Fig 2: Targeting CD24 and its inhibitor gene NDRG2 by shRNAs improved chemotherapy sensitivity in vitro and in vivo(A, B, C and D) After seeding in matrigel for 5-days, HCC1806 cells were treated with 20 nM docetaxel (DTX) or 1.5 μM doxorubicin (DXR) for 4-days. HCC1937 cells were treated with 0.6 μM docetaxel. P-values of the compared groups were calculated using unpaired t-tests for cell total area. (E) These data show the working scheme for treatments used in tumor xenograft experiments. (F, G, H) These data show results of experiments with five tumors in each treatment group. (I) These data show statistical analyses of tumor shrinkage in the three pairs of experiments. P-values of each paired group were calculated with paired sample t-tests. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent SEM. Scale bar, 200 μm.

Fig 3: Interactions between Bcl-2, TGF-βR1 and ATM signaling in TNBC cells treated with either docetaxel (DTX) or doxorubicin (DRX)(A) and (B) Western blot results. Docetaxel: 6.4 μM; doxorubicin: 4 μM; cyclophosphamide: 2 μM. The results showed that doxorubicin reduced p-Bcl-2 in HCC1937 and HCC1806 and docetaxel increased p-Bcl-2 in the same cell lines. Cyclophosphamide had no significant effect on p-Bcl-2 in the two cell lines. HCC38 had a relatively higher basal level of TGF-βR1 compared to HCC1937. The bands of p-Bcl-2, Bcl-2 and β-actin in HCC1806 treated with DTX and HCC1937 treated with DXR have been shown in Figure 5. They were shown here again for a different comparison. (C) Cells were treated with 5 μM Bcl-2 inhibitor ABT-737. The results showed that Bcl-2 inhibitor stimulated p-TGF-βR1 and p-ATM, suggesting that Bcl-2 is an inhibitor of TGF-βR1 and ATM in the three cell lines. (D) Cells were treated with 5 μM TGF-βR1 inhibitor LY 364947. The results showed that TGF-βR1 inhibitor suppressed p-ATM and TGF-βR1 is stimulatory to p-ATM. (E) The Western blot results of autophagy marker-LC3B. The results showed that CD24 knockdown increased LC3B expression and NDRG2 knockdown eliminated LC3B expression. (F) Proposed diagram to summarize contrasting effects of doxorubicin and docetaxel on critical TNBC cell signaling pathways.

Fig 4: High expression of CD24 in archival tumor specimens from TNBC patients treated with taxane-based chemotherapy associate with poor prognosisKaplan-Meier survival curves are shown in relation to CD24 expression in archival tumor specimens represented in a TNBC Microarray (TMA). (A) Overall survival analysis of all patients who received either doxorubicin- or taxane-based treatments. (B), (C) Data show that the survival analysis was limited to only patients who received taxane-based regimens. (B, overall survival analysis; C, disease-free survival analysis). (D) Representative CD24 IHC staining of TNBC TMA is shown. The specificity of CD24 staining was confirmed using TNBC cells stained with the same antibodies and IHC reagent kit (data not shown). (E, F) Representative CD24 staining of tumor samples from 4 of the 9 patients noted in Supplementary Table 5 is shown. Scale bar, 200 μm.

Fig 5: Bcl-2, TGF-βR1 and ATM signaling assessed by Western blot and FACS in selected TNBC cell lines(A) Cells were treated with 10 μM ATM inhibitor KU60019. (B) FACS results showed that 10 μM ATM inhibitor increased CD24 expression in MDA-MB-231 cells; and 4 μM doxorubicin reduced CD24 expression. (C) Selected cells were transfected with control vector, CD24 shRNA or NDRG2 shRNA. Knockdown of NDRG2 caused dramatic p-ATM increase in HCC1806 and MDA231 cells (D) The cells were treated with docetaxel 6.4 μM or doxorubicin 4 μM. Both docetaxel and doxorubicin increased p-TGF-βR1 in the three cell lines. Doxorubicin reduced p-Bcl-2 in HCC1937 and docetaxel elevated p-Bcl-2 in HCC1806 and HCC38.