Introduction The p functions are ubiquitously

Introduction The p53 functions are ubiquitously altered in cancer cells by mutations/perturbation of its signaling pathways, and loss of p53 activity is a prerequisite for cancer development. Mutant p53 is thought to play a pivotal role in promoting invasion, favoring cancer cell exit from the primary tumor site and dissemination, ultimately leading to metastasis formation (Gadea et al., 2007; Muller et al., 2009; Roger et al., 2010; Vinot et al., 2008). Recent reports have documented a p53 role in stem cell homeostasis and pluripotency. Wild-type (WT) p53 counteracts somatic cell reprogramming (Hong et al., 2009; Kawamura et al., 2009; Liu et al., 2009; Utikal et al., 2009), whereas mutant p53 stimulates induced pluripotent stem (iPS) cell formation (Sarig et al., 2010). Depletion of p53 significantly increases cell reprogramming efficacy and facilitates iPS cell generation (Kawamura et al., 2009). Consequently, p53 might be considered as the guardian of the pituitary adenylate cyclase-activating peptide and also of reprogramming. All these functions are associated with full-length p53 (i.e., the TAp53α isoform). However, the TP53 gene encodes at least 12 different physiological isoforms (TAp53 [α, β, and γ], Δ40p53 [α, β, and γ], Δ133p53 [α, β, and γ], and Δ160p53 [α, β, and γ]) (Bourdon, 2007) via several mechanisms: alternative promoters (the TA and Δ133 isoforms), alternative intron splicing (intron 2: Δ40 isoforms and intron 9: α, β, and γ isoforms), and alternative translational initiation sites (Δ40 and Δ160 isoforms). The TAp53α isoform is the best described and classically mentioned in the literature as p53. Basically, p53 isoforms can be divided into two groups as follows: (1) long isoforms that contain the transactivation domain (TA and Δ40), and (2) short isoforms without the transactivation domain (Δ133 and Δ160). Furthermore, the β and γ isoforms do not contain the canonical C-terminal oligomerization domain, but an additional domain with unknown function(s) (Khoury and Bourdon, 2011). The p53 isoforms modify p53 transcriptional activity in many processes, such as cell-cycle progression, programmed cell death, replicative senescence, cell differentiation, viral replication, and angiogenesis (Aoubala et al., 2011; Bernard et al., 2013; Bourdon et al., 2005; Marcel et al., 2012; Terrier et al., 2011, 2012). Importantly, p53 isoforms are specifically deregulated in human tumors (Machado-Silva et al., 2010). However, the functions of p53 isoforms in cancer stem cell (CSC) homeostasis have never been explored. Here, we show that the Δ133p53β isoform is specifically involved in promoting cancer cell stemness. Overexpression of Δ133p53β in human breast cancer cell lines stimulated mammosphere formation and the expression of key pluripotency and stemness regulators (SOX2, OCT3/4, and NANOG and CD24/CD44), but not C-MYC. Furthermore, using MDA-MB-231-based cell lines, we show that increased expression of Δ133p53 isoforms correlates with the increased metastatic potential and with mammosphere formation. Finally, incubation of MCF-7 and MDA-MB-231 cells with the anti-cancer drug etoposide also promoted cell stemness in a Δ133p53-dependent manner. Our results demonstrate that short p53 isoforms positively regulate CSC potential regardless of any p53 mutation. Consequently, WT TP53, which is considered a tumor suppressor gene, also can act as an oncogene through Δ133p53β expression.
Results
Discussion In this work, by modulating p53 isoform expression in breast cancer cell lines, we show that Δ133p53 isoforms have a role in regulating their stemness potential. Surprisingly, depletion of all p53 isoforms in MCF-7 cells significantly reduced mammosphere formation (a hallmark of CSC potential), although previous reports indicate that TAp53α hinders cell reprogramming. Conversely, selective depletion of TAp53 and Δ40p53 isoforms with the Sh2 shRNA did not affect mammosphere formation, suggesting that Δ133p53 isoforms are responsible for this activity. We then confirmed this hypothesis by showing that mammosphere formation was strongly reduced upon knockdown of these small isoforms (Figure 1). Similarly, depletion of the β isoforms had a deleterious effect on the capacity of MCF-7 cell to form mammospheres, while depletion of the α isoforms did not have any effect. Moreover, all changes in p53 isoform expression, particularly Δ133p53, were associated with variations in the expression of SOX2, OCT3/4, and NANOG, key cell pluripotency/reprogramming genes, but not of C-MYC (Figures 1 and 2). Furthermore, the finding that Δ133p53β isoform specifically promoted mammosphere formation and increased the proportion of CD44+/CD24− cells indicates that this isoform positively regulates CSC potential in MCF-7 breast cancer cells. Indeed, our data show that Δ133p53β expression positively correlates with SOX2, OCT3/4, and NANOG expression, genes responsible for cell pluripotency induction and maintenance (Figure 2). Finally, using a breast cell model of tumor aggressiveness, we show that higher metastatic potential and chemoresistance are coupled with increased expression of the Δ133p53 isoforms, CSC stemness, and increased expression of key pluripotency/reprogramming genes (Figures 3 and 4).