MiR-600 acts as a bimodal switcher that regulates breast cancer stem cell fate through WNT signaling
ABSTRACT: Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define bCSC-state are unclear. We have performed concurrent human miRNome-wide gain- and loss-of-function screens to identify switcher miRNAs controling the choice between bCSC self-renewal and differentiation. These analysis enlightened miR-600 whose silencing resulted in bCSC expansion. Mechanistically, miR-600 targets the stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. To explore further miR-600 interactions and WNT-pathway, SUM159 cell line constructions were made and FACS-sorted to select the bCSCs. We compared gene expression profiles from native, miR-600 'over-expressed', miR-600'knock-down' and siSCD1 bCSCs. We showed that in the absence of miR-600, WNT signaling is maintained active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT proteins and promotes bCSC differentiation. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decision and influences tumor progression.
Project description:Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define bCSC-state are unclear. We have performed concurrent human miRNome-wide gain- and loss-of-function screens to identify switcher miRNAs controling the choice between bCSC self-renewal and differentiation. These analysis enlightened miR-600 whose silencing resulted in bCSC expansion. Mechanistically, miR-600 targets the stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. To explore further miR-600 interactions and WNT-pathway, SUM159 cell line constructions were made and FACS-sorted to select the bCSCs. We compared gene expression profiles from native, miR-600 'over-expressed', miR-600'knock-down' and siSCD1 bCSCs. We showed that in the absence of miR-600, WNT signaling is maintained active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT proteins and promotes bCSC differentiation. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decision and influences tumor progression.
Project description:Breast cancer stem cells (BCSCs) are considered to be responsible for recurrence in breast cancer. The 68?kDa Src-associated protein in mitosis (Sam68) has been linked to the development and progression of breast cancer; however, the posttranscriptional regulation and role of Sam68 in BCSC self-renewal remain unclear.Sam68 was ectopically overexpressed or knocked down using a siRNA; the self-renewal potential of breast cancer cell lines was assessed using flow cytometry, in vitro mammosphere culture and a xenograft model in NOD/SCID mice. Activation of beta-catenin was assessed by immunohistochemical staining, Western blotting, and luciferase reporter gene assays. The ArrayExpress dataset GSE45666 was used to identify conserved microRNAs downregulated in breast cancer; real-time PCR, Western blotting, luciferase reporter assay, and xenografted tumor model were used to confirm miR-204 regulated Sam68.We found that endogenous Sam68 expression correlated positively with the self-renewal potential of breast cancer cell lines. Overexpression of Sam68 promoted, whereas knockdown reduced, breast cancer cell self-renewal potential in vitro and tumorigenicity in vivo. The Wnt/beta-catenin pathway was identified as a functional mediator of Sam68-induced self-renewal in SKBR-3 and MCF-7 cells. Furthermore, miR-204 was found to be frequently downregulated in human breast cancer and confirmed to directly target Sam68; miR-204 inhibited the self-renewal of breast cancer cell lines by targeting and suppressing Sam68.Our study reveals that Sam68 is regulated by miR-204 and may play an important role in the self-renewal of BCSCs via activating the Wnt/beta-catenin pathway. Sam68 may represent a novel therapeutic target for breast cancer.
Project description:Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.
Project description:Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray-based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA-128-3p (miR-128-3p). Herein, this study aimed to characterize the tumour-suppressive role of miR-128-3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44? CD24-/low cells were selected for subsequent experiments. After verification of the target relationship between miR-128-3p and NEK2, the relationship among miR-128-3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR-128-3p on proliferation, migration, invasion and self-renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain- and loss-of-function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down-regulation of miR-128-3p. Overexpression of miR-128-3p was found to inhibit proliferation, migration, invasion, self-renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down-regulating NEK2. In summary, this study indicates that miR-128-3p inhibits the stem-like cell features of BCSCs via inhibition of the Wnt signalling pathway by down-regulating NEK2, which provides a new target for breast cancer treatment.
Project description:Breast cancer stem cells (BCSCs) are the minor population of breast cancer (BC) cells that exhibit several phenotypes such as migration, invasion, self-renewal, and chemotherapy as well as radiotherapy resistance. Recently, BCSCs have been more considerable due to their capacity for recurrence of tumors after treatment. Recognition of signaling pathways and molecular mechanisms involved in stemness phenotypes of BCSCs could be effective for discovering novel treatment strategies to target BCSCs. This review introduces BCSC markers, their roles in stemness phenotypes, and the dysregulated signaling pathways involved in BCSCs such as mitogen-activated protein (MAP) kinase, PI3K/Akt/nuclear factor kappa B (NF?B), TGF-?, hedgehog (Hh), Notch, Wnt/?-catenin, and Hippo pathway. In addition, this review presents recently discovered molecular mechanisms implicated in chemotherapy and radiotherapy resistance, migration, metastasis, and angiogenesis of BCSCs. Finally, we reviewed the role of microRNAs (miRNAs) in BCSCs as well as several other therapeutic strategies such as herbal medicine, biological agents, anti-inflammatory drugs, monoclonal antibodies, nanoparticles, and microRNAs, which have been more considerable in the last decades.
Project description:Cancer stem cells (CSCs; also known as tumor-initiating cells) are essential effectors of tumor progression due to their self-renewal capacity, differentiation potential, tumorigenic ability and resistance to chemotherapy, all of which contribute to cancer relapse, metastasis and a poor prognosis. Breast cancer stem cells (BCSCs) have been identified to be involved in the processes of BC initiation, growth and recurrence. MicroRNAs (miRNAs) are a class of non-coding small RNAs of 19-23 nucleotides in length that regulate gene expression at the post-transcriptional level through various mechanisms, and serve critical roles in cancer progression. miRNAs have been demonstrated to elicit effects on BCSCs characteristics via the targeting of oncogenes or tumor suppressor genes. The present study focused on the effect of miRNAs on BCSC, including BCSC formation, self-renewal and differentiation, by which miRNAs may inhibit BCSC invasion and metastasis, modulate clonogenicity and tumorigenicity of BCSCs as well as regulate chemotherapy resistance to BC. Through an improved understanding of the association between BCSCs and miRNAs, a novel and safer therapeutic target for BC may be identified.
Project description:MicroRNAs (miRNAs/miRs) negatively regulate gene expression and participate in various cellular processes. miRNA dysregulation is associated with cancer progression. The present study aimed to identify the miRNAs that participate in breast cancer tumorigenesis and determine the mechanism that underlies this. miRNA microarray data analysis and validation assays indicated that miR-376c-3p was downregulated in breast tumour tissues and breast cancer stem cells (BCSCs) compared with adjacent non-cancerous tissues and MCF-10A cells, respectively. Ras-related protein Rab-2A (RAB2A) was predicted as a target of miR-376c-3p, which was confirmed by conducting further experiments. miR-376c-3p regulated the BCSC population and the expression of stem cell regulatory genes by targeting RAB2A. By performing mammosphere, Cell Counting Kit-8, colony formation and transwell invasion assays, it was demonstrated that miR-376c-3p also inhibited BCSC self-renewal, proliferation and invasion by regulating RAB2A expression. Using a xenograft mouse model, it was revealed that miR-376c-3p overexpression suppressed breast cancer growth in vivo. In conclusion, the results indicated that miR-376c-3p targeted RAB2A to regulate BCSC fate and properties; therefore, miR-376c-3p may serve as a potential therapeutic target for breast cancer.
Project description:BACKGROUND:The mechanism underlying breast cancer stem cell (BCSCs) characteristics remains to be fully elucidated. Accumulating evidence implies that long noncoding RNAs (lncRNAs) play a pivotal role in regulating BCSCs stemness. METHODS:LncRNA LUCAT1 expression was assessed in breast cancer tissues (n?=?151 cases) by in situ hybridization. Sphere-formation assay and colony formation assay were used to detect cell self-renewal and proliferation, respectively. RNA immunoprecipitation, RNA pull down and luciferase reporter assays were used to identify LUCAT1 and TCF7L2 as the direct target of miR-5582-3p. The activity of the Wnt/?-catenin pathway was analyzed by TOP/FOP-Flash reporter assays, western blot and immunohistochemistry (IHC). RESULTS:This study found LUCAT1 expression was related to tumor size (p?=?0.015), lymph node metastasis (p?=?0.002) and TNM staging (p?<?0.001). High LUCAT1 expression indicated a shorter overall survival (p?=?0.006) and disease-free survival (p?=?0.011). Furthermore, LUCAT1 was more expressed in BCSCs than in breast cancer cells (BCCs) by lncRNA microarray chips. LUCAT1 up-regulation promoted proliferation of BCCs, while LUCAT1 down-regulation inhibited self-renewal of BCSCs. MiR-5582-3p was directly bound to LUCAT1 and TCF7L2 and negatively regulated their expression. LUCAT1 affected Wnt/?-catenin pathway. CONCLUSIONS:LUCAT1 might be a significant biomarker to evaluate prognosis in breast cancer. LUCAT1 increased stem-like properties of BCCs and stemness of BCSCs by competitively binding miR-5582-3p with TCF7L2 and enhancing the Wnt/?-catenin pathway. The LUCAT1/miR-5582-3p/TCF7L2 axis provides insights for regulatory mechanism of stemness, and new strategies for clinical practice.
Project description:Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, display poor prognosis and exhibit resistance to conventional therapies, partly due to an enrichment in breast cancer stem cells (BCSCs). Here, we investigated the role of the cyclooxygenase-2 (COX-2), a downstream target of TGF?, in regulating BCSCs in TNBC. Bioinformatics analysis revealed that COX-2 is highly expressed in TNBC and that its expression correlated with poor survival outcome in basal subtype of breast cancer. We also found TGF?-mediated COX-2 expression to be Smad3-dependent and to be required for BCSC self-renewal and expansion in TNBCs. Knocking down COX-2 expression strikingly blocked TGF?-induced tumorsphere formation and TGF?-induced enrichment of the two stem-like cell populations, CD24lowCD44high and ALDH+ BCSCs. Blocking COX-2 activity, using a pharmacological inhibitor also prevented TGF?-induced BCSC self-renewal. Moreover, we found COX-2 to be required for TGF?-induced expression of mesenchymal and basal breast cancer markers. In particular, we found that TGF?-induced expression of fibronectin plays a central role in TGF?-mediated breast cancer stemness. Together, our results describe a novel role for COX-2 in mediating the TGF? effects on BCSC properties and imply that targeting the COX-2 pathway may prove useful for the treatment of TNBC by eliminating BCSCs.
Project description:BCSCs are “seed cells” of distant metastasis. ESAM is highly expressed in breast cancer tissues, and its expression is reduced in miR-7-treated BCSC-driven tumor. miR-7 overexpression decreases the BCSC metastasis by inhibiting ESAM, which could be a therapeutic target for breast cancer therapy.