Heather E. Kleiner

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

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.

All-Trans Retinoic Acid Suppresses Stat3 Signaling during Skin Carcinogenesis

Squamous cell carcinoma (SCC) of the skin is the most clinically aggressive form of nonmelanoma skin cancer. We have determined the effects of all-trans retinoic acid (ATRA), a naturally occurring chemopreventive retinoid, on signal transducer and activator of transcription 3 (Stat3) signaling during the development of skin SCC. Stat3 is a transcription factor that plays a critical role in cell proliferation and survival, and it is constitutively active in several malignant cell types. We have previously shown that Stat3 is required for the initiation, promotion, and progression of skin SCC. ATRA is a highly efficient suppressor of tumor formation in the two-stage mouse skin carcinogenesis model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/Erk signaling pathway. In this study, we have determined the pattern of Stat3 phosphorylation throughout the course of the two-stage protocol, both in the presence and absence of ATRA. We have used both SENCAR mice and K5.Stat3C transgenic mice, which express the Stat3C protein, a constitutively active form of Stat3, in the skin. Using Western blotting and immunohistochemical staining with phosphospecific antibodies, we show that coadministration of ATRA suppressed the 12-O-tetradecanoylphorbol-13-acetate–induced phosphorylation of Stat3 in both models, but was only able to suppress tumor formation in the SENCAR mice. Surprisingly, ATRA actually enhanced tumor formation in 12-O-tetradecanoylphorbol-13-acetate–treated K5.Stat3C mice. We hypothesize that ATRA blocks tumor formation, at least in part, by targeting events upstream of Stat3, such as the B-Raf/Mek/Erk pathway, and that in the K5.Stat3C mice, in which Stat3 activity is constitutive, it cannot suppress tumor formation.

Modifiers of Cytochrome(s) P450

Cytochromes P450 (CYPs) belong to a superfamily of enzymes that have different, but overlapping, substrate specificities and tissue distribution. The highest concentration of CYPs is in the liver endoplasmic reticulum, but P450 is found in most other tissues of the body. CYPs are heme-containing enzymes that can either detoxify or bioactivate xenobiotics (foreign chemicals). The following seven types of reactions are catalyzed by CYPs: i) hydroxylation of an aliphatic or aromatic carbon; ii) epoxidation of a double bond; iii) heteroatom (S-, N-, and I-) oxygenation and N-hydroxylation; iv) heteroatom (O-, S-, and N-) dealkylation; v) oxidative group transfer; vi) cleavage of esters; and vii) dehydrogenation (1). In humans, the predominant isoform of P450 in the liver is CYP1A2. Other human liver P450s include CYP 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 (reviewed in ref. 1). CYP 1 A 1 is expressed in extrahepatic sites including human lung, the intestines, the skin, lymphocytes, and the placenta. Human CYP 1 B 1 catalyzes the activation of a number of diverse pro-carcinogens (2), and is expressed in a variety of extra-hepatic sites, including steroid-responsive and steroidogenic tissues (3–5).

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

Correction to Kleiner HE, Krishnan P, Tubbs J, Smith M, Meschonat C, Shi R, Lowery-Nordberg M, Adegboyega P, Unger M, Cardelli J et al: Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer. J Exp Clin Cancer Res 2009, 28:5.