Dysregulation of cell cycle control caused by overexpression of the oncogene pp32r1 (ANP32C) and the Tyr>His mutant pp32r1Y140H.

The pp32 (ANP32A) gene acts as a tumor suppressor while its closely related homologue pp32r1 (ANP32C) is oncogenic and is overexpressed in breast, prostate and pancreatic tumors. The transduction of p53wt cell lines (ACHN and HeLa) with pp32r1 or pp32r1Y140H lentivirus increased the proliferation of p53wt cell lines compared to the untransduced control cells while transduction of the p53(R248W) MiaPaCa2 cell line had no effect. Cell cycle analysis of transduced ACHN cells by PI staining and BrdU incorporation illustrated a pronounced shift toward the S-phase of the cell cycle in cells overexpressing the pp32r1 and pp32r1Y140H proteins. Confocal microscopy and western blotting demonstrated that pp32r1 and the pp32r1Y140H mutant protein reside predominantly in the cytoplasm in constrast to pp32 which is a nuclear/cytoplasmic shuttling protein. To determine the effects of pp32r1 or pp32r1Y140H overexpression at the proteomic level we performed a comprehensive proteome analysis on ACHN, ACHN-pp32r1 and ACHN-pp32r1Y140H cell lysates using the isotope-coded protein label (ICPL) method. Among those proteins with >40% regulation were Macrophage Capping protein (CAPG) and Chromodomain Helicase DNA binding protein 4 (CHD4) proteins which were significantly upregulated by pp32r1 and pp32r1Y140H overexpression. This increase in CHD4 also appears to influence a number of cell cycle regulator genes including; p53, p21 and cyclinD1 as judged by western blotting. Silencing of CHD4 in ACHN-pp32r1Y140H cells using specific shRNA reverted the cell cycle dysregulation caused by pp32r1Y140H expression to that of the untransduced ACHN cell line, suggesting that CHD4 is the prominent effector of the pp32r1/pp32r1Y140H phenotype.

Pharmacologically controlling protein-protein interactions through epichaperomes for therapeutic vulnerability in cancer.

Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks.

Fusion of lysosomes to plasma membrane initiates radiation-induced apoptosis.

Diverse stresses, including reactive oxygen species (ROS), ionizing radiation, and chemotherapies, activate acid sphingomyelinase (ASMase) and generate the second messenger ceramide at plasma membranes, triggering apoptosis in specific cells, such as hematopoietic cells and endothelium. Ceramide elevation drives local bilayer reorganization into ceramide-rich platforms, macrodomains (0.5-5-µm diameter) that transmit apoptotic signals. An unresolved issue is how ASMase residing within lysosomes is released extracellularly within seconds to hydrolyze sphingomyelin preferentially enriched in outer plasma membranes. Here we show that physical damage by ionizing radiation and ROS induces full-thickness membrane disruption that allows local calcium influx, membrane lysosome fusion, and ASMase release. Further, electron microscopy reveals that plasma membrane "nanopore-like" structures (∼100-nm diameter) form rapidly due to lipid peroxidation, allowing calcium entry to initiate lysosome fusion. We posit that the extent of upstream damage to mammalian plasma membranes, calibrated by severity of nanopore-mediated local calcium influx for lysosome fusion, represents a biophysical mechanism for cell death induction.

Overexpression of the pp32r1 (ANP32C) oncogene or its functional mutant pp32r1Y140H confers enhanced resistance to FTY720 (Finguimod).

pp32r1 (ANP32C) is oncogenic and has been shown to be overexpressed in tumors of the breast, prostate, and pancreas. In this work we show that pp32 family proteins are able to bind to the sphingosine analog FTY720 (Finguimod). Molecular docking studies highlight that a conserved residue F136 is likely to be a key determinant of the FTY720 binding site on the pp32 leucine-rich repeat domain. Transduction of the renal carcinoma cell line ACHN or cervical cancer cell line HeLa with lentivirus expressing the oncogenic family member pp32r1 or a pp32r1Y140H functional mutant illustrated an enhanced resistance to FTY720 induced apoptosis. These findings highlight that certain cancers overexpressing pp32r1 or pp32r1 mutants are likely to demonstrate enhanced resistance to FTY720 treatment.