The HtrA3 protease promotes drug‐induced death of lung cancer cells by cleavage of the X‐linked inhibitor of apoptosis protein (XIAP)

Abstract

HtrA3 is a proapoptotic protease shown to promote drug‐induced cytotoxicity in lung cancer cells and proposed to have an antitumor effect. However, at the molecular level, the role of HtrA3 in cell death induction is poorly understood. There are two HtrA3 isoforms, a long and a short one, termed HtrA3L and HtrA3S. By performing pull down assays, co‐immunoprecipitation and ELISA, we showed that HtrA3 formed complexes with the X‐linked inhibitor of apoptosis protein (XIAP). The recombinant HtrA3 variants ΔN‐HtrA3L and ‐S, lacking the N‐terminal regions that are not essential for protease activity, cleaved XIAP with a comparable efficiency, though ΔN‐HtrA3S was more active in the presence of cellular extract, suggesting the existence of an activating factor. Immunofluorescence and proximity ligation assays indicated that HtrA3 partially co‐localized with XIAP. Exogenous ΔN‐HtrA3L/S promoted apoptotic death of lung cancer cells treated with etoposide and caused a significant decrease of cellular XIAP levels, in a way dependent on HtrA3 proteolytic activity. These results collectively indicate that both HtrA3 isoforms stimulate drug‐induced apoptotic death of lung cancer cells via XIAP cleavage and thus help to understand the molecular mechanism of HtrA3 function in apoptosis and in cancer cell death caused by chemotherapy.

HtrA4 Protease Promotes Chemotherapeutic-Dependent Cancer Cell Death

“…It should be noted that the ΔN-HtrA4 variant contains the region required for trimerization [12] and forms trimers in solution [12,34]. We found that under normal conditions, the full-length HtrA4 formed clusters in cytoplasm and also partially colocalized with mitochondria (confirmed by statistical analysis) (Figure 2A, Table 1), while the ΔN-HtrA4 formed a diffused pattern, typical for cytoplasmic proteins, resembling the pattern observed before for HtrA1/3 [10,27,29,30]. We expect that the HtrA-containing punctate structures other than mitochondria are the ER and Golgi involved in the HtrA4 protein transport outside the cell.…”

“…The HtrA4 protein possesses a similar signal secretory peptide as the HtrA1/3, and it has been previously shown to be exported to medium of cultured cells [18]. However, since the HtrA1/3 were found to function not only in cellular matrix but also to play important roles inside the cell [10,29,30,31], we checked cellular localization of HtrA4. We found that indeed, HtrA4 was secreted into the medium by the cultured MCF7 breast cancer cells, which contained a relatively high level of endogenous HtrA4 (Figure 1A and Figure S1).…”

“…Viral particles were produced by co-transfecting 10 μg of the pTW_H4-GFP or pTW_∆H4-GFP plasmid, based on pBabe puro (Clontech, Inc., Mountain View, CA, USA), and 5 μg of the envelope protein-expressing plasmid pCMV-VSV-G (Cell Biolabs, Inc., San Diego, CA, USA) into packaging cells GP2-293 (Clontech, Inc., Mountain View, CA, USA), using CalPhos™ Mammalian Transfection Kit (Clontech, Inc., Mountain View, CA, USA), according to the manufacturer’s protocol as described in [10]. To obtain cells with inducible HtrA4 expression, the Retro-X™ Tet-On Advanced Inducible Expression System (Clontech, Inc., Mountain View, CA, USA) was used.…”

“…The HtrA1–3 proteases function as a protein control guard by removing misfolded and aberrant proteins to avoid the accumulation of toxic aggregates [2,3,4]. Moreover, the HtrA1–3 play an important role as pro-apoptotic proteases, which induce cell death mainly by interaction with the X-linked inhibitor of apoptosis protein (XIAP) [5,6,7,8,9,10,11]. It was observed that changed levels of these proteins were connected with neurodegenerative disorders and cancer whose development is closely connected to cell death by apoptosis [3,4].…”

A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3

“…The known physiological substrates of HtrA3 are limited, most of which include proteoglycans [23,24], XIAP and a few cytoskeletal proteins [61,62]. These interactions suggest that HtrA3 functions are regulated by complex multiple pathways involving its protease activity and the mechanisms of substrate specificity and cleavage may, therefore, play a significant role.…”