Fig. 8: The expression of p-AXL and AXL positively correlates with the expression of KLF5 in human TNBC specimens. Moreover, DCC-2036 increases the sensitivity of TNBC chemotherapy by decreasing BCSCs.
A Representative immunohistochemical staining images of p-AXL, AXL, and KLF5 protein in TNBC specimens, in which the expression of p-AXL, AXL, and KLF5 proteins was indicated by mild positive (+), moderate positive (++), and strong positive (+++), respectively. Left: Scale bars, 200 μm (magnification 40×); Right: Scale bars, 50 μm (magnification 100×). B The expression levels of p-AXL, AXL, and KLF5 in TNBC samples were positively correlated. The statistical significance was determined by Spearman correlation analysis. R-value is the correlation coefficient. *P < 0.05, **P < 0.01. C The cell viability analysis of MDA-MB-231 and the doxorubicin-resistant MDA-MB-231 cells after treating with different concentrations of doxorubicin and DCC-2036 (n = 3). D The number and size of mammospheres formed following treatment with doxorubicin (10 nM), DCC-2036 (5 μM), and a combination of doxorubicin and DCC-2036 in MDA-MB-231 cells. Scale bars, 100 μm (magnification 40×); Scale bars, 100 μm (magnification 100×). The histogram represents the mean value of three experiments. The statistical significance was determined by Student’s t-test. **P < 0.01, ***P < 0.001. E Schematic representations of the inhibitory effect of DCC-2036 on triple-negative breast cancer stem cells through AXL-KLF5 positive feedback loop. In the progression of TNBC, AXL can activate Akt, which subsequently increases GSK3β (S9) phosphorylation and leads to the GSK3β inactivation. However, GSK3β-mediated KLF5 (S303) phosphorylation level promotes the KLF5 ubiquitination by SCFFbw7E3 ubiquitin ligase and degradation by 26 S proteasome. Thus, KLF5 into the nucleus promotes the expressions of stemness-associated transcription factors, and in turn, KLF5 binds to the AXL promoter region (−171 to −162 bp), which forms a positive feedback regulation mechanism further to facilitate tumorigenesis. DCC-2036 inhibits the expression of KLF5 via the AXL-Akt-GSK3β signal axis and further disrupts the AXL-KLF5 positive feedback loop. Ultimately, DCC-2036 inhibits TNBC stem cells and suppresses TNBC development.
