The prosurvival IKK-related kinase IKKϵ integrates LPS and IL17A signaling cascades to promote Wnt-dependent tumor development in the intestine

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkϵ in Wnt-driven tumor development. We found that Ikkϵ was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikkϵ in b-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkϵ to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkϵ was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding proinflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkϵ-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkϵ phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkϵ to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. � 2016 American Association for Cancer Research

Functional and spatial proteomics profiling reveals intra- and intercellular signaling crosstalk in colorectal cancer

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology—a strategy that is based on perturbing primary tumor cells from cancer patients—could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC.Molecular tumour pathology - and tumour genetic

Mesenchymal Cells in Colon Cancer

Mesenchymal cells in the intestine comprise a variety of cell types of diverse origins, functions, and molecular markers. They provide mechanical and structural support and have important functions during intestinal organogenesis, morphogenesis, and homeostasis. Recent studies of the human transcriptome have revealed their importance in the development of colorectal cancer, and studies from animal models have provided evidence for their roles in the pathogenesis of colitis-associated cancer and sporadic colorectal cancer. Mesenchymal cells in tumors, called cancer-associated fibroblasts, arise via activation of resident mesenchymal cell populations and the recruitment of bone marrow–derived mesenchymal stem cells and fibrocytes. Cancer-associated fibroblasts have a variety of activities that promote colon tumor development and progression; these include regulation of intestinal inflammation, epithelial proliferation, stem cell maintenance, angiogenesis, extracellular matrix remodeling, and metastasis. We review the intestinal mesenchymal cell-specific pathways that regulate these processes, with a focus on their roles in mediating interactions between inflammation and carcinogenesis. We also discuss how increasing our understanding of intestinal mesenchymal cell biology and function could lead to new strategies to identify and treat colitis-associated cancers. © 2017 AGA Institut

Concurrent video-rate color and near-infrared fluorescence laparoscopy.

The visual identification and demarcation of tumors and tumor margins remains challenging due to the low optical contrast of cancer cells over surrounding tissues. Fluorescence molecular imaging was recently considered clinically for improving cancer detection during open surgery. We present herein a next step in the development of fluorescence molecular guidance by describing a novel video-rate imaging laparoscope capable of concurrently recording color and near-infrared fluorescence images and video. Video-rate operation is based on graphics processing unit-based image processing. We examine the optical characteristics of the system developed and provide performance metrics related to intra-operative endoscopic guidance, showcased on phantoms and endoscopic color and fluorescence molecular imaging of tumors in a mouse model of the human disease

Selective <em>in vivo</em> imaging of tumors with a tumor cell-specific Hsp70 peptide-based probe.

Although in vivo targeting of tumors using fluorescently-labeled probes has greatly gained in importance over the last few years, most of the clinically applied reagents lack tumor cell specificity. Our novel tumor cell-penetrating peptide-based probe (TPP) recognizes an epitope of Hsp70 that is exclusively present on the cell surface of a broad variety of human and mouse tumors and metastases, but not on normal tissues. Due to the rapid turn-over rate of membrane-Hsp70, fluorescently-labeled TPP is continuously internalized into syngeneic, spontaneous, chemically/genetically induced and xenograft tumors following intravenous administration, thereby enabling site-specific labeling of primary tumors and metastases. In contrast to the commercially available non-peptide small molecule alpha v beta3-integrin antagonist IntegriSense (trademark), TPP exhibits a significantly higher tumor-to-background contrast and stronger tumor-specific signal intensity in all tested tumor models. Moreover, in contrast to IntegriSense (trademark), TPP reliably differentiates between tumor cells and cells of the tumor microenvironment, such as tumor-associated macrophages and fibroblasts which were found to be membrane-Hsp70 negative. Therefore, TPP provides a useful tool for multimodal imaging of tumors and metastases that might help to improve our understanding of tumorigenesis and allow the establishment of improved diagnostic procedures and more accurate therapeutic monitoring. TPP might also be a promising platform for tumor-specific drug delivery and other Hsp70- based targeted therapies

Card9-dependent IL-1&beta; regulates IL-22 production from group 3 innate lymphoid cells and promotes colitis-associated cancer.

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell-specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL-1&beta; production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis-associated cancer (CAC) model. Card9 -/- mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL-1&beta; generation and defective IL-1&beta; controlled IL-22 production from group 3 innate lymphoid cells. Consistent with the key role of immune-derived IL-22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9 -/- mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9-controlled, ILC3-mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9-mediated innate immunity in inflammation-associated carcinogenesis

Glutathione peroxidase 4 prevents necroptosis in mouse erythroid precursors.

Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis since imbalanced production of ROS may lead to oxidative stress and cell death. The anti-oxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress-induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that it is essential for preventing RIP3 dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation. This results in necroptosis, which occurs independently of TNF&alpha; activation. While genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4 deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as so far unrecognized unconventional upstream signaling activators of RIP3-dependent necroptosis

IKKα controls ATG16L1 degradation to prevent ER stress during inflammation

Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation

Olfactomedin 4 associates with expression of differentiation markers but not with properties of cancer stemness, EMT nor metastatic spread in colorectal cancer.

Tumor stem cells play a pivotal role in carcinogenesis and metastatic spread in colorectal cancer (CRC). Olfactomedin 4 (OLFM4) is co-expressed with the established stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 at the bottom of intestinal crypts and has been suggested as a surrogate for cancer stemness and a biomarker in gastrointestinal tumors associated with prognosis. Therefore, it was the aim of the present study to clarify whether OLFM4 is involved in carcinogenesis and metastatic spread in CRC. We used a combined approach of functional assays using forced OLFM4 overexpression in human CRC cell lines, xenograft mice, and an immunohistochemical approach using patient tissues to investigate the impact of OLFM4 on stemness, canonical Wnt signaling, properties of metastasis and differentiation as well as prognosis. OLFM4 expression correlated weakly with tumor grade in one patient cohort (metastasis collection: p&nbsp;= 0.05; pooled analysis of metastasis collection and survival collection: p&nbsp;= 0.19) and paralleled the expression of differentiation markers (FABP2, MUC2, and CK20) (p&nbsp;= 0.002) but did not correlate with stemness-associated markers. Further analyses in CRC cells lines as well as xenograft mice including forced overexpression of OLFM4 revealed that OLFM4 neither altered the expression of markers of stemness nor epithelial-mesenchymal transition, nor did OLFM4 itself drive proliferation, migration, or colony formation, which are all prerequisites of carcinogenesis and tumor progression. In line with this, we found no significant correlation between OLFM4 expression, metastasis, and patient survival. In summary, expression of OLFM4 in human CRC seems to be characteristic of differentiation marker expression in CRC but is not a driver of carcinogenesis nor metastatic spread

ROS production and NF-κB activation triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation

The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation