miR-135b promotes proliferation and metastasis by targeting APC in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. The aim of our study was to investigate the functional role of microRNA-135b (miR-135b) in TNBC. A real-time polymerase chain reaction assay was used to quantify miR-135b expression levels in 90 paired TNBC tissue and adjacent normal tissue samples. Wound-healing and transwell assays were performed to evaluate the effects of miR-135b expression on the migration and invasion of TNBC cells. Luciferase reporter and western blot analyses were used to verify whether the mRNA encoding APC is a major target of miR-135b. In the current study, we found that miR-135b was highly expressed in TNBC tissue and cells, and the expression levels were correlated with lymph node status and TNM stage. In TNBC cells, the ectopic expression of miR-135b promoted cell proliferation and invasion in vitro. In addition, our study proved that the overexpression of miR-135b significantly suppressed APC expression by targeting the 3'-untranslated region of APC, whereas enhanced APC expression could partially abrogate the miR-135b-mediated promotion of carcinogenic traits in TNBC cells. Taken together, our study demonstrated that miR-135b expression promoted the proliferation and invasion of TNBC by downregulating APC expression, indicating that miR-135b may serve as a promising target for the treatment of TNBC patients.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2.

BACKGROUND/AIMS: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. METHODS: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. RESULTS: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3'-untranslated region (3'-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. CONCLUSIONS: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.

Silencing of Prrx2 Inhibits the Invasion and Metastasis of Breast Cancer both In Vitro and In Vivo by Reversing Epithelial-Mesenchymal Transition.

BACKGROUND/AIMS: Epithelial-mesenchymal transition (EMT) is recognized as a crucial mechanism in breast cancer progression and metastasis. Paired-related homeobox 2 (Prrx2) has been identified as a new EMT inducer in cancer, but the underlying mechanisms are still poorly understood. METHODS: The expression of Prrx2 was assessed by immunohistochemistry in breast cancer tissues to evaluate the clinicopathological significance of Prrx2, as well as the correlation between Prrx2 and EMT. Short hairpin RNA knockdown of Prrx2 was used to examine cellular effects of Prrx2, detecte the expression of Wnt/ß-catenin signaling and EMT-associated proteins, and observe cell proliferation, invasion and migration abilities in vitro and in vivo. RESULTS: Clinical association studies showed that Prrx2 expression was related to tumor size, lymph node metastasis, tumor node metastasis stages, EMT and poor survival. Results also showed that knockdown of Prrx2 could alter cell morphology, suppressed the abilities of cell proliferation, invasion and migration in breast cancer. Moreover, silencing of Prrx2 induced the mesenchymal-epithelial transition and prevented nuclear translocation of ß-catenin, inhibited wnt/ß-catenin signaling pathway. CONCLUSION: Our study indicated that Prrx2 may be an important activator of EMT in human breast cancer and it can serve as a molecular target of therapeutic interventions for breast cancer.

miR-655 suppresses epithelial-to-mesenchymal transition by targeting Prrx1 in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR-655 was down-regulated in TNBC, and its expression levels were associated with molecular-based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR-655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR-655 not only induced the up-regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR-655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR-655 significantly suppressed Prrx1, as demonstrated by Prrx1 3'-untranslated region luciferase report assay. Our study demonstrated that miR-655 inhibits the acquisition of the EMT phenotype in TNBC by down-regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.

Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired-related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial-mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up-regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal-like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/ß-catenin signaling pathway and induced the mesenchymal-epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.

[Anti-apoptotic mechanism for p21(waf1) in human basal like breast cancer cell line HCC1937].

OBJECTIVE: To explore the anti-apoptotic mechanism of p21(waf1) in human basal like breast cancer cell line HCC1937. METHODS: There were 3 groups, i.e. experimental group HCC1937 with lentivirus -p21(waf1)-shRNA-RFP, control group 1 HCC1937 without lentivirus and control group 2 HCC1937 with lentivirus -RFP. The p21(waf1) and bim mRNA and protein expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. And apoptosis of HCC1937 in different groups was assayed by terminal deoxynucleotidyl transferase mediated C-dUTP nick end labeling (TUNEL). RESULTS: After interference with lentivirus-p21(waf1)-shRNA-RFP, p21(waf1) mRNA and protein expressions declined significantly in the experimental group versus the control groups (experiment group: 0.260 ± 0.004, 0.293 ± 0.006, control group 1: 0.879 ± 0.028, 0.483 ± 0.071, control group 2: 0.870 ± 0.025, 0.469 ± 0.047, all P < 0.01). The bim mRNA and protein expressions increased. And there was significant difference between the experiment and control groups (experiment group: 0.420 ± 0.013, 0.355 ± 0.007, control group 1: 0.258 ± 0.005, 0.142 ± 0.012, control group 2: 0.259 ± 0.002, 0.147 ± 0.013, all P < 0.001); apoptotic index increased (experiment group: 0.279 ± 0.012, control group 1: 0.145 ± 0.008, control group 2: 0.148 ± 0.012, both P < 0.001). CONCLUSION: In human basal-like breast cancer cell line HCC1937, p21(waf1) exerts anti-apoptotic activity by inhibiting the expression of bim, a mediator of mitochondrial apoptosis.

[Retracted] TGF­ß1 induces peritoneal fibrosis by activating the Smad2 pathway in mesothelial cells and promotes peritoneal carcinomatosis.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the fluorescence microscopy data shown in Fig. 6A and B were strikingly similar to data appearing in different form in Fig. 7 in a previously published paper [Lv Z­D, Na D, Liu F­N, Du Z­M, Sun Z, Li Z, Ma X­Y, Wang Z­N and Xu H­M: Induction of gastric cancer cell adhesion through transforming growth factor­beta1­mediated peritoneal fibrosis. J Exp Clin Cancer Res 29: 139, 2010], which featured some of the same authors, although the data were shown to portray results obtained under different experimental conditions. Furthermore, the data in Fig. 7A for the 'TGF­ß1' and the 'TGF­ß1 + siRNAcon' experiments contained an overlapping section, such that these data appeared to have been derived from the same original source, even though they were intended to show the results from differently performed experiments. Owing to the fact that the contentious data in the above article had already been published prior to its submission to International Journal of Molecular Medicine, and due to a lack of overall confidence in the presented data, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 29: 373­379, 2012; DOI: 10.3892/ijmm.2011.852].

[Regulation of p14(ARF) expression and induction of cell apoptosis with c-myc in a p53-independent pathway].

OBJECTIVE: To explore the regulation of p14(ARF) expression and induction of cell apoptosis with the mutant and wild-type c-myc genes in a p53-independent pathway of signal transduction. METHODS: The mutant and wild-type c-myc genes were transfected by lentivirus into HCC1937 to form the stable over-expression cell lines. Uninfected cells and lentivirus-infected ones carrying no c-myc gene acted as blank and infection controls respectively. And c-myc and p14(ARF) mRNA and protein, proliferation and apoptosis in HCC1937 with mutant and wild-type c-myc were detected by reverse transcription (RT)-PCR, Western blotting, thiazolyl blue tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling (TUNEL) respectively. RESULTS: After the lentivirus-mediated gene transfer, c-myc mRNA and protein expression increased in the mutant and wild-type groups. p14(ARF) mRNA and protein increased in the wild-type group and the mutant group and there were significant difference between them with blank and infection controls (mutant groups: 0.560 ± 0.010, 0.154 ± 0.011, wild-type groups: 0.651 ± 0.010, 0.382 ± 0.013, both P < 0.05). The group of mutant and wild-type c-myc could promote the proliferation of cell growth. And c-myc was more effective to induce apoptosis in the wild-type group as compared with the mutant group (7.1% ± 0.7% vs 3.2% ± 0.4%, P < 0.05). CONCLUSIONS: In a p53-independent pathway, the over-expression of wild-type c-myc obviously up-regulates the expression of p14(ARF). And cell apoptosis may be induced through the regulation of p14(ARF)-related gene, keep balance of proliferative promotion and apoptosis induction. When there is a loss-of-function of mutant c-myc, tumorigenicity increases via a disturbed balance of proliferative promotion and apoptosis induction.

[Effect of peritoneal fibrosis induced by transforming growth factor-beta 1 on the adhesion of gastric cancer cell].

OBJECTIVE: To elucidate the effect of transforming growth factor-beta 1 (TGF-ß1) on peritoneal fibrosis and the regulation of gastric cancer adhering to mesothelial cells. METHODS: The peritoneal mesothelial cell line of HMrSV5 was used to determine the role of TGF-ß1 in the regulation of gastric cancer cell adhering to mesothelial cells. And the mRNA and protein expressions of collagen III and fibronectin were detected by adhesion assay, Western blot, immunofluorescent staining and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: (1) The treatment of 5 ng/ml TGF-ß1 could induce the expressions of collagen III and fibronectin in mesothelial cells at 24, 48 and 72 h (P < 0.01). (2) As compared with the controls, the percentages of adhered HGC-27 and HSC-39 gastric cancer cells significantly increased under the treatment of TGF-ß1 for 24 and 72 h. The increased adhesion percentages of HGC-27 were 65% ± 5% and 124% ± 11% (P < 0.05) while those of HSC-39 85% ± 9% and 146% ± 17% respectively (P < 0.05). (3) Arginyl-glycyl-aspartic acid (RGD) (knockdown of minimal sites for cell-binding domain of extracellular matrix) decreased the number of cancer cells adhering to mesothelial cells under the stimulation of TGF-ß1. And the decreased adhesion percentage of HGC-27 was 65% ± 8% (P < 0.05). CONCLUSIONS: TGF-ß1 significantly stimulates the expressions of collagen III and fibronectin in mesothelial cells. And it is associated with the increased adhesion of gastric cancer cell and offers a favorable environment for the dissemination of gastric cancer.

[Effects of transfected pIRES-p21(waf1)-p27(kip1) gene on the duplication of centrosomes and the proliferation of breast cancer cell line MCF-7].

OBJECTIVE: To explore the role of transfected pIRES-p21(waf1)-p27(kip1) gene on the centrosome duplication and cell proliferation of MCF-7, a breast cancer cell line. METHODS: The pIRES-p21(waf1), pIRES-p27(kip1) and pIRES-p21(waf1)-p27(kip1) genes were transfected into the MCF-7 cells by lipofection. The effect on proliferation was evaluated by MTT assay and cell growth curve was drawn. The cell cycle was analyzed by flow cytometry. The centrosome duplication was detected by using indirect immunofluorescence microscopy. RESULTS: After transfected 24 hours, the p21(waf1) and p27(kip1) protein expressions were significantly increased as compared with untransfected MCF-7 cells (P < 0.01), and cell growth was obviously inhibited and resulted in an accumulation of cells in G(1) (P < 0.01), presenting that the proportion of cells in G(1) phase was obviously increased from(47.28 +/- 2.25)% to (69.52 +/- 3.21)% of p21(waf1) transfected cells, (60.83 +/- 3.02)% of p27(kip1) transfected cells, and (78.37 +/- 2.83)% of p21 (waf1)-p27(kip1) transfected cells. The proportion of cells which contained unnormal centrosomes was obviously decreased, from (13.47 +/- 0.33)% to (5.07 +/- 0.38)%, (6.28 +/- 0.35)%, (3.47 +/- 0.23)%, respectively. CONCLUSION: The transfer of p21(waf1) and p27(kip1) genes could inhibit the growth of human breast carcinoma cells and the unnormal duplication of centrosomes. p21(waf1) had a really synergy with p27(kip1) in these effects, suggesting p21(waf1)-p27(kip1) combined gene can inhibit the genesis and development of breast cancer and might have potential clinical significance as therapeutic agents of breast cancer.

[Association of cytokine gene polymorphisms with cachexia related to sporadic female breast cancer].

OBJECTIVE: To investigate the relation of the frequency of tumor necrosis factor (TNF)-alpha - 308, TNF-beta + 252, IL-1 beta + 3954, and IL-10 - 1082 gene polymorphisms to female breast cancer. METHODS: Peripheral blood samples were collected from 102 breast cancer patients with cachexia and 120 breast cancer patients without cachexia. Biallelic polymorphisms were performed by analyzing the incision enzyme-digested DNA fragment obtained using PCR. RESULTS: The allele frequencies of TNF-beta + 252, IL-1 beta + 3954, and IL-10 -1082 in the patients with cachexia were comparable with those of the patients without cachexia (all P > 0.05). The patients with cachexia showed a significantly higher prevalence of TNF2 than the patients without cachexia (20.6 % vs 10.0 %, P = 0.027). Logistic regression analysis indicated TNF2 as a risk factor for cachexia in breast cancer ( OR = 2.333, 95% CI: 1.085-5.017). CONCLUSION: TNF2 plays an important role in the susceptibility of cachexia in breast cancer.

NKD1 down-regulation is associated with poor prognosis in breast invasive ductal carcinoma.

As a negative modulator of the canonical Wnt signaling pathway, Naked1 (NKD1) is widely expressed in many normal tissues. However, the expression and clinicopathological significance of NKD1 in patients with breast cancer is still unclear. The aim of this study was to evaluate NKD1 expression in breast cancer and to investigate the question of whether reduced expression of NKD1 may have any pathological significance in breast cancer development or progression. In this study, we performed western blotting and immunohistochemistry to evaluate the expression of NKD1 and relevance with clinicopathological factors in the breast invasive ductal carcinoma. Reduction of NKD1 was significantly correlated with lymph node metastasis, histological grade and ER expression in breast cancer. Patients with negative NKD1 expression had significantly lower cumulative postoperative 5 year survival rate than those with positive NKD1 expression. This interpretation is in keeping with the results obtained from our in vitro experiments on MDA-MB-231 cells, we demonstrated that upregulation of NKD1 expression by infect with an adenovirus containing a NKD1 vector significantly reduced the migration of breast cancer cells. These data suggest that NKD1 plays an important role in invasion in human breast cancer and it appears to be a potential prognostic marker for patients with breast cancer.

Curcumin induces apoptosis in breast cancer cells and inhibits tumor growth in vitro and in vivo.

Curcumin has shown therapeutic and/or adjuvant therapeutic effects on the treatment of some patients with breast cancer. However, its mechanisms of action are largely unknown. This study was designed to investigate its antitumor effect and underlying mechanisms in human breast cancer MDA-MB-231 and MCF-7 cells. The MTT assay was used to evaluate cell viability, and flow cytometry, acridine orange staining and transmission electron microscopy were used to detect apoptosis for cultured cells. The protein expression in cells was evaluated by western blot analysis. Breast tumors were established by subcutaneous injection of MDA-MB-231 cells in nude BALB/c mice, and curcumin was administered to the mice. The size of tumors was monitored and the weight of tumors was examined. The exposure of breast cancer cells to curcumin resulted in growth inhibition and the induction of apoptosis in a dose-dependent manner. We also found that the expression of Bcl-2 protein decreased and the expression of Bax protein increased which lead to an increase of the Bax/Bcl-2 ratio. In mice bearing MDA-MB-231 xenograft tumors, administration of curcumin showed a significant decrease of tumor volumes and tumor weight compared with the control. Our results showed that curcumin exhibited antitumor effects in breast cancer cells with an induction of apoptosis.

CXCL12 chemokine expression suppresses human breast cancer growth and metastasis in vitro and in vivo.

Chemokine receptors are now known to play an important role in cancer growth and metastasis. However, there is little information regarding chemokine expression in breast cancer. The aim of this study was to evaluate CXCL12 expression in breast cancer and to investigate the question of whether reduced expression of CXCL12 may have any pathological significance in breast cancer development or progression. In this study, we performed western blotting and immunohistochemistry to evaluate the expression of CXCL12 and relevance with clinicopathological factors in the invasive ductal carcinoma. Reduction of CXCL12 was significantly correlated with tumor size, lymph node metastasis, TNM stage and Her-2 expression in breast cancer. Patients with negative CXCL12 expression had significantly lower cumulative postoperative 5 year survival rate than those with positive CXCL12 expression. In addition, we demonstrated that upregulation of CXCL12 expression by infection with an adenovirus containing a CXCL12 vector significantly inhibited cell growth and reduced the migration of breast cancer cells. Furthermore, animal studies revealed that nude mice injected with the Ad-CXCL12 cell lines featured a lighter weight than the control cell lines. These data suggest that CXCL12 plays an important role in cell growth and invasion in human breast cancer and it appears to be a potential prognostic marker for patients with breast cancer.

TGF-ß1 induces peritoneal fibrosis by activating the Smad2 pathway in mesothelial cells and promotes peritoneal carcinomatosis.

Peritoneal dissemination is one of the main causes of death in gastric cancer patients. Our previous study demonstrated that peritoneal fibrosis induced by transforming growth factor-ß1 (TGF-ß1) may provide a favorable environment for the dissemination of gastric cancer. The role of Smad3 in the development of dermal fibrosis, subcapsular cataract, and peritoneal fibrosis has been reported. However, the potential role of Smad2 in the development of fibrosis is unclear. The objective of this study was to determine the effect of Smad2 in peritoneal fibrosis, induced by TGF-ß1, on dissemination of gastric cancer. Here we demonstrate that TGF-ß1 significantly stimulated the expression of collagen III and fibronectin in mesothelial cells through the Smad2 signal transduction pathway, but knockdown of the Smad2 gene by silencing siRNA partially inhibited these effects. This inhibition was associated with a depressed adhesion and invasiveness of gastric cancer cells. We conclude that peritoneal fibrosis induced by TGF-ß1 is dependent on Smad2 signaling and may provide a hospitable environment for carcinomatosis.