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Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis.

Wang HC, Chiang WF, Huang HH, Shen YY, Chiang HC - BMC Cancer (2014)

Bottom Line: We observed similar results in phosphatase-dead SHP2 C459S mutant expressing cells.Enhanced invasiveness was associated with significant upregulation of E-cadherin, vimentin, Snail/Twist1, and matrix metalloproteinase-2 in the highly invasive clones.In addition, we determined that SHP2 activity is required for the downregulation of phosphorylated ERK1/2, which modulates the downstream effectors, Snail and Twist1 at a transcript level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Environmental Health and Occupational Medicine, National Health Research Institutes, No,35, Keyan Road, Zhunan, 35053 Miaoli County, Taiwan. hcchiang@nhri.org.tw.

ABSTRACT

Background: Tumor invasion and metastasis represent a major unsolved problem in cancer pathogenesis. Recent studies have indicated the involvement of Src-homology 2 domain-containing tyrosine phosphatase 2 (SHP2) in multiple malignancies; however, the role of SHP2 in oral cancer progression has yet to be elucidated. We propose that SHP2 is involved in the progression of oral cancer toward metastasis.

Methods: SHP2 expression was evaluated in paired oral cancer tissues by using immunohistochemical staining and real-time reverse transcription polymerase chain reaction. Isogenic highly invasive oral cancer cell lines from their respective low invasive parental lines were established using a Boyden chamber assay, and changes in the hallmarks of the epithelial-mesenchymal transition (EMT) were assessed to evaluate SHP2 function. SHP2 activity in oral cancer cells was reduced using si-RNA knockdown or enforced expression of a catalytically deficient mutant to analyze migratory and invasive ability in vitro and metastasis toward the lung in mice in vivo.

Results: We observed the significant upregulation of SHP2 in oral cancer tissues and cell lines. Following SHP2 knockdown, the oral cancer cells markedly attenuated migratory and invasion ability. We observed similar results in phosphatase-dead SHP2 C459S mutant expressing cells. Enhanced invasiveness was associated with significant upregulation of E-cadherin, vimentin, Snail/Twist1, and matrix metalloproteinase-2 in the highly invasive clones. In addition, we determined that SHP2 activity is required for the downregulation of phosphorylated ERK1/2, which modulates the downstream effectors, Snail and Twist1 at a transcript level. In lung tissue sections of mice, we observed that HSC3 tumors with SHP2 deletion exhibited significantly reduced metastatic capacity, compared with tumors administered control si-RNA.

Conclusions: Our data suggest that SHP2 promotes the invasion and metastasis of oral cancer cells. These results provide a rationale for further investigating the effects of small-molecule SHP2 inhibitors on the progression of oral cancer, and indicate a previously unrecognized SHP2-ERK1/2-Snail/Twist1 pathway that is likely to play a crucial role in oral cancer invasion and metastasis.

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Characteristics of highly invasive clone, HSC3-Inv4 derived from parental HSC3 cells. (A) Bright file microscopy images of HSC3 parental and HSC3 Inv 4 (20×, Upper panels). Cells were stained with E-cadherin and images were taken under fluorescence at 60× (Lower panels). (B) Expressions of E-cadherin and vimentin were analyzed by Western blot with indicated antibodies; GAPDH as a loading control. (C) Increased Snail (Upper panel) and Twist1 (Middle panel) transcript levels were observed in HSC3-Inv4 and HSC3-Inv8 compared to HSC3 parental cells. Experiments were done at least in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with the adjacent normal in each case. Western blot shows the expression level of Snail and Twist1 in HSC3-parental, HSC3-Inv4 and HSC3-Inv8 cells (Lower panel). (D) Status of MMP-2 secretion on highly invasive clones. Medium collected from HSC3 parental, HSC-Inv4 and HSC3-Inv8 cells were subjected to MMP-2 secretion analysis. Significantly increased amounts of MMP-2 were seen in selected sub-cell lines compared to parental cells. (E) SHP2 depletion resulted in decreased MMP-2 secretion in HSC3 parental, HSC3-Inv4 and HSC3-Inv8 cells.
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Figure 3: Characteristics of highly invasive clone, HSC3-Inv4 derived from parental HSC3 cells. (A) Bright file microscopy images of HSC3 parental and HSC3 Inv 4 (20×, Upper panels). Cells were stained with E-cadherin and images were taken under fluorescence at 60× (Lower panels). (B) Expressions of E-cadherin and vimentin were analyzed by Western blot with indicated antibodies; GAPDH as a loading control. (C) Increased Snail (Upper panel) and Twist1 (Middle panel) transcript levels were observed in HSC3-Inv4 and HSC3-Inv8 compared to HSC3 parental cells. Experiments were done at least in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with the adjacent normal in each case. Western blot shows the expression level of Snail and Twist1 in HSC3-parental, HSC3-Inv4 and HSC3-Inv8 cells (Lower panel). (D) Status of MMP-2 secretion on highly invasive clones. Medium collected from HSC3 parental, HSC-Inv4 and HSC3-Inv8 cells were subjected to MMP-2 secretion analysis. Significantly increased amounts of MMP-2 were seen in selected sub-cell lines compared to parental cells. (E) SHP2 depletion resulted in decreased MMP-2 secretion in HSC3 parental, HSC3-Inv4 and HSC3-Inv8 cells.

Mentions: As shown in Figure 3A, we evaluated the changes in EMT-associated E-cadherin and vimentin in highly invasive oral cancer cells. Our results indicated that the majority of the parental HSC3 cells were polygonal in shape (Figure 3A, left upper panel); whereas, the HSC3-Inv4 cells were rather spindle shaped (Figure 3A, right upper panel), with downregulated of E-cadherin protein and upregulated of vimentin protein (Figure 3B). When we evaluated the levels of the transcripts of EMT regulators Snail/Twist1, we observed significant upregulation of Snail/Twist1 mRNA expression levels in the highly invasive clones generated from the HSC3 cells (Figure 3C).We then tested the medium from the highly invasive clones to evaluate the secretion of MMP-2. As shown in Figure 3D, increased MMP-2 secretion from oral cancer cells significantly correlated with increased cell invasion. While we analyzed the medium from SHP2-depleted cells, we observed significantly reduced MMP-2 (Figure 3E). Collectively, these results suggested that SHP2 exerts its function in several critical stages that contribute to the acquirement of invasiveness during oral cancer metastasis.


Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis.

Wang HC, Chiang WF, Huang HH, Shen YY, Chiang HC - BMC Cancer (2014)

Characteristics of highly invasive clone, HSC3-Inv4 derived from parental HSC3 cells. (A) Bright file microscopy images of HSC3 parental and HSC3 Inv 4 (20×, Upper panels). Cells were stained with E-cadherin and images were taken under fluorescence at 60× (Lower panels). (B) Expressions of E-cadherin and vimentin were analyzed by Western blot with indicated antibodies; GAPDH as a loading control. (C) Increased Snail (Upper panel) and Twist1 (Middle panel) transcript levels were observed in HSC3-Inv4 and HSC3-Inv8 compared to HSC3 parental cells. Experiments were done at least in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with the adjacent normal in each case. Western blot shows the expression level of Snail and Twist1 in HSC3-parental, HSC3-Inv4 and HSC3-Inv8 cells (Lower panel). (D) Status of MMP-2 secretion on highly invasive clones. Medium collected from HSC3 parental, HSC-Inv4 and HSC3-Inv8 cells were subjected to MMP-2 secretion analysis. Significantly increased amounts of MMP-2 were seen in selected sub-cell lines compared to parental cells. (E) SHP2 depletion resulted in decreased MMP-2 secretion in HSC3 parental, HSC3-Inv4 and HSC3-Inv8 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Characteristics of highly invasive clone, HSC3-Inv4 derived from parental HSC3 cells. (A) Bright file microscopy images of HSC3 parental and HSC3 Inv 4 (20×, Upper panels). Cells were stained with E-cadherin and images were taken under fluorescence at 60× (Lower panels). (B) Expressions of E-cadherin and vimentin were analyzed by Western blot with indicated antibodies; GAPDH as a loading control. (C) Increased Snail (Upper panel) and Twist1 (Middle panel) transcript levels were observed in HSC3-Inv4 and HSC3-Inv8 compared to HSC3 parental cells. Experiments were done at least in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with the adjacent normal in each case. Western blot shows the expression level of Snail and Twist1 in HSC3-parental, HSC3-Inv4 and HSC3-Inv8 cells (Lower panel). (D) Status of MMP-2 secretion on highly invasive clones. Medium collected from HSC3 parental, HSC-Inv4 and HSC3-Inv8 cells were subjected to MMP-2 secretion analysis. Significantly increased amounts of MMP-2 were seen in selected sub-cell lines compared to parental cells. (E) SHP2 depletion resulted in decreased MMP-2 secretion in HSC3 parental, HSC3-Inv4 and HSC3-Inv8 cells.
Mentions: As shown in Figure 3A, we evaluated the changes in EMT-associated E-cadherin and vimentin in highly invasive oral cancer cells. Our results indicated that the majority of the parental HSC3 cells were polygonal in shape (Figure 3A, left upper panel); whereas, the HSC3-Inv4 cells were rather spindle shaped (Figure 3A, right upper panel), with downregulated of E-cadherin protein and upregulated of vimentin protein (Figure 3B). When we evaluated the levels of the transcripts of EMT regulators Snail/Twist1, we observed significant upregulation of Snail/Twist1 mRNA expression levels in the highly invasive clones generated from the HSC3 cells (Figure 3C).We then tested the medium from the highly invasive clones to evaluate the secretion of MMP-2. As shown in Figure 3D, increased MMP-2 secretion from oral cancer cells significantly correlated with increased cell invasion. While we analyzed the medium from SHP2-depleted cells, we observed significantly reduced MMP-2 (Figure 3E). Collectively, these results suggested that SHP2 exerts its function in several critical stages that contribute to the acquirement of invasiveness during oral cancer metastasis.

Bottom Line: We observed similar results in phosphatase-dead SHP2 C459S mutant expressing cells.Enhanced invasiveness was associated with significant upregulation of E-cadherin, vimentin, Snail/Twist1, and matrix metalloproteinase-2 in the highly invasive clones.In addition, we determined that SHP2 activity is required for the downregulation of phosphorylated ERK1/2, which modulates the downstream effectors, Snail and Twist1 at a transcript level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Environmental Health and Occupational Medicine, National Health Research Institutes, No,35, Keyan Road, Zhunan, 35053 Miaoli County, Taiwan. hcchiang@nhri.org.tw.

ABSTRACT

Background: Tumor invasion and metastasis represent a major unsolved problem in cancer pathogenesis. Recent studies have indicated the involvement of Src-homology 2 domain-containing tyrosine phosphatase 2 (SHP2) in multiple malignancies; however, the role of SHP2 in oral cancer progression has yet to be elucidated. We propose that SHP2 is involved in the progression of oral cancer toward metastasis.

Methods: SHP2 expression was evaluated in paired oral cancer tissues by using immunohistochemical staining and real-time reverse transcription polymerase chain reaction. Isogenic highly invasive oral cancer cell lines from their respective low invasive parental lines were established using a Boyden chamber assay, and changes in the hallmarks of the epithelial-mesenchymal transition (EMT) were assessed to evaluate SHP2 function. SHP2 activity in oral cancer cells was reduced using si-RNA knockdown or enforced expression of a catalytically deficient mutant to analyze migratory and invasive ability in vitro and metastasis toward the lung in mice in vivo.

Results: We observed the significant upregulation of SHP2 in oral cancer tissues and cell lines. Following SHP2 knockdown, the oral cancer cells markedly attenuated migratory and invasion ability. We observed similar results in phosphatase-dead SHP2 C459S mutant expressing cells. Enhanced invasiveness was associated with significant upregulation of E-cadherin, vimentin, Snail/Twist1, and matrix metalloproteinase-2 in the highly invasive clones. In addition, we determined that SHP2 activity is required for the downregulation of phosphorylated ERK1/2, which modulates the downstream effectors, Snail and Twist1 at a transcript level. In lung tissue sections of mice, we observed that HSC3 tumors with SHP2 deletion exhibited significantly reduced metastatic capacity, compared with tumors administered control si-RNA.

Conclusions: Our data suggest that SHP2 promotes the invasion and metastasis of oral cancer cells. These results provide a rationale for further investigating the effects of small-molecule SHP2 inhibitors on the progression of oral cancer, and indicate a previously unrecognized SHP2-ERK1/2-Snail/Twist1 pathway that is likely to play a crucial role in oral cancer invasion and metastasis.

Show MeSH
Related in: MedlinePlus