Dr. Weeks’ Comment: For the past 15 years P53 was considered a tumor suppressor gene. That was the holy grail of oncology – to stimulate the gene that suppressed tumors. Now we see that P53 also contributes to cancer STEM cell development. Like chemotherapy and radiation – two films of conventional cancer treatment which Dr. Max Wicha and other esteemed colleagues claim “make your cancer worse” (by stimulating cancer STEM cells despite killing the less dangerous cancer TUMOR cells), we now see that stimulating P53 also may make your cancer worse by turning on the genes to suppress cancer TUMOR cells while dangerously stimulating cancer STEM cell progression.
“…In summary, our study shows that Î”133p53Î² plays a critical role in supporting the CSC potential of breast cancer cells…”
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 genome and also of reprogramming.
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, Terrier et al., 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 Î”133p53Î² isoform is specifically involved in promoting cancer cell stemness. Overexpression of Î”133p53Î² 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 notC-MYC. Furthermore, using MDA-MB-231-based cell lines, we show that increased expression of Î”133p53 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 Î”133p53-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 Î”133p53Î² expression…..
Chemotherapy Treatment of Breast Cancer Cell Lines Upregulates the Expression of Î”133p53 Isoforms and Activates Key Reprogramming Genes
Topoisomerase II inhibitors (etoposide-VP16 and doxorubicin) are frequently used as adjuvant chemotherapy treatment for several cancer types alone or in combination with other drugs (cisplatin most frequently). Topoisomerase II inhibitors induce double-strand DNA breaks, a genotoxic stress that strongly activates p53 signaling. Upregulation of TAp53 should be beneficial due to its ability to induce cell-cycle arrest, apoptosis, and to negatively regulate cell reprogramming. We thus assessed whether etoposide could affect Î”133p53 expression and CSC potential in breast cancer cell lines. Increasing concentrations of etoposide resulted in TAp53Î± stabilization in MCF-7. As expected, p21 expression (positively regulated by p53) was increased, whereas C-MYC expression (negatively regulated by p53) was reduced (Figure 4A), as also confirmed by qRT-PCR quantification (Figure 4B). Moreover, qRT-PCR and western blot analysis showed that, upon etoposide treatment, Î”133p53 isoforms (Figures 4C and 4D) as well as OCT3/4, NANOG, and SOX2 (Figure 4E) were strongly upregulated in a dose-dependent manner. This last result is particularly intriguing because TAp53Î±, which is considered as a negative regulator of pluripotency/reprogramming genes, is stabilized and transcriptionally active….. we evaluated the effect of etoposide treatment on mammosphere formation in Sh2-transduced MCF-7 cells. Etoposide treatment in control cells (active TAp53) significantly reduced mammosphere formation, whereas it did not have any significant effect in Sh2-transduced cells (Figure 4G). Moreover, Î”133p53 level was correlated with the expression of reprogramming genes (Figures 4H and 4I). These data indicate that TAp53 and Î”133p53Î² have an antagonistic action in sphere formation.
…..These data suggest that, in human breast cancer cells, the topoisomerase II inhibitor etoposide increases Î”133p53 expression, resulting in the activation of the reprogramming genes NANOG, SOX2, and OCT3/4.
In this work, by modulating p53 isoform expression in breast cancer cell lines, we show that Î”133p53 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 TAp53Î± hinders cell reprogramming. Conversely, selective depletion of TAp53 and Î”40p53 isoforms with the Sh2 shRNA did not affect mammosphere formation, suggesting that Î”133p53 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 Î² isoforms had a deleterious effect on the capacity of MCF-7 cell to form mammospheres, while depletion of the Î± isoforms did not have any effect. Moreover, all changes in p53 isoform expression, particularly Î”133p53, 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 Î”133p53Î² isoform specifically promoted mammosphere formation and increased the proportion of CD44+/CD24âˆ’ cells indicates that this isoform positively regulates CSC potential in MCF-7 breast cancer cells. Indeed, our data show that Î”133p53Î² 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 Î”133p53 isoforms, CSC stemness, and increased expression of key pluripotency/reprogramming genes (Figures 3 and 4).
Importantly, our results show that TAp53 (Î±, Î², and Î³) silencing does not affect CSC formation, whereas Î”133p53 (Î±, Î², and Î³) silencing does, indicating that the CSC potential is mainly regulated by Î”133p53 activity rather than by TAp53. These findings challenge the prominent role of full-length p53 (TAp53Î±) in CSC regulation and clearly indicate that most of the p53-mediated regulation of the CSC potential is via the short p53 isoforms.
In summary, our study shows that Î”133p53Î² plays a critical role in supporting the CSC potential of breast cancer cells. Mutations of TP53 are considered the main mechanism for inhibiting its tumor suppressor activity. Here, we demonstrate that p53 mutations are not necessary to block p53 tumor suppressor activity, because expression of specific p53 isoforms, particularly Î”133p53Î², is sufficient to increase CSC activity. Our study challenges the paradigm that TP53 always acts as a tumor suppressor by showing that Î”133p53Î² antagonizes TAp53Î± to promote CSC potential.