Melissa H. Y. Chang

βIII-tubulin (encoded by TUBB3) expression is associated with therapeutic resistance and aggressive disease in non–small cell lung cancer (NSCLC), but the basis for its pathogenic influence is not understood. Functional and differential proteomics revealed that βIII-tubulin regulates expression of proteins associated with malignant growth and metastases. In particular, the adhesion-associated tumor suppressor maspin was differentially regulated by βIII-tubulin. Functionally, βIII-tubulin suppression altered cell morphology, reduced tumor spheroid outgrowth, and increased sensitivity to anoikis. Mechanistically, the PTEN/AKT signaling axis was defined as a critical pathway regulated by βIII-tubulin in NSCLC cells. βIII-Tubulin blockage in vivo reduced tumor incidence and growth. Overall, our findings revealed how βIII-tubulin influences tumor growth in NSCLC, defining new biologic functions and mechanism of action of βIII-tubulin in tumorigenesis. Cancer Res; 75(2); 415–25. ©2014 AACR.

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16^INK4a and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16^INK4a on EC function and to identify molecular changes associated with repression of p16^INK4a. In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16^INK4a showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16^INK4a-negative and a p16^INK4a-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16^INK4a expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16^INK4a expression. This corresponded with aberrant cytoskeletal architectures among p16^INK4a-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16^INK4a-positive cells. A direct link between p16^INK4a repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16^INK4a. siRNA-mediated repression of p16^INK4a significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16^INK4a are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies.

Antimitotic agents that interfere with the tubulin/microtubule system are important in the treatment of a range of cancers. Natural product tubulin-binding agents such as the Vinca alkaloids have proven highly effective in the treatment of leukemia. Improved understanding of the mechanisms of action of these and related drugs has led to the identification of distinct binding sites on tubulin that cause inhibition of spindle microtubule dynamics, mitotic arrest and cell death. Despite the efficacy of these agents, treatment failure caused by the emergence of drug resistant leukemic cells is a significant clinical problem. Alterations in the cellular target of tubulin-binding agents have been strongly implicated in resistance to these agents. This review will focus on the microtubule cytoskeleton and its role in drug resistance in leukemia. The identification of novel protein pathways involved in drug response and the development of new drugs targeted against microtubules, offers opportunities to treat resistant disease, improve outcome and potentially reduce toxicity for leukemia patients.

LAMP-1 (lysosome-associated membrane protein), a major glycoprotein present in the lysosomal membrane, constitutes up to 50% of total membrane proteins. LAMP-1, expressed at the plasma membrane, is reported to be the major molecule expressing the sialyl-Lewis X antigen. Two forms of LAMP-1 exist; the full-length LAMP-1 [LAMP-1 (+Tail)] has a highly glycosylated lumenal domain, a membrane-spanning domain and a short cytoplasmic tail, and the truncated LAMP-1 [LAMP-1 (−Tail)] contains only the lumenal domain. Soluble LAMP-1 (±Tail) has been reported in circulation. LAMP-1 at the cell surface has been shown to interact with E-selectin and galectin and is proposed to function in cell–cell interactions. However, the functional role(s) of soluble LAMP-1 in circulation is unclear. To investigate the functional role of soluble LAMP-1 in circulation, recombinant LAMP-1 (−Tail) and LAMP-1 (+Tail) were produced in HT1080 cells. Two immune-quantification assays were developed to distinguish between the LAMP-1 forms. The interaction and aggregation properties of the different LAMP-1 forms were investigated using the immune-quantification assays. Only LAMP-1 (+Tail) was found to aggregate and interact with plasma proteins. Plasma proteins that interact with LAMP-1 were isolated by affinity chromatography with either the recombinant LAMP-1 (−Tail) or a synthesized peptide consisting of the 14 amino acids of the LAMP-1 cytoplasmic tail. Transthyretin was found to interact with the cytoplasmic tail of LAMP-1. Transthyretin exists as a homotetramer in plasma, as such may play a role in the aggregation of LAMP-1 in circulation.