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Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment.

Zhao H, Agazie YM - BMC Cancer (2015)

Bottom Line: The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes.The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression.Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and The Marry Babb Randolph Cancer Center School of Medicine, West Virginia University, Morgantown, WV, 26506, USA. ttangela168@gmail.com.

ABSTRACT

Background: The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes. Since the basal-like and triple-negative breast cancer (BTBC) is characterized by dysregulation of multiple tyrosine kinase oncogenes, we wanted to determine the importance of SHP2 in BTBC cell lines.

Methods: Short hairpin RNA-based and dominant-negative expression-based SHP2 inhibition techniques were used to interrogate the functional importance of SHP2 in BTBC cell biology. In addition, cell viability and proliferation assays were used to determine hormone dependency for growth and sensitivity to anti-estrogen treatment.

Results: We show that inhibition of SHP2 in BTBC cells induces luminal-like epithelial morphology while suppressing the mesenchymal and invasive property. We have termed this process as basal-to-luminal transition (BLT). The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression. Furthermore, the occurrence of BLT led to estrogen receptor alpha (ERα) expression, hormone dependency, and sensitivity to tamoxifen treatment.

Conclusions: Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy. Therefore, inhibition of SHP2 may provide a therapeutic benefit in basal-like and triple-negative breast cancer.

No MeSH data available.


Related in: MedlinePlus

Effect of SHP2 silencing on expression of EMT proteins. A) Silencing SHP2 expression down regulated the levels of fibronectin (FN), Snail and vimentin (Vim). B) Proteasome inhibition with Mg-132 restores Snail and fibronectin levels, but not vimentin. C) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB231 cells after stabilization with Mg-132 and inhibition of new protein synthesis with cyclohexamide (CHA in a time-course (TC) fashion. D) Band density measurement of FN and Snail levels from three independent experiments performed as in C. E) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB468 cells. The experiments were performed as in C. F) Band density measurement of FN and Snail levels from three independent experiments performed as in E. G) Effect of SHP2 silencing on expression and secretion of MMP2 and MMP9 in MDA-MB231 cells. H) Effect of SHP2 silencing on expression and secretion of MMP9 in MDA-MB468 cells.
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Fig3: Effect of SHP2 silencing on expression of EMT proteins. A) Silencing SHP2 expression down regulated the levels of fibronectin (FN), Snail and vimentin (Vim). B) Proteasome inhibition with Mg-132 restores Snail and fibronectin levels, but not vimentin. C) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB231 cells after stabilization with Mg-132 and inhibition of new protein synthesis with cyclohexamide (CHA in a time-course (TC) fashion. D) Band density measurement of FN and Snail levels from three independent experiments performed as in C. E) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB468 cells. The experiments were performed as in C. F) Band density measurement of FN and Snail levels from three independent experiments performed as in E. G) Effect of SHP2 silencing on expression and secretion of MMP2 and MMP9 in MDA-MB231 cells. H) Effect of SHP2 silencing on expression and secretion of MMP9 in MDA-MB468 cells.

Mentions: The retardation of cell migration and the blockade of invasiveness induced by SHP2 silencing implied loss of the mesenchymal phenotype. To further validate these observations, total cell lysates prepared from the 3D matrigel cultures were analyzed for expression of the mesenchymal markers, Snail, fibronectin (FN), and Vimentin. Consistent with the observed phenotypic changes, the levels of Snail, FN, and Vimentin were significantly reduced in the SHP2-silenced cells (Figure 3A), confirming that SHP2 is essential for the maintenance of the mesenchymal property in BTBC cells. Assuming that one of the mechanisms for SHP2 in promoting the expression of EMT proteins could be through protein stability, we conducted proteasome inhibition studies with Mg-132 treatment. As shown in Figure 3B, treating cells with Mg-132 restored FN and Snail, but not vimentin. These findings suggest that SHP2 promotes the stability of FN and Snail, but its positive effect on vimentin might be at the level of transcription.Figure 3


Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment.

Zhao H, Agazie YM - BMC Cancer (2015)

Effect of SHP2 silencing on expression of EMT proteins. A) Silencing SHP2 expression down regulated the levels of fibronectin (FN), Snail and vimentin (Vim). B) Proteasome inhibition with Mg-132 restores Snail and fibronectin levels, but not vimentin. C) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB231 cells after stabilization with Mg-132 and inhibition of new protein synthesis with cyclohexamide (CHA in a time-course (TC) fashion. D) Band density measurement of FN and Snail levels from three independent experiments performed as in C. E) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB468 cells. The experiments were performed as in C. F) Band density measurement of FN and Snail levels from three independent experiments performed as in E. G) Effect of SHP2 silencing on expression and secretion of MMP2 and MMP9 in MDA-MB231 cells. H) Effect of SHP2 silencing on expression and secretion of MMP9 in MDA-MB468 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359540&req=5

Fig3: Effect of SHP2 silencing on expression of EMT proteins. A) Silencing SHP2 expression down regulated the levels of fibronectin (FN), Snail and vimentin (Vim). B) Proteasome inhibition with Mg-132 restores Snail and fibronectin levels, but not vimentin. C) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB231 cells after stabilization with Mg-132 and inhibition of new protein synthesis with cyclohexamide (CHA in a time-course (TC) fashion. D) Band density measurement of FN and Snail levels from three independent experiments performed as in C. E) Analysis of FN and Snail levels in the control and the SHP2-silenced MDA-MB468 cells. The experiments were performed as in C. F) Band density measurement of FN and Snail levels from three independent experiments performed as in E. G) Effect of SHP2 silencing on expression and secretion of MMP2 and MMP9 in MDA-MB231 cells. H) Effect of SHP2 silencing on expression and secretion of MMP9 in MDA-MB468 cells.
Mentions: The retardation of cell migration and the blockade of invasiveness induced by SHP2 silencing implied loss of the mesenchymal phenotype. To further validate these observations, total cell lysates prepared from the 3D matrigel cultures were analyzed for expression of the mesenchymal markers, Snail, fibronectin (FN), and Vimentin. Consistent with the observed phenotypic changes, the levels of Snail, FN, and Vimentin were significantly reduced in the SHP2-silenced cells (Figure 3A), confirming that SHP2 is essential for the maintenance of the mesenchymal property in BTBC cells. Assuming that one of the mechanisms for SHP2 in promoting the expression of EMT proteins could be through protein stability, we conducted proteasome inhibition studies with Mg-132 treatment. As shown in Figure 3B, treating cells with Mg-132 restored FN and Snail, but not vimentin. These findings suggest that SHP2 promotes the stability of FN and Snail, but its positive effect on vimentin might be at the level of transcription.Figure 3

Bottom Line: The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes.The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression.Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and The Marry Babb Randolph Cancer Center School of Medicine, West Virginia University, Morgantown, WV, 26506, USA. ttangela168@gmail.com.

ABSTRACT

Background: The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes. Since the basal-like and triple-negative breast cancer (BTBC) is characterized by dysregulation of multiple tyrosine kinase oncogenes, we wanted to determine the importance of SHP2 in BTBC cell lines.

Methods: Short hairpin RNA-based and dominant-negative expression-based SHP2 inhibition techniques were used to interrogate the functional importance of SHP2 in BTBC cell biology. In addition, cell viability and proliferation assays were used to determine hormone dependency for growth and sensitivity to anti-estrogen treatment.

Results: We show that inhibition of SHP2 in BTBC cells induces luminal-like epithelial morphology while suppressing the mesenchymal and invasive property. We have termed this process as basal-to-luminal transition (BLT). The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression. Furthermore, the occurrence of BLT led to estrogen receptor alpha (ERα) expression, hormone dependency, and sensitivity to tamoxifen treatment.

Conclusions: Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy. Therefore, inhibition of SHP2 may provide a therapeutic benefit in basal-like and triple-negative breast cancer.

No MeSH data available.


Related in: MedlinePlus