Background/Aims: Regorafenib is recommended by guidelines and trials as a sequential second-line therapy following progression on first-line sorafenib or lenvatinib in hepatocellular carcinoma (HCC). However, efficacy is limited, highlighting the urgent need to screen suitable patients and develop sensitization strategies. Methods: Acquired sorafenib- or lenvatinib-resistant (SR or LR) HCC cell lines and organoids were established. Genome-wide CRISPR library screen was performed in SR or LR cell strains to identify synthetic lethal targets of regorafenib. RNA-seq and FITC-regorafenib efflux assay were used to elucidate ERBB3-driven downstream signaling. Preclinical mouse models of cell line- and patient-derived xenografts and clinical cohorts of HCC patients were employed to validate the efficacy of ERBB3-guided patient stratification. Results: Screening with CRISPR library, we showed that inhibition of ERBB3 was synthetic lethal with regorafenib in SR or LR cell strains and organoids. Mechanistically, SR or LR triggered feedback activation of ERBB3 signaling and mediated regorafenib efflux via ERBB3-HIF1A-ABCB1 cascade pathway, limiting sensitivity to regorafenib. Moreover, ERBB3-low tumors following SR or LR exhibited significant sensitivity to regorafenib, suggesting its potential as a predictive biomarker to screen optimal candidates for sequential therapy. Seribantumab, an ERBB3-targeting monoclonal antibody, inhibited ERBB3-HIF1A-ABCB1 cascade, and its combination with regorafenib exerted marked synergistic anti-tumor effects on ERBB3-high tumors resistant to sorafenib or lenvatinib both in vitro and in vivo. Conclusions This study revealed that ERBB3 was a key resistance factor driving limited efficacy to sequential regorafenib, but also an effective therapeutic target whose inhibition enhanced regorafenib sensitivity after SR or LR.

Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created.>80%of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183, P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970, P=0.004), day three (F=16.738, P=0.004), day four (F=5.414, P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138, P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679, P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827, P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.

Objective　To investigate the level of cell adhesion molecule sialyl-LeX expression in colorectal carcinoma and its relation with carcinogenesis, differentiation, metastasis and prognosis. Methods　Sialyl-LeX expression and its optical density in colorectal carcinoma (n=90) and remote normal mucosa (n=30) were quantitatively studied with microwave-LSAB immunohistochemical method combined with image analysis technique. Fifty-three patients were followed up. Results　 The weaker staining in remote normal colorectal mucosa was observed in very limited parts of some deep crypts. Positive rate of sialyl-LeX expression was only 16.7%(5/30). The positive expression of sialyl-LeX was observed in 83 of 90 patients with colorectal carcinoma(92.2%). The apical cytoplasma of cancer tubules, the luminal contents, and the cytoplasma of the cancer cells were strongly stained. The mean integral optical density of sialyl-LeX positive cell in poorly differentiated adenocarcinoma was significantly higher than that in highly differentiated and mucinous ones (P<0.01). It was markedly higher in patients with positive lymphatic nodes than that in negative ones (P<0.01). With followed-up for longer than 5 years, it was much lower in the alive cases than that in the dead (P<0.01). Conclusion　These findings indicate that changes of sialyl-LeX expression and its optical density is related to carcinogenesis, differentiation, invasion and metastasis of colorectal carcinoma. It may be a good predicter for the prognosis of patients with colorectal carcinoma.

Objective To investigate the expression and distribution of sialyl Lewis X in colorectal carcinoma and its relationship with carcinogenesis, progression and metastatic proclivity.Methods Microwave LSAB immunohistochemical technique was used to detect the expression of sialyl Lewis X in colorectal carcinoma and in normal mucosa. Immunoelectromicroscopic localization of sialyl Lewis X labelled by colloidal gold was also observed.Results The positive rate of sialyl Lewis X expression in primary colorectal cancer was 92 2%(83/90), and 16 7%(5/30) in normal mucosa. The positive rate was 100% in patients with lymphatic nodes metastasis, compared with that of negative nodes of 82 1% ( P

Objective To investigate the level of cell adhesion molecules LeX , sialyl-LeX, LeA and sialyl-LeA expression in colorectal carcinoma and its relation with carcinogenesis, differentiation, metastasis and prognosis.Methods The expression forms of LeX,sialyl-LeX,LeA and sialyl-LeA were studied by immunohistochemistry method with catalysis signal amplification (CSA)in 90 carcinomas and 30 normal mucosal specimens of colon and rectum, as well as in 51 metastatic lymph nodes.Fifty-three patients were followed up. Results Their antigens were expressed in normal mucosa distant from carcinoma lesions with the following frequencies: LeX, 93 3% (28/30); sialyl-LeX, 16 7% (5/30); LeA, 93 3% (28/30); and sialyl-LeA, 90 0% (27/30), whereas in colorectal carcinomas, the expression frequency rate were: LeX, 90 0% (81/90); sialyl-LeX, 94 4% (85/90); LeA, 78 9% (71/90); and sialyl-LeA, 80 0%(72/90). In metastatic lesion of lymphode LeX, sialyl-LeX, LeA and sialyl-LeA were expressed in 68 7% (35/51),96 1% (49/51),66 7% (34/51) and 66 7% (34/51), respectively.Conclusions The alteration of Lewis-related carbohydrate antigens in cancer cell membranes, including sialylation and/or aberrant glycosylation,these alteration may be related to metastatic behavior. These results suggested that sialyl-LeX may be used for estimating infiltration, metastasis and prognosis as a tumor-associated antigen in colorectal carcinoma.