Objectives: Gastric cancer (GC) is one of the most malignant tumors. Mounting studies highlighted gastric cancer stem cells (GCSCs) were responsible for the failure of treatment due to recurrence and drug resistance of advanced GC. However, targeted therapy against GCSC for improving GC prognosis suffered from lack of suitable models and molecular targets in terms of personalized medicine. To address this issue, two patient-derived GC cell lines SD209 and SD292 with cancer stem cells (CSCs) such as phenotype were isolated for establishing targeted therapy aiming at critical metastatic signaling in GC. Materials and Methods: The primary patient-derived GCSCs were established from parts of GC tissues for characterization of stem cells (SCs) phenotype at both cellular and molecular levels. Western blot and Immunohistochemistry (IHC) were performed for identifying the deregulated signaling in GC tissue. Immunofluorescence was used for analyzing proliferating and SC markers in GCSC attached on fibroblast. Acridine orange and propidium iodide analyses were performed for the survival of GCSC in suspensions. Results: In the culture environments of both SD209 and SD292, a lot of mesenchymal fibroblasts spread and crowd together on which a lot of cell clumps, suspected as GCSC, were firmly attached. In the IHC analysis, the GCSC stemness genes CD44 and Ep-CAM increased in tumor tissues of SD209, whereas Nanog-1 and octamer-binding transcription factor 3 (OCT-3) increased in that of SD292. By immunofluorescent analysis of a proliferation marker Ki67, the growth of SD209 and SD292 on mesenchymal fibroblasts was found to be reduced by dasatinib, the inhibitor of the Src kinase whose activity was upregulated in tumor tissues of both GCs. Dasatinib also suppressed the expression of Nanog-1 and OCT-3 in SD292 attached on mesenchymal fibroblasts. Conclusion: This study may provide a base for targeted therapy against GCSCs/GCs progression in future preclinical/clinical settings.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer with poor prognosis. The deregulation of a lot of oncogenic signaling molecules, such as receptor tyrosine kinases (RTKs), has been found to be associated with CCA progression. However, RTKs-based target therapy showed limited improvement suggesting a need to search for alternative targets for preventing CCA progression. To address this issue, we screened the oncogenic signal molecules upregulated in surgical tissues of CCAs. Interestingly, over-expression of hydrogen peroxide inducible clone-5 (Hic-5) coupled with over-activation of Src, AKT, JNK were observed in 50% of the cholangiocarcinoma with metastatic potential. To investigate whether these molecules may work together to trigger metastatic signaling, their up-and-down relationship was examined in a well-established cholangiocarcinoma cell line, HuCCT1. Src inhibitors PP1 (IC50, 13.4 μM) and dasatinib (IC50, 0.1 μM) significantly decreased both phosphorylated AKT (phosphor-AKT Thr450) and Hic-5 in HuCCT1. In addition, a knockdown of Hic-5 effectively suppressed activation of Src, JNK, and AKT. These implicated a positive cross-talk occurred between Hic-5 and Src for triggering AKT activation. Further, depletion of Hic-5 and inhibition of Src suppressed HuccT1 cell migration in a dose-dependent manner. Remarkably, prior transfection of Hic-5 siRNA for 24 h followed by treatment with PP1 or dasatinib for 24 h resulted in additive suppression of HuCCT1 migration. This suggested that a promising combinatory efficacy can be achieved by depletion of Hic-5 coupled with inhibition of Src. In the future, target therapy against CCA progression by co-targeting Hic-5 and Src may be successfully developed in vivo.

Cholangiocarcinoma (CCA) is a malignant neoplasm of the bile ducts, being the second most common type of cancer in the liver, and most patients are diagnosed at a late stage with poor prognosis. Targeted therapy aiming at receptors tyrosine kinases (RTKs) such as c-Met or EGFR have been developed but with unsatisfactory outcomes. In our recent report, we found several oncogenic molecules downstream of RTKs, including hydrogen peroxide clone-5 (Hic-5), Src, AKT and JNK, were elevated in tissues of a significant portion of metastatic CCAs. By inhibitor studies and a knockdown approach, these molecules were found to be within the same signal cascade responsible for the migration of HuCCT1 cells, a conventionally used CCA cell line. Herein, we also found Src inhibitor dasatinib and Hic-5 siRNA corporately suppressed HuCCT1 cell invasion. Moreover, dasatinib inhibited the progression of the HuCCT1 tumor on SCID mice skin coupled with decreasing the expression of Hic-5 and EGFR and the activities of Src, AKT and JNK. In addition, we found a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and several cytoskeletal molecules such as tubulin and cofilin were dramatically decreased after a long-term treatment of the HuCCT1 tumor with a high dose of dasatinib. Specifically, GAPDH was shown to be a downstream effector of the Hic-5/Src/AKT cascade involved in HuCCT1 cell migration. On the other hand, TFK1, another CCA cell line without Hic-5 expression, exhibited very low motility, whereas an ectopic Hic-5 expression enhanced the activation of Src and AKT and marginally increased TFK1 migration. In the future, it is tempting to investigate whether cotargeting Src, Hic-5 and/or GAPDH is efficient for preventing CCA progression in future clinical trials.

Background/Objectives: Gastric cancer (GC) is one of the devastating cancers with frequent metastasis. The non-receptor tyrosine kinase Src may trigger oncogenic signaling, which is a critical target for preventing GC progression. Another potential GC target is the programmed death-1 ligand (PD-L1), which is one of the immune checkpoint molecule responsible for immunosuppression. Moreover, PD-L1 may directly influence cancer progression by regulating tumor-intrinsic events. Interestingly, Src has intimate relationship with tumor-intrinsic PD-L1 in terms of signal transduction. In this study, whether tumor-intrinsic PD-L1 interacts with Src to regulate GC progression was investigated. Methods: Western blot was used for analyzing expression of PD-L1 and activity of Src. Trans-well migration and invasion assays were for investigating whether PD-L1 and Src are required for GC progression in vitro. Lipid nanoparticles SNALPs carrying PD-L1 siRNA was for in vivo knockdown of PD-L1. Results: Experiments using Src inhibitors coupled with PD-L1 knockdown indicated PD-L1 and Src be both upstream and downstream of each other in conventional AGS and patient-derived SD110 GC cells. By scavenging reactive oxygen species (ROS) using an antioxidant dithiothreitol (DTT), both Src activity and PD-L1 expression were found to be ROS signaling-dependent. The ROS-mediated PD-L1/Src signaling was required for migration and invasion of AGS and SD110. Co-targeting Src and PD-L1 suppressed migration of AGS and SD110 more efficiently than single targeting. In the SCID mice, in vivo knockdown of PD-L1 reduced Src activity and metastasis of SD110 tumor from stomach to liver. Conclusions: co-targeting Src and tumor intrinsic PD-L1 might be a promising therapeutic approach in GC therapy.