Page 130 - 2019_05-HaematologicaMondo-web

I. Spinello et al.
fected HPCs with specific-CD147 interfering small RNAs (CD147-siRNA) or with a non-targeting control siRNA (c- siRNA). Silencing of CD147 mRNA decreased CD147 pro- tein levels in transfected (CD147-siRNA)-HPCs, as com- pared to (c-siRNA)-HPCs of control (Figure 1D); then, transfected-HPCs were grown under culture conditions, to allow their selective G or Mo proliferation and differenti- ation. We observed that the decreased expression of CD147 impaired the proliferation of both G and Mo dif- ferentiating (CD147-siRNA)-HPCs, as compared to con- trol (c-siRNA)-HPCs (Figure 1E), without any significant modulation of G or Mo specific antigen expression on these cells (data not shown) and without any significant induction of apoptosis (data not shown). Clonogenic G and Mo progenitor assays performed with transfected-HPCs, showed a significant decrease in the number of CFU-GM and CFU-M colonies obtained from (CD147-siRNA)- HPCs, compared with the number of colonies obtained from (c-siRNA)-HPCs (Figure 1F). Altogether, our data indicate that CD147 plays a role in HPC proliferation and clonogenic activity.
Over-expressed in AML, CD147 is down-regulated during differentiation of leukemic cells
By analyzing CD147 mRNA expression levels in pri- mary leukemic blast cells obtained from 48 patients with different subtypes of AML,29 we found that CD147 is over-expressed in all AML subtypes, in particular in the AML-M3 subtype, as compared to normal CD34+ HPCs (Figure 2A, left panel), in line with the data from AML samples generated by the TCGA Research Network (Figure 2A, right panel).
Then, we also examined CD147 expression in several AML cell lines induced to terminal differentiation. All leukemic cell lines analyzed over-expressed the fully gly- cosylated mature CD147 protein, biologically active (Figure 2B, HG-CD147), like also its mRNA, as compared to normal CD34+ HPCs (Figure 2C). We found that CD147 expression is down-regulated during vitamin D3-induced Mo differentiation of U937 cells and ATRA-induced G dif- ferentiation of HL-60 and NB4 cell lines, at both mRNA and protein levels during terminal differentiation (Figure 2D-F and Online Supplementary Figure S1C-H), similarly to Mo and G terminal differentiation of normal HPCs (Figure 1A-C). However, CD147 expression is not affected by ATRA treatment of the NB4-R4 cell line (Figure 2G and Online Supplementary Figure S1I and L), indicating that induction of cell differentiation is required for CD147 downregulation.
AC-73 inhibits leukemic cell proliferation by blocking ERK/STAT3 signaling and induces autophagy
To investigate whether a high level of CD147 can pro- mote cell proliferation, we used AC-73 to inhibit CD147 function in normal and leukemic cells.
First, we examined the effect of AC-73 treatment on G and Mo differentiation of normal HPCs. AC-73 used at 5 μM moderately decreased cell growth without affecting cell cycling or differentiation of HPCs (Figure 3A-C). Morphology analysis of HPCs at different days of culture supported these observations, showing that AC-73-treat- ed cells regularly progress along G and Mo differentiation and do not show any significant morphology abnormality or delay in differentiation (Figure 3D).
We analyzed the effects of chronic AC-73 administra-
tion on leukemic cell growth, apoptosis and viability of AML cell lines. Leukemic cells were treated from 1 to 3 days, with various AC-73 doses (2.5, 5 and 10 mM) to determine their sensitivity to AC-73 treatment. We observed a time- and dose-dependent effect of AC-73 on leukemic cell growth, with growth inhibition of all leukemic cell lines treated (Figure 4A and Online Supplementary Figure S2B). We could not detect any effect of AC-73 on cell growth and apoptosis of the Chinese Hamster Ovary (CHO) cells (data not shown) used as a CD147-negative cell line (Online Supplementary Figure S2A, left panel).
NB4 and NB4-R4 cell lines were more sensitive to AC- 73 treatment than U937 and HL-60 cell lines. Indeed, a higher significant decrease in cell growth (Figure 4A), apoptosis (Figure 4B), and viability (Figure 4C and D) was observed in NB4 and NB4-R4 cells treated with low-dose AC-73 (2.5 mM) than in U937 and HL-60 lines (2 days) (Figure 4D). MV4-11, Kasumi-1 cells were the most resist- ant to AC-73 treatment (Online Supplementary Figure S2B- E) when compared to other cell lines (Figure 4A-D). Significant cell growth inhibition and sensitivity of MV4- 11 and Kasumi-1 cells occurred after treatment with high concentration of AC-73 (5 and 10 mM) (Online Supplementary Figure S2B-D) or when cells were treated for longer (3-4 days) (Online Supplementary Figure S2E).
AC-73 treatment had no significant effect on cell cycle distribution in the leukemic cell lines tested as compared to control cells (data not shown), suggesting that AC-73 decreases the cell growth rate but does not inhibit the pro- gression of cells in the cell cycle. Analysis of CD11b, CD14 and CD15 expression levels in leukemic cell lines treated for 3 days with 5 mM AC-73 showed no significant effect of AC-73 on leukemic cell differentiation (data not shown). By analyzing the effects of AC-73 (5 mM) on clono- genic growth of U937 and NB-4 cells we found that AC- 73 decreases the in vitro colony formation of both U937 and NB-4 cell lines, indicating an inhibitory effect of AC- 73 on the clonogenetic capacity of leukemic cells (Online Supplementary Figure S2F).
Altogether, despite the different sensitivities to AC-73 manifested by the different leukemic cell lines, probably related to the specific molecular alteration, such as PML/RARA, FLT3-ITD or RUNX1/RUNX1T1, of these cell lines, AC-73 exhibits potent growth inhibition and cyto- toxic activity on leukemic cells only at high doses. Because low doses of AC-73 inhibit leukemic cell proliferation, but do not cause cell death via apoptosis or cell cycle arrest, we investigated the possibility that AC-73 treatment induces autophagy in leukemic cells. First, we assessed the effect of AC-73 on the level of the autophagic indicator LC3 by western blotting in U937 and NB4 cells. Our results showed a dose-dependent effect of AC-73 on the increase of LC3-II/LC3-I ratio in leukemic cells as com- pared to control (-) cells (Figure 4E), indicating induction of autophagy.31 Then we monitored autophagy flux by flow cytometry analysis in live leukemic cells treated for 72 hours (h) by AC-73 compared to control cells. Our data demonstrated that AC-73 induces dose-dependent autophagy (Figure 4F) in CD147-expressing leukemic cells (Figure 4G), but not in CHO cells (Online Supplementary Figure S2A, right panels), again indicating that the biological effects induced by AC-73 require CD147 expression on target cells. Considering that AC-73 suppresses CD147/ERK1/2/STAT3/MMP-2 pathways in hepatocellu-
976
haematologica | 2019; 104(5)