PPP1CA contributes to the senescence program induced by oncogenic Ras

Abstract

Ectopic expression of conditional murine p53 (p53val135) and oncogenic ras is enough to induce a senescent-like growth arrest at the restrictive temperature. We took advantage of this cellular system to identify new key players in the ras/p53-induced senescence. Applying a retroviral-based genetic screen, we obtained an antisense RNA fragment against PPP1CA, the catalytic subunit of protein phosphatase 1alpha, whose loss of function bypasses ras/p53-induced growth arrest and senescence. Expression of a specific short hairpin (sh)RNA against PPP1CA impairs the p53-dependent induction of p21 after DNA damage and blocks the subsequent pRb dephosphorylation, thus bypassing p53-induced arrest. We found that oncogenic ras promotes an increase in the intracellular level of ceramides together with an increase in the PPP1CA protein levels. Addition of soluble ceramide to the cells induced a senescence phenotype that is blocked through PPP1CA downregulation by specific shRNA. Analysis of human tumors suggests that one of the PPP1CA alleles might be lost in a high percentage of carcinomas such as kidney and colorectal. The overexpression of two out of five PPP1CA alternative spliced variants reduced tumor cell growth and the downregulation of the protein to hemizygosity increased the anchorage-independent growth. We propose that oncogenic stress induced by ras causes ceramide accumulation, therefore, increasing PPP1CA activity, pRb dephosphorylation and onset of the p53-induced arrest, contributing to tumor suppression.

Spinophilin acts as a tumor suppressor by regulating Rb phosphorylation

“…To determine whether the loss of Spn also affected the PPP1CA level under stress conditions, we expressed oncogenic Ras in the cells; this has previously been reported to increase the PPP1CA level. 23,27 The expression of oncogenic Ras induced a 50% increase in the PPP1CA level in the WT MEFs but not in the Spn (-/-) MEFs (Fig. 1C and D).…”

“…However, similar to PPP1CA overexpression, 21,23 SPN overexpression resulted in growth inhibition in culture independently of the status of Rb and p53. This may be due to PP1-target proteins other than Rb, whose phosphorylation is thought to enable cells to replicate DNA, such as DNApolα or TopoII.…”

“…21 The targeting of Rb by PP1 contributes to the dephosphorylation and subsequent activation of Rb, shutting down the G 1 /S phases of the cell cycle. 21,22 Furthermore, the PP1a-mediated dephosphorylation of Rb contributes to the senescent phenotype induced by oncogenic Ras 23 and the constitutive inactivation of Rb by maintaining highly phosphorylated Rb contributes to cell cycle deregulation and tumorigenesis. [24][25][26] Therefore, SPN may contribute to tumorigenesis by regulating the functions of PP1 and/or Rb.…”

“…In addition, it has been shown that low levels of Rb activity activate the E2F family of transcription factors, which, in turn, activate p19 ARF , thereby contributing to p53 upregulation. [37][38][39][40] To explore this possibility, we first tested the effect of shRNA-mediated SPN silencing in cells expressing the Val135 thermosensitive p53 mutant (p53(-/-) ts cells) 23 (Fig. 4A).…”

“…55 However, complete loss of PP1a is also deleterious for the cells, 21 and only partial loss of activity has been associated with tumorigenesis. 23 PP1 does not only regulate the cell cycle, and Rb is not its only substrate. Therefore, SPN loss would be expected to have pleiotropic effects that are not solely related to Rb, but which may be equally important for tumor cell growth/survival.…”