Cortactin Is Overexpressed In Neoplastic Cells of Patients with B-Cell Chronic Lymphocytic Leukemia

Abstract 2436

Cortactin is an ubiquitous actin-binding protein that is encoded by EMS1 gene, localized in chromosome 11q13 region. Cortactin is expressed in nearly all mammalian tissues and it was observed to migrate in two different bands with an apparent molecular weight of 80–85kDa, where p85 isoform originates from the p80 because of tyrosine phosphorylation by various different tyrosine kinases. Cortactin mostly localizes to dynamic actin structures and has been implicated in the regulation of several actin-dependent processes, including lamellipodium protrusion, trafficking of the key invadopodia proteases and intracellular transducer downstream of kinase-mediated cell signalling upon phosphorylation. The identification of two alternative splice variants affecting the F-actin binding domain of human cortactin have been described by van Rossum et al., e.g. SV1-cortactin, lacking the 6th repeat (exon 11), and SV2-cortactin, lacking the 5th and 6th repeats (exons 10 and 11). When compared with cells expressing WT-cortactin mRNA, cells expressing SV1- and SV2-cortactin mRNA differ significantly in their ability to bind and cross-link F-actin, promoting actin polymerization in vitro and cell migration. The protein cortactin was found overexpressed in several tumors such as esophageal squamous cell carcinoma, head and neck squamous cell carcinoma and gastric carcinoma. This overexpression increases tumor aggressiveness, possibly through promotion of tumor invasion and metastasis. This protein was originally identified as a substrate for the protein Src kinase Lyn, that plays a key role in CLL disease pathogenesis and progression.

In the present study we investigated the expression of cortactin in B cells from 93 patients with B-CLL and 14 healthy controls. We found that this protein was significantly overexpressed in patients (1.10±1.32 SD) with respect to normal control (0.17±0.17 SD, p<0.01; Student's t-test). We also investigated the correlation between level of cortactin and presence of somatic hypermutations (SHM) in the immunoglobulin heavy-chain variable region (IgV_H), since the absence of SHM is relevant in terms of reduced survival and responsiveness to chemotherapy. We found that unmutated patients (n=38, 41%), with unfavourable prognosis, showed higher levels of cortactin (1.42±1.63 SD) with respect to mutated ones (n=55, 59%; 0.93±1.14 SD). The differences in cortactin expression levels between unmutated, mutated and normal subjects was statistically significant (p<0.01, ANOVA test). Moreover, Western blotting data demonstrated that in 44% of our patients, neoplastic B lymphocytes express the protein cortactin with a molecular weight of 80/85kDa never found in normal B lymphocytes, that have a protein with a molecular weight of 70/75kDa (Fisher's exact test, p<0.01). The presence of proteins with different molecular weight was confirmed by two-dimensional polyacrylamide gel electrophoresis (2DE) that allowed to separate proteins according to their molecular weight and to their pI (isoeletrict point). Finally the origin of proteins with different molecular weight was investigated at level of mRNA expression. By semi-quantitative RT-PCR analysis on mRNA using primers that flank the region encoding the entire acting binding domain, in B cells purified from 7 normal controls and 21 patients with CLL we studied the expression of mRNA splice variants. We found that 53% of patients expressed the WT mRNA while none of controls was WT, these latter expressing the SV1 isoform only (100%). Results of our SDS-PAGE, 2DE and RT-PCR analyses suggest that WT mRNA codifies for the p80 protein while the SV1 mRNA codifies for the p70 one and that the p85 and p75 isoforms originate from the p80 and p70 respectively as a consequence of post-transcriptional modifications.

These findings might suggest that the overexpression of cortactin in leukemic B cells play a role in the pathogenesis and aggressiveness of the neoplastic clone in B-CLL.