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Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

Hausmann S, Brandt E, Köchel C, Einsele H, Bargou RC, Seggewiss-Bernhardt R, Stühmer T - PLoS ONE (2015)

Bottom Line: SGK3 was expressed in all MM cell lines and in all primary MM samples tested.Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation.We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, Division of Hematology and Oncology, University Hospital of Würzburg, Würzburg, Germany.

ABSTRACT
Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

No MeSH data available.


Related in: MedlinePlus

SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in primary MM cells.Shown are Western blots prepared from frozen pellets of primary MM cells purified by CD138 microbead selection. The material was used to load two gels, with the representative β-actin control belonging to the same blot on which PI3K-p110α, P-FOXO1/3A, P-Akt (Thr308) and pan-Akt were also stained. Of note, the phospho-Akt (Thr308) antibody also stained a slightly larger band in most primary MM samples (marked by "?") that was not visible in any MM cell line. This band runs slightly higher, though, than the SGK3 band as stained on the parallel blot. Staining for P-PRAS40 (CST; no. 2997) was performed after staining for P-GSK-3β. Since both antibodies were raised in rabbit the latter signal reappeared in the P-PRAS40 blot.
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pone.0122689.g002: SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in primary MM cells.Shown are Western blots prepared from frozen pellets of primary MM cells purified by CD138 microbead selection. The material was used to load two gels, with the representative β-actin control belonging to the same blot on which PI3K-p110α, P-FOXO1/3A, P-Akt (Thr308) and pan-Akt were also stained. Of note, the phospho-Akt (Thr308) antibody also stained a slightly larger band in most primary MM samples (marked by "?") that was not visible in any MM cell line. This band runs slightly higher, though, than the SGK3 band as stained on the parallel blot. Staining for P-PRAS40 (CST; no. 2997) was performed after staining for P-GSK-3β. Since both antibodies were raised in rabbit the latter signal reappeared in the P-PRAS40 blot.

Mentions: SGK3 was also found to be present in all primary MM samples tested (note: the apparently very strong signal in sample pMM-6 is in part the result of rearward smearing off of a strong band that runs slightly lower than full-length SGK3) (Fig 2). Although the phosphorylation levels of PI3K system components showed strong differences between the individual primary samples, the presence of phospho-Akt was more stringently correlated with phosphorylation of downstream substrates than in MM cell lines (Fig 2). Taken together, these analyses showed that SGK3 expression in MM cells appears to be ubiquitous, but that its presence is not obviously correlated to a particular activity pattern of potential downstream substrates.


Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

Hausmann S, Brandt E, Köchel C, Einsele H, Bargou RC, Seggewiss-Bernhardt R, Stühmer T - PLoS ONE (2015)

SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in primary MM cells.Shown are Western blots prepared from frozen pellets of primary MM cells purified by CD138 microbead selection. The material was used to load two gels, with the representative β-actin control belonging to the same blot on which PI3K-p110α, P-FOXO1/3A, P-Akt (Thr308) and pan-Akt were also stained. Of note, the phospho-Akt (Thr308) antibody also stained a slightly larger band in most primary MM samples (marked by "?") that was not visible in any MM cell line. This band runs slightly higher, though, than the SGK3 band as stained on the parallel blot. Staining for P-PRAS40 (CST; no. 2997) was performed after staining for P-GSK-3β. Since both antibodies were raised in rabbit the latter signal reappeared in the P-PRAS40 blot.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383545&req=5

pone.0122689.g002: SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in primary MM cells.Shown are Western blots prepared from frozen pellets of primary MM cells purified by CD138 microbead selection. The material was used to load two gels, with the representative β-actin control belonging to the same blot on which PI3K-p110α, P-FOXO1/3A, P-Akt (Thr308) and pan-Akt were also stained. Of note, the phospho-Akt (Thr308) antibody also stained a slightly larger band in most primary MM samples (marked by "?") that was not visible in any MM cell line. This band runs slightly higher, though, than the SGK3 band as stained on the parallel blot. Staining for P-PRAS40 (CST; no. 2997) was performed after staining for P-GSK-3β. Since both antibodies were raised in rabbit the latter signal reappeared in the P-PRAS40 blot.
Mentions: SGK3 was also found to be present in all primary MM samples tested (note: the apparently very strong signal in sample pMM-6 is in part the result of rearward smearing off of a strong band that runs slightly lower than full-length SGK3) (Fig 2). Although the phosphorylation levels of PI3K system components showed strong differences between the individual primary samples, the presence of phospho-Akt was more stringently correlated with phosphorylation of downstream substrates than in MM cell lines (Fig 2). Taken together, these analyses showed that SGK3 expression in MM cells appears to be ubiquitous, but that its presence is not obviously correlated to a particular activity pattern of potential downstream substrates.

Bottom Line: SGK3 was expressed in all MM cell lines and in all primary MM samples tested.Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation.We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, Division of Hematology and Oncology, University Hospital of Würzburg, Würzburg, Germany.

ABSTRACT
Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

No MeSH data available.


Related in: MedlinePlus