Dataset Information

SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer.

ABSTRACT: Understanding the mechanism by which cancer cells enhance stemness facilitates cancer therapies. Here, we revealed that a stem cell transcription factor, SALL4, functions to enhance stemness in basal-like breast cancer cells. We used shRNA-mediated knockdown and gene overexpression systems to analyze gene functions. To evaluate stemness, we performed a sphere formation assay. In SALL4 knockdown cells, the sphere formation ability was reduced, indicating that SALL4 enhances stemness. CD44 is a membrane protein and is known as a stemness factor in cancer. CD44 splicing variants are involved in cancer stemness. We discovered that SALL4 modulates CD44 alternative splicing through the upregulation of KHDRBS3, a splicing factor for CD44. We cloned the KHDRBS3-regulated CD44 splicing isoform (CD44v), which lacks exons 8 and 9. CD44v overexpression prevented a reduction in the sphere formation ability by KHDRBS3 knockdown, indicating that CD44v is positively involved in cancer stemness. In addition, CD44v enhanced anoikis resistance under the control of the SALL4 - KHDRBS3 network. Basal-like breast cancer is an aggressive subtype among breast cancers, and there is no effective therapy so far. Our findings provide molecular targets for basal-like breast cancer therapy. In the future, this study may contribute to the establishment of drugs targeting cancer stemness.

SUBMITTER: Matsumoto Y

PROVIDER: S-EPMC5806117 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer.

Matsumoto Yoshiaki Y Itou Junji J Sato Fumiaki F Toi Masakazu M

Cancer medicine 20180122 2

Understanding the mechanism by which cancer cells enhance stemness facilitates cancer therapies. Here, we revealed that a stem cell transcription factor, SALL4, functions to enhance stemness in basal-like breast cancer cells. We used shRNA-mediated knockdown and gene overexpression systems to analyze gene functions. To evaluate stemness, we performed a sphere formation assay. In SALL4 knockdown cells, the sphere formation ability was reduced, indicating that SALL4 enhances stemness. CD44 is a me ...[more]

PMID: 29356399

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Project description:INTRODUCTION: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells. METHODS: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion. RESULTS: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen. CONCLUSION: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.

Project description:IntroductionHuman breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44+/CD24- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes.MethodsDouble-staining immunohistochemistry was used to quantify CD44 and CD24 expression in 240 human breast tumors for which information on other tumor markers and clinical characteristics was available. Gene expression data were also accessible for a cohort of the material.ResultsA considerable heterogeneity in CD44 and CD24 expression was seen both between and within tumors. A complete lack of both proteins was evident in 35% of the tumors, while 13% contained cells of more than one of the CD44+/CD24-, CD44-/CD24+ and CD44+/CD24+ phenotypes. CD44+/CD24- cells were detected in 31% of the tumors, ranging in proportion from only a few to close to 100% of tumor cells. The CD44+/CD24- phenotype was most common in the basal-like subgroup--characterized as negative for the estrogen and progesterone receptors as well as for HER2, and as positive for cytokeratin 5/14 and/or epidermal growth factor receptor, and particularly common in BRCA1 hereditary tumors, of which 94% contained CD44+/CD24- cells. The CD44+/CD24- phenotype was surprisingly scarce in HER2+ tumors, which had a predominantly CD24+ status. A CD44+/CD24- gene expression signature was generated, which included CD44 and alpha6-integrin (CD49f) among the top-ranked overexpressed genes.ConclusionWe demonstrate an association between basal-like and particularly BRCA1 hereditary breast cancer and the presence of CD44+/CD24- cells. Not all basal-like tumors and very few HER2+ tumors, however, contain CD44+/CD24- cells, emphasizing that a putative tumorigenic ability may not be confined to cells of this phenotype and that other breast cancer stem cell markers remain to be identified.

Dataset Information

SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer.

Publications

SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer.

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