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The BRAF kinase domain promotes the development of gliomas in vivo.

Shin CH, Grossmann AH, Holmen SL, Robinson JP - Genes Cancer (2015)

Bottom Line: They also lacked the necrosis and vascular proliferation seen in BRAFV600E-driven tumors.The BRAF-KD-expressing astrocytes showed elevated MAPK signaling, albeit at reduced levels compared to the BRAF(V600E) mutant.Pharmacologic inhibition of MEK and PI3K inhibited cell growth and induced apoptosis in astrocytes expressing BRAF-KD.

View Article: PubMed Central - PubMed

Affiliation: Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, Utah, USA ; Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, Utah, USA.

ABSTRACT
In-frame BRAF fusions have been observed in over 80% of sporadic pilocytic astrocytomas. In each fusion, the N-terminal autoinhibitory domain of BRAF is lost, which results in constitutive activation via the retained C-terminal kinase domain (BRAF-KD). We set out to determine if the BRAF-KD is sufficient to induce gliomas alone or in combination with Ink4a/Arf loss. Syngeneic cell lines demonstrated the transforming ability of the BRAF-KD following Ink4a/Arf loss. In vivo, somatic cell gene transfer of the BRAF-KD did not cause tumors to develop; however, gliomas were detected in 21% of the mice following Ink4a/Arf loss. Interestingly, these mice demonstrated no obvious symptoms. Histologically the tumors were highly cellular and atypical, similar to BRAF(V600E) tumors reported previously, but with less invasive borders. They also lacked the necrosis and vascular proliferation seen in BRAFV600E-driven tumors. The BRAF-KD-expressing astrocytes showed elevated MAPK signaling, albeit at reduced levels compared to the BRAF(V600E) mutant. Pharmacologic inhibition of MEK and PI3K inhibited cell growth and induced apoptosis in astrocytes expressing BRAF-KD. Our findings demonstrate that the BRAF-KD can cooperate with Ink4a/Arf loss to drive the development of gliomas and suggest that glioma development is determined by the level of MAPK signaling.

No MeSH data available.


Related in: MedlinePlus

Analysis of the expression and functional activity of the BRAF-KD in the context of Ink4a/Arf-deficiencyA: Western blot of Ink4a/Arf-deficient astrocytes (MC41+) infected with BRAF-KD (KD) and BRAFV600E (FLVE) with corresponding uninfected, negative control astrocytes (MC41−), and positive control avian fibroblasts (+). B: BRAF-KD expression causes MAPK activation as measured by P-Erk in BRAF-KD transfected human 293FT cells (+) compared to mock transfected cells (−). C: Relative proliferation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells. The mean of three replicates is shown with error bars for standard error. D: Colony formation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells in soft agar. Error bars reflect standard error of three replicates.
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Figure 2: Analysis of the expression and functional activity of the BRAF-KD in the context of Ink4a/Arf-deficiencyA: Western blot of Ink4a/Arf-deficient astrocytes (MC41+) infected with BRAF-KD (KD) and BRAFV600E (FLVE) with corresponding uninfected, negative control astrocytes (MC41−), and positive control avian fibroblasts (+). B: BRAF-KD expression causes MAPK activation as measured by P-Erk in BRAF-KD transfected human 293FT cells (+) compared to mock transfected cells (−). C: Relative proliferation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells. The mean of three replicates is shown with error bars for standard error. D: Colony formation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells in soft agar. Error bars reflect standard error of three replicates.

Mentions: We previously demonstrated the transformative capacity of N-TVA/Ink4a/Arf−/− astrocytes infected with RCASBP(A) viruses containing CRAFß22W, BRAFV600E or KRASG12D, which form numerous colonies in soft agar [16]. To assess the transforming potential of the BRAF kinase domain BRAF-KD, we infected N-TVA/Ink4a/Arf−/− astrocytes with RCASBP(A)BRAF-KD and the full length BRAF with the V600E mutation, RCASBP(A)BRAFV600E (hereafter BRAF-FLVE). Gene delivery and expression was confirmed by immunoblotting (Figure 2A). Analysis of phosphorylated Erk 1/2 (P-Erk) demonstrated elevated MAPK activation in cells expressing BRAF-KD (Figure 2B). Ink4a/Arf-deficient astrocytes expressing BRAF-KD or BRAF-FLVE proliferated more rapidly than the negative control; however, there was no significant difference between proliferation of BRAF-KD or BRAF-FLVE cells (P=0.29; Figure 2C). Whereas Ink4a/Arf-deficient astrocytes did not form colonies in soft agar, expression of BRAF-KD or our positive control BRAF-FLVE, induced colonies demonstrating their ability to transform cells in the context of altered Rb and/or p53 signaling (Figure 2D). Interestingly, there was a statistically significant difference between the number of colonies formed by BRAF-KD and BRAF-FLVE expression (P=0.04).


The BRAF kinase domain promotes the development of gliomas in vivo.

Shin CH, Grossmann AH, Holmen SL, Robinson JP - Genes Cancer (2015)

Analysis of the expression and functional activity of the BRAF-KD in the context of Ink4a/Arf-deficiencyA: Western blot of Ink4a/Arf-deficient astrocytes (MC41+) infected with BRAF-KD (KD) and BRAFV600E (FLVE) with corresponding uninfected, negative control astrocytes (MC41−), and positive control avian fibroblasts (+). B: BRAF-KD expression causes MAPK activation as measured by P-Erk in BRAF-KD transfected human 293FT cells (+) compared to mock transfected cells (−). C: Relative proliferation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells. The mean of three replicates is shown with error bars for standard error. D: Colony formation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells in soft agar. Error bars reflect standard error of three replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362480&req=5

Figure 2: Analysis of the expression and functional activity of the BRAF-KD in the context of Ink4a/Arf-deficiencyA: Western blot of Ink4a/Arf-deficient astrocytes (MC41+) infected with BRAF-KD (KD) and BRAFV600E (FLVE) with corresponding uninfected, negative control astrocytes (MC41−), and positive control avian fibroblasts (+). B: BRAF-KD expression causes MAPK activation as measured by P-Erk in BRAF-KD transfected human 293FT cells (+) compared to mock transfected cells (−). C: Relative proliferation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells. The mean of three replicates is shown with error bars for standard error. D: Colony formation of Ink4a/Arf-deficient astrocytes infected with BRAF-KD (KD) or BRAFV600E (FLVE) compared to uninfected, control cells in soft agar. Error bars reflect standard error of three replicates.
Mentions: We previously demonstrated the transformative capacity of N-TVA/Ink4a/Arf−/− astrocytes infected with RCASBP(A) viruses containing CRAFß22W, BRAFV600E or KRASG12D, which form numerous colonies in soft agar [16]. To assess the transforming potential of the BRAF kinase domain BRAF-KD, we infected N-TVA/Ink4a/Arf−/− astrocytes with RCASBP(A)BRAF-KD and the full length BRAF with the V600E mutation, RCASBP(A)BRAFV600E (hereafter BRAF-FLVE). Gene delivery and expression was confirmed by immunoblotting (Figure 2A). Analysis of phosphorylated Erk 1/2 (P-Erk) demonstrated elevated MAPK activation in cells expressing BRAF-KD (Figure 2B). Ink4a/Arf-deficient astrocytes expressing BRAF-KD or BRAF-FLVE proliferated more rapidly than the negative control; however, there was no significant difference between proliferation of BRAF-KD or BRAF-FLVE cells (P=0.29; Figure 2C). Whereas Ink4a/Arf-deficient astrocytes did not form colonies in soft agar, expression of BRAF-KD or our positive control BRAF-FLVE, induced colonies demonstrating their ability to transform cells in the context of altered Rb and/or p53 signaling (Figure 2D). Interestingly, there was a statistically significant difference between the number of colonies formed by BRAF-KD and BRAF-FLVE expression (P=0.04).

Bottom Line: They also lacked the necrosis and vascular proliferation seen in BRAFV600E-driven tumors.The BRAF-KD-expressing astrocytes showed elevated MAPK signaling, albeit at reduced levels compared to the BRAF(V600E) mutant.Pharmacologic inhibition of MEK and PI3K inhibited cell growth and induced apoptosis in astrocytes expressing BRAF-KD.

View Article: PubMed Central - PubMed

Affiliation: Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, Utah, USA ; Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, Utah, USA.

ABSTRACT
In-frame BRAF fusions have been observed in over 80% of sporadic pilocytic astrocytomas. In each fusion, the N-terminal autoinhibitory domain of BRAF is lost, which results in constitutive activation via the retained C-terminal kinase domain (BRAF-KD). We set out to determine if the BRAF-KD is sufficient to induce gliomas alone or in combination with Ink4a/Arf loss. Syngeneic cell lines demonstrated the transforming ability of the BRAF-KD following Ink4a/Arf loss. In vivo, somatic cell gene transfer of the BRAF-KD did not cause tumors to develop; however, gliomas were detected in 21% of the mice following Ink4a/Arf loss. Interestingly, these mice demonstrated no obvious symptoms. Histologically the tumors were highly cellular and atypical, similar to BRAF(V600E) tumors reported previously, but with less invasive borders. They also lacked the necrosis and vascular proliferation seen in BRAFV600E-driven tumors. The BRAF-KD-expressing astrocytes showed elevated MAPK signaling, albeit at reduced levels compared to the BRAF(V600E) mutant. Pharmacologic inhibition of MEK and PI3K inhibited cell growth and induced apoptosis in astrocytes expressing BRAF-KD. Our findings demonstrate that the BRAF-KD can cooperate with Ink4a/Arf loss to drive the development of gliomas and suggest that glioma development is determined by the level of MAPK signaling.

No MeSH data available.


Related in: MedlinePlus