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.

Expression of SKP2 Protein in Non-small Cell Lung Carcinoma

Objective： To study the expressive characteristics of SKP2 protein in non-small cell lung carcinoma and it is affection to NSCLC patients＇ prognosis. Methods： The expression of SKP2 protein was detected in 89 NSCLC, 5 benign lung neoplasmas, 5 normal bronchus and lung tissues by Tissue Chip and immunohistochemistry technology. Results： The positive rate of SKP2 protein staining was （23.52±13.57）% in NSCLC tissues, significantly higher than that in benign lung neoplasmas, normal brochus and lung tissues （2.91±1.27）% （P=0.0000〈0.001）. The expressive level of SKP2 protein in NSCLC tissues was closely related to cell differentiation （PI=0.000〈0.001）, but not to age, sex, smoking history, pathological type, site, size, lymph node metastasis and TNM stage （each Pl〉0.05）. The survival analysis displayed that the NSCLC patients＇ 5 years survival rate was lower in positive expression group than that in negative expression group （P1=0.042/0.031〈0.05; r=-0.186, P2=0.000〈0.001）. Conclusion： The positive expression of SKP2 protein may play an enhancement role in the occurrence and development of NSCLC. Moreover, it may be a bad indicator to NSCLC patients＇ prognosis.

Expression of SKP2 Protein in Lung Carcinoma

Objective： To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis. Methods： The expression of SKP2 protein was detected in 89 non small cell lung carcinoma, 13 small cell lung carcinoma, 10 lung benign lesion tissues by Tissue Chip and Immunohistochemistry technology. Results： The positive rate of SKP2 protein staining was （23.52±13.57）% in non small cell lung carcinoma and （53.85±12.26）% in small cell lung carcinoma, which were significantly higher than （2.91±1.27）% in lung benign lesion tissues. It was highest in small cell lung carcinoma and lowest in lung benign lesion tissues, with a significant difference between them （P=0.000）. The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation, lymph node metastasis and pathological types, but not to age, sex, smoking history, tumor site and size, and TNM staging. The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was lower in SKP2 protein positive expression group than that in negative expression group （P1=0.003/0.002; r=-0.275, P2=0.005）. Conclusion. The positive expression of SKP2 protein is higher in lung carcinoma than in lung benign lesion tissues, in particular, much higher in small cell lung carcinoma. In lung carcinoma, its expressive level was closely related to cell differentiation, lymph node metastasis and pathological types. Moreover, it may be an independent factor to prognosis of patients with lung carcinoma.

口腔疣状癌中survivin与BCL-2、Skp2与P27两组蛋白表达特点及相关性研究

目的:研究口腔疣状癌(OVC)组织中survivin(生存蛋白)与BCL-2(B淋巴瘤/白血病-2基因)、Skp2(S期激酶蛋白-2)与P27(激酶抑制蛋白)两组蛋白的表达特点及其相关性。方法:以本院2012-01-2015-01期间收治的34例口腔疣状癌患者作为本次研究资料,全部患者均在本院接受手术治疗。将术中取得的口腔疣状癌组织、癌旁组织,以及2015年1月间在本院因良性病变行病理检查患者的正常口腔黏膜组织作为研究标本,测定3种口腔黏膜组织中survivin、BCL-2、Skp2、P27的蛋白表达水平并进行对比分析,分析不同类型、不同分期的口腔疣状癌组织中4项蛋白表达的差异性,研究survivin与BCL-2、Skp2与P27之间的相关性,并据此研究4项蛋白表达水平对于口腔疣状癌诊断治疗的意义。结果:口腔疣状癌组织中Survivin、BCL-2、Skp2、P27表达阳性率均显著高于癌旁组织与正常组织,P<0.01;经进一步两两对比分析,癌旁组织中4项蛋白表达的阳性率与正常组织无明显差异,P>0.05。Survivin在口腔疣状癌组织中的阳性表达与BCL-2的阳性表达呈显著正相关性,P<0.01,0<r<1;Skp2在口腔疣状癌组织中的阳性表达与P27的阳性表达呈显著负相关性,P<0.01,-1<r<0。不同肿瘤类型、不同分期口腔疣状癌组织中,Survivin、BCL-2、Skp2、P27阳性表达率差异具有统计学差异性,P<0.05。结论:survivin、BCL-2、Skp2、P27在口腔疣状癌中均可见特异性表达,Survivin与BCL-2在口腔疣状癌组织中的阳性表达以及Skp2与P27在OVC癌组织中的阳性表达均具有明确相关性。

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.

The steady-state level of CDK4 protein ms regulated by antagonistic actions between PAQR4 and SKP2 and involved in tumorigenesis

CDK4 is crucial for Gl-to-S transition of cell cycle. It is well established that ubiquitin-mediated degradations of CDK inhibitors and cycUns are pivotal for the timely and unidirectional progression of cell cycle. However, how CDK4 itself is modulated by ubiquitin-mediated degradation has been elusive. Here we report that the steady-state level of CDK4 is controlled by PAQR4, a member of the progestin and adipoQ receptor family, and SKP2, an E3 ubiquitin ligase. Knockdown of PAQR4 leads to reduction of cell proliferation, accompanied by reduced protein level of CDK4. PAQR4 reduces polyubiquitination and degradation of CDK4. PAQR4 interacts with the C-terminal lobe of CDK4. On the other hand, SKP2 also interacts with the C-terminal lobe of CDK4 and enhances polyubiquitination and degradation of CDK4. importantly, PAQR4 and SKP2 bind to the same region in CDK4, and PAQR4 competes with SKP2 for the binding, thereby abrogating SKP2-mediated ubiquitination of CDK4. Using a two-stage DMBA/TPA-induced skin cancer model, we find that PAQR4-deleted mice are resistant to chemical carcinogen-induced tumor formation. Collectively, our findings reveal that the steady-state level of CDK4 is controlled by the antagonistic actions between PAQR4 and SKP2, contributing to modulation of cell proliferation and tumorigenesis.

Skp2、p27及PTEN在胰腺癌组织中的表达及其意义

大多数恶性肿瘤的形成过程中都存在细胞周期的紊乱。细胞S期激酶相关蛋白2（S-phase kinase associated protein2，Skp2）是泛素连接酶SCF（skp1—cull—F—box protein，SCF）的底物识别亚单位，主要通过识别p27进而介导其泛素化降解来调控细胞周期，是细胞周期由G。期进入S期所必需的。许多研究发现，skp2与肿瘤的发生、发展及预后有密切关系，且skp2蛋白的高表达往往伴随着p27蛋白的低表达。