Microenvironment regulates the expression of miR-21 and tumor suppressor genes PTEN, PIAS3 and PDCD4 through ZAP-70 in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironment, being the BCR pathway one key player in this crosstalk. Among proteins participating, ZAP-70 enhances response to microenvironmental stimuli. MicroRNA-21 (miR-21) is overexpressed in diverse neoplasias including CLL, where it has been associated to refractoriness to fludarabine and to shorter time to progression and survival. To further elucidate the role of ZAP-70 in the biology of CLL, we studied its involvement in miR-21 regulation. MiR-21 expression was higher in CLL cells with high ZAP-70. Ectopic expression of ZAP-70 induced transcription of miR-21 via MAPK and STAT3, which subsequently induced downregulation of tumor suppressors targeted by miR-21. The co-culture of primary CLL cells mimicking the microenvironment induced ZAP-70 and miR-21 expression, as well as downregulation of miR-21 targets. Interestingly, the increase in miR-21 after co-culture was significantly impaired by ibrutinib, indicating that the BCR signaling pathway is involved in its regulation. Finally, survival of CLL cells induced by the co-culture correlated with miR-21 upregulation. In conclusion, stimuli from the microenvironment regulate miR-21 and its targeted tumor suppressor genes via a signaling pathway involving ZAP-70, thus contributing to the cytoprotection offered by the microenvironment particularly observed in CLL cells expressing ZAP-70.

Figure 1

Expression of miR-21 is significantly higher in patients with CLL with high expression of ZAP-70. Expression levels of miR-21 were measured by QRT-PCR in PBMC from 33 patients diagnosed with CLL with high ZAP-70 (N = 17) or low ZAP-70 (N = 16), as indicated at the bottom of the histogram. *P < 0.05.

Figure 2

ZAP-70 protein overexpression enhances the induction of miR-21 expression after BCR stimulation in B cells via MAPK and STAT3 activation. (a–b) Ramos stable transfectants were stimulated with 5 µg/mL F(ab’)₂ anti-IgM for 4 and 12 hours. Expression levels of primary miR-21 (a) and miR-21 (b) were measured by QRT-PCR. (c–d) Ramos GFP-ZAP-70 cells were stimulated with 5 µg/mL F(ab’)₂ anti-IgM for 5 minutes after pre-incubation for 1 hour with 5 µM or 10 µM LY294002 for Akt inhibition or 50 µM or 100 µM PD98059 for ERK1/2 inhibition. Immunobloting analysis confirmed the inhibition of phosphorylation of Akt and ERK1/2 (Jurkat cells treated with PV were used as positive control) (c) and expression levels of miR-21 were measured by QRT-PCR after 12 hours (d). (e–f) Ramos stable transfectants were stimulated with 5 µg/mL F(ab’)₂ anti-IgM for 5 minutes and pre-treated 1 hour with 5 µM JSI-124 for STAT3 inhibition. Immunobloting analysis confirmed the inhibition of STAT3 phosphorylation (Jurkat cells treated with PV were used as positive control) (e) and expression levels of miR-21 were measured by QRT-PCR after 48 hours (f). *P < 0.05, **P < 0.005.

Figure 3

The tumor suppressor genes PTEN, PDCD4 and PIAS3, targeted by miR-21, are downregulated after BCR stimulation. (a,b) Ramos stable transfectants were stimulated with 5 µg/mL F(ab’)₂ anti-IgM for 5 minutes and 48 hours. Enhanced STAT3 and ERK1/2 phosphorylation was observed in IgM-BCR stimulated cells for 5 minutes. PTEN, PIAS3 and PDCD4 downregulation was observed in IgM-BCR stimulated cells after 48 hours. (c,d) Gene set enrichment analysis (GSEA) for Gabriely_miR-21_targets gene set (N = 289). The panel shows GSEA of Ramos GFP-ZAP-70 stimulated with 5 µg/mL F(ab’)₂ anti-IgM for 4 hours versus Ramos GPF-ZAP-70 unstimulated (c). The enrichment plot shows GSEA of Ramos GFP-ZAP-70 versus Ramos GFP cells, both stimulated with 5 µg/mL F(ab’)₂ anti-IgM for 4 hours (d). ES: enrichment score.

Figure 4

Co-culture of primary CLL cells with BMSC, CD40L and CpG ODN induces the expression of miR-21 and downregulation of the miR-21 targets PTEN, PDCD4 and PIAS3. (a) Primary CLL cells from 40 patients were cultured in suspension or in co-culture for 48 hours and miR-21 expression was measured by QRT-PCR. (b) Primary CLL cells from 22 patients were cultured in suspension or in co-culture for 48 hours. PTEN, PDCD4 and PIAS3 expression was determined by QRT-PCR. (c–d) Relative gene expression comparison between matched samples from peripheral blood (PB) and lymph nodes (LN) from 17 treatment-naive CLL patients, using public data from Y.Herishanu et al., Blood 2011. mRNA expression level of miR-21 (c) and PTEN (d) were analyzed and compared by Wilcoxon matched pairs signed rank test. (e) PBMC from 13 patients were pre-treated with 5 µM PCI-32765 for 1 hour and cultured in suspension or in co-culture for 48 hours. Expression levels of miR-21 were measured by QRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.0001.

Figure 5

Co-culture of primary CLL cells with BMSC, CD40L and CpG ODN increases survival, proliferation and miR-21 expression. Survival and miR-21 increase are significantly and positively correlated. PBMC from 11 patients diagnosed with CLL were maintained in suspension or in co-culture for 48 hours. (a) Linear regression analysis of the correlation between fold changes of viability (viability in co-culture versus suspension) and fold changes of miR-21 mRNA expression (miR-21 expression in co-culture versus suspension). (b) Linear regression analysis of the correlation between fold changes of proliferation (proliferation in co-culture versus suspension) and fold changes of miR-21 mRNA expression (miR-21 expression in co-culture versus suspension). r² is the coefficient of determination.