Chronic infection and associated inflammation have long been suspected to promote human carcinogenesis. Recently, certain gut bacteria, including some in the genus, have been implicated in playing a role in human colorectal cancer development. However, the species and subspecies involved and their oncogenic mechanisms remain to be determined. We sought to identify the specific spp. and ssp. in clinical colorectal cancer specimens by targeted sequencing of 16S ribosomal RNA gene. Five spp. were identified in clinical colorectal cancer specimens. Additional analyses confirmed that ssp. was the most prevalent subspecies in human colorectal cancers. We also assessed inflammatory cytokines in colorectal cancer specimens using immunoassays and found that expression of the cytokines IL17A and TNFα was markedly increased but IL21 decreased in the colorectal tumors. Furthermore, the chemokine (C-C motif) ligand 20 was differentially expressed in colorectal tumors at all stages. In co-culture assays, ssp. induced CCL20 protein expression in colorectal cancer cells and monocytes. It also stimulated the monocyte/macrophage activation and migration. Our observations suggested that infection with ssp. in colorectal tissue could induce inflammatory response and promote colorectal cancer development. Further studies are warranted to determine if ssp. could be a novel target for colorectal cancer prevention and treatment..

Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.

Analysis of knockout animals indicates that 30 ,5 0 cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2 þ / þ and Prkg2 À / À mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2 þ / þ mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2 þ / þ but not Prkg2 À / À mice. Treatment of Prkg2 À / À mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.

Activation of cGMP-dependent protein kinase (PKG) has antitumor effects in colon cancer cells but the mechanisms are not fully understood. The present study has examined the regulation of β-catenin/TCF signaling, since this pathway has been highlighted as central to the antitumor effects of PKG. We show that PKG activation in SW620 cells results in reduced β-catenin expression and a dramatic inhibition of TCF-dependent transcription. PKG did not affect protein stability, nor did it increase phosphorylation of the amino-terminal Ser33/37/Thr41 residues that are known to target β-catenin for degradation. However, we found that PKG potently inhibited transcription from a luciferase reporter driven by the human CTTNNB1 promoter, and this corresponded to reduced β-catenin mRNA levels. While PKG was able to inhibit transcription from both the CTNNB1 and TCF reporters, the effect on protein levels was less consistent. Ectopic PKG had a marginal effect on β-catenin protein levels in SW480 and HCT116 but was able to inhibit TCF-reporter activity by over 80%. Investigation of alternative mechanisms revealed that cJun N-terminal kinase (JNK) activation was required for the PKG-dependent regulation of TCF activity. PKG activation caused β-catenin to bind to FOXO4 in colon cancer cells, and this required JNK. Activation of PKG was also found to increase the nuclear content of FOXO4 and increase the expression of the FOXO target genes MnSOD and catalase. FOXO4 activation was required for the inhibition of TCF activity since FOXO4-specific siRNA completely blocked the inhibitory effect of PKG. These data illustrate a dual inhibitory effect of PKG on TCF activity in colon cancer cells that involves reduced expression of β-catenin at the transcriptional level, and also β-catenin sequestration by FOXO4 activation.

Background:Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are key regulators of angiogenesis, affecting endothelial cell survival and function. However, the effect of VEGF-VEGFR signalling on tumour cell function is not well understood. Our previous studies in colorectal cancer (CRC) cells have demonstrated an intracrine VEGF/VEGFR1 signalling mechanism that mediates CRC cell survival and chemo-sensitivity. Since extracellular VEGF signalling regulates migration of endothelial cells and various tumour cells, we attempted to determine whether intracrine VEGF signalling affects CRC cell motility.Methods:Migration and invasion of CRC cells, with and without VEGF or VEGFR1 depletion, were assayed using transwell migration chambers. Changes in cell morphology, epithelial-mesenchymal transition (EMT) markers, and markers of cell motility were assessed by immunostaining and western blot.Results:Depletion of intracellular VEGF and VEGFR1 in multiple CRC cell lines led to strong inhibition of migration and invasion of CRC cells. Except for Twist, there were no significant differences in markers of EMT between control and VEGF/VEGFR1-depleted CRC cells. However, VEGF/VEGFR1-depleted CRC cells demonstrated a significant reduction in levels of phosphorylated focal adhesion kinase and its upstream regulators pcMET and pEGFR.Conclusions:Inhibition of intracrine VEGF signalling strongly inhibits CRC cell migration and invasion by regulating proteins involved in cell motility.

The effects of vascular endothelial growth factor-A (VEGF-A/VEGF) and its receptors on endothelial cells function has been studied extensively, but their effects on tumor cells are less well defined. Studies of human colorectal cancer (CRC) cells where the VEGF gene has been deleted suggest an intracellular role of VEGF as a cell survival factor. In this study, we investigated the role intracrine VEGF signaling in CRC cell survival. In human CRC cells, RNAi-mediated depletion of VEGF decreased cell survival and enhanced sensitivity to chemotherapy. Unbiased reverse phase protein array studies and subsequent validation experiments indicated that impaired cell survival was a consequence of disrupted AKT and ERK1/2 (MAPK3/1) signaling, as evidenced by reduced phosphorylation. Inhibition of paracrine or autocrine VEGF signaling had no effect on phospho-AKT or phospho-ERK1/2 levels, indicating that VEGF mediates cell survival via an intracellular mechanism. Notably, RNAi-mediated depletion of VEGF receptor VEGFR1/FLT1 replicated the effects of VEGF depletion on phospho-AKT and phospho-ERK1/2 levels. Together, these studies show how VEGF functions as an intracrine survival factor in CRC cells, demonstrating its distinct role in CRC cell survival.

In recent years, several antitumor signaling pathways mediated by the cGMP-dependent protein kinases have been identified in colon cancer cells. This review aims to present the mounting evidence in favor of cGMP/protein kinase G (PKG) signaling as a therapeutic strategy in colon cancer. The homeostatic and tumor suppressive effects of cGMP in the intestine are uncontested, but the signaling details are not understood. PKG is the central cGMP effector, and can block proliferation and tumor angiogenesis by inhibiting β-catenin/TCF and SOX9 signaling. Therapeutic activation of cGMP/PKG offers a promising avenue for the prevention and treatment of colon cancer, but additional preclinical studies are needed to fully understand the potential of this system.

The regulation of colorectal cancer cell survival pathways remains to be elucidated. Previously, it was demonstrated that endothelial cells (EC) from the liver (liver parenchymal ECs or LPEC), the most common site of colorectal cancer metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in colorectal cancer cells. However, the paracrine effects of LPECs on other colorectal cancer cellular functions have not been investigated. Here, results showed that CM from LPECs increased cell growth and chemoresistance by activating AKT in colorectal cancer cells Using an unbiased receptor tyrosine kinase array, it was determined that human epidermal growth factor receptor 3 (ERBB3/HER3) was activated by CM from LPECs, and it mediated AKT activation, cell growth, and chemoresistance in colorectal cancer cells. Inhibition of HER3, either by an inhibitor AZD8931 or an antibody MM-121, blocked LPEC-induced HER3-AKT activation and cell survival in colorectal cancer cells. In addition, CM from LPECs increased tumor growth in a xenograft mouse model. Furthermore, inhibiting HER3 with AZD8931 significantly blocked tumor growth induced by EC CM. These results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in colorectal cancer cells. This study suggested a potential of treating patients with metastatic colorectal cancer with HER3 antibodies/inhibitors that are currently being assessed in clinical trials for various cancer types.