Cortactin, another player in the Lyn signaling pathway, is over-expressed and alternatively spliced in leukemic cells from patients with B-cell chronic lymphocytic leukemia

Abstract

Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.

Figure 1.

Evaluation of cortactin expression level in patients and healthy subjects. (A) Expression of protein cortactin was evaluated by Western blotting analysis in B CD19⁺CD5⁺ lymphocytes of 106 CLL patients and 15 normal CD19⁺ controls. Figure reports one representative healthy subject (control B CD19⁺ cells #1) and six representative B CLL patients (CLL B CD19⁺CD5⁺ cells #1, #2, #3, #4, #5, #6). (B) Densitometry analysis (arbitrary unit) of cortactin level in CLL patients versus controls. All isoforms of cortactin, presented in Western blotting results of Figure 1A, were measured densitometrically. (C) Evaluation of cortactin expression was assessed at mRNA levels by real-time RT-PCR. (D) Representative confocal microscopy analysis in normal B CD19⁺ (n=5) and B CD19⁺CD5⁺ (n=15) cells of cortactin protein (Alexa-488, green). Original magnification, 60X. Fluorescence was detected using the UltraView LCI confocal system (Perkin Elmer) equipped with a fluorescence filter set for excitation at 488 nm.

Figure 2.

Evaluation of cortactin by flow cytometry. (A). Cortactin expression was evaluated in normal CD19⁺ cells (n=10) and neoplastic CD19⁺CD5⁺ lymphocytes (n=40) by flow cytometry. In Figure are reported one representative plot of healthy control and one of representative patient. The left panel shows the dot plot, the right panel reports the overlay histogram plot of MFI. (B). Mean of cortactin ΔMFI evaluated in patients (CD19⁺CD5⁺ CLL) and in controls (CD19⁺ controls). (C). Level of cortactin was evaluated by real-time RT-PCR in T lymphocytes isolated from patients (n=5) and healthy controls (n=5). (D). Cortactin expression was evaluated in B CD19⁺ and T CD5⁺ lymphocytes of patients (n=15) and T CD5⁺ lymphocytes of normal control (n=10) by flow cytometry. One representative plot of healthy control and one of representative patient are reported. The left panel shows the dot plot, the right panel reports the overlay histogram plot of MFI. (E). ΔMFI of cortactin was evaluated in T CD5⁺ and in B CD19⁺ of patients and controls.

Figure 3.

Expression of different cortactin isoforms in patients and healthy controls. (A) Results of cortactin Western blotting analysis in platelets (Plts), monocytes (Mo), NK and T cells from healthy subjects, and cell lines K562, Jurkat and Raji. (B) Percentage of patients and controls expressing the p70/75 or p80/85 form of cortactin. Percentages were calculated by direct count. (C) Densitometry evaluation from Western blotting analysis of p80/85 forms of cortactin expressed in patients (group 2) and of p70/75 forms expressed in patients (group 1) and in healthy subjects (controls).

Figure 4.

Investigation of cortactin forms phosphorylation. (A) B cells (5×10⁵) were lysed and subject to 2DE electrophoresis (upper panel) and western blotting analysis (lower panel). (B) Results of Western blotting analysis for Tyr421 phosphorylated cortactin in the cell line Jurkat and K562, in 3 representative patients (CLL#86, #90, #08) and one control (Ctr). Middle panel reports quantification of phosphorylated cortactin in Tyr421, in the lower panel is reported quantification level of total cortactin.

Figure 5.

Investigation of cortactin phosphorylation by Src kinase Lyn. Patient cells (5×10⁵) were lysed and subject to 2DE electrophoresis and Western blotting analysis before (alone) and after treatment with the PP2 inhibitor (PP2, 30′). (A) Evaluation by Western blotting of Lyn phosphorylation level in Tyr396, before and after PP2 treatment. (B) Effect of PP2 treatment on total tyrosine phosphorylation evaluated by 2DE electrophoresis. (C) Effect of PP2 treatment on cortactin migration in 2DE electrophoresis.

Figure 6.

Exon map and alternative splicing variants of the acting binding domain of the human EMS1/cortactin gene. (A) Exon map of the acting binding repeat domain of human EMS1 gene in relation to the protein sequence derived from the cDNA nucleotide sequence. The boxes (1-61/2) represent the 37 amino acid repeat motifs in the actin binding domain (motifs). The exons encoding the different motifs are indicated in the upper panel (exon). The position of primer sets used in this study for PCR is indicated. (B) Expression of WT and SV1 –cortactin mRNA variants (upper panel), using primers F-R on cDNA derived from the cell line K562, platelets and B lymphocytes of one representative healthy control (n=7) and 2 representative CLL patients (n=21), were compared to protein forms in Western blotting analysis (lower panel). (C) Frequency for WT cortactin mRNA in patients with respect to controls. K562: cell line; Plts: normal platelets; N: normal B lymphocytes; CLL: neoplastic B lymphocytes from patients with CLL.