ABSTRACT
Liposarcomas are separated into clinicopathological entities with a characteristic morphological spectrum and mutually exclusive genetic alterations. Therefore, the rare occurrence of cases with combined patterns of well-differentiated liposarcoma and myxoid liposarcoma designated as mixed-type liposarcoma pose a conceptual problem. Moreover, this feature may have consequences for treatment choice and prognosis. Here, we have dissected the molecular relation of tumor components in cases of mixed-type liposarcoma. On the basis of heterogeneous preoperative magnetic resonance image (MRI) features, eight cases of mixed-type liposarcoma were selected. Preoperative biopsy samples and resection specimens were analyzed including molecular and immunohistochemical analysis on all components. As controls, cases with homogeneous MRI features and uniform aspects of myxoid liposarcoma (n = 5), round cell liposarcoma (n = 5) and well-differentiated liposarcoma (n = 5) were studied. All patients with heterogeneous MRI features showed morphological components of myxoid liposarcoma and well-differentiated liposarcoma. Real-time polymerase chain reaction showed FUS-DDIT3 fusion in both components in five of eight cases in the absence (zero of five) of MDM2 and CDK4 amplification. In three of eight patients, MDM2 and/or CDK4 were overexpressed, and amplification was shown by multiplex ligation-dependent probe amplification (MLPA) in the absence of myxoid liposarcoma translocations. All control patients showed a molecular pattern consistent with their morphological features. Therefore, mixed-type liposarcomas should not be regarded as collision tumors, but as an extreme variant of the morphological spectrum within a single biological entity, explaining the biological contradiction of mixed-type liposarcoma. For treatment stratification, detailed classification including molecular support should be performed in tumors with heterogeneous MRI features.
Subject(s)
Biomarkers, Tumor/genetics , Liposarcoma, Myxoid/classification , Liposarcoma, Myxoid/pathology , Magnetic Resonance Imaging , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Cohort Studies , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Female , Follow-Up Studies , Humans , Immunoenzyme Techniques , Liposarcoma, Myxoid/genetics , Male , Middle Aged , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Prognosis , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Messenger/genetics , RNA-Binding Protein EWS , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Retroperitoneal Neoplasms/genetics , Retroperitoneal Neoplasms/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Treatment decisions and prognosis assessment for liposarcoma is based on a classification that depends on morphological and genetic features. Revisions by experienced referral pathologists are often advocated. METHODS: The process of histopathological classification in referring hospitals and subsequently in a referral center in relation to molecular biological information is evaluated. A total of 331 consecutive liposarcoma patients were evaluated for the added value of histological review at time of referral. Subsequently, cases were reclassified with implementation of present-day molecular information. For all patients, complete data on staging, treatment, and follow-up were available. RESULTS: Upon histological revision, 15/54 (28%) diagnoses were reclassified in the first decade, 14/65 (22%) in the second, and 14/53 (26%) in the last decade. Molecular biological analysis enabled well-differentiated liposarcoma with or without dedifferentiated component to be better recognized as such and distinguished from myxoid liposarcoma and pleomorphic liposarcoma. Inclusion of cytogenetic information resulted in reclassification after revision in 4/18 (22%) cases in the first decade, 10/38 (26%) cases in the second decade, and 19/75 (25%) cases in the last decade. CONCLUSIONS: This study indicates that liposarcomas are heterogeneous tumors. Expert assessment and implementation of molecular biological analysis are valuable for adequate classification as a basis for treatment decisions.
Subject(s)
Liposarcoma/classification , Liposarcoma/diagnosis , Chromosomes, Human, Pair 12/genetics , Clinical Laboratory Techniques , Cohort Studies , Cyclin-Dependent Kinase 4/genetics , DNA, Neoplasm/genetics , Diagnosis, Differential , Female , Humans , Liposarcoma/therapy , Male , Oncogene Proteins, Fusion/genetics , Polymerase Chain Reaction , Prognosis , Survival RateABSTRACT
Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein-coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) from the plasma membrane. Knockdown of phospholipase Cbeta3 (PLCbeta3) inhibits PtdIns(4,5)P(2) hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P(2) depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P(2) is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCbeta3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P(2)-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor-PLC coupling. Our results show that PtdIns(4,5)P(2) is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCbeta3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5)P(2).
Subject(s)
Cell Communication , Connexin 43/metabolism , Gap Junctions/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Humans , Hydrolysis , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Models, Neurological , Phospholipase C beta , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Structure, Tertiary , Rats , Signal Transduction , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , Zonula Occludens-1 ProteinABSTRACT
Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine kinase that is usually inactivated by serine phosphorylation in response to extracellular cues. However, GSK-3 can also be activated by tyrosine phosphorylation, but little is known about the upstream signaling events and tyrosine kinase(s) involved. Here we describe a G protein signaling pathway leading to GSK-3 activation during lysophosphatidic acid (LPA)-induced neurite retraction. Using neuronal cells expressing the LPA(1) receptor, we show that LPA(1) mediates tyrosine phosphorylation and activation of GSK-3 with subsequent phosphorylation of the microtubule-associated protein tau via the G(i)-linked PIP(2) hydrolysis-Ca(2+) mobilization pathway. LPA concomitantly activates the Ca(2+)-dependent tyrosine kinase Pyk2, which is detected in a complex with GSK-3beta. Inactivation or knockdown of Pyk2 inhibits LPA-induced (but not basal) tyrosine phosphorylation of GSK-3 and partially inhibits LPA-induced neurite retraction, similar to what is observed following GSK-3 inhibition. Thus, Pyk2 mediates LPA(1)-induced activation of GSK-3 and subsequent phosphorylation of microtubule-associated proteins. Pyk2-mediated GSK-3 activation is initiated by PIP(2) hydrolysis and may serve to destabilize microtubules during actomyosin-driven neurite retraction.
Subject(s)
Focal Adhesion Kinase 2/metabolism , Glycogen Synthase Kinase 3/metabolism , Neurites/physiology , Receptors, Lysophosphatidic Acid/metabolism , Animals , Calcium/metabolism , Enzyme Activation , Focal Adhesion Kinase 2/antagonists & inhibitors , Focal Adhesion Kinase 2/genetics , Hydrolysis , Lysophospholipids/pharmacology , Mice , Neurites/enzymology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphorylation , Rats , Receptors, Lysophosphatidic Acid/agonists , Tyrosine/metabolism , tau Proteins/metabolismABSTRACT
p116Rip, originally identified as a binding partner of activated RhoA, is an actin-binding protein that interacts with the regulatory myosin-binding subunit (MBS) of myosin-II phosphatase and is essential for Rho-regulated cytoskeletal contractility. Here, we have examined the role of p116Rip in RhoA-mediated activation of the transcription factor SRF. We show that p116Rip oligomerizes via its C-terminal coiled-coil domain and, when overexpressed, inhibits RhoA-induced SRF activation without affecting RhoA-GTP levels. Mutant forms of p116Rip that fail to oligomerize or bind to MBS are still capable of inhibiting SRF activity. Our results suggest that p116Rip interferes with RhoA-mediated transcription through its ability to disassemble the actomyosin cytoskeleton downstream of RhoA.
Subject(s)
Microfilament Proteins/physiology , Serum Response Factor/antagonists & inhibitors , rhoA GTP-Binding Protein/antagonists & inhibitors , Amino Acid Sequence , Animals , Cells, Cultured , Humans , Mice , Microfilament Proteins/genetics , Molecular Sequence Data , Mutation , Myosin Light Chains/metabolism , Protein Structure, Tertiary , Serum Response Element/genetics , Serum Response Factor/metabolism , Transcription, Genetic , rhoA GTP-Binding Protein/metabolismABSTRACT
Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.