Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Abstract

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.

Accelerated Senescence of Cancer Stem Cells: A Failure to Thrive or a Route to Survival?

“…This was downstream of activated TP53, as both p21 and partly OCT4A became downregulated after TP53 RNAi silencing ( Figure 2C) [45]. Further study [46] revealed that p53-activated OCT4A down-regulated p21CIP1, moderating its expression and preventing cells from precipitating terminal senescence or apoptosis (Figure 2D), thus providing the opportunity for repair. Downregulation of p21 via OCT4 was previously shown in ESC [47].…”

“…The bizarre duality of OCT4A with p21CIP1 was subdued and cell cycle in the PA1 clones returned to normal, on days 7-14 (Figure 2A), while silencing of stress-activated OCT4A prevented recovery [46]. This transient undecided state in G2 arrest, between true senescence and true self-renewal, was thus lasting as long as wtTP53 was activated by the DNA DSB.…”

“…Moreover, such stress-activated OCT4A was transiently disconnected from its self-renewal partners (SOX2 and NANOG), as those protein levels were low and/or not activated alongside Oct4A [46]. Therefore, the autoregulatory and feedforward loops seen in ESC [48] were not present, potentially due to the known down-regulation of the Nanog gene promoter by activated p53 [49] and/or the down-regulation of NANOG by high levels of OCT4A [50].…”

“…It stresses the importance of another possible player in this "undecided" stage between senescence and self-renewal, of transcriptional noise. (A) Cells were cytospun, ixed and stained for DNA image cytometry as previously described [46]. DNA content was determined for at least 200 cells in each condition and is represented as a percentage.…”

“…P53 is activated and simultaneously induces p21CIP1 and OCT4A. OCT4A moderates the expression of p21CIP1 preventing apoptosis or terminal senescence [45,46]. Activated p53 also downregulates the promoter of Nanog gene [49].…”