Deshan Zhou

Mucinous colorectal adenocarcinoma (MCA) is characterized by a great mount of extracellular mucus fundamentally composed of Mucin2 (MUC2) which is significantly correlated with the high malignancy and strong invasive ability of MCA. However, rare is known about the underlying mechanism of the mucus accumulation in MCA. Our latest study demonstrated that SCF/c-KIT signaling was highly activated in MCA patients and mouse model, which up-regulated MUC2 transcription. In the present study, we paid a special interest in whether and how SCF/cKIT signaling promoted mucus secretion by using wild-type (WT) C57BL mice and their littermates who harbor mutational c-kit gene (Wads(m/m)), clinical colorectal cancer (CRC) samples, as well as human CRC cell lines. Our results clearly showed that the inner mucus layer of colon was thinner and the intracellular mucin residual was more in Wads(m/m) mice than those in WT mice by Alcian blue and PAS staining, suggesting that the mucus secretion process was crippled when SCF/c-KIT signaling was hypo-activated. Inhibiting SCF/c-KIT signaling by Imatinib also resulted in weakened mucus secretion in WT mice. Intraperitoneal administration of MANS which competitively inhibits the activity of the vesicular transport protein MARCKS efficiently reduced mucus secretion in colonic goblet cells of WT mice. Significantly, phosphorylated MARCKS (p-MARCKS) was overtly decreased in colonic mucosa of Wads(m/m) mice compared with WT mice, indicating that SCF/c-KIT signaling-regulated mucus secretion was probably mediated by MARCKS activation. Similar results were obtained in MCA patients and mouse model. Moreover, SCF/c-KIT signaling was activated or inhibited in HT-29 and LS174T CRC cells, which potently increased or decreased MARCKS activity, respectively. Finally, we found that PKC delta, a known kinase for MARCKS, was activated in WT and MCA mice along with MARCKS. Inhibition or activation of SCF/c-KIT signaling resulted in decreased or increased PKC delta activity respectively in vitro. In conclusion, we demonstrated that SCF/c-KIT signaling can promote the mucus secretion by activating PKC delta-MARCKS, which provided a new insight into understanding the mechanism of mucus secretion of goblet cells and MCA development.

Diabetic gastroparesis is a common complication of diabetes mellitus (DM) that is characterized by decreased serum insulin and insulin-like growth factor-1 (IGF-1). Despite the fact that insulin treatment not glycemic control potently accelerated gastric emptying in type 1 DM patients, the role of insulin/InsR and IGF-1/IGF-1R signaling in diabetic gastroparesis remains incompletely elucidated. In the present study, type 1 DM mice were established and treated with insulin or Voglibose for 8 weeks. The gastric emptying was delayed from DM week 4 when the gastric InsR and IGF-1R were declined. Meanwhile, the gastric choline acetyltransferase (ChAT) was significantly reduced and the myenteric cholinergic neurones and their fibers were significantly diminished. The production of stem cell factor (SCF) was dramatically repressed in the gastric smooth muscles in DM week 6. TWereafter, interstitial cells of Cajal (ICC) were clearly lost and their networks were impaired in DM week 8. Significantly, compared with Voglibose, an 8-week treatment with insulin more efficiently delayed diabetic gastroparesis development by protecting the myenteric cholinergic neurones and ICC. In conclusion, diabetic gastroparesis was an aggressive process due to the successive damages of myenteric cholinergic neurones and ICC by impairing the insulin/InsR and IGF-1/IGF-1R signaling. Insulin therapy in the early stage may delay diabetic gastroparesis.

Gastrointestinal motility disorders (GMDs) are attributed to loss of interstitial cells of Cajal (ICC), whose survival and function are deeply dependent on the activation of KIT/SCF signalling. Based on the facts that gastrointestinal distention is common in GMD patients and SCF produced by smooth muscle cells (SMCs) is usually decreased before ICC loss, we considered a possible contribution of persistent gastrointestinal distention/stretch to SCF deficiency. In this study, chronic colonic distention mouse model, diabetic gastrointestinal paresis mouse model, cultured mouse colonic SMCs and colon specimens from Hirschsprung's disease patients were used. The results showed that SCF was clearly decreased in distent colon of mice and patients, and microRNA array and real‐time PCR indicated a concomitant increase of miR‐34c in distent colon. A negative regulation of miR‐34c on SCF expression was confirmed by luciferase reporter assays together with knock‐down and overexpression of miR‐34c in cultured colonic SMCs. Using EMSA and ChIP assays, we further consolidated that in response to persistent stretch, the transcription factor AP‐1/c‐Jun was highly activated in colonic SMCs and significantly promoted miR‐34c transcription by binding to miR‐34c promoter. Knock‐down or overexpression of AP‐1/c‐Jun in cultured colonic SMCs leads to down‐ or up‐regulation of miR‐34c, respectively. In addition, the activation of AP‐1/c‐Jun was through ERK1/2 signalling provoked by Ca²⁺ overload in colonic SMCs that were subject to persistent stretch. In conclusion, our data demonstrated that persistent distention/stretch on colonic SMCs could suppress SCF production probably through Ca²⁺‐ERK‐AP‐1‐miR‐34c deregulation, resulting in ICC loss or impairment and GMD progress.

Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca²⁺ overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca²⁺ overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca²⁺ overload was originated from extracellular Ca²⁺ influx and endoplasmic reticulum (ER) Ca²⁺ release identified by treating with different Ca²⁺ channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca²⁺ overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.

Erythropoietin-producing hepatocellular A1 (EPHA1) is the first member of the EPH superfamily. Its abnormal expression has been reported in various cancer types. However, the contribution of EPHA1 to the regulation of colorectal cancer cell behaviour remains unknown. In this study, we investigated the expression profile of EPHA1 in human colorectal cancer and its effect on the adhesion and motility of colorectal cancer cells. We used human colorectal cancer specimens and the colorectal adenocarcinoma cell line HRT18 for this purpose. Our cohort screening data showed that in patients with colorectal cancer, low expression of EPHA1 gene is correlated with a remarkably reduced survival. After EPHA1 is knocked-down in colorectal cancer cells using a clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR-CAS9) genomic editing system, we observed an increase in the spreading and adhesion of HRT18 cells. Moreover, protein array data indicated that the extracellular-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signaling pathways were activated as a consequence. Inhibition of ERK and JNK proteins with specific inhibitors led to suppression of migration of the colorectal cancer cells. EPHA1 suppresses spreading and adhesion of HRT18 colorectal cancer cells through deactivation of ERK and JNK signaling pathways.

Tumour suppressor miR-34c deficiency resulted from hyper-methylation in its promoter is believed to be one of the main causes of colorectal cancer (CRC). Till date, miR-34c has been validated as a direct target of p53; but previous evidence suggested other transcription factor(s) must be involved in miR-34c transcription. In the present study, we in the first place identified a core promoter region (−1118 to −883 bp) of pre-miR-34c which was embedded within a hyper-methylated CpG island. Secondly, E2F1 promoted miR-34c transcription by physical interaction with the miR-34c promoter at site −897 to −889 bp. The transcriptional activating effect of E2F1 on miR-34c was in a p53 independent manner but profoundly promoted in the presence of p53 with exposure to 5-aza-2′-deoxycytidine (DAC). Thirdly, stem cell factor (SCF), a miR-34c target, was specifically reduced upon an introduction of E2F1 which lead to suppression of CRC cell proliferation. The E2F1-suppressed cell proliferation was partially abrogated by additional miR-34c inhibitor, indicating that the anti-proliferation effect of E2F1 was probably through activating miR-34c-SCF axis. Finally, SCF/KIT signalling increased E2F1 production by reducing its proteosomal degradation dependent on PI3K/Akt-GSK3β pathway. In conclusion, our results suggested the existence of E2F1-miR-34c-SCF negative feedback loop which was interrupted by the hyper-methylation of miR-34c promoter in CRC cells and increased cell proliferation.

Silence of the tumor suppressor miR-34c is implicated in the development of colorectal cancer (CRC). For the past few years, Resveratrol (Res) has been introduced to oncotherapies alone or with traditional chemotherapeutic drugs. However, the study of molecular mechanism involved in the anti-CRC effect of Res is still ongoing. The anti-CRC effect of Res alone or with Oxaliplatin (Oxa) was determined by cell viability assay, soft agar colony formation assay, flow cytometry and real-time cellular analyzer in HT-29 (p53 + ) and HCT-116 (p53 − ) CRC cell lines. Expressions of miR-34c and its targets were detected by qPCR and/or western blot. To evaluate the role of miR-34c in anti-CRC effect by Res alone or with Oxa, miR-34c was up or down-regulated by lentiviral mediation or specific inhibitor, respectively. To investigate how miR-34c was increased by Res, the methylation status of miR-34c promoter was detected by MSP. The tumor bearing mouse model was established by subcutaneous injection of HCT-116 cells to assess anti-CRC effect of Res alone or with Oxa in vivo. IL-6 and TNF-α in xenografts were detected by ELISA. Res inhibited cell viability, proliferation, migration and invasion as well as promoted apoptosis both in HT-29 and HCT-116 CRC cells. The anti-CRC effect of Res was partially but specifically through up-regulating miR-34c which further knocked down its target KITLG; and the effect was enhanced in the presence of p53 probably through inactivating PI3K/Akt pathway. Besides, Res sensitized CRC cells to Oxa in a miR-34c dependent manner. The xenograft experiments showed that exposure to Res or Oxa suppressed tumor growth; and the efficacy was evidently augmented by the co-treatment of Res and Oxa. Likewise, miR-34c level was elevated in xenografts of Res-treated mice while the KITLG was decreased. Finally, Res clearly reduced IL-6 in xenografts. Res suppressed CRC by specifically activating miR-34c-KITLG in vitro and in vivo; and the effect was strengthened in the presence of p53. Besides, Res exerted a synergistic effect with Oxa in a miR-34c dependent manner. We also suggested that Res-increased miR-34c could interfere IL-6-triggered CRC progression.

C-kit signaling promotes proliferation and invasion of colorectal mucinous adenocarcinoma in a murine model C-kit signaling promotes proliferation and invasion of colorectal mucinous adenocarcinoma in a murine model