Cadherin-17 Research Articles

Silencing of cadherin-17 enhances apoptosis and inhibits autophagy in colorectal cancer cells.

Cadherin-17 (CDH17), a structurally unique member of the non-classical cadherin family, is associated with poor survival, cell proliferation, and metastasis in colorectal cancer. However, the role of CDH17 in the apoptosis and autophagy of colorectal cancer cells remains unclear. Here, we aimed to investigate the effect of CDH17 knockdown on autophagy and apoptosis in colorectal cancer cells. We inhibited CDH17 expression in KM12SM and KM12C colorectal cancer cells by RNA interference and found that silencing of CDH17 significantly inhibited cell viability and increased apoptosis in KM12SM and KM12C cells. In addition, silencing of CDH17 significantly increased the expression of cleaved caspase-3 and Bax and decreased the expression of Bcl-2. Concurrently, silencing of CDH17 significantly inhibited the conversion of LC3-I to LC3-II and decreased the formation of LC3+ autophagic vacuoles and the accumulation of acidic vesicular organelles, indicating that autophagy was significantly inhibited in KM12SM and KM12C cells. Additionally, treatment with the autophagy-specific activator rapamycin attenuated apoptosis in CDH17-knockdown cells and as indicated by decreased caspase-3 activity, decreased expression of cleaved caspase-3 and Bax, and increased expression of Bcl-2. In conclusion, CDH17 silencing induced apoptosis and inhibited autophagy in KM12SM and KM12C cells, and this autophagy protected the cells from apoptotic cell death.

Determination of Cadherin-17 in Tumor Tissues of Different Metastatic Grade Using a Single Incubation-Step Amperometric Immunosensor.

This paper reports the development of an amperometric immunosensing platform for the determination of cadherin-17 (CDH-17), an atypical adhesion protein involved in the progression, metastatic potential, and survival of high prevalence gastric, hepatocellular, and colorectal tumors. The methodology developed relies on the efficient capture and enzymatic labeling of the target protein on the magnetic microparticles (MBs) surface using commercial antibodies and amperometric transduction at screen-printed carbon electrodes (SCPEs) through the HRP/H2O2/HQ system. The developed immunosensing platform allows the selective determination of the target protein at low ng mL-1 level (LOD of 1.43 ng mL-1) in 45 min and using a single incubation step. The electrochemical immunosensor was successfully used for the accurate determination of the target protein in a small amount (0.5 μg) of raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade.

Metformin Ameliorates Renal Fibrosis in Mice with Unilateral Ureteral Obstruction and Inhibits TGF-ß1-Induced Upregulation of Cadherin-6 in Renal Proximal Tubule Epithelial Cells

Background: Metformin has been reported of nephroprotective effects by suppressing TGF-β1-induced renal tubulointerstitial fibrosis. The mechanisms have not been fully clarified. Cadherin-6(CDH6) is a TGF-β1 target gene related to epithelial-to-mesenchymal transition(EMT). This study aimed to examine the effects of metformin on TGF-β1-induced expression of CDH6 and renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and HK-2 human renal proximal tubule epithelial cells. Methods: Six-week-old male Kunming mice(n=18) were randomly divided into three groups: 1. Sham: sham-operated mice; 2. UUO: mice underwent UUO; 3. MET+UUO: UUO mice received metformin by gavage for 21 days after UUO. Kidneys were harvested for histopathologic examination and urine was collected for creatinine evaluation. HK-2 cells were stimulated with either TGF-β1 and/or metformin. RT-qPCR and Western Blot were used to evaluate mRNA and protein level respectively. Results: Masson’s Trichrome staining showed significant tubulointerstitial accumulation of extracellular matrix in obstructed kidneys, which could be reduced by metformin. Meanwhile, urinary creatinine was increased with metformin treatment. Upregulation of CDH6, COL1A1, PAI-1 mRNA level was observed in obstructed kidneys, which was reduced in MET+UUO group. Immunohistochemistry showed increased expression of CDH6, α-SMA in obstructed kidneys, which was alleviated in metformin treated group. In HK-2 cells, TGF-β1 upregulated mRNA level of CDH6, vimentin, PAI-1 and protein level of CDH6. Pretreatment with metformin attenuated TGF-β1-induced upregulation of CDH6, vimentin, PAI-1 mRNA level. Conclusion: Metformin prevents renal fibrosis partly through inhibition of TGF-β1-induced increase of CDH6 expression and EMT in renal proximal tubule epithelial cells. Disclosure D. Liang: None. S. Liu: None. Z. Song: None. W. Liang: None. R. Chang: None. Y. Li: None.

CDH17 alters MMP-2 expression via canonical NF-κB signalling in human gastric cancer

Gastric cancer (GC), one of the most common cancers of the digestive system, results in high morbidity and mortality, but the molecular mechanisms underlying GC remain largely unknown. Cadherin-17 (CDH17) is a nonclassical member of the cadherin (CDH) superfamily of calcium-dependent proteins. Despite recent advances in the understanding of CDH17 biology, the mechanism of CDH17 in GC proliferation, migration, and invasion has not been extensively studied. In the present study, we observed that CDH17 expression was increased in GC tissues compared with para-carcinoma tissues and was correlated with lymph node metastasis and the AJCC stage. Additionally, a significant correlation was found between CDH17 protein expression and the number of blood and lymph vessels in GC tissues. Furthermore, in vitro suppression of CDH17 expression using short-interfering RNA (siRNA) decreased AGS cell proliferation, migration and invasion. Conversely, overexpression of CDH17 through plasmid transfection enhanced the malignant activity of AGS cells. Moreover, CDH17 increased the matrix metallopeptidase 2 (MMP-2) levels via the canonical nuclear factor-kappaB (NF-κB) pathway. Our findings offer new insights into the mechanism of the CDH17/NF-κB/MMP-2 axis, and the associated signalling pathways might represent novel targets for the treatment of GC.

The cyclin-dependent kinase inhibitor flavopiridol (alvocidib) inhibits metastasis of human osteosarcoma cells.

Osteosarcoma is the most common primary malignant neoplasm of bone and typically occurs in children and young adults. As a highly metastatic malignancy, 15–20% of osteosarcoma patients are diagnosed after the tumor has already metastasized (typically to the lungs), which translates to 5-year survival rates of <40%. Here, we tested the effect of the cyclin-dependent kinase (CDK) inhibitor flavopiridol (alvocidib) in U2OS, SaOS-2, SJSA-1, and 143B osteosarcoma tumor cells in vitro and in vivo. Our results show that flavopiridol can drastically decrease survival in these osteosarcoma cell lines at nanomolar concentrations and induce mitotic catastrophe in p53-null osteosarcomas. We also performed transcriptome analysis (RNA-seq) of flavopiridol-treated osteosarcoma cells, which revealed significant changes in genes coding for proteins involved in cell-cell and cell-matrix adhesions, including cadherin 3 (CDH3) and 4 (CDH4). These transcriptional changes translated to a striking reduction in the ability of osteosarcoma cells to migrate and invade in vitro. Further, in vivo assessment of the effects of flavopiridol on osteosarcoma metastasis resulted in a significant reduction in the number of lung metastases in mice treated with flavopiridol at concentrations that are physiologically tolerable. This study suggests that flavopiridol, likely in combination with other cytotoxic chemotherapeutic agents, may be a promising drug for the treatment of osteosarcoma.

Combined overexpression of cadherin 6, cadherin 11 and cluster of differentiation 44 is associated with lymph node metastasis and poor prognosis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a highly invasive lesion that frequently metastasizes to the cervical lymph nodes and is associated with a poor prognosis. Several adhesion factors, including cadherin 6 (CDH6), cadherin 11 (CDH11) and cluster of differentiation 44 (CD44), have been reported to be involved in the invasion and metastasis of multiple types of cancer. Therefore, the aim of the present study was to determine the expression of CDH6, CDH11 and CD44 in tumor tissues from patients with OSCC, and whether this was associated with the metastasis and survival of OSCC. The mRNA expression of the human tumor metastasis-related cytokines was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in OSCC tumors with or without lymph node metastasis (n=10/group). The expression of CDH6, CDH11 and CD44 in 101 OSCC and 10 normal oral mucosa samples was examined by immunohistochemical staining. The association between overall and disease-specific survival times of patients with OSCC and the expression of these three proteins was evaluated using Kaplan-Meier curves and the log-rank test. RT-qPCR results indicated that the mRNA expression of CDH6, CDH11 and CD44 was increased in OSCC patients with lymph node metastasis (2.93-, 2.01- and 1.92-fold; P<0.05). Overexpression of CDH6, CDH11 and CD44 was observed in 31/35 (89%), 25/35 (71%) and 31/35 (89%) patients, respectively. The number of OSCC patients with lymph node metastasis exhibiting CDH6, CDH11 and CD44 overexpression was significantly higher than the number of patients without lymph node metastasis exhibiting overexpression of these proteins (P=0.017, P=0.038 and P=0.007, respectively). OSCC patients with high co-expression of CDH6, CDH11 and CD44 exhibited lower disease-specific survival times (P=0.047; χ2=3.933) when compared with OSCC patients with low co-expression of these adhesion factors. CDH6, CDH11 and CD44 serve important roles in OSCC metastasis and the combined use of these factors as biomarkers may improve the accuracy of the prediction of cancer metastases and prognosis.

A novel endogenous damage signal, glycyl tRNA synthetase, activates multiple beneficial functions of mesenchymal stem cells.

During tissue repair, the injury site releases various bioactive molecules as damage signals to actively recruit stem cells to the damaged region. Despite convincing evidence that mesenchymal stem cells (MSCs) can sense damage signals and promote repair processes, the identity of these signals and how these signals regulate stem cell-mediated tissue repair remain unknown. Glycyl tRNA synthetase (GRS) is a ubiquitously expressed enzyme that catalyzes the first step of protein synthesis in all organisms. In addition to this canonical function, we identified for the first time that GRS is released by damaged tissues or cells in response to various injury signals and may function as a damage signal that activates the proliferative, differentiation, and migratory potential of MSCs, possibly through its identified receptor, cadherin-6 (CDH-6). Binding between GRS and CDH-6 activates survival signals, such as those of the PI3K/Akt and/or FAK/ERK1/2 pathways. More importantly, we also found that MSCs stimulated with GRS show significantly improved homing and differentiation potential and subsequent in vivo therapeutic effects, in a liver fibrosis animal model. Collectively, our findings provide compelling evidence for a novel function of GRS in enhancing the multiple beneficial functions of stem cells via a non-canonical mechanism as a damage signal.

MiR-223-3p Inhibits Human Osteosarcoma Metastasis and Progression by Directly Targeting CDH6.

Cadherin-6 (CDH6) is aberrantly expressed in cancer and closely associated with tumor progression. However, the functions of CDH6 in human osteosarcoma and the molecular mechanisms underlying CDH6 in osteosarcoma oncogenesis remain poorly understood. In this work, we assessed the role of CDH6 in human osteosarcoma and identified that the expression of CDH6 was closely related with the overall survival and poor prognosis of osteosarcoma patients. MicroRNAs (miRNAs) have been implicated as important epigenetic regulators during the progression of osteosarcoma. Using dual-luciferase reporter assays, we showed that miR-223-3p suppresses CDH6 expression by directly binding to the 3′ UTR of CDH6. miR-223-3p overexpression significantly inhibited cell invasion, migration, growth, and proliferation by suppressing the CDH6 expression in vivo and in vitro. Besides, CDH6 overexpression in the miR-223-3p-transfected osteosarcoma cells effectively rescued the inhibition of cell invasion, migration, growth, and proliferation mediated by miR-223-3p. Additionally, Kaplan-Meier analysis suggests that the expression of miR-223-3p predicts favorable clinical outcomes for osteosarcoma patients. Moreover, the expression of miR-223-3p was downregulated in osteosarcoma patients and was negatively associated with the expression of CDH6. Collectively, these data highlight that miR-223-3p/CDH6 axis is an important novel pleiotropic regulator and could early predict the metastatic potential in human osteosarcoma treatments.

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms.

Synergistic Cytotoxic Effect on Gastric Cancer Cells of an Immunotoxin Cocktail in Which Antibodies Recognize Different Epitopes on CDH17.

Cadherin-17 (CDH17) is highly expressed in gastric cancer and is thus considered to be a good target for antibody therapy. CDH17 is classified as a nonclassical cadherin, in that it is composed of seven extracellular cadherin domains. We generated anti-CDH17 monoclonal antibodies (mAbs) which recognize the extracellular domain of CDH17. Competitive assay using AGS, a gastric cancer cell line, cells revealed that five selected anti-CDH17 mAbs recognize different epitopes on CDH17. As AGS cells were shown to exhibit broad expression pattern of CDH17 by flow cytometry, we separated three clones with a low (10,000/cell), medium (50,000/cell), and high (200,000/cell) expression level, designating them as AGSlow, AGSmed, and AGShigh, respectively. The mAbs, coupled with saporin, exhibited effective cytotoxicity to AGShigh, but poor cytotoxicity to AGSlow. By contrast, the immunotoxin cocktail using the three clones D2101, D2005, and D2008, which recognize different epitopes, exhibited efficient cytotoxicity, even to the AGSlow group. The effect of the immunotoxin cocktail is synergistic, as the combination index was demonstrated to be below 1.0, as calculated by the method of Chou and Talalay using CalcuSyn software. These results suggest that the immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which expand the applicable range of immunotoxin therapy for cancer.

Monoclonal Antibodies Directed against Cadherin RGD Exhibit Therapeutic Activity against Melanoma and Colorectal Cancer Metastasis.

Purpose: New targets are required for the control of advanced metastatic disease. We investigated the use of cadherin RGD motifs, which activate the α2β1integrin pathway, as targets for the development of therapeutic monoclonal antibodies (mAb).Experimental Design: Cadherin 17 (CDH17) fragments and peptides were prepared and used for immunization and antibody development. Antibodies were tested for inhibition of β1 integrin and cell adhesion, proliferation, and invasion assays using cell lines from different cancer types (colorectal, pancreatic, melanoma, and breast cancer). Effects of the mAbs on cell signaling were determined by Western blot analysis. Nude mice were used for survival analysis after treatment with RGD-specific mAbs and metastasis development.Results: Antibodies against full-length CDH17 failed to block the binding to α2β1 integrin. However, CDH17 RGD peptides generated highly selective RGD mAbs that blocked CDH17 and vascular-endothelial (VE)-cadherin-mediated β1 integrin activation in melanoma and breast, pancreatic, and colorectal cancer cells. Antibodies provoked a significant reduction in cell adhesion and proliferation of metastatic cancer cells. Treatment with mAbs impaired the integrin signaling pathway activation of FAK in colorectal cancer, of JNK and ERK kinases in colorectal and pancreatic cancers, and of JNK, ERK, Src, and AKT in melanoma and breast cancer. In vivo, RGD-specific mAbs increased mouse survival after inoculation of melanoma and colorectal cancer cell lines to cause lung and liver metastasis, respectively.Conclusions: Blocking the interaction between RGD cadherins and α2β1 integrin with highly selective mAbs constitutes a promising therapy against advanced metastatic disease in colon cancer, melanoma, and, potentially, other cancers. Clin Cancer Res; 24(2); 433-44. ©2017 AACRSee related commentary by Marshall, p. 253.

A CDH3 Mutation is Segregated in an Iranian Family with Congenital Hypotrichosis and Juvenile Macular Dystrophy

Backgrounds Hypotrichosis with juvenile macular dystrophy (HJMD) is a rare genetic disorder caused from mutations in the Cadherin 3 (CDH3) gene. Results In the present study, we reported an Iranian family with three affected members born to a consanguineous parent. Mutational analysis using whole exome sequencing has revealed a nucleotide change in CDH3 gene (NM_001793:exon8:c.830delG) which leads to a frame-shift mutation (p.G277Afs*20). No intra-familial phenotypic variation was found. Conclusion Identification of disease-causing mutation in this family facilitated the effective genetic counseling and prenatal diagnosis.

RETRACTED ARTICLE: Cadherin 6 is activated by Epstein–Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy, which is notorious among head-and-neck cancers with its metastatic feature. Epstein–Barr virus (EBV) infection plays a fundamental role in NPC development with the mechanism is not well understood. Here we demonstrate that EBV oncoprotein LMP1 drives EMT and metastasis of NPC by reactivating the adhesion molecule, cadherin 6 (CDH6), which normally occurs in embryogenesis with unknown role in NPC. CDH6 was found to be upregulated in LMP1-positive NPC tissues, and was identified as a target of the epithelium-specific miR-203. LMP1-activated NF-κB transcriptionally repressed the miR-203 expression by binding to the promoter region of miR-203 gene. CDH6 activation in turn induced EMT and promoted metastasis in NPC. CDH6 depletion, NF-κB inhibitor and miR-203 overexpression were able to impair the EMT effects. The miR-203 downregulation in NPC tissues was strongly associated with metastasis clinically. The CDH6 activator, Runt-related transcription factor 2 (RUNX2), was also activated by EBV in the event. For both CDH6 and RUNX2 are components at TGF-β downstream, CDH6 became a node protein for the interplay of multiple signalings including NF-κB and TGF-β. Therefore, the switch-on of miR-203 was important for nasopharyngeal epithelial cells to maintain normal phenotype. This study demonstrates that EBV has evolved sophisticated strategies by driving epithelial cells to obtain malignant features, particularly in NPC metastasis, providing novel biomarkers for the therapy and prognosis of EBV-associated NPC.

Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma.

A previous study demonstrated that cytokeratin 19 (CK19) expression in hepatocellular carcinoma (HCC) is an indicator of HCC invasiveness, including lymph node metastasis (LNM), tumor infiltration/non-encapsulation and poor prognosis. The exact mechanism by which CK19 expression results in poor prognosis remains unclear. Through the use of an Affymetrix U133A oligonucleotide microarray [20 patients with hepatitis B virus (HBV)-HCC], it was demonstrated that cadherin 17 (CDH17) significantly correlated with CK19 expression (R2, 0.867; P<0.001) in HBV-HCC. Immunohistochemical analysis (114 patients with HBV-HCC) also demonstrated a significant correlation between CK19 and CDH17 expressions in primary tumor tissue (R2, 0.414; P<0.001). In addition, CK19 and CDH17 expressions levels revealed a significant association with LNM (P<0.001). Cox regression multivariate analysis demonstrated that indocyanine green retention at 15 min >10% and CDH17 expression were independent prognostic factors for disease free survival (P=0.010 and 0.002, respectively). In vitro studies showed that epidermal growth factor can induce the expression of both CK19 and CDH17, and CDH17 in turn can enhance the expression of CK19 in HCC. In summary, this study demonstrated that the early recurrence and poor prognosis of CK19(+) HCC may be due to the expression of CDH17, a gene known to be associated with vascular invasion, tumor metastasis, and advanced tumor stage of HCC. Thus, novel therapeutics by targeting CDH17 may be beneficial for CK19(+) HCC.

Discovery and Optimization of HKT288, a Cadherin-6-Targeting ADC for the Treatment of Ovarian and Renal Cancers.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Plasma expression of the intestinal metaplasia markers CDH17 and TFF3 in patients with gastric cancer.

Few reliable blood markers are available for detecting gastric cancer, mainly owing to the heterogeneity of the cancer. To establish gastric cancer diagnostic markers, we evaluated the levels of plasma cadherin 17 (CDH17) and trefoil factor 3 (TFF3), which are secretory proteins and known markers for intestinal metaplasia (IM), in patients with gastric cancer. The protein expression level was analyzed in blood plasma samples from 111 gastric cancer patients and 44 healthy individuals, using a sandwich ELISA kit, followed by statistical analyses. Overall, the plasma levels of CDH17 and TFF3 were not significantly different between groups (p= 0.160 and p= 0.113, respectively). However, CDH17 expression was significantly elevated in patients with stage II and III gastric cancers compared to that in healthy controls (p= 0.023 and p= 0.037, respectively). In contrast, TFF3 levels were significantly elevated in patients with stage I (p= 0.001) and T1 gastric cancer (p= 0.013). The sensitivity and specificity of CDH17 were 66.7 and 61.4%, respectively (cutoff point: 0.189 ng/mL); for TFF3, these values were 62.2 and 56.8%, respectively (cutoff point: 5.215 ng/mL). These findings indicate that secretory protein markers for metaplastic lineages can be used as blood markers for gastric cancer.

Abstract 3089: Combining MEK and HDAC inhibitors as a therapeutic strategy to promote differentiation of colorectal cancer

Abstract The five-year survival rate for patients with metastatic colorectal cancer is less than 15%, necessitating an urgent need to develop novel therapeutic strategies for this disease. Loss of differentiation is associated with worse overall survival in colorectal cancer, suggesting that strategies to re-induce differentiation may provide clinical benefit. Differentiation therapy is effective in the treatment of acute promyelocytic leukemia but whether this approach can be efficacious in treating colorectal cancer is unknown. Inhibitors of the MAPK signaling pathway including the MEK inhibitor Trametinib can induce markers of differentiation in colorectal cancer cells. Trametinib also induces expression of Cdx-2, a known driver of colonic cell differentiation. Similarly, histone deacetylase inhibitors (HDACi) induce markers of differentiation in multiple tumor cell lines, including colon cancer cells. The aim of this study was to assess the potential therapeutic benefit of combining MEK (Trametinib) and HDAC (Panobinostat) inhibitors to further promote differentiation in colorectal cancer cells, and to elucidate the mechanistic basis for this effect. Combination treatment of HT29 and T84 cells with Trametinib and Panobinostat significantly enhanced mRNA and protein expression of the differentiation markers Cadherin 17 (CDH17) and Keratin 20 (KRT20), compared to either agent alone. Trametinib also induced expression of Cdx-2, an effect that was enhanced upon combination treatment with Panobinostat. Notably, Cdx-2 knockdown attenuated CDH17 and KRT20 induction by the combination, establishing a direct role for Cdx-2 in differentiation induction by the Trametinib/Panobinostat combination. Concomitant with inducing differentiation, the drug combination also invoked more apoptosis than either agent alone. To validate these findings in vivo, HT29 cells grown as xenografts were treated with Trametinib and Panobinostat alone and in combination. Mice treated with the combination had significantly smaller tumours than mice treated with vehicle or either agent alone. Consistent with the in vitro findings, immunostaining of the tumour xenografts demonstrated a strong increase in CDH17, KRT20 and Cdx2 expression upon treatment with the drug combination. Collectively, this study highlights a drug combination strategy to re-induce differentiation for the treatment of colorectal cancers. Citation Format: Laura J. Jenkins, Ian Y. Luk, Janson W. Tse, Jennifer Mooi, Amardeep S. Dhillon, John M. Mariadason. Combining MEK and HDAC inhibitors as a therapeutic strategy to promote differentiation of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2017-3089

Combination of cadherin-17 and SATB homeobox 2 serves as potential optimal makers for the differential diagnosis of pulmonary enteric adenocarcinoma and metastatic colorectal adenocarcinoma.

BackgroundPulmonary enteric adenocarcinoma (PEAC), a rare type of non-small cell lung cancer, has similar histological and immunohistochemical morphology to colorectal adenocarcinoma. Cadherin-17 (CDH17) and SATB homeobox 2 (SATB2) immunoexpression have recently been demonstrated in colorectal adenocarcinoma. In this study, we evaluated the value of CDH17 and SATB2 in the diagnosis of pulmonary enteric adenocarcinoma and metastatic colorectal adenocarcinoma.MethodsA total of 13 PEAC cases and 27 metastatic colorectal adenocarcinoma cases were enrolled in our cohort study. We analyzed the expressions of CK7, CK20, CDX-2, villin, cadherin-17 (CDH17), and SATB homeobox 2 (SATB2) using immunohistochemistry. Staining intensity and percentage of positive-staining cells were recorded. Sensitivity and specificity values for immunostains, individually and in combination, were computed and compared.ResultsCombining CDH17 and SATB2 resulted in high sensitivity (76.92%) and specificity (100%). In our study, the use of CK7+, napsin A+, TTF-1+, napsin A+TTF-1+ in combination with CDH17-/SATB2- had a higher area under the curve compared to the combination CDH17-/SATB2-. However, no significant differences were observed between the combination CDH17-/SATB2- and other combinations (P>0.05).ConclusionsIn combination, CDH17 and SATB2 serve as potential optimal markers for the differential diagnosis of PEAC and metastatic colorectal adenocarcinoma.

Serine peptidase inhibitor Kazal type 1 (SPINK1) as novel downstream effector of the cadherin-17/β-catenin axis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide. Previously, we reported that cadherin-17 (CDH17) and its related CDH17/β-catenin axis may be responsible for inducing HCC in a subset of patients exhibiting CDH17 over-expression. Here we aimed at obtaining a better understanding of the CDH17-related HCC biology and to obtain further indications for the design of targeted therapies in CDH17 over-expressing HCC patients. We found that SPINK1 acts as a downstream effector of the CDH17/β-catenin axis in HCC. In addition, we found that SPINK1 expression exhibited a positive correlation with CDH17 expression in human HCCs and was over-expressed in up to 70% of the tumors. We identified SPINK1 as a downstream effector of the CDH17/β-catenin axis using a spectrum of in vitro assays, including gene expression modulation and inhibitor assays, bioinformatics analyses and luciferase reporter assays. These in vitro results were validated in primary human HCCs, including the observation that alteration in β-catenin expression (a core component of the CDH17/β-catenin axis) in tumors affects SPINK1 serum levels in HCC patients. Similar to CDH17, SPINK1 expression in HCC cells was found to be associated with specific tumor-related properties via activating the c-Raf/MEK/ERK pathway. Our current data substantiate our knowledge on the role of CDH17 in the biology of HCC and suggest that components of the CDH17/β-catenin axis may serve as therapeutic targets in CDH17 over-expressing HCC patients.

The roles of cadherin 17 in interleukin-10-induced invasion of gastric cancer cells

Objective To explore the roles and mechanisms of Cadherin 17 (CDH17) in interleukin-10-induced invasion of gastric cancer cells. Methods We detected the invasion of MKN-45 gastric cancer cells induced by interleukin-10 through Transwell assay. CDH17 small interfering RNA (siRNA) was constructed and transfected in MKN-45 cells to inhibit CDH17 expression. We then compared the difference in cell invasion with or without CDH17 expression and detected the activation of corresponding signaling pathway by immunoblotting in response to interleukin-10. We additionally detected invasion of MKN-45 cell after inactivation of signaling by using its specific inhibitor. Results Western blotting and transwell assay revealed that interleukin-10 (100 ng/ml) significantly induced expression of CDH17 in MKN-45 cells (P=0.021) and promoted its invasion (10 250±1 500 vs. 2 750±450, P=0.019) as well as activated phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway (P=0.041). After successful inhibition of CDH17 expression by siRNA, the interleukin-10-indueced invasion of MKN-45 cells was significantly reduced (10 250±1 500 vs. 2 325±300, P=0.027) and the activation of PI3K/Akt signaling pathway was also suppressed. Furthermore, preincubation of Ly294002 (10 nmol/L), the specific inhibitor of PI3K/Akt signaling pathway, not only prohibit the activation of PI3K/Akt signaling pathway but also obviously diminished interleukin-10-induced invasion of MKN-45 cells (2 050±400 vs. 11 500±1 750, P=0.016). Conclusion Interleukin-10-promoted invasion of MKN-45 gastric cancer cells is mediated by CDH17 through PI3K/Akt signaling pathway. Key words: Interleukin-10; Gastric cancer cells; Cadherin 17; Phosphoinositide-3-kinase signaling; Invasion