Background: Chemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells’ fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu’s cytotoxicity in vitro and in vivo. Methods: Cell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice. Results: Curcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells. Conclusion: Pre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.

Metastasis is the main cause of death in individuals with cancer. Immune checkpoint blockade (ICB) can potentially reverse CD8⁺ cytotoxic T lymphocytes (CTLs) dysfunction, leading to significant remission in multiple cancers. However, the mechanism underlying the development of CTL exhaustion during metastatic progression remains unclear. Here, we established an experimental pulmonary metastasis model with melanoma cells and discovered a critical role for melanoma‐released exosomes in metastasis. Using genetic knockdown of nSMase2 and Rab27a, 2 key enzymes for exosome secretion, we showed that high levels of effector‐like tumor‐specific CD8⁺ T cells with transitory exhaustion, instead of terminal exhaustion, were observed in mice without exosomes; these cells showed limited inhibitory receptors and strong proliferation and cytotoxicity. Mechanistically, the immunosuppression of exosomes depends on exogenous PD‐L1, which can be largely rescued by pretreatment with antibody blockade. Notably, we also found that exosomal PD‐L1 acts as a promising predictive biomarker for ICB therapies during metastasis. Together, our findings suggest that exosomal PD‐L1 may be a potential immunotherapy target, suggesting a new curative therapy for tumor metastasis.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most frequent malignant tumors originated from digestive system around the world and the treatment was limited by the unclear mechanism. DNA polymerase epsilon 2, accessory subunit (POLE2) is involved in DNA replication, repair, and cell cycle control, whose association with ESCC is still not clear. Methods: In this study, the expression level of POLE2 in ESCC tissues was detected by IHC. The POLE2 knockdown cell line was constructed, identified by qPCR and western blot and used for detecting cellular functions and constructing xenotransplantation mice model. MTT Assay, colony formation assay, flow cytometry, wound-healing assay and Transwell assay were used to detected cell proliferation, apoptosis and migration. Results: We firstly identified that the expression of POLE2 was overexpressed in ESCC. Moreover, the high expression of POLE2 can predict the tumor deterioration and poor prognosis of ESCC patients. Additionally, downregulation of POLE2 was involved in ESCC progression by promoting proliferation, migration, and inhibiting apoptosis in vitro. In vivo studies proved that POLE2 was positively correlated with ESCC tumor formation, which was consistent with the results in vitro. We also illuminated that POLE2 knockdown upregulated pro-apoptotic proteins (Bax, Caspase3, CD40L, FasL, IGFBP-5 and P21) and downregulated anti-apoptotic proteins (CLAP-2, IGF-I and sTNF-R2). In addition, POLE2 was involved in ESCC via targeting PI3K/Akt, Cyclin D1 signaling pathway. Conclusions: Therefore, POLE2 was proved to be involved in the development of ESCC, which may be a potential therapeutic target and bring new breakthroughs in the treatment of ESCC.

Background and objectives: The optimal therapeutic method for patients with superficial esophageal squamous cell carcinoma (sESCC) remains to be established. Methods: Clinical data of all the patients from 2002 to 2014 who underwent curative esophagectomy and three-field lymphadenectomy for thoracic sESCC were collected based on a prospectively-maintained database. The pattern of lymph node metastasis was analyzed based on the depth of tumor invasion, tumor location and surgical fields. Results: The involved lymph node region was associated to the tumor location, however, upper mediastinal and perigastric region was the most vulnerable region. The incidence of lymph node metastasis increased with the depth of tumor invasion. No lymph node involvement was found in tumors invading proper mucosa (M2), while the pattern of positive lymph nodes in tumors invading the deepest 1/3 submucosa was similar to that in advanced ESCC. Lymphatic invasion, tumor location and upper mediastinal lymph node involvement were independent predictors for cervical lymph node metastasis. For patients without lymphatic invasion, the positive predictive value of upper mediastinal lymph node metastasis for positive cervical lymph node was low (0 similar to 25%), while the negative predictive value was extremely high, wherever the tumor located (93.8 similar to 100%). Conclusions: Tumors invading till proper mucosa was the best indication for endoscopic mucosa resection. Mediastinal-abdominal lymphadenectomy was essential for sESCC invading beyond proper mucosa. For those without lymphatic invasion, cervical lymphadenectomy might be avoided in case of negative upper mediastinal lymph node.