| Nowadays,breast cancer is the most prevalent malignancy and the leading cause of cancer-related death among women world-wide.Of breast cancers,triple-negative breast cancer(TNBC),which is Estrogen receptor(ER)and progesterone receptor(PR)-negative and does not over-express the human epidermal growth factor receptor-2(HER-2),is highly aggressive and frequently experience distant metastasis.Clinical management of TNBC is challenging owing to the limited treatment options.Therefore,novel and effective targets and drugs are urgently needed to improve outcome of patients with TNBC.The emergence of breast cancer stem cells(BCSCs)highlight the potentiality to target BCSCs as another promising therapeutic strategy to combat TNBC.Of the cells in the tumor’s bulk,a small population termed cancer stem cells is a subset of stem-like cancer cells which are able to self-renew and maintain the undifferentiated state.Recently,CSCs have been reported to play a pivotal role in the tumor development,progression and metastasis therefore they have created great excitement in the research field.Objective:Previous studies have confirmed the role of NANOG in the stemness maintenance in the embryonic stem cells(ESCs)and normal stem cells.Interestingly,NANOG has been found to act as a negative prognosis indicator in breast cancer.Given the close relationship between ESCs and CSCs,the functional role of NANOG in CSCs,especially in CSCs of the TNBC,is remain unknown.Therefore,in an attempt to provide a novel therapeutic target for TNBC,we evaluated the expression and functional role of NANOG in TNBC.Methods:1)Firstly,mammosphere culture platform was established in our laboratory.And the NANOG expression in TNBC cells and BCSCs were also evaluated.2)We knockdown the NANOG expression using the lentivirus-mediated RNAi technology.We also analyzed the effect of NANOG depletion on stemness of TNBC cells in vitro and in vivo.3)Additionally,we further analyzed the molecular mechanisms underlying the regulatory effect of NANOG on CSCs with the aid of ChIP assay.PP,a novel WNT inhibitor,was also used to validate the involved regulatory network of NANOG.4)At last,in order to provide a more concise and accurate therapeutic target for CSC therapy in TNBC,the NANOG pseudogenes were examined.It have been determined only two of the eleven daughters of NANOG(NANOG1 and NANOGp8)is expressed in cancer cell.However,the dominant pseudogene in triple-negative breast cancer,especially in the CSCs of TNBC is elusive.Results:1)With the aid of flow-cytometry assay,we demonstrated that CD44~+/CD24~-CSCs was enriched in the mammospheres and TNBC cell lines compared with those counterparts.Considering the aggressive phenotype of TNBC,we speculated the CSC might drive the malignant activities of it.2)Furthermore,real-time PCR and western-blot assay demonstrated that elevated NANOG expression in the mammospheres,triple-negative breast cancer cell lines such as MADA-MB-231,MDA-MB-468 and BT549.These data,taken together,indicated that NANOG regulated the biological properties of TNBC via regulating the stemness of the CSCs.3)We demonstrated that NANOG depletion significantly decreased the CD44~+/CD24~-population as well as the expression of stemness-related markers including SOX2,OCT4 and KLF4.At the function level,self-renewal and mammosphere-formation ability were also impaired.In vivo,the limiting dilution assay also supported that NANOG was critical for the stemness maintenance of CSCs in TNBC.In addition,wound-healing assay and transwell assay were also employed to analyzed the impact of NANOG knockdown on the metastatic potential on the CSCs in TNBC,and our results showed that NANOG also activates the metastatic potential via inducing the epithelial-mesenchymal transition(EMT)process.4)We noticed that CK1α,an important component of WNT pathway,decreased after NANOG depletion.And ChIP-PCR assay also verified the interaction between NANOG and CK1α.Thus,our postulate is that NANOG modulates the CSCs activities via interacting with CK1α.In order to further support our hypothesis,pyrvinium pamoate(PP)which is the selectively inhibitor of CK1 was used.We found PP not only successfully phenocopied the NANOG knockdown on the CSCs properties in vivo and in vitro,but also significantly decreased the metastatic potential of breast cancer cells.Together,these observations indicate that NANOG drives CSCs stemness through the NANOG-CK1α-WNT pathway.5)Our PCR sequencing assay and AlwNI digestion assay revealed that NANOGp8but not NANOG1 predominantly expressed in both cancer cells and CSCs in triple-negative breast cancer.And therapeutic strategies targeting NANOGp8 holds the potential to eliminate the CSCs in TNBC. |
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