MYC and DNMT3A-mediated DNA methylation represses microRNA-200b in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with a poor prognosis. The microRNA-200 (miR-200) family has been associated with breast cancer metastasis. However, the epigenetic mechanisms underlying miR-200b repression in TNBC are not fully elucidated. In this study, we found that MYC proto-oncogene, bHLH transcription factor (MYC) and DNA methyltransferase 3A (DNMT3A) were highly expressed in TNBC tissues compared with other breast cancer subtypes, while miR-200b expression was inhibited significantly. We demonstrated that MYC physically interacted with DNMT3A in MDA-MB-231 cells. Furthermore, we demonstrated that MYC recruited DNMT3A to the miR-200b promoter, resulting in proximal CpG island hypermethylation and subsequent miR-200b repression. MiR-200b directly inhibited DNMT3A expression and formed a feedback loop in TNBC cells. MiR-200b overexpression synergistically repressed target genes including zinc-finger E-box-binding homeobox factor 1, Sex determining region Y-box 2 (SOX2), and CD133, and inhibited the migration, invasion and mammosphere formation of TNBC cells. Our findings reveal that MYC can collaborate with DNMT3A on inducing promoter methylation and miR-200b silencing, and thereby promotes the epithelial to mesenchymal transition and mammosphere formation of TNBC cells.

Fibronectin protects lung cancer cells against docetaxel-induced apoptosis by promoting Src and caspase-8 phosphorylation.

Fibronectin is involved in orchestrating many diverse cellular behaviors, including adhesion, invasion, differentiation, and proliferation and recently has also been shown to participate in the development of chemoresistance. In this study, we found that fibronectin expression was inversely correlated with clinical responses to docetaxel treatment in non-small cell lung cancer patients. Subsequently, we showed that fibronectin pretreatment could enhance cell viability and reduce apoptosis in docetaxel-treated lung cancer cells because fibronectin induced phosphorylated Src and caspase-8, rendering the later inactive, thus inhibiting docetaxel-induced apoptosis. The inhibition of apoptosis by fibronectin was found to be enhanced by Src overexpression and reversed by Src knockdown in lung cancer cells. Further investigation revealed that a downregulation of phospho-Src via treatment with a Src kinase inhibitor could also abolish fibronectin activity and recover docetaxel-induced apoptosis. Molecular studies revealed that this reversion was due to decreased phospho-Src levels rather than a reduction in total Src expression. Inhibition of phospho-Src reduced phospho-caspase-8 and promoted caspase-8 activity, restoring apoptosis following docetaxel and fibronectin co-treatment. Finally, xenografts experiments demonstrated that fibronectin promoted lung cancer cell proliferation during docetaxel treatment in vivo. Our findings indicate that fibronectin promotes Src and caspase-8 phosphorylation in lung cancer cells, which decreases caspase-8 activation and protects tumor cells from docetaxel-induced apoptosis. Therefore, the fibronectin/Src/caspase-8 pathway may play a crucial role in docetaxel resistance in lung cancer.

A miR-26a/E2F7 feedback loop contributes to tamoxifen resistance in ER-positive breast cancer.

Tamoxifen (TAM) resistance is a substantial challenge in the treatment of estrogen receptor (ER)-positive breast cancer. Previous studies have revealed an important role of microRNA (miRNA/miR)-26a in TAM resistance in breast cancer. However, the mechanism underlying the regulatory effects of miR-26a on TAM resistance remains to be elucidated. The expression levels of miR-26a in ER-positive breast cancer were detected by reverse transcription-quantitative polymerase chain reaction. E2F transcription factor 7 (E2F7) and MYC proto-oncogene, bHLH transcription factor (MYC) levels were detected by western blotting. The present study demonstrated that miR-26a expression was reduced in ER-positive breast cancer compared with in normal breast tissues, whereas E2F7 expression was significantly elevated. Furthermore, an inverse correlation between miR-26a and E2F7 expression was detected in ER-positive breast cancer. The results indicated that miR-26a directly inhibited E2F7 expression through translational inhibition and indirectly inhibited MYC expression partly via E2F7 repression. E2F7, in turn, decreased miR-26a expression via MYC-induced transcriptional inhibition of miRNAs. Furthermore, transfection with miR-26a mimics increased the expression of its host genes (CTD small phosphatase like and CTD small phosphatase 2), whereas ectopic E2F7 expression abrogated the effects of miR-26a. These findings indicated that miR-26a and E2F7 may form a double-negative feedback loop, resulting in downregulation of miR-26a and upregulation of E2F7 in ER-positive breast cancer. Both miR-26a knockdown and E2F7 overexpression conferred resistance to TAM in MCF-7 cells. Conversely, miR-26a overexpression and E2F7 silencing resensitized MCF-7 resistant cells to TAM. These findings revealed that a feedback loop between miR-26a and E2F7 may promote TAM resistance in ER-positive breast cancer.

[miR-101 inhibits the proliferation and migration of breast cancer cells via downregulating the expression of DNA methyltransferase 3a].

OBJECTIVE: To investigate the effect of microRNA-101 (miR-101) on the proliferation and migration of breast cancer cells and its possible mechanism. METHODS: The expressions of miR-101 and DNA methyltransferase 3a (DNMT3a) in breast cancer tissues, corresponding normal breast tissues, breast cancer cells and normal breast cells were detected by real-time quantitative PCR. The lentiviral vectors containing miR-101 and shRNA-DNMT3a sequences were transfected into MDA-MB-231 cells to regulate the expressions of miR-101 and DNMT3a. The expressions of DNMT3a and E-cadherin were determined by Western blotting. The proliferation and migration abilities of MDA-MB-231 cells were evaluated by MTT assay and wound healing assay, respectively. RESULTS: Breast cancer tissues and breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-361, HCC70) showed lower miR-101 expression and higher DNMT3a expression than the adjacent normal breast tissues and normal breast cell line. The overexpression of miR-101 resulted in downregulation of DNMT3a and restoration of E-cadherin. Besides, knockdown of DNMT3a by shRNA increased E-cadherin expression. MTT assay and would healing assay showed that miR-101 overexpression significantly inhibited the proliferation and migration abilities of MDA-MB-231 cells. CONCLUSION: miR-101 may inhibit MDA-MB-231 cell proliferation and migration by repressing DNMT3a expression and up-regulating E-cadherin expression.

CDH1 promoter methylation correlates with decreased gene expression and poor prognosis in patients with breast cancer.

The E-cadherin gene (CDH1) is associated with poor prognosis and metastasis in patients with breast cancer, and methylation of its promoter is correlated with decreased gene expression. However, there is currently no direct evidence that CDH1 promoter methylation indicates poor prognosis in patients with breast cancer. In the present study, methylation-specific polymerase chain reaction (PCR) was applied to detect the methylation status of the CDH1 promoter in 137 primary breast cancer, 85 matched normal breast tissue and 13 lung metastasis specimens. Reverse transcription-quantitative PCR was used to assess the relative expression levels of CDH1 mRNA, and correlation analysis between CDH1 methylation status, and gene expression, clinicopathological characteristics and patient survival was performed. Methylation of CDH1 was identified in 40.9% (56/137) of primary breast cancer specimens, 61.5% (8/13) of lung metastasis specimens and none of the matched normal breast specimens. The downregulation of CDH1 mRNA and E-cadherin protein expression were identified to be significantly correlated with CDH1 methylation (P<0.05). In addition, CDH1 methylation was significantly associated with lymph node metastasis and estrogen receptor status of patients (P<0.05). In univariate analyses, patients with CDH1 methylation exhibited poor overall survival (OS) and disease-free survival (DFS; P<0.05). Furthermore, multivariate analyses revealed that CDH1 methylation was an independent prognostic factor predicting poor OS (HR, 1.737; 95% CI, 0.957-3.766; P=0.041) and DFS (HR, 2.018; 95% CI, 2.057-3.845; P=0.033) in patients with breast cancer. Therefore, the present study suggests that CDH1 promoter methylation may be correlated with breast carcinogenesis and indicates poor prognosis in patients with breast cancer.

LCL161 increases paclitaxel-induced apoptosis by degrading cIAP1 and cIAP2 in NSCLC.

BACKGROUND: LCL161, a novel Smac mimetic, is known to have anti-tumor activity and improve chemosensitivity in various cancers. However, the function and mechanisms of the combination of LCL161 and paclitaxel in non-small cell lung cancer (NSCLC) remain unknown. METHODS: Cellular inhibitor of apoptotic protein 1 and 2 (cIAP1&2) expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry. The correlations between cIAP1&2 expression and clinicopathological characteristics, prognosis were analyzed. Cell viability and apoptosis were measured by MTT assays and Flow cytometry. Western blot and co-immunoprecipitation assay were performed to measure the protein expression and interaction in NF-kB pathway. siRNA-mediated gene silencing and caspases activity assays were applied to demonstrate the role and mechanisms of cIAP1&2 and RIP1 in lung cancer cell apoptosis. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of LCL161 alone or in combination with paclitaxel. RESULTS: The expression of cIAP1 and cIAP2 in Non-small cell lung cancer (NSCLC) tumors was significantly higher than that in adjacent normal tissues. cIAP1 was highly expressed in patients with late TNM stage NSCLC and a poor prognosis. Positivity for both cIAP1 and cIAP2 was an independent prognostic factor that indicated a poorer prognosis in NSCLC patients. LCL161, an IAP inhibitor, cooperated with paclitaxel to reduce cell viability and induce apoptosis in NSCLC cells. Molecular studies revealed that paclitaxel increased TNFα expression, thereby leading to the recruitment of various factors and the formation of the TRADD-TRAF2-RIP1-cIAP complex. LCL161 degraded cIAP1&2 and released RIP1 from the complex. Subsequently, RIP1 was stabilized and bound to caspase-8 and FADD, thereby forming the caspase-8/RIP1/FADD complex, which activated caspase-8, caspase-3 and ultimately lead to apoptosis. In nude mouse xenograft experiments, the combination of LCL161 and paclitaxel degraded cIAP1,2, activated caspase-3 and inhibited tumor growth with few toxic effects. CONCLUSION: Thus, LCL161 could be a useful agent for the treatment of NSCLC in combination with paclitaxel.

OTUD7B and NIK expression in non-small cell lung cancer: Association with clinicopathological features and prognostic implications.

PURPOSE: To investigate the correlation among OTUD7B and NIK expression and the clinicopathological characteristics in NSCLC patients. METHODS: One hundred and twenty patients were involved in this study. We detected OTUD7B and NIK expression by immunohistochemistry and analyzed their correlation with clinicopathological data. RESULTS: The expression of OTUD7B and NIK were negatively correlated in NSCLC tumor samples (rs=-0.421, P<0.001). The higher expression of OTUD7B was associated with smaller tumor size(P=0.018), less lymph node metastasis (P=0.012) and earlier TNM stage(P=0.039), while the higher expression of NIK was only related to more lymph node metastasis(P=0.031) and later TNM stage(P=0.011). MMP-9 was negatively correlated with OTUD7B and positively correlated with NIK. In addition, the high expression of OTUD7B was associated with good prognosis of NSCLC patients (log-rank=6.714, P=0.0096), and a high OTUD7B/low NIK index can predict an even better prognosis (log-rank=11.794, P=0.0006). Moreover, the multivariate Cox regression analysis showed that OTUD7B rather than NIK is an independent marker of overall survival in NSCLC patients(HR=1.602, 95% CI 1.009-2.544, P=0.046). CONCLUSIONS: OTUD7B and NIK may play important roles in the development of lung cancer. The combination of OTUD7B and NIK expression may be a good index for predicting the prognosis of NSCLC.

Let-7a regulates mammosphere formation capacity through Ras/NF-κB and Ras/MAPK/ERK pathway in breast cancer stem cells.

Breast cancer stem cells (BCSCs) have the greatest potential to maintain tumorigenesis in all subtypes of tumor cells and were regarded as the key drivers of tumor. Recent evidence has demonstrated that BCSCs contributed to a high degree of resistance to therapy. However, how BCSCs self renewal and tumorigenicity are maintained remains obscure. Herein, our study illustrated that overexpression of let-7a reduced cell proliferation and mammosphere formation ability of breast cancer stem cells(BCSCs) in a KRas-dependent manner through different pathways in vitro and in vivo. To be specific, we provided the evidence that let-7a was decreased, and reversely the expression of KRas was increased with moderate expression in early stages (I/II) and high expression in advanced stages (III/IV) in breast cancer specimens. In addition, the negative correlation between let-7a and KRas was clearly observed. In vitro, we found that let-7a inhibited mammosphere-forming efficiency and the mammosphere-size via NF-κB and MAPK/ERK pathway, respectively. The inhibitory effect of let-7a on mammosphere formation efficiency and the size of mammospheres was abolished after the depletion of KRas. On the contrary, enforced expression of KRas rescued the effect of let-7a. In vivo, let-7a inhibited the growth of tumors, whereas the negative effect of let-7a was rescued after overexpressing KRas. Taken together, our findings suggested that let-7a played a tumor suppressive role in a KRas-dependent manner.

MiR-208a stimulates the cocktail of SOX2 and ß-catenin to inhibit the let-7 induction of self-renewal repression of breast cancer stem cells and formed miR208a/let-7 feedback loop via LIN28 and DICER1.

MiR-208a stimulates cardiomyocyte hypertrophy, fibrosis and ß-MHC (ß-myosin heavy chain) expression, being involved in cardiovascular diseases. Although miR-208a is known to play a role in cardiovascular diseases, its role in cancer and cancer stem cells (CSCs) remains uncertain. We identified an inverse relationship between miR-208a and let-7a in breast cancer specimens, and found that SOX2, ß-catenin and LIN28 are highly expressed in patients with advanced breast cancer opposed to lesser grades. Further, we isolated ALDH1+ CSCs from ZR75-1 and MDA-MB-231 (MM-231) breast cancer cell lines to test the role of miR-208a in breast CSCs (BrCSCs). Our studies showed that overexpression of miR-208a in these cells strongly promoted the proportion of ALDH1+ BrCSCs and continuously stimulated the self-renewal ability of BrCSCs. By using siRNAs of SOX2 and/or ß-catenin, we found that miR-208a increased LIN28 through stimulation of both SOX2 and ß-catenin. The knockdown of either SOX2 or ß-catenin only partially attenuated the functions of miR-208a. Let-7a expression was strongly inhibited in miR-208a overexpressed cancer cells, which was achieved by miR-208a induction of LIN28, and the restoration of let-7a significantly inhibited the miR-208a induction of the number of ALDH1+ cells, inhibiting the propagations of BrCSCs. In let-7a overexpressed ZR75-1 and MM-231 cells, DICER1 activity was significantly inhibited with decreased miR-208a. Let-7a failed to decrease miR-208a expression in ZR75-1 and MM-231 cells with DICER1 knockdown. Our research revealed the mechanisms through which miR-208a functioned in breast cancer and BrCSCs, and identified the miR-208a-SOX2/ß-catenin-LIN28-let-7a-DICER1 regulatory feedback loop in regulations of stem cells renewal.