S. Iochmann

Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion.

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells.

TFPI-2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour-stromal cell interactions.

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α1 integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.

Monitoring of tumour progression using bioluminescence imaging and computed tomography scanning in a nude mouse orthotopic model of human small cell lung cancer

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI‐2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI‐2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI‐2 in tumour progression, TFPI‐2 was overexpressed in NCI‐H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI‐2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI‐H209 cells expressing TFPI‐2. We also demonstrated that TFPI‐2 upregulation in NCI‐H209 cells decreased MMP expression, particularly by downregulating MMP‐1 and MMP‐3. Moreover, TFPI‐2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP‐1 was predominant in SCLC tissues and was inversely expressed with TFPI‐2 in 35% of cases. These results suggest that downregulation of TFPI‐2 expression could favour the development of SCLC.

Monitoring of tumor progression using bioluminescence imaging in a nude mice orthotopic model of human small cell lung cancer

Introduction Lung cancer is the main cause of cancer death throughout the world and a clinically relevant animal model of human small cell lung carcinoma (SCLC) should be useful to study the molecular aspects of the tumor progression and test the efficiency of new therapeutic agents. In this study, we generated a reproductible and reliable nude mice orthotopic model of human SCLC based on NCI-H209 tumor cells genetically modified to express firefly luciferase. Methods NCI-H209 cells were transfected with pCMV-luc plasmid and clones highly expressing luciferase were isolated and amplified. Cells were analyzed for long-term bioluminescent stability and a clone was subcutaneously passaged twice in vivo to enhance tumorigenicity. Cells resuspended in Matrigel ® and/or EDTA RPMI medium containing a Tc 99M colloid were implanted intrabronchially using a catheter inserted into the trachea and positioned into the right main bronchus using interventional imaging. Punctual deposition of cells was then assessed by scintigraphy. Tumor progression was then followed using bioluminescence imaging. Results Only tumor nodules were observed into lung and trachea when cells were implanted with EDTA Lung tumor invading parenchyma were present in 40% of the mice with Matrigel ® and improved to 75% with EDTA and Matrigel ® . The growth of the primary tumor was sensitively and non-invasively followed and quantified by bioluminescence imaging using a CCD-camera. This tool allows a real-time monitoring of tumor progression on the same animals over a 2-12 week period. Combination of 3D bioluminescence imaging and computed tomography scanning was used to further document tumor location and measurement. Subsequently, the histological analysis of tissue sections confirmed the presence of a lung tumor. Conclusion Our nude mice orthotopic model resembles various stages human small cell lung carcinoma, and then could be useful for evaluating new therapeutic strategies.

054 Le Tissue Factor Pathway lnhibitor-2, un nouveau gène suppresseur de tumeur ?

Introduction Le TFPI-2 ( Tissue Factor Pathway Inhibitor-2 ), un inhibiteur de proteases a serine et notamment de la plasmine, peut limiter l’activation des metalloproteases responsables de la degradation des matrices extracellulaires et ainsi reduire l’invasion tumorale. Nous avons etudie l’expression du TFPI-2 dans le cancer bronchopulmonaire non a petites cellules (CBNPC) et evalue l’impact de l’inactivation du TFPI-2 sur l’invasion des cellules tumorales NCI-H460. Methodes Par PCR en temps reel, nous avons mesure et compare l’expression des ARNm du TFPI-2 au sein de prelevements apparies, dans la zone tumorale et la partie adjacente non atteinte, obtenus de 59 patients operes d’un CBNPC. Parallelement, la proteine TFPI-2 a ete localisee en immunohistochimie a l’aide d’un anticorps polyclonal anti-TFPI-2. Le promoteur du gene du TFPI-2 a ete sequence et sa regulation par methylation analysee apres traitement par le bisulfite de sodium et sequencage. Le potentiel invasif des cellules H460, apres inactivation des transcrits du TFPI-2 par des ARN interferents, a ete etudie a l’aide d’inserts recouverts de Matrigel™. Resultats Une expression plus faible des transcrits du TFPI-2 a ete retrouvee au sein du tissu tumoral par rapport a celle mesuree dans la zone non atteinte dans 37 % des cas avec une localisation de la proteine dans le stroma et notamment dans les macrophages. Cette diminution de l’expression, observee dans 50 % des tumeurs avec metastases ganglionnaires, est correlee a une hypermethylation du promoteur qui contient un ilot CpG s’etalant de la region promotrice a l’exon 2 ( Br J Cancer 2005 ; 92 : 775-83; Biol Chem 2003 ; 384 : 1029-34). Parallelement nous avons montre que l’inactivation de plus de 90 % des ARNm du TFPI-2 par une strategie d’ARN interference diminue l’expression de la proteine et double le potentiel invasif des cellules tumorales pulmonaires H460. Conclusions Nous avons montre que la diminution de l’expression du TFPI-2 pouvait etre associee, dans le CBNPC, a une hypermethylation de son promoteur. Cette repression transcriptionnelle du gene pourrait favoriser la progression tumorale comme en temoigne l’augmentation du potentiel invasif des cellules H460 apres inactivation specifique de l’ARNm du TFPI-2. L’ensemble de ces elements permettrait de classer le TFPI-2 parmi les genes suppresseurs de tumeur et cet inhibiteur de proteases pourrait constituer, dans le futur, une cible potentielle en therapie anticancereuse.