Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr 27;7(1):1220.
doi: 10.1038/s41598-017-00916-z.

Deficiency of CCN5/WISP-2-Driven Program in breast cancer Promotes Cancer Epithelial cells to mesenchymal stem cells and Breast Cancer growth

Amlan Das  1   2   3 , Kakali Dhar  1   4   5 , Gargi Maity  1   2 , Sandipto Sarkar  1   6 , Arnab Ghosh  1   4 , Inamul Haque  1   4 , Gopal Dhar  1   4   7 , Snigdha Banerjee  8   9 , Sushanta K Banerjee  10   11   12   13
Affiliations

Deficiency of CCN5/WISP-2-Driven Program in breast cancer Promotes Cancer Epithelial cells to mesenchymal stem cells and Breast Cancer growth

Amlan Das et al. Sci Rep. .

Abstract

Breast cancer progression and relapse is conceivably due to tumor initiating cells (TICs)/cancer stem cells. EMT (epithelial-mesenchymal-transition)-signaling regulates TICs' turnover. However, the mechanisms associated with this episode are unclear. We show that, in triple-negative-breast cancer (TNBC) cells enriched with TICs, CCN5 significantly blocks cellular growth via apoptosis, reversing EMT-signaling and impairing mammosphere formation, thereby blocking the tumor-forming ability and invasive capacity of these cells. To corroborate these findings, we isolated tumor-initiating side populations (SP) and non-side population (NSP or main population) from MCF-7 cell line, and evaluated the impact of CCN5 on these subpopulations. CCN5 was overexpressed in the NSP but downregulated in the SP. Characteristically, NSP cells are ER-α positive and epithelial type with little tumorigenic potency, while SP cells are very similar to triple-negative ones that do not express ER-α- and Her-2 and are highly tumorigenic in xenograft models. The overexpression of CCN5 in SP results in EMT reversion, ER-α upregulation and delays in tumor growth in xenograft models. We reasoned that CCN5 distinguishes SP and NSP and could reprogram SP to NSP transition, thereby delaying tumor growth in the xenograft model. Collectively, we reveal how CCN5-signaling underlies the driving force to prevent TNBC growth and progression.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
CCN5 overexpression is associated with good prognosis of human breast cancer and CCN5-treatment suppresses growth and invasive phenotypes of TNBC cells. (A) Plots generated by SurvExpress program to analyze samples of breast invasive carcinoma patients from TCGA show the expression levels of CCN5. Low- and high-risks groups are shown in green and red, respectively. (B) Kaplan-Meier overall survival curves of CCN5 signatures were constructed by using the SurvExpress program and the P-value resulting from a t-test of the difference. Low- and high-risks groups are shown in green and red, respectively. (C) Effect of hrCCN5 or vehicle alone (control) on cell survival of TNBC cells (i.e., MDA-MB-231 and HCC-70) was measured after 48 h using anchorage-dependent growth (ADG)/Colony/focus forming assay. We determined the plating efficiency (PE) (left panel) and survival fraction (SF) (right panel) in both cell lines as described in Materials and Methods. Upper panel is the representative images of ADG in different treatment setups. Bar graphs indicate PE (left) and SF (right) in hrCCN5-treated or control samples. Error bars indicate mean ± SD, and represent at least three independent experiments. (D) Dose-dependent effect of hrCCN5 on apoptotic cell death was measured in TNBC cells after 48 h using cell-death detection ELISA kits. Error bars indicate mean ± SD, and represent at least three independent experiments. (E) Representative Western blots and quantification (bargraph) of Bax/BCl-2 ratio in the lysates of hrCCN5-treated or vehicle-treated MDA-MB-231 cells. β-actin was used as loading controls. Error bars indicate mean ± SD, and represent at least three independent experiments. (F) Effect of hrCCN5 or vehicle alone on aggressive malignant phenotypes of TNBC cells was measured by anchorage-independent growth (AIG) using fluorescence-based soft agar colony forming assay. Error bars indicate mean ± SD, and represent at least three independent experiments. (G) Detection of morphological alteration from mesenchymal to epithelial type in MDA-MB-231 cells following hrCCN5 treatment using Phalloidin-FITC. (H) Representative Western blots of EMT markers in the lysates of hrCCN5-treated or vehicle-treated MDA-MB-231 cells. β-actin was used as loading controls. Statistical significance was determined using long rank test, two-way ANOVA and two-tailed unpaired Student’s t-test. All the photographs are cropped from original figures.
Figure 2
Figure 2
CCN5-treatment suppresses sphere-forming ability and self-renewal of tumor initiating cell (TICs) population of TNBC cells. (A,B) Effect of hrCCN5 or vehicle alone (control) on the mammosphere forming ability of MDA-MB-231 and HCC-70 was determined at different time points indicated in the Figure. Photographs illustrate structure and size of spheres in hrCCN5-treated and control cells. Bar graph represents the distribution of mammospheres of different sizes (left) and area (right) in treated and untreated groups. Scale bar = 100 μm. Error bars indicate mean ± SD, and represent at least three independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test.
Figure 3
Figure 3
CCN5 treatment prevents in vitro migration of TNBC cells. (A) The schematic representation of experimental design. Cells were treated with hrCCN5 (250 ng/ml) or vehicle for 7 days and then cells (10,000 cell/well) were seeded on the upper chamber of the modified Boyden chamber in the presence (PT + T) or absence (C or PT) of hrCCN5 for overnight migration towards serum. C, control, T, treatment and PT, pre-treated. Individual figures (i.e., T-flask and petri-disk) were obtained from ScientificSlides suite, a Microsoft PowerPoint based software. (B) The bar graph represents the quantification of migration efficiency in hrCCN5-treated and untreated groups. The extent of migration was measured according to the protocol indicated in the Materials and Methods section. Data show mean ± SD, and are representative of at least three independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test.
Figure 4
Figure 4
Differential expression of epithelial and mesenchymal markers in side population (SP) and non-side population (NSP) in MCF-7 cell line. (A,B) Western blot analysis and quantification of epithelial and mesenchymal/stem cell markers in SP and NSP cells isolated from MCF-7 cell line. Data show mean ± SD, and are representative of at least three independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test. All the photographs are cropped from original figures.
Figure 5
Figure 5
Expression profiles of ER-α, Her-2 and CCN5 in MCF-7 heterogeneous cell populations. (A) ER-α Status. Detection and quantification (bargraph) of ER-α protein level in NSP and SP cells isolated from MCF-7 cells using Western blot analysis. β-actin is used as a loading control. (B) Her-2 status. Detection and quantification (bargraph) of ER-α protein level in NSP and SP cells isolated from MCF-7 cells using Western blot analysis. β-actin is used as a loading control. (C) CCN5 status. Western blot analysis (upper and middle left panels), Northern Blot analysis (Upper and middle right panels) and qRT-PCR (lower panel) for the quantification of CCN5 expression in NSP and SP cells isolated from MCF-7 cell line. Data show mean ± SD, and are representative of at least three independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test. All the photographs are cropped from original figures.
Figure 6
Figure 6
CCN5 overexpression suppresses EMT program in SP of MCF-7 cells. (A,B) Western blot analysis and quantification of epithelial (A) and mesenchymal (B) markers in SP cells stably transfected with vectors containing CCN5 gene or vector alone. Data show mean ± SD, and are representative of at least three independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test. All the photographs are cropped from original figures.
Figure 7
Figure 7
CCN5 regulation of ER-α in subpopulation of MCF-7 cells. (A) The Western blot analysis represents the expression of pER-α in NSP cells treated with anti-CCN5 antibody treatment (500 ng/ml) or PBS and SP cells treated with hrCCN5 protein (250 ng/ml) treatment or PBS for 72 h. GAPDH was used as a loading control. (B) Florescence microscopy for ER-α in NSP cells with or without CCN5 antibody (500 ng/ml) treatment and SP cells with or without hrCCN5 protein (250 ng/ml) treatment for 72 hours. ER-α was stained by indirect immunofluorescence, using a FITC-conjugated secondary antibody. α-Tubulin was stained using Alexa Fluor 555-conjugated antibody. First, second and third columns show staining for ER-α, DAPI and α-Tubulin respectively. Merge images of ER-α and α-Tubulin (fourth column) document lower expression of ER-α upon anti-CCN5 antibody treatment in MCF7-NSP cells and elevated ER-α expression after hrCCN5 protein treatment in MCF7-SP cells. Scale bar = 50 μm.
Figure 8
Figure 8
CCN5 prevents in vitro SP cell proliferation, migration and tumor regression in vivo. (A) Cell growth analysis. Cell growth analysis of NSP and SP in the presence or absence of hrCCN5 (250 ng/ml) was measured in different time points as indicated in the Figure by cell counting. Error bars indicate mean ± SD, and represent at least three independent experiments. (B) In vitro migration assay. NSP, SP and CCN5-transfeted SP (SPCCN5(T)) cells were seeded on the upper chamber of the Boyden chamber and allowed them to migrate towards serum overnight. The extent of migration was measured according to the protocol indicated in the Materials and Methods section. Data show mean ± SD and are representative of at least eight independent experiments. (C) Representative subcutaneous tumor xenograft nude mouse model exhibiting tumor growth by SP cells within 3–5 days following inoculation while no sign of tumor growth in the NSP cell-inoculation site. (D) Tumor growth curves of NSP, SP and SPCCN5(T) cells. Growth curve was plotted by measuring the relative tumor volume in different time points as indicated in the graph. Error bars represent means ± SD (n = 5 mice per group). *p < 0.001(SP vs SPCCN5(T)), **p < 0.0001 (SP vs NSP), ***p < 0.0001 (SPCCN5(T) vs NSP). Statistical significance was determined using two-tailed unpaired Student’s t-test.
Figure 9
Figure 9
Diagram illustrating CCN5 regulation of molecular markers associated with cell growth, EMT and stemness in TNBC cells. Blue indicates upregulated proteins and orange indicates downregulated proteins. Individual figures were obtained from ScientificSlides suite, a Microsoft PowerPoint based software.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Polyak K. Heterogeneity in breast cancer. The Journal of clinical investigation. 2011;121:3786–3788. doi: 10.1172/JCI60534. - DOI - PMC - PubMed
    1. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–890. doi: 10.1016/j.cell.2009.11.007. - DOI - PubMed
    1. Blick T, et al. Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in human breast cancer. Journal of mammary gland biology and neoplasia. 2010;15:235–252. doi: 10.1007/s10911-010-9175-z. - DOI - PubMed
    1. Bill R, Christofori G. The relevance of EMT in breast cancer metastasis: Correlation or causality? FEBS letters. 2015;589:1577–1587. doi: 10.1016/j.febslet.2015.05.002. - DOI - PubMed
    1. Barriere G, Riouallon A, Renaudie J, Tartary M, Rigaud M. Mesenchymal and stemness circulating tumor cells in early breast cancer diagnosis. BMC cancer. 2012;12:114. doi: 10.1186/1471-2407-12-114. - DOI - PMC - PubMed

Publication types

Substances