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. 2011;6(6):e20701.
doi: 10.1371/journal.pone.0020701. Epub 2011 Jun 8.

Evidence that aberrant expression of tissue transglutaminase promotes stem cell characteristics in mammary epithelial cells

Anupam Kumar  1 , Hui Gao, Jia Xu, James Reuben, Dihua Yu, Kapil Mehta
Affiliations

Evidence that aberrant expression of tissue transglutaminase promotes stem cell characteristics in mammary epithelial cells

Anupam Kumar et al. PLoS One. 2011.

Abstract

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population, but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation, increased ability of cells to form mammospheres, and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells, which express high basal levels of TG2, shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together, these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TG2 expression confers CD44high/CD24low phenotype in non-transformed and transformed breast epithelial cells.
(A) Immunoblot analysis (left panel) showing the expression of TG2 in vector- (MCF10A-Vec), TG2-lentiviral infected MCF10A cells (MCF10A-TG2) and MCF10A-TG2 cells infected with either control- (sh-control) or TG2-shRNA (shTG2). FACS analysis (Right panel) of cell-surface markers CD44 and CD24 in the cells described in left panel. (B) Immunoblot analysis (left panel) showing basal expression of TG2 in drug-sensitive (MCF-7) and drug-resistant (MCF7/RT) breast cancer cells and in MCF7/RT cells after their transfection with control or TG2-shRNA. FACS analysis (right panel) for cell-surface markers CD44 and CD24 in cells described in left panel. (C) Immunoblot analysis (left panel) showing basal expression of TG2 in highly aggressive MDA-231 breast cancer cells before and after their transfection with control- or TG2-shRNA. FACS analysis (right panel) for CD44 and CD24 antigen expression in cells described in left panel.
Figure 2
Figure 2. TG2 expression promotes mammosphere-forming ability in breast epithelial cells.
(A) Phase-contrast images of mammospheres formed after 10 days' culture of indicated cells in the mammosphere medium. (B) Quantification of mammospheres formed by cells (top panel) and immunoblot analysis for TG2 expression (bottom panel) in cells described in (A). (C) Quantification of mammospheres formed by MCF10A-TG2, MCF7/RT and MDA-231 cells transfected with either control (sh-contol) or TG2 (sh-TG2) shRNA. The data shown are average number of mammospheres formed/1000 seeded cells ± SEM from triplicate values of a representative experiment performed three times with similar results.
Figure 3
Figure 3. TG2 expression enhances the self-renewal ability of breast epithelial cells.
To test the self-renewal ability, we assessed the sphere-initiation efficiency of serially passaged cells cultured as mammospheres for up to four generations. Number of mammospheres formed by (A) MCF10A-Vec, (B) MCF10A-TG2, (C) MCF-7 and (D) MCF-7/RT cells at different passages (M1 to M4). (E) Immunoblot analysis showing the expression of TG2 in MCF-7/RT cells transfected with either control or TG2 shRNA. Number of mammospheres formed by control- (F) and TG2-shRNA (G)-transfected MCF-7/RT cells during different passages (M1 to M4). The data shown are average number of mammospheres formed/1000 seeded cells ± SEM from triplicate values of experiments repeated twice with similar results. Please note 10-fold difference in y-axis scales for cells lacking TG2 (0–35) and TG2 expressing cells (0–350).
Figure 4
Figure 4. TG2 induced stem cellness is dynamic in nature.
(A) CD44high/CD24high cells and CD44high/CD24low cells were separated by FACS. (B) Phase-contrast images of morphology (left) and mammospheres (right) obtained after seeding the CD44high/CD24high (top) and CD44high/CD24low (bottom) cells from (A). Magnification x100; inset, X500 (C) Phase-contrast images of morphology (left) and mammospheres (right) obtained by seeding CD44high/CD24high (top) and CD44high/CD24low (bottom) cells at the 5th passage (p5). (D) Quantification of mammospheres formed by p1 and p5 cells from the sorted CD44high/CD24high and CD44high/CD24low populations. The data shown are average number of mammospheres formed/1000 seeded cells ±SEM from triplicate values of experiments repeated twice with similar results (E) Immunoblot analysis for TG2 expression in p1 and p5 cells from the sorted CD44high/CD24high and CD44high/CD24low populations.
Figure 5
Figure 5. TG2 expression does not affect the differentiation ability of mammary stem cells.
Mammospheres formed by indicated cell type were subsequently grown for 12 days in Matrigel containing prolactin (2 µg/ml). Differentiated structures were immunostained for basal (CD49f/integrin α6) and luminal (Muc1) cell markers. (A) Phase-contrast images of secondary structures formed by Matrigel cultures of indicated mammospheres in the presence of prolactin. (B). The differentiated structures from 10A-Vec and 10A-TG2-derived mammospheres show the presence of luminal (red), basal (green), and bipotent (yellow) cells. MDA-231 mammospheres, in contrast, under these conditions failed to differentiate into secondary structures (A) and did not show luminal marker expression but showed only low basal cell marker expression.
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