Precise definition of PTEN C-terminal epitopes and its implications in clinical oncology

Abstract

Anti-PTEN monoclonal antibodies (mAb) are arising as important tools for immunohistochemistry (IHC) and protein quantification routine analysis in clinical oncology. Although an effort has been made to document the reliability of tumor tissue section immunostaining by anti-PTEN mAb, and to standardize their IHC use in research and in the clinical practice, the precise topological and biochemical definition of the epitope recognized by each mAb has been conventionally overlooked. In this study, six commercial anti-PTEN mAb have been validated and characterized for sensitivity and specificity by IHC and FISH, using a set of prostate and urothelial bladder tumor specimens, and by immunoblot, using PTEN positive and PTEN negative human cell lines. Immunoblot precise epitope mapping, performed using recombinant PTEN variants and mutations, revealed that all mAb recognized linear epitopes of 6–11 amino acid length at the PTEN C-terminus. Tumor-associated or disease-associated mutations at the PTEN C-terminus did not affect subcellular localization or PIP3 phosphatase activity of PTEN in cells, although resulted in specific loss of reactivity for some mAb. Furthermore, specific mimicking-phosphorylation mutations at the PTEN C-terminal region also abolished binding of specific mAb. Our study adds new evidence on the relevance of a precise epitope mapping in the validation of anti-PTEN mAb for their use in the clinics. This will be substantial to provide a more accurate diagnosis in clinical oncology based on PTEN protein expression in tumors and biological fluids.

The mechanism of full activation of tumor suppressor PTEN at the phosphoinositide-enriched membrane

“…In the cytoplasm, PTEN exists as a monomer and can even form a homodimer in vitro (Heinrich et al, 2015). The disordered CTT is key to the regulation of the monomeric PTEN state in the cytosol through the phosphorylation of the serine-threonine cluster (Ser380, Thr382, Thr383, and Ser385) located in it (Bolduc et al, 2013;Masson et al, 2016;Masson and Williams, 2020;Mingo et al, 2019;Vazquez et al, 2000). Cytosolic PTEN monomers exhibit a Figure 1.…”

The mechanism of full activation of tumor suppressor PTEN at the phosphoinositide-enriched membrane

“…Previous PTEN studies showed that affinity to the membrane is reduced by the following: cancer-associated mutations (Masson and Williams, 2020) inhibit recruitment to the plasma membrane, thus abolishing its interactions with phospholipids (Nguyen et al, 2015); phosphorylation of the CTT serine-threonine cluster impairs PTEN membrane binding ability, resulting in a closed protein conformation (Bolduc et al, 2013;Malaney et al, 2013;Masson and Williams, 2020;Rahdar et al, 2009;Ross and Gericke, 2009); CTT deletion decreases protein stability leading to tumor development (Georgescu et al, 1999;Sun et al, 2014); and a mutation in the PBD (K13E) leads to PTEN inactivation (Masson and Williams, 2020;Nguyen et al, 2015;Walker et al, 2004). In the cytosol, monomeric PTEN is in the inactive state with its PBD bound to the phosphatase domain and the phosphorylated CTT interfering with the membrane binding interface (Bolduc et al, 2013;Chen et al, 2016;Malaney et al, 2013;Masson et al, 2016;Masson and Williams, 2020;Mingo et al, 2019;Rahdar et al, 2009;Ross and Gericke, 2009;Vazquez et al, 2000) (Figure 7), whereby the inactive, autoinhibited PTEN is in the "closedclosed" conformation.…”

MMADHC premature termination codons in the pathogenesis of cobalamin D disorder: Potential of translational readthrough reconstitution

“…Cell nuclei were stained with Hoechst 33342 (ThermoFisher Scientific). For immunoblot, COS-7 cells were processed as described [ 26 ]. Briefly, cells were washed with PBS and lysed in ice-cold M-PER lysis buffer (ThermoFisher Scientific) supplemented with PhosSTOP phosphatase inhibitor and complete protease inhibitor cocktails (Roche).…”