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.

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.

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.

ZIC5 Drives Melanoma Aggressiveness by PDGFD-Mediated Activation of FAK and STAT3.

Insights into mechanisms of drug resistance could extend the efficacy of cancer therapy. To probe mechanisms in melanoma, we performed siRNA screening of genes that mediate the development of neural crest cells, from which melanocytes are derived. Here, we report the identification of ZIC5 as a mediator of melanoma drug resistance. ZIC5 is a transcriptional suppressor of E-cadherin expressed highly in human melanoma. ZIC5 enhanced melanoma cell proliferation, survival, drug resistance, in vivo growth and metastasis. Microarray analysis revealed that ZIC5 downstream signaling included PDGFD and FAK activation, which contributes to drug resistance by enhancing STAT3 activation. Silencing of ZIC5 or PDGFD enhanced the apoptotic effects of BRAF inhibition and blocked survival of melanoma cells resistant to BRAF inhibitors. Furthermore, inhibition of FAK or STAT3 suppressed expression of ZIC5, which was positively regulated by PDGFD, FAK, and STAT3 in a positive feedback loop. Taken together, our results identify ZIC5 and PDGFD as candidate therapeutic targets to overcome drug resistance in melanoma. Cancer Res; 77(2); 366-77. ©2016 AACR.

Phospholipase C δ1 in macrophages negatively regulates TLR4-induced proinflammatory cytokine production and Fcγ receptor-mediated phagocytosis.

Macrophages are key players in the innate immune response. Turnover of phosphoinositides (PI), particularly phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2), has been implicated in macrophage functions such as toll-like receptor (TLR)-mediated cytokine production and phagocytosis. However, PI metabolizing enzymes responsible for macrophage functions are not well defined. The phospholipase C (PLC) family of enzymes is critical in PI(4,5)P2 turnover. In this study, we investigated the role of PLCδ1, a prototype PLC, in macrophages on the expression of inflammation-associated genes and phagocytosis. Lipopolysaccharides (LPS) signal through TLR4 to produce proinflammatory cytokines such as interleukin (IL)-1ß. LPS stimulation of both RAW264.7 murine macrophages and murine bone marrow-derived macrophages resulted in lower PLCδ1 mRNA and protein expression levels, compared to that in the control. Using chemical inhibitor compounds, we demonstrated that the up-regulation of p38 MAPK activity led to down-regulation of PLCδ1 mRNA expression in macrophages. PLCδ1 reduction by RNAi or gene deletion resulted in greater LPS-induced IL-1ß expression than that observed in the control siRNA-treated cells, without increasing TLR4 cell surface expression. PLCδ1 also negatively regulated LPS-induced cell spreading. Analysis of Fcγ receptor-mediated phagocytosis demonstrated an increased phagocytosis index after PLCδ1 knockdown in RAW264.7 cells. Conversely, overexpression of PLCδ1 reduced phagocytosis whereas catalytic inactive PLCδ1 had no effect. Altered levels of PLCδ1 affected the binding of opsonized latex beads with cells, rather than the phagocytic activity. Taken together, the data suggest that PLCδ1 negatively regulates LPS-induced production of IL-1ß and Fcγ receptor-mediated phagocytosis in macrophages.

H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPß binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

JMJD1A is a signal-sensing scaffold that regulates acute chromatin dynamics via SWI/SNF association for thermogenesis.

Histone 3 lysine 9 (H3K9) demethylase JMJD1A regulates ß-adrenergic-induced systemic metabolism and body weight control. Here we show that JMJD1A is phosphorylated at S265 by protein kinase A (PKA), and this is pivotal to activate the ß1-adrenergic receptor gene (Adrb1) and downstream targets including Ucp1 in brown adipocytes (BATs). Phosphorylation of JMJD1A by PKA increases its interaction with the SWI/SNF nucleosome remodelling complex and DNA-bound PPARγ. This complex confers ß-adrenergic-induced rapid JMJD1A recruitment to target sites and facilitates long-range chromatin interactions and target gene activation. This rapid gene induction is dependent on S265 phosphorylation but not on demethylation activity. Our results show that JMJD1A has two important roles in regulating hormone-stimulated chromatin dynamics that modulate thermogenesis in BATs. In one role, JMJD1A is recruited to target sites and functions as a cAMP-responsive scaffold that facilitates long-range chromatin interactions, and in the second role, JMJD1A demethylates H3K9 di-methylation.

Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in CRC predict the ineffectiveness of EGF receptor-targeted therapy. Previous transcriptional microarray analysis suggests the association between phospholipase Cδ1 (PLCδ1) expression and KRAS mutation status in CRC. However, both the roles and the regulatory mechanisms of PLCδ1 in CRC are not known. Here, we found that the expression of PLCδ1, one of the most basal PLCs, is down-regulated in CRC specimens compared with normal colon epithelium by immunohistochemistry. Furthermore, we examined the roles of PLCδ1 in CRC cell lines that harbor an activating KRAS mutation. Ectopic expression of PLCδ1 in CRC cells induced the expression of E-cadherin, whereas knockdown of PLCδ1 repressed the expression of E-cadherin. Moreover, the overexpression of PLCδ1 suppressed the expression of several mesenchymal genes and reduced cell motility, invasiveness, and in vivo tumorigenicity of SW620 CRC cells. We also showed that PLCδ1 expression is repressed by the KRAS/mitogen-activated protein kinase kinase (MEK) pathway. Furthermore, PLCδ1 suppressed the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 through E-cadherin induction in CRC cells, suggesting the presence of a negative regulatory loop between KRAS/MEK/ERK signaling and PLCδ1. These data indicate that PLCδ1 has tumor-suppressive functions in CRC through E-cadherin induction and KRAS/MEK/ERK signal attenuation.

Saracatinib impairs the peritoneal dissemination of diffuse-type gastric carcinoma cells resistant to Met and fibroblast growth factor receptor inhibitors.

Diffuse-type gastric carcinomas (DGC) exhibit more aggressive progression and poorer prognosis than intestinal-type and other gastric carcinomas. To identify potential therapeutic targets, we examined protein tyrosine phosphorylation in a panel of DGC and other gastric cancer cell lines. Protein tyrosine phosphorylation was significantly enhanced or altered in DGC cell lines compared with that in other gastric cancer cell lines. Affinity purification and mass spectrometry analysis of tyrosine-phosphorylated proteins identified Met as a protein that is preferentially expressed and phosphorylated in DGC cell lines. Unexpectedly, Met inhibitors blocked cell growth, Met downstream signaling and peritoneal dissemination in vivo in only a subset of cell lines that exhibited remarkable overexpression of Met. Likewise, only cell lines with overexpression of fibroblast growth factor receptor 2 (FGFR2) or phosphorylation of FRS2 were sensitive to an FGFR2 inhibitor. A Src inhibitor saracatinib impaired growth in cell lines that are insensitive to both Met and FGFR2 inhibitors. Saracatinib also effectively impaired peritoneal dissemination of Met-independent and FGFR2-independent SGC cells. Moreover, DGC cell lines exhibited nearly mutually exclusive susceptibility to Met, FGFR and Src inhibitors. These results suggest that DGC have distinct sensitivities to molecular target drugs and that targeting Src is beneficial in the treatment of DGC insensitive to Met and FGFR inhibition.