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MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

Vecchione A, Fassan M, Anesti V, Morrione A, Goldoni S, Baldassarre G, Byrne D, D'Arca D, Palazzo JP, Lloyd J, Scorrano L, Gomella LG, Iozzo RV, Baffa R - Oncogene (2008)

Bottom Line: Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein.We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively.Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

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MITOSTATIN promotes mitochondrial fragmentation and clumping independently of Drp1(a–d) Representative confocal images of mt-dsRED fluorescence in HeLa cells transfected with mt-dsRED (a) or co-transfected with mt-dsRED and MITOSTATIN (b), dominant negative Drp1 (Drp1K38A, c), and MITOSTATIN plus Drp1K38A (d). Bar, 10 µm. (e) morphometric analysis of mitochondrial fragmentation induced by overexpression of MITOSTATIN. Data represent mean ± SE of 5 independent experiments.
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Figure 3: MITOSTATIN promotes mitochondrial fragmentation and clumping independently of Drp1(a–d) Representative confocal images of mt-dsRED fluorescence in HeLa cells transfected with mt-dsRED (a) or co-transfected with mt-dsRED and MITOSTATIN (b), dominant negative Drp1 (Drp1K38A, c), and MITOSTATIN plus Drp1K38A (d). Bar, 10 µm. (e) morphometric analysis of mitochondrial fragmentation induced by overexpression of MITOSTATIN. Data represent mean ± SE of 5 independent experiments.

Mentions: Because of confocal images of subcellular localization suggested that MITOSTATIN protein is localized at the mitochondrial level, we analyzed MITOSTATIN effect on normal mitochondrial morphology. Mitochondrial shape results from the balance between fusion and fission, regulated by a family of “mitochondria-shaping” proteins impinging on both sides of the equilibrium. Core components in mammals include the pro-fusion proteins optic atrophy 1, mitofusins 1 and 2; and the pro-fission ones Fis1 and dynamin related protein 1 (Drp1) (Cereghetti et al., 2006). We wished to verify if high levels of MITOSTATIN were associated with changes in normal mitochondrial morphology. To this end we expressed a mitostatin-V5 fusion protein together with the fluorescent protein dsRED targeted to the mitochondrial matrix (mt-dsRED) (Dimmer et al., 2008) and acquired confocal images of the mitochondrial reticulum in HeLa cells (Figure 3a–d). High levels of MITOSTATIN induced marked changes in mitochondrial morphology, including fragmentation of the highly branched network of HeLa cells and clumping of the fragmented organelles in the perinuclear region. Since mitochondrial fragmentation can depend on the activation of the core fission machinery, we tested if a dominant negative mutant of Drp1 could interfere with the observed changes. The dominant negative Drp1K38A (Smirnova et al., 1998) further elongated the mitochondrial network of HeLa cells and it blocked fragmentation induced by MITOSTATIN, as further confirmed by a morphometric analysis (Figure 3e). However, Drp1K38A had no effect on perinuclear clusterization of mitochondria observed in cells expressing high levels of MITOSTATIN, suggesting that the two observed phenotypes can be functionally dissected. Of note, since these experiments were carried in the presence of the broad caspase inhibitor zVAD-fmk, it is unlikely that the observed changes reflect the activation by MITOSTATIN of the apoptotic cascade, often associated with mitochondrial fragmentation and clustering. In conclusion, fragmentation induced by MITOSTATIN depends on the core mitochondrial fission machinery, while it can induce perinuclear clustering of the organelle occur when fission is blocked.


MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

Vecchione A, Fassan M, Anesti V, Morrione A, Goldoni S, Baldassarre G, Byrne D, D'Arca D, Palazzo JP, Lloyd J, Scorrano L, Gomella LG, Iozzo RV, Baffa R - Oncogene (2008)

MITOSTATIN promotes mitochondrial fragmentation and clumping independently of Drp1(a–d) Representative confocal images of mt-dsRED fluorescence in HeLa cells transfected with mt-dsRED (a) or co-transfected with mt-dsRED and MITOSTATIN (b), dominant negative Drp1 (Drp1K38A, c), and MITOSTATIN plus Drp1K38A (d). Bar, 10 µm. (e) morphometric analysis of mitochondrial fragmentation induced by overexpression of MITOSTATIN. Data represent mean ± SE of 5 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2628456&req=5

Figure 3: MITOSTATIN promotes mitochondrial fragmentation and clumping independently of Drp1(a–d) Representative confocal images of mt-dsRED fluorescence in HeLa cells transfected with mt-dsRED (a) or co-transfected with mt-dsRED and MITOSTATIN (b), dominant negative Drp1 (Drp1K38A, c), and MITOSTATIN plus Drp1K38A (d). Bar, 10 µm. (e) morphometric analysis of mitochondrial fragmentation induced by overexpression of MITOSTATIN. Data represent mean ± SE of 5 independent experiments.
Mentions: Because of confocal images of subcellular localization suggested that MITOSTATIN protein is localized at the mitochondrial level, we analyzed MITOSTATIN effect on normal mitochondrial morphology. Mitochondrial shape results from the balance between fusion and fission, regulated by a family of “mitochondria-shaping” proteins impinging on both sides of the equilibrium. Core components in mammals include the pro-fusion proteins optic atrophy 1, mitofusins 1 and 2; and the pro-fission ones Fis1 and dynamin related protein 1 (Drp1) (Cereghetti et al., 2006). We wished to verify if high levels of MITOSTATIN were associated with changes in normal mitochondrial morphology. To this end we expressed a mitostatin-V5 fusion protein together with the fluorescent protein dsRED targeted to the mitochondrial matrix (mt-dsRED) (Dimmer et al., 2008) and acquired confocal images of the mitochondrial reticulum in HeLa cells (Figure 3a–d). High levels of MITOSTATIN induced marked changes in mitochondrial morphology, including fragmentation of the highly branched network of HeLa cells and clumping of the fragmented organelles in the perinuclear region. Since mitochondrial fragmentation can depend on the activation of the core fission machinery, we tested if a dominant negative mutant of Drp1 could interfere with the observed changes. The dominant negative Drp1K38A (Smirnova et al., 1998) further elongated the mitochondrial network of HeLa cells and it blocked fragmentation induced by MITOSTATIN, as further confirmed by a morphometric analysis (Figure 3e). However, Drp1K38A had no effect on perinuclear clusterization of mitochondria observed in cells expressing high levels of MITOSTATIN, suggesting that the two observed phenotypes can be functionally dissected. Of note, since these experiments were carried in the presence of the broad caspase inhibitor zVAD-fmk, it is unlikely that the observed changes reflect the activation by MITOSTATIN of the apoptotic cascade, often associated with mitochondrial fragmentation and clustering. In conclusion, fragmentation induced by MITOSTATIN depends on the core mitochondrial fission machinery, while it can induce perinuclear clustering of the organelle occur when fission is blocked.

Bottom Line: Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein.We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively.Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

Show MeSH
Related in: MedlinePlus