Human-specific elimination of epithelial Siglec-XII suppresses the risk of inflammation driven colorectal cancers.

Carcinomas are common in humans but rare among closely related "great apes". Plausible explanations, including human-specific genomic alterations affecting the biology of sialic acids are proposed, but causality remains unproven. Here, an integrated evolutionary genetics-phenome-transcriptome approach studied the role of SIGLEC12 gene (encodes Siglec-XII) on epithelial transformation and cancer. Exogenous expression of the protein in cell lines and genetically engineered mice recapitulated ~30% of the human population in whom the protein is expressed in a form that cannot bind ligand due to a fixed, homozygous, human-universal missense mutation. Siglec-XII null cells/mice recapitulated the remaining ~70% of the human population in whom an additional polymorphic frameshift mutation eliminates the entire protein. Siglec-XII expression drove several pro-oncogenic phenotypes in cell lines, and increased tumor burden in mice challenged with chemical carcinogen and inflammation. Transcriptomic studies yielded a 29-gene signature of Siglec-XII-positive disease and when used as a computational tool for navigating human datasets, pinpointed with surprising precision that SIGLEC12 expression (model) recapitulates a very specific type of colorectal carcinomas (disease) that is associated with mismatch-repair defects and inflammation, disproportionately affects European-Americans, and carries a better prognosis. They revealed a hitherto unknown evolutionary genetic mechanism for an ethnic/environmental predisposition of carcinogenesis.

Long-term exposure to house dust mites accelerates lung cancer development in mice.

BACKGROUND: Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1ß (IL-1ß), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1ß signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1ß, and CCL2 neutralization, or ICS treatment. CONCLUSIONS: Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

Lp(a), oxidized phospholipids and oxidation-specific epitopes are increased in subjects with keloid formation.

BACKGROUND: Keloid formation following trauma or surgery is common among darkly pigmented individuals. Since lipoprotein(a) [Lp(a)] has been postulated to have a putative role in wound healing, and also mediates atherosclerotic cardiovascular disease, it was assessed whether Lp(a), its associated oxidized phospholipids and other oxidation-specific biomarkers were associated with keloid formation. METHODS: This case-control study included darkly pigmented individuals of African ancestry, 100 with keloid scarring and 100 non-keloid controls. The lipid panel, hsCRP, Lp(a), oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB), IgG and IgM apoB-immune complexes and IgG and IgM autoantibodies to a malondialdehyde mimotope (MDA-mimotope) were measured. Immunohistochemistry of keloid specimens was performed for both Lp(a) and OxPL staining. RESULTS: Cases and controls were well matched for age, sex and lipid profile. Mean Lp(a) (57.8 vs. 44.2 mg/dL; P = 0.01, OxPL-apoB 17.4 vs. 15.7 nmol/L; P = 0.009) and IgG and IgM apoB-immune complexes and IgG and IgM MDA-mimotope levels were significantly higher in keloid cases. Keloid tissue stained strongly for OxPL. CONCLUSION: Darkly pigmented individuals of African ancestry with keloids have higher plasma levels of Lp(a), OxPL-apoB and oxidation-specific epitopes. The commonality of excessive wound healing in keloids and chronic complications from coronary revascularization suggests avenues of investigation to define a common mechanism driven by Lp(a) and the innate response to oxidized lipids.

Targeting glycan sulfation in a CD11c+ myeloid population inhibits early KRAS-mutant lung neoplasia.

Early lung carcinoma development may be modulated by innate host cellular mechanisms that promote tumor growth and invasion. We recently identified how a loss-of-function mutation in the glycan sulfating enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in heparan sulfate biosynthesis) targeted to antigen presenting cells (APCs) may augment acquired anti-tumor T cell immune mechanisms. Crossing this mutation (Ndst1f/f CD11cCre+) onto a model of inducible spontaneous Kras mutant lung cancer [CCSP-rtTA; (tetO7) CMV-Kras-G12D] allowed us to examine how the APC mutation affects the formation and growth of early lung carcinoma. We examined early bronchocentric adenoma formation in the model, and the frequency of such events was significantly reduced on the mutant background. This was associated with significant reductions in tumor associated FOXP3+ cellular infiltration and CD163+ M2-type macrophage infiltration. The findings evolved prior to effector CD8+ T cell infiltration into tumors. The impact of this unique glycan under-sulfating mutation on inhibiting early Kras G12D mutant bronchocentric adenoma formation along with a cellular phenotype of inhibited tumor infiltration by cells involved in suppressive T-regulatory cell signaling (FOXP3+ cells) or tumor-permissive M2 macrophage functions (CD163+ cells) provides insight on how glycan targeting may modulate innate cellular mechanisms during early lung tumor development. The findings may also impact the future design of host-centered immunologic anti-tumor therapeutic strategies.

Human-specific polymorphic pseudogenization of SIGLEC12 protects against advanced cancer progression.

Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec-XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full-length protein expression in ~60%-70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec-XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec-XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2-dependent cancers. Immunohistochemistry was used to detect Siglec-XII expression on tissue microarrays. PC-3 prostate cancer cells were transfected with Siglec-XII and transcription of genes enriched with Siglec-XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec-XII expressors versus non-expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec-XII was detected as expected in ~30%-40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late-stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec-XII protein recruits Shp2-related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

Evaluation of IL-17D in Host Immunity to Group A Streptococcus Infection.

IL-17D is a cytokine that belongs to the IL-17 family and is conserved in vertebrates and invertebrates. In contrast to IL-17A and IL-17F, which are expressed in Th17 cells, IL-17D is expressed broadly in nonimmune cells. IL-17D can promote immune responses to cancer and viruses in part by inducing chemokines and recruiting innate immune cells such as NK cells. Although bacterial infection can induce IL-17D in fish and invertebrates, the role of mammalian IL-17D in antibacterial immunity has not been established. To determine whether IL-17D has a role in mediating host defense against bacterial infections, we studied i.p. infection by group A Streptococcus (GAS) in wild-type (WT) and Il17d -/- mice. Compared with WT animals, mice deficient in IL-17D experienced decreased survival, had greater weight loss, and showed increased bacterial burden in the kidney and peritoneal cavity following GAS challenge. In WT animals, IL-17D transcript was induced by GAS infection and correlated to increased levels of chemokine CCL2 and greater neutrophil recruitment. Of note, GAS-mediated IL-17D induction in nonimmune cells required live bacteria, suggesting that processes beyond recognition of pathogen-associated molecular patterns were required for IL-17D induction. Based on our results, we propose a model in which nonimmune cells can discriminate between nonviable and viable GAS cells, responding only to the latter by inducing IL-17D.

DDX5 promotes oncogene C3 and FABP1 expressions and drives intestinal inflammation and tumorigenesis.

Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid-binding protein 1 (FABP1). However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that the RNA-binding protein DDX5 binds to the mRNA transcripts of C3 and Fabp1 to augment their expressions posttranscriptionally. Knocking out DDX5 in epithelial cells protected mice from intestinal tumorigenesis and dextran sodium sulfate (DSS)-induced colitis. Identification of DDX5 as a common upstream regulator of tissue-specific oncogenic molecules provides an excellent therapeutic target for intestinal diseases.

Human species-specific loss of CMP-N-acetylneuraminic acid hydroxylase enhances atherosclerosis via intrinsic and extrinsic mechanisms.

Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD-risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr-/- mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr-/- mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a "xeno-autoantigen" via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc "xeno-autoantibodies" potentiate chronic inflammation ("xenosialitis"). Cmah-/-Ldlr-/- mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah-/-Ldlr-/- mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.

MST1R kinase accelerates pancreatic cancer progression via effects on both epithelial cells and macrophages.

The MST1R (RON) kinase is overexpressed in >80% of human pancreatic cancers, but its role in pancreatic carcinogenesis is unknown. In this study, we examined the relevance of Mst1r kinase to Kras driven pancreatic carcinogenesis using genetically engineered mouse models. In the setting of mutant Kras, Mst1r overexpression increased acinar-ductal metaplasia (ADM), accelerated the progression of pancreatic intraepithelial neoplasia (PanIN), and resulted in the accumulation of (mannose receptor C type 1) MRC1+, (arginase 1) Arg+ macrophages in the tumor microenvironment. Conversely, absence of a functional Mst1r kinase slowed PanIN initiation, resulted in smaller tumors, prolonged survival and a reduced tumor-associated macrophage content. Mst1r expression was associated with increased production of its ligand Mst1, and in orthotopic models, suppression of Mst1 expression resulted in reduced tumor size, changes in macrophage polarization and enhanced T cell infiltration. This study demonstrates the functional significance of Mst1r during pancreatic cancer initiation and progression. Further, it provides proof of concept that targeting Mst1r can modulate pancreatic cancer growth and the microenvironment. This study provides further rationale for targeting Mst1r as a therapeutic strategy.

Inhibition of Nuclear PTEN Tyrosine Phosphorylation Enhances Glioma Radiation Sensitivity through Attenuated DNA Repair.

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera.

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.

Preventive Inhibition of Liver Tumorigenesis by Systemic Activation of Innate Immune Functions.

Liver cancer has become the second most deadly malignant disease, with no efficient targeted or immune therapeutic agents available yet. While dissecting the roles of cytoplasmic signaling molecules in hepatocarcinogenesis using an inducible mouse gene targeting system, Mx1-cre, we identified a potent liver tumor-inhibitory effect of synthetic double-stranded RNA (dsRNA), polyinosinic-polycytidylic acid (pIC), an inducer of the Mx1-cre system. Injection of pIC at the pre-cancer stage robustly suppressed liver tumorigenesis either induced by chemical carcinogens or by Pten loss and associated hepatosteatosis. The immunostimulatory dsRNA inhibited liver cancer initiation, apparently by boosting multiple anti-tumor activities of innate immunity, including induction of immunoregulatory cytokines, activation of NK cells and dendritic cells, and reprogramming of macrophage polarization. This study paves the way for the development of preventive and early interfering strategies for liver cancer to reduce the rapidly increasing incidences of liver cancer in an ever-growing population with chronic liver disorders.

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen.

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E.

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

Dual Shp2 and Pten Deficiencies Promote Non-alcoholic Steatohepatitis and Genesis of Liver Tumor-Initiating Cells.

The complexity of liver tumorigenesis is underscored by the recently observed anti-oncogenic effects of oncoproteins, although the mechanisms are unclear. Shp2/Ptpn11 is a proto-oncogene in hematopoietic cells and antagonizes the effect of tumor suppressor Pten in leukemogenesis. In contrast, we show here cooperative functions of Shp2 and Pten in suppressing hepatocarcinogenesis. Ablating both Shp2 and Pten in hepatocytes induced early-onset non-alcoholic steatohepatitis (NASH) and promoted genesis of liver tumor-initiating cells likely due to augmented cJun expression/activation and elevated ROS and inflammation in the hepatic microenvironment. Inhibiting cJun partially suppressed NASH-driven liver tumorigenesis without improving NASH. SHP2 and PTEN deficiencies were detected in liver cancer patients with poor prognosis. These data depict a mechanism of hepato-oncogenesis and suggest a potential therapeutic strategy.

Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec-5 and Siglec-14.

The intracellular chaperone heat-shock protein 70 (Hsp70) can be secreted from cells, but its extracellular role is unclear, as the protein has been reported to both activate and suppress the innate immune response. Potential immunomodulatory receptors on myelomonocytic lineage cells that bind extracellular Hsp70 are not well defined. Siglecs are Ig-superfamily lectins on mammalian leukocytes that recognize sialic acid-bearing glycans and thereby modulate immune responses. Siglec-5 and Siglec-14, expressed on monocytes and neutrophils, share identical ligand-binding domains but have opposing signaling functions. Based on phylogenetic analyses of these receptors, we predicted that endogenous sialic acid-independent ligands should exist. An unbiased screen revealed Hsp70 as a ligand for Siglec-5 and Siglec-14. Hsp70 stimulation through Siglec-5 delivers an anti-inflammatory signal, while stimulation through Siglec-14 is pro-inflammatory. The functional consequences of this interaction are also addressed in relation to a SIGLEC14 polymorphism found in humans. Our results demonstrate that an endogenous non-sialic acid-bearing molecule can be either a danger-associated or self-associated signal through paired Siglecs, and may explain seemingly contradictory prior reports on extracellular Hsp70 action.

On the apparent rarity of epithelial cancers in captive chimpanzees.

Malignant neoplasms arising from epithelial cells are called carcinomas. Such cancers are diagnosed in about one in three humans in 'developed' countries, with the most common sites affected being lung, breast, prostate, colon, ovary and pancreas. By contrast, carcinomas are said to be rare in captive chimpanzees, which share more than 99% protein sequence homology with humans (and possibly in other related 'great apes'-bonobos, gorillas and orangutans). Simple ascertainment bias is an unlikely explanation, as these nonhuman hominids are recipients of excellent veterinary care in research facilities and zoos, and are typically subjected to necropsies when they die. In keeping with this notion, benign tumours and cancers that are less common in humans are well documented in this population. In this brief overview, we discuss other possible explanations for the reported rarity of carcinomas in our closest evolutionary cousins, including inadequacy of numbers surveyed, differences in life expectancy, diet, genetic susceptibility, immune responses or their microbiomes, and other potential environmental factors. We conclude that while relative carcinoma risk is a likely difference between humans and chimpanzees (and possibly other 'great apes'), a more systematic survey of available data is required for validation of this claim.

Advanced Technologies in Sialic Acid and Sialoglycoconjugate Analysis.

Although the structural diversity of sialic acid (Sia) is rapidly expanding, understanding of its biological significance has lagged behind. Advanced technologies to detect and probe diverse structures of Sia are absolutely necessary not only to understand further biological significance but also to pursue medicinal and industrial applications. Here we describe analytical methods for detection of Sia that have recently been developed or improved, with a special focus on 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac), N-glycolylneuraminic acid (Neu5Gc), deaminoneuraminic acid (Kdn), O-sulfated Sia (SiaS), and di-, oligo-, and polysialic acid (diSia/oligoSia/polySia) in glycoproteins and glycolipids. Much more attention has been paid to these Sia and sialoglycoconjugates during the last decade, in terms of regulation of the immune system, neural development and function, tumorigenesis, and aging.