J Breast Cancer.  2019 Dec;22(4):522-532. 10.4048/jbc.2019.22.e51.

Kinesin Family Member 11 Enhances the Self-Renewal Ability of Breast Cancer Cells by Participating in the Wnt/β-Catenin Pathway

• Pei YY
• Li GC
• Ran J
• Wan XH
• Wei FX
• Wang L

• KMID: 2470890
• DOI: http://doi.org/10.4048/jbc.2019.22.e51

Abstract

PURPOSE
Our previous studies have shown that kinesin family member 11 (KIF11) is markedly overexpressed in human breast cancer cells or tissues and positively correlated with distant metastasis and prognosis in patients with breast cancer, suggesting an important role in the regulation of cancer stem cells. Herein, we examined the role of KIF11 in breast cancer stem cells.
METHODS
In the current study, we validated our previous findings through analysis of data collected in The Cancer Genome Atlas. Endogenous KIF11 was stably silenced in MCF-7 and SKBR-3 cells. Flow cytometry was used to measure the proportion of side-population (SP) cells. Mammosphere culture and tumor implantation experiments in immunodeficient mice were used to assess the self-renewal ability of breast cancer cells. Real-time polymerase chain reaction, western blot, immunofluorescence staining, luciferase reporter assays and Wnt agonist treatment were conducted to investigate the signaling pathways regulated by KIF11.
RESULTS
We found that the expression level of KIF11 was positively correlated with stem cell-enrichment genes. The proportion of SP cells was significantly reduced in KIF11-silenced cells. Silencing endogenous KIF11 not only reduced the size and number of mammospheres in vitro, but also reduced the ability of breast cancer cells to form tumors in mice. Simultaneously, we found that KIF11 was involved in regulating the activation of the Wnt/β-catenin signaling pathway.
CONCLUSION
Endogenous KIF11 enhances the self-renewal of breast cancer cells by activating the Wnt/β-catenin signaling pathway, thereby enhancing the characteristics of breast cancer stem cells.

Keyword

Breast neoplasms; Kinesin; Cell self renewal; Stem cells; Beta-catenin

MeSH Terms

Animals
beta Catenin
Blotting, Western
Breast Neoplasms*
Breast*
Cell Self Renewal
Flow Cytometry
Fluorescent Antibody Technique
Genome
Humans
In Vitro Techniques
Kinesin*
Luciferases
Mice
Neoplasm Metastasis
Neoplastic Stem Cells
Prognosis
Real-Time Polymerase Chain Reaction
Stem Cells
Kinesin
Luciferases
beta Catenin

Figure

• 

  Figure 1 Upregulated KIF11 positively affects stem cell-regulating genes. (A) The RNA level of KIF11 in 1,092 breast cancer tissues (Tumor) compared with 111 adjacent normal bladder epithelial samples (Normal) in the TCGA profile validated the upregulation of KIF11 in breast cancer. (B) The RNA level of KIF11 was upregulated in every subtype of breast cancer. (C) Gene set enrichment analysis plots of TCGA data show KIF11 levels positively affected stem cell-regulating genes. KIF11 = kinesin family member 11; SEM = standard error of the mean; TCGA = The Cancer Genome Atlas; HER2 = human epidermal growth factor receptor 2; NS = nonsense. *p < 0.05; †p < 0.001.

• 

  Figure 2 Silencing KIF11 reduces the proportion of stem cells in breast cancer cell lines. (A) The mRNA expression level of KIF11 was successfully decreased in the indicated cells. (B) The protein expression of KIF11 was successfully decreased in the indicated cells. (C) The mRNA level of the indicated genes was detected using real-time polymerase chain reaction. (D) Flow cytometry analysis of Hoechst 33342-effluxed cells show that the proportion of SP cells was significantly decreased in the KIF11-silenced cells. KIF11 = kinesin family member 11; mRNA = messenger RNA; Oct4 = organic cation/carnitine transporter4; ALDH1 = aldehyde dehydrogenase 1; CD44 = cluster of differentiation 44. *p < 0.05.

• 

  Figure 3 Silencing KIF11 attenuates the self-renewal ability of breast cancer cells. (A, B) Representative micrographs and colony numbers in the mammosphere culture assay show that silencing KIF11 reduced mammosphere formation in MCF-7 and SKBR-3 cells. (C) Images of excised tumors from nonobese diabetic/severe combined immunodeficiency disease mice at 28 days after injection with the indicated cells show that KIF11-silenced SKBR-3 cells formed fewer and smaller tumors than vector-transfected SKBR-3 cells. Each bar represents the mean±standard error of the mean of 3 independent experiments. KIF11 = kinesin family member 11. *p < 0.05; †p < 0.001.

• 

  Figure 4 Silencing KIF11 inactivates Wnt/β-catenin signaling. (A) Gene set enrichment analysis plots of The Cancer Genome Atlas data show that the expression of KIF11 was positively correlated with the gene characteristics of Wnt activation. (B) Real-time polymerase chain reaction analysis shows that TCF1 and CCND1 expression decreased sharply in KIF11-silenced cells. (C) Immunofluorescence staining show distribution of β-catenin from nucleus to cytoplasm in KIF11-silenced cells (×400). (D) Analysis of the β-catenin reporter gene, based on TCF4 and LEF1 genes, showed that silencing KIF11 significantly reduced the activation activity of β-catenin in cells. (E, F) Representative micrographs and colony numbers in the mammosphere culture assay show that Wnt agonists neutralized the inhibition of KIF11 downregulation in mammosphere formation. (G) Luciferase reporter assay of TCF/LEF transcriptional activity shows that Wnt agonists significantly neutralized the inhibition of KIF11 downregulation on intracellular β-catenin-mediated transcriptional activity. KIF11 = kinesin family member 11; DAPI = 4′,6-diamidino-2-phenylindole; TCF = transcription factor T-cell factor; LEF = lymphoid enhancer-binding factor; CCND1 = cyclin D1; mRNA = messenger RNA. *p < 0.05.

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