DNAJB4 suppresses breast cancer progression and promotes tumor immunity by regulating the Hippo signaling pathway.

PURPOSE: Breast cancer is the most common cancer worldwide. Low DNAJB4 expression levels are strongly correlated with poor prognosis in breast cancer patients. However, the molecular mechanism by which DNAJB4 regulates breast cancer progression is unclear. METHODS: The expression of DNAJB4 was validated in human breast cancer tissues, normal human breast tissues, and breast cancer cell lines. CCK-8, colony-forming, and wound healing assays were used to assess the biological effect of DNAJB4 overexpression on cell proliferation and migration in MCF-7 cell lines. Bioinformatic analysis was used to identify the DNAJB4 related pathways in breast cancer. Epithelial-mesenchymal transition (EMT)-related biomarkers and Hippo pathway components were quantified by Western blots. Luciferase and Western blot assays were used to validate which miRNA regulates DNAJB4. In addition, the effects of DNAJB4 on in vivo tumor growth were assessed in xenograft models. RESULTS: DNAJB4 was expressed at low levels in human breast cancer tissues and breast cancer cell lines and correlated with poor prognosis. DNAJB4 overexpression significantly inhibited cell proliferation and migration in vitro by activating the Hippo pathway. The dual-luciferase assay showed that hsa-miR-183-5p targeted DNAJB4. Moreover, the effects of DNAJB4 could be reversed by miR-183-5p. In addition, the expression of DNAJB4 was strongly correlated with immune infiltration levels. Notably, DNAJB4 overexpression markedly enhanced CD4 + and CD8 + T cells and reduced PD-L1 levels in 4T1 tumors via the Hippo pathway, which retarded tumor growth in a subcutaneous xenograft tumor mouse model of 4T1 cells. CONCLUSIONS: The present study demonstrated that DNAJB4 overexpression inhibited the malignant biological behavior of breast cancer by regulating the Hippo pathway and tumor immunosuppressive environment.

Identification of PTK2 as an adverse prognostic biomarker in breast cancer by integrated bioinformatics and experimental analyses.

PTK2 is highly expressed in many cancers and is involved in cell growth, survival, migration, and invasion. However, the prognostic value of PTK2 and its potential function remain unclear in breast cancer. Therefore, we performed a comprehensive analysis of multiple public databases to explore the roles of PTK2. By integrating multiple datasets, we found that PTK2 mRNA expression in breast cancer tissue was higher than that in normal breast tissue or adjacent tissue. High PTK2 expression was associated with lymph node metastasis stage, tumor stage, breast cancer type, age, TP53 mutation, and gender and significantly predicted a poor survival outcome in breast cancer patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results suggested that PTK2 and co-expressed genes participated in the cell cycle. Immune infiltration analysis clarified that high PTK2 expression was positively correlated with infiltrating levels of CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. The DNA methylation of PTK2 in breast cancer tissues was higher than that in normal tissues, and high PTK2 methylation was correlated with poor prognosis in breast cancer patients. Furthermore, 16 possible ceRNA networks related to PTK2 were constructed for breast cancer. Additionally, PTK2 knockdown could suppress the proliferation and migration ability of MCF-7 cells. These results suggest that PTK2 can be used as a prognostic biomarker for breast cancer.

The involvement of high succinylation modification in the development of prostate cancer.

Objective: Succinylation modification of the lysine site plays an important role in tumorigenesis and development, but it is rarely reported in prostate cancer (PCa), so this study aims to elucidate its expression in and clinical correlation with PCa. Methods: A total of 95 tumor, 3 normal and 52 paired adjacent tissue of PCa were involved for succinylation stanning. 498 PCa samples with 20 succinylation modification-related genes from TCGA were downloaded for model construction. Statistical methods were employed to analyze the data, including Non-Negative Matrix Factorization (NMF) algorithm, t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and Cox regression analysis. Results: The pan-succinyllysine antibody stanning indicated that tumor tissues showed higher succinyllysine level than adjacent tissues (p<0.001). Gleason grade and PDL1 expression levels were significantly different (p<0.001) among the high, medium and low succinylation staining scores. The types of PCa tissue were divided into four clusters using RNA-seq data of 20 succinylation-related genes in TCGA database. Clinical characterize of age, PSA level, and pathological stage showed differences among four clusters. The expression of succinylation-related genes (KAT5, SDHD and GLYATL1) and PCa related genes (PDL1, AR and TP53) were significantly different in 52 matched tumor and adjacent tissues (p<0.001). GLYATL1 and AR gene expression was significantly related to the pathological stage of PCa. Conclusion: Succinylation was significantly increased in PCa tissues and was closely related to Gleason grade and PD-L1 expression. Model construction of 20 genes related to succinylation modification showed that the later the pathological stage of PCa, the higher the level of succinylation modification.

Effect of cell density on the malignant biological behavior of breast cancer by altering the subcellular localization of ANXA2 and its clinical implications

To investigate the subcellular localization of ANXA2 in breast cancer of different cell densities in humans and its relationship with the clinicopathological features of patients. To investigate the differences in ANXA2 subcellular localization in MDA-MB-231 cells of different cell densities. To compare the proliferation, invasion, and migration ability of MDA-MB-231 cells under different ANXA2 subcellular localization.MethodsImmunohistochemistry was applied to detect the subcellular localization of ANXA2 in tissue sections of 60 breast cancer patients, and the association with ANXA2 subcellular localization was verified in conjunction with cell density. To investigate the relationship between cell density and clinicopathological data of breast cancer patients. To establish high- and low-density models of MDA-MB-231 breast cancer cell lines and verify the subcellular localization of ANXA2 using immunofluorescence and observation under confocal microscopy. The proliferation, migration, and invasion ability of MDA-MB-231 cells under different subcellular localization of ANXA2 were detected and compared using CCK-8 assay and Transwell assay. After changing the subcellular localization of ANXA2 in high-density MDA-MB-231 cells with PY-60, changes in biological behaviors of the compared MDA-MB-231 cells were observed. Two different 4T1 cell lines with high and low densities were implanted subcutaneously in nude mice to observe the effects of different cell densities on tumor growth in nude mice.ResultsThe clinical data showed that breast cancer with high cell density had higher T stage and higher TNM stage, and the cell density was positively correlated with breast cancer mass size. ANXA2 was mainly localized to the cell membrane when the cell density of breast cancer cells was high and to the cytoplasm when the cell density was low. (AU)

Effect of cell density on the malignant biological behavior of breast cancer by altering the subcellular localization of ANXA2 and its clinical implications.

OBJECTIVE: To investigate the subcellular localization of ANXA2 in breast cancer of different cell densities in humans and its relationship with the clinicopathological features of patients. To investigate the differences in ANXA2 subcellular localization in MDA-MB-231 cells of different cell densities. To compare the proliferation, invasion, and migration ability of MDA-MB-231 cells under different ANXA2 subcellular localization. METHODS: Immunohistochemistry was applied to detect the subcellular localization of ANXA2 in tissue sections of 60 breast cancer patients, and the association with ANXA2 subcellular localization was verified in conjunction with cell density. To investigate the relationship between cell density and clinicopathological data of breast cancer patients. To establish high- and low-density models of MDA-MB-231 breast cancer cell lines and verify the subcellular localization of ANXA2 using immunofluorescence and observation under confocal microscopy. The proliferation, migration, and invasion ability of MDA-MB-231 cells under different subcellular localization of ANXA2 were detected and compared using CCK-8 assay and Transwell assay. After changing the subcellular localization of ANXA2 in high-density MDA-MB-231 cells with PY-60, changes in biological behaviors of the compared MDA-MB-231 cells were observed. Two different 4T1 cell lines with high and low densities were implanted subcutaneously in nude mice to observe the effects of different cell densities on tumor growth in nude mice. RESULTS: The clinical data showed that breast cancer with high cell density had higher T stage and higher TNM stage, and the cell density was positively correlated with breast cancer mass size. ANXA2 was mainly localized to the cell membrane when the cell density of breast cancer cells was high and to the cytoplasm when the cell density was low. The CCK-8 assay showed that the proliferation rate of MDA-MB-231 cells increased (P < 0.05) after shifting the subcellular localization of ANXA2 from the cell membrane to the cytoplasm. Transwell invasion assay and Transwell migration assay showed that the invasion and migration ability of MDA-MB-231 cells increased significantly after the subcellular localization of ANXA2 was transferred from the cell membrane to the cytoplasm (P < 0.05). The animal experiments showed that high-density breast cancer cells could promote the growth of subcutaneous tumors in nude mice relative to low-density breast cancer cells. CONCLUSION: Cell density can regulate the subcellular localization of ANXA2, and changes in the subcellular localization of ANXA2 are accompanied by the changes in the biological behavior of breast cancer.

Targeted Intervention of NF2-YAP Signaling Axis in CD24-Overexpressing Cells Contributes to Encouraging Therapeutic Effects in TNBC.

Triple-negative breast cancer (TNBC) cells have not been usefully classified, and no targeted therapeutic plans are currently available, resulting in a high recurrence rate and metastasis potential. In this research, CD24high cells accounted for the vast majority of TNBC cells, and they were insensitive to Taxol but sensitive to ferroptosis agonists and effectively escaped phagocytosis by tumor-associated macrophages. Furthermore, the NF2-YAP signaling axis modulated the expression of ferroptosis suppressor protein 1 (FSP1) and CD24 in CD24high cells, with subsequent ferroptotic regulation and macrophage phagocytosis. In addition, a precision targeted therapy system was designed based on the pH level and glutathione response, and it can be effectively used to target CD24high cells to induce lysosomal escape and drug burst release through CO2 production, resulting in enhanced ferroptosis and macrophage phagocytosis through FSP1 and CD24 inhibition mediated by the NF2-YAP signaling axis. This system achieved dual antitumor effects, ultimately promoting cell death and thus inhibiting TNBC tumor growth, with some tumors even disappearing. The composite nanoprecision treatment system reported in this paper is a potential strategic tool for future use in the treatment of TNBC.

The association between molecular type and prognosis of patients with stage IV breast cancer: an observational study based on SEER database.

BACKGROUND: Molecular subtype, the basis for personalized treatment of breast cancer, is of great value in evaluating prognosis and guiding treatment of early-stage breast cancer. However, its value in stage IV patients remains unclear. In this study, we investigated the association between molecular subtype and prognosis of de novo stage IV breast cancer using Surveillance, Epidemiology, and End Results (SEER) database with the purpose to provide evidence for optimal therapeutic options for breast cancer patients. METHODS: We retrospectively analyzed de novo stage IV breast cancer patients with the SEER Program data from 2010 to 2015. Characteristics of patients with different molecular subtypes were compared by chi-square test and survival curves for breast cancer specific survival (BCSS) according to subtypes were plotted by Kaplan-Meier method. The Cox proportional hazards model was performed to search for independent prognostic factors in stage IV breast cancer patients. RESULTS: A total of 11,974 patients were included in this study, among which 7,100 (59.30%) patients were of HR+/HER2-, 2,093 (17.48%) of HR+/HER2+, 1,139 (9.51%) of HR-/HER2+ and 1,642 (13.71%) of HR-/HER2-. Multivariate Cox analysis revealed that molecular subtype, age, race, marital status, grade, surgery and chemotherapy were independent prognostic factors for BCSS in de novo stage IV patients. Taking HR+/HER2- patients as reference, HR+/HER2+ patients had better BCSS (HR =0.81, 95% CI: 0.75-0.88, P<0.001), HR-/ HER2- patients had worse BCSS (HR =1.42, 95% CI: 1.29-1.46, P<0.001) and HR-/HER2+ patients had no significant difference (HR =1.03, 95% CI: 0.98-1.08, P=0.188). In patients with different single organ metastases, the prognosis of HR+/HER2+ subtype was the best (except brain metastasis), while that of HR-/HER2- subtype was the worst. CONCLUSIONS: Molecular subtypes were closely associated with the prognosis of de novo stage IV breast cancer. Among the four subtypes, HR+/HER2+ patients had the best prognosis while HR-/HER2- patients had the worst. The prognosis of patients with different single organ metastases was the same, but in patients with brain metastases, HR+/HER2+ ones did not have a significantly better prognosis than other subtypes except triple-negative type.

miR-498 inhibits autophagy and M2-like polarization of tumor-associated macrophages in esophageal cancer via MDM2/ATF3.

Structured abstract Aim: To elucidate the effect of miRNA (miR)-498 on autophagy and M2-like macrophage polarization in esophageal cancer. Methods: Autophagy was evaluated in esophageal cancer. Macrophage markers specific for M1- or M2-like phenotype were determined. The binding relationships between miR-498 and MDM2, MDM2 and ATF3 were analyzed. Results: miR-498 was downregulated in esophageal cancer and was associated with disease-free and overall patient survival. Enhanced miR-498 reduced LC3I conversion to LC3II and increased p62 accumulation in KYSE-150 cells, and increased macrophage polarization to M2-like phenotype in KYSE-150 and TAM co-culture. miR-498 inhibited MDM2-mediated ATF3 degradation, thus suppressing autophagy and M2-like polarization of macrophages in esophageal cancer. Conclusion: miR-498 may inhibit autophagy and M2-like polarization of macrophages to suppress esophageal cancer via MDM2/ATF3.

Lay abstract In this study, we aimed to elucidate the therapeutic mechanism of miRNA (miR)-498 in autophagy and macrophage polarization to M2-like phenotype in esophageal cancer. This study reports lower miR-498 expression in esophageal cancer tissues compared with adjacent normal tissues. According to the experimental results, miR-498 negatively targets MDM2 by binding to its 3'UTR, which leads to attenuated ubiquitination and degradation of ATF3 induced by MDM2. Specifically, overexpressed miR-498 reduces ratio of LC3II (a marker that is commonly utilized to detect cell autophagy) to LC3I and increases p62 (a common cargo receptor for autophagy) accumulation in KYSE-150 cells, and elevates macrophage polarization to M2-like phenotype by depressing MDM2-mediated ATF3 degradation. The present study deepened our understanding of the causes of esophageal cancer and provided at least three novel therapeutic targets for the development of effective targeted therapy for esophageal cancer.

Systemic analysis of the expression levels and prognosis of breast cancer-related cadherins.

Cadherins form connection between cells, facilitate communication, and serve as essential agents in the progression of multiple cancers. Over 100 cadherins have been identified and they are mainly divided into four groups: classical cadherins (CDHs), protocadherins (PCDHs), desmosomal (DSC), and cadherin-related proteins. Accumulating evidence has indicated that several members of the cadherins are involved in breast cancer development. Nevertheless, the expression profiles and corresponding prognostic outcomes of these breast cancer-related cadherins are yet to be analyzed. Here, we examined the expression levels and prognostic potential of these breast cancer-related cadherins from the specific databases viz. oncomine, gene expression profiling interactive analysis, human protein atlas, UALCAN, Kaplan-Meier Plotter, and cBioPortal. We found that the CDH2/11 levels were higher in breast cancer tissues, compared to healthy breast tissues, whereas with CDH3-5, PCDH8/10, and DSC3, the levels were lower in the former than in the latter. Additionally, for CDH1/6/13/17/23, PCDH7, and FAT4, trancript level alterations between breast cancer and healthy tissues varied across different databases. The CDH1 protein levels were elevated in breast cancer tissues versus healthy breast tissues, whereas the protein levels of CDH3/11 and PCDH8/10 were reduced in breast cancer, compared to healthy breast tissues. For CDH15 and CDH23, the expression levels paralleled tumor stage. Survival analysis, using the Kaplan-Meier Plotter database, demonstrated that elevated CDH1-3 levels correlated with diminished relapse-free survival in breast cancer patients. Alternately, enhanced CDH4-6/15/17/23, PCDH10, DSC3, and FAT4 levels estimated a rise in relapse-free survival of breast cancer patients. These data suggest CDH1-3 to be a promising target for breast cancer precision therapy and CDH4-6/15/17/23, PCDH10, DSC3, and FAT4 to be novel biomarkers for breast cancer prognosis.

Silence of lncRNA MIAT-mediated inhibition of DLG3 promoter methylation suppresses breast cancer progression via the Hippo signaling pathway.

As the foremost common female malignancy, breast cancer (BC) poses a significant public health stumbling block. Although treatment protocols have improved over the years, the overall prognosis of BC remains unsatisfactory. Extensive investigations have taken place into long non coding RNAs (lncRNAs) pertaining to their involvement in carcinogenesis. The current study in connection with bioinformatics tools aimed to identify the myocardial infarction associated transcript (MIAT) as a BC-related differentially expressed lncRNA in an attempt to elucidate the effect of MIAT in BC cells. MIAT was initially overexpressed while DLG3 was down-regulated in BC. BC cells were subsequently treated with si-MIAT or/and si-DLG3, after which the expressions of DLG3 and the Hippo signaling pathway-related proteins were evaluated to analyze their regulatory mechanism in BC, which indicated that MIAT inhibition up-regulated DLG3 and activated the Hippo signaling pathway to suppress proliferation and promote apoptosis of BC cells. MS-PCR and RIP assays demonstrated that MIAT bound to the methylation proteins DNMT1, DNMT3A and DNMT3B, promoted the methylation of CpG islands in DLG3 promoter and inhibited the DLG3 expression. Moreover, our data suggested that DLG3 could bind to MST2 and regulate LAST1, which prevented the nuclear translocation of YAP. The in vitro results were further verified via the in vivo findings. Taken together, the central findings of our study demonstrate that MIAT silencing inhibits BC progression by means of up-regulating DLG3 via activation of the Hippo signaling pathway, highlighting a novel potential therapeutic target for the treatment of the BC.

IL-6 Triggers the Migration and Invasion of Oestrogen Receptor-Negative Breast Cancer Cells via Regulation of Hippo Pathways.

Breast cancer is one of the major challenges for women's health. However, the role and mechanisms of interleukins (ILs) on the progression of breast cancer are not well illustrated. Our present study revealed that the expressions of IL-6 and IL-8 were significantly increased in oestrogen receptor-negative (ER-) breast cancer cells. Increased expression of IL-6 was observed in 83.9% (26/31) ER- breast cancer tissues as compared with their matched adjacent normal tissues. In vitro studies indicated that IL-6 can significantly promote the migration and invasion of ER- breast cancer cells via increasing the dephosphorylation, nuclear translocation and transcriptional activities of YAP in breast cancer cells. Knockdown of YAP can attenuate IL-6-induced migration and invasion of cancer cells, suggesting that YAP plays an essential role in IL-6-induced malignancy of breast cancer cells. Furthermore, IL-6 treatment also decreased the phosphorylation of LATS1/2. The knockdown of LATS1/2 synergistically suppressed si-IL-6-induced deactivation of YAP. Targeted inhibition of IL-6/YAP can significantly suppress the invasion of ER- breast cancer cells. Collectively, our study revealed that IL-6 can trigger the malignancy of breast cancer cells via activation of YAP signals. Targeted inhibition of IL-6/YAP might be a novel therapeutic approach for the treatment of ER- breast cancer.

Curcumol attenuates epithelial-mesenchymal transition of nasopharyngeal carcinoma cells via TGF-ß1.

The current study aimed to identify the effect and primary mechanism of Curcumol on the migration of nasopharyngeal carcinoma (NPC) cells in vitro and in vivo. Curcumol was dissolved in absolute ethyl alcohol and the experiment was performed in NPC 5­8F cells in vitro and in vivo. The effect of different concentrations of Curcumol on cell migration was determined using wound healing and Transwell assays. A cell counting kit­8 (CCK­8) assay was also performed in order to determine cell viability. Flow cytometry was used to detect the effect of Curcumol on apoptosis. The expression of epithelial­mesenchymal transition (EMT)­associated proteins and genes was evaluated by western blotting, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and ELISA. In addition, the antitumor activity of Curcumol was investigated in female BALB/C nude mice with orthotopic tumor implants. The results indicated that cell apoptosis was increased and the viability of NPC 5­8F cells was decreased following treatment with Curcumol at doses of 0.1, 0.2 and 0.4 µM/ml. The results of in vivo experiments indicated that tumor growth and weight were decreased following Curcumol administration. Furthermore, the results of western blotting and RT­qPCR demonstrated that Curcumol altered the level of E­cadherin and N­cadherin in a dose­dependent manner in vivo. Curcumol also regulated the secretion of protein markers in the serum that were associated with EMT and TGF­ß1 in the 5­8F xenograft mouse model. Thus, the results indicated that Curcumol induced TGF­ß1­mediated EMT arrest by regulating E­cadherin and N­cadherin, which may prevent further development of NPC.

Cdc37 facilitates cell survival of colorectal carcinoma via activating the CDK4 signaling pathway.

Cell division cycle 37 (Cdc37) is an important partner for heat shock protein 90 (HSP90), assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Given its influence on cell growth pathways, Cdc37 has been discussed as a potential intermediate in carcinogenesis. However, to date, the potential functional roles and molecular mechanisms by which Cdc37 regulates cell survival in colorectal carcinoma (CRC) remain unclear. Here, we investigated the expression of Cdc37 and its clinical significance in CRC, and systematically explored the role and the underlying mechanism of Cdc37 in CRC cell survival both in vitro and in vivo. Our results showed that Cdc37 was remarkably up-regulated in CRC, which facilitated cell survival mainly by promoting cell proliferation, G1-S transition, and inhibiting cell apoptosis. Our data further indicated that Cdc37 increased the stability of cyclin-dependent kinase 4 (CDK4) to activate the retinoblastoma 1 (RB1) signaling pathway, followed by increased expression of Bcl-2 and Bcl-xL, which ultimately promoted cell survival in CRC. Moreover, knockdown of CDK4 reversed the Cdc37-mediated effect in promoting the progression of CRC. Our findings showed that Cdc37 played a critical role in promoting CRC cell survival by increasing CDK4 stability to activate the RB1 signaling pathway. Thereby, Cdc37 might serve as a potential therapeutic target in CRC patients.

FAT4 functions as a tumor suppressor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is often associated with biologic behavior with frequent distant metastasis. FAT tumor suppressor homolog 4 (FAT4), a cadherin-related protein, is involved in a variety of biological processes as a tumor suppressor; however, the role of FAT4 in TNBC is still unclear. The aim of our study was to identify the role of FAT4 in TNBC and examine the underlying molecular mechanisms. The expression of FAT4 was evaluated by immunohistochemistry, western blotting, and qRT-PCR in a series of TNBC tissues. The effects of FAT4 on the ability of cell proliferation, migration, and invasion were assessed by MTT assay and migration and invasion assays. We demonstrated that the repression of FAT4 by shRNA could promote TNBC progression. Taken together, our findings provide evidence for a role of the FAT4 cluster as a tumor suppressor in TNBC patients and may serve as potential novel targets for the treatment of TNBC.

MiR-940 Inhibited Cell Growth and Migration in Triple-Negative Breast Cancer.

BACKGROUND Breast cancer is the main type of cancer in women, and triple-negative breast cancer (TNBC) is a unique subtype of breast cancer. The expression of miR-940 has been shown to play an important role in various cancers; however, the role of miR-940 in TNBC remains unknown. MATERIAL AND METHODS The expression of miR-940 in TNBC tissues or cells were tested by qRT-PCR; the expression of miR-940 in cells were overexpressed by miR-940 mimics, and suppressed by anti-miR-940. Bioinformatics algorithms from TargetScanHuman were used to predict the target genes of miR-940. The interaction between miR-940 and ZNF24 was confirmed by dual luciferase assays. The protein level was assayed by Western blot. RESULTS TNBC tissues and cells showed lower miR-940 levels. CONCLUSIONS MiR-940 inhibited cellular proliferation and migration in TNBC.

Systematic analyses of key genes and pathways in the development of invasive breast cancer.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a common type of non-invasive breast cancer and can sometimes progress into invasive breast cancer (IBC). Identification of the critical genes and biological processes specifically and/or commonly changed in DCIS or IBC can help us understand more about breast cancer development and provide more critical targets and signal transduction pathways for the diagnosis and treatments for breast cancer patients. AIM AND METHODS: We aimed to gain more understanding about the whole process of IBC development, especially in the early stage. Here we systematically analyzed an online breast cancer patient database to identify those significantly changed genes and biological processes in epithelium from normal stage to DCIS stage or from DCIS stage to IBC stage. RESULTS: 344 specific genes, such as FN1, AURKA and HSPA8, were found to be significantly changed (both upregulated and downregulated) in DCIS group in comparison with normal tissue group, which represents the gene profile changes in early stage of breast cancer development. Meanwhile, 304 specific genes were significantly changed (both upregulated and downregulated) in IBC group in comparison with normal tissue group, which represents the gene profile changes in late stage of breast cancer development. Importantly, seven genes were identified to have consistent changes in both early stage and late stage, indicating they might play "driving" roles in the breast cancer development. Of these 7 genes, 5 have been shown to be involved in breast cancer progression by previous studies, which demonstrates the validity of our analyses. Notably, DNAPTP3 was identified for the first time to play an oncogenic role in breast cancer development. In the GO term analyses, cell cycle genes was found to play more important roles in the early stage while biological adhesion was indicated to be more specifically involved in late stage of breast cancer development. SIGNIFICANCE: Our systematic analyses provide better understanding of the unique gene profiles and biological processes during the breast cancer development and identify more potentially important targets for future studies, such as DNAPTP3.

Knockdown of ubiquitin protein ligase E3A affects proliferation and invasion, and induces apoptosis of breast cancer cells through regulation of annexin A2.

The present study used RNA interference (RNAi) to study how the expression of annexin A2 was affected by ubiquitin protein ligase E3A (UBE3A). In addition, the proliferation, apoptosis and invasiveness of BT-549 breast cancer cells was studied following knockdown of UBE3A. Three pairs of small interfering RNA (siRNA) fragments targeting UBE3A were designed and transfected into the BT-549 cells. The effects of silencing UBE3A were detected by reverse transcription-polymerase chain reaction and western blotting, and the same methods were used to detect the expression levels of annexin A2. Cell proliferation was determined using the Cell Counting kit-8, and flow cytometry and a Transwell chamber assay were used to assess the rate of cell apoptosis and invasion, respectively. Following transfection with the three siRNAs targeting UBE3A for 72 h, the mRNA expression levels of UBE3A were downregulated, as compared with those in the untreated groups, and siRNA1 was the shown to be the most effective siRNA for silencing UBE3A expression. The protein expression levels were concordant with the mRNA expression levels of UBE3A. In addition, the mRNA and protein expression levels of annexin A2 were downregulated. Cellular proliferation and invasion of the siRNA1 group was inhibited as compared with those in the untreated groups, and apoptosis of UBE3A-siRNA1 cells was increased as compared with that in the untreated groups. The results of the present study indicated that UBE3A may regulate the expression of annexin A2, resulting in promotion of proliferation and invasion and suppression of apoptosis in BT-549 cells.

[Inhibition of Hep-2 cell apoptosis after annexin A5 knockdown].

OBJECTIVE: To study the effect of annexin A5 on the apoptosis of laryngeal cancer cells. METHODS: Special siRNAs were used to knock annexinA5 down in Hep-2 cell, and RT-PCR and Western blot were applied to identify the efficacy of RNA interference. The flow cytometry assay was performed to detect the Hep-2 cell apoptosis. RESULTS: RT-PCR analysis showed that the relative mRNA expression of annexin A5 in siRNA group, negative control group, Lipofectamine 2000 group and blank control group were 0.70 ± 0.03, 1.18 ± 0.05, 1.17 ± 0.06 and 1.23 ± 0.07. The relative mRNA expression of annexin A5 in siRNA group was significantly decreased than contrast groups(t = -14.77, t = -13.23, t = -12.99, P < 0.05).In Western blot assay, the trend of protein expression level was consistent with the mRNA expression levels of annexin A5. The relative levels of proteins in siRNA group, negative control group, Lipofectamine 2000 group and blank control group were shown 1.21 ± 0.03, 3.88 ± 0.06, 3.87 ± 0.02 and 3.95 ± 0.08. The relative protein expression of annexin A5 in siRNA group was significantly decreased than contrast groups(t = -70.34, t = -150.62, t = -56.32, P < 0.05). At the same time in flow cytometry the apoptotic rate of siRNA group, negative control group, Lipofectamine 2000 group and blank control group were 4.43% ± 0.12%, 13.67% ± 0.22%, 13.66% ± 0.12% and 13.35% ± 0.13%, the difference between the siRNA group and contrast groups was statistically significant(t = -62.50, t = -14.16, t = -11.47, P < 0.05).So after RNA interference, expression of annexin A5 decreased, and the results in the apoptosis inhibition of Hep-2 cell. CONCLUSION: Annexin A5 promotes apoptosis of Hep-2 cells, and it may be a potential therapeutic target for the laryngeal cancer.

FBXL20 acts as an invasion inducer and mediates E-cadherin in colorectal adenocarcinoma.

The mechanisms eliciting colorectal adenocarcinoma are not well understood and the FBXL20 gene is problematic as it exhibits an abnormal expression in colorectal cancer cells. In the present study a recombinant plasmid, pReceiver-M03-FBL20 expression plasmid was constructed, which overexpressed FBXL20; this was transfected into Lovo cells to form Lovo-FBL20 cells. The FBXL20 expression level was examined by quantitative polymerase chain reaction (qPCR) and western blot analysis. The cell viability and invasion capacity were measured using cell counting kit 8, Transwell chamber and wound healing assays, respectively. The associated genes, including E-cadherin, ß-catenin, c-Myc, SET nuclear oncogene, protein phosphatase-2A, Axin, p53 and caspase 3, were detected by qPCR and western blotting. It was demonstrated that the FBXL20 expression level was markedly upregulated in the Lovo-FBL20 cells transfected with pReceiver-M03-FBL20 expression plasmid, compared with that of the Lovo cells. In addition, the cell viability and invasion capacity of the Lovo-FBL20 cells were significantly increased. These increases correlated with a significant upregulation in the expression level of ß-catenin and c-Myc, and a downregulated expression level of E-cadherin. The results of the present study indicate that FBXL20 may mediate the ubiquitin degradation of E-cadherin resulting in an increased invasive ability of malignant cells.

E3 ubiquitin ligases in regulating stress fiber, lamellipodium, and focal adhesion dynamics.

Recent discoveries have unveiled the roles of a complicated network of E3 ubiquitin ligases in regulating cell migration machineries. The E3 ubiquitin ligases Smurf1 and Cul/BACURD ubiquitinate RhoA to regulate stress fiber formation and cell polarity, and ASB2α ubiquitinates filamins to modulate cytoskeletal stiffness, thus regulating cell spreading and cell migration. HACE1, XIAP, and Skp1-Cul1-F-box bind to Rac1 and cause its ubiquitination and degradation, thus suppressing lamellipodium protrusions, while PIAS3, a SUMO ligase, activates Rac1 to promote lamellipodium dynamics. Smurf1 also enhances Rac1 activation but it does not ubiquitinate Rac1. Both Smurf1 and HECTD1 regulate focal adhesion (FA) assembly and (or) disassembly through ubiquitinating the talin head domain and phosphatidylinositol 4 phosphate 5-kinase type I γ (PIPKIγ90), respectively. Thus, E3 ubiquitin ligases regulate stress fiber formation, cell polarity, lamellipodium protrusions, and FA dynamics through ubiquitinating the key proteins that control these processes.