Hyaluronic acid regulates normal intestinal and colonic growth in mice

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.     

ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer

Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.     

Logarithmic expansion of LGR5+ cells in human colorectal cancer

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5⁺ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5⁺ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.     

Overlapping expression of microRNAs in human embryonic colon and colorectal cancer

MicroRNAs (miRNAs) are essential for regulating cell differentiation and maintaining the pluripotent state of stem cells. Although dysregulation of specific miRNAs has been associated with certain types of cancer, to date no evidence has linked miRNA expression in embryonic and tumor tissues. We assessed the expression of mature miRNAs in human embryonic colon tissue, and in colorectal cancer and paired normal colon tissue. Overlapping miRNA expression was detected between embryonic colonic mucosa and colorectal cancer. We have found that the miR-17-92 cluster and its target, E2F1, exhibit a similar pattern of expression in human colon development and colonic carcinogenesis, regulating cell proliferation in both cases. In situ hybridization confirmed the high level of expression of miR-17-5p in the crypt progenitor compartment. We conclude that miRNA pathways play a major role in both embryonic development and neoplastic transformation of the colonic epithelium.     

Role of Intracellular and Extracellular MicroRNA-92a in Colorectal Cancer

Colorectal cancer is one of the leading causes of cancer-related death worldwide. Previous studies have shown that miR-92a has an oncogenic function in several cancers and that its up-regulation is correlated with malignant clinicopathologic behaviors of colorectal cancer. It also has been suggested that circulating miR-92a in patients' plasma can be a potential biomarker for colorectal cancer. However, the precise roles of intracellular and extracellular miR-92a are not yet understood. In this study, we examined the expression levels of miR-92a in colorectal tumors (38 cancer specimens and 56 adenoma specimens) and paired adjacent nontumorous tissues. Increased expression of miR-92a was frequently observed in the cancers compared with that in the adenomas and was correlated with advanced clinical stages, tumor depth, and size. We also demonstrated that the levels of miR-92a within microvesicles (MVs) in the plasma of mice bearing colon cancer xenografts were significantly increased compared with those in control mice. One of the roles of intracellular and extracellular miR-92a was shown to be down-regulation of Dickkopf-3 (Dkk-3), a presumed tumor suppressor gene. Within the colon cancer cells, suppression of Dkk-3 by miR-92a contributed to the cell proliferation. Extracellular miR-92a packed within MVs secreted by colon cancer cells was delivered into endothelial cells and contributed to the proliferation and motility of these cells through down-regulation of the same target gene, Dkk-3. These data suggest that intracellular and extracellular miR-92a had important roles in tumor growth and the tumor microenvironment in colorectal cancer.     

TNFAIP8 protein functions as a tumor suppressor in inflammation-associated colorectal tumorigenesis

Tumor necrosis factor-α-induced protein 8 (TNFAIP8 or TIPE) is a member of the TNFAIP8 family. While TIPE was broadly considered to be pro-cancerous, its precise roles in carcinogenesis especially those of the intestinal tract are not clear. Here, we show that genetic deletion of TIPE in mice exacerbated chemical-induced colitis and colitis-associated colon cancer. Loss of TIPE exacerbated inflammatory responses and inflammation-associated dysbiosis, leading to the activation of NF-κB and STAT3, and it also accelerated dysplasia, DNA damage and proliferation of intestinal epithelial cells. We further show that colon microbiota were essential for increased tumor growth and progression in Tipe^−/− mice. The tumor suppressive function of TIPE originated primarily from the non-hematopoietic compartment. Importantly, TIPE was downregulated in human colorectal cancers, and patients with low levels of Tipe mRNA were associated with reduced survival. These results indicate that TIPE serves as an important modulator of colitis and colitis-associated colon cancer.Subject terms: Cancer microenvironment, Chronic inflammation     

CYP27A1 and CYP24 expression as a function of malignant transformation in the colon.

Vitamin D deficiency is strongly associated with the risk of developing colorectal cancer (CRC). Because of the propensity of bioactive 1,25-dihydroxyvitamin D3 to cause toxic hypercalcemia, considerable effort has been directed to identifying safer drugs while retaining the efficacy of the parent compound. However, vitamin D precursors do not present toxicity concerns and may be sufficient for CRC chemoprevention or chemotherapy, providing the appropriate enzymes are present in colonic epithelia. We previously showed that CYP27B1 is present at equally high levels in the colon and CRC irrespective of differentiation but was not present in metastases. In this study we used quantitative immunohistochemistry to show that CYP27A1, converting D3 to 25-hydroxycholecalciferol, is present in increasing concentrations in the nuclei of normal colonic epithelia, aberrant crypt foci (ACF), and adenomatous polyps. Whereas total cellular CYP27A1 remains high in CRC and lymph node metastases, the amount of enzyme present in the nuclei decreases with tumor cell dedifferentiation while rising in the cytoplasm. Similarly, increasing amounts of the deactivating enzyme CYP24 are present in the nuclei of normal colonic epithelia, ACFs, and adenomatous polyps. Although the amount of total CYP24 decreases slightly in CRC as a function of tumor cell dedifferentiation and metastasis, location of this enzyme shifts almost entirely from the nuclear compartment to the cytoplasmic compartment. These data indicate that non-toxic vitamin D precursors should be sufficient for CRC chemoprevention, but that neither vitamin D nor its precursors may be sufficient for CRC chemotherapy.     

The congenital chloride diarrhea gene is expressed in seminal vesicle, sweat gland, inflammatory colon epithelium, and in some dysplastic colon cells

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder of intestinal electrolyte transportation caused by mutations in the anion transporter protein encoded by the down-regulated in adenoma (DRA), or CLD, gene. In this study, in situ hybridization and immunohistochemistry were performed to investigate the expression of CLD in extraintestinal normal epithelia and in intestinal inflammatory and neoplastic epithelia. The expression of the closely related anion transporter diastrophic dysplasia sulfate transporter, DTDST, was also examined and compared with that of CLD in colon. The only extraintestinal tissues showing CLD expression were eccrine sweat glands and seminal vesicles. In inflammatory bowel disease and ischemic colitis, expression of CLD mRNA in colon epithelium was similar to histologically normal colon epithelium, but the protein was found deeper in crypts, including proliferative epithelial cells. In intestinal tumors, the expression pattern of CLD was dependent on the differentiation status of the tissue studied: epithelial polyps with no or minor dysplasia showed abundant expression, whereas adenocarcinomas were negative. The DTDST gene was abundantly expressed in the upper crypt epithelium of colonic mucosa.     

Anti-metastatic potential of human Vδ1+ γδ T cells in an orthotopic mouse xenograft model of colon carcinoma

The role of human intraepithelial Vδ1⁺ γδ T cell cytotoxic effectors in the immune surveillance against metastatic colon cancer has never been addressed, despite their reported capacity to infiltrate colon carcinomas and to kill colonic cancer cells in vitro. We previously showed that Vδ1⁺ γδ T cells are enriched in blood in response to cytomegalovirus (CMV) infection, and that such increase may be protective against epithelial cancers. The objective of the present study was to investigate whether CMV-induced Vδ1⁺ γδ T lymphocytes could inhibit the propagation of human colon tumors in vivo, in order to evaluate their immunotherapeutic potential in this context. Even though metastases are an important cause of death in various cancers including colorectal cancer (CRC), the anti-metastatic effect of immune effectors has been poorly analyzed. To this purpose, we set up a reliable model of metastatic colon cancer through orthotopic implantation of luciferase-expressing human HT29 cells in immunodeficient mice. Using bioluminescence imaging to follow the outcome of colonic cancer cells, we showed that a systemic treatment with CMV-induced Vδ1⁺ γδ T cells could not only inhibit primary colon tumor growth but also the emergence of secondary tumor foci in the lungs and liver. Finally, our data lead to propose that Vδ1⁺ γδ T lymphocytes may directly influence the appearance of metastases independently from their control of primary tumor size. These findings, which extend our previous work, pave the road for the potential manipulation of Vδ1⁺ γδ T lymphocytes in novel anti-CRC immunotherapeutic protocols.     

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis

The Ras/mitogen-activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual-specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal-regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium-induced colitis, as opposed to wild-type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc ^Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage-independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.     

SMYD2 targets RIPK1 and restricts TNF-induced apoptosis and necroptosis to support colon tumor growth

SMYD2 is a histone methyltransferase, which methylates both histone H3K4 as well as a number of non-histone proteins. Dysregulation of SMYD2 has been associated with several diseases including cancer. In the present study, we investigated whether and how SMYD2 might contribute to colorectal cancer. Increased expression levels of SMYD2 were detected in human and murine colon tumor tissues compared to tumor-free tissues. SMYD2 deficiency in colonic tumor cells strongly decreased tumor growth in two independent experimental cancer models. On a molecular level, SMYD2 deficiency sensitized colonic tumor cells to TNF-induced apoptosis and necroptosis without affecting cell proliferation. Moreover, we found that SMYD2 targeted RIPK1 and inhibited the phosphorylation of RIPK1. Finally, in a translational approach, pharmacological inhibition of SMYD2 attenuated colonic tumor growth. Collectively, our data show that SMYD2 is crucial for colon tumor growth and inhibits TNF-induced apoptosis and necroptosis.Subject terms: Colon cancer, Apoptosis     

slc26a3 (dra)-deficient mice display chloride-losing diarrhea, enhanced colonic proliferation, and distinct up-regulation of ion transporters in the colon

Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters.     

Four Jointed Box 1 Promotes Angiogenesis and Is Associated with Poor Patient Survival in Colorectal Carcinoma

Angiogenesis, the recruitment and re-configuration of pre-existing vasculature, is essential for tumor growth and metastasis. Increased tumor vascularization often correlates with poor patient outcomes in a broad spectrum of carcinomas. We identified four jointed box 1 (FJX1) as a candidate regulator of tumor angiogenesis in colorectal cancer. FJX1 mRNA and protein are upregulated in human colorectal tumor epithelium as compared with normal epithelium and colorectal adenomas, and high expression of FJX1 is associated with poor patient prognosis. FJX1 mRNA expression in colorectal cancer tissues is significantly correlated with changes in known angiogenesis genes. Augmented expression of FJX1 in colon cancer cells promotes growth of xenografts in athymic mice and is associated with increased tumor cell proliferation and vascularization. Furthermore, FJX1 null mice develop significantly fewer colonic polyps than wild-type littermates after combined dextran sodium sulfate (DSS) and azoxymethane (AOM) treatment. In vitro, conditioned media from FJX1 expressing cells promoted endothelial cell capillary tube formation in a HIF1-α dependent manner. Taken together our results support the conclusion that FJX1 is a novel regulator of tumor progression, due in part, to its effect on tumor vascularization.