MicroRNA-155 influences B-cell receptor signaling and associates with aggressive disease in chronic lymphocytic leukemia

Abstract

High-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of ζ-chain-associated protein of 70 kD. CLL with high-level miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5(bright)CXCR4(dim) cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5(dim)CXCR4(bright) cells of the same patient. Treatment of CLL or normal B cells with CD40-ligand or B-cell-activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL.

© 2014 by The American Society of Hematology.

Figures

Figure 1

Relationship between expression levels of miR-155, ZAP-70, IGHV mutation status, and TFS. (A) In the training dataset, Kaplan-Meier curves depicting the TFS probability over time from diagnosis of patients who were segregated into 2 groups (miR-155-Lo or miR-155-Hi) according to the relative amounts of miR-155 expressed by the blood CLL cells of each patient. (B,C) Kaplan-Meier curves depicting the TFS probability over time from diagnosis of patients segregated by miR-155 and ZAP-70 status (B), or miR-155 and IGHV mutation status (C). (D) In the validation dataset, Kaplan-Meier curves depicting the TFS probability over time from diagnosis for patients who were segregated into 2 groups (miR-155-Lo or miR-155-Hi) based upon whether or not the blood CLL cells expressed miR-155 above the training set–defined threshold for high miR-155HG. (E,F) In the validation dataset, Kaplan-Meier curves depicting the TFS probability over time from diagnosis for patients segregated by miR-155 and ZAP70 status (E) or miR-155 and IGHV status (F). Statistical significance was determined by log-rank test (P < .05). P values for the comparisons between subgroups are indicated below the graph for panels B, C, E, and F.

Figure 2

Expression of SHIP1 protein in CLL cells that expressed high vs low levels of miR-155. (A) Expression of SHIP1 in CLL cells that expressed high (n = 21) or low (n = 24) levels of miR-155, as indicated at the bottom of the histogram. The height of each column corresponds to the mean MFIR of cells stained for SHIP1 in each subgroup. Error bars indicate the standard deviation of the mean. Statistical significance was determined by unpaired Student t test (P < .05). (B) Expression of SHIP1 in representative CLL samples, CLL#1 (Rai stage 1 at diagnosis, mutated IGHV, ZAP-70–negative, Δ peak anti-µ–induced MFIR = 7) or CLL#2 (Rai stage 2 at diagnosis, mutated IGHV, ZAP-70–positive, Δ peak anti-µ–induced MFIR = 32), after transfection with mimic-ct (control microRNA) or miR-155 microRNA, as indicated at the bottom of each panel. (C) Expression of SHIP1 in representative CLL samples, CLL#3 (Rai stage 2 at diagnosis, unmutated IGHV, ZAP-70–positive, Δ peak anti-µ–induced MFIR = 4.2) or CLL#4 (Rai stage 0 at diagnosis, mutated IGHV, ZAP-70–negative, Δ peak anti-µ–induced MFIR = 1.4), after transfection with oligo-ct (control microRNA) or miR-155 inhibitor, as indicated at the bottom of each panel. In (B) and (C) data are presented from immunoblot analyses (top panels) or flow cytometry (bottom panels), respectively. After the immunoblots were probed with anti-SHIP1, they were stripped and probed with anti–β-actin to monitor the uniformity of protein loading. The numbers between the immunoblot panels provide density ratios of the SHIP1 band relative to that of the β-actin band, normalized with respect to the ratio observed in control-treated samples. The histograms in the bottom panel depict the mean MFIR of CLL cells stained for SHIP1 after transfection with (B) mimic-ct or miR-155 mimic or (C) oligo-ct or miR-155 inhibitor, as indicated at the bottom of each histogram. Statistical significance was determined by paired Student t test (P < .05).

Figure 3

Anti-µ–induced calcium flux in CLL cells that express high vs low levels of miR-155. (A) The height of each column in the histogram describes the increase in fluorescence intensity after anti-µ stimulation of CLL cells that expressed high miR-155 vs low miR-155, as indicated at the bottom of the histogram. (B) Anti-µ–induced calcium mobilization in CLL cells after transfection with mimic-ct (top graph) or miR-155 (lower graph). The relative mean fluorescence intensity in intracellular calcium is plotted as a function of time. The arrow labeled “IgM” indicates the time at which the anti-µ was added to the cells. In the histogram at the bottom, the height of each column corresponds to the mean increase of fluorescence intensity after anti-µ stimulation for samples transfected with either mimic-ct or miR-155 microRNA, as indicated at the bottom by a “+”. (C) Anti-µ–induced calcium mobilization in CLL cells after transfection with oligo-ct or miR-155 inhibitor. The relative mean fluorescence intensity in intracellular calcium is plotted as in panel B. The height of each column in the bottom histogram corresponds to the increase of fluorescence intensity after anti-µ stimulation for samples respectively transfected with either control-ct or miR-155 inhibitor, as indicated at the bottom by a “+”. (D) The height of each column in the histogram describes the increase in fluorescence intensity after anti-µ stimulation of ZAP-70–negative CLL cells with high miR-155 or low miR-155, as indicated at the bottom of the histogram. Statistical significance was determined by the unpaired Student t test (P < .05). IgM, immunoglobulin M.

Figure 4

Expression of miR-155, SHIP1, and sensitivity to anti-µ in subpopulations of CLL cells isolated from the blood mononuclear cells of the same patient. (A) Flow cytometric analyses of representative unsorted CLL cells (left), isolated CD5brightCXCR4dim CLL cells (upper right), or isolated CD5dimCXCR4bright CLL cells (lower right). Contour plots (10% probability) depict the fluorescence of CLL cells stained with fluorochrome-conjugated mononuclear antibodies specific for human CD5 (y-axis) or human CXCR4 (x-axis). (B) Expression of miR-155 in CLL cells that expressed high levels of CD5 and low levels of CXCR4, or low levels of CD5 and high levels of CXCR4. The height of each column in the histogram indicates the fold increase of miR-155 copy number of sorted CD5brightCXCR4dim cells relative to that of sorted CD5dimCXCR4bright cells, as indicated at the bottom. Statistical significance was determined by paired Student t test (P < .05). (C) Expression of SHIP1 in sorted CD5brightCXCR4dim or CD5dimCXCR4bright CLL cells of representative samples (CLL1 [left 2 lanes] or CLL2 [right 2 lanes]), as indicated at the bottom. Probing for β-actin was used to monitor for equal loading of sample lysates. (D) Anti-µ–induced calcium mobilization in CLL cells that expressed high levels of CD5 and low levels of CXCR4, or low levels of CD5 and high levels of CXCR4. The relative mean fluorescence intensity in intracellular calcium is plotted in Figure 3B,C for each cell subset. The height of each column in the histogram on the right corresponds to the increase of fluorescence intensity after anti-µ stimulation for sorted CD5brightCXCR4dim or CD5dimCXCR4bright cells, as indicated at the bottom. Statistical significance was determined by unpaired Student t test (P < .05).

Figure 5

Stimulation of CLL cells with CD154 can induce expression miR-155, downregulation of SHIP1, and enhanced BCR signaling. (A) Expression of miR-155 in CLL cells without (−) or with (+) CD154 stimulation. The height of each column in the histogram corresponds to the fold increase in expression of miR-155 in CLL cells stimulated with CD154 (+) relative to that in CLL cells that had not been so stimulated (−), as indicated at the bottom of the histogram. We determined statistical significance using the paired Student t test (P < .05). (B) Expression of SHIP1 in CLL cells without (−) or with (+) CD154 stimulation, as indicated at the bottom of the histogram. The height of each column corresponds to the mean MFIR for SHIP1. (C) Anti-µ–induced calcium mobilization in CLL cells without (Ct, top panel) or with (CD154, lower panel) CD154 stimulation. The relative mean fluorescence intensity in intracellular calcium is plotted as a function of time. The arrow labeled “IgM” indicates the time at which the anti-µ was added to the cells (left panel). The histogram to the right depicts the mean increase of fluorescence intensity of CLL cells following stimulation with anti-µ without (−) or with (+) prior stimulation with CD154, as indicated at the bottom of the histogram. (D) Anti-µ–induced calcium mobilization in CLL cells with (+) or without (−) CD154 stimulation after the cells were transfected with a control oligonucleotide (oligo-ct) or miR-155 inhibitor. The height of each column in the histogram corresponds to the mean increase of fluorescence intensity after treatment with anti-µ of CLL cells. We determined statistical significance using 1-way analyses of variance (P < .05).

Figure 6

Model of the phenotype of CLL cells exiting the lymph node into the blood or exiting the blood into the LN. Within the PCs of the lymph node, CLL cells are stimulated via factors in the microenvironment (eg, CD154 or BAFF/APRIL), where there is upregulation of miR-155 and downregulation of INPP5D. The CLL cells that recently have exited the LN express relatively low levels of CXCR4 (CXCR4Dim), high levels of CD5 (CD5Bright), high levels of miR-155 (miR-155High), and low levels of SHIP1 (SHIP1Low), and have high responsiveness to BCR ligation with anti-µ (BCR-signalingHigh). Conversely, the cells that may exit the blood for the LN are CXCR4BrightCD5Dim and have relatively low levels of miR-155 (miR-155Low), high levels of SHIP1 (SHIP1High), and relatively low responsiveness to BCR ligation with anti-µ (BCR-signalingLow). The arrows indicate the direction of trafficking from the blood to the lymph node and then back to the blood.