Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue.

Invariant natural killer T cells (iNKT cells) are lipid-sensing innate T cells that are restricted by the antigen-presenting molecule CD1d and express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, as well as their targets in adipose tissue, are unknown. Here we found that iNKT cells in adipose tissue had a unique transcriptional program and produced interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lacked PLZF but expressed the transcription factor E4BP4, which controlled their IL-10 production. The adipose iNKT cells were a tissue-resident population that induced an anti-inflammatory phenotype in macrophages and, through the production of IL-2, controlled the number, proliferation and suppressor function of regulatory T cells (Treg cells) in adipose tissue. Thus, iNKT cells in adipose tissue are unique regulators of immunological homeostasis in this tissue.

A murine model of Lyme disease demonstrates that Borrelia burgdorferi colonizes the dura mater and induces inflammation in the central nervous system.

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice have been used to define the kinetics of B. burgdorferi infection and host immune responses in joints and heart, however similar studies are lacking in the CNS of these animals. A tractable animal model for investigating host-Borrelia interactions in the CNS is key to understanding the mechanisms of CNS pathogenesis. Therefore, we characterized the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of mice during early and subacute infection. Using fluorescence-immunohistochemistry, intravital microscopy, bacterial culture, and quantitative PCR, we found B. burgdorferi routinely colonized the dura mater of C3H mice, with peak spirochete burden at day 7 post-infection. Dura mater colonization was observed for several Lyme disease agents including B. burgdorferi, B. garinii, and B. mayonii. RNA-sequencing and quantitative RT-PCR showed that B. burgdorferi infection was associated with increased expression of inflammatory cytokines and a robust interferon (IFN) response in the dura mater. Histopathologic changes including leukocytic infiltrates and vascular changes were also observed in the meninges of infected animals. In contrast to the meninges, we did not detect B. burgdorferi, infiltrating leukocytes, or large-scale changes in cytokine profiles in the cerebral cortex or hippocampus during infection; however, both brain regions demonstrated similar changes in expression of IFN-stimulated genes as observed in peripheral tissues and meninges. Taken together, B. burgdorferi is capable of colonizing the meninges in laboratory mice, and induces localized inflammation similar to peripheral tissues. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines is unique to the brain parenchyma, and provides insight into the potential mechanisms of CNS pathology associated with this important pathogen.

Loss of Slfn3 induces a sex-dependent repair vulnerability after 50% bowel resection.

Bowel resection accelerates enterocyte proliferation in the remaining gut with suboptimal absorptive and digestive capacity because of a proliferation-associated decrease in functional differentiation markers. We hypothesized that although schlafen 3 (Slfn3) is an important regulator of enterocytic differentiation, Slfn3 would have less impact on bowel resection adaptation, where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers from resected and postoperative bowel of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and were even longer in Slfn3KO mice. Mitotic marker, Phh3+, and the proliferation markers Lgr5, FoxL1, and platelet-derived growth factor-α (PDGFRα) were increased after resection in male WT, but this was blunted in male Slfn3KO mice. Cell-shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, whereas Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice, whereas other Slfn family members 1, 2, 5, 8, and 9 had varied expressions that were affected also by sex difference and loss of Slfn3. Differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased, suggesting that enterocytic differentiation effort is incompatible with rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Therefore, Slfn3 may be an important regulator not only of "normal" enterocytic differentiation but also in response to bowel resection.NEW & NOTEWORTHY The differentiating stimulus of Slfn3 signaling restrains an increase in mucosal mass after bowel resection, and there is a Slfn3-sex interaction regulating differentiation gene expression and intestinal adaptation. This current study highlights the combinatory effects of gender and Slfn3 genotype on the gene expression changes that contribute to the adaptation in intestinal cellular milleu (i.e. villus and crypt structure) which are utilized to compensate for the stress-healing response that the animals display in intestinal adaptation.

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation.

BACKGROUND/AIMS: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. METHODS: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. RESULTS: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. CONCLUSIONS: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

Cognate interaction with iNKT cells expands IL-10-producing B regulatory cells.

Successful induction of B-cell activation and memory depends on help from CD4+ T cells. Invariant natural killer T (iNKT) cells (glycolipid-specific, CD1d-restricted innate lymphocytes) provide both cognate (direct) and noncognate (indirect) helper signals to enhance B-cell responses. Both forms of iNKT-cell help induce primary humoral immune responses, but only noncognate iNKT-cell help drives humoral memory and plasma cells. Here, we show that iNKT cognate help for B cells is fundamentally different from the help provided by conventional CD4+ T cells. Cognate iNKT-cell help drives an early, unsustained germinal center B-cell expansion, less reduction of T follicular regulatory cells, an expansion of marginal zone B cells, and early increases in regulatory IL-10-producing B-cell numbers compared with noncognate activation. These results are consistent with a mechanism whereby iNKT cells preferentially provide an innate form of help that does not generate humoral memory and has important implications for the application of glycolipid molecules as vaccine adjuvants.

Current Immunotherapy Treatments of Primary Breast Cancer Subtypes.

Breast cancer receives the most funding when compared to any other cancer type, according to a global study conducted by The Lancet. Nevertheless, this malignancy remains the most diagnosed cancer among women and relies heavily on a neoadjuvant treatment regimen of chemotherapy and targeted therapy. After standard treatment, 25-30% of breast cancer patients still develop disease recurrence and must undergo cytoreductive debulking surgery followed by intensive chemotherapy. An array of targeted therapies are currently being utilized and developed to alleviate negative side effects, eradicate cancer growth, and diminish disease recurrence. Immunotherapy is a promising cancer therapy that upregulates one's immune system to stimulate a therapeutic effect and is utilized for cancer management among other ailments such as immunodeficiencies, hypersensitivity reactions, autoimmune diseases, inflammatory disorders, tissue and organ transplantation, and infectious diseases. This review highlights the five primary subtypes of breast cancer, provides a brief history of immunotherapy, evaluates the current landscape of treating breast cancer with immunotherapy, analyzes selected ongoing or recently completed immunotherapy clinical trials for hormone receptor-positive, HER2-enriched, and triple-negative breast cancer, and examines future trends for the treatment of breast cancer with immunotherapeutic techniques. This review provides a formal summary categorized by breast cancer subtype rather than types of immunotherapeutic treatment.

Focal Adhesion Kinase and Colony Stimulating Factors: Intestinal Homeostasis and Innate Immunity Crosstalk.

Thousands struggle with acute and chronic intestinal injury due to various causes. Epithelial intestinal healing is dependent on phenotypic transitions to a mobile phenotype. Focal adhesion kinase (FAK) is a ubiquitous protein that is essential for cell mobility. This phenotype change is mediated by FAK activation and proves to be a promising target for pharmaceutical intervention. While FAK is crucial for intestinal healing, new evidence connects FAK with innate immunity and the importance it plays in macrophage/monocyte chemotaxis, as well as other intracellular signaling cascades. These cascades play a part in macrophage/monocyte polarization, maturation, and inflammation that is associated with intestinal injury. Colony stimulating factors (CSFs) such as macrophage colony stimulating factor (M-CSF/CSF-1) and granulocyte macrophage colony stimulating factor (GM-CSF/CSF-2) play a critical role in maintaining homeostasis within intestinal mucosa by crosstalk capabilities between macrophages and epithelial cells. The communication between these cells is imperative in orchestrating healing upon injury. Diving deeper into these connections may allow us a greater insight into the role that our immune system plays in healing, as well as a better comprehension of inflammatory diseases of the gut.

Vil-Cre specific Slfn3KO mice exhibit sex-specific differences in lung, stomach, cecum, kidney, and proximal colon differentiation markers and Slfn family members expression levels.

The Schlafen (Slfn) family proteins are critical regulators of cell proliferation, induction of immune responses, differentiation, self-restoration, and cell cycle progression. Rodent Slfn3 and human ortholog SLFN12 are critical in the regulation of intestinal epithelial differentiation. Following previous work utilizing Vil-Cre epithelial-specific Slfn3 knockout (VC-Slfn3KO) mice to evaluate Slfn3's role in small intestinal epithelial differentiation, we sought to characterize and distinguish the effects of Slfn3 loss on Slfn family member mRNA expression and differentiation markers for other epithelial cells in the lung, stomach, cecum, and proximal colon. Quantitative PCR analysis of Slfn1, 2, 4, 5, 8, and 9 and multiple differentiation markers were evaluated. We observed gender-specific effects with the loss of Slfn3 on the other Slfn family members and epithelial differentiation markers expression. Lung Slfn4 and 5 were increased only in male VC-Slfn3KO while lung Slfn2 and 8 were decreased only in female VC-Slfn3KO compared to controls. Slfn1, 2, 4, and 9 were increased in the gastric mucosa of male VC-Slfn3KO mice compared to controls. Slfn5 was reduced in female VC-Slfn3KO proximal colonic mucosa compared to controls. Lung AT1 cell differentiation marker Hopx mRNA expression was decreased and Ager was increased in VC-Slfn3KO male mice compared to controls. Lung AT2 differentiation markers and surfactant genes Sftpc and Sftpd were decreased in male VC-Slfn3KO mice. Stomach transcription factors, Lgr5 and Notch1 were increased in male VC-Slfn3KO. Tff1 secretory protein gene was decreased in female VC-Slfn3KO mice. Sucrase isomaltase was greatly increased in male VC-Slfn3KO mice in both cecal and proximal colonic mucosa, but glucose transporter Glut2 was decreased only in the cecum of female VC-Slfn3KO. The changes induced by VC-Slfn3KO in the expression of epithelial differentiation markers and other Schlafen proteins in various target tissues, indicate a complex regulation of gene expression that is sex-dependent.

Schlafen 12 Slows TNBC Tumor Growth, Induces Luminal Markers, and Predicts Favorable Survival.

The Schlafen 12 (SLFN12) protein regulates triple-negative breast cancer (TNBC) growth, differentiation, and proliferation. SLFN12 mRNA expression strongly correlates with TNBC patient survival. We sought to explore SLFN12 overexpression effects on in vivo human TNBC tumor xenograft growth and performed RNA-seq on xenografts to investigate related SLFN12 pathways. Stable SLFN12 overexpression reduced tumorigenesis, increased tumor latency, and reduced tumor volume. RNA-seq showed that SLFN12 overexpressing xenografts had higher luminal markers levels, suggesting that TNBC cells switched from an undifferentiated basal phenotype to a more differentiated, less aggressive luminal phenotype. SLFN12-overexpressing xenografts increased less aggressive BC markers, HER2 receptors ERBB2 and EGFR expression, which are not detectable by immunostaining in TNBC. Two cancer progression pathways, the NAD signaling pathway and the superpathway of cholesterol biosynthesis, were downregulated with SLFN12 overexpression. RNA-seq identified gene signatures associated with SLFN12 overexpression. Higher gene signature levels indicated good survival when tested on four independent BC datasets. These signatures behaved differently in African Americans than in Caucasian Americans, indicating a possible biological difference between these races that could contribute to the worse survival observed in African Americans with BC. These results suggest an increased SLFN12 expression modulates TNBC aggressiveness through a gene signature that could offer new treatment targets.

Schlafen Family Intra-Regulation by IFN-α2 in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a poor prognosis and no targeted therapy for treatment. The Schlafen gene family, particularly SLFN12, critically mediates TNBC biology. Higher expression of SLFN12 correlates with decreased TNBC viability and increased chemosensitivity and patient survival, yet no treatment is known to upregulate SLFN12 in TNBC. We hypothesized that Interferon-α (IFN-α2) upregulates SLFN12 in TNBC, subsequently reducing cell viability. We utilized short hairpin adenovirus to knockout SLFN12 (AdvShSLFN12) in MDA-MB-231, Hs-578T, and BT-549 TNBC cells. Cells were treated with AdvShSLFN12 and IFN-α2. After treatment, TNBC cell viability, SLFN family mRNA, and protein expression were analyzed. Treating TNBC cells with IFN-α2 increased SLFN12 expression and reduced cell viability. However, when AdvShSLFN12 knocked down SLFN12 during IFN-α2 treatment, TNBC cell viability was still reduced. We, therefore, investigated the potential involvement of other SLFN members IFN-α2 effects on cell viability. IFN-α2 increased SLFN5, SLFN12-Like, and SLFN14 but not SLFN11 or SLFN13. During AdvShSLFN12 + IFN-α2 treatment, the expressions of SLFN5, SLFN12-Like, and SLFN14 further increased. However, when siRNA knocked down SLFN5, SLFN12-Like, and SLFN14, the IFN-α2 reduction in viability was blunted. Although the interpretation of these results may be limited by the potential interactions between different siRNAs, these data suggest a complex regulatory signaling cascade among SLFN family members. Targeting this cascade to manipulate SLFN levels may, in the future, offer the potential to manipulate the chemosensitivity of TNBC tumors.

A novel drug-like water-soluble small molecule Focal Adhesion Kinase (FAK) activator promotes intestinal mucosal healing.

Non-steroidal anti-inflammatory drugs (NSAIDs) injure the proximal and distal gut by different mechanisms. While many drugs reduce gastrointestinal injury, no drug directly stimulates mucosal wound healing. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, induces epithelial sheet migration. We synthesized and evaluated a water-soluble FAK-activating small molecule, M64HCl, with drug-like properties. Monolayer wound closure and Western blots measured migration and FAK phosphorylation in Caco-2 â€‹cells, in vitro kinase assays established FAK activation, and pharmacologic tests assessed drug-like properties. 30 â€‹mg/kg/day M64HCl was administered in two murine small intestine injury models for 4 days. M64HCl (0.1-1000 â€‹nM) dose-dependently increased Caco-2 FAK-Tyr 397 phosphorylation, without activating Pyk2 and accelerated Caco-2 monolayer wound closure. M64HCl dose-responsively activates the FAK kinase domain vs. the non-salt M64, increasing the Vmax of ATP-binding. Pharmacologic tests suggested M64HCl has drug-like properties and is enterally absorbed. M64HCl 25 â€‹mg/kg/day continuous infusion promoted healing of ischemic jejunal ulcers and indomethacin-induced small intestinal injury in C57Bl/6 mice. M64HCl-treated mice exhibited smaller ulcers 4 days after ischemic ulcer induction or indomethacin injury. Renal histology and plasma creatinine were normal. Mild hepatic inflammatory changes and ALT elevation were similar among M64HCl-treated mice and controls. M64HCl was concentrated in kidney and gastrointestinal mucosa and functional nephrectomy studies suggested predominantly urinary excretion. Little toxicity was observed in vitro or in single-dose mouse toxicity studies until >1000x higher than effective concentrations. M64HCl, a water-soluble FAK activator, promotes epithelial restitution and intestinal mucosal healing and may be useful to treat gut mucosal injury.

SLFN12 Over-expression Sensitizes Triple Negative Breast Cancer Cells to Chemotherapy Drugs and Radiotherapy.

BACKGROUND/AIM: Schlafen 12 (SLFN12) expression correlates with survival in triple negative breast cancer (TNBC). SLFN12 slows TNBC proliferation and induces TNBC differentiation, but whether SLFN12 affects the tumoral response to chemotherapy or radiation is unknown. MATERIALS AND METHODS: We over-expressed SLFN12 in MDA-MB-231 cells using two different lentiviral vectors. We assessed viable cell numbers via crystal violet assay after treatment with carboplatin, paclitaxel, olaparib, zoledronic acid, camptothecin, or cesium irradiation. CHK1 and CHK2 phosphorylation was assessed by western blot and the effects of inhibiting CHK1/CHK2 by AZD7762 were examined. Key findings were confirmed in Hs578t and BT549 TNBC cells after adenoviral SLFN12 over-expression. RESULTS: SLFN12 over-expression increased TNBC sensitivity to radiation, carboplatin, paclitaxel, zoledronic acid, and camptothecin, but not to olaparib. SLFN12 over-expression decreased CHK1 and CHK2 phosphorylation after treatment with the DNA damaging agent camptothecin (CPT). The CHK1/CHK2 inhibitor diminished the significant cytotoxicity difference between over-expression and baseline SLFN12 levels in response to carboplatin. CONCLUSION: SLFN12 increases TNBC sensitivity to DNA-damaging agents at least in part by reducing CHK1/2 phosphorylation. This may contribute to improved survival in patients whose TNBC over-expresses SLFN12. Therefore, SLFN12 levels may be used to customize or predict radiotherapy and chemotherapy effects in TNBC.

RNA Sequencing of Intestinal Enterocytes Pre- and Post-Roux-en-Y Gastric Bypass Reveals Alteration in Gene Expression Related to Enterocyte Differentiation, Restitution, and Obesity with Regulation by Schlafen 12.

BACKGROUND: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. METHODS: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6-9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. RESULTS: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. CONCLUSIONS: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS).

Schlafens: Emerging Proteins in Cancer Cell Biology.

Schlafens (SLFN) are a family of genes widely expressed in mammals, including humans and rodents. These intriguing proteins play different roles in regulating cell proliferation, cell differentiation, immune cell growth and maturation, and inhibiting viral replication. The emerging evidence is implicating Schlafens in cancer biology and chemosensitivity. Although Schlafens share common domains and a high degree of homology, different Schlafens act differently. In particular, they show specific and occasionally opposing effects in some cancer types. This review will briefly summarize the history, structure, and non-malignant biological functions of Schlafens. The roles of human and mouse Schlafens in different cancer types will then be outlined. Finally, we will discuss the implication of Schlafens in the anti-tumor effect of interferons and the use of Schlafens as predictors of chemosensitivity.

Microbiome diversity declines while distinct expansions of Th17, iNKT, and dendritic cell subpopulations emerge after anastomosis surgery.

BACKGROUND: Anastomotic failure causes morbidity and mortality even in technically correct anastomoses. Initial leaks must be prevented by mucosal reapproximation across the anastomosis. Healing is a concerted effort between intestinal epithelial cells (IECs), immune cells, and commensal bacteria. IEC TLR4 activation and signaling is required for mucosal healing, leading to inflammatory factor release that recruits immune cells to limit bacteria invasion. TLR4 absence leads to mucosal damage from loss in epithelial proliferation, attenuated inflammatory response, and bacteria translocation. We hypothesize after anastomosis, an imbalance in microbiota will occur due to a decrease in TLR4 expression and will lead to changes in the immune milieu. RESULTS: We isolated fecal content and small intestinal leukocytes from murine, Roux-en-Y and end-to-end anastomoses, to identify microbiome changes and subsequent alterations in the regulatory and pro-inflammatory immune cells 3 days post-operative. TLR4+ IECs were impaired after anastomosis. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. A distinct TCRßhi CD4+ T cells subset after anastomosis was 10-20-fold greater than in control mice. 84% were Th17 IL-17A/F+ IL-22+ and/or TNFα+. iNKT cells were increased and TCRßhi. 75% were iNKT IL-10+ and 13% iNKTh17 IL-22+. Additionally, Treg IL-10+ and IL-22+ cells were increased. A novel dendritic cell subset was identified in anastomotic regions that was CD11bhi CD103mid and was 93% IL-10+. CONCLUSIONS: This anastomotic study demonstrated a decrease in IEC TLR4 expression and microbiome diversity which then coincided with increased expansion of regulatory and pro-inflammatory immune cells and cytokines. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.

Vil-Cre specific Schlafen 3 knockout mice exhibit sex-specific differences in intestinal differentiation markers and Schlafen family members expression levels.

The intestinal epithelium requires self-renewal and differentiation in order to function and adapt to pathological diseases such as inflammatory bowel disease, short gut syndrome, and ulcers. The rodent Slfn3 protein and the human Slfn12 analog are known to regulate intestinal epithelial differentiation. Previous work utilizing a pan-Slfn3 knockout (KO) mouse model revealed sex-dependent gene expression disturbances in intestinal differentiation markers, metabolic pathways, Slfn family member mRNA expression, adaptive immune cell proliferation/functioning genes, and phenotypically less weight gain and sex-dependent changes in villus length and crypt depth. We have now created a Vil-Cre specific Slfn3KO (VC-Slfn3KO) mouse to further evaluate its role in intestinal differentiation. There were increases in Slfn1, Slfn2, Slfn4, and Slfn8 and decreases in Slfn5 and Slfn9 mRNA expression that were intestinal region and sex-specific. Differentiation markers, sucrase isomaltase (SI), villin 1, and dipeptidyl peptidase 4 and glucose transporters, glucose transporter 1 (Glut1), Glut2, and sodium glucose transporter 1 (SGLT1), were increased in expression in VC-Slfn3KO mice based on intestinal region and were also highly female sex-biased, except for SI in the ileum was also increased for male VC-Slfn3KO mice and SGLT1 was decreased for both sexes. Overall, the variations that we observed in these VC-Slfn3KO mice indicate a complex regulation of intestinal gene expression that is sex-dependent.

ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain.

Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP-PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27-FAK interaction. In human Caco-2 cells, Zn27-stimulated FAK-Tyr-397 phosphorylation despite PTP-PEST inhibition and did not affect PTP1B-FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full-length 125 kDa FAK and its 35 kDa kinase domain. The ATP-competitive FAK inhibitor PF573228 reduced basal and ZN27-stimulated FAK phosphorylation in Caco-2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose-dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (Vmax ) of FAK. The apparent Km of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the Vmax of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing.

Loss of Schlafen3 influences the expression levels of Schlafen family members in ileum, thymus, and spleen tissue.

BACKGROUND: The Schlafen (Slfn) family proteins are important for regulation of cell growth, cell differentiation and cell cycle progression. We sought to distinguish Slfn family expression in Slfn3 knockout (KO) mice after RNA sequencing analysis of Slfn3KO vs. wildtype (WT) mice revealed varying expressions of Slfn family in ileal mucosa. METHODS: Quantitative PCR analysis of Slfn members was evaluated in ileal mucosa, thymus and spleen tissue since Slfn family members have roles in differentiating intestinal and immune cells. RESULTS: Ileal mucosa of Slfn3KO mice displayed a decrease in Slfn3, 4, 8 and 9 while Slfn1 and 5 increased in mRNA expression vs. WT mice. Thymic tissue had a Slfn9 increase and a Slfn4 decrease while splenic tissue had a Slfn8 and Slfn9 increase in Slfn3KO mice vs. WT mice. These differential expressions of Slfn members could indicate a feedback regulatory mechanism within the Slfn family. Indeed, MATCH™ tool from geneXplain predicted that all Slfn members have regions in their promoters for the Kruppel-like factor-6 transcription factor. In addition, NFAT related factors, ING4, ZNF333 and KLF4 are also predicted to bind in up to 6 of the 8 Slfn promoters. This study further describes a possible autoregulatory mechanism amongst the Slfn family members which could be important in how they regulate the differentiation of various cell types.

Schlafen 12 Is Prognostically Favorable and Reduces C-Myc and Proliferation in Lung Adenocarcinoma but Not in Lung Squamous Cell Carcinoma.

Schlafen 12 (SLFN12) is an intermediate human Schlafen that induces differentiation in enterocytes, prostate, and breast cancer. We hypothesized that SLFN12 influences lung cancer biology. We investigated survival differences in high versus low SLFN12-expressing tumors in two databases. We then adenovirally overexpressed SLFN12 (AdSLFN12) in HCC827, H23, and H1975 cells to model lung adenocarcinoma (LUAD), and in H2170 and HTB-182 cells representing lung squamous cell carcinoma (LUSC). We analyzed proliferation using a colorimetric assay, mRNA expression by RT-qPCR, and protein by Western blot. To further explore the functional relevance of SLFN12, we correlated SLFN12 with seventeen functional oncogenic gene signatures in human tumors. Low tumoral SLFN12 expression predicted worse survival in LUAD patients, but not in LUSC. AdSLFN12 modulated expression of SCGB1A1, SFTPC, HOPX, CK-5, CDH1, and P63 in a complex fashion in these cells. AdSLFN12 reduced proliferation in all LUAD cell lines, but not in LUSC cells. SLFN12 expression inversely correlated with expression of a myc-associated gene signature in LUAD, but not LUSC tumors. SLFN12 overexpression reduced c-myc protein in LUAD cell lines but not in LUSC, by inhibiting c-myc translation. Our results suggest SLFN12 improves prognosis in LUAD in part via a c-myc-dependent slowing of proliferation.

ZINC4085554 inhibits cancer cell adhesion by interfering with the interaction of Akt1 and FAK.

Perioperative or circulatory forces enhance disseminated cancer cell adhesiveness by modulating focal adhesion kinase (FAK)-Akt1 interaction. Selectively blocking FAK-Akt1 interaction by a peptide derived from the FAK-Four-point-one, ezrin, radixin, moesin (FERM) domain reduces colon cancer cell adhesion in vitro and in mice. A preliminary in silico screening identified two small molecules resembling a peptide that may inhibit pressure-stimulated SW620 cancer cell adhesion to collagen I. The present study selected ZINC4085554 for further study to validate its proposed mechanism of action, using human SW620 colon cancer cells as a model system. At 25 and 50 µM, ZINC4085554 inhibited the pressure-stimulated adhesion of SW620 colon cancer cells to collagen I. This molecule prevented pressure-stimulated FAK-Tyr-397 phosphorylation; however, it did not affect Akt1-Ser-473 phosphorylation, indicating that ZINC4085554 acts downstream of Akt1, while Akt-Thr-308 remains unchanged in the presence of pressure and or ZINC4085554. Indeed, ZINC4085554 inhibited FAK-Akt1 interaction in response to increased extracellular pressure, consistent with the proposed mechanism. ZINC4085554 did not inhibit FAK-Tyr-397 phosphorylation in response to cell adhesion to collagen I, indicating the specificity of the inhibitory effects towards force-stimulated pathways. Finally, the present study confirmed that ZINC4085554 at 50 µM prevented pressure-activation of adhesion to surgical wounds in vivo in parallel to its ablation of intracellular signaling. In summary, ZINC4085554 is a small molecule mimicking part of the structure of FAK that reduces cancer cell adhesion by impairing pressure-stimulated FAK-Akt1 interaction and its downstream consequences. ZINC4085554 does not inhibit conventional outside-in FAK signaling and may be less toxic than global FAK inhibitors, and ZINC4085554 may be an important step towards the inhibition of metastasis.