A systems biology dynamical model of mammalian G1 cell cycle progression.

The current dogma of G(1) cell-cycle progression relies on growth factor-induced increase of cyclin D:Cdk4/6 complex activity to partially inactivate pRb by phosphorylation and to sequester p27(Kip1)-triggering activation of cyclin E:Cdk2 complexes that further inactivate pRb. pRb oscillates between an active, hypophosphorylated form associated with E2F transcription factors in early G(1) phase and an inactive, hyperphosphorylated form in late G(1), S and G(2)/M phases. However, under constant growth factor stimulation, cells show constitutively active cyclin D:Cdk4/6 throughout the cell cycle and thereby exclude cyclin D:Cdk4/6 inactivation of pRb. To address this paradox, we developed a mathematical model of G(1) progression using physiological expression and activity profiles from synchronized cells exposed to constant growth factors and included a metabolically responsive, activating modifier of cyclin E:Cdk2. Our mathematical model accurately simulates G(1) progression, recapitulates observations from targeted gene deletion studies and serves as a foundation for development of therapeutics targeting G(1) cell-cycle progression.

Bayesian Network Inference Modeling Identifies TRIB1 as a Novel Regulator of Cell-Cycle Progression and Survival in Cancer Cells.

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G1-S transition. The model identified known components of the cell-cycle machinery, such as CCND1, CCNE2, and CDC25A, as well as revealed novel regulators of G1-S transition, IER2, TRIB1, TRIM27. Experimental validation of model predictions confirmed 10 of 12 predicted genes to have a role in G1-S progression. Further analysis showed that TRIB1 regulated the cyclin D1 promoter via NFκB and AP-1 sites and sensitized cells to TRAIL-induced apoptosis. In clinical specimens of breast cancer, TRIB1 levels correlated with expression of NFκB and its target genes (IL8, CSF2), and TRIB1 copy number and expression were predictive of clinical outcome. Together, our results establish a critical role of TRIB1 in cell cycle and survival that is mediated via the modulation of NFκB signaling. Cancer Res; 77(7); 1575-85. ©2017 AACR.

Specific immunological detection of the (V+C)(-) fibronectin isoform.

The population of fibronectins in adult mammalian cartilage includes high levels of a cartilage-specific (V+C)(-) isoform which lacks the V, III-15, and I-10 segments and thus contains a novel junction between protein segments III-14 and I-11. We report production of a monoclonal antibody specific for (V+C)(-) fibronectin without cross-recognition of V(+)C(+) and V(-)C(+) isoforms found in plasma and other tissues. Presentation of epitope to this antibody requires the III-14/I-11 junction, but the epitope itself extends beyond 14 amino acids immediately surrounding the junction site and involves a conformational change in III-14 and/or the N-terminal portion of I-11. The antibody, designated Mab 5D10 anti (V+C)(-), displays specificity for (V+C)(-) fibronectin from multiple mammalian species including humans and utility in immunoblots, immunohistochemistry, and ELISA.

Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition.

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.

The cartilage-specific fibronectin isoform has a high affinity binding site for the small proteoglycan decorin.

Binding of fibronectin to the small proteoglycan decorin plays an important role in cell differentiation and cell migration. The cartilage-specific (V+C)(-) fibronectin isoform, in which nucleotides that normally encode the protein segments V, III(15), and I(10) are spliced out, is one of the major splice variants present in cartilage matrices. Full-length and truncated cDNA constructs were used to express recombinant versions of fibronectin. Results demonstrated that the (V+C)(-) isoform has a higher affinity for decorin. Dissociation constants for decorin and fibronectin interaction were calculated to be 93 nm for the V(+)C(+) isoform and 24 nm and 223 nm for (V+C)(-) fibronectin. Because heparin competed with decorin competitively, binding of decorin to fibronectin likely occurs at a heparin-binding region. We propose that alternative splicing of the V and C regions changes the global conformation of fibronectin in such a way that it opens an additional decorin-binding site. This conformational change is responsible for the higher affinity of the (V+C)(-) fibronectin isoform for decorin.