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Comparative Study
. 2015 Apr 20;6(11):9018-30.
doi: 10.18632/oncotarget.3379.

Cell type of origin as well as genetic alterations contribute to breast cancer phenotypes

Divya Bhagirath  1 , Xiangshan Zhao  1 , William W West  2   3 , Fang Qiu  4 , Hamid Band  1   2   5   6   7   3 , Vimla Band  1   3
Affiliations
Comparative Study

Cell type of origin as well as genetic alterations contribute to breast cancer phenotypes

Divya Bhagirath et al. Oncotarget. .

Abstract

Breast cancer is classified into different subtypes that are associated with different patient survival outcomes, underscoring the importance of understanding the role of precursor cell and genetic alterations in determining tumor subtypes. In this study, we evaluated the oncogenic phenotype of two distinct mammary stem/progenitor cell types designated as K5+/K19- or K5+/K19+ upon introduction of identical combinations of oncogenes-mutant H-Ras (mRas) and mutant p53 (mp53), together with either wild-type ErbB2(wtErbB2) or wild-type EGFR (wtEGFR). We examined their tumor forming and metastasis potential, using both in-vitro and in-vivo assays. Both the combinations efficiently transformed K5+/K19- or K5+/K19+ cells. Xenograft tumors formed by these cells were histologically heterogeneous, with variable proportions of luminal, basal-like and claudin-low type components depending on the cell types and oncogene combinations. Notably, K5+/K19- cells transformed with mRas/mp53/wtEGFR combination had a significantly longer latency for primary tumor development than other cell lines but more lung metastasis incidence than same cells expressing mRas/mp53/wtErbB2. K5+/K19+ cells exhibit shorter overall tumor latency, and high metastatic potential than K5+/K19- cells, suggesting that these K19+ progenitors are more susceptible to oncogenesis and metastasis. Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors.

Keywords: breast cancer; metastasis; stem cells; transformation; xenograft.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Transformation of K5+/K19− or K5+/K19+ cells with different gene combination
(A) K5+/K19− or K5+/K19+ cell lines over-expressing mutant p53, mutant Ras, wild type ErbB2 and wild type EGFR in triple oncogene combinations were analyzed by Western Blotting. β-Actin was used as loading control. (B) Anchorage independent growth assay of K5+/K19− and K5+/K19+ cells with vector or triple gene combinations. Mean ± S.D of a representative experiment done in triplicate is shown. Three independent experiments were done. (C) Representative images (magnification 40X) of colonies from K5+/K19− and K5+/K19+ cells with vector or triple oncogene combination are shown here.
Figure 2
Figure 2. In-vitro self-renewal and differentiation of transformed K5+/K19− or K5+/K19+
(A) and (B). Control or transformed K5+/K19− (A) or K5+/K19+ (B) cells were grown in DFCI-2 (differentiation) medium in Matrigel. Acini were trypsinized and stained with PE-Cy5 conjugated anti-CD49f and FITC conjugated anti-MUC1 and subjected to FACS analysis. (C) Representative images (magnification 4X) of tumorspheres from K5+/K19− and K5+/K19+ cells with vector or triple oncogene combinations are shown. (D) For tumorsphere-formation assay indicated cell lines were cultured in low-attachment plates in MEGM media for 3 weeks. Spheres ≥ 200 μm were quantified. Mean +/− SD of a representative experiment done in 6 replicates is shown.
Figure 3
Figure 3. Transformed K5+/K19− or K5+/K19+ cells give rise to distinct tumors
(A) Representative images of H&E staining of tumor sections (magnification 20X) from K5+/K19− and K5+/K19+ cells over-expressing mRas/mp53/wtErbB2 (upper panel) or mRas/mp53/wtEGFR (Lower panel). (B) Images from different tumors at magnification 20X. Immunohistochemical staining of tumor sections with anti-CK5 (Basal/Stem), anti-MUC1 (Luminal), anti-αSMA (Myoepithelial) anti-vimentin (Stem/myoepithelial) and claudin4 (for claudin-low) antibodies. (C) Representative image of tumors from K5+/K19− orK5+/K19+ cells double immunostained with anti-αSMA (green) and anti-vimentin (red) show presence of claudin-low (SMA+/vimentin+) areas within different tumors. (D) Same tumor sections were double immunostained E-Cadherin (green) and vimentin (red) show presence of luminal like (E-Cadherin+) areas within different tumors. DAPI (blue) shows nucleus.
Figure 4
Figure 4. Transformed K5+/K19− or K5+/K19+ cells retain stem cell characteristics in-vivo
(A) Immunostaining with lineage specific markers of tumors and mammary duct like structures with varying degree of hyperplasia originating from K5+/K19+ cells over-expressing mRas/mp53/wtEGFR. Yellow arrowheads with H indicate human mammary ductal structures, white arrowheads with M indicate mouse mammary ducts and arrow with T shows tumor. (B) Immunofluorescence staining of the same tumor section with anti-vimentin (red), anti-αSMA (green) antibodies at magnifications 20X (left panel) and 40X (right panel). White arrowhead indicate mouse ductal structure, yellow arrowheads indicate co-expression in human ductal structures.
Figure 5
Figure 5. In-vivo tumor and metastasis formation from transformed K5+/K19− or K5+/K19+ cells
(A) Kaplan-Meier plot for probability of no tumor in mice injected with K5+/K19− and K5+/K19+ cells over-expressing mRas/mp53/wtErbB2 or mRas/mp53/wtEGFR from the second experiment. (B) Kaplan-Meier plot for probability of no metastasis in above mentioned mice. (C) Representative image of mice with or without lung metastasis as shown by IVIS luciferase imaging. (D) Representative image of lung metastatic lesions formed by different cell types with either mRas/mp53/wtErbB2 (upper panel) or mRas/mp53/wtEGFR (Lower panel). (E) Representative image of liver metastasis as seen in mice injected with K5+/K19− cells over-expressing mRas/mp53/wtEGFR.
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