Alterations of P53 and PTEN Genes Are Associated with Increased TF Expression and Reduced Survival in Non Small Cell Lung Cancer

Introduction: Tissue Factor (TF) is the main initiator of blood coagulation and also a signalling protein that regulates cancerous cell migration, angiogenesis and metastasis. TF is frequently synthesized by many tumor cells and TF gene expression can be modulated by tumor suppressor genes (TSG) such as P53 and PTEN. Moreover, heparanase is an endo-b-D-glucuronidase also implicated in tumor development and was recently described to enhance TF expression and activity. Finally, another TSG, LKB1 that regulates the mTOR pathway with P53 and PTEN, has also been recently shown to modulate cancer differentiation and metastasis.

Aim of the study: To look for a relationship between TF and heparanase genes expression in NSCLC and to investigate whether these genes can be regulated by P53, PTEN and LKB1.

Material and Methods: TF and heparanase mRNA levels were measured by real-time PCR in lung tumors surgically removed in 53 Caucasian patients with NSCLC. Direct sequencing of exons 5–8 of P53 was performed on genomic DNA. Mutations of PTEN and LKB1 were screened by multiplex ligation dependant probe amplification using specific SALSA-MLPA kits (MRC-Holland, Amsterdam, Holland). Results were analyzed and compared to clinicopathological features using the Mann Whitney U-test and Fisher’s exact test. Moreover, survival analysis was evaluated using the Kaplan Meier method and differences assessed by the Log-rank test.

Results: TF mRNA levels were variable from one sample to another (median: 11540 copies/10⁸ copies of 18S RNA; range: 10–310 000) but significantly higher in T3–T4 tumors (19150 vs 8530 copies in T1–T2 tumors; p=0.04) and in stages III–IV of NSCLC (17710 vs 9780 copies in stages 1–2; p=0.03) as previously reported (Regina et al, J Thorac Oncol, 2008). Mutations of P53, LKB1 and PTEN were identified in 37.7 % (20/53), 49 % (26/53) and in 37.7 % (20/53) of tumors, respectively. TF mRNA levels were higher in samples mutated for P53 (median in P53^Mut: 15655 vs 6225 copies/10⁸ copies of 18S RNA in P53^WT tissues; p=0.02), and also in cancerous tissues in whom PTEN mutations had been evidenced (median in PTEN^Mut: 17370 vs 8530 copies in PTEN^WT; p=0.03). On the other hand, TF gene expression was unaffected in tumors when LKB1 mutations were detected alone but it dramatically increased if combined genetic alterations were present, with a number of mRNA copies varying from 3500 when no mutation was detected to 11600, 15800, and 78 000 copies when 1, 2 and 3 TSG were mutated (Spearman correlation p=0.01). Heparanase mRNA levels measured in lung tumors were also variable (median: 2922 copie/10⁸ copies of 18S RNA; range: 80–39750), without any correlation with TF gene expression, TSG mutations and clinicopathological features. In contrast, the median survival time was shorter in patients with tumor TF mRNA levels above median value (26 vs 66 months; Hazard ratio: 1.96; CI95%:1.01–3.8; p=0.04) and when P53 was mutated (33 vs 43 months; Hazard ratio: 1.5; CI95%:1.02–2.5; p=0.05).

Conclusion: This study provides evidence that combined oncogene events affecting TSG, such as P53, PTEN and LKB1, dramatically affect TF gene expression in NSCLC, this process contributing to cancer progression. TSG directly stimulate the mTOR pathway that could also be activated by TF/VIIa/XA complexes, favoring tumor cell migration.