It is well known that genetic mutations can drive drug resistance and lead to tumor relapse. Here, we focus on alternate mechanisms—those without mutations, such as phenotypic plasticity and stochastic cell-to-cell variability that can also evade drug attacks by giving rise to drug-tolerant persisters. The phenomenon of persistence has been well-studied in bacteria and has also recently garnered attention in cancer. We draw a parallel between bacterial persistence and resistance against androgen deprivation therapy in prostate cancer (PCa), the primary standard care for metastatic disease. We illustrate how phenotypic plasticity and consequent mutation-independent or non-genetic heterogeneity possibly driven by protein conformational dynamics can stochastically give rise to androgen independence in PCa, and suggest that dynamic phenotypic plasticity should be considered in devising therapeutic dosing strategies designed to treat and manage PCa.

Significance: The onset of androgen resistance is a major impediment in treating prostate cancer (PCa). However, the underlying molecular mechanisms are not fully understood. Here, we integrate multiple biophysical approaches that report conformational preferences of the intrinsically disordered protein (IDP) Prostate-Associated Gene 4 (PAGE4) with human cancer biology and nonlinear dynamics. Based on our biophysical, biochemical, and cellular data, a mathematical model is presented that suggests a mechanism by which phosphorylation-induced conformational changes in PAGE4 can switch from an androgen-dependent phenotype to an androgen-independent phenotype in PCa cells. The study underscores how IDPs that engage in multiple “promiscuous” interactions due to their conformational dynamics when overexpressed or aberrantly expressed can stochastically orchestrate phenotypic heterogeneity in PCa.

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)–CD3 complex comprising an αβ TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αβ TCR engagement by peptide–major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR–CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and β chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cβ FG loop projecting from the Vβ/Cβ interface; 2) a cluster of Cβ residues near the Cβ αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR β chain enables allosteric communication between the TCR-binding sites for pMHC and CD3.

Prostate-associated Gene-4 (PAGE4) is an intrinsically disordered Cancer/Testis Antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death while its over-expression leads to a growth advantage of prostate cancer cells [Zeng et al. (2011) J. Biol. Chem. 286, 13985-13994]. Phosphorylation of PAGE4 at T51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at T51. The population of transient turn-like structures increases upon phosphorylation in an approximately 20-residue acidic region centered on T51. This central region thus becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N- and C-termini. While flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at T51, most likely due to masking effects of the more compact phosphorylated state. Thus, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble with large functional consequences. The changes in the structural ensemble induced by post-translational modifications are similar conceptually to the conformational switching events seen in some marginally stable (metamorphic) folded proteins in response to mutation or environmental triggers.