Chronic myeloid leukemia-derived exosomes promote tumor growth through an autocrine mechanism

Abstract

Background: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder in which leukemic cells display a reciprocal t(9:22) chromosomal translocation that results in the formation of the chimeric BCR-ABL oncoprotein, with a constitutive tyrosine kinase activity. Consequently, BCR-ABL causes increased proliferation, inhibition of apoptosis, and altered adhesion of leukemic blasts to the bone marrow (BM) microenvironment. It has been well documented that cancer cells can generate their own signals in order to sustain their growth and survival, and recent studies have revealed the role of cancer-derived exosomes in activating signal transduction pathways involved in cancer cell proliferation. Exosomes are small vesicles of 40-100 nm in diameter that are initially formed within the endosomal compartment, and are secreted when a multivesicular body (MVB) fuses with the plasma membrane. These vesicles are released by many cell types including cancer cells, and are considered messengers in intercellular communication. We have previously shown that CML cells released exosomes able to affect the tumor microenvironment.

Results: CML cells, exposed up to one week, to exosomes showed a dose-dependent increased proliferation compared with controls. Moreover, exosome treatment promotes the formation of LAMA84 colonies in methylcellulose. In a CML xenograft model, treatment of mice with exosomes caused a greater increase in tumor size compared with controls (PBS-treated mice). Real time PCR and Western Blot analysis showed, in both in vitro and in vivo samples, an increase in mRNA and protein levels of anti-apoptotic molecules, such as BCL-w, BCL-xl, and survivin, and a reduction of the pro-apoptotic molecules BAD, BAX and PUMA. We also found that TGF- β1 was enriched in CML-exosomes. Our investigations showed that exosome-stimulated proliferation of leukemia cells, as well as the exosome-mediated activation of an anti-apoptotic phenotype, can be inhibited by blocking TGF-β1 signaling.

Conclusions: CML-derived exosomes promote, through an autocrine mechanism, the proliferation and survival of tumor cells, both in vitro and in vivo, by activating anti-apoptotic pathways. We propose that this mechanism is activated by a ligand-receptor interaction between TGF-β1, found in CML-derived exosomes, and the TGF- β1 receptor in CML cells.

Figure 1

CML-Derived Exosomes Promote in Vitro and in Vivo Tumor Growth. Cell growth was evaluated by BrdU assay (a). For the experiment LAMA84 cells were incubated for 48, 72, or 96 hours, or 1 week, with increasing doses of LAMA84-exosomes (5–10–20 μg/ml). The values were plotted as a percentage of the control (untreated cells); each point represents the mean ± SD for three independent experiments. (b) The addition of 1, 5, 10, 20 or 50 μg/ml of LAMA84 exosomes increased the area of CML colonies in methylcellulose compared with controls. Asterisks indicate a statistically significant values in comparison to control (Ctrl) (*p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001). (c) LAMA84 cells were injected subcutaneously in NOD/SCID mice, and mice were treated as described in Material and Methods. Comparison of the median tumor weight was used as an index of the proliferative efficacy of CML-derived exosomes.

Figure 2

Anti-Apoptotic and Pro-Survival Effect of CML-Derived Exosomes on Gene Expression. (a) Real-time PCR analysis shows that treatment of LAMA84 with LAMA84 exosomes up to 1 week increases the mRNA expression of the anti-apoptotic genes survivin, BCL-xl, and BCL-w, while it decreases the mRNA levels of the pro-apoptotic genes BAD, BAX and PUMA. (b) Similar effects were observed in samples from mice treated with exosomes compared with control mice, treated with PBS (black bar). Asteriks indicate a significant difference in comparison to control (Ctrl) (**p ≤ 0.01; *** p ≤ 0.001).

Figure 3

Anti-Apoptotic and Pro-Survival Effect of CML-Derived Exosomes on Protein Expression. (a) Western blot analysis shows that treatment of LAMA84 with 10 μg/ml of LAMA84 exosomes for 72 or 96 hours increases the expression of the anti-apoptotic molecules BCL-xl, BCL-w, and survivin, while it decreases the expression of the pro-apoptotic molecules BAD and BAX. (b) Protein levels of the same molecules were analyzed in the tumor biopsies of mice treated with exosomes (Exo 1 and Exo 2) and control mice treated with PBS (Ctrl 1 and Ctrl 2). Blots were stripped and subsequently reprobed with an antibody against β -actin to ensure equal loading. (c) Representative photomicrographs showing 5-μm-thick paraffin-embedded sections of tumor biopsy specimens obtained from control mice (Ctrl) and exosome-treated mice (Exo) stained for BAX and BCL-w.

Figure 4

CML Exosomes Trigger the in Vitro and in Vivo Activation of ERK/ Akt/ NF-Kb Pathways. (a) Western blot analysis shows that treatment of LAMA84 with 10 μg/ml of LAMA84 exosomes for 72 or 96 hours triggers the phosphorylation of ERK and Akt without altering the total level of these proteins, and increases the expression of NF-kB. (b) A comparable effect can be observed in CML xenografts treated or not with exosomes.

Figure 5

TGF-β1 Is Expressed on CML Exosomes. (a) Western blot analysis shows that LAMA84-derived exosomes express TGF- β1. Loading was based on equal protein amount (upper panel). LAMA84 cells express TGF- β1 receptor (lower panel). (b) After 72 or 96 hours of treatment of LAMA84 with 10 μg/ml of LAMA84-exosomes there is an increase in the phosphorylation of the downstream target of TGF-β1, SMAD 2/3; the treatment does not alter the total level of the protein.

Figure 6

Inhibition of TGF-β1 Receptor Signaling Reverses Exosome-Mediated Effects on CML Cells. (a) Cell growth was evaluated by BrdU assay. LAMA84 cells were incubated for 72, 96 hours, or 1 week, with increasing doses of LAMA84-exosomes (10–20 μg/ml) plus 10 or 20 μM of SB. The values were plotted as a percentage of the control (untreated cells); each point represents the mean ± SD of three independent experiments. (b) Colony formation assay shows that the co-treatment of LAMA84 with 10 or 20 μg/ml of exosomes plus 10 or 20 μM of SB reverses exosome-mediated increase of LAMA84 colonies area (** p ≤ 0.01; ***p ≤ 0.001). (c) Western blot analysis shows that treatment of LAMA84 with 10 μg/ml of exosomes plus 10 μM of SB for 72 or 96 hours decreases the phosphorylation of ERK, Akt and SMAD 2/3 without altering total protein level. The co-treatment with SB reverses exosome-mediated decrease of the pro-apoptotic molecules (BAD, BAX) as well as the increase of the anti-apoptotic proteins (BCL-xl, BCL-w, survivin).

Figure 7

Exosomal TGF- β1 inhibition reverses exosome- mediated increase of proliferation and colonies area of LAMA84 cells. (a) Cell growth was evaluated by BrdU assay. LAMA84 cells were incubated for 72, 96 hours, or 1 week, with LAMA84-exosomes (10–20 μg/ml) pretreated or not with neutralizing anti TGF-β1 antibodies. The values were plotted as a percentage of the control (untreated cells); each point represents the mean ± SD of three independent experiments. (b) Colony formation assay shows that the co-treatment of LAMA84 with 10 or 20 μg/ml of exosomes pretreated with neutralizing anti TGF-β1 antibody reverses exosome-mediated increase of LAMA84 colonies area. Asterisks indicate a statistically significant values (** p ≤ 0.01; ***p ≤ 0.001).

Figure 8

Working Hypothesis on Exosome-mediated Autocrine Loop. CML derived exosomes promote proliferation and survival of leukemic cells through the establishment of an autocrine signaling loop mediated by exosome-associated TGF-β1.