Abstract 5236: Potential loss of p53/p21 function in a murine lung adenocarcinoma cell line prevents TGFβ1-induced dormancy despite p38α/β activation and DEC2 and p27 induction
Authors: Carlini Maria J. , Bragado Paloma , Vazquez Paula F. , de Kier Joffe Elisa D. Bal , Puricelli Lydia I. , Aguirre-Ghiso Julio A. 
Published: August 2012

Disseminated tumor cells can reside in target organs in a state of dormancy (growth arrest) for prolonged periods before developing into overt lesions. Studies using the HEp3 model of head and neck squamous cell carcinoma (HNSCC) showed that a low ERK:p38 signaling ratio predicts for cellular dormancy while the opposite prompts active proliferation. Further, p38 activation induces the expression of p53 and the tumor/metastasis suppressor BHLHB3/DEC2, while inhibiting c-jun and FoxM1 expression. Also, at least p53 and DEC2 are positively regulated by transforming growth factor beta (TGFβ). Preliminary data studying the murine lung adenocarcinoma cell line LP07 suggested that TGFβ1 can only delay primary tumor growth with no effect on spontaneous metastasis development. These studies motivated us to test whether the effects of TGFβ1 signaling in the LP07 model might be short-circuited to prevent the activation of a tumor cell dormancy program as that described in HNSCC. Flow cytometry analysis showed that TGFβ1 treatment mediated arrest in G0/G1 phase of the cell cycle in LP07 cells, which was consistent with p27 upregulation observed by Western blot. Concomitantly, TGFβ1 activated the transcriptions factors Smad2/3 and inhibited proliferation, all these events blocked by an inhibitor of TGFβ-receptor serine kinase activity. Consistent with these data, LP07 cells exhibited a high ERK:p38 ratio that was lowered by TGFβ1, primarily due to a strong p38 activation. Accordingly, we observed in vivo growth inhibition of TGFβ1-pretreated cells upon subcutaneous implantation in syngeneic BALB/c mice. However, sustained in vivo growth arrest was not observed. We also found by qPCR an increased level of DEC2 mRNA in TGFβ1-treated cells. LP07 cells overexpressing DEC2 or a constitutive active mutant of p38 had increased p27 expression as assessed by Western blot, suggesting that the p38-&gt;DEC2 pathway can be re-activated. Upon treatment of the cells with a MEK1/2 inhibitor, p27 was also upregulated and phospho-histone H3 was downregulated. p53 and p21 were refractory to modulation by these treatments, even following overexpression of wild type p53. This suggests that p53 function might be disrupted and/or that p21 may be epigenetically silenced in this cell line. In conclusion, we have found that the metastatic LP07 cell line displays an ERK/p38 ratio and downstream signaling that predict for an efficient escape from dormancy. While the p38-&gt;DEC2-p27 pathway could be restored by establishing a low ERK/p38 signaling ratio, absence of functional p53 and p21 induction most likely explains why TGFβ1 only attenuates tumor growth. If p53 and p21 (also a target of DEC2) are silenced by DNA promoter methylation it is possible that restoration of p21 promoter sensitivity to DEC2 using 5-Aza-C may allow for reprogramming LP07 cells into a prolonged dormant state.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5236. doi:1538-7445.AM2012-5236