Fundamental control of grade-specific colorectal cancer morphology by Src regulation of ezrin-centrosome engagement.

The phenotypic spectrum of colorectal cancer (CRC) is remarkably diverse, with seemingly endless variations in cell shape, mitotic figures and multicellular configurations. Despite this morphological complexity, histological grading of collective phenotype patterns provides robust prognostic stratification in CRC. Although mechanistic understanding is incomplete, previous studies have shown that the cortical protein ezrin controls diversification of cell shape, mitotic figure geometry and multicellular architecture, in 3D organotypic CRC cultures. Because ezrin is a substrate of Src tyrosine kinase that is frequently overexpressed in CRC, we investigated Src regulation of ezrin and morphogenic growth in 3D CRC cultures. Here we show that Src perturbations disrupt CRC epithelial spatial organisation. Aberrant Src activity suppresses formation of the cortical ezrin cap that anchors interphase centrosomes. In CRC cells with a normal centrosome number, these events lead to mitotic spindle misorientation, perturbation of cell cleavage, abnormal epithelial stratification, apical membrane misalignment, multilumen formation and evolution of cribriform multicellular morphology, a feature of low-grade cancer. In isogenic CRC cells with centrosome amplification, aberrant Src signalling promotes multipolar mitotic spindle formation, pleomorphism and morphological features of high-grade cancer. Translational studies in archival human CRC revealed associations between Src intensity, multipolar mitotic spindle frequency and high-grade cancer morphology. Collectively, our study reveals Src regulation of CRC morphogenic growth via ezrin-centrosome engagement and uncovers combined perturbations underlying transition to high-grade CRC morphology. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mechanistic Insights into Colorectal Cancer Phenomics from Fundamental and Organotypic Model Studies.

Colorectal cancer (CRC) diagnosis and prognostic stratification are based on histopathologic assessment of cell or nuclear pleomorphism, aberrant mitotic figures, altered glandular architecture, and other phenomic abnormalities. This complexity is driven by oncogenic perturbation of tightly coordinated spatiotemporal signaling to disrupt multiple scales of tissue organization. This review clarifies molecular and cellular mechanisms underlying common CRC histologic features and helps understand how the CRC genome controls core aspects of tumor aggressiveness. It further explores a spatiotemporal framework for CRC phenomics based on regulation of living cells in fundamental and organotypic model systems. The review also discusses tissue homeostasis, considers distinct classes of oncogenic perturbations, and evolution of cellular or multicellular cancer phenotypes. It further explores the molecular controls of cribriform, micropapillary, and high-grade CRC morphology in organotypic culture models and assesses relevant translational studies. In addition, the review delves into complexities of morphologic plasticity whereby a single molecular signature generates heterogeneous cancer phenotypes, and, conversely, morphologically homogeneous tumors show substantive molecular diversity. Principles outlined may aid mechanistic interpretation of omics data in a setting of cancer pathology, provide insight into CRC consensus molecular subtypes, and better define principles for CRC prognostic stratification.

Apc(MIN) modulation of vitamin D secosteroid growth control.

A central paradox of vitamin D biology is that 1alpha,25-(OH)(2) D(3) exposure inversely relates to colorectal cancer (CRC) risk despite a capacity for activation of both pro- and anti-oncogenic mediators including osteopontin (OPN)/CD44 and E-cadherin, respectively. Most sporadic CRCs arise from adenomatous polyposis coli (APC) gene mutation but understanding of its effects on vitamin D growth control is limited. Here we investigate effects of the Apc(Min/+) genotype on 1alpha,25-(OH)(2) D(3) regulation of OPN/CD44/E-cadherin signalling and intestinal tumourigenesis, in vivo. In untreated Apc(Min/+) versus Apc(+/+) intestines, expression levels of OPN and its CD44 receptor were increased, whereas E-cadherin tumour suppressor signalling was attenuated. Treatment by 1alpha,25-(OH)(2) D(3) or rationally designed analogues (QW or BTW) enhanced OPN but inhibited expression of CD44, the OPN receptor implicated in cell growth. These treatments also enhanced E-cadherin tumour suppressor activity, characterized by inhibition of beta-catenin nuclear localization, T-cell factor 1 and c-myelocytomatosis protein expression in Apc(Min/+) intestine. All secosteroids suppressed Apc(Min/+)-driven tumourigenesis although QW and BTW had lower calcium-related toxicity. Taken together, these data indicate that the Apc(Min/+) genotype modulates vitamin D secosteroid actions to promote functional predominance of E-cadherin tumour suppressor activity within antagonistic molecular networks. APC heterozygosity may promote favourable tissue- or tumour-specific conditions for growth control by vitamin D secosteroid treatment.

Vitamin D receptor modulates the neoplastic phenotype through antagonistic growth regulatory signals.

Vitamin D receptor (VDR) can modulate functionally antagonistic growth regulatory pathways, involving beta-catenin/E-cadherin on one hand and osteopontin (OPN) on the other. This study investigates effects of VDR ligand treatment on the balance of these discordant signals and on associated cell behavior. Treatment of Rama 37 or SW480 cells by 1alpha,25-(OH)(2) D(3) or analogs suppressed beta-catenin/Lef-1/Tcf signaling and upregulated E-cadherin, consistent with a cancer-inhibitory action. Conversely, treatment also increased transcription of OPN that may be implicated in tumor progression. Molecular crosstalk was observed between the antagonistic VDR-dependent signals, in that beta-catenin/Lef-1/Tcf molecules modulated VDR activation of OPN. Treatment effects on cell growth were related to a constitutive balance of OPN and E-cadherin expression. No growth effects were observed in Rama 37 cells that have low OPN and high E-cadherin expression. Conversely, treatment of Rama 37 stably transfected subclones that had high OPN and/or low level E-cadherin induced small but significant increases of cell attachment to fibronectin, anchorage-independent growth or invasion. This study shows that relative expression levels of key VDR downstream genes may influence growth regulation by 1alpha,25-(OH)(2) D(3) or analogs. These findings may be relevant to the cell- or tissue-specificity of vitamin D growth regulation.

PTEN controls glandular morphogenesis through a juxtamembrane ß-Arrestin1/ARHGAP21 scaffolding complex.

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein ß-Arrestin1. Because ß-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42 -dependent morphogenic processes through a ß-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs ß-Arrestin1 membrane localization, ß-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by ß-Arrestin1 KD or inhibition of ß-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of ß-Arrestin1, ARHGAP21 and Cdc42.

Vitamin D3 suppresses morphological evolution of the cribriform cancerous phenotype.

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3,theactive form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted "Swiss cheese-like" cribriform morphology (CM) comprising multiple abnormal "back to back" lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.

Overexpression of p65/RelA potentiates curcumin-induced apoptosis in HCT116 human colon cancer cells.

Curcumin, the yellow pigment in the spice turmeric, has potent chemopreventive activities that involve diverse molecular pathways. It is widely believed that curcumin pro-apoptotic properties are mediated by downregulation of NF kappa B (NFkappaB). The p65/RelA subunit of NFkappaB may influence cell death, in part by activation of NFkappaB anti-apoptotic target genes including X-linked inhibitor of apoptosis (XIAP), A20, bcl-xL and inhibition of sustained activation of c-Jun N-terminal kinase (JNK). We have shown previously that curcumin inhibits NFkappaB, activates JNK and promotes apoptosis in HCT116 colorectal cancer cells. Here, we show that forced overexpression of p65 does not affect curcumin-induced JNK activation. Indeed, overexpression of p65 enhanced curcumin-mediated apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and poly(ADP-ribose) polymerase (PARP) cleavage. This potentiating effect of p65 upon curcumin-mediated apoptosis was reversed by transfection of cells with an IkappaB super-repressor (DeltaNIkappaB). Curcumin treatment inhibited expression of NFkappaB anti-apoptotic target genes in mock-transfected and in p65-overexpressing HCT116 cells, although expression levels remained higher in the latter. Taken together, these results show that curcumin-mediated activation of JNK or induction of apoptosis does not require inhibition of p65. Furthermore, curcumin/p65 synergy in promotion of apoptosis cannot be attributed to active repression of NFkappaB anti-apoptotic genes.

The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation.

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.

Methylprednisolone increases urinary nitrate concentrations and reduces subclinical renal injury during infrarenal aortic ischemia reperfusion.

OBJECTIVE: This study tests the hypothesis that methylprednisolone may influence eNOS activity in renal arterial and venous vascular beds and impede subclinical renal injury. SUMMARY BACKGROUND DATA: Acute renal failure is a major complication of cardiovascular surgery. Renal damage arises in part from excessive vasoconstriction mediated by an imbalance of vasoconstrictive ET-1 and vasodilatory NO produced by eNOS. While methylprednisolone may reduce subclinical renal injury as measured by urinary N-acetyl-beta-D-glucosaminidase (beta-NAG), its effects upon eNOS activity in renal arterial and venous vascular beds, reflected by urinary nitrate levels, is unclear. METHODS: A porcine model of normotensive, euvolemic infrarenal aortic ischaemia-reperfusion was used. Forty-two pigs underwent a 60-minute laparotomy followed by 150 minutes of infrarenal ischemia and 180 minutes of reperfusion. Animals were randomized to receive methylprednisolone 30 mg/kg or placebo after induction of general anesthesia. Urinary beta-NAG levels were assessed as an index of subclinical renal injury, whereas urinary nitrate was assessed as an indicator of eNOS activity in renal arterial and venous vascular beds. RESULTS: Methylprednisolone treatment did not influence mean arterial, central venous, or pulmonary artery wedge pressures but suppressed plasma IL-6 levels. After the ischemia-induced rise from preanaesthetic baseline levels, urinary beta-NAG levels declined to significantly lower values in the MP group, indicative of MP renal protection (P < 0.05). Conversely, urinary nitrate levels indicative of vascular e-NOS activity remained significantly and persistently higher in MP-treated animals (P < 0.05). CONCLUSION: This study, in a porcine model of renal ischaemia-reperfusion injury, shows the benefits of methylprednisolone pretreatment in enhancing urinary nitrate levels indicative of vascular eNOS activity and the reduction of urinary beta-NAG levels, which represent subclinical renal injury.

Chemopreventive properties of curcumin.

Inhibition of defined molecular steps of tumorigenesis by natural nontoxic compounds may be an efficient means to tackle the population cancer burden. Extensive research has addressed the chemotherapeutic potential of curcumin (diferuloylmethane), a relatively nontoxic plant-derived polyphenol. This review considers the following properties of curcumin: anticancer effects in animal model systems; metabolism; biological structure and pharmacokinetics; biological properties implicated in chemoprevention; antioxidant properties; influences upon Phase I and II carcinogen-metabolizing enzymes; signal transduction properties and the neoplastic phenotype; apoptosis evasion, cell proliferation, de-differentiation, migration and invasion and clinical studies. This review will summarize the unique properties of curcumin that may be exploited for successful clinical cancer prevention.

Modulation of N-methyl-N-nitrosourea-induced crypt restricted metallothionein immunopositivity in mouse colon by a non-genotoxic diet-related chemical.

Red meat consumption is associated with endogenous metabolic generation of mutagenic N-nitroso compounds (NOC) and may be implicated in causation of colorectal cancer. Assessment of a biologically relevant dose of NOCs is hampered by imperfect understanding of NOC interactions with other dietary components. This study tests the hypothesis that NOC effects upon mutational biomarkers in mouse colon may be modulated by a non-genotoxic diet-related compound. N-methyl-N-nitrosourea (MNU) and undegraded lambda carrageenan (lambdaCgN) were selected as test chemicals, representing a NOC and a non-genotoxic agent, respectively. Study end-points included (i) DNA adduct formation and (ii) metallothionein (MT) crypt restricted immunopositivity indices (MTCRII) which are considered representative of crypt stem cell mutations. Frequency and size of MT immunopositive foci as well as total number of MT immunopositive crypts were assessed. Biologically effective doses of MNU and lambdaCgN were determined in model validation studies and the agents were then tested alone and in combination. Continuous lambdaCgN treatment for 10 weeks induced significantly greater colonic mucosal injury than a drinking water control. In combined treatment regimens, lambdaCgN treatment did not significantly affect MNU-induced DNA adduct formation. However, combinations of lambdaCgN with MNU significantly increased MTCRII in excess of those induced by MNU alone. Recurrent or continuous lambdaCgN regimens had greater interactive effects with MNU upon MTCRII than short-term lambdaCgN treatment. This study has shown that exposure to a non-genotoxic diet-related compound (lambdaCgN) modulates the effective NOC dosimetry for induction of MT crypt restricted immunopositivity.