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MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Prickaerts P, Niessen HE, Dahlmans VE, Spaapen F, Salvaing J, Vanhove J, Geijselaers C, Bartels SJ, Partouns I, Neumann D, Speel EJ, Takihara Y, Wouters BG, Voncken JW - PLoS ONE (2015)

Bottom Line: Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others.In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus.In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands.

ABSTRACT
Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

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Modulation of MK3-levels leads to M/SAPK signalling imbalance.(A) Proliferation profiles of U-2OS/shMK3 cells (left panel; filled squares)) and HeLa/shMK3 cells (right panel; filled squares) versus control (shcon; open circles). (B) Proliferation profiles of TIG3 cells expressing MK3 mutants MK3P28S (MK3P28SOE; filled triangles) or MK3E105A (MK3E105AOE; open triangles) at 1 week post-transduction (left panel) and 4 weeks post-transduction (right panel). The dotted lines in the graphs represent the MK3WT proliferation profiles, as depicted in S2A Fig. Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Protein expression profiles of GST-tagged MK3 (GST:MK3), endogenous MK3 (MK3endo), ERK, phosphorylated ERK (pERK) P38, TP53 and p16INK4A (P16) in TIG3 cells at indicated timepoints post-transduction (corresponding to Fig 4B); loading controls: b-Actin (bAct).
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pone.0118840.g006: Modulation of MK3-levels leads to M/SAPK signalling imbalance.(A) Proliferation profiles of U-2OS/shMK3 cells (left panel; filled squares)) and HeLa/shMK3 cells (right panel; filled squares) versus control (shcon; open circles). (B) Proliferation profiles of TIG3 cells expressing MK3 mutants MK3P28S (MK3P28SOE; filled triangles) or MK3E105A (MK3E105AOE; open triangles) at 1 week post-transduction (left panel) and 4 weeks post-transduction (right panel). The dotted lines in the graphs represent the MK3WT proliferation profiles, as depicted in S2A Fig. Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Protein expression profiles of GST-tagged MK3 (GST:MK3), endogenous MK3 (MK3endo), ERK, phosphorylated ERK (pERK) P38, TP53 and p16INK4A (P16) in TIG3 cells at indicated timepoints post-transduction (corresponding to Fig 4B); loading controls: b-Actin (bAct).

Mentions: As the parental U-2OS and HeLa cell lines both express moderate levels of MK3 (cf. S5B Fig), we studied the effect of RNAi-mediated MK3 depletion on cell proliferation. Remarkably, both U-2OS and HeLa cells proliferated faster in the absence of MK3 (Fig 6A). Thus, in contrast to the negative effects of gain and loss of MK3 function on proliferative capacity in in normal human fibroblasts, MK3WT overexpression and ablation produce opposing effects in cancer cell models.


MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Prickaerts P, Niessen HE, Dahlmans VE, Spaapen F, Salvaing J, Vanhove J, Geijselaers C, Bartels SJ, Partouns I, Neumann D, Speel EJ, Takihara Y, Wouters BG, Voncken JW - PLoS ONE (2015)

Modulation of MK3-levels leads to M/SAPK signalling imbalance.(A) Proliferation profiles of U-2OS/shMK3 cells (left panel; filled squares)) and HeLa/shMK3 cells (right panel; filled squares) versus control (shcon; open circles). (B) Proliferation profiles of TIG3 cells expressing MK3 mutants MK3P28S (MK3P28SOE; filled triangles) or MK3E105A (MK3E105AOE; open triangles) at 1 week post-transduction (left panel) and 4 weeks post-transduction (right panel). The dotted lines in the graphs represent the MK3WT proliferation profiles, as depicted in S2A Fig. Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Protein expression profiles of GST-tagged MK3 (GST:MK3), endogenous MK3 (MK3endo), ERK, phosphorylated ERK (pERK) P38, TP53 and p16INK4A (P16) in TIG3 cells at indicated timepoints post-transduction (corresponding to Fig 4B); loading controls: b-Actin (bAct).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390245&req=5

pone.0118840.g006: Modulation of MK3-levels leads to M/SAPK signalling imbalance.(A) Proliferation profiles of U-2OS/shMK3 cells (left panel; filled squares)) and HeLa/shMK3 cells (right panel; filled squares) versus control (shcon; open circles). (B) Proliferation profiles of TIG3 cells expressing MK3 mutants MK3P28S (MK3P28SOE; filled triangles) or MK3E105A (MK3E105AOE; open triangles) at 1 week post-transduction (left panel) and 4 weeks post-transduction (right panel). The dotted lines in the graphs represent the MK3WT proliferation profiles, as depicted in S2A Fig. Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Protein expression profiles of GST-tagged MK3 (GST:MK3), endogenous MK3 (MK3endo), ERK, phosphorylated ERK (pERK) P38, TP53 and p16INK4A (P16) in TIG3 cells at indicated timepoints post-transduction (corresponding to Fig 4B); loading controls: b-Actin (bAct).
Mentions: As the parental U-2OS and HeLa cell lines both express moderate levels of MK3 (cf. S5B Fig), we studied the effect of RNAi-mediated MK3 depletion on cell proliferation. Remarkably, both U-2OS and HeLa cells proliferated faster in the absence of MK3 (Fig 6A). Thus, in contrast to the negative effects of gain and loss of MK3 function on proliferative capacity in in normal human fibroblasts, MK3WT overexpression and ablation produce opposing effects in cancer cell models.

Bottom Line: Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others.In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus.In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands.

ABSTRACT
Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

Show MeSH
Related in: MedlinePlus