of hus1. For that purpose, we created a null allele of hus1 by imprecise excision of a P element found 2 kb from the 3' of the hus1 gene. We found that hus1 mutant flies are viable, but the females are sterile. We determined that hus1 mutant flies are sensitive to hydroxyurea and methyl methanesulfonate but not to X-rays, suggesting that hus1 is required for the activation of an S-phase checkpoint. We also found that hus1 is not required for the G2-M checkpoint and for post-irradiation induction of apoptosis. We subsequently studied the role of hus1 in activation of the meiotic checkpoint and found that the hus1 mutation suppresses the dorsal-ventral pattering defects caused by mutants in DNA repair enzymes. Interestingly, we found that the hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in DNA repair enzymes. These results demonstrate that hus1 is essential for the activation of the meiotic checkpoint and that hus1 is also required for the organization of the oocyte DNA, a function that might be independent of the meiotic checkpoint.