Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial-mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan-CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia-inducible factor-1α and CEACAM5 but increased E-cadherin expression. Mitochondrial genes and proteins were induced upon pan-CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis-promoting role of CD44 isoform 4.

Specificity of viscumin revised. As probed with a printed glycan array.

Viscumin, a lectin used in anti-cancer therapy, was originally considered as ßGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galß1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral ß-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a ß-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

Differences in Medium-Induced Conformational Plasticity Presumably Underlie Different Cytotoxic Activity of Ricin and Viscumin.

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

Low expression of CD24 is associated with poor survival in colorectal cancer.

In this study we analyzed expression of CD24 in a cohort of colorectal cancer patients using immunohistochemistry staining of CD24. We found a significant association between absence or low expression of CD24 (10% of membranous and 55% of cytoplasmic staining) and shortened patient survival. Protein localization played a crucial role in the prognosis: membranous form was the major and prognostic one in primary tumors, while cytoplasmic expression was elevated in liver metastases compared to the primary tumors and contained prognostic information. Then, using The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) RNA-seq data, we showed that CD24 mRNA level was two-fold decreased in primary colorectal cancers compared to adjacent normal mucosa. Like the protein staining data, ten percent of patients with the lowest mRNA expression levels of CD24 in primary tumors had reduced survival compared to the ones with higher expression. To explain these findings mechanistically, shRNA-mediated CD24 knockdown was performed in HT-29 colorectal cancer cells. It resulted in the increase of cell migration in vitro, no changes in proliferation and apoptosis, and a slight decrease in cell invasion. As increased cell migration is a hallmark of metastasis formation, this finding corroborates the association of a decreased CD24 expression with poor prognosis. Differential gene expression analysis revealed upregulation of genes involved in cell migration in the group of patients with low CD24 expression, including integrin subunit α3 and α3, ß3 subunits of laminin 332. Further co-expression analysis identified SPI1, STAT1 and IRF1 transcription factors as putative master-regulators in this group.

HIF-Dependent NFATC1 Activation Upregulates ITGA5 and PLAUR in Intestinal Epithelium in Inflammatory Bowel Disease.

Intestinal epithelial cells exist in physiological hypoxia, leading to hypoxia-inducible factor (HIF) activation and supporting barrier function and cell metabolism of the intestinal epithelium. In contrast, pathological hypoxia is a common feature of some chronic disorders, including inflammatory bowel disease (IBD). This work was aimed at studying HIF-associated changes in the intestinal epithelium in IBD. In the first step, a list of genes responding to chemical activation of hypoxia was obtained in an in vitro intestinal cell model with RNA sequencing. Cobalt (II) chloride and oxyquinoline treatment of both undifferentiated and differentiated Caco-2 cells activate the HIF-signaling pathway according to gene set enrichment analysis. The core gene set responding to chemical hypoxia stimulation in the intestinal model included 115 upregulated and 69 downregulated genes. Of this set, protein product was detected for 32 genes, and fold changes in proteome and RNA sequencing significantly correlate. Analysis of publicly available RNA sequencing set of the intestinal epithelial cells of patients with IBD confirmed HIF-1 signaling pathway activation in sigmoid colon of patients with ulcerative colitis and terminal ileum of patients with Crohn's disease. Of the core gene set from the gut hypoxia model, expression activation of ITGA5 and PLAUR genes encoding integrin α5 and urokinase-type plasminogen activator receptor (uPAR) was detected in IBD specimens. The interaction of these molecules can activate cell migration and regenerative processes in the epithelium. Transcription factor analysis with the previously developed miRGTF tool revealed the possible role of HIF1A and NFATC1 in the regulation of ITGA5 and PLAUR gene expression. Detected genes can serve as markers of IBD progression and intestinal hypoxia.

Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy.

The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, ß-, and γ-chains (αßγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.

Evaluation of the long-term skeletal effect induced by teratogen 5-aza-2'deoxycytidine on offspring of high (C3H/HeJ) and low (C57BL/6J) bone mass phenotype mice.

The long term skeletal effects of antenatal exposure to teratogen 5-deoxy-2'-cytidine (5-AZA) were studied using two inbred strains, C3H/HeJ (C3H, with inherently stronger bones) and C57Bl/6J (C57, with weaker bones). We previously reported that in-utero exposure to 5-AZA resulted in loss of bone quality in 3- and 6-mo-old C3H offspring. In this study, we further examined whether the long-term effects of an acute teratogenic exposure are still evident in older mice. Bone phenotypes of 12 mo-old mice exposed to a single injection of 5-AZA on day 10 of their mother's pregnancy were evaluated by micro-computed tomography and compared to the untreated controls. The main observation of this study is that 5-AZA-induced loss of bone length was registered in 12-mo-old C57 and C3H males. As expected, we did not find differences in the 3rd lumbar vertebra since in-utero exposure to 5-AZA was shown to affect the limb buds but not the axial skeleton. Trajectory of changes in bone phenotypes from ages 3 mo through 6 mo to 12 mo was also compared; 5-AZA-exposed C57 males had consistently lower femoral length and trabecular BMD than age-matched controls. In summary, by characterizing teratogen-exposed C57 and C3H mice, we further confirmed that the adaptive response to antenatal insults continue into mid-life of the mice as well as there is a sex-specificity of these responses.