Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer

Abstract

BackgroundEukaryotic initiation factor 4E (eIF4E) is elevated in many cancers and is a prognostic indicator in breast cancer. Many pro-tumorigenic proteins are selectively translated via eIF4E, including c-Myc, cyclin D1, ornithine decarboxylase (ODC), vascular endothelial growth factor (VEGF) and Tousled-like kinase 1B (TLK1B). However, western blot analysis of these factors in human breast cancer has been limited by the availability of fresh frozen tissue and the labor-intensive nature of the multiple assays required. Our goal was to validate whether formalin-fixed, paraffin-embedded tissues arranged in a tissue microarray (TMA) format would be more efficient than the use of fresh-frozen tissue and western blot to test multiple downstream gene products.ResultsBreast tumor TMAs were stained immunohistochemically and quantitated using the ARIOL imaging system. In the TMAs, eIF4E levels correlated strongly with c-Myc, cyclin D1, TLK1B, VEGF, and ODC. Western blot comparisons of eIF4E vs. TLK1B were consistent with the immunohistochemical results. Consistent with our previous western blot results, eIF4E did not correlate with node status, ER, PR, or HER-2/neu.ConclusionWe conclude that the TMA technique yields similar results as the western blot technique and can be more efficient and thorough in the evaluation of several products downstream of eIF4E.

Overexpression of eIF4F components in meningiomas and suppression of meningioma cell growth by inhibiting translation initiation

“…Loss of merlin abnormally activates several mitogenic signals, such as the PI3K/AKT/mTOR pathway (Ammoun et al, 2008; Jacob et al, 2008). Indeed, meningiomas often exhibit elevated levels of phospho-AKT (p-AKT), which can promote protein biosynthesis by stimulating p70 S6-kinase, leading to phosphorylation of the S6 ribosomal protein (Sorrells et al, 1999; De Benedetti and Graff, 2004; Holland et al, 2004; Anjum and Blenis, 2008; Menon and Manning, 2008; Kleiner et al, 2009; Ma and Blenis, 2009; Silvera et al, 2010; Li et al, 2012). Other important signaling proteins downstream of the PI3K/AKT/mTOR pathway include the 4E-binding protein (4E-BP) translational repressors; upon phosphorylation, these proteins are inactivated and protein biosynthesis is facilitated.…”

“…As uncontrolled growth of tumor cells often requires a high degree of protein translation, increased expression of the eIF4F components has been reported in several cancer types (Sorrells et al, 1999; De Benedetti and Graff, 2004; Kleiner et al, 2009; Silvera et al, 2010; Li et al, 2012). Overexpression of eIF4E promotes cell transformation (Lazaris-Karatzas et al, 1990) and frequently correlates with high tumor grade and poor patient prognosis (Li et al, 1997; Berkel et al, 2001; Li et al, 2012).…”

Targeting Mnks for Cancer Therapy

“…Elevated levels of eIF4E have been found in many types of tumours and cancer cell lines including cancers of the colon, breast, bladder, lung, prostate, gastrointestinal tract, head and neck, Hodgkin's lymphomas and neuroblastomas, but not in typical benign lesions [8, 10, 13-20]. A role for eIF4E as a prognostic marker has also been suggested for certain cancers and the involvement of eIF4E in metastasis has been considered [8, 10, 12, 21, 22].…”

“…A role for eIF4E as a prognostic marker has also been suggested for certain cancers and the involvement of eIF4E in metastasis has been considered [8, 10, 12, 21, 22]. Further evidence supporting a role for eIF4E in malignancy has been provided by studies where expression of antisense RNA to eIF4E in HeLa cells suppressed proliferation and altered cellular morphology [23].…”

Establishment of a mammary carcinoma cell line from Syrian hamsters treated with N-methyl-N-nitrosourea

“…All sections were deparaffinized, lightly counterstained with hematoxylin and eosin (H&E) and mounted with Permount and cover-slipped. Immunohistochemical staining was performed using an automated processor (BioGenex I6000 Automated Staining System, San Ramon, CA) as described previously [16] under the following conditions: sections were incubated with either rabbit polyclonal anti-HER-2/neu (Abcam; Cambridge, MA) at 1:100 dilution, mouse monoclonal anti-pancytokeratin (Abcam) at 1:300 dilution, mouse monoclonal anti-cytokeratin 5 and 6 (Ventana Medical Systems; Tucson, AZ) at 1:100 dilution, mouse monoclonal anti-p63 (Ventana) at 1:100 dilution for 1 h. After incubation with secondary antibody (MultiLink-BioGenex Super Sensitive Link-Label IHC Detection System) for 30 min, sections were incubated with horseradish peroxidase label (BioGenex) for 15 min and exposed to diaminobenzidine (DAB) substrate for 3 min. Positive and negative (no primary antibody) controls were included in every experiment.…”