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Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

Blaskovich MA, Yendluri V, Lawrence HR, Lawrence NJ, Sebti SM, Springett GM - PLoS ONE (2013)

Bottom Line: Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down.Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function.Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America.

ABSTRACT
Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

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Related in: MedlinePlus

LPAAT-β expression and siRNA knockdown in human pancreatic cancer cells.(A) A panel of human pancreatic cancer cell lines shows differential expression of LPAAT-β protein. (B) Knockdown efficiency of siRNA transfection of AsPC-1, MiaPaCa2, and Panc-1 cells with LPAAT-β siRNA (LP-1 and LP-2) compared to mock transfection (m) and non-targeting (nt) siRNA controls.
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pone-0078632-g001: LPAAT-β expression and siRNA knockdown in human pancreatic cancer cells.(A) A panel of human pancreatic cancer cell lines shows differential expression of LPAAT-β protein. (B) Knockdown efficiency of siRNA transfection of AsPC-1, MiaPaCa2, and Panc-1 cells with LPAAT-β siRNA (LP-1 and LP-2) compared to mock transfection (m) and non-targeting (nt) siRNA controls.

Mentions: LPAAT-β protein was found to be expressed in several pancreatic adenocarcinoma cell lines similar to its previously reported expression in gynecologic cancer cells (Figure 1A) [14]. For our work, we chose three pancreatic cancer cell lines, with high (AsPC-1), intermediate (MiaPaCa2), and low (Panc-1) levels of LPAAT-β protein expression. We used three siRNA constructs targeting LPAAT-β to knock down the expression of LPAAT-β protein, LP-1, LP-2 and LP-4. All three siRNAs exhibited concentration- and time-dependent inhibition. LP-1 and LP-4 (25 nM) inhibited the expression of LPAAT-β by greater than 80% after 72 hours (Figure 1B, Figure S1). LP-2 (25 nM) inhibited expression by greater than 60% after 72 hours of treatment (Figure 1B).


Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

Blaskovich MA, Yendluri V, Lawrence HR, Lawrence NJ, Sebti SM, Springett GM - PLoS ONE (2013)

LPAAT-β expression and siRNA knockdown in human pancreatic cancer cells.(A) A panel of human pancreatic cancer cell lines shows differential expression of LPAAT-β protein. (B) Knockdown efficiency of siRNA transfection of AsPC-1, MiaPaCa2, and Panc-1 cells with LPAAT-β siRNA (LP-1 and LP-2) compared to mock transfection (m) and non-targeting (nt) siRNA controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814986&req=5

pone-0078632-g001: LPAAT-β expression and siRNA knockdown in human pancreatic cancer cells.(A) A panel of human pancreatic cancer cell lines shows differential expression of LPAAT-β protein. (B) Knockdown efficiency of siRNA transfection of AsPC-1, MiaPaCa2, and Panc-1 cells with LPAAT-β siRNA (LP-1 and LP-2) compared to mock transfection (m) and non-targeting (nt) siRNA controls.
Mentions: LPAAT-β protein was found to be expressed in several pancreatic adenocarcinoma cell lines similar to its previously reported expression in gynecologic cancer cells (Figure 1A) [14]. For our work, we chose three pancreatic cancer cell lines, with high (AsPC-1), intermediate (MiaPaCa2), and low (Panc-1) levels of LPAAT-β protein expression. We used three siRNA constructs targeting LPAAT-β to knock down the expression of LPAAT-β protein, LP-1, LP-2 and LP-4. All three siRNAs exhibited concentration- and time-dependent inhibition. LP-1 and LP-4 (25 nM) inhibited the expression of LPAAT-β by greater than 80% after 72 hours (Figure 1B, Figure S1). LP-2 (25 nM) inhibited expression by greater than 60% after 72 hours of treatment (Figure 1B).

Bottom Line: Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down.Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function.Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America.

ABSTRACT
Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

Show MeSH
Related in: MedlinePlus