Platelet-derived growth factor B induces senescence and transformation in normal human fibroblasts

PDGFB constitutive expression induces both senescence and transformation in normal human diploid fibroblasts

To investigate if PDGFB oncogenic growth factor might impact oncogene induced senescence in normal human dermal fibroblasts, we constitutively expressed it. To this end, we have infected fibroblasts with a control or a PDGFB encoding retroviral vector and validated its constitutive expression (Figure 1A). Its constitutive expression resulted in a slight decrease in the growth of these cells when compared to their control counterparts (Figure 1B), suggesting that the PDGFB expression does not induce oncogene induced senescence. Nevertheless, a microscopic analysis enabled us to distinguish two distinct cell populations with striking morphology dissimilarities.

The first population of PDGFB expressing cells displayed features of senescence cells. Indeed they displayed a flattened morphology (Figure 1C), an increase Senescence Associated β Galactosidase (SA-β-Gal) activity (Figure 1D) and an increase Senescence Associated Heterochromatin Foci (SAHF) (Figure 1E). They also displayed an increase expression of Sprouty2 (Figure 1F) and p16 (Figure 1G) mRNA, 2 genes whose expression increased in senescent cells.

The second population of PDGFB expressing cells also displayed an altered morphology when compared to control cells. However, they did not display any characteristics of senescent cells but instead these cells had lost their contact inhibition: a feature of transformed cells (Figure 2A). Cellular transformation was further supported by the ability of PDGFB expressing cells to form colonies whereas the control cells stopped growing when reaching confluence (Figure 2B). Furthermore, PDGFB expressing cells were found to grow and to form colonies in soft agar when compared to control cells (Figure 2C). Together, these data show that PDGFB constitutive expression results in 2 distinct and “opposite” phenotypes in fibroblasts.

Some cellular oncogenes have been shown to induce both senescence and transformation, but theses processes generally appeared in a sequential manner; a mitogenic effect during the first days followed by senescence activation and subsequent proliferation arrest [13]. Interestingly, here, these 2 populations were not seen in a sequential manner but instead were triggered simultaneously a few days after infection. Importantly, these 2 populations are maintained with passages explaining the slow growth rate of the PDGFB expressing cells (Figure 1B). To maintain both populations, senescent cells have to be generated by the transformed cells during passages suggesting that PDGFB induced transformation is subject to stochastic events that have a tendency to disturb the homeostasis of the transformed cells, and then leading to senescence. In the meantime, the PDGFB induced senescent population might exert an intrinsic inhibition towards the PDGFB induced transformed population explaining that the transformed cells do not out grow.

Favoring an escape from PDGFB induced senescence increases PDGFB induced transformation

We next wanted to know whether the senescent population limits the ability of PDGFB to transform the cells. To this end, we wished to eliminate the PDGFB induced senescent population by decreasing p53 expression, a key player of the senescence response [6, 14]. We then ectopically expressed PDGFB with or without an shRNA targeting the p53 protein (Figure 3A). As expected, p53 knockdown strongly decreased the appearance of the senescence population in the PDGFB expressing cells as measured by loss of the SA-β-Gal positive cells (Figure 3B) and the decreased SAHF positive cells (Figure 3C). As hypothesized, loss of the senescent population in p53 knockdown cells allowed the transformed population to increase as seen by the increase cells with the transformed morphology (Figure 3D), the increase number of cells forming colonies and loosing the contact inhibition (Figure 3E), and the increase ability to form clones in soft agar (Figure 3F). These results support the view that the low senescence response induced by the PDGFB limits the growth of the PDGFB transformed cells.

Senescence markers are observed in PDGFB dependent human tumor samples

Dermatofibrosarcoma protuberans (DFSP) is a rare and locally aggressive skin sarcoma that appears at different age, starting at childhood. This type of tumor can grow and form large lesion, but it barely forms metastasis. DFSP displays in most if not all cases a chromosomal rearrangement resulting in the production of COL1A1/PDGFB precursor chimera [15]. The COL1A1/PDGFB precursor is next processed to the oncoprotein PDGFB, which is thought to be at the origin of DFSP [11]. Because our in vitro results suggest that PDGFB triggers senescence, we decided to investigate whether these human PDGFB depending tumors, with limited aggressiveness, might display some senescence features. The two senescence markers examined, Sprouty2 (Figure 4A) and p16 (Figure 4B), were found strongly up regulated in DFSP when compared to normal tissues. This supports the view that an increase of PDGFB signaling might result in the appearance of tumors composed of transformed and senescent cells, limiting the intrinsic aggressiveness of the tumors. Interestingly, the very rare aggressive version of the DFSP, the DFSP Fibrosarcomatous, may displays p53 loss of function [16]. This is in agreement with the idea that p53 loss might decrease the ability of PDGFB to induce senescence and potentially DFSP aggressiveness.

Our results also point out that a single oncogene such as the PDGFB, which according to our data is a weak senescence inducer, might trigger transformation with a minimal number of genetic events in fibroblasts. This result is rather unexpected at first sight. Indeed, classical cellular oncogenes such as the ones in the RAS-RAF-MEK pathway, that are strong oncogene induced senescence inducer, need to cooperate with other genetic events, such as loss of function of the p53 and Rb pathways, to transform normal human cells [17, 18]. In this study, we thus describe that the PDGFB oncogenic growth factor is able to activate cellular transformation of primary fibroblasts. Nevertheless, low level of senescence is a self limiting response that decreases the transformed phenotype. The price to pay for PDGF induced transformation, in a context of limited genetic events, is that although the transformed cell population grows, this growth is slow, probably limited by the effects of neighboring senescent cells.

Cell Culture

Human Dermal Fibroblast (Lonza) and virus producing GP293 cells (Clontech) were cultured in DMEM (Invitrogen). All medium were supplemented with 10% FBS (Lonza), 1% penicillin/streptomycin (Invitrogen), 0.36% gentamycin (Invitrogen).

Vectors

pBABE/PDGFB and empty pBABE were provided from Addgene and were described in [19]. pSR/shp53 retroviral vector encoding an shRNA directed against p53 was described in [20].

Transfection and infection

GP293 cells were transfected by PEI reagents according to the manufacturer's recommendations (Euromedex). Two days after transfection, the viral supernatant mixed with fresh medium (1/2) and polybrene (final concentration: 8 μg/mL) was used to infect target cells. HDF cells were infected for a period of 7 hours. Importantly, the infection protocols were designed so that practically all cells were infected, as judged by the results of infection with a GFP control. One day post-infection, cells were eventually selected with puromycin at the final concentration of 250 ng/ml.

RNA extraction, retro-transcription and PCR

Total RNA extraction was performed using a phenol–chloroform method using TriReagent (Sigma-Aldrich, Saint Louis, MO, USA). The synthesis of cDNA was performed from 3 μg of total RNA using the First-Strand cDNA Synthesis Kit (GE Healthcare, Chalfont St Giles, UK). The RT reaction was diluted 1/60 and used as cDNA template for qPCR analysis. TaqMan quantitative PCR analysis was carried out on a LightCycler 2.0 System (Roche Applied Science). PCR mixtures contained LightCycler TaqMan mix, 200nM primers and 1.67μl of cDNA template in a 6.67μl reaction volume. Housekeeping gene ACTB was used for normalization of PDGF-B, p16/INK4a and SPRY2 mRNA expression in each sample type. Real-time PCR intron-spanning assays were designed using the ProbeFinder software (Roche Applied Science).

Colony formation assays

Colony formation assays were carried out in 6 or 12-well plates. Thirty thousand or 60.000 HDF cells were seeded in 6 or 12-well plates respectively. Ten to forteen days after seeding, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.05 % crystal violet (Sigma-Aldrich).

SA-β-Gal analysis

SA-β-Gal analyses were performed as in [21]. At least 5 different fields were counted for each condition representing at least 500 events.

Senescence-associated heterochromatin foci (SAHF) staining

For senescence-associated heterochromatin foci analysis [22], cells were fixed with formaldehyde 4 %, wash with PBS and stained with Hoechst (Sigma). DNA staining was examined under a Zeiss fluorescence microscope.

Soft-agar assays

To measure anchorage-independent growth, cells were detached with trypsin and re-suspended in growth medium. Base agar was prepared with 0.75 % low-melting agarose (Lonza) in growth medium. The top agar contained the suspension of cells in 0.45% low-melting agarose. Plates were incubated for 4 weeks at 37°C and colonies were counted under a bright light microscope.