BackgroundLiver cancer stem cells (LCSCs) contribute to hepatocellular carcinoma (HCC) development, metastasis, and drug resistance. MSI2 and Notch1 signaling are involved in the maintenance of CSCs. However, it is unknown whether MSI2 and Notch1 are involved in the maintenance of CD44v6+ LCSCs. Therefore, we investigated the clinical significance and function of MSI2 and its relationship with Notch1 signaling in the maintenance of stemness properties in CD44v6+ LCSCs.MethodsThe expression of MSI2 and CD44v6 were detected by fresh specimens and a HCC tissue microarray. The tissue microarray containing 82 HCC samples was used to analyze the correlation between CD44v6 and MSI2. CD44v6+/− cells were isolated using microbeads sorting. We explored the roles of MSI2 and Notch1 signaling in CD44v6+ LCSCs by sphere formation assay, transwell assay, clone formation assay in vitro, and xenograft tumor models in vivo. A Notch RT2 PCR Array, Co-immunoprecipitation, and RNA-immunoprecipitation were used to further investigate the molecular mechanism of MSI2 in activating Notch1 signaling.ResultsHere, we found MSI2 expression was positively correlated with high CD44v6 expression in HCC tissues, and further correlated with tumor differentiation. CD44v6+ cells isolated from HCC cell lines exhibited increased self-renewal, proliferation, migration and invasion, resistance to Sorafenib and tumorigenic capacity. Both MSI2 and Notch1 signaling were elevated in sorted CD44v6+ cells than CD44v6- cells and played essential roles in the maintenance of stemness of CD44v6+ LCSCs. Mechanically, MSI2 directly bound to Lunatic fringe (LFNG) mRNA and protein, resulting in Notch1 activation.ConclusionsOur results demonstrated that MSI2 maintained the stemness of CD44v6+ LCSCs by activating Notch1 signaling through the interaction with LFNG, which could be a potential molecular target for stem cell-targeted therapy for liver cancer.
Purpose
Both cancer-associated fibroblasts (CAFs) and liver cancer stem cells (LCSCs) play an important part in the tumorigenesis, development and metastasis of hepatocellular carcinoma (HCC). Moreover, the stem-like properties in HCC cells could be promoted by CAFs. However, the mechanism remains largely unknown.
Patients and methods
We used conditioned medium (CM) of CAFs to culture Huh7 cells. Stemness of the cells was then examined mainly by sphere formation assay while stemness-associated genes including Nanog, Sox2 and Oct4 were measured by Western blotting. Immunofluorescence staining, Transmission Electron Microscope as well as Western blotting were performed to detect the level of autophagy in Huh7 cells.
Results
Increased level of stemness and autophagy was observed in HCC cells cultured in CAFs-CM compared to the control group. Activation of CAFs-induced autophagic flux could be inhibited by Chloroquine (CQ), which can accumulate LC3-II protein and increase punctate distribution of LC3 localization. Treatment of HCC cells with CQ effectively reversed the CAF-induced stemness, invasion, and metastasis ability in these cells. In vivo, Huh7 cells inoculated together with CAFs developed significantly larger tumors than Huh7 cells injected alone. Moreover, blockage of autophagy in Huh7 cells by CQ greatly reduced the growth of xenografted tumors of Huh7 cells combined with CAFs.
Conclusion
These results reveal that CAFs are capable of promoting stemness and metastasis of HCC cells and blocking autophagy could markedly attenuate the stemness enhanced by CAFs, suggesting that targeting autophagy in HCC could be an effective strategy in HCC treatment.
INTRODUCTION: The ability of carbohydrate antigen 19-9 (CA19-9) to differentiate pancreatic cancer from other benign pancreatic lesions is unsatisfactory. This study explored the diagnostic value of KRAS gene mutations and plasma circulating tumor DNA (ctDNA) in patients with pancreatic cancer.
METHODS:The prospective cohort study comprised 149 consecutive patients with solid pancreatic lesions who underwent endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). KRAS subtype mutations were analyzed by digital droplet PCR (ddPCR) in EUS-FNA histopathology tissue samples, and blood samples were sent for plasma ctDNA analysis. The final diagnosis was based on surgical resection pathology or follow-up for at least 2 years.
RESULTS:Adding KRAS mutation ddPCR increased the sensitivity and accuracy of EUS-FNA from 71.4% to 91.6% (P < 0.001) and 75.8% to 88.6% (P < 0.001), respectively. By comparison, the sensitivities of circulating biomarkers ctDNA and CA19-9 were only 35.2% and 71.2%. The area under the curve of the receiver operating characteristic curve (AUC) of EUS-FNA and KRAS ddPCR combination was >0.90 for distinguishing pancreatic cancer from benign lesions, whereas the AUC of EUS-FNA and CA19-9 combination was 0.83. The median survival time was significantly shorter in patients with G12D KRAS mutations than that in patients with other mutations (180 vs 240 days, P < 0.001).DISCUSSION:FNA tissue sample KRAS mutation analysis in tissues significantly improves the diagnostic accuracy of cyto/histopathological evaluation in EUS-FNA samples. The combination of EUS-FNA and tissue sample KRAS ddPCR provided a more accurate method for pancreatic cancer diagnosis, superior to the combination of EUS-FNA and CA19-9/ctDNA. G12D KRAS mutations in pancreatic cancer were independently associated with poor overall survival.
Purpose:
Because many hepatocellular carcinoma (HCC) cases develop from fibrotic or cirrhotic livers, fibroblasts are abundant in the microenvironment of HCC. Although the contribution of cancer-associated fibroblasts (CAFs) to the progression of HCC is well established, the role of fibroblasts has not been comprehensively revealed.
Patients and methods:
The RayBio Human Cytokine Antibody Array was used to elucidate the role of peri-tumor fibroblasts (PTFs) in promoting malignant properties of HCC. IL-6 and STAT3 signaling were inhibited in both HCC cell lines and non-tumor L-02 liver cells to further determine its role in the progression of HCC. Moreover, the expression of IL-6 and pTyr705 STAT3 was detected in HCC samples and peri-tumor liver tissues by immunohistochemical staining.
Results:
PTFs not only promoted the proliferation, invasion, and metastasis of liver cancer cells, but also stimulated the permanent malignant transformation of human non-tumor L-02 liver cells, resulting in hepatocarcinogenesis in vivo. The RayBio Human Cytokine Antibody Array indicated that PTFs secreted a higher level of soluble IL-6 than CAFs. IL-6 derived from PTFs greatly activated STAT3 Tyr705 phosphorylation in both non-tumor L-02 cells and HCC cells. IL-6-neutralizing antibody and STAT3 Tyr705 phosphorylation inhibitor, cryptotanshinone, largely abolished the positive effects of PTFs on HCC carcinogenesis and progression. Moreover, high expression of pTyr705 STAT3 in peri-tumor tissues was significantly correlated with tumor recurrence rate after three years in a postsurgical follow-up with patients with HCC.
Conclusion:
These results indicated that PTFs induce carcinogenesis and development of HCC via IL-6 and STAT3 signaling.
Cancer‐associated fibroblasts (CAFs) are heterogeneous stromal cells present in the tumor microenvironment (TME), which play a critical role in gastric cancer (GC) progression. Here, we examined a subset of CAFs with high podoplanin (PDPN) expression, which is correlated with tumor metastasis and poor survival in GC patients. Animal models of gastric cancer liver metastasis monitored by micro‐PET/CT confirmed that periostin (POSTN) derived from PDPN(+) CAFs regulated CAFs’ pro‐migratory ability. Mechanistically, PDPN(+) CAFs secreted POSTN to modulate cancer stem cells (CSCs) through FAK/AKT phosphorylation. Furthermore, POSTN could also activate FAK/YAP signaling in GC cells to produce increased amounts of IL‐6, which in turn induced phosphorylation of PI3K/AKT in PDPN(+) CAFs. Prolonged PI3K/AKT pathway activation in PDPN(+) CAFs maintains the production of POSTN and the effect on CSC enrichment and GC cell migration. In conclusion, our study demonstrated a positive feedback loop between PDPN(+) CAFs and CSCs during GC progression and suggested a selective target for GC treatment.
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