The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes

Abstract

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying-or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.

Oncogenic Ras: A double-edged sword for human epidermal stem and transient amplifying cells

“…2). 45 Indeed, TA-keratinocytes Figure 2. Ras-V12 overexpression and early morphological changes in keratinocyte cultures Primary keratinocyte cultures display 5-10% of holoclones, high p63 and Bmi-1 levels whereas p16INK4a is undetectable.…”

“…41 Oncogenic Ras-expression outcome might depend on the clonogenic potential of the recipient human keratinocyte. 45 Indeed, Ras-V12-overexpression induces senescence in freshly isolated primary human keratinocyte cultures, but some cells escape senescence and proliferate indefinitely. Ras-overexpression in TA-or SC-enriched cultures shows that p16…”

“…Of note, the regulators of clonal conversion markedly increase already before bypass of senescence, in keeping with a deregulation of the machinery for selfrenewing of normal SCs. 45 Indeed, DNp63a isoform Figure 3. Growth suppression evasion, morphological changes and tumorigenicity in vivo Empty-vector-transduced primary cultures (blue line) undergo around 100 cell generations before senescence.…”

“…Immortalization is also sustained by activation of ERK1/2 and Akt pathways. 45 pRb inactivation might contribute to tumor progression not only through the loss of cell cycle control but also through the upregulation of ZEB factors that are modulators of E-cadherin and vimentin, key players of EMT. 49 Interestingly, also p63 negatively regulates ZEB1 and vimentin expression in keratinocytes whereas their targets, belonging to miR-200 family, can be regulated by p53 and Akt pathways.…”

“…3). 45 Although resistant Ras-V12-induced senescence subpopulation do not display cell transformation features in vitro, they have enhanced ability to drive tumor formation even 7 day after transduction. 45 …”