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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

Histological analyses of peripheral nervous system phenotype. Standard hematoxylin-eosin staining (HE) and immunohistochemical (IHC) staining were performed on all peripheral nervous system tissue sections. (a) Representative HE and IHC staining of peripheral nerves taken from a Dhh-Cre; Ptenflox/flox; Cnp-EGFR (ΔPten/C-EGFR) experimental and Dhh-Cre; Ptenflox/+; Cnp-EGFR (Pten-het/C-EGFR) control mice using antibodies against the proliferative marker (Ki67), Schwann cell/oligodendrocyte lineage marker (S100ß), activated Ras/Mapk/Erk signaling by phospho-Erk1/2 (pErk), activated Pi3k/Akt signaling by phospho-Akt (pAkt) detection, and activated mTor signaling by phospho-S6 (pS6). Negative controls, sections incubated without the primary antibody gave no significant signal above background (data not shown). (b) Semiquantitative analysis of proliferative peripheral nerve cells in various control and experimental cohorts. Representative peripheral nerves were isolated from each cohort and IHC stained using the Ki67 proliferative marker. The number of Ki67-positive peripheral nerve cells was counted and shown as a percentage of total cells per counted field of view at 20x magnification (mean ± standard deviation). Peripheral nerves were taken from ΔPten/C-EGFR experimental mice, Dhh-Cre; Ptenflox/flox (ΔPten) and Pten-het/C-EGFR control mice. No Ki67-positive cells were detected in sciatic nerves isolated from FVB/N mice (Supplementary Figure 1). n, number of mice from each cohort; N.S.: nonsignificance between indicated cohorts; P: unpaired t-test.
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fig3: Histological analyses of peripheral nervous system phenotype. Standard hematoxylin-eosin staining (HE) and immunohistochemical (IHC) staining were performed on all peripheral nervous system tissue sections. (a) Representative HE and IHC staining of peripheral nerves taken from a Dhh-Cre; Ptenflox/flox; Cnp-EGFR (ΔPten/C-EGFR) experimental and Dhh-Cre; Ptenflox/+; Cnp-EGFR (Pten-het/C-EGFR) control mice using antibodies against the proliferative marker (Ki67), Schwann cell/oligodendrocyte lineage marker (S100ß), activated Ras/Mapk/Erk signaling by phospho-Erk1/2 (pErk), activated Pi3k/Akt signaling by phospho-Akt (pAkt) detection, and activated mTor signaling by phospho-S6 (pS6). Negative controls, sections incubated without the primary antibody gave no significant signal above background (data not shown). (b) Semiquantitative analysis of proliferative peripheral nerve cells in various control and experimental cohorts. Representative peripheral nerves were isolated from each cohort and IHC stained using the Ki67 proliferative marker. The number of Ki67-positive peripheral nerve cells was counted and shown as a percentage of total cells per counted field of view at 20x magnification (mean ± standard deviation). Peripheral nerves were taken from ΔPten/C-EGFR experimental mice, Dhh-Cre; Ptenflox/flox (ΔPten) and Pten-het/C-EGFR control mice. No Ki67-positive cells were detected in sciatic nerves isolated from FVB/N mice (Supplementary Figure 1). n, number of mice from each cohort; N.S.: nonsignificance between indicated cohorts; P: unpaired t-test.

Mentions: Histopathological and immunohistochemical (IHC) analyses of peripheral nervous system tissues taken from ΔPten/C-EGFR experimental animals demonstrated high-grade PNSTs compared with hyperplasia to low-grade PNSTs seen in Pten-het/C-EGFR animals by histology and Ki67 staining criteria as defined [20, 21] (Figures 3(a) and 3(b)). Mast cells were detected in these enlarged peripheral nerves by toluidine blue staining (data not shown). Enlarged peripheral nerves taken from ΔPten control animals were low-grade PNSTs as previously observed [9]. Enlarged peripheral nerves taken from representative experimental and control animals were positive for S100ß staining, consistent with nerve association (Figure 3(a)). Enlarged peripheral nerves taken from ΔPten/C-EGFR and Pten-het/C-EGFR were Ki67-positive at varying intensities, indicative of cell proliferation (Figure 3(a)). Analyses for proliferation demonstrated significant differences (P < 0.01) in the number of Ki67-positive cells in ΔPten/C-EGFR animals with high-grade PNSTs when compared with other cohorts with hyperplasia/low-grade to low-grade tumors seen in Pten-het/C-EGFR and ΔPten animals, respectively, (Figure 3(b) and Supplementary Figure 1: available online at doi:10.1155/2012/620834). Importantly, the histological and immunohistochemical features of the rapidly growing tumors are consistent with genetically engineered mouse model (GEMM) high-grade PNST and mimic human sporadic MPNST [20]. In a representative tumor sample, we detected hypercellularity, haphazard cell arrangement, poor cell differentiation, variable nuclear pleomorphism, and high mitotic index (Supplementary Figure 2). In addition, all GEMM-PNSTs showed association between nerves and scattered S100β-positive cells (Figure 3(a) and Supplementary Figure 2), typical of GEMM high-grade PNSTs.


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)

Histological analyses of peripheral nervous system phenotype. Standard hematoxylin-eosin staining (HE) and immunohistochemical (IHC) staining were performed on all peripheral nervous system tissue sections. (a) Representative HE and IHC staining of peripheral nerves taken from a Dhh-Cre; Ptenflox/flox; Cnp-EGFR (ΔPten/C-EGFR) experimental and Dhh-Cre; Ptenflox/+; Cnp-EGFR (Pten-het/C-EGFR) control mice using antibodies against the proliferative marker (Ki67), Schwann cell/oligodendrocyte lineage marker (S100ß), activated Ras/Mapk/Erk signaling by phospho-Erk1/2 (pErk), activated Pi3k/Akt signaling by phospho-Akt (pAkt) detection, and activated mTor signaling by phospho-S6 (pS6). Negative controls, sections incubated without the primary antibody gave no significant signal above background (data not shown). (b) Semiquantitative analysis of proliferative peripheral nerve cells in various control and experimental cohorts. Representative peripheral nerves were isolated from each cohort and IHC stained using the Ki67 proliferative marker. The number of Ki67-positive peripheral nerve cells was counted and shown as a percentage of total cells per counted field of view at 20x magnification (mean ± standard deviation). Peripheral nerves were taken from ΔPten/C-EGFR experimental mice, Dhh-Cre; Ptenflox/flox (ΔPten) and Pten-het/C-EGFR control mice. No Ki67-positive cells were detected in sciatic nerves isolated from FVB/N mice (Supplementary Figure 1). n, number of mice from each cohort; N.S.: nonsignificance between indicated cohorts; P: unpaired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539440&req=5

fig3: Histological analyses of peripheral nervous system phenotype. Standard hematoxylin-eosin staining (HE) and immunohistochemical (IHC) staining were performed on all peripheral nervous system tissue sections. (a) Representative HE and IHC staining of peripheral nerves taken from a Dhh-Cre; Ptenflox/flox; Cnp-EGFR (ΔPten/C-EGFR) experimental and Dhh-Cre; Ptenflox/+; Cnp-EGFR (Pten-het/C-EGFR) control mice using antibodies against the proliferative marker (Ki67), Schwann cell/oligodendrocyte lineage marker (S100ß), activated Ras/Mapk/Erk signaling by phospho-Erk1/2 (pErk), activated Pi3k/Akt signaling by phospho-Akt (pAkt) detection, and activated mTor signaling by phospho-S6 (pS6). Negative controls, sections incubated without the primary antibody gave no significant signal above background (data not shown). (b) Semiquantitative analysis of proliferative peripheral nerve cells in various control and experimental cohorts. Representative peripheral nerves were isolated from each cohort and IHC stained using the Ki67 proliferative marker. The number of Ki67-positive peripheral nerve cells was counted and shown as a percentage of total cells per counted field of view at 20x magnification (mean ± standard deviation). Peripheral nerves were taken from ΔPten/C-EGFR experimental mice, Dhh-Cre; Ptenflox/flox (ΔPten) and Pten-het/C-EGFR control mice. No Ki67-positive cells were detected in sciatic nerves isolated from FVB/N mice (Supplementary Figure 1). n, number of mice from each cohort; N.S.: nonsignificance between indicated cohorts; P: unpaired t-test.
Mentions: Histopathological and immunohistochemical (IHC) analyses of peripheral nervous system tissues taken from ΔPten/C-EGFR experimental animals demonstrated high-grade PNSTs compared with hyperplasia to low-grade PNSTs seen in Pten-het/C-EGFR animals by histology and Ki67 staining criteria as defined [20, 21] (Figures 3(a) and 3(b)). Mast cells were detected in these enlarged peripheral nerves by toluidine blue staining (data not shown). Enlarged peripheral nerves taken from ΔPten control animals were low-grade PNSTs as previously observed [9]. Enlarged peripheral nerves taken from representative experimental and control animals were positive for S100ß staining, consistent with nerve association (Figure 3(a)). Enlarged peripheral nerves taken from ΔPten/C-EGFR and Pten-het/C-EGFR were Ki67-positive at varying intensities, indicative of cell proliferation (Figure 3(a)). Analyses for proliferation demonstrated significant differences (P < 0.01) in the number of Ki67-positive cells in ΔPten/C-EGFR animals with high-grade PNSTs when compared with other cohorts with hyperplasia/low-grade to low-grade tumors seen in Pten-het/C-EGFR and ΔPten animals, respectively, (Figure 3(b) and Supplementary Figure 1: available online at doi:10.1155/2012/620834). Importantly, the histological and immunohistochemical features of the rapidly growing tumors are consistent with genetically engineered mouse model (GEMM) high-grade PNST and mimic human sporadic MPNST [20]. In a representative tumor sample, we detected hypercellularity, haphazard cell arrangement, poor cell differentiation, variable nuclear pleomorphism, and high mitotic index (Supplementary Figure 2). In addition, all GEMM-PNSTs showed association between nerves and scattered S100β-positive cells (Figure 3(a) and Supplementary Figure 2), typical of GEMM high-grade PNSTs.

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