Identification of lysosome-targeting drugs with anti-inflammatory activity as potential invasion inhibitors of treatment resistant HER2 positive cancers.

Purpose: Most HER2 positive invasive cancers are either intrinsic non-responsive or develop resistance when treated with 1st line HER2 targeting drugs. Both 1st and 2nd line treatments of HER2 positive cancers are aimed at targeting the HER2 receptor directly, thereby strongly limiting the treatment options of HER2/ErbB2 inhibition resistant invasive cancers.

Methods: We used phenotypic high throughput microscopy screening to identify efficient inhibitors of ErbB2-induced invasion using 1st line HER2 inhibitor trastuzumab- and pertuzumab-resistant, p95-ErbB2 expressing breast cancer cells in conjunction with the Prestwick Chemical Library®.

AMPK-dependent phosphorylation is required for transcriptional activation of TFEB and TFE3.

Increased macroautophagy/autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK (AMP-activated protein kinase) activation and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) inhibition to maintain energy homeostasis and cell survival. TFEB (transcription factor E3) and TFE3 (transcription factor binding to IGHM enhancer 3) are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by MTORC1-dependent multisite phosphorylation.

DNA-dependent protein kinase regulates lysosomal AMP-dependent protein kinase activation and autophagy.

Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase.

Release of transcriptional repression via ErbB2-induced, SUMO-directed phosphorylation of myeloid zinc finger-1 serine 27 activates lysosome redistribution and invasion.

HER2/ErbB2 activation turns on transcriptional processes that induce local invasion and lead to systemic metastasis. The early transcriptional changes needed for ErbB2-induced invasion are poorly understood. Here, we link ErbB2 activation to invasion via ErbB2-induced, SUMO-directed phosphorylation of a single serine residue, S27, of the transcription factor myeloid zinc finger-1 (MZF1).

KIAA1524/CIP2A promotes cancer growth by coordinating the activities of MTORC1 and MYC.

KIAA1524/CIP2A/cancerous inhibitor of protein phosphatase 2A is a cancer-promoting protein that stabilizes the MYC proto-oncogene protein by inhibiting its dephosphorylation. Our recent report demonstrates that KIAA1524/CIP2A supports cancer cell growth also at the level of the mechanistic target of rapamycin complex 1 (MTORC1), a key signaling module that drives cell growth by stimulating protein synthesis and inhibiting autophagy. KIAA1524/CIP2A suppresses MTORC1-associated protein phosphatase 2A (PP2A) activity in an allosteric manner thereby stabilizing the phosphorylation of MTORC1 substrates and keeping the cell in an anabolic mode.

CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation.

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy.

Apoptosis and autophagy: Targeting autophagy signalling in cancer cells -'trick or treats'?

Macroautophagy (hereafter referred to as autophagy) is a tightly regulated lysosome-dependent catabolic pathway. During this process, cytosolic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes to become autolysosomes, where their contents are degraded. Autophagy contributes to the maintenance of the cellular energy homeostasis, to the clearance of damaged organelles and to adaptation to environmental stresses.

PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response.

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation.

A sigma-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction.

CIP2A inhibits PP2A in human malignancies.

Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, we describe a cellular inhibitor of PP2A with oncogenic activity.

Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg.

Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA.

Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection.

We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The alpha-catalytic subunit of CK2 (CK2alpha) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatography-tandem mass spectrometry.

Detection of the potyviral genome-linked protein VPg in virions and its phosphorylation by host kinases.

The multifunctional genome-linked protein (VPg) of Potato virus A (PVA; genus Potyvirus) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species Solanum commersonii and delays systemic infection, which is not observed in tobacco plants.

Proteolytic processing of potyviral proteins and polyprotein processing intermediates in insect and plant cells.

Processing of the polyprotein encoded by Potato virus A (PVA; genus Potyvirus) was studied using expression of the complete PVA polyprotein or its mutants from recombinant baculoviruses in insect cells. The time-course of polyprotein processing by the main viral proteinase (NIaPro) was examined with the pulse-chase method. The sites at the P3/6K1, CI-6K2 and VPg/NIaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate.