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

Bhagirath D, Zhao X, West WW, Qiu F, Band H, Band V - Oncotarget (2015)

Bottom Line: Both the combinations efficiently transformed K5+/K19- or K5+/K19+ cells.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.Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: It has been shown that loss of function of the tumor suppressor p53 enhances self-renewal ability of mammary stem cells [19]. Similarly, other studies have shown that EGFR [20, 21], ErbB2 [22] or Ras [23, 24] play an important role in mammary stem cell self-renewal. We have previously shown that upon immortalization (pre-neoplastic transformation) with certain oncogenes, the stem/progenitor cell lines lose their ability to differentiate into myoepithelial cells [25]. Therefore, in this study we evaluated the impact of triple oncogene combinations on stem cell self-renewal and differentiation. We have previously shown that K5+/K19− and K5+/K19+ mammary stem/progenitor cell lines are bi-potent stem/uncommitted progenitors and are able to differentiate into both luminal and myoepithelial cells under appropriate differentiating conditions [6]. Furthermore, when grown in 3D matrigel culture and subjected to differentiating media DFCI-2, the stem/progenitor cells are preferentially induced into luminal differentiation [25]. Notably, when we subjected K5+/K19− and K5+/K19+ cells expressing triple oncogene combinations to differentiation using in-vitro 3D matrigel culture, we observed an increased CD49f+ (marker for stem cell) fraction (97% vs. 86% for K5+/K19− and 96% vs. 92% for K5+/K19+) and decrease in MUC1+ (marker for luminal differentiation) fraction (0.4%, 0.1% vs. 0.9% for K5+/K19− and 0.5%, 0.2% vs. 1.2% for K5+/K19+) in transformed lines vs. their controls (Figure 2A, 2B). These results indicate that oncogene-mediated transformation of mammary stem/progenitor cells reduces their ability to differentiate. Next, we assessed the ability of oncogene-transformed vs. vector control mammary stem/progenitor cell lines to form tumorspheres in ultra-low attachment plates, a commonly used assay to determine cancer stem cell self-renewal abilities [26–29]. Vector expressing cells showed formation of small spheres (< 200 μm) and were not counted (Figure 2C). Notably, cells expressing the triple combinations of oncogenes exhibited a significant increase in number of tumorsphere (> 200 μm in size) formed (Figure 2C, 2D). Both the transformed K5+/K19− or K5+/K19+cells formed secondary tumorspheres upon re-plating (Supplementary Figure S1A) and the efficiency for tertiary tumorsphere formation was substantially enriched for cells over-expressing oncogene combination mRas/mp53/wtEGFR (Supplementary Figure S1B) demonstrating increased self-renewal capabilities of these transformed cell lines. Taken together, these results demonstrate an increase in stem cell property of triple oncogene transformed derivatives.


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

Bhagirath D, Zhao X, West WW, Qiu F, Band H, Band V - Oncotarget (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496199&req=5

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.
Mentions: It has been shown that loss of function of the tumor suppressor p53 enhances self-renewal ability of mammary stem cells [19]. Similarly, other studies have shown that EGFR [20, 21], ErbB2 [22] or Ras [23, 24] play an important role in mammary stem cell self-renewal. We have previously shown that upon immortalization (pre-neoplastic transformation) with certain oncogenes, the stem/progenitor cell lines lose their ability to differentiate into myoepithelial cells [25]. Therefore, in this study we evaluated the impact of triple oncogene combinations on stem cell self-renewal and differentiation. We have previously shown that K5+/K19− and K5+/K19+ mammary stem/progenitor cell lines are bi-potent stem/uncommitted progenitors and are able to differentiate into both luminal and myoepithelial cells under appropriate differentiating conditions [6]. Furthermore, when grown in 3D matrigel culture and subjected to differentiating media DFCI-2, the stem/progenitor cells are preferentially induced into luminal differentiation [25]. Notably, when we subjected K5+/K19− and K5+/K19+ cells expressing triple oncogene combinations to differentiation using in-vitro 3D matrigel culture, we observed an increased CD49f+ (marker for stem cell) fraction (97% vs. 86% for K5+/K19− and 96% vs. 92% for K5+/K19+) and decrease in MUC1+ (marker for luminal differentiation) fraction (0.4%, 0.1% vs. 0.9% for K5+/K19− and 0.5%, 0.2% vs. 1.2% for K5+/K19+) in transformed lines vs. their controls (Figure 2A, 2B). These results indicate that oncogene-mediated transformation of mammary stem/progenitor cells reduces their ability to differentiate. Next, we assessed the ability of oncogene-transformed vs. vector control mammary stem/progenitor cell lines to form tumorspheres in ultra-low attachment plates, a commonly used assay to determine cancer stem cell self-renewal abilities [26–29]. Vector expressing cells showed formation of small spheres (< 200 μm) and were not counted (Figure 2C). Notably, cells expressing the triple combinations of oncogenes exhibited a significant increase in number of tumorsphere (> 200 μm in size) formed (Figure 2C, 2D). Both the transformed K5+/K19− or K5+/K19+cells formed secondary tumorspheres upon re-plating (Supplementary Figure S1A) and the efficiency for tertiary tumorsphere formation was substantially enriched for cells over-expressing oncogene combination mRas/mp53/wtEGFR (Supplementary Figure S1B) demonstrating increased self-renewal capabilities of these transformed cell lines. Taken together, these results demonstrate an increase in stem cell property of triple oncogene transformed derivatives.

Bottom Line: Both the combinations efficiently transformed K5+/K19- or K5+/K19+ cells.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.Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA.

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.

No MeSH data available.


Related in: MedlinePlus