SHP2 promotes proliferation of breast cancer cells through regulating Cyclin D1 stabilityviathe PI3K/AKT/GSK3β signaling pathway

Objective:The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner.Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer.Nevertheless,the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined.Methods:The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases.The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry.CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells.Cell-counting kit-8,colony formation,cell cycle,and EdU incorporation assays,as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation.Quantitative RT-PCR,western blotting,immunofluorescence staining,and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation.Results:High SHP2 expression is correlated with poor prognosis in patients with breast cancer.SHP2 is required for the proliferation of breast cancer cellsin vitro and tumor growthin vivo through regulation of Cyclin D1 abundance,thereby accelerating cell cycle progression.Notably,SHP2 modulates the ubiquitin–proteasome-dependent degradation of Cyclin D1viathe PI3K/AKT/GSK3βsignaling pathway.SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3β.GSK3βthen mediates phosphorylation of Cyclin D1 at threonine 286,thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin–proteasome system.Conclusions:Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation.SHP2 may therefore potentially serve as a therapeutic target for breast cancer.

Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells

Apigenin （4＇,5,7-trihydroxyflavone） is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles （AVOs） by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-Ⅱ, the processed form of LC3-Ⅰ, was increased. Treatment with the autophagy inhibitor, 3-methyladenine （3-MA）, significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

Crucial role of Anxa2 in cancer progression:highlights on its novel regulatory mechanism

Anxa2 is the most studied member of the calcium-mediated phospholipid-binding protein family annexins and is a biomarker in cancers.In this review,we listed clinical findings and confirmed the value of Anxa2 in early diagnosis and prognostic prediction due to its overexpression and adverse effect on the outcome in most tumors.Anxa2 plays a pivotal role in cancer cell proliferation,migration,invasion,metastasis,and treatment resistance.Improved understanding of its cancer-promoting function might make it an ideal target for cancer therapy.Here,we systematically summarized the mechanism of Anxa2 in regulating epithelialmesenchymal transition(EMT),cytoskeleton dynamicity,cell cycle,apoptosis,angiogenesis,and immunology by using various tumor models.These data emphasize the potential of Anxa2 for targeted intervention in tumors.Altering Anxa2 expression,neutralizing the cell surface Anxa2,or inhibiting its activation,such as through Tyr23 phosphorylation,could be considered based on the regulatory mechanism of Anxa2 in tumor progression.

Proteasome Inhibitors Sensitize Hepatocellular Carcinoma Cells to TRAIL

OBJECTIVE To investigate the effect of proteasome inhibition on the sensitivity of carcinoma cells to TRAIL-inducing apoptosis, and to study the mechanism of the response. METHODS Human hepatocellular carcinoma cells, pretreated with the proteasome inhibitor, MG132, were cotreated with TRAIL. Western blot assays, immunoprecipitation and RT-PCR were performed to test the expression of the Bcl-2 family proteins and Bax mRNA. RESULTS We found that (i) proteasome inhibition sensitized the human hepatocellular carcinoma cells to TRAIL; and (ii) resulted in Bax accumulation before release of cytochrome C and induction of apoptosis. These results were associated with the ability of proteasome inhibitors to overcome Bcl-2-mediated antiapoptotic function; (iii) Bax is regulated by an ubiquitin/proteasome-dependent degradation pathway. CONCLUSION Proteasome inhibition sensitized hepatocellular carcinoma cells to TRAIL by the inhibition of the ubiquitin/proteasome-mediated Bax degradation pathway.

In Vivo Selection of Phage Sequences and Characterization of Peptide-specific Binding to Breast Cancer Cells

OBJECTIVE To screen specific polypeptide target binding to breast cancer xenogra s in vivo from a phage-displayed peptide library in order to provide peptide sequences for breast cancer tumor-targeting diagnosis and therapy. METHODS A mouse model for carrying breast cancer xenografts was established using Tientsin Albinao II mice(TA II).A 12-peptide library was biopanned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue(liver).The distribution of phages was detected by immunohistochemical staining. RESULTS Phage homing to breast cancer was enriched through 4 rounds of biopanning,being 14-fold of that recovered from liver tissue.A peptide sequence,ASANPFPTKALL was characterized by randomly picked-up clones which appeared most frequently. Immunohistochemical staining revealed phage localization in cancer xenografts 40 min after injection of the enriched phages. When a specific phage was tested individually,the phage reclaimed from breast cancer xenografts was 14 times as those from control tissues. CONCLUSION Tumor-specific homing peptides may provide an effective tool for breast cancer target therapy.The in vivo phage display selection technique employed in this study was feasible and applicable to screening peptides that home to breast cells.

Down-Regulation of CXCR4 Expression by siRNA Inhibits Invasive Ability of Breast Cancer Cells

OBJECTIVE To investigate the efficiency of gene silencing by CXCR4- siRNAs (small interfering RNA), and to examine the invasive ability and the expression of other metastatic-associated genes in siRNA-treated breast cancer cells. METHODS Three siRNAs were designed and cloned into the pSilenc ? 3.1-H1 neo vector. The reconstructed plasmids were purified and trans- fected into the T47D breast cancer cell line, which highly expressed CXCR4. The amount of CXCR4 expression in the transfected cells was measured by flow cytometry and Real-time PCR. Cell invasive ability was evaluated us- ing 24-well Matrigel invasion chambers. In addition, the expression of other metastatic-associated genes, such as E-cad, IGFBP-5, FN and MMP-2, was assessed by Real-time PCR. RESULTS The suppression rates of CXCR4 mRNA expression reached 95.7%, 85.9% and 98.3%compared with control-siRNA cells in the 3 CXCR4- siRNA T47D cells respectively. FCM assays for CXCR4 protein expression showed a similar inhibitory effect. The invasion indexes of these CXCR4- siRNA cells were 0.037, 0.290 and 0.188 respectively compared with control- siRNA cells. After treatment of the cells with CXCR4-siRNA, the expression of E-cad showed an upward tendency and that of IGFBP-5 had a downward trend, while alteration in expression of FN and MMP2 varied without a con- sistant effect. CONCLUSION CXCR4 plays an important role in modulating migra- tion of human breast cancer cells. Small interfering RNA can significantly silence the CXCR4 gene in the human T47D breast cancer cell line. The results of this study strengthen the need for further research on novel gene therapy against breast cancer metastasis.

The Effect of Twist Expression on Angiogenesis in Hepatocellular Carcinoma

OBJECTIVE Hepatocellular carcinoma (HCC) is a hypervascular tumor for which angiogenesis plays an important role in its progression. The aim of this study was to investigate the expression of TWIST and VEGF and determine their roles in angiogenesis of HCC. METHODS Expression Twist and VEGF mRNA was determined by real-time RT-PCR in 30 pairs of hepatocellular carcinoma and matched non-cancerous tissues. Immunohistochemistry was carried out to analyze the protein expression of Twist and VEGF in 40 hepatocellular carcinoma cases. Staining of endothelial cells for CD34 was used to evaluate the microvessel density (MVD). RESULTS We found that the HCC specimens showing positive Twist expression in tumor cells had a higher microvessel density than those without Twist expression. Furthermore, we found that overexpression of the Twist protein positively correlated with up-regulation of VEGF in the HCC tissues (r=0.479, P=0.002). CONCLUSION Our results demonstrate that Twist may play an important role in the angiogenesis of HCC and a high-level of Twist expression may be related to the malignant potential of tumor cells.