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TGF-β induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer.

Singha PK, Pandeswara S, Geng H, Lan R, Venkatachalam MA, Saikumar P - Genes Cancer (2014)

Bottom Line: Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-β.Moreover, signaling alterations produced by high TMEPAI were associated with oncogenic Snail expression and lung metastases.Together, our findings suggest that TMEPAI has dually critical roles to promote TGF-β dependent cancer cell growth and metastasis.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Pathology, UT Health Science Center at San Antonio, TX.

ABSTRACT
TMEPAI (transmembrane prostate androgen-induced) is amplified at genomic, transcript and protein levels in triple-negative breast cancers and promotes TGF-β dependent growth, motility and invasion. Tumor promotion by TMEPAI depends on two different but related actions on TGF-β signaling. Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-β. Secondly, increased expression of TMEPAI decreases PTEN (phosphatase and tensin homolog) abundance, and thereby increases TGF-β dependent tumor promotive PI3K/Akt signaling. These actions of TMEPAI give rise to increased cell proliferation and motility. Moreover, signaling alterations produced by high TMEPAI were associated with oncogenic Snail expression and lung metastases. Finally, an inverse correlation between TMEPAI and PTEN levels was confirmed in triple negative breast cancer tumor samples. Together, our findings suggest that TMEPAI has dually critical roles to promote TGF-β dependent cancer cell growth and metastasis. Thus, redirected TGF-β signaling through TMEPAI may play a pivotal role in TGF-β mediated tumor promotion.

No MeSH data available.


Related in: MedlinePlus

TMEPAI sequesters R-SmadsA. Schematic representation of point mutations in SIM and PY motifs of human TMEPAI. SIM mutant (186PPNR189 → PAAR) and PY mutant (158PPPY161 → PPPA and 229PPTY232 → PPTA) were created by site-directed mutagenesis. B. Relative luciferase activity from MDA-MB-231 cells transiently transfected with 12X CAGA-Luc reporter and expression plasmids of TMEPAI and its mutants (See Materials section for details). Cells were treated without or with TGF-β (2ng/ml) for 16 h. C. MDA-MB-231 cells were transfected with pcDNA or pHis-TMEPAI (wild type and PY or SIM mutants) along with Flag-tagged Smad2 or Smad3 expression vectors. 24 hours later, cells lysates were collected and analyzed by Western blotting. D. Proteins from above lysates were captured by Co2+- chelate matrix and then analyzed for TMEPAI and R-Smads.
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Figure 4: TMEPAI sequesters R-SmadsA. Schematic representation of point mutations in SIM and PY motifs of human TMEPAI. SIM mutant (186PPNR189 → PAAR) and PY mutant (158PPPY161 → PPPA and 229PPTY232 → PPTA) were created by site-directed mutagenesis. B. Relative luciferase activity from MDA-MB-231 cells transiently transfected with 12X CAGA-Luc reporter and expression plasmids of TMEPAI and its mutants (See Materials section for details). Cells were treated without or with TGF-β (2ng/ml) for 16 h. C. MDA-MB-231 cells were transfected with pcDNA or pHis-TMEPAI (wild type and PY or SIM mutants) along with Flag-tagged Smad2 or Smad3 expression vectors. 24 hours later, cells lysates were collected and analyzed by Western blotting. D. Proteins from above lysates were captured by Co2+- chelate matrix and then analyzed for TMEPAI and R-Smads.

Mentions: To confirm TMEPAI sequestration of R-Smads is essential for its anti-TGF-β activity as reported by Watanabe et al [20], we generated SIM (Smad interacting motif) and PY (NEDD4 WW domain interacting motif) mutants in TMEPAI (Fig.4A). While wild type TMEPAI completely suppressed Smad dependent 12XCAGA reporter activity, SIM mutant has no effect (Fig.4B). However, it was surprising that the PY mutant, which abolished the interaction between TMEPAI and NEDD4 [17], inhibited 12XCAGA reporter activity only partially instead of completely (Fig.4B). We speculated that PY mutations could have altered the conformation of TMEPAI such that interaction of SIM domain with R- Smads may be affected. Expression of Smad2 and Smad3 proteins were lower in cells that were transfected with wild type TMEPAI vector than in pcDNA or TMEPAI mutant vectors (Fig.4C). Both R-Smads were pulled down by wild type TMEPAI and PY mutant but not SIM mutant (Fig.4D). However, PY mutant was slightly less efficient than wild type TMEPAI in pulling down R-Smads (Fig.4D), which is consistent with our speculation above.


TGF-β induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer.

Singha PK, Pandeswara S, Geng H, Lan R, Venkatachalam MA, Saikumar P - Genes Cancer (2014)

TMEPAI sequesters R-SmadsA. Schematic representation of point mutations in SIM and PY motifs of human TMEPAI. SIM mutant (186PPNR189 → PAAR) and PY mutant (158PPPY161 → PPPA and 229PPTY232 → PPTA) were created by site-directed mutagenesis. B. Relative luciferase activity from MDA-MB-231 cells transiently transfected with 12X CAGA-Luc reporter and expression plasmids of TMEPAI and its mutants (See Materials section for details). Cells were treated without or with TGF-β (2ng/ml) for 16 h. C. MDA-MB-231 cells were transfected with pcDNA or pHis-TMEPAI (wild type and PY or SIM mutants) along with Flag-tagged Smad2 or Smad3 expression vectors. 24 hours later, cells lysates were collected and analyzed by Western blotting. D. Proteins from above lysates were captured by Co2+- chelate matrix and then analyzed for TMEPAI and R-Smads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4209604&req=5

Figure 4: TMEPAI sequesters R-SmadsA. Schematic representation of point mutations in SIM and PY motifs of human TMEPAI. SIM mutant (186PPNR189 → PAAR) and PY mutant (158PPPY161 → PPPA and 229PPTY232 → PPTA) were created by site-directed mutagenesis. B. Relative luciferase activity from MDA-MB-231 cells transiently transfected with 12X CAGA-Luc reporter and expression plasmids of TMEPAI and its mutants (See Materials section for details). Cells were treated without or with TGF-β (2ng/ml) for 16 h. C. MDA-MB-231 cells were transfected with pcDNA or pHis-TMEPAI (wild type and PY or SIM mutants) along with Flag-tagged Smad2 or Smad3 expression vectors. 24 hours later, cells lysates were collected and analyzed by Western blotting. D. Proteins from above lysates were captured by Co2+- chelate matrix and then analyzed for TMEPAI and R-Smads.
Mentions: To confirm TMEPAI sequestration of R-Smads is essential for its anti-TGF-β activity as reported by Watanabe et al [20], we generated SIM (Smad interacting motif) and PY (NEDD4 WW domain interacting motif) mutants in TMEPAI (Fig.4A). While wild type TMEPAI completely suppressed Smad dependent 12XCAGA reporter activity, SIM mutant has no effect (Fig.4B). However, it was surprising that the PY mutant, which abolished the interaction between TMEPAI and NEDD4 [17], inhibited 12XCAGA reporter activity only partially instead of completely (Fig.4B). We speculated that PY mutations could have altered the conformation of TMEPAI such that interaction of SIM domain with R- Smads may be affected. Expression of Smad2 and Smad3 proteins were lower in cells that were transfected with wild type TMEPAI vector than in pcDNA or TMEPAI mutant vectors (Fig.4C). Both R-Smads were pulled down by wild type TMEPAI and PY mutant but not SIM mutant (Fig.4D). However, PY mutant was slightly less efficient than wild type TMEPAI in pulling down R-Smads (Fig.4D), which is consistent with our speculation above.

Bottom Line: Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-β.Moreover, signaling alterations produced by high TMEPAI were associated with oncogenic Snail expression and lung metastases.Together, our findings suggest that TMEPAI has dually critical roles to promote TGF-β dependent cancer cell growth and metastasis.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Pathology, UT Health Science Center at San Antonio, TX.

ABSTRACT
TMEPAI (transmembrane prostate androgen-induced) is amplified at genomic, transcript and protein levels in triple-negative breast cancers and promotes TGF-β dependent growth, motility and invasion. Tumor promotion by TMEPAI depends on two different but related actions on TGF-β signaling. Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-β. Secondly, increased expression of TMEPAI decreases PTEN (phosphatase and tensin homolog) abundance, and thereby increases TGF-β dependent tumor promotive PI3K/Akt signaling. These actions of TMEPAI give rise to increased cell proliferation and motility. Moreover, signaling alterations produced by high TMEPAI were associated with oncogenic Snail expression and lung metastases. Finally, an inverse correlation between TMEPAI and PTEN levels was confirmed in triple negative breast cancer tumor samples. Together, our findings suggest that TMEPAI has dually critical roles to promote TGF-β dependent cancer cell growth and metastasis. Thus, redirected TGF-β signaling through TMEPAI may play a pivotal role in TGF-β mediated tumor promotion.

No MeSH data available.


Related in: MedlinePlus