TEX19 increases the levels of CDK4 and promotes breast cancer by disrupting SKP2-mediated CDK4 ubiquitination.

BACKGROUND: Globally, breast cancer in women is the fifth leading cause of cancer death. There is an urgent need to explore the molecular mechanism of breast cancer proliferation and metastasis. METHOD: TCGA database analysis was used to analyze genes expression in breast cancer and normal samples and the association between gene expression and prognosis. Immunohistochemical staining, qPCR and western blotting was sued to detected gene expression. The cell function tests were conducted to investigate the effects of TEX19 and CDK4 with abnormal expression on cell proliferation, migration, apoptosis, cell cycle, and colony formation. Bioinformatics analysis methods combined with CHX tracking experiment and Co-IP experiment were performed to screen and verify the downstream molecule and regulatory mechanism of TEX19. Besides, subcutaneous tumorigenesis model in nude mice was constructed. RESULTS: TEX19 was significantly upregulated in breast cancer, and the TEX19 level was related to tumor invasion and prognosis. TEX19 knockdown inhibited the proliferation and migration of breast cancer cells, increased cell apoptosis, and blocked the cell cycle in the G2 phase. Besides, TEX19 suppressed the growth of tumors in the body. Mechanically, TEX19 upregulated the level of CDK4 protein, which depended on the E3 ubiquitin ligase SKP2. Specifically, TEX19 knockdown and SKP2 protein overexpression destroyed the stability of CDK4 protein and enhanced the ubiquitination of CDK4 protein. Additionally, CDK4 knockdown inhibited the proliferation, migration, and colony formation of breast cancer cells, and alleviated the promotion of TEX19 overexpression on the proliferation and migration of breast cancer cell. CONCLUSION: TEX19 and CDK4 were upregulated in breast cancer, and TEX19 increased the level of CDK4 protein by influencing SKP2-mediated ubiquitination of CDK4, thereby promoting the progression of breast cancer.

Targeting tumorous Circ-E-Cadherinencoded C-E-Cad inhibits the recruitment and function of breast cancer-associated myeloid-derived suppressor cells.

We previously demonstrated that the C-E-cad protein encoded by circ-E-cadherin promotes the self-renewal of glioma stem cells. The expression pattern of C-E-cad in breast cancer and its potential function in the tumor microenvironment are unclear. The expression of circ-E-cadherin and C-E-cad was detected in breast cancer specimens. The influence of C-E-cad expression on MDSCs was assessed using FACS and in vivo tumorigenesis experiments. The synergistic effect of anti-C-E-cad and anti-PD-1 antibodies was validated in vivo. circ-E-cadherin and the encoded protein C-E-cad were found to be upregulated in breast cancer vs. normal samples. C-E-cad promotes the recruitment of MDSCs, especially PMN-MDSCs. C-E-cad activates EGFR signaling in tumor cells and promotes the transcription of CXCL8; moreover, C-E-cad binds to MDSCs and maintains glycolysis in PMN-MDSCs. Targeting C-E-cad enhanced anti-PD-1 efficiency. Our data suggested that C-E-cad is markedly overexpressed in breast cancer and promotes MDSC recruitment and survival. Targeting C-E-cad increases the efficacy of immune checkpoint inhibitor therapy.

GSG2 facilitates the progression of human breast cancer through MDM2-mediated ubiquitination of E2F1.

BACKGROUND: Breast cancer (BC) has posed a great threat to world health as the leading cause of cancer death among women. Previous evidence demonstrated that germ cell-specific gene 2 (GSG2) was involved in the regulation of multiple cancers. Thus, the clinical value, biological function and underlying mechanism of GSG2 in BC were investigated in this study. METHODS: The expression of GSG2 in BC was revealed by immunohistochemistry (IHC), qPCR and western blotting. Secondly, the biological function of GSG2 in BC was evaluated by MTT assay, flow cytometry, Transwell assay and wound healing assay. Furthermore, the potential molecular mechanism of GSG2 regulating the progression of BC by co-immunoprecipitation (Co-IP) and protein stability detection. RESULTS: Our data indicated that GSG2 was frequently overexpressed in BC. Moreover, there was a significant correlation between the GSG2 expression and the poor prognosis of BC patients. Functionally, GSG2 knockdown inhibited the malignant progression of BC characterized by reduced proliferation, enhanced apoptosis and attenuated tumor growth. Migration inhibition of GSG2 knockdown BC cells via epithelial-mesenchymal transition (EMT), such as downregulation of Vimentin and Snail. In addition, E2F transcription factor 1 (E2F1) was regarded as a target protein of GSG2. Downregulation of E2F1 attenuated the promoting role of GSG2 on BC cells. Mechanistically, knockdown of GSG2 accelerated the ubiquitination of E2F1 protein, which was mediated by E3 ubiquitin ligase MDM2. CONCLUSIONS: GSG2 facilitated the development and progression of BC through MDM2-mediated ubiquitination of E2F1, which may be a promising candidate target with potential therapeutic value.

Hypoxic Breast Cancer Cell-Derived Exosomal SNHG1 Promotes Breast Cancer Growth and Angiogenesis via Regulating miR-216b-5p/JAK2 Axis.

BACKGROUND: Hypoxia is an important process that involved in the tumor microenvironment. In addition, hypoxic tumor cell-derived exosomes could promote tumor growth and angiogenesis. Thus, we aimed to investigate whether exosomes could regulate tumor development and progression under hypoxia in breast cancer. METHODS: The level of SNHG1 in hypoxic breast cancer cells and exosomes derived from hypoxic breast cancer cells was determined by real-time qPCR assay. Bioinformatics prediction and dual-luciferase reporter assays were used to determine the interaction between SNHG1, miR-216b-5p and JAK2. RESULTS: We found that comparing with exosomes derived from normoxia breast cancer cells, exosomes derived from hypoxic breast cancer cells could promote the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs). In addition, SNHG1 level was significantly upregulated in exosomes derived from hypoxic breast cancer cells. Moreover, exosome-mediated delivery of SNHG1 siRNA3 markedly reversed the effects of exosome-mediated delivery of SNHG1 on HUVECs. Mechanically, SNHG1 could increase the level of JAK2 by competitively binding to miR-216b-5p. Additionally, exosome-mediated delivery of SNHG1 was found to promote breast cancer growth in vivo. CONCLUSION: Collectively, our study revealed that exosomal SNHG1 from hypoxic breast cancer cells could promote tumor angiogenesis and growth via regulating miR-216b-5p/JAK2 axis, suggesting that SNHG1 may serve as a potential therapeutic target for breast cancer.

Triptolide inhibits vascular endothelial growth factor-mediated angiogenesis in human breast cancer cells.

Triptolide has been demonstrated to induce tumor cell apoptosis. However, the role of triptolide in breast cancer angiogenesis remains unclear. The present study aimed to investigate the function of triptolide in breast cancer and the molecular mechanisms underlying this. The results revealed that triptolide could significantly decrease the expression of vascular endothelial growth factor A (VEGFA) in Hs578T and MDAMB231 breast cancer cells. Furthermore, human umbilical vein endothelial cells were used to perform tube formation and bromodeoxyuridine incorporation assays, which demonstrated an antiangiogenic effect of triptolide. In addition, the effect of triptolide in vivo was examined in a xenograft mouse model, which determined that VEGFA, cluster of differentiation 31 and anti-proliferation marker protein Ki67 expression in tumor sections was decreased in the triptolide treatment group compared with the control group. Western bolt analysis was performed to investigate the phosphorylation of extracellular signal-related kinase (ERK)1/2 and RAC-α serine/threonine-protein kinase after triptolide treatment, and it's effect on hypoxia inducible factor (HIF)1-α expression. The results demonstrated that triptolide suppressed ERK1/2 activation and HIF1-α expression. Furthermore, overexpression of HIF1-α could partially abrogate the inhibitory effect of triptolide on VEGFA expression. These results suggest that triptolide inhibits breast cancer cell angiogenesis in vitro and in vivo through inhibiting the ERK1/2-HIF1-α-VEGFA axis.

Knockdown of ZEB1 suppressed the formation of vasculogenic mimicry and epithelial-mesenchymal transition in the human breast cancer cell line MDA-MB-231.

Breast cancer is a common malignant tumor in women. It has been suggested that a type of microcirculation pattern that does not rely on host microvascular endothelial cells known as vasculogenic mimicry (VM) may contribute to the poor effect of anti­angiogenesis treatment on some patients with breast cancer. However, the formation and regulatory mechanism of VM in breast cancer are unclear and still require further investigation. The present study examined whether decreasing the expression of zinc finger E­box binding homeobox (ZEB1) using siRNA can inhibit the formation of VM in Triple Negative Breast Cancer (TNBC), and its specific function and molecular mechanism. mRNA and protein expression were detected by RT­qPCR and western blotting. Invasion assay and tube formation assay were also performed. The results demonstrated that ZEB1 small hairpin (sh)RNA inhibited the formation of VM. Knockdown of ZEB1 markedly inhibited the expression of vimentin in MDA­MB­231 cells and markedly increased the expression of E­cadherin. It was suggested that ZEB1 shRNA may have inhibited the epithelial­mesenchymal transition (EMT). In addition, ZEB1 shRNA inhibited the invasion of MDA­MB­231 cells and suppressed the expression of fetal liver kinase 1 (flk­1). The flk­1 inhibitor Semaxanib inhibited the formation of VM; thus, ZEB1 shRNA inhibited EMT and cell invasion, and may have inhibited the formation of VM through flk­1. The present study contributed further understanding on the theory of tumor angiogenesis and provided theoretical basis for novel targeted therapy of TNBC.

HPV Status and Its Correlation with BCL2, p21, p53, Rb, and Survivin Expression in Breast Cancer in a Chinese Population.

Despite recent evidence, the role of human papillomavirus (HPV) in breast carcinogenesis is controversial. The correlations of HPV infection with the clinicopathological features of breast cancer and the expression of cell cycle/apoptosis-associated proteins have not been well elucidated. In this study, we sought to determine the prevalence of high-risk HPVs (HR-HPVs) infection and BCL2, p21, p53, Rb, and survivin expression in breast cancer patients and to investigate the relationship of HPV with these cancer-related proteins, in an attempt to clarify the potential mechanism of HPV in breast cancer pathogenesis. HPV presence in 81 fresh breast cancer tissues was determined by hybrid capture 2 (HC2) assay, and expression of BCL2, p21, p53, Rb, and survivin was detected by immunohistochemistry. Here we showed that fourteen (17.3%) patients were HR-HPV positive. HPV infection demonstrated no significant correlation with the clinicopathological characteristics of breast cancer. HPV-positive tumors showed significantly higher BCL2 and lower p53 expression than HPV-negative tumors. Expression of p21, Rb, and survivin was not associated with HPV status. Our results suggest a possible role of HR-HPV in breast cancer carcinogenesis, in which BCL2 and p53 may be involved.

Loss of microRNA-145 expression is involved in the development and prognosis of breast cancer complicated by type 2 diabetes mellitus.

OBJECTIVE: To explore the relationships of the expression of miR-145 to the clinicopathological characteristics and prognosis of patients with breast cancer complicated by type 2 diabetes mellitus (T2DM). METHODS: A total of 257 female patients with breast cancer were enrolled for our experiment, including 140 patients with simple breast cancer (control group) and 117 patients with breast cancer complicated by T2DM (observation group). Patients were treated with modified radical mastectomy supplemented with radiotherapy, chemotherapy and endocrine therapy. qRT-PCR was used for the detection of miR-145 expression in patients of both groups. Follow-up lasted 13-60 months. RESULTS: The relative expression of miR-145 in the observation group was significantly lower than that in the control group (p<0.05). The expression of miR-145 in patients with breast cancer complicated by T2DM was related to the history of diabetes, tumor node metastasis (TNM) stage, tumor size, lymph node metastasis (LNM), estrogen receptor (ER) status, and HER2 (all p<0.05). The median disease-free survival (DFS) was significantly longer and the 5-year DFS rate significantly higher in the high-expression group than in the low-expression group. History of diabetes, TNM stage, tumor size, LNM, ER status, and HER2 were risk factors for patients with breast cancer complicated by T2DM (all p<0.05). CONCLUSIONS: Loss of miR-145 expression is related to the development of breast cancer complicated by T2DM, and low miR-145 expression might be an adverse prognostic factor in patients with this disease.

Nitidine chloride inhibits proliferation and induces apoptosis in colorectal cancer cells by suppressing the ERK signaling pathway.

Nitidine chloride (NC) is a natural bioactive phytochemical alkaloid that has displayed anticancer activity in various types of cancer. However, no evidence has been reported for the direct effect of NC on CRC cell proliferation and apoptosis, and the underling mechanisms to be fully elucidated. The present study aimed to investigate the influence of NC on the apoptosis and proliferation of CRC cells. The viability and proliferation of CRC cells was measured by MTT assay and a [3H] thymidine uptake assay. Apoptosis was measured using a flow cytometric apoptosis assay and TUNEL staining. The expression levels of apoptotic­regulated proteins in addition to extracellular signal­regulated kinase (ERK) were measured by western blot analysis following stimulation with NC. The results indicated that NC inhibited the proliferation of HCT116 cells in a dose­ and time­dependent manner. Additionally, apoptotic induction by NC treatment was confirmed. Furthermore, NC was demonstrated to significantly upregulate the expression of Bax, p53, cleaved caspase­3 and ­9 and downregulate the expression of Bcl­2. Treatment with NC reduced the phosphorylation of ERK and by using an ERK inhibitor, U0126, the roles of NC in apoptotic induction and the inhibition of proliferation were further demonstrated. These results demonstrated that NC inhibited the proliferation and induced the apoptosis of CRC cells via the ERK signaling pathway.

Detection of high-risk human papillomaviruses in fresh breast cancer samples using the hybrid capture 2 assay.

The etiology of breast cancer remains unknown and the role of human papillomavirus (HPV) in breast carcinogenesis is controversial. This study investigated the prevalence of high-risk HPV infections in Chinese women with breast cancer and the possible relationship between high-risk HPV infection and the clinicopathological characteristics of breast cancer. Tumor cells from 224 fresh breast cancer samples and 37 fresh breast fibroadenomas were collected for hybrid capture 2 (HC2) assay. HC2 was the only technique approved by the United States Food and Drug Administration for screening for high-risk HPV infection in 2008. The prevalence of high-risk HPV infection in breast cancer samples was 21.4%, which was slightly higher than the 16.2% observed in breast fibroadenomas. Age and menopausal status were not risk factors for high-risk HPV infection among breast cancer patients. The clinical and pathological characteristics of breast cancer showed no significant correlation with high-risk HPV infection. Although the prevalence of 13 subtypes of high-risk HPV infections was similar in breast cancer and nonmalignant breast samples, the presence of high-risk HPVs in both malignant and benign breast samples implies that a possible causal role in breast cancer carcinogenesis could not be ruled out. Clarifying the possible link between high-risk HPVs and breast cancer might benefit women vaccinated against HPV and could decrease the incidence of HPV-related breast cancer.

Lymphangiogenic characteristics of triple negativity in node-negative breast cancer.

PURPOSE: Triple-negativity breast cancer (TNBC), being negative for the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor 2, represents a subgroup of breast cancer with a poor clinical outcome. The aim of the study was to determine whether TNBC is associated with lymphangiogenesis in node-negative breast carcinomas. METHODS: The authors investigated the clinicopathologic characteristics, lymphatic vessel density (LVD), and expression of 2 lymphangiogenic factors, vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor D (VEGF-D), in 21 lymph node-negative TNBCs and 70 lymph node-negative non-TNBCs. RESULTS: TNBC correlated with younger age (below 35 year) and higher histological grade. It also correlated with a higher intratumoral and peritumoral LVD, positive lymphatic invasion, and positive VEGF-C and VEGF-D expression. CONCLUSIONS: For the first time, this study indicated a link between triple-negativity breast cancer and lymphangiogenesis. Lymphangiogenesis might help explain the special malignant phonotype of breast cancer, and lymphangiogenesis inhibitors might be a novel choice for triple-negativity breast cancer patients.

Paclitaxel-doxorubicin sequence is more effective in breast cancer cells with heat shock protein 27 overexpression.

BACKGROUND: Cancer cells with overexpression of heat shock protein 27 (HSP27) are resistant to chemotherapeutic drug doxorubicin (Dox). Paclitaxel (Pacl) was reported to suppress HSP27 expression in ovarian and uterine cancer cells. The purposes of this study were to investigate whether Pacl inhibits the expression of HSP27 in breast cancer cells, whether Pacl can sensitize breast cancer cells with HSP27 overexpression to Dox, and to define a more effective schedule for the combination of Dox with Pacl. METHODS: The HSP27 high-expressing human breast cancer cell lines, MCF-7 and MDA-MB-435, and the HSP27 low-expressing cell line, MDA-MB-231, were used in this study. The level of HSP27, topoisomerase (Topo) IIalpha and beta expression were assessed by Western blotting. The cytotoxic activities of Dox, Pacl and combination of these two drugs were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and flow cytometric assays. RESULTS: Pacl (0.1 micromol/L) inhibited HSP27 expression by approximately 2-fold in MCF-7 and MDA-MB-435 cells, while up-regulating the level of topo IIalpha and beta. In contrast, expression of HSP27 in MDA-MB-231 did not change significantly following Pacl treatment. There were synergistic effects in both treatment sequences (Pacl-Dox and Dox-Pacl) when Pacl was combined with Dox. Compared with those treated with the Dox-Pacl sequence, the Pacl-Dox sequence had a stronger effect in cancer cells with HSP27 overexpression, as MCF-7 and MDA-MB-435 treated with the Pacl-Dox sequence had lower viabilities and a higher apoptotic rate. CONCLUSIONS: Paclitaxel significantly decreases the level of HSP27 in breast cancer cells overexpressing HSP27. In combination therapies, the Pacl-Dox sequence is more effective in clearing breast cancer cells with high HSP27 expression compared with the Dox-Pacl sequence.