Osmotically evoked PLCδ1-dependent translocation of ΔN-TRPV1 channels in rat supraoptic neurons.

Osmoregulation is an essential homeostatic process that requires constant release of vasopressin during sustained increases in plasma osmolality. The magnocellular neurosecretory cells (MNCs) respond to increases in external osmolality through increases in the activity of ΔN-TRPV1 channels, which leads to increased action potential firing and vasopressin release. We show that sustained exposure of acutely isolated rat and mouse MNCs to hypertonic solutions (90 min) causes a reversible translocation of ΔN-TRPV1 channels from internal stores to the plasma membrane that depends on the activation of phospholipase C and on SNARE-dependent exocytosis. ΔN-TRPV1 channel translocation is absent in MNCs isolated from transgenic mice lacking the PLCδ1 isoform, suggesting that PLCδ1 is essential for triggering this process. The translocation of ΔN-TRPV1 channels to the cell surface could contribute to the maintenance of MNC excitability during sustained increases in osmolality. Our data therefore have important implications for the mechanisms underlying mammalian osmoregulation.

Tissue factor-induced fibrinogenesis mediates cancer cell clustering and multiclonal peritoneal metastasis.

Peritoneal metastasis is one of the most frequent causes of death in several types of advanced cancers; however, the underlying molecular mechanisms remain largely unknown. In this study, we exploited multicolor fluorescent lineage tracking to investigate the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor displayed multicolor fluorescence regardless of metastasis sites, indicating that it consists of multiclonal cancer cell populations. Multicolored cancer cell clusters form within the peritoneal cavity and collectively attach to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients induces cancer cell clustering, which is inhibited by anticoagulants. Cancer cell clusters formed in vitro and in vivo are associated with fibrin formation. Furthermore, tissue factor knockout in cancer cells abrogates cell clustering, peritoneal attachment, and peritoneal metastasis. Thus, we propose that cancer cells activate the coagulation cascade via tissue factor to form fibrin-mediated cell clusters and promote peritoneal attachment; these factors lead to the development of multiclonal peritoneal metastasis and may be therapeutic targets.

Downregulation of protein kinase C gamma reduces epithelial property and enhances malignant phenotypes in colorectal cancer cells.

Loss of epithelial integrity is associated with colorectal cancer (CRC) aggressiveness. Protein kinase C (PKC) is frequently implicated in human cancers, but the role of PKCγ in CRC remains poorly understood. Here, we show that PKCγ, a conventional PKC, is expressed in normal colonic epithelium, but this is lower in dedifferentiated CRC. PKCγ expression was downregulated by SNAI1 overexpression, and low PKCγ expression was associated with poor prognosis in patients with CRC. Transient or stable knockdown of PKCγ reduced E-cadherin expression in CRC cells. PKCγ knockdown enhanced proliferation, anchorage-independent cell growth, resistance to anti-cancer drugs, and in vivo tumor growth of DLD-1 cells. We have also identified phosphorylation substrates for PKCγ. Among them, ARHGEF18, a RhoA activator that stabilizes cell-cell junctions, was phosphorylated and stabilized by PKCγ. Thus, these results suggest that the downregulation of PKCγ decreases the epithelial property of CRC cells and enhances its malignant phenotypes.

Patulin and LL-Z1640-2 induce apoptosis of cancer cells by decreasing endogenous protein levels of Zic family member 5.

Zic family member 5 (ZIC5) is a transcription factor that promotes the survival of several cancer cell types. As ZIC5 is expressed at minimal levels in normal human adult tissues, it is a potential therapeutic target. In this study, we screened a chemical library containing 3398 compounds that includes pre-existing drugs and compounds with known effects to identify ZIC5 inhibitors. In the first screening, 18 hit compounds decreased GFP intensity in melanoma A375 cells overexpressing GFP-tagged ZIC5. In the second screening, five compounds that attenuated ZIC5 protein levels in A375 cells were identified. Among them, LL-Z1640-2 and patulin selectively induced apoptosis in melanoma cells expressing ZIC5, while only inducing very low levels of apoptosis in normal human melanocytes, which have no detectable ZIC5 expression. LL-Z1640-2 and patulin also induced apoptosis in BRAF inhibitor-resistant melanoma, pancreatic cancer, cholangiocarcinoma and colorectal cancer cells. LL-Z1640-2- and patulin-mediated suppression of melanoma proliferation were rescued by ZIC5 overexpression. These results suggest that LL-Z1640-2 and patulin are promising compounds that decrease ZIC5 expression to induce apoptosis in cancer cells.

Zic family member 5 promotes survival in human pancreatic cancer and cholangiocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) are malignant tumors with poor prognosis because of the limited effectiveness of traditional chemotherapy and few effective molecular therapeutic agents. Here, we determined the essential roles of Zic family member 5 (ZIC5) in the survival of PDAC and CCA cells. The results showed that ZIC5 is strongly expressed in PDAC and CCA tissues, while ZIC5 expression is barely observed in most normal human adult tissues. Furthermore, ZIC5 expression is related to poor prognosis of patients with PDAC. ZIC5 knockdown via small interfering RNA decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3), a protein that is associated with PDAC and CCA aggressiveness. However, ZIC5 knockdown induced cell death regardless of STAT3 activation, which is promoted by interleukin (IL) -6, a factor associated with inflammation. Furthermore, knockdown of ZIC5 in PDAC and CCA cells additively or synergistically induced apoptosis with the anti-cancer drug gemcitabine. Thus, ZIC5 constitutes a potential therapeutic target for the treatment of PDAC and CCA.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics.

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

Homeostatic membrane tension constrains cancer cell dissemination by counteracting BAR protein assembly.

Malignancy is associated with changes in cell mechanics that contribute to extensive cell deformation required for metastatic dissemination. We hypothesized that the cell-intrinsic physical factors that maintain epithelial cell mechanics could function as tumor suppressors. Here we show, using optical tweezers, genetic interference, mechanical perturbations, and in vivo studies, that epithelial cells maintain higher plasma membrane (PM) tension than their metastatic counterparts and that high PM tension potently inhibits cancer cell migration and invasion by counteracting membrane curvature sensing/generating BAR family proteins. This tensional homeostasis is achieved by membrane-to-cortex attachment (MCA) regulated by ERM proteins, whose disruption spontaneously transforms epithelial cells into a mesenchymal migratory phenotype powered by BAR proteins. Consistently, the forced expression of epithelial-mesenchymal transition (EMT)-inducing transcription factors results in decreased PM tension. In metastatic cells, increasing PM tension by manipulating MCA is sufficient to suppress both mesenchymal and amoeboid 3D migration, tumor invasion, and metastasis by compromising membrane-mediated mechanosignaling by BAR proteins, thereby uncovering a previously undescribed mechanical tumor suppressor mechanism.

Ubiquitination-dependent and -independent repression of target genes by SETDB1 reveal a context-dependent role for its methyltransferase activity during adipogenesis.

The lysine methyltransferase SETDB1, an enzyme responsible for methylation of histone H3 at lysine 9, plays a key role in H3K9 tri-methylation-dependent silencing of endogenous retroviruses and developmental genes. Recent studies have shown that ubiquitination of human SETDB1 complements its catalytic activity and the silencing of endogenous retroviruses in human embryonic stem cells. However, it is not known whether SETDB1 ubiquitination is essential for its other major role in epigenetic silencing of developmental gene programs. We previously showed that SETDB1 contributes to the formation of H3K4/H3K9me3 bivalent chromatin domains that keep adipogenic Cebpa and Pparg genes in a poised state for activation and restricts the differentiation potential of pre-adipocytes. Here, we show that ubiquitin-resistant K885A mutant of SETDB1 represses adipogenic genes and inhibits pre-adipocyte differentiation similar to wild-type SETDB1. We show this was due to a compensation mechanism for H3K9me3 chromatin modifications on the Cebpa locus by other H3K9 methyltransferases Suv39H1 and Suv39H2. In contrast, the K885A mutant did not repress other SETDB1 target genes such as Tril and Gas6 suggesting SETDB1 represses its target genes by two mechanisms; one that requires its ubiquitination and another that still requires SETDB1 but not its enzyme activity.

PLCδ1 Plays Central Roles in the Osmotic Activation of ΔN-TRPV1 Channels in Mouse Supraoptic Neurons and in Murine Osmoregulation.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENT Magnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+ influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.

PLEKHA5 regulates the survival and peritoneal dissemination of diffuse-type gastric carcinoma cells with Met gene amplification.

Met gene amplification has been found in a subset of malignant carcinomas, including diffuse-type gastric carcinoma (DGC), which has a poor prognosis owing to rapid infiltrative invasion and frequent peritoneal dissemination. Met is considered a promising therapeutic target for DGC. However, DGC cells with Met gene amplification eventually acquire resistance to Met inhibitors. Therefore, identification of alternate targets that mediate Met signaling and confer malignant phenotypes is critical. In this study, we conducted a phosphoproteomic analysis of DGC cells possessing Met gene amplification and identified Pleckstrin Homology Domain Containing A5 (PLEKHA5) as a protein that is tyrosine-phosphorylated downstream of Met. Knockdown of PLEKHA5 selectively suppressed the growth of DGC cells with Met gene amplification by inducing apoptosis, even though they had acquired resistance to Met inhibitors. Moreover, PLEKHA5 silencing abrogated the malignant phenotypes of Met-addicted DGC cells, including peritoneal dissemination in vivo. Mechanistically, PLEKHA5 knockdown dysregulates glycolytic metabolism, leading to activation of the JNK pathway that promotes apoptosis. These results indicate that PLEKHA5 is a novel downstream effector of amplified Met and is required for the malignant progression of Met-addicted DGC.

Phosphatidylinositol-specific phospholipase C enhances epidermal penetration by Staphylococcus aureus.

Staphylococcus aureus (S. aureus) commonly colonizes the human skin and nostrils. However, it is also associated with a wide variety of diseases. S. aureus is frequently isolated from the skin of patients with atopic dermatitis (AD), and is linked to increased disease severity. S. aureus impairs the skin barrier and triggers inflammation through the secretion of various virulence factors. S. aureus secretes phosphatidylinositol-specific phospholipase C (PI-PLC), which hydrolyses phosphatidylinositol and cleaves glycosylphosphatidylinositol-anchored proteins. However, the role of S. aureus PI-PLC in the pathogenesis of skin diseases, including AD, remains unclear. In this study, we sought to determine the role of S. aureus PI-PLC in the pathogenesis of skin diseases. PI-PLC was observed to enhance the invasion and persistence of S. aureus in keratinocytes. Besides, PI-PLC promoted the penetration of S. aureus through the epidermal barrier in a mouse model of AD and the human organotypic epidermal equivalent. Furthermore, the loss of PI-PLC attenuated epidermal hyperplasia and the infiltration of Gr-1+ cells and CD4+ cells induced by S. aureus infection in the mouse model of AD. Collectively, these results indicate that PI-PLC eases the entry of S. aureus into the dermis and aggravates acanthosis and immune cell infiltration in infected skin.

Phosphatidylinositol 4,5-bisphosphate is localized in the plasma membrane outer leaflet and regulates cell adhesion and motility.

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

Phospholipase Cγ1 is required for normal irritant contact dermatitis responses and sebaceous gland homeostasis.

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte-specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.

Epidermal loss of phospholipase Cδ1 attenuates irritant contact dermatitis.

Irritant contact dermatitis (ICD) is one of the most common inflammatory skin diseases caused by exposure to chemical irritants. Since chemical irritants primarily damage keratinocytes, these cells play a pivotal role in ICD. One of the phosphoinositide-metabolizing enzymes, phospholipase C (PLC) δ1, is abundantly expressed in keratinocytes. However, the role of PLCδ1 in ICD remains to be clarified. Here, we found that croton oil (CrO)-induced ear swelling, a feature of ICD, was attenuated in keratinocyte-specific PLCδ1 knockout mice (PLCδ1 cKO mice). Dendritic epidermal T cells (DETCs), which have a protective role against ICD, were activated in the epidermis of the PLCδ1 cKO mice. In addition, the skin of CrO-treated PLCδ1 cKO mice showed increased infiltration of Gr1+CD11b+ myeloid cells. Of note, elimination of Gr1+CD11b+ myeloid cells restored CrO-induced ear swelling in PLCδ1 cKO mice to a similar level as that in control mice. Taken together, our results strongly suggest that epidermal loss of PLCδ1 protects mice from ICD through induction of Gr1+CD11b+ myeloid cells and activation of DETCs.

Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

The BRAF inhibitor PLX4032 is effective in treating BRAF-mutated melanoma; however, because drug resistance develops in most cases, it is critical to develop a new strategy for inhibiting drug-resistant melanoma growth. The melanoma-associated membrane glycoprotein CD63 is involved in cell proliferation and metastasis. Here, we found that cell surface CD63 suppresses the proliferation of human melanoma cells and PLX4032-resistant cells. Endogenous CD63 protein levels were negatively correlated with PLX4032 resistance of human melanoma cell lines. CD63 overexpression in these cells, in which endogenous CD63 levels are low, suppressed cell proliferation under PLX4032 treatment. The cell surface levels and average molecular mass of CD63 were increased with PLX4032 treatment because of the up-regulated polylactosamine modification caused by induced ß1,3- N-acetylglucosaminyltransferase 2 expression, which is involved in polylactosamine synthesis. Forced cell surface localization of CD63 led to reduced melanoma cell proliferation without PLX4032 treatment. CD63 overexpression in PLX4032-resistant cells, in which CD63 levels were lower and cell surface polylactosamine levels were higher than those in parental cells, effectively suppressed proliferation. Our study shows the potential of CD63 to sensitize melanoma cells to PLX4032 and to reduce the proliferation of PLX4032-resistant cells.-Kudo, K., Yoneda, A., Sakiyama, D., Kojima, K., Miyaji, T., Yamazaki, M., Yaita, S., Hyodo, T., Satow, R., Fukami, K. Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells.

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.

Identification of zinc finger protein of the cerebellum 5 as a survival factor of prostate and colorectal cancer cells.

Identification of specific drug targets is very important for cancer therapy. We recently identified zinc finger protein of the cerebellum 5 (ZIC5) as a factor that promotes melanoma aggressiveness by platelet-derived growth factor D (PDGFD) expression. However, its roles in other cancer types remain largely unknown. Here we determined the roles of ZIC5 in prostate cancer (PCa) and colorectal cancer (CRC) cells. Results showed that ZIC5 was highly expressed in CRC and dedifferentiated PCa tissues, whereas little expression was observed in relevant normal tissues. Knockdown of ZIC5 decreased proliferation of several PCa and CRC cell lines with induction of cell death. ZIC5 knockdown significantly suppressed PDGFD expression transcriptionally, and PDGFD suppression also decreased proliferation of PCa and CRC cell lines. In addition, suppression of ZIC5 or PDGFD expression decreased levels of phosphorylated focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) which are associated with PCa and CRC aggressiveness. Furthermore, knockdown of ZIC5 or PDGFD enhanced death of PCa and CRC cells induced by the anti-cancer drugs docetaxel or oxaliplatin, respectively. These results suggest that ZIC5 and PDGFD promote survival of PCa and CRC cells by enhancing FAK and STAT3 activity, and that the roles of ZIC5 are consistent across several cancer types.

Group III phospholipase A2 promotes colitis and colorectal cancer.

Lipid mediators play pivotal roles in colorectal cancer and colitis, but only a limited member of the phospholipase A2 (PLA2) subtypes, which lie upstream of various lipid mediators, have been implicated in the positive or negative regulation of these diseases. Clinical and biochemical evidence suggests that secreted PLA2 group III (sPLA2-III) is associated with colorectal cancer, although its precise role remains obscure. Here we have found that sPLA2-III-null (Pla2g3 -/-) mice are highly resistant to colon carcinogenesis. Furthermore, Pla2g3 -/- mice are less susceptible to dextran sulfate-induced colitis, implying that the amelioration of colonic inflammation by sPLA2-III ablation may underlie the protective effect against colon cancer. Lipidomics analysis of the colon revealed significant reduction of pro-inflammatory/pro-tumorigenic lysophosholipids as well as unusual steady-state elevation of colon-protective fatty acids and their oxygenated metabolites in Pla2g3 -/- mice. Overall, our results establish a role of sPLA2-III in the promotion of colorectal inflammation and cancer, expand our understanding of the divergent roles of multiple PLA2 enzymes in the gastrointestinal tract, and point to sPLA2-III as a novel druggable target for colorectal diseases.

Actinin-1 and actinin-4 play essential but distinct roles in invadopodia formation by carcinoma cells.

Invadopodia are ventral membrane protrusions formed by cancer cells that degrade the extracellular matrix (ECM) during tumor invasion and metastasis. Formation of invadopodia is initiated by the assembly of actin filaments (F-actin) that results from the coordinated activation of several actin regulatory proteins. Actinin-1 and actinin-4 are actin bundling proteins expressed in non-muscle cells and actinin-4 is preferentially associated with malignant phenotypes of carcinoma cells. In this study, we investigated the role of actinin-1 and -4 in invadopodia formation. Expression of both actinin-1 and -4 tended to be higher in invasive and metastatic breast carcinoma cell lines than in non-invasive ones. Immunofluorescence analysis revealed that actinin-1 and -4 colocalized at core actin structures of invadopodia. Time-lapse imaging showed that appearance of both actinins at invadopodia is concomitant with the assembly of F-actin. Knockdown of either actinin-1 or actinin-4 suppressed the formation of invadopodia and degradation of the ECM by carcinoma cells. Interestingly, overexpression of actinin-4, but not actinin-1, significantly promoted the formation of invadopodia and this activity required the actin binding domains and the unique N-terminal motif that exists only in actinin-4. These results demonstrate that both actinin-1 and actinin-4 participate in the assembly of F-actin at invadopodia. Additionally, actinin-4 may have a selective advantage in accelerating invadopodia-mediated invasion of carcinoma cells.

Phospholipase C δ1 negatively regulates autophagy in colorectal cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Kirsten rat sarcoma viral oncogene homolog (KRAS) is frequently mutated in CRC, and KRAS mutations promote cell motility, growth, and survival. We previously revealed that the expression of phospholipase C (PLC) δ1, one of the most basal PLCs, is down-regulated in colon adenocarcinoma, and that the KRAS signaling pathway suppresses PLCδ1 expression. Although recent studies revealed that KRAS mutations activate autophagy in cancer cells, a relation between PLCδ1 and autophagy remains unclear. Here, we found that PLCδ1 overexpression suppresses the formation of autophagosomes, which are key structures of autophagy, whereas endogenous PLCδ1 knockdown increases autophagosome formation in CRC cells. We also showed that PLCδ1 overexpression promotes cell death under nutrient deprivation. Furthermore, PLCδ1 overexpression suppresses the autophagy induced by the anti-cancer drug oxaliplatin and promotes cell death under oxaliplatin treatment. These data suggest that PLCδ1 negatively regulates autophagy, and PLCδ1 suppression contributes to the tolerance of CRC cells harboring KRAS mutations to nutrient deprivation and anti-cancer drug treatment.