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Papel del microambiente en la respuesta de células de leucemia linfocítica crónica a trióxido de arsénico: mecanismos de resistencia

AutorAmigo-Jiménez, Irene
DirectorGarcía-Pardo, Angeles
Palabras claveLLC
Drug resistance
Fecha de publicación17-jun-2015
EditorCSIC - Centro de Investigaciones Biológicas (CIB)
ResumenIntroduction Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant CD5+ B lymphocytes (CLL cells) in the peripheral blood. The progression of the disease is determined by CLL cell infiltration in lymphoid organs, mainly lymph nodes and bone marrow, where they receive survival signals from common components of these tissue microenvironments, such as MMP-9 and stromal cells. These survival signals impair therapy, indeed, despite the efforts made, CLL remains an incurable disease, becoming crucial to continue searching for new therapeutic options based on the contribution of the microenvironment to the disease progression. Arsenic trioxide (ATO) is successfully employed for the treatment of acute promyelocytic leukemia and is being trialed for other hematological malignancies, including CLL. Indeed, ATO induces apoptosis in all CLL cases and could constitute an efficient therapy for this disease. Objectives In this context, the work that makes up this Doctoral Thesis is organized around three main objectives: - Molecular characterization of the ATO effect in CLL cells. - Determination of the role of MMP-9 in the CLL cell response to ATO. - Analysis of the influence of other molecules and signaling pathways induced by the microenvironment in the CLL cell response to ATO. Methodology We used B lymphocytes from the peripheral blood of 47 CLL patients (CLL cells) and MEC-1 cells (CLL-derived), both the parental cell line as MMP-9 stable transfectants. We used also co-cultures of CLL cells and bone marrow stromal cells (BMSCs), as well as BMSC-conditioned medium. The effect of drugs (ATO and Fludarabine) was determined by flow cytometry, using Annexin V-FITC/propidium iodide, and by the MTT assay. The global gene expression profile of MEC-1 cells in response to ATO was analyzed by RNA microarrays. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression and association was analyzed by Western blotting and immunoprecipitation, respectively. Gene silencing was performed by nucleofection of specific siRNAs. Statistical analyses were performed using the two-tailed Student's t-test and a p value of ≤ 0.05 was considered significant. Results The gene expression profile induced by ATO in CLL cells reflects an antioxidant and defensive response. Indeed, among the most significantly enriched Gene Ontology terms in the group of upregulated genes were found regulation of apoptosis, response to wounding and response to oxidative stress. Since the main effect of ATO in CLL cells is the induction of apoptosis, we were interested in the genes related to regulation of this process (HMOX1, HSPA1B, CLU, TNFRSF9, GCLM, NQO1, SQSTM1, CTSB and MMP9), particularly in those encoding HMOX-1 and MMP-9 due to their roles on oxidative stress regulation and CLL cell survival, respectively. We have studied the role of HMOX-1 and MMP-9 in the CLL cell response to ATO and found that this agent upregulates HMOX-1 (mRNA and protein) in CLL cells and this seem to favor its cytotoxic effect, thus suggesting a proapoptotic role for HMOX-1 in response to ATO. On the other hand, ATO also upregulates MMP-9 (mRNA and protein) and its localization at the surface of early apoptotic cells, being both processes dependent on apoptosis onset. These results suggest a role for MMP-9 in the CLL cell response to these drugs. Indeed, isolated or stromal MMP-9 induce CLL cell resistance to ATO and fludarabine, which is also achieved by overexpressing MMP-9 in MEC-1 cells and is reversed by MMP9 gene silencing. The molecular mechanism underlying MMP-9-induced drug resistance involves modulation of the balance between the anti- and pro-apoptotic Bcl-2 family members.
Culturing CLL cells on BMSCs also protects CLL cells from the ATO-induced apoptosis and this protection is overcome by blocking MMP-9 or α4β1 and/or αLβ2 integrins. The BMSC-conditioned medium also partially protects CLL cells against ATO-induced apoptosis, indicating that both cell-cell interactions and soluble factors are contributing to BMSC-induced resistance to ATO in CLL cells. We have studied the molecular mechanism involves in this protection and found that BMSCs induce PI3K/Akt, PKC, Lyn and ERK activation, as well as NF-κB and STAT3, and also upregulates Mcl-1 and Bcl-xL in CLL cells. Using inhibitors of these pathways, we demonstrated that PI3K and PKC are involved in the stroma-induced resistance to ATO. Further, blocking PI3Kδ or PKCβ isoforms with Idelalisib or Sotrastaurin, respectively, inhibits Akt phosphorylation, NF-κB/STAT3 activation and Mcl-1 upregulation and restores CLL cell sensibility to ATO. Likewise, MCL1 gene silencing also overcomes the protecting effect of stroma and confirms the key role of this protein in the mechanism of stroma-induced resistance to ATO in CLL cells. Conclusions In CLL cells, ATO induces expression of genes related with an antioxidant and defensive response. HMOX-1 seems to favor the cytotoxic effect of ATO, while MMP-9 promotes cell survival via modulation of the balance between the pro- and anti-apoptotic members of the Bcl-2 family. BMSCs also induce resistance to ATO in CLL cells and this protection is overcame by blocking MMP-9 or the integrins α4β1 or αLβ2. The molecular mechanism underlying this resistance involves activation of the PI3Kδ/PKCβ→Akt→NF-κB/STAT3→Mcl-1, and blocking PI3Kδ and PKCβ restores sensibility to ATO. Taken together, our results indicate that combination of ATO with specific inhibitors for MMP-9 and/or PI3Kδ and PKCβ may constitute an efficient alternative/complementary therapy for CLL.
Descripción182 p.-56 fig.-12 tab.
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