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The growth of LNCaP, a human prostate adenocarcinoma cell line, and MCF-7, a human breast adenocarcinoma cell line, is initially hormone dependent. We previously demonstrated that LNρ0-8 and MCFρ0, derived from LNCaP and MCF-7 by depleting mitochondrial DNA (mtDNA), exhibited hormone-independent growth that led to progressed phenotypes. Here, we demonstrate that LNρ0-8 and MCFρ0 have invasive characters as evaluated by the ability of invasion through the extracellular matrix (ECM) in vitro. In addition, the induction of vimentin and the repression of E-cadherin expression in ρ0 cells indicate that they are mesenchymal cells. Since LNρ0-8 and MCFρ0 were derived from epithelial cancer cell lines, LNCaP and MCF-7 must have lost epithelial features and gained the mesenchymal phenotype by epithelial-mesenchymal transition (EMT) during the mtDNA depletion. In the ρ0 cell lines, the Raf/MAPK signaling cascade was highly activated together with the expressions of transforming growth factor-beta (TGF-β) and type I TGF-β receptor (TGF-βRI). EMT requires cooperation of TGF-β signaling with activation of the Raf/MAPK cascade, suggesting that EMT was induced in mtDNA depleted cells resulting in the acquisition of progressive tumor features, such as higher invasiveness and loss of hormone dependent growth. Our results indicate that decreasing mtDNA content induces EMT, enabling the progressive phenotypes observed in cancer. (Cancer Sci 2008; 99: 1584–1588)
It is known that prostate and breast cancers originate in hormone-dependent forms. However, they recur as hormone-independent phenotypes during cancer progression. The mechanisms involved in this phenotypic change are not completely understood. Previously, we have demonstrated that C4-2 cells, established from LNCaP cells (a human prostate adenocarcinoma cell line) in an androgen-deprived environment, and 4-hydoroxy tamoxifen (antiestrogen)–resistant MCF-7 cells (a human mammary adenocarcinoma cell line), have a greatly reduced amount of mtDNA. Moreover, mtDNA depleted LNCaP (designated as LNρ0-8 cells) and MCF-7 (designated as MCFρ0 cells) changed from androgen-dependent to androgen-independent, and from antiestrogen-susceptible to antiestrogen-resistant, respectively. MtDNA transferred ρ0 cells (cybrids) were recovered from hormone-independent to hormone-dependent forms. Collectively, these facts suggest that mtDNA regulates hormone-dependent growth.(1,2)
Epithelial cells are the cells that cover surfaces, line a cavity, perform secretion, transport, or regulate functions. They (1) have cohesive interactions among cells; (2) facilitate the formation of continuous cell layers; (3) possess three membrane domains: apical, lateral, and basal; (4) are connected through tight junctions between apical and lateral domains; (5) have an apicobasal polarized distribution of the various organelles and cytoskeleton components; and (6) lack mobility as individual cells with respect to their local environment. Mesenchymal cells are the cells of mesodermal origin that are capable of developing into connective tissues, blood, and lymphatic and blood vessels. In contrast to epithelial cells, mesenchymal cells (1) have loose or no interactions among each other, so a continuous cell layer cannot be formed; (2) have no clear apical and lateral membranes; (3) possess no apcicobasal polarized distribution of organelles and cytoskeleton components; and (4) are motile cells that may even have invasive properties.(3–5)
The EMT represents the transition of epithelial cells to a mesenchymal phenotype, and it features a loss of epithelial cell markers such as junctional and cell–cell adhesion proteins. In development, EMT is observed in mesoderm formation and in emigration of neural crest cells from the neural tube in avian and in mammalian embryo. In adults it has been implicated that EMT plays an important role in tumor formation and progression to metastatic carcinomas.(6) The autocrine TGF-β loop cooperating with oncogenic ras activation is required for the maintenance of EMT in epithelial cells and for metastasis in a mouse model.(7)
In this report, we demonstrate that LNρ0-8 and MCFρ0 are of the mesenchymal phenotype by invasive features with induction of vimentin and suppression of E-cadherin expression. In these cells, the Raf/MAPK signaling cascade was highly activated together with TGF-β1 and TGF-βRI expression. Those results suggest that EMT was induced in mitochondrial DNA–depleted cells, and that resulted in the acquisition of progressive features, such as increased invasiveness and hormonally deregulated cell growth. The association between altered mtDNA copy number, especially a decrease(8–11) with cancer initiation and progression, has been reported. Our results provide a mechanism for the cancer progression, suggesting that a decrease in mtDNA induces EMT, thereby shifting the cancer cells into a progressive phenotype.
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Although we have found that the down-regulation of E-cadherin is due to hypermethylation of its promoter region in LNρ0-8 and MCFρ0 cells,(12) the transcription factor is responsible for the expression of vimentin in both ρ0 cells. A couple of molecules and pathways are thought to be involved in EMT induction in addition to the cooperation of TGF-β signaling with Raf/MAPK. Transcription factors, such as SNAIL and TWIST, are proposed to enhance vimentin transcription and down-regulate E-cadherin.(16,17) In the E-cadherin promoter, there is a repressor element called E-pal, where SNAIL binds to suppress E-cadherin expression.(18) SNAIL is regulated by GSK3β through WNT signaling,(19) and hypoxia is proposed to regulate EMT through SNAIL.(20) In the case of LNρ0-8 and MCFρ0 cells, we could not detect any up-regulation of Wnt signaling, indicating that SNAIL may not be involved in the EMT induced by reduction of mtDNA content (data not shown). Hypoxia cannot stabilize hypoxia inducible factor-1-alpha (HIF-1α) in ρ0 cells,(21) although there are contradictions about the hypoxia response in ρ0 cells.(22) Therefore the involvement of HIF-1α is also unlikely. Thus TGF-β signaling with Raf/MAPK signaling may play a significant role in EMT in ρ0 cells, though the transcriptional regulation of vimentin should be more precisely investigated.
Since inhibitors of mitochondrial respiratory chains and uncouplers can induce MAPK activation,(23) the mechanisms underlying Raf/MAPK activation by mtDNA depletion have been implicated. We speculate that the alteration of mitochondrial membrane potential, cytosolic calcium concentration, shift of ATP generation from respiration to glycolysis, redox regulation, O2 sensoring, or other mitochondrial changes may up-regulate Raf/MAPK signaling.
Here, we have demonstrated that mtDNA-depleted cell lines acquire invasive features and hormonal-independent growth through EMT caused by activation of the Raf/MAP kinase pathway together with TGF-β signal transduction. Indeed, we have proven that mtDNA content was deregulated in prostate cancer cells compared with normal adjacent cells using the laser microdissection coupled with real-time PCR.(24) In addition, breast,(9) renal,(11) and liver(8,10) cancers reportedly have reduced mtDNA contents, while head and neck,(25,26) lung,(26) thyroid,(9) and pancreas(27) cancers have increased. It is possible that abnormality of p53 observed in many cancers deregulate mtDNA contents. This idea is supported by the fact that p53 accumulates in mitochondria in a transcription-independent manner(28) and maintains mtDNA stability;(29) and/or that environmental pressures for disturbing the maintenance of mtDNA can also cause mtDNA decrease. For example, tamoxifen causes mtDNA decrease in mammary cancer cells and hepatocytes,(2,30) and androgen ablation causes mtDNA decrease in prostate cancer.(1) ROS, by products of respiration in mitochondria, can also damage mtDNA.
The role of EMT in development has been widely accepted; however, the role of EMT in cancer progression remains a big debate because it has not been proven in vivo; the criteria for the identification of carcinoma (cancer derived from epithelial cells) and sarcoma (cancer derived from mesenchymal cells) have been pathologically well defined and are not thought to be convertible, and the mixed type of the tumor, known as sarcomatoid carcinoma, is rarely found. Here, we propose that a decrease in mtDNA by any number of mechanisms including ROS, hypoxia, and chemotherapeutic agents, induce EMT to result in a progressed phenotype. After escaping from a growth disadvantage due to the environmental pressure by metastasis or deregulation from hormone dependence, mtDNA copy number can then be increased.(2,31) Finally the cells may morphologically regain epithelial features (MET) at metastatic sites. That is the possible reason that we can not find EMT in vivo. Our idea will be supported by the identification of increased or reduced mtDNA content in vivo. We propose the theory that cancers achieve their growth advantage/progression via EMT/MET induced by the alteration of mtDNA amounts.