ABSTRACT
BACKGROUND: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. RESULTS: We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells. CONCLUSIONS: Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.
Subject(s)
Phosphorylation , Receptors, Progesterone , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Shape , Energy Metabolism , Glycolysis , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Mitochondria/metabolism , Neoplasms , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Progesterone/biosynthesis , Receptors, Progesterone/metabolismABSTRACT
Mammalian embryos develop in a low oxygen environment. The transcription factor hypoxia inducible factor 1a (HIF1α) is a key element in the cellular response to hypoxia. Complete deletion of Hif1α from the mouse conceptus causes extensive placental, vascular and heart defects, resulting in embryonic lethality. However the precise role of Hif1α in each of these organ systems remains unknown. To further investigate, we conditionally-deleted Hif1α from mesoderm, vasculature and heart individually. Surprisingly, deletion from these tissues did not recapitulate the same severe heart phenotype or embryonic lethality. Placental insufficiency, such as occurs in the complete Hif1α null, results in elevated cellular hypoxia in mouse embryos. We hypothesized that subjecting the Hif1α conditional null embryos to increased hypoxic stress might exacerbate the effects of tissue-specific Hif1α deletion. We tested this hypothesis using a model system mimicking placental insufficiency. We found that the majority of embryos lacking Hif1α in the heart died when exposed to non-physiological hypoxia. This was a heart-specific phenomenon, as HIF1α protein accumulated predominantly in the myocardium of hypoxia-stressed embryos. Our study demonstrates the vulnerability of the heart to lowered oxygen levels, and that under such conditions of non-physiological hypoxia the embryo absolutely requires Hif1α to continue normal development. Importantly, these findings extend our understanding of the roles of Hif1α in cardiovascular development.
Subject(s)
Gene Expression Regulation, Developmental , Gene-Environment Interaction , Heart/embryology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Alleles , Animals , Cell Hypoxia , Cell Nucleus/metabolism , Cell Proliferation , Endothelial Cells/cytology , Female , Gene Deletion , Genotype , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitosis , Myocardium/metabolism , Oxygen/metabolism , Phenotype , Placenta/metabolism , PregnancyABSTRACT
Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of chemotherapeutics in bacterially derived 400 nm minicells. We discovered that minicells can be packaged with therapeutically significant concentrations of chemotherapeutics of differing charge, hydrophobicity, and solubility. Targeting of minicells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. This affects highly significant tumor growth inhibition and regression in mouse xenografts and case studies of lymphoma in dogs despite administration of minute amounts of drug and antibody; a factor critical for limiting systemic toxicity that should allow the use of complex regimens of combination chemotherapy.
