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Conditional Inactivation of Pten with EGFR Overexpression in Schwann Cells Models Sporadic MPNST.

Keng VW, Watson AL, Rahrmann EP, Li H, Tschida BR, Moriarity BS, Choi K, Rizvi TA, Collins MH, Wallace MR, Ratner N, Largaespada DA - Sarcoma (2012)

Bottom Line: The genetic mechanisms involved in the transformation from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis-type-1- (NF1-)associated or sporadic malignant peripheral nerve sheath tumors (MPNSTs) remain unclear.It is hypothesized that many genetic changes are involved in transformation.We tested if these two genes cooperate in the evolution of PNSTs.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA ; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA ; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA ; Brain Tumor Program, University of Minnesota, Minneapolis, MN 55455, USA ; Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.

ABSTRACT
The genetic mechanisms involved in the transformation from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis-type-1- (NF1-)associated or sporadic malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. It is hypothesized that many genetic changes are involved in transformation. Recently, it has been shown that both phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) play important roles in the initiation of peripheral nerve sheath tumors (PNSTs). In human MPNSTs, PTEN expression is often reduced, while EGFR expression is often induced. We tested if these two genes cooperate in the evolution of PNSTs. Transgenic mice were generated carrying conditional floxed alleles of Pten, and EGFR was expressed under the control of the 2',3'-cyclic nucleotide 3'phosphodiesterase (Cnp) promoter and a desert hedgehog (Dhh) regulatory element driving Cre recombinase transgenic mice (Dhh-Cre). Complete loss of Pten and EGFR overexpression in Schwann cells led to the development of high-grade PNSTs. In vitro experiments using immortalized human Schwann cells demonstrated that loss of PTEN and overexpression of EGFR cooperate to increase cellular proliferation and anchorage-independent colony formation. This mouse model can rapidly recapitulate PNST onset and progression to high-grade PNSTs, as seen in sporadic MPNST patients.

No MeSH data available.


Related in: MedlinePlus

Knockdown of PTEN and overexpression of EGFR cooperate in vitro to oncogenically transform immortalized human Schwann cells. (a) PiggyBac (PB) constructs used to knock down PTEN (PB-shPTEN) and/or overexpress EGFR (PB-EGFR) in HSC2λ immortalized human Schwann cells. CAG: cytomegalovirus early enhancer element and chicken beta-actin promoter; PGK: phosphoglycerate kinase; EEF1A1: eukaryotic translation elongation factor 1 alpha 1 promoter; IRES: internal ribosome entry site; Gfp: green fluorescent protein; pA: polyadenylation signal; Puro: puromycin resistance gene; triangles: PB-specific inverted terminal repeat sequences. Quantitative PCR analysis demonstrating that PTEN mRNA levels are reduced (b) and EGFR mRNA levels are increased (c) when these constructs are stably transfected into HSC2λ cells. (d) MTS proliferation assay shows that PTEN knockdown or EGFR overexpression alone do not change the rate of proliferation compared to control transfected cells, but when combined significantly increase cellular proliferation. (e) Soft agar colony formation assay demonstrates that PTEN knockdown moderately increases colony formation, but in the context of EGFR overexpression, reduction in PTEN significantly increases the number of colonies formed. *P < 0.05 and **P < 0.0001, unpaired t-test; mean ± standard deviation.
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fig5: Knockdown of PTEN and overexpression of EGFR cooperate in vitro to oncogenically transform immortalized human Schwann cells. (a) PiggyBac (PB) constructs used to knock down PTEN (PB-shPTEN) and/or overexpress EGFR (PB-EGFR) in HSC2λ immortalized human Schwann cells. CAG: cytomegalovirus early enhancer element and chicken beta-actin promoter; PGK: phosphoglycerate kinase; EEF1A1: eukaryotic translation elongation factor 1 alpha 1 promoter; IRES: internal ribosome entry site; Gfp: green fluorescent protein; pA: polyadenylation signal; Puro: puromycin resistance gene; triangles: PB-specific inverted terminal repeat sequences. Quantitative PCR analysis demonstrating that PTEN mRNA levels are reduced (b) and EGFR mRNA levels are increased (c) when these constructs are stably transfected into HSC2λ cells. (d) MTS proliferation assay shows that PTEN knockdown or EGFR overexpression alone do not change the rate of proliferation compared to control transfected cells, but when combined significantly increase cellular proliferation. (e) Soft agar colony formation assay demonstrates that PTEN knockdown moderately increases colony formation, but in the context of EGFR overexpression, reduction in PTEN significantly increases the number of colonies formed. *P < 0.05 and **P < 0.0001, unpaired t-test; mean ± standard deviation.

Mentions: Cultured normal Schwann cells were derived from a healthy individual's sciatic nerve [24] and immortalized by overexpressing both human telomerase reverse transcriptase (TERT) and mouse cyclin-dependent kinase 4 (Cdk4) to allow for in vitro studies-immortalized human Schwann cells referred to as HSC2λ hereafter (H. Li & M. R. Wallace, manuscript in preparation). The plasmid vectors shown in Figure 5(a) along with a plasmid containing piggyBac 7 (PB7) transposase [25] under the control of a cytomegalovirus promoter (construct not shown) were transfected into HSC2λ cells using the Neon transfection system, according to manufacturer's protocol (Invitrogen). PiggyBac (PB) transposon expression constructs contain the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter to drive expression of a PTEN shRNA and/or EGFR cDNA and include a puromycin resistance gene and green fluorescent protein (Gfp) cDNA for selection purposes. The PB-control vector contains the Luciferase and Gfp reporter genes under the control of the cytomegalovirus early enhancer element and chicken beta-actin (CAG) promoter. Following transfection, cells underwent selection in standard DMEM full media containing 4 μg/mL puromycin (Invitrogen). Transposon-mediated gene transfer was validated by Gfp expression and quantitative PCR.


Conditional Inactivation of Pten with EGFR Overexpression in Schwann Cells Models Sporadic MPNST.

Keng VW, Watson AL, Rahrmann EP, Li H, Tschida BR, Moriarity BS, Choi K, Rizvi TA, Collins MH, Wallace MR, Ratner N, Largaespada DA - Sarcoma (2012)

Knockdown of PTEN and overexpression of EGFR cooperate in vitro to oncogenically transform immortalized human Schwann cells. (a) PiggyBac (PB) constructs used to knock down PTEN (PB-shPTEN) and/or overexpress EGFR (PB-EGFR) in HSC2λ immortalized human Schwann cells. CAG: cytomegalovirus early enhancer element and chicken beta-actin promoter; PGK: phosphoglycerate kinase; EEF1A1: eukaryotic translation elongation factor 1 alpha 1 promoter; IRES: internal ribosome entry site; Gfp: green fluorescent protein; pA: polyadenylation signal; Puro: puromycin resistance gene; triangles: PB-specific inverted terminal repeat sequences. Quantitative PCR analysis demonstrating that PTEN mRNA levels are reduced (b) and EGFR mRNA levels are increased (c) when these constructs are stably transfected into HSC2λ cells. (d) MTS proliferation assay shows that PTEN knockdown or EGFR overexpression alone do not change the rate of proliferation compared to control transfected cells, but when combined significantly increase cellular proliferation. (e) Soft agar colony formation assay demonstrates that PTEN knockdown moderately increases colony formation, but in the context of EGFR overexpression, reduction in PTEN significantly increases the number of colonies formed. *P < 0.05 and **P < 0.0001, unpaired t-test; mean ± standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Knockdown of PTEN and overexpression of EGFR cooperate in vitro to oncogenically transform immortalized human Schwann cells. (a) PiggyBac (PB) constructs used to knock down PTEN (PB-shPTEN) and/or overexpress EGFR (PB-EGFR) in HSC2λ immortalized human Schwann cells. CAG: cytomegalovirus early enhancer element and chicken beta-actin promoter; PGK: phosphoglycerate kinase; EEF1A1: eukaryotic translation elongation factor 1 alpha 1 promoter; IRES: internal ribosome entry site; Gfp: green fluorescent protein; pA: polyadenylation signal; Puro: puromycin resistance gene; triangles: PB-specific inverted terminal repeat sequences. Quantitative PCR analysis demonstrating that PTEN mRNA levels are reduced (b) and EGFR mRNA levels are increased (c) when these constructs are stably transfected into HSC2λ cells. (d) MTS proliferation assay shows that PTEN knockdown or EGFR overexpression alone do not change the rate of proliferation compared to control transfected cells, but when combined significantly increase cellular proliferation. (e) Soft agar colony formation assay demonstrates that PTEN knockdown moderately increases colony formation, but in the context of EGFR overexpression, reduction in PTEN significantly increases the number of colonies formed. *P < 0.05 and **P < 0.0001, unpaired t-test; mean ± standard deviation.
Mentions: Cultured normal Schwann cells were derived from a healthy individual's sciatic nerve [24] and immortalized by overexpressing both human telomerase reverse transcriptase (TERT) and mouse cyclin-dependent kinase 4 (Cdk4) to allow for in vitro studies-immortalized human Schwann cells referred to as HSC2λ hereafter (H. Li & M. R. Wallace, manuscript in preparation). The plasmid vectors shown in Figure 5(a) along with a plasmid containing piggyBac 7 (PB7) transposase [25] under the control of a cytomegalovirus promoter (construct not shown) were transfected into HSC2λ cells using the Neon transfection system, according to manufacturer's protocol (Invitrogen). PiggyBac (PB) transposon expression constructs contain the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter to drive expression of a PTEN shRNA and/or EGFR cDNA and include a puromycin resistance gene and green fluorescent protein (Gfp) cDNA for selection purposes. The PB-control vector contains the Luciferase and Gfp reporter genes under the control of the cytomegalovirus early enhancer element and chicken beta-actin (CAG) promoter. Following transfection, cells underwent selection in standard DMEM full media containing 4 μg/mL puromycin (Invitrogen). Transposon-mediated gene transfer was validated by Gfp expression and quantitative PCR.

Bottom Line: The genetic mechanisms involved in the transformation from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis-type-1- (NF1-)associated or sporadic malignant peripheral nerve sheath tumors (MPNSTs) remain unclear.It is hypothesized that many genetic changes are involved in transformation.We tested if these two genes cooperate in the evolution of PNSTs.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA ; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA ; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA ; Brain Tumor Program, University of Minnesota, Minneapolis, MN 55455, USA ; Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.

ABSTRACT
The genetic mechanisms involved in the transformation from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis-type-1- (NF1-)associated or sporadic malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. It is hypothesized that many genetic changes are involved in transformation. Recently, it has been shown that both phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) play important roles in the initiation of peripheral nerve sheath tumors (PNSTs). In human MPNSTs, PTEN expression is often reduced, while EGFR expression is often induced. We tested if these two genes cooperate in the evolution of PNSTs. Transgenic mice were generated carrying conditional floxed alleles of Pten, and EGFR was expressed under the control of the 2',3'-cyclic nucleotide 3'phosphodiesterase (Cnp) promoter and a desert hedgehog (Dhh) regulatory element driving Cre recombinase transgenic mice (Dhh-Cre). Complete loss of Pten and EGFR overexpression in Schwann cells led to the development of high-grade PNSTs. In vitro experiments using immortalized human Schwann cells demonstrated that loss of PTEN and overexpression of EGFR cooperate to increase cellular proliferation and anchorage-independent colony formation. This mouse model can rapidly recapitulate PNST onset and progression to high-grade PNSTs, as seen in sporadic MPNST patients.

No MeSH data available.


Related in: MedlinePlus