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I. Spinello et al.
lar events by interacting with various binding partners, such as tumor- and inflammation-associated molecules including integrins, monocarboxylate transporters (MCTs), cyclophilins, caveolin-1, and E-selectin, explain- ing its significant role in the pathogenesis of several dis- eases.3-6,10 CD147 overexpression and more recently its co- expression with MCTs11,12 are regarded as unfavorable prognostic factors in cancers associated with hypoxia, a common feature of solid tumors, but also a major compo- nent of the bone marrow (BM) microenvironment, crucial in leukemia progression.13,14 However, in contrast to solid tumors, the function of CD147 remains poorly defined in leukemia.
Recent studies have shown growing interest in the CD147 molecule in AML15,16 and in some hematologic neoplasia, in particular in multiple myeloma (MM), where CD147 expression levels have a prognostic value and are required for the proliferation of MM cells.17-19 Moreover, CD147 is over-expressed in erythroid cells of myelodys- plastic syndrome (MDS) with 5q deletion.18 Here, we show that CD147 is expressed in normal CD34+ hematopoietic progenitor cells (HPCs) and down-regulat- ed during monocytic and granulocytic differentiation of HPCs. We then show that CD147 is over-expressed in blasts pertaining to different subtypes of AML and pro- motes leukemic cell proliferation. Interestingly, we report that CD147 is expressed at the level of CD34+CD371+ AML cells, previously described for their leukemia-initiat- ing properties.20
Recently, the small-molecule AC-73 has been proposed as a specific inhibitor for CD147.21 First, we checked that the response to AC-73 treatment is not involved in an off- target mechanism in leukemic cells. Then, we analyzed the effects of CD147 inhibition by AC-73 in AML cell lines and in primary leukemic blasts. We found that AC- 73 inhibits leukemic cell proliferation by suppressing the ERK/STAT3 activation pathway, known to play a role in AML cell proliferation and survival,22 but also by activating autophagy, an essential phenomenon for hematopoietic stem cell (HSC) maintenance, resistance to stress, survival and differentiation, the machinery of which might be dis- rupted in AMLs.23-25 Next, we analyzed whether AC-73 enhanced the sensitivity of leukemic cells to conventional chemotherapeutic agents. We used arabinosylcytosine (Ara-C), one of the most active cytotoxic agents in myeloid leukemia, and arsenic trioxide (ATO), an active anti-proliferative agent used in the treatment of patients with acute promyelocytic leukemia (APL) (AML-M3)1,2,26 [although with low efficacy in AML lacking the t(15;17) translocation], and also an inducer of autophagy.25,27 We found that AC-73 used in vitro in combination with Ara-C or ATO, increases the effects of these agents.
Altogether, our data suggest that CD147 plays a key role in leukemic cell proliferation and represents a potential therapeutic target in AML patients, and that AC-73 is a new promising inhibitor that could be used in combina- tion with conventional chemotherapeutic agents as a novel treatment strategy in AML.
Methods
Cell cultures
Human cord blood (CB) was obtained from healthy donors after informed consent. Leukemic blasts were isolated from BM
obtained from patients with newly diagnosed AML, using Ficoll- Hypaque density gradient. Informed consent was obtained from patients in accordance with the Declaration of Helsinki. This study was approved by the local ethical committees of the Istituto Superiore di Sanità and the University of Tor Vergata, Rome, Italy.
Cord blood CD34+ HPC purification, unilineage monocytic (Mo) and granulocytic (G) differentiation and morphological analyses were performed,28,29 as described in the Online Supplementary Methods.
Human primary AML blasts were maintained in culture in Iscove medium supplemented with 10% FCS, GM-CSF (10 ng/mL), SCF (50 ng/mL), IL-3 (10 ng/mL) (PeproTech Inc., Rocky Hill, NJ, USA), as described.29
Human AML cell lines used in our study were: U937 as a model of AML-M5, NB4 and HL-60 as models of AML-M3 and AML- M4, respectively; NB4-R4 as AML-M3 resistant to all-trans retinoic acid (ATRA) treatment; MV4-11 as AML-M2 mutated for FLT3- ITD; Kasumi-1 as AML-M2 with the t(8;21) translocation. All cell lines were grown in RPMI medium supplemented with 10% FCS (Gibco, Carlsbad, CA, USA).
Cell growth, cell cycle profile, viability and apoptosis analysis were performed, as described in the Online Supplementary Methods. Flow cytometry, western blot and quantitative real-time RT- PCR analysis were as described in the Online Supplementary
Methods.
Knockdown of CD147 expression by RNA interference, clono- genic assays and colony formation assays were performed, as described30 in the Online Supplementary Methods.
Cyto-ID autophagy detection was performed using the Cyto- ID assay (Enzo Life Sciences ENZ-51031-K200) as described in the Online Supplementary Methods.
AC-73 treatment of leukemic cells used alone or in combination with chemotherapeutic agents
AC-73 (3-{2-[([1,1’-biphenyl]-4-ylmethyl) amino]-1-hydrox- yethyl}phenol) (Specs ID number AN-465/42834501, Specs, Zoetermeer, the Netherlands) was dissolved in 20% DMSO (Sigma, St. Louis, MO, USA) and diluted in DMEM, with a final DMSO concentration of no more than 0.2% for all in vitro stud- ies.21 In leukemic cell lines, dose-response and time-course analy- sis were performed using AC-73 at 1.0, 2.5, 5.0 and 10 mM from 1 to 4 days of treatment; results were compared with 0.2% DMSO- treated leukemic cells, indicated as control leukemic cells. AC-73 was added in cultures every 2 days to maintain its activity. Combinations of treatment were performed using AC-73 (2.5 mM) alone over 24 hours and then by adding Ara-C (0.01; 0.1 and 1.0 μM) or ATO (0.1; 0.01 and 1.0 μM) for another 1 (for NB4 and NB4-R4 cells) or 2 (for U937, HL-60, MV4-11 and Kasumi-1 cells) days. Cell viability assays were performed to evaluate the effect of AC-73 alone or in combination on cell growth and viability of these cells.
Analysis of The Cancer Genome Atlas data
Datasets of The Cancer Genome Atlas (TCGA) Research Network 2008, were processed and obtained directly from the public access data portal (http://tcga-data.nci.nih.gov/).
Statistical analysis
Student t-test was applied to assess statistical significance of dif- ferences between multiple/group of experiments. Data were ana- lyzed using GraphPad Prism software. For univariate survival analysis, Kaplan-Meier plots with a log-rank test were presented using the overall survival data of AML patients from the TGCA. Additional information is provided in the Online Supplementary Appendix.
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