Figure 5. EPO activates JAK/STAT signaling in breast TICs, and JAK inhibition is a potential therapeutic target in breast cancer.

(A) SUM149 cells were FACS sorted into TIC (CD44⁺CD24^–EpCAM⁺) and non-TIC (not CD44⁺CD24^–EpCAM⁺) populations, treated with PBS or EPO (1 IU/ml) for 10 minutes, and immunoblotted with the indicated antibodies. (B) Gene-expression profiles were generated from total RNA of sorted SUM149 TICs (CD44⁺CD24^–EpCAM⁺), treated with PBS or EPO (1IU/ml) for 16 hours,and subjected to ssGSEA for the indicated JAK/STAT and cancer stem cell gene signatures. (C) Mammosphere formation of CD44⁺CD24^–EpCAM⁺ SUM149 cells cultured at hypoxia (2% O₂) in the presence of control antibody (anti-HA) or anti-EPO antibody at the indicated dilutions. (D) Mammosphere formation of CD44⁺CD24^–EpCAM⁺ SUM149 cells cultured at hypoxia (2% O₂) in the presence of control antibody (anti-HA) or 2 indicated anti-EPOR antibodies (1:200). (E) 20,000 (CD44⁺CD24^–EpCAM⁺) SUM149 cells were plated in duplicate in the presence of EPO (10 IU/ml) and vehicle or EPO and the JAK inhibitor TG101348 at the indicated concentrations. Spheres were counted on day 5. (F) Orthotopic MMTV-Wnt1 tumors were grown to 5 mm in length and width (average tumor volume of all groups = 60 mm³) and treated with vehicle (NT), carboplatin (Carbo), TG101348 (TG), or the combination of carboplatin and TG101348. Caliper measurements were taken weekly. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (G) Hematocrit (HCT) measurements were done at baseline and at end point in the indicated treatment groups.