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8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis.

Rodrigues-Ferreira S, Di Tommaso A, Dimitrov A, Cazaubon S, Gruel N, Colasson H, Nicolas A, Chaverot N, Molinié V, Reyal F, Sigal-Zafrani B, Terris B, Delattre O, Radvanyi F, Perez F, Vincent-Salomon A, Nahmias C - PLoS ONE (2009)

Bottom Line: ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy.Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis.Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Inserm U567, CNRS UMR8104, Paris, France.

ABSTRACT

Background: Breast cancer is a heterogeneous disease that is not totally eradicated by current therapies. The classification of breast tumors into distinct molecular subtypes by gene profiling and immunodetection of surrogate markers has proven useful for tumor prognosis and prediction of effective targeted treatments. The challenge now is to identify molecular biomarkers that may be of functional relevance for personalized therapy of breast tumors with poor outcome that do not respond to available treatments. The Mitochondrial Tumor Suppressor (MTUS1) gene is an interesting candidate whose expression is reduced in colon, pancreas, ovary and oral cancers. The present study investigates the expression and functional effects of MTUS1 gene products in breast cancer.

Methods and findings: By means of gene array analysis, real-time RT-PCR and immunohistochemistry, we show here that MTUS1/ATIP3 is significantly down-regulated in a series of 151 infiltrating breast cancer carcinomas as compared to normal breast tissue. Low levels of ATIP3 correlate with high grade of the tumor and the occurrence of distant metastasis. ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy. Functional studies indicate that silencing ATIP3 expression by siRNA increases breast cancer cell proliferation. Conversely, restoring endogenous levels of ATIP3 expression leads to reduced cancer cell proliferation, clonogenicity, anchorage-independent growth, and reduces the incidence and size of xenografts grown in vivo. We provide evidence that ATIP3 associates with the microtubule cytoskeleton and localizes at the centrosomes, mitotic spindle and intercellular bridge during cell division. Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis.

Conclusions: Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome. ATIP3 re-expression limits tumor cell proliferation in vitro and in vivo, suggesting that this protein may represent a novel useful biomarker and an interesting candidate for future targeted therapies of aggressive breast cancer.

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ATIP3 inhibits cancer cell proliferation.A. ATIP3 silencing in MDA-MB-468 cells transfected with control siRNA or specific ATIP3 siRNA#1 or siRNA#2 for 72 hours. Left panel: real-time RT-PCR using ATIP3-specific primers relative to EEF1G, normalized to non transfected cells (NT). Middle panel: immunoblotting with anti-MTUS1 antibodies showing ATIP3 at 170 kDa, and reprobing with anti-alpha-tubulin antibodies for internal control. Right panel: MTT assay. Results are expressed as percent of MTT incorporation in control siRNA-transfected cells at time 96 hours. Shown are the results of two representative experiments out of four performed in quadruplicate. **p<0.0001. B. Cell proliferation in stable MDA-MB-231 transfectants. Left panel : immunoblotting of total cell lysates from MDA-MB-231 cells non transfected (NT) or stably expressing GFP or GFP-ATIP3 (clone 3A1) at levels comparable to those in MDA-MB-468. Blots were probed with anti-MTUS1 (upper panel), anti-GFP (middle panel), anti-alpha-tubulin antibodies (lower panel). Arrows indicate the migration of GFP-ATIP3 (200 kDa), ATIP3 (170 kDa), GFP (25 kDa), alpha-tubulin (50 kDa). Middle and right panels: measurement of BrdU incorporation and MTT assay. Shown is one representative experiment out of three performed in quadruplicate. **p<0.0001; *p<0.001. C. Colony formation of GFP- or GFP-ATIP3-transfected MCF7 cells plated at different densities. Shown is one representative experiment out of five performed in duplicate. Right panel : quantification of the number of colonies per well. **p<0.0001. D. Immunoblotting of total lysates from MCF7 cells left untransfected (NT) or stably transfected with GFP or GFP-ATIP3 (clones HC1 and HC6), revealed as in (B). Middle panel : MCF7 stable transfectants grown in soft agar for four weeks. Shown are photomicrographs of one representative experiment out of three performed in triplicate. A bar represents 50 µm (upper panel) and 10 µm (lower panel). Right panel : quantification of the number of colonies per field counted under an inverted microscope. **p<0.0001.
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pone-0007239-g002: ATIP3 inhibits cancer cell proliferation.A. ATIP3 silencing in MDA-MB-468 cells transfected with control siRNA or specific ATIP3 siRNA#1 or siRNA#2 for 72 hours. Left panel: real-time RT-PCR using ATIP3-specific primers relative to EEF1G, normalized to non transfected cells (NT). Middle panel: immunoblotting with anti-MTUS1 antibodies showing ATIP3 at 170 kDa, and reprobing with anti-alpha-tubulin antibodies for internal control. Right panel: MTT assay. Results are expressed as percent of MTT incorporation in control siRNA-transfected cells at time 96 hours. Shown are the results of two representative experiments out of four performed in quadruplicate. **p<0.0001. B. Cell proliferation in stable MDA-MB-231 transfectants. Left panel : immunoblotting of total cell lysates from MDA-MB-231 cells non transfected (NT) or stably expressing GFP or GFP-ATIP3 (clone 3A1) at levels comparable to those in MDA-MB-468. Blots were probed with anti-MTUS1 (upper panel), anti-GFP (middle panel), anti-alpha-tubulin antibodies (lower panel). Arrows indicate the migration of GFP-ATIP3 (200 kDa), ATIP3 (170 kDa), GFP (25 kDa), alpha-tubulin (50 kDa). Middle and right panels: measurement of BrdU incorporation and MTT assay. Shown is one representative experiment out of three performed in quadruplicate. **p<0.0001; *p<0.001. C. Colony formation of GFP- or GFP-ATIP3-transfected MCF7 cells plated at different densities. Shown is one representative experiment out of five performed in duplicate. Right panel : quantification of the number of colonies per well. **p<0.0001. D. Immunoblotting of total lysates from MCF7 cells left untransfected (NT) or stably transfected with GFP or GFP-ATIP3 (clones HC1 and HC6), revealed as in (B). Middle panel : MCF7 stable transfectants grown in soft agar for four weeks. Shown are photomicrographs of one representative experiment out of three performed in triplicate. A bar represents 50 µm (upper panel) and 10 µm (lower panel). Right panel : quantification of the number of colonies per field counted under an inverted microscope. **p<0.0001.

Mentions: The functional consequence of ATIP3 underexpression was investigated by transfection of specific siRNA into the ATIP3-positive MDA-MB-468 cell line. Two different sequences of siRNA successfully silenced ATIP3 expression in MDA-M468 cells both at the mRNA and protein level (Fig. 2A, left panels). As shown in Fig. 2A (right panel), ATIP3 knock-down by both siRNAs led to a significant increase (66.4±5.3% and 63±10.2% upon transfection of siRNA#1 and siRNA#2, respectively) in MDA-MB-468 breast cancer cell proliferation, pointing to an anti-proliferative effect of ATIP3.


8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis.

Rodrigues-Ferreira S, Di Tommaso A, Dimitrov A, Cazaubon S, Gruel N, Colasson H, Nicolas A, Chaverot N, Molinié V, Reyal F, Sigal-Zafrani B, Terris B, Delattre O, Radvanyi F, Perez F, Vincent-Salomon A, Nahmias C - PLoS ONE (2009)

ATIP3 inhibits cancer cell proliferation.A. ATIP3 silencing in MDA-MB-468 cells transfected with control siRNA or specific ATIP3 siRNA#1 or siRNA#2 for 72 hours. Left panel: real-time RT-PCR using ATIP3-specific primers relative to EEF1G, normalized to non transfected cells (NT). Middle panel: immunoblotting with anti-MTUS1 antibodies showing ATIP3 at 170 kDa, and reprobing with anti-alpha-tubulin antibodies for internal control. Right panel: MTT assay. Results are expressed as percent of MTT incorporation in control siRNA-transfected cells at time 96 hours. Shown are the results of two representative experiments out of four performed in quadruplicate. **p<0.0001. B. Cell proliferation in stable MDA-MB-231 transfectants. Left panel : immunoblotting of total cell lysates from MDA-MB-231 cells non transfected (NT) or stably expressing GFP or GFP-ATIP3 (clone 3A1) at levels comparable to those in MDA-MB-468. Blots were probed with anti-MTUS1 (upper panel), anti-GFP (middle panel), anti-alpha-tubulin antibodies (lower panel). Arrows indicate the migration of GFP-ATIP3 (200 kDa), ATIP3 (170 kDa), GFP (25 kDa), alpha-tubulin (50 kDa). Middle and right panels: measurement of BrdU incorporation and MTT assay. Shown is one representative experiment out of three performed in quadruplicate. **p<0.0001; *p<0.001. C. Colony formation of GFP- or GFP-ATIP3-transfected MCF7 cells plated at different densities. Shown is one representative experiment out of five performed in duplicate. Right panel : quantification of the number of colonies per well. **p<0.0001. D. Immunoblotting of total lysates from MCF7 cells left untransfected (NT) or stably transfected with GFP or GFP-ATIP3 (clones HC1 and HC6), revealed as in (B). Middle panel : MCF7 stable transfectants grown in soft agar for four weeks. Shown are photomicrographs of one representative experiment out of three performed in triplicate. A bar represents 50 µm (upper panel) and 10 µm (lower panel). Right panel : quantification of the number of colonies per field counted under an inverted microscope. **p<0.0001.
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pone-0007239-g002: ATIP3 inhibits cancer cell proliferation.A. ATIP3 silencing in MDA-MB-468 cells transfected with control siRNA or specific ATIP3 siRNA#1 or siRNA#2 for 72 hours. Left panel: real-time RT-PCR using ATIP3-specific primers relative to EEF1G, normalized to non transfected cells (NT). Middle panel: immunoblotting with anti-MTUS1 antibodies showing ATIP3 at 170 kDa, and reprobing with anti-alpha-tubulin antibodies for internal control. Right panel: MTT assay. Results are expressed as percent of MTT incorporation in control siRNA-transfected cells at time 96 hours. Shown are the results of two representative experiments out of four performed in quadruplicate. **p<0.0001. B. Cell proliferation in stable MDA-MB-231 transfectants. Left panel : immunoblotting of total cell lysates from MDA-MB-231 cells non transfected (NT) or stably expressing GFP or GFP-ATIP3 (clone 3A1) at levels comparable to those in MDA-MB-468. Blots were probed with anti-MTUS1 (upper panel), anti-GFP (middle panel), anti-alpha-tubulin antibodies (lower panel). Arrows indicate the migration of GFP-ATIP3 (200 kDa), ATIP3 (170 kDa), GFP (25 kDa), alpha-tubulin (50 kDa). Middle and right panels: measurement of BrdU incorporation and MTT assay. Shown is one representative experiment out of three performed in quadruplicate. **p<0.0001; *p<0.001. C. Colony formation of GFP- or GFP-ATIP3-transfected MCF7 cells plated at different densities. Shown is one representative experiment out of five performed in duplicate. Right panel : quantification of the number of colonies per well. **p<0.0001. D. Immunoblotting of total lysates from MCF7 cells left untransfected (NT) or stably transfected with GFP or GFP-ATIP3 (clones HC1 and HC6), revealed as in (B). Middle panel : MCF7 stable transfectants grown in soft agar for four weeks. Shown are photomicrographs of one representative experiment out of three performed in triplicate. A bar represents 50 µm (upper panel) and 10 µm (lower panel). Right panel : quantification of the number of colonies per field counted under an inverted microscope. **p<0.0001.
Mentions: The functional consequence of ATIP3 underexpression was investigated by transfection of specific siRNA into the ATIP3-positive MDA-MB-468 cell line. Two different sequences of siRNA successfully silenced ATIP3 expression in MDA-M468 cells both at the mRNA and protein level (Fig. 2A, left panels). As shown in Fig. 2A (right panel), ATIP3 knock-down by both siRNAs led to a significant increase (66.4±5.3% and 63±10.2% upon transfection of siRNA#1 and siRNA#2, respectively) in MDA-MB-468 breast cancer cell proliferation, pointing to an anti-proliferative effect of ATIP3.

Bottom Line: ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy.Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis.Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Inserm U567, CNRS UMR8104, Paris, France.

ABSTRACT

Background: Breast cancer is a heterogeneous disease that is not totally eradicated by current therapies. The classification of breast tumors into distinct molecular subtypes by gene profiling and immunodetection of surrogate markers has proven useful for tumor prognosis and prediction of effective targeted treatments. The challenge now is to identify molecular biomarkers that may be of functional relevance for personalized therapy of breast tumors with poor outcome that do not respond to available treatments. The Mitochondrial Tumor Suppressor (MTUS1) gene is an interesting candidate whose expression is reduced in colon, pancreas, ovary and oral cancers. The present study investigates the expression and functional effects of MTUS1 gene products in breast cancer.

Methods and findings: By means of gene array analysis, real-time RT-PCR and immunohistochemistry, we show here that MTUS1/ATIP3 is significantly down-regulated in a series of 151 infiltrating breast cancer carcinomas as compared to normal breast tissue. Low levels of ATIP3 correlate with high grade of the tumor and the occurrence of distant metastasis. ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy. Functional studies indicate that silencing ATIP3 expression by siRNA increases breast cancer cell proliferation. Conversely, restoring endogenous levels of ATIP3 expression leads to reduced cancer cell proliferation, clonogenicity, anchorage-independent growth, and reduces the incidence and size of xenografts grown in vivo. We provide evidence that ATIP3 associates with the microtubule cytoskeleton and localizes at the centrosomes, mitotic spindle and intercellular bridge during cell division. Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis.

Conclusions: Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome. ATIP3 re-expression limits tumor cell proliferation in vitro and in vivo, suggesting that this protein may represent a novel useful biomarker and an interesting candidate for future targeted therapies of aggressive breast cancer.

Show MeSH
Related in: MedlinePlus