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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

Silencing SHP2 expression induces morphological changes in BTBC cells. A) The expression of SHP2 was effectively silenced with two independent shRNA (shRNA-1 and shRNA-2) sequences. P: parental cells; C: control cells; sh-1: shRNA-1 cells; sh-2: shRNA-2 cells. (B) Pictures of parental, control and SHP2-silenced cells showing morphological changes induced by SHP2 silencing in the MDA-MB231 cells. A picture of the MCF-10A cells is included to show morphological similarity to the SHP2-silenced cells. C) Analysis of ectopic SHP2 expression; V: vector alone; WT: wild type SHP2; CS: C459S-SHP2. D) Pictures of MDA-MB231 and MDA-MB468 cells expressing vector alone, WT-SHP2 or C459S-SHP2 to show morphological changes. Note that expression of dominant-negative SHP2 (C459S-SHP2) induces morphological changes reminiscent of SHP2-silnced cells.
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Fig1: Silencing SHP2 expression induces morphological changes in BTBC cells. A) The expression of SHP2 was effectively silenced with two independent shRNA (shRNA-1 and shRNA-2) sequences. P: parental cells; C: control cells; sh-1: shRNA-1 cells; sh-2: shRNA-2 cells. (B) Pictures of parental, control and SHP2-silenced cells showing morphological changes induced by SHP2 silencing in the MDA-MB231 cells. A picture of the MCF-10A cells is included to show morphological similarity to the SHP2-silenced cells. C) Analysis of ectopic SHP2 expression; V: vector alone; WT: wild type SHP2; CS: C459S-SHP2. D) Pictures of MDA-MB231 and MDA-MB468 cells expressing vector alone, WT-SHP2 or C459S-SHP2 to show morphological changes. Note that expression of dominant-negative SHP2 (C459S-SHP2) induces morphological changes reminiscent of SHP2-silnced cells.

Mentions: To determine the functional significance of SHP2 in BTBC, we chose the MDA-MB231 and the MDA-MB468 breast cancer cell lines which are known to be basal-like and triple-negative [24,25]. These cells harbor several genetic abnormalities commonly discovered in BTBC, including activating Ras mutation in the MDA-MB231, elevated EGFR expression and p53 mutation in both [24,26], and PTEN homo-deletion and EGFR gene amplification in the MDA-MB468 cells [24,27]. In addition, both cell lines show elevated SHP2 expression [28]. Hence, they are appropriate for testing the importance of SHP2 in BTBC. The expression of SHP2 was silenced with two independent shRNA sequences that were previously shown to be specific and devoid of off-target effects [18,21]. Cells expressing luciferase shRNA were used as controls in this study. As shown in Figure 1A, each shRNA effectively silenced SHP2 expression. Microscopic examination of confluent cells grown in 2D cultures revealed that the SHP2 silenced cells had acquired an epithelial morphology characteristic of luminal epithelial cells while the parental and the control cells showed the expected elongated and spindle-shaped mesenchymal morphology. In other words, silencing SHP2 expression induced morphological changes that are comparable to the MCF-10 cells, the immortalized and non-tumorigenic breast epithelial cells commonly used as “normal” controls (Figure 1B). Morphological changes induced by SHP2 silencing were obvious even in non-confluent cells (Additional file 1: Figure S1A). Similar results were obtained in the MDA-MB468 cells (Additional file 1: Figure S1B and C).Figure 1


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)

Silencing SHP2 expression induces morphological changes in BTBC cells. A) The expression of SHP2 was effectively silenced with two independent shRNA (shRNA-1 and shRNA-2) sequences. P: parental cells; C: control cells; sh-1: shRNA-1 cells; sh-2: shRNA-2 cells. (B) Pictures of parental, control and SHP2-silenced cells showing morphological changes induced by SHP2 silencing in the MDA-MB231 cells. A picture of the MCF-10A cells is included to show morphological similarity to the SHP2-silenced cells. C) Analysis of ectopic SHP2 expression; V: vector alone; WT: wild type SHP2; CS: C459S-SHP2. D) Pictures of MDA-MB231 and MDA-MB468 cells expressing vector alone, WT-SHP2 or C459S-SHP2 to show morphological changes. Note that expression of dominant-negative SHP2 (C459S-SHP2) induces morphological changes reminiscent of SHP2-silnced cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359540&req=5

Fig1: Silencing SHP2 expression induces morphological changes in BTBC cells. A) The expression of SHP2 was effectively silenced with two independent shRNA (shRNA-1 and shRNA-2) sequences. P: parental cells; C: control cells; sh-1: shRNA-1 cells; sh-2: shRNA-2 cells. (B) Pictures of parental, control and SHP2-silenced cells showing morphological changes induced by SHP2 silencing in the MDA-MB231 cells. A picture of the MCF-10A cells is included to show morphological similarity to the SHP2-silenced cells. C) Analysis of ectopic SHP2 expression; V: vector alone; WT: wild type SHP2; CS: C459S-SHP2. D) Pictures of MDA-MB231 and MDA-MB468 cells expressing vector alone, WT-SHP2 or C459S-SHP2 to show morphological changes. Note that expression of dominant-negative SHP2 (C459S-SHP2) induces morphological changes reminiscent of SHP2-silnced cells.
Mentions: To determine the functional significance of SHP2 in BTBC, we chose the MDA-MB231 and the MDA-MB468 breast cancer cell lines which are known to be basal-like and triple-negative [24,25]. These cells harbor several genetic abnormalities commonly discovered in BTBC, including activating Ras mutation in the MDA-MB231, elevated EGFR expression and p53 mutation in both [24,26], and PTEN homo-deletion and EGFR gene amplification in the MDA-MB468 cells [24,27]. In addition, both cell lines show elevated SHP2 expression [28]. Hence, they are appropriate for testing the importance of SHP2 in BTBC. The expression of SHP2 was silenced with two independent shRNA sequences that were previously shown to be specific and devoid of off-target effects [18,21]. Cells expressing luciferase shRNA were used as controls in this study. As shown in Figure 1A, each shRNA effectively silenced SHP2 expression. Microscopic examination of confluent cells grown in 2D cultures revealed that the SHP2 silenced cells had acquired an epithelial morphology characteristic of luminal epithelial cells while the parental and the control cells showed the expected elongated and spindle-shaped mesenchymal morphology. In other words, silencing SHP2 expression induced morphological changes that are comparable to the MCF-10 cells, the immortalized and non-tumorigenic breast epithelial cells commonly used as “normal” controls (Figure 1B). Morphological changes induced by SHP2 silencing were obvious even in non-confluent cells (Additional file 1: Figure S1A). Similar results were obtained in the MDA-MB468 cells (Additional file 1: Figure S1B and C).Figure 1

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