Huangzhen Wang

MiR-208a stimulates the cocktail of SOX2 and β-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 β-MHC (β-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, β-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 β-catenin, we found that miR-208a increased LIN28 through stimulation of both SOX2 and β-catenin. The knockdown of either SOX2 or β-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/β-catenin-LIN28-let-7a-DICER1 regulatory feedback loop in regulations of stem cells renewal.

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.

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.

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

BackgroundLCL161, 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.MethodsCellular 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.ResultsThe 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.ConclusionThus, LCL161 could be a useful agent for the treatment of NSCLC in combination with paclitaxel.

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.

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.