Fascin Is Critical for the Maintenance of Breast Cancer Stem Cell Pool Predominantly via the Activation of the Notch Self-Renewal Pathway.

An emerging dogma shows that tumors are initiated and maintained by a subpopulation of cancer cells that hijack some stem cell features and thus referred to as "cancer stem cells" (CSCs). The exact mechanism that regulates the maintenance of CSC pool remains largely unknown. Fascin is an actin-bundling protein that we have previously demonstrated to be a major regulator of breast cancer chemoresistance and metastasis, two cardinal features of CSCs. Here, we manipulated fascin expression in breast cancer cell lines and used several in vitro and in vivo approaches to examine the relationship between fascin expression and breast CSCs. Fascin knockdown significantly reduced stem cell-like phenotype (CD44hi /CD24lo and ALDH+ ) and reversal of epithelial to mesenchymal transition. Interestingly, expression of the embryonic stem cell transcriptional factors (Oct4, Nanog, Sox2, and Klf4) was significantly reduced when fascin expression was down-regulated. Functionally, fascin-knockdown cells were less competent in forming colonies and tumorspheres, consistent with lower basal self-renewal activity and higher susceptibility to chemotherapy. Fascin effect on CSC chemoresistance and self-renewability was associated with Notch signaling. Activation of Notch induced the relevant downstream targets predominantly in the fascin-positive cells. Limiting-dilution xenotransplantation assay showed higher frequency of tumor-initiating cells in the fascin-positive group. Collectively, our data demonstrated fascin as a critical regulator of breast CSC pool at least partially via activation of the Notch self-renewal signaling pathway and modification of the expression embryonic transcriptional factors. Targeting fascin may halt CSCs and thus presents a novel therapeutic approach for effective treatment of breast cancer. Stem Cells 2016;34:2799-2813 Video Highlight: https://youtu.be/GxS4fJ_Ow-o.

Barnawi R ，Al-Khaldi S ，Majed Sleiman G ，Sarkar A ，Al-Dhfyan A ，Al-Mohanna F ，Ghebeh H ，Al-Alwan M ... -  《-》

β1 Integrin is essential for fascin-mediated breast cancer stem cell function and disease progression.

Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, β1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.

Barnawi R ，Al-Khaldi S ，Colak D ，Tulbah A ，Al-Tweigeri T ，Fallatah M ，Monies D ，Ghebeh H ，Al-Alwan M ... -  《-》

LncRNA-Hh Strengthen Cancer Stem Cells Generation in Twist-Positive Breast Cancer via Activation of Hedgehog Signaling Pathway.

Cancer stem cells (CSCs) are a subpopulation of neoplastic cells with self-renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial-mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non-coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT-associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist-positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs-associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh-GLI1 pathway associated lncRNA-Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere-formation efficiency (MFE) and the self-renewal capacity in vitro, and oncogenicity in vivo in Twist-positive breast cancer cells are elevated. lncRNA-Hh silence in Twist-positive breast cells attenuates the activated Shh-GLI1 signaling and decreases the CSC-associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist-induced EMT cells to gain the CSC-like stemness properties.

Zhou M ，Hou Y ，Yang G ，Zhang H ，Tu G ，Du YE ，Wen S ，Xu L ，Tang X ，Tang S ，Yang L ，Cui X ，Liu M ... -  《-》

Fascin, an actin-bundling protein, promotes breast cancer progression in vitro.

Fascin, an actin-cross-linking protein, is up-regulated in breast cancer and correlates with a more aggressive disease. This study was conducted to elucidate the effects of manipulating fascin in breast cancer cells on the metastasis-associated events, including proliferation, adhesion, invasion, epithelial-mesenchymal transition (EMT) and enrichment of a CD44(+) /CD24(-) subpopulation that show some stem/progenitor cell properties. Western blot analysis of a panel of breast cancer cell lines revealed high expression of fascin in MDA-MB-435 and MDA-MB-231 cells but revealed no or low expression in MDA-MB-453, Her-18 and T47D. Gain-of-function and loss-of-function studies in breast cancer cells demonstrated that forced expression of fascin promoted cell proliferation assessed by the MTT assay, decreased cellular adhesion to fibronectin and potentiated the invasive capacity in the Transwell chamber invasion assay. Conversely, down-regulation of fascin via small interfering RNA increased cell adhesion and facilitated cell proliferation and invasion. In addition, fascin participated in the EMT and modulated the proportion of the CD44(+) /CD24(-) subpopulation in breast cancer cells. In conclusion, our data highlight an important role for fascin in breast cancer progression in vitro through orchestrating a variety of cellular events associated with metastasis, and thus, targeting this gene might have therapeutic implications.

Xing P ，Li JG ，Jin F ，Zhao TT ，Liu Q ，Dong HT ，Wei XL ... -  《-》

miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regulation and sustained AKT activation.

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT-PCR analysis. One of the most altered miRNAs was miR-10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR-10b in MCF-7 cells (miR-10b-OE cells) promoted higher self-renewal and expression of stemness and epithelial-mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR-10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self-renewal. Bioinformatics analyses identified several potential miR-10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self-renewal. The targeting of PTEN by miR-10b was confirmed using a luciferase reporter, qRT-PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR-10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self-renewal ability of CSCs and breast cancer cell lines overexpressing miR-10b. In conclusion, miR-10b regulates the self-renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.

Bahena-Ocampo I ，Espinosa M ，Ceballos-Cancino G ，Lizarraga F ，Campos-Arroyo D ，Schwarz A ，Maldonado V ，Melendez-Zajgla J ，Garcia‐Lopez P ... -  《-》