Runx2 disruption promotes immortalization and confers resistance to oncogene-induced senescence in primary murine fibroblasts

Abstract

The Runx genes play paradoxical roles in cancer where they can function either as dominant oncogenes or tumor suppressors according to context. We now show that the ability to induce premature senescence in primary murine embryonic fibroblasts (MEF) is a common feature of all three Runx genes. However, ectopic Runx-induced senescence contrasts with Ras oncogene-induced senescence, as it occurs directly and lacks the hallmarks of proliferative stress. Moreover, a fundamental role for Runx function in the senescence program is indicated by the effects of Runx2 disruption, which renders MEFs prone to spontaneous immortalization and confers an early growth advantage that is resistant to stress-induced growth arrest. Runx2(-/-) cells are refractory to H-Ras(V12)-induced premature senescence, despite the activation of a cascade of growth inhibitors and senescence markers, and are permissive for oncogenic transformation. The aberrant behavior of Runx2(-/-) cells is associated with signaling defects and elevated expression of S-G(2)-M cyclins and their associated cyclin dependent kinase activities that may override the effects of growth inhibitory signals. Coupling of stress responses to the cell cycle represents a novel facet of Runx tumor suppressor function and provides a rationale for the lineage-specific effects of loss of Runx function in cancer.

Figure 1

Runx2 and Runx3 induce a senescence-like growth arrest in WT MEFs that is distinct from H-Ras^V12. (A) Cells were transduced with a vector containing Runx2 or a control vector containing the PURO-selectable gene. Growth curves showing viable cell numbers of Runx2 (dashed line) and vector control (solid line) cells are shown. Similar results were obtained in 2 independent experiments. Senescence-associated (SA) β-galactosidase staining of matched day 8 cultures are displayed. Photographs are at the same magnification. (B) Parallel growth curves and senescence-associated (SA) β-galactosidase staining in WT MEFs transduced with a vector containing Runx3 (dashed line) and (C) mutant H-Ras^V12 (dashed line).

Figure 2

Runx2^−/− fibroblasts are prone to secondary events that predispose to immortalisation. (A) 3T3 passage culture was performed on five WT and six Runx2^−/− lines. Cells were plated at 3.0 × 10⁵/T25 flask and passaged every third day reseeding 3.0 × 10⁵ cells into a fresh flask. The increase in total viable cell numbers was calculated at every passage and added to a cumulative count over time. Secondary events that predispose to immortalisation occurred between passage 8 and 10. (B)​ Runx2^−/− fibroblasts display an early growth advantage in vitro. Growth curve of two Runx2^−/− lines compared to two WT lines seeded at passage 4. The experiment was repeated on six littermate matched Runx2^−/− and WT lines with essentially identical results. (C) The percentage of BrdUrd incorporation in three Runx2^−/− and three WT lines seeded at passage 5 and labelled for 3h. A comparison is shown for NIH3T3 cells labelled for 1h (hatched bar).

Figure 3

Loss of Runx2 conveys a growth advantage that is resistant to certain cellular stresses but not to TGFβ mediated growth inhibition. (A) Total viable cell counts at day seven from six WT and six Runx2^−/− lines plated in triplicate at passages 3, 5 and 7. (B)​ Runx2^−/− fibroblasts are more resistant to UVC induced cell death than WT controls. Two Runx2^−/− and two WT cultures were plated in triplicate in 60mm dishes and irradiated with 5, 10 and 20 J/m² UVC irradiation. Live / dead cell counts were performed by trypan blue exclusion after 24h in culture. Similar results were obtained in 2 independent experiments. (C) The growth of Runx2^−/− and WT fibroblasts was inhibited by TGFβ. 5ng/ml, 0.5ng/ml or 0.05ng/ml TGFβ was added to triplicate wells on days 1, 3, 5 & 6 and total viable cell counts calculated on day 7 by trypan blue exclusion. Control cultures were fed fresh media in parallel. The histogram is a representative of two independent experiments. Replicate experiments on 3 independent Runx2^−/− and WT lines performed with 5ng/ml TGFβ gave essentially identical results.

Figure 4

Runx2^−/− cells expressing H-Ras^V12 fail to undergo senescence. (A)​ Runx2^−/− and WT cells were transduced with a vector encoding H-Ras^V12 or a control vector containing only the PURO selectable gene. Passage 4 growth curves over 14 days showing viable cell numbers. Similar results were obtained in 4 independent experiments using littermate matched Runx2^−/− and WT lines. B) Senescence associated β-galactosidase activity of Runx2^−/− cells transduced with a vector encoding murine Runx2. Transduction with the control vector containing only the PURO selectable gene failed to generate positively stained cultures. Photographs are at the same magnification. (C) Colony formation of Runx2^−/− fibroblasts retrovirally transduced with H-Ras^V12. Cells were transduced with a retrovirus encoding H-Ras^V12 or a control vector containing only the PURO selectable gene and analysed for oncogenic transformation. Numbers were determined for agar colonies > 0.1mm derived from plating 10³ cells per 60mm dish and fed every 3-4 days for 5 weeks. Error bars indicate the average number of colonies derived from 3 independent dishes.

Figure 5

Loss of Runx2 does not block the induction of growth arrest pathways by Ras but facilitates transit through S/G2/M. (A) Total protein was extracted from early passage (p4) littermate matched Runx2^−/− and WT lines transduced with H-Ras^V12 (R) or the PURO (P) vector control and probed against antibodies to phospho-p38MAP kinase or p38MAP kinase as a loading control. The western blot is representative of two independent experiments. (B) Western blot analysis as in (A) probed against antibodies to p16^INK4a, p19^ARF, p21^WAF1 and p53. Actin was used as a loading control. (C) Cell cycle analysis of H-Ras^V12 transduced WT and Runx2^−/− MEFs is shown as the percentage of propidium iodide stained cells with 2N (top) and 4N (bottom) DNA content at day 13 following transduction. Two independent littermate matched WT and Runx2^−/− cultures are grouped (1 & 2). Error bars represent triplicate samples. Similar results from two independent experiments. (D) Cytometry density plots of Runx2^−/− (top) and WT (bottom) cells transduced with H-Ras^V12 which were labelled with BrdUrd for 15 hours. Percentage of BrdUrd incorporation is given based on triplicate samples. Identical results were seen with 2 independent WT and Runx2^−/− samples.

Figure 6

Loss of Runx2 confers increased expression of S/G2/M cyclins and associated cyclin-dependent kinase activities. (A) Cyclin D1 expression is refractory to loss of Runx2 but remains sensitive to induction by H-Ras^V12. cDNA was prepared from littermate matched Runx2^−/− and WT cultures transduced with H-Ras^V12 or the PURO control vector and plated for 0 to 13 days in culture. Cyclin D1 expression was assayed at each timepoint by relative quantification to endogenous control hprt and calibrated to the day 0 WT sample. (B) – (D) Loss of Runx2 confers increased expression of cyclin A2 (B), cyclin B1 (C) and cyclin E1 (D) and sustains downstream cyclin dependent kinase activity. cDNA was analysed from the same data set as described in (A). The data is displayed as raw RQ values and is representative of two independent experiments on two littermate matched Runx2^−/− and WT lines. Parallel cultures were harvested for immunoprecipitation (IP) kinase assays of total cyclin A, cyclin B1 and cyclin E associated kinase activities. Cell extracts were immunoprecipitated with antibodies specific for the relevant cyclin and the precipitates analysed for kinase activity using Histone H1(32kDa) as a substrate. The 25kDa and 37kDa molecular weight markers are marked with an asterisk. The data is representative of 2 independent experiments.