Figure 7. Roles of Src Activation and PTEN Inactivation in Conferring Resistance to Trastuzumab by rHuEPO.

(A) Effects of rHuEPO and trastuzumab on association between Src and HER2. MCF7-HER18 cells were cultured in the presence or absence of 20 nM trastuzumab overnight in low serum medium and then stimulated with 10 U/ml rHuEPO or not for 30 minutes. The cell lysates were subjected to immunoprecipitation of HER2 with an anti-HER2 antibody, followed by Western blot analysis with the indicated antibodies.

(B) Phosphorylation of PTEN upon rHuEPO stimulation. MCF7-HER18 cells were treated as described in (A). Cell lysates were prepared and subjected to immunoprecipitation for PTEN, followed by Western blot analysis with the indicated antibodies.

(C) Correlation of rHuEPO-induced phosphorylation of PTEN with its antagonizing effects on trastuzumab-mediated inhibition of cell signaling. MCF7-HER18 cells were treated as described in (A). Cell lysates were subjected to Western blot analysis with the indicated antibodies.

(D) Dependence of rHuEPO-induced cell signaling on Src activity. MCF7-HER18 cells were left untreated or treated with 5 μM or 25 μM PP2 (Src inhibitor) in 0.5% FBS medium overnight and then stimulated with 10 U/ml rHuEPO or not for 30 minutes, followed by cell lysis and Western blot analysis with the indicated antibodies.

(E) Effects of rHuEPO and trastuzumab on Src and CK2α association. MCF7-HER18 cells were treated as described in (A). Cell lysates were prepared and subjected to immunoprecipitation of Src, followed by Western blot analysis of the immunoprecipitates with the indicated antibodies.

(F) Roles of Src and CK2α in rHuEPO-mediated phosphorylation of Akt and Erk. MCF7-HER18 cells were transfected with siRNA for expression knockdown of CK2α, Src, or CK2α and Src, as indicated. After 72 hours, the cells were stimulated with 10 U/ml rHuEPO or not for 30 minutes, followed by cell lysis and Western blot analysis with the indicated antibodies.

(G) Schematic of our working model. Trastuzumab binds to HER2 expressed by breast cancer cells, preventing activation of HER2-mediated cell signaling (dashed lines). rHuEPO binds to EpoR expressed by the same breast cancer cells, leading to activation of EpoR-associated protein tyrosine kinase Jak2 and subsequent activation of Src and inactivation of PTEN. The thick arrows indicate the pathways identified in the current study. The thick arrow with a question mark indicates a need for further confirmation. All of the experiments (A–F) were repeated at least once with similar findings. See also Figure S5.