Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Thymidine phosphorylase facilitates retinoic acid inducible gene-I induced endothelial dysfunction.

Activation of nucleic acid sensors in endothelial cells (ECs) has been shown to drive inflammation across pathologies including cancer, atherosclerosis and obesity. We previously showed that enhancing cytosolic DNA sensing by inhibiting three prime exonuclease 1 (TREX1) in ECs led to EC dysfunction and impaired angiogenesis. Here we show that activation of a cytosolic RNA sensor, Retinoic acid Induced Gene 1 (RIG-I) diminishes EC survival, angiogenesis and triggers tissue specific gene expression programs. We discovered a RIG-I dependent 7 gene signature that affects angiogenesis, inflammation and coagulation. Among these, we identified the thymidine phosphorylase TYMP as a key mediator of RIG-I induced EC dysfunction via its regulation of a subset of interferon stimulated genes. Our RIG-I induced gene signature was also conserved in the context of human diseases - in lung cancer vasculature and herpesvirus infection of lung endothelial cells. Pharmacological or genetic inhibition of TYMP rescues RIG-I induced EC death, migration arrest and restores sprouting angiogenesis. Interestingly, using RNAseq we identified a gene expression program that was RIG-I induced but TYMP dependent. Analysis of this dataset indicated that IRF1 and IRF8 dependent transcription is diminished in RIG-I activated cells when TYMP is inhibited. Functional RNAi screen of our TYMP dependent EC genes, we found that a group of 5 genes - Flot1, Ccl5, Vars2, Samd9l and Ube2l6 are critical for endothelial cell death mediated by RIG-I activation. Our observations identify mechanisms by which RIG-I drives EC dysfunction and define pathways that can be pharmacologically targeted to ameliorate RIG-I induced vascular inflammation.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

Analysis of the Equilibrium Phase in Immune-Controlled Tumors Provides Hints for Designing Better Strategies for Cancer Treatment.

When it comes to improving cancer therapies, one challenge is to identify key biological parameters that prevent immune escape and maintain an equilibrium state characterized by a stable subclinical tumor mass, controlled by the immune cells. Based on a space and size structured partial differential equation model, we developed numerical methods that allow us to predict the shape of the equilibrium at low cost, without running simulations of the initial-boundary value problem. In turn, the computation of the equilibrium state allowed us to apply global sensitivity analysis methods that assess which and how parameters influence the residual tumor mass. This analysis reveals that the elimination rate of tumor cells by immune cells far exceeds the influence of the other parameters on the equilibrium size of the tumor. Moreover, combining parameters that sustain and strengthen the antitumor immune response also proves more efficient at maintaining the tumor in a long-lasting equilibrium state. Applied to the biological parameters that define each type of cancer, such numerical investigations can provide hints for the design and optimization of cancer treatments.

A Distinct Innate Immune Signature of Early Onset Colorectal Cancer.

Despite a decrease in the prevalence of colorectal cancer (CRC) over the last 40 y, the prevalence of CRC in people under 50 y old is increasing around the globe. Early onset (≤50 y old) and late onset (≥65 y old) CRC appear to have differences in their clinicopathological and genetic features, but it is unclear if there are differences in the tumor microenvironment. We hypothesized that the immune microenvironment of early onset CRC is distinct from late onset CRC and promotes tumor progression. We used NanoString immune profiling to analyze mRNA expression of immune genes in formalin-fixed paraffin-embedded surgical specimens from patients with early (n = 40) and late onset (n = 39) CRC. We found three genes, SAA1, C7, and CFD, have increased expression in early onset CRC and distinct immune signatures based on the tumor location. After adjusting for clinicopathological features, increased expression of CFD and SAA1 were associated with worse progression-free survival, and increased expression of C7 was associated with worse overall survival. We also performed gain-of-function experiments with CFD and SAA1 in s.c. tumor models and found that CFD is associated with higher tumor volumes, impacted several immune genes, and impacted three genes in mice that were also found to be differentially expressed in early onset CRC (EGR1, PSMB9, and CXCL9). Our data demonstrate that the immune microenvironment, characterized by a distinct innate immune response signature in early onset CRC, is unique, location dependent, and might contribute to worse outcomes.

Cutaneous Squamous Cell Carcinoma Development Is Associated with a Temporal Infiltration of ILC1 and NK Cells with Immune Dysfunctions.

NK cells and tissue-resident innate lymphoid cells (ILCs) are innate effectors found in the skin. To investigate their temporal dynamics and specific functions throughout the development of cutaneous squamous cell carcinoma (cSCC), we combined transcriptomic and immunophenotyping analyses in mouse and human cSCCs. We identified an infiltration of NK cells and ILC1s as well as the presence of a few ILC3s. Adoptive transfer of NK cells in NK cell‒ and ILC-deficient Nfil3-/- mice revealed a role for NK cells in early control of cSCC. During tumor progression, we identified a population skewing with the infiltration of atypical ILC1 secreting inflammatory cytokines but reduced levels of IFN-γ at the papilloma stage. NK cells and ILC1s were functionally impaired, with reduced cytotoxicity and IFN-γ secretion associated with the downregulation of activating receptors. They also showed a high degree of heterogeneity in mouse and human cSCCs with the expression of several markers of exhaustion, including TIGIT on NK cells and PD-1 and TIM-3 on ILC1s. Our data show an enrichment in inflammatory ILC1 at the precancerous stage together with impaired antitumor functions in NK cells and ILC1 that could contribute to the development of cSCC and thus suggest that future immunotherapies should take both ILC populations into account.

Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development.

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC.

Differential regulation of microRNA-15a by radiation affects angiogenesis and tumor growth via modulation of acid sphingomyelinase.

Activation of acid sphingomyelinase (SMPD1) and the generation of ceramide is a critical regulator of apoptosis in response to cellular stress including radiation. Endothelial SMPD1 has been shown to regulate tumor responses to radiation therapy. We show here that the SMPD1 gene is regulated by a microRNA (miR), miR-15a, in endothelial cells (ECs). Standard low dose radiation (2 Gy) upregulates miR-15a and decreases SMPD1 levels. In contrast, high dose radiation (10 Gy and above) decreases miR-15a and increases SMPD1. Ectopic expression of miR-15a decreases both mRNA and protein levels of SMPD1. Mimicking the effects of high dose radiation with a miR-15a inhibitor decreases cell proliferation and increases active Caspase-3 & 7. Mechanistically, inhibition of miR-15a increases inflammatory cytokines, activates caspase-1 inflammasome and increases Gasdermin D, an effector of pyroptosis. Importantly, both systemic and vascular-targeted delivery of miR-15a inhibitor decreases angiogenesis and tumor growth in a CT26 murine colorectal carcinoma model. Taken together, our findings highlight a novel role for miR mediated regulation of SMPD1 during radiation responses and establish proof-of-concept that this pathway can be targeted with a miR inhibitor.

PAR2-dependent activation of GSK3ß regulates the survival of colon stem/progenitor cells.

Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3ß (GSK3ß) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3ß. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2 Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3ß activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3ß activation implicates an arrestin/PP2A/GSK3ß complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3ß nonactive form, strengthening the role of PAR2 in GSK3ß activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3ß as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.