and its regulation in lung cancer cells
Evžen Křepela
Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University in Prague, Czech Republic
Background: To kill cancer cells, human cytotoxic lymphocytes (CTLs) and natural killer (NK) cells use the multistep and complex granzyme-perforin mechanism.
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This cell death mode functions via the secretory immunological synapse (SIS) that is formed between the immune killer cell(s) and the tumour cell that had been recognized as a target (Galandrini et al., 2013; Voskoboinik et al., 2015). Human CTLs and NK cells express five different granzymes: A, B, H, K and M. These serine proteases are vectorially released into SIS from polarized cytotoxic granules (secretory lysosomes) of synapsing CTLs and NK cells. The released granzymes are subsequently internalized into the cytosol of target tumour cells via a process dependent on the Ca2+-binding glycoprotein perforin, which is co-secreted from synapsing CTLs and NK cells into SIS (Voskoboinik et al., 2015). The entry route of granzymes into the cytosol goes through the polyperforin pores formed in the plasma membrane and/or in the non-acidifying endosomes (gigantosomes) of target tumour cells, which engulf the granzymes together with perforin monomers via endocytosis. Although cytotoxic activities have been ascribed to all human granzymes, only granzyme B has been unequivocally proven to exert multiple cell death activities (Roušalová and Křepela, 2010b; Joeckel and Bird 2014a).
Proteolytic targets of granzyme B within tumour cells during the granzyme B-induced apoptosis comprise several tens of proteins, which have different subcellular localization and belong to different functional groups (Roušalová and Křepela, 2010b; Joeckel and Bird 2014b; Jacquemin et al., 2015). The multiplicity of the granzyme B-mediated proteolytic attacks is important to overcome the apoptotic threshold of cancer cells since it can lead to significant amplification and robustness of the apoptotic process. The intracellular proteolytic targets of granzyme B that contribute to amplification of its cell death activities include procaspase-3 and -7, Bid and Mcl-1 proteins, and the inhibitory/chaperone subunit A of DNA fragmentation factor. Recent developments in the recombinant human granzyme B-based biopharmaceuticals suggest that these proapoptotic drugs could be promising tools for specific killing of tumour cells (Caldas et al., 2006; Kurschus and Jenne, 2010; Oberoi et al., 2013; Lu et al., 2015).
Tumour cells use several mechanisms to prevent cytosolic entry of granzymes from CTLs and NK cells. They include inhibition of the chemokine-mediated lymphocyte trafficking into the tumours (da Silva et al., 2015), extracellular acidification that blocks the formation of polyperforin pores (Praper et al., 2010), autophagy of gigantosomes during hypoxia leading to degradation of endocytosed granzyme B (Viry et al., 2014), and the emperitosis- and activation -induced death of immune killer cells (Wang et al., 2013; Bird et al., 2014). Importantly, many tumour cell types are also capable to abolish the cytosolic and nuclear death activities of granzyme B via its inactivation by a specific, fast-reacting and irreversible protein inhibitor serpinB9 (Roušalová and Křepela, 2010b). There is evidence that hypoxia and the extracellular acidic stress, which often occur in the microenvironment of many solid tumours, can induce overexpression of SERPINB9 gene in both stem and non-stem cancer cells (Li et al., 2009; Hjelmeland et al., 2011). Due to frequent overexpression of serpinB9 in lung cancer cells and
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tumours, this serpin could be a significant protectant of lung cancer cells against the immune-mediated death (Roušalová and Křepela, 2010a; Soriano et al., 2012).
In the present study we investigated the expression of serpinB9 (SB9) and its involvement in the inhibition of granzyme B (GrB) in human lung cancer cell lines and non-small cell lung carcinoma (NSCLC) tumours and matched lungs from surgically treated patients. We analysed the GrB-mediated activation of procaspase-3 and the cytosolic inhibition of the GrB-generated caspase-3 activity, the expression status of SB9 in NSCLC and small cell lung carcinoma (SCLC) cells and other human cancer cell lines, the role of cytosolic SB9 in the inhibition of GrB-mediated induction of caspase-3-like activity, and the formation of a stable GrB·SB9 complex. In order to get insight into the regulation of SERPINB9 gene expression in NSCLC cells we examined the relationship between the expression of SB9 and EGFR mRNAs and we studied the effect of 5-aza-2’-deoxycytidine (ADC) on the level of SB9 mRNA expression.
Methods: The samples of NSCLC tumours were obtained from surgically treated lung cancer patients and were histopathologically classified according to the WHO criteria 2004. The study was approved by the Ethical Committee of the Hospital Bulovka, Prague. NSCLC cell lines (CALU-1, SKMES-1, NCI-H520, A549, SKLU-1, COLO-699, LXF-289, COR-L23, LCLC-103H, NCI-H1299), SCLC cell lines (NCI-H69, NCI-H82, NCI-H146, NCI-H209, NCI-H345, NCI-H378, NCI-H446), glioblastoma cell lines (U87, U118, U138, U251, U373, T98G) and breast cancer cell lines (T47D, MCF-7, MDA-MB-231, SK-BR-3) were grown in culture as described (Roušalová et al., 2010a; Bušek et al., 2012). The indicated NSCLC cells were also cultured in the presence of 10 mM of ADC for 72 hours.
The expression of SB9 and EGFR mRNAs and β-actin mRNA (a reference
transcript; Roušalová et al., 2010a), were quantitated by coupled real-time RT-PCR.
In the assays of SB9 mRNA and EGFR mRNA expression, the used forward and reverse primers and TaqMan probe had the following sequences, respectively:
5´-GGAATGAACCGTTTGACGAA-3’, 5‘-TTTCCACCGTGCTGA GCT-3´, and 5´-(6-FAM)CGCACCTCGCCCACGTGG(TAMRA)-3´, and 5´-CAGCGCTAC CTTGTCATTCAG-3´, 5´-GGTTGCACTCAGAGAGCTCAG-3´, and
5´-(6-FAM)ATGAGG TACTCGTCGGCATCCACCA(TAMRA)-3´. The expression of procaspase-3 (PC-3) and SB9 proteins was assessed by Western blot-ECL analysis using specific antibodies and the GrB-mediated induction of caspase-3-like activity was measured with Ac-DEVD-AFC as the fluorogenic caspase substrate (Křepela et al., 2004; Roušalová et al., 2010a). The formation of a stable GrB·SB9 complex in cytosol from NSCLC cells was examined by Western blot-ECL analysis using recombinant human GrB and specific antibodies (Roušalová et al., 2010a).
Results: The full GrB-mediated PC-3 activation in cytosolic extracts from NSCLC cells and tissues and lungs required both the proteolytic processing
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of PC-3 and the thiol-assisted reduction of PC-3 and/or caspase-3 (CS-3). At higher protein concentrations, the GrB-generated CS-3-like activity was lower in NSCLC tumours as compared to matched lungs, suggesting increased level of a GrB inhibitor in the tumours, which had significantly higher PC-3 protein expression. Although all tested NSCLC cells expressed SB9 mRNA and protein, these tumour cells could be classified according to their SB9 level as high and low SB9 expressors. Certain analysed tumour cell types have substantially different expression of SB9, e.g. lung cancer cells, which mostly showed high SB9 mRNA expression, and glioblastoma cells, which expressed extremely low SB9 mRNA levels. On the other hand, among tumour cells of the same type both the high and low SB9 expressors could be found, e.g. in NSCLC cells and breast cancer cells.
In NSCLC cells, the level of SB9 protein showed a significant inverse correlation with the GrB-mediated induction of CS-3-like activity. Human recombinant GrB formed with SB9 in the cytosol from NSCLC cells a denaturation-resistant complex GrB·SB9, indicating that the endogenous SB9 is a functional GrB inhibitor. The levels of SB9 mRNA and EGFR mRNA showed a positive correlation in NSCLC cell lines, suggesting that the increase of EGFR expression may promote the expression of SB9. Treatment of cultured NSCLC cells lines with 5-aza-2’-deoxycytidine, an inhibitor of DNA methyltransferases, induced high increase of SB9 mRNA expression in 8 of 10 tested NSCLC cells lines. This suggests that DNA methylation could be involved in silencing of SERPINB9 gene expression in NSCLC cells.
Conclusions: The present study suggest that the majority of lung cancer cells is endowed with high expression of the functional granzyme B inhibitor serpinB9. This phenotypic property may significantly increase their apoptotic threshold against the apoptotic attack of immune killer cells such as CTLs and NK cells. The high expression of serpinB9 in lung carcinomas may represent an obstacle for their effective immunotherapy and possibly also for targeted biomolecular therapy using recombinant granzyme B-based biopharmaceuticals.
Acknowledgements: The author would like to thank to prof. MUDr. Aleksi Šedo, DrSc. and doc. MUDr. Jiří Vachtenheim, CSc. for providing RNA samples isolated from glioblastoma cells and small cell lung carcinoma cells, respectively.
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