Thioridazine reverses trastuzumab resistance in gastric cancer by inhibiting S-phase kinase associated protein 2-mediated aerobic glycolysis

BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance.While S-phase kinase associated protein 2(Skp2)overexpression has been implicated in the malignant progression of GC,its role in regulating trastuzumab resistance in this context remains uncertain.Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products,there has been a lack of successful commercialization of drugs specifically targeting Skp2.AIM To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment.METHODS Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells.Q-PCR,western blot,and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression.A cell counting kit-8 assay,flow cytometry,a amplex red glucose/glucose oxidase assay kit,and a lactate assay kit were utilized to measure the proliferation,apoptosis,and glycolytic activity of GC cells in vitro.A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo.RESULTS The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab.Thioridazine demonstrated the ability to directly bind to Skp2,resulting in a reduction in Skp2 expression at both the transcriptional and translational levels.Moreover,thioridazine effectively inhibited cell proliferation,exhibited antiapoptotic properties,and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways.The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo,surpassing the efficacy of either monotherapy.CONCLUSION Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance,particularly when used in conjunction with lapatinib.This compound has potential benefits for patients with Skp2-proficient tumors.

Epidermal growth factor upregulates Skp2/Cks1 and p27^(kip1) in human extrahepatic cholangiocarcinoma cells

AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.

Oncogenic Role of Skp2 and p27^(Kip1) in Intraductal Proliferative Lesions of the Breast

Objective To investigate whether the connection of p27 Kip1 to S-phase kinase-associated protein 2 (Skp2) plays an oncogenic role in intraductal proliferative lesions of the breast. Methods Here we investigated the mechanism involved in association of Skp2's degradation of p27 Kip1 with the breast carcinogenesis by immunohistochemical method through detection of Skp2 and p27 Kip1 protein levels in 120 paraffin-embedded tissues of intraductal proliferative lesions including usual ductal hyperplasia (UDH, n=30), atypical ductal hyperplasia (n=30), flat epithelial atypia (FEA, n=30), and ductal carcinoma in situ (DCIS, n=30). Moreover, the expression status of Skp2 and p27 Kip1 in 30 cases of the normal breast paraffin-embedded tissues were explored. Results The DCIS group was with the highest Skp2 level and the lowest p27 Kip1 level, and the UDH group was with the lowest Skp2 level and the highest p27 Kip1 level. Both Skp2 and p27 Kip1 levels in the DCIS group were significantly different from those in the UDH group (all P<0.01). The levels of Skp2 and p27 Kip1 in the FEA group were significantly different from both the DCIS and UDH groups (all P<0.05). p27 Kip1 was negatively correlated with Skp2 in both the UDH group (r=-0.629, P=0.026) and DCIS group (r=-0.893, P=0.000). Conclusion Overexpression of Skp2 might be the mechanism underlying p27 Kip1 over degradation.

Molecular mechanism of Skp2 in promoting cervical cancer HeLa cell proliferation

Objective: To explore the impact of s-phase kinase-associated protein 2 (Skp2) on cervical cancer cell proliferation and the relationship between Skp2 and expression of cell regulation factors and transcription factors. Methods: RNAi technology was used to silence Skp2 gene in HeLa cells. After interference, RT-PCR was used for detection of Skp-2 mRNA, and Western blotting and flow cytometry were used for protein expression analysis. Results: siRNA significantly inhibited HeLa cell proliferation (P<0.05) and increased HeLa apoptosis, and G1/G0 phase cells were increased significantly (P<0.01). Skp2 siRNA transfected HeLa cells effectively reduced Skp2 protein levels, while p27 and p-p53 protein levels were increased significantly. RT-PCR results showed that after interference Skp2 mRNA, c-myc mRNA and cyclin A mRNA expressions decreased significantly compared with those in control group (P<0.01), and p27mRNA expression level was significantly higher (P<0.01). Conclusion: The change of Skp2 expression affects the expression of the cell cycle protein, thus affecting proliferation and apoptosis of HeLa cells. Skp2 protein plays an important role in the progression of cervical cancer; yet the specific mechanism still needs further study.

Correlation of BRD4 and Skp2 expression levels in ultrasound-guided thyroid nodule fine needle aspiration biopsy tissue with the pathological features of nodules

Objective:To study the correlation of bromodomain-containing protein 4(BRD4)and S-phase kinase-associated protein 2(Skp2)expression levels in ultrasound-guided thyroid nodule fine needle aspiration biopsy tissue with the pathological features of nodules.Methods:The tissues obtained in ultrasound-guided thyroid nodule fine needle aspiration biopsy in our hospital between March 2015 and March 2018 was selected and divided into malignant group and benign group according to the pathological results,and the expression levels of BRD4,Skp2,proliferation genes and angiogenesis genes were detected.Results:The BRD4 and Skp2 mRNA expression in thyroid nodules of the malignant group were significantly higher than those of the benign group,and the BRD4 and Skp2 mRNA expression in the malignant group of thyroid nodules with TNM III-IV,capsular invasion and lymph node metastasis were significantly higher than those in the thyroid nodules with TNM I-II,without capsular invasion and without lymph node metastasis;cyclin D1(CCND1),β-catenin,proliferation cell nuclear antigen(PCNA),vascular endothelial growth factor(VEGF),endothelial cell specific molecule-1(ESM-1),Survivin and cyclooxygenase 2(COX2)mRNA expression in thyroid nodules of the malignant group were obviously higher than those of the benign group and positively correlated with BRD4 and Skp2 while cyclin G2(CCNG2)and endostatin(ES)mRNA expression were significantly lower than those of the benign group and negatively correlated with BRD4 and Skp2.Conclusion:The high expression of BRD4 and Skp2 in malignant thyroid nodules is correlated with the pathological changes and can change the expression of proliferation genes and angiogenesis genes.