Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

Abstract

We have investigated mechanisms of mitochondrial stressinduced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca 2+ ] c , and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFb1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-X L were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFb1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondriato-nucleus signaling pathway operating through increased [Ca 2+ ] c plays an important role in cancer progression and metastasis. Oncogene (2002Oncogene ( ) 21, 7839 -7849. doi:10.1038 Keywords: mitochondria; calcium signaling; cathepsin L; TGFb; tumor invasion; MAP kinases IntroductionThe role of mitochondria in carcinogenesis was initially suggested by Warburg et al. (1926;Warburg, 1956), based on the observation that number of experimentally induced rodent tumors exhibit reduced respiration-coupled oxidative metabolism and increased glycolysis. Since then, altered mitochondrial morphology, as well as changes in mitochondrial enzyme patterns and membrane transport systems, have been described in several tumor types (Zafar et al., 1982). Cell fusion studies (Jonasson et al., 1977;Howell and Sager, 1978;Giguere and Morais, 1981) also suggest that unknown cytoplasmic elements may play roles in carcinogenesis. Fusion between normal cytoplasm and karyoplasts from malignant phenotypes resulted in the ablation of tumorigenicity (Israel and Schaeffer, 1987) and conversely, the cytoplasm of the malignant phenotype could transfer the malignancy to the karyoplasts from normal cells (Israel and Schaeffer, 1988). Studies using mitochondrial DNA (mtDNA)-depleted tumor cells to evaluate the role of mtDNA, and thus mitochondrial function in tumorigenicity have yielded mixed results (Giguere and Morais, 1981;Morais et al., 1994;Cavalli et al., 1997;Cavalli and Liang, 1998;Hofhaus and Gattermann, 1999). Cavalli et al. (1997) found diminished tumor formation by glioblastoma cells following depletion of mtDNA (r 0 cells) with ethidium bromide treatment, while Morais et al. (1994) found increased capacity to p...

Role of endoplasmic reticulum stress in drug‐induced toxicity

“…1999; Amuthan et al. 2002). Since numerous drugs are able to impair mitochondrial function (Labbe et al.…”

Cancer as a Metabolic Disease

“…A prolonged RTG response, however, would leave the nuclear genome vulnerable to instability and mutability [112,117,119]. Mitochondrial dysfunction also increases levels of cytoplasmic calcium, the multi-drug resistance phenotype, production of reactive oxygen species, and abnormalities in iron-sulfur complexes, which together would further accelerate aberrant RTG signaling and genome mutability [85,106,107,110,111,[120][121][122]. Chronic tissue inflammation could further damage mitochondria, which would accelerate these processes [123,124].…”

“…This mostly involves an energy transition from oxidative phosphorylation to substrate level phosphorylation. Similar systems are also expressed in mammalian cells [110][111][112][113]. Prolonged or continued activation of the retrograde response, however, can have dire consequences on nuclear genome stability and function.…”

“…The primary role of the RTG response is to coordinate the synthesis of ATP through glycolysis alone or through a combination of glycolysis and glutaminolysis when respiratory function is impaired [110,111]. The RTG response would be essential for maintaining a stable ΔG' ATP for cell viability during periods when respiration is impaired.…”

“…The retrograde (RTG) response is the general term for mitochondrial signaling and involves cellular responses to changes in the functional state of mitochondria [106][107][108][109][110]. Although the RTG response has been most studied in yeast, mitochondrial stress signaling is an analogous response in mammalian cells [110,111]. Expression of multiple nuclear genes controlling energy metabolism is profoundly altered following impairment in mitochondrial energy homeostasis [112,113].…”