Transcription factor EHF interacting with coactivator AJUBA aggravates malignancy and acts as a therapeutic target for gastroesophageal adenocarcinoma.

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

Exosome-derived lnc-HOXB8-1:2 induces tumor-associated macrophage infiltration to promote neuroendocrine differentiated colorectal cancer progression by sponging hsa-miR-6825-5p.

INTRODUCTION: Neuroendocrine differentiation (NED) in colorectal cancer (CRC) cells has been known for decades, and our previous meta-analysis indicated that CRC patients with neuroendocrine differentiation have a lower 5-year survival rate. In recent years, an increasing number of studies have found that exosome-derived long non-coding RNAs (lncRNAs) play important roles in cancer progression and metastasis. However, the functions and mechanism of exosome-derived lncRNAs in CRC with neuroendocrine differentiation are not yet fully clear. MATERIALS AND METHODS: The clinical significance of NED was assessed in a retrospective study of 105 patients. Next-generation sequencing and bioinformatics analysis were conducted to select lnc-HOXB8-1:2 for further study. Using immunohistochemistry, qRT-PCR, western blot, transwell assay, immunofluorescence assay, fluorescence in situ hybridization assay and dual-luciferase reporter assay, the oncogenic role of exosome-derived lnc-HOXB8-1:2 was determined in CRC with NED. The mechanism underlying the lnc-HOXB8-1:2/hsa-miR-6825-5p/CXCR3 axis was also explored. RESULTS: NED was a risk factor for the progression and mortality of CRC. lnc-HOXB8-1:2, derived from exosomes secreted by neuroendocrine differentiated colon cancer cells, was identified in our study. The proportion of M2 macrophages and the migration and invasion capacities of tumor-associated macrophages (TAMs) markedly increased after the addition of neuroendocrine differentiated CRC cell-derived exosomes. More excitingly, the expression of lnc-HOXB8-1:2 and the protein level of CXCR3 were also upregulated in TAMs. The lnc-HOXB8-1:2/hsa-miR-6825-5p/CXCR3 axis was predicted via miRanda software and confirmed by the dual-luciferase reporter assay. Furthermore, the increased expression of lnc-HOXB8-1:2 was accompanied by downregulation of hsa-miR-6825-5p expression and upregulation of CXCR3 protein levels. Overexpression of hsa-miR-6825-5p also reduced CXCR3 expression. CONCLUSION: lnc-HOXB8-1:2 in exosomes derived from neuroendocrine differentiated CRC cells acted as a ceRNA competitively binding hsa-miR-6825-5p to upregulate CXCR3 expression and leading to TAM infiltration and M2 polarization, which promotes neuroendocrine differentiated CRC progression.

Methylated septin 9 gene is an important prognostic marker in stage II and stage III colorectal cancer for evaluating local recurrence or distant metastasis after surgery.

BACKGROUND: Abnormal hypermethylation of the septin 9 gene was an inchoate incident in some cancers. Though latest several researches had paid attention to its value in prognosis, the consequences were not distinctly, especially in colorectal cancer (CRC) with stage II and stage III. PURPOSE: The aim of this research was to pick up the prognostic value of the methylated septin 9 gene (mSEPT9) in CRC patients, particularly in TNM stage II-III. METHODS: Blood samples before surgery were obtained from 144 CRC patients, of which there were 94 with stage II and stage III. mSEPT9 was considered positive when the cycle number of the peak reaction (Ct) was lower than the threshold value (41.0) for two times during three times PCR test. mSEPT9 and other relative factors of prognosis were estimated by survival analysis. The level of septin 9 in tissues was tested by immunohistochemical (IHC). RESULTS: Stage II and stage III patients with mSEPT9 positive (mSEPT9+) had a lower disease-free survival (DFS) rate than those with mSEPT9 negative (mSEPT9-) (2-year DFS rates, 52.1% vs 73.9%, P = 0.014). In multivariate regression analysis, mSEPT9 was also an independent predictor of prognosis (HR = 2.741, P = 0.009). The risk of local recurrence or distant metastasis in CRC patients after surgery was mSEPT9+ with stage III, mSEPT9- with stage III/mSEPT9+ with stage II, and mSEPT9- with stage II (P = 0.001), from highest to lowest. In addition, mSEPT9 was strongly associated with TNM staging, tumor immersion depth, distant metastasis, differentiation degree, vascular invasion and microsatellite. When we explored the associations between septin 9 protein level revealed by IHC and other elements, recurrence/progression (R = - 0.523, P = 0.001), mSEPT9 status (R = - 0.451, P = 0.004) and T stage (R = - 0.375, P = 0.017) showed significant correlations. CONCLUSIONS: Positive mSEPT9 is a poor prognostic marker for CRC patients in stage II and III. It is also a powerful complement to TNM staging in predicting postoperative DFS of CRC patients of stage II and III.

Immune Infiltration and Clinical Outcome of Super-Enhancer-Associated lncRNAs in Stomach Adenocarcinoma.

Super-enhancers (SEs) comprise large clusters of enhancers that highly enhance gene expression. Long non-coding RNAs (lncRNAs) tend to be dysregulated in cases of stomach adenocarcinoma (STAD) and are vital for balancing tumor immunity. However, whether SE-associated lncRNAs play a role in the immune infiltration of STAD remains unknown. In the present study, we identified SE-associated lncRNAs in the H3K27ac ChIP-seq datasets from 11 tumor tissues and two cell lines. We found that the significantly dysregulated SE-associated lncRNAs were strongly correlated with immune cell infiltration through the application of six algorithms (ImmuncellAI, CIBERSORT, EPIC, quantiSeq, TIMER, and xCELL), as well as immunomodulators and chemokines. We found that the expression of SE-associated lncRNA TM4SF1-AS1 was negatively correlated with the proportion of CD8+ T cells present in STAD. TM4SF1-AS1 suppresses T cell-mediated immune killing function and predicts immune response to anti-PD1 therapy. ChIP-seq, Hi-C and luciferase assay results verified that TM4SF1-AS1 was regulated by its super-enhancer. RNA-seq data showed that TM4SF1-AS1 is involved in immune and cancer-related processes or pathways. In conclusion, SE-associated lncRNAs are involved in the tumor immune microenvironment and act as indicators of clinical outcomes in STAD. This study highlights the importance of SE-associated lncRNAs in the immune regulation of STAD.

Circular RNA circHERC4 as a novel oncogenic driver to promote tumor metastasis via the miR-556-5p/CTBP2/E-cadherin axis in colorectal cancer.

BACKGROUND: The main cause of death in colorectal cancer patients is metastasis. Accumulating evidences suggest that circRNA plays pivotal roles in cancer initiation and development. However, the underlying molecular mechanisms of circRNAs that orchestrate cancer metastasis remain vague and need further clarification. METHODS: Two paired CRC and adjacent normal tissues were used to screen the upregulated circRNAs by circRNA-seq; then, cell invasion assay was applied to confirm the functional invasion-related circRNAs. According to the above methods, circHERC4 (hsa_circ_0007113) was selected for further research. Next, we investigated the clinical significance of circHERC4 in a large cohort of patients with CRC. The oncogenic activity of circHERC4 was investigated in both CRC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying circHERC4 as a malignant driver. RESULTS: We demonstrated that circHERC4 was aberrantly elevated in CRC tissues (P < 0.001), and was positively associated with lymph node metastasis and advanced tumor grade (P < 0.01). Notably, the expression of circHERC4 was associated with worse survival in patients with CRC. Silencing of circHERC4 significantly inhibited the proliferation and migration of two highly aggressive CRC cell lines and reduced liver and lung metastasis in vivo. Mechanistically, we revealed that circHERC4 inactivated the tumor suppressor, miR-556-5p, leading to the activation of CTBP2/E-cadherin pathway which promotes tumor metastasis in CRC. CONCLUSIONS: CircHERC4 exerts critical roles in promoting tumor aggressiveness through miR-556-5p/CTBP2/E-cadherin pathway and is a prognostic biomarker of the disease, suggesting that circHERC4 may serve as an exploitable therapeutic target for patients with CRC.

The mechanisms of colorectal cancer cell mesenchymal-epithelial transition induced by hepatocyte exosome-derived miR-203a-3p.

BACKGROUND: Liver metastasis is the most common cause of death in patients with colorectal cancer (CRC). Phosphatase of regenerating liver-3 induces CRC metastasis by epithelial-to-mesenchymal transition, which promotes CRC cell liver metastasis. Mesenchymal-to-epithelial transition (MET), the opposite of epithelial-to-mesenchymal transition, has been proposed as a mechanism for the establishment of metastatic neoplasms. However, the molecular mechanism of MET remains unclear. METHODS: Using Immunohistochemistry, western blotting, invasion assays, real-time quantitative PCR, chromatin immunoprecipitation, luciferase reporter assays, human miRNA arrays, and xenograft mouse model, we determined the role of hepatocyte exosome-derived miR-203a-3p in CRC MET. RESULTS: In our study, we found that miR-203a-3p derived from hepatocyte exosomes increased colorectal cancer cells E-cadherin expression, inhibited Src expression, and reduced activity. In this way miR-203a-3p induced the decreased invasion rate of CRC cells. COCLUSION: MiR-203a-3p derived from hepatocyte exosomes plays an important role of CRC cells to colonize in liver.

Identification of a 3-gene signature for predicting the prognosis of stage II colon cancer based on microsatellite status.

BACKGROUND: Though colon cancer (CC) is one of the most malignant tumors across the world, CC patients with microsatellite instability-high (MSI-H) in stage II seem to have a better prognosis. However, the molecular mechanisms underlying the phenomena haven't been elucidated yet. METHODS: This study enrolled 322 CCs with known microsatellite status from GSE143985, GSE39582 and GSE92921 in the Gene Expression Omnibus (GEO) database. Robust rank aggregation (RRA) analysis, univariate Cox regression analysis and multivariate Cox stepwise regression analysis were performed to identify genes and construct risk score signature. Kaplan-Meier and receiver operating characteristic (ROC) curves analyses were used to evaluate the prognostic value of the signature. The potential mechanisms underlying this signature were assessed in the Metascape database, gene set enrichment analysis (GSEA) and immune infiltration analysis. RESULTS: RRA analysis identified 40 differently expressed genes (DEGs). A 3-gene risk score signature (MKQ signature) associated with disease-free survival (DFS) was generated. DFS was significantly longer in CC patients with lower than higher scores (P=0.0046). The areas under curves (AUCs) of the time-dependent ROC curves of MKQ signature at 1-, 3- and 5-year DFS were 1, 0.963 and 0.961 respectively. Recurrence-free survival (RFS) was significantly longer in patients in GSE39582 with lower than higher risk scores (P=0.032). The AUCs for 1-, 3- and 5-year RFS in GSE39582 were 0.63, 0.618 and 0.583, respectively, validating the value of the MKQ signature. Functional annotation and GSEA revealed that the MKQ signature was associated with multiple immune-related pathways. Immune cell infiltration was found to differ in patients differing in the MKQ signature. CONCLUSIONS: Gene expression and microsatellite status identified a 3-gene signature (MKQ signature) that could facilitate risk-stratified management in patients with stage II CC. Dysregulation of MSMB, KRT23, and QPRT can serve as prognostic markers in stage II CC.

Mesenchymal and Phosphatase of Regenerating Liver-3 Status in Circulating Tumor Cells May Serve as a Crucial Prognostic Marker for Assessing Relapse or Metastasis in Postoperative Patients With Colorectal Cancer.

INTRODUCTION: Circulating tumor cells (CTCs) and phosphatase of regenerating liver-3 (PRL-3) have been considered to be significant prognostic indicators in metastatic colorectal cancer (CRC). This study discusses the prognostic significance of mesenchymal CTCs with PRL-3 (M+ PRL-3+ CTCs) in postoperative patients with CRC. METHODS: We detected CTC subtypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) and PRL-3 in CTCs from the peripheral blood samples of 156 patients. Receiver operating characteristic curve analysis, Kaplan-Meier analysis, and Cox proportional hazards regression analysis were performed to identify the prognostic value of mesenchymal CTCs with PRL-3+. Immunohistochemistry was used to detect the expression of PRL-3 in tumor tissues from some of the patients to explore the connection between CTCs and tissues. RESULTS: All CTCs were positive in all samples, both mesenchymal CTCs and PRL-3-positive cells. The count of mesenchymal and PRL-3+ CTCs was significantly associated with recurrence, and the optimal cutoff value was 2 (area under the curve = 0.690, P < 0.001). In addition, these patients had a significantly shorter median disease-free survival than those who did not fulfill the criteria (8.5 vs 24 months, P < 0.001) according to multivariable and multinomial logistic regression. Immunohistochemistry was applied to explore the associations between PRL-3 expression and significant prognostic risk factors, including recurrence (R = 0.566; P < 0.001), and M+ PRL-3+ status in CTCs (R = 0.452; P = 0.001). DISCUSSION: The status of M+ PRL-3+ in CTCs may serve as a crucial prognostic marker for assessing clinical outcomes in CRC.

Interaction with tumor­associated macrophages promotes PRL­3­induced invasion of colorectal cancer cells via MAPK pathway­induced EMT and NF­κB signaling­induced angiogenesis.

Protein phosphatase of regenerating liver­3 (PRL­3) is considered to be metastasis­associated phosphatase and is associated with a poor prognosis. Additionally, tumor­associated macrophages (TAMs) participate in cancer progression. A previous study demonstrated that PRL­3 promotes invasion and metastasis by inducing TAM infiltration. However, the underlying mechanism has not been elucidated. In the present study, western blot analysis, polymerase chain reaction, immunohistochemistry, ELISA, mouse model experiments and functional experiments were performed to confirm that the interaction between TAMs and colorectal cancer (CRC) cells induced epithelial­mesenchymal transition (EMT)­associated features in CRC cells by activating mitogen­activated protein kinase (MAPK) pathways in TAMs and upregulating the expression of interleukin (IL)­6 and IL­8. The neutralization of IL­6 and IL­8 reduced EMT and the invasive and migratory abilities of CRC cells. Therefore, IL­6 and IL­8 were considered important factors in EMT, and in CRC invasion and metastasis. In addition, increased angiogenesis was observed after TAMs were co­cultured with CRC cells that overexpress PRL­3. Vascular endothelial growth factor­A was significantly upregulated, and the nuclear factor­κB (NF­κB) signaling pathway was activated in CRC cells after co­culture. Moreover, nude mice injected with CRC cells with high PRL­3 expression levels tended to generate larger xenografts. Immunohistochemistry results from xenografted CRC cells overexpressing PRL­3 also confirmed the activation of MAPK pathways in xenografts. Overall, the findings indicate that PRL­3 promotes CRC cell invasion and metastasis by activating MAPK pathways in TAMs to initiate the EMT, and PRL­3 promotes angiogenesis by activating the NF­κB pathway in CRC cells.

CCL26 Participates in the PRL-3-Induced Promotion of Colorectal Cancer Invasion by Stimulating Tumor-Associated Macrophage Infiltration.

Both phosphatase of regenerating liver-3 (PRL-3) and tumor-associated macrophages (TAM) influence cancer progression. Whether PRL-3 plays a critical role in colorectal cancer invasion and metastasis by inducing TAM infiltration remains unclear. In the current study, we investigated the effects of chemokine ligand 26 (CCL26) on TAM infiltration and colorectal cancer invasion and the underlying mechanism in colorectal cancer cells by overexpressing or silencing PRL-3. We found that PRL-3 upregulated CCL26 expression correlatively and participated in cell migration, according to the results of gene ontology analysis. In addition, IHC analysis results indicated that the PRL-3 and CCL26 levels were positively correlated and elevated in stage III and IV colorectal cancer tissues and were associated with a worse prognosis in colorectal cancer patients. Furthermore, we demonstrated that CCL26 induced TAM infiltration by CCL26 binding to the CCR3 receptor. When LoVo-P and HT29-C cells were cocultured with TAMs, CCL26 binding to the CCR3 receptor enhanced the invasiveness of LoVo-P and HT29-C cells by mobilizing intracellular Ca2+of TAMs to increase the expression of IL6 and IL8. In addition, IHC results indicated that protein levels of CCR3 and TAMs counts were higher in stage III and IV colorectal cancer tissues and correlated with CCL26. Moreover, similar results were observed in vivo using mice injected with LoVo-P and HT29-C cells. These data indicate that PRL-3 may represent a potential prognostic marker that promotes colorectal cancer invasion and metastasis by upregulating CCL26 to induce TAM infiltration. Mol Cancer Ther; 17(1); 276-89. ©2017 AACR.

PRL-3 improves colorectal cancer cell proliferation and invasion through IL-8 mediated glycolysis metabolism.

Phosphatase of regenerating liver-3 (PRL-3) has been found to be overexpressed in liver metastases of colorectal cancer and rarely expressed in primary tumors, which plays an important role in the metastasis of colorectal cancer cells. Metabolism reprogramming has been found to be a hallmark of cancer cells, and aerobic glycolysis is a metabolic adaption for cancer cells and promotes cell proliferation. However, the association between PRL-3 and glycolysis in colorectal cancer cells is not well understood. In the present study, we explored the association between PRL-3 and glycolysis. We found that PRL-3 improved colorectal cancer cell glucose assumption, lactate production and reduced intracellular ROS levels. Besides, PRL-3 improved the expression of Glut1, HK2, PKM2 and LDHA, which are important glycolysis related molecules and enzymes. Moreover, we explored IL-8 mediated enhancement of glycolysis by PRL-3. More importantly, the proliferation and invasion of colorectal cancer cells were enhanced significantly by PRL-3 through improving glycolysis. Taken together, these results implicated the important role of PRL-3 in glycolysis metabolism through improving IL-8 secretion in colorectal cancer cells, and PRL-3 mediated glycolysis contributed to the promotion of cancer metastasis.

Methyl Sartortuoate Inhibits Colon Cancer Cell Growth by Inducing Apoptosis and G2/M-Phase Arrest.

The potential anti-neoplastic activity of terpenoids is of continued interest. In this study, we investigate whether methyl sartortuoate, a terpenoid isolated from soft coral, induced cell cycle arrest and apoptosis in a human colon cancer cell line. Culture studies found that methyl sartortuoate inhibited colon cancer cell (LoVo and RKO) growth and caused apoptotic death in a concentration- and time-dependent manner, by activation of caspase-8, caspase-9, caspase-3, p53 and Bax, and inactivation of B-cell lymphoma 2 (Bcl-2) apoptosis regulating proteins. Methyl sartortuoate treatment led to reduced expression of cdc2 and up-regulated p21 and p53, suggesting that Methyl sartortuoate induced G2-M arrest through modulation of p53/p21/cdc2 pathways. Methyl sartortuoate also up-regulated phospho-JNK and phospho-p38 expression levels. This resulted in cell cycle arrest at the G2-M phase and apoptosis in LoVo and RKO cells. Treatment with the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 prevented methyl sartortuoate-induced apoptosis in LoVo cells. Moreover, methyl sartortuoate also prevented neoplasm growth in NOD-SCID nude mice inoculated with LoVo cells. Taken together, these findings suggest that methyl sartortuoate is capable of leading to activation of caspase-8, -9, -3, increasing p53 and Bax/Bcl-2 ratio apoptosis through MAPK-dependent apoptosis and results in G2-M phase arrest in LoVo and RKO cells. Thus, methyl sartortuoate may be a promising anticancer candidate.

Tumor-associated macrophage-derived IL-6 and IL-8 enhance invasive activity of LoVo cells induced by PRL-3 in a KCNN4 channel-dependent manner.

BACKGROUND: Tumor-associated macrophages (TAMs) are known to promote cancer progression and metastasis through the release of a variety of cytokines. Phosphatase of regenerating liver (PRL-3) has been considered as a marker of colorectal cancer (CRC) liver metastasis. Our previous research suggests that PRL-3 can enhance the metastasis of CRC through the up-regulation of intermediate-conductance Ca2+-activated K+ (KCNN4) channel, which is dependent on the autocrine secretion of tumor necrosis factor-alpha (TNF-α). However, whether TAMs participate in the progression and metastasis of CRC induced by PRL-3 remains unknown. METHODS: We used flow cytometry, coculture, western blotting, invasion assays, real-time quantitative PCR, chromatin immunoprecipitation, luciferase reporter assays, and immunofluorescence staining to determine the effect of TAMs on the ability of PRL-3 to promote invasiveness of CRC cells. RESULTS: In this study, we found that TAMs facilitated the metastasis of CRC induced by PRL-3. When TAMs were cocultured with CRC cells, the expression of KCNN4 was increased in TAMs and the invasion of CRC cells was enhanced. Furthermore, cytokines that were secreted by TAMs, such as IL-6 and IL-8, were also significantly increased. This response was attenuated by treating TAMs with the KCNN4 channel-specific inhibitor, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), which suggested that KCNN4 channels may be involved in inducing the secretion of IL-6 and IL-8 by TAMs and improving CRC cell invasiveness. Moreover, the expression of KCNN4 channels in TAMs was regulated through the NF-κB signal pathway, which is activated by TNF-α from CRC cells. Immunofluorescence analysis of colorectal specimens indicated that IL-6 and IL-8 double positive cells in the stroma showed positive staining for the TAM marker CD68, suggesting that TAMs produce IL-6 and IL-8. Increased numbers of these cells correlated with higher clinical stage. CONCLUSIONS: Our findings suggested that TAMs participate in the metastasis of CRC induced by PRL-3 through the TNF-α mediated secretion of IL-6 and IL-8 in a paracrine manner.