Tumor-suppressive role of the musculoaponeurotic fibrosarcoma gene in colorectal cancer.

Somatic cell reprogramming using the microRNAs miR-200c, miR-302s, and miR-369s leads to increased expression of cyclin-dependent kinase inhibitors in human colorectal cancer (CRC) cells and suppressed tumor growth. Here, we investigated whether these microRNAs inhibit colorectal tumorigenesis in CPC;Apc mice, which are prone to colon and rectal polyps. Repeated administration of microRNAs inhibited polyp formation. Microarray analysis indicated that c-MAF, which reportedly shows oncogene-like behavior in multiple myeloma and T cell lymphoma, decreased in tumor samples but increased in microRNA-treated normal mucosa. Immunohistochemistry identified downregulation of c-MAF as an early tumorigenesis event in CRC, with low c-MAF expression associated with poor prognosis. Of note, c-MAF expression and p53 protein levels were inversely correlated in CRC samples. c-MAF knockout led to enhanced tumor formation in azoxymethane/dextran sodium sulfate-treated mice, with activation of cancer-promoting genes. c-MAF may play a tumor-suppressive role in CRC development.

Hyper-Progressive Disease: The Potential Role and Consequences of T-Regulatory Cells Foiling Anti-PD-1 Cancer Immunotherapy.

Antibody-mediated disruption of the programmed cell death protein 1 (PD-1) pathway has brought much success to the fight against cancer. Nevertheless, a significant proportion of patients respond poorly to anti-PD-1 treatment. Cases of accelerated and more aggressive forms of cancer following therapy have also been reported. Termed hyper-progressive disease (HPD), this phenomenon often results in fatality, thus requires urgent attention. Among possible causes of HPD, regulatory T-cells (Tregs) are of suspect due to their high expression of PD-1, which modulates Treg activity. Tregs are a subset of CD4+ T-cells that play a non-redundant role in the prevention of autoimmunity and is functionally dependent on the X chromosome-linked transcription factor FoxP3. In cancer, CD4+FoxP3+ Tregs migrate to tumors to suppress anti-tumor immune responses, allowing cancer cells to persist. Hence, Treg accumulation in tumors is associated with poor prognosis. In mice, the anti-tumor efficacy of anti-PD-1 can be enhanced by depleting Tregs. This suggests Tregs pose resistance to anti-PD-1 therapy. In this article, we review the relevant Treg functions that suppress tumor immunity and the potential effects anti-PD-1 could have on Tregs which are counter-productive to the treatment of cancer, occasionally causing HPD.

miR-4711-5p regulates cancer stemness and cell cycle progression via KLF5, MDM2 and TFDP1 in colon cancer cells.

BACKGROUND: It is important to establish cancer stem cell (CSC)-targeted therapies to eradicate cancer. As it is a CSC marker, we focused on Kruppel-like factor 5 (KLF5) in this study. METHODS: We searched for candidate microRNAs (miRNAs) that inhibited KLF5 expression by in silico analyses and screened them in colon cancer cell lines. RESULTS: We identified one promising miRNA, miR-4711-5p, that downregulated KLF5 expression by direct binding. This miRNA suppressed cell proliferation, migration and invasion ability, as well as stemness, including decreased stem cell marker expression, reactive oxygen species activity and sphere formation ability. MiR-4711-5p inhibited the growth of DLD-1 xenografts in nude mice with no adverse effects. We found that miR-4711-5p provoked G1 arrest, which could be attributed to direct binding of miR-4711-5p to TFDP1 (a heterodimeric partner of the E2F family). Our findings also suggested that direct binding of miR-4711-5p to MDM2 could upregulate wild-type p53, leading to strong induction of apoptosis. Finally, we found that miR-4711-5p had a potent tumour-suppressive effect compared with a putative anti-oncomiR, miR-34a, in tumour cell cultures derived from five patients with colorectal cancer. CONCLUSIONS: Our data suggest that miR-4711-5p could be a promising target for CSC therapy.

E-cadherin-Fc chimera protein matrix enhances cancer stem-like properties and induces mesenchymal features in colon cancer cells.

Cancer stem cells (CSC) are a subpopulation of tumor cells with properties of high tumorigenicity and drug resistance, which lead to recurrence and poor prognosis. Although a better understanding of CSC is essential for developing cancer therapies, scarcity of the CSC population has hindered such analyses. The aim of the present study was to elucidate whether the E-cadherin-Fc chimera protein (E-cad-Fc) enhances cancer stem-like properties because studies show that soluble E-cadherin stimulates human epithelial growth factor receptor (EGFR) and downstream signaling pathways that are reported to play a crucial role in CSC. For this purpose, we used ornithine decarboxylase (ODC)-degron-transduced (Degron(+)) KM12SM cells as a CSC model that retains relatively low CSC properties. Compared to cultures without E-cad-Fc treatment, we found that E-cad-Fc treatment further suppressed proteasome activity and largely enhanced cancer stem-like properties of ODC-degron-transduced KM12SM cells. These results include increased expression of stem cell markers Lgr5, Bmi-1, SOX9, CD44, and CD44v9, aldehyde dehydrogenase (ALDH), and enhancement of robust spheroid formation, and chemoresistance to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP). These effects could be attributed to activation of the EGFR pathway as identified by extensive phosphorylation of EGFR, ERK, PI3K, AKT, and mTOR. In SW480 cells, E-cad-Fc matrix induced some CSC markers such as CD44v9 and ALDH. We also found that E-cad-Fc matrix showed high efficiency of inducing mesenchymal changes in colon cancer cells. Our data suggest that the E-cad-Fc matrix may enhance CSC properties such as enhancement of chemoresistance and sphere formation.

Sox2 is associated with cancer stem-like properties in colorectal cancer.

Sox2 is known as the undifferentiated cell marker. Recent studies have shown that Sox2 may also be involved in the maintenance of cancer stem cells (CSCs) in skin and bladder cancers. In this study, we aimed to clarify the role of Sox2 in colorectal CSCs. Sox2 expression was measured in colon cancer cells and colorectal clinical samples by qRT-PCR and western blot analysis. To visualize the active Sox2 mRNA production, we generated a Sox2 promoter-dependent DsRed fluorescence emission system. Colon cancer cell lines and colorectal tumor tissues generally expressed the Sox2 protein. Knockdown of Sox2 by siRNA led to increased proliferative activity in Caco2 cells. Kaplan-Meier survival curves showed that the group with high Sox2 mRNA expression had a worse prognosis for relapse-free survival (RFS) than the low expression group (P = 0.045, median follow-up 60.0 months). Time-lapse image analysis revealed that most DsRed+ cells exhibited typical asymmetric cell division and had higher CSC marker expressions. The DsRed+ cells exhibited chemoresistance and they grew slower in vitro, yet they established rather larger tumors in vivo. Our data suggest that Sox2 may be a potential biomarker for colorectal CSCs.

Mesothelial cells facilitate cancer stem­like properties in spheroids of ovarian cancer cells.

Ovarian cancer is characterized by widespread peritoneal dissemination with ascites. Spheroids observed in the ascites of ovarian cancer patients are a mixture of cancer cells and mesothelial cells. In the present study, we evaluated whether mesothelial cells exfoliated from the peritoneum facilitate tumor spheroid formation and give rise to cancer stem­like properties in ovarian cancer cells. Spheroids from the CAOV3 and A2780 ovarian cancer cell lines grew much larger in co­culture with mesothelial cells than in monoculture under 3D conditions. The spheroids in co­culture displayed high Ki­67 expression in the peripheral zone and low expression in the central zone area. The expression of CD133 emerged in the inner portion of spheroids at later time­points (96 and 168 h), indicating that cancer cells expanded to the inner spheroid and acquired stem cell­properties. The mRNA levels of cancer stem cell markers Dclk­1, CD44 and Bmi­1 significantly increased in co­cultured CAOV3 and mesothelial cells compared to CAOV3 cells alone. Furthermore, the mesothelial cells promoted the tumorigenesis and growth of the CAOV3 cells in a mouse xenograft model compared to cancer cells alone. In conclusion, mesothelial cells promoted spheroid formation by ovarian cancer cells and facilitated cancer stem­like properties.

A miR-29b Byproduct Sequence Exhibits Potent Tumor-Suppressive Activities via Inhibition of NF-κB Signaling in KRAS-Mutant Colon Cancer Cells.

We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence "MIRTX." MIRTX directly targeted the 3'-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977-87. ©2018 AACR.

The glycogen synthase 2 gene (Gys2) displays parallel evolution between Old World and New World fruit bats.

Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism.