C9orf10/Ossa regulates the bone metastasis of established lung adenocarcinoma cell subline H322L-BO4 in a mouse model.

Lung cancer frequently metastasizes to the bones. An in vivo model is urgently required to identify potential therapeutic targets for the prevention and treatment of lung cancer with bone metastasis. We established a lung adenocarcinoma cell subline (H322L-BO4) that specifically showed metastasis to the leg bones and adrenal glands. This was achieved by repeated isolation of metastatic cells from the leg bones of mice. The cells were intracardially injected into nude mice. Survival was prolonged for mice that received H322L-BO4 cells versus original cells (H322L). H322L-BO4 cells did not exhibit obvious changes in general in vitro properties associated with the metastatic potential (e.g., cell growth, migration, and invasion) compared with H322L cells. However, the phosphorylation of chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa) was increased in H322L-BO4 cells. This result confirmed the increased anchorage independence through C9orf10/Ossa-mediated activation of Src family tyrosine kinase. Reduction of C9orf10/Ossa by shRNA reduced cells' metastasis to the leg bone and prolonged survival in mice. These findings indicate that H322L-BO4 cells can be used to evaluate the effect of candidate therapeutic targets against bone metastatic lung cancer cells. Moreover, C9orf10/Ossa may be a useful target for treatment of lung cancer with bone metastasis.

Ruthenium Pyrazole Complexes: A Family of Highly Active Metallodrugs for Photoactivated Chemotherapy.

Ruthenium complexes bearing bis pyrazole (pzH) ligands, cis-[Ru(bpy)2(R-pzH)2]2+ (bpy = 2,2'-bipyridine, R = -H, -Cl), were examined as photoactivated anticancer prodrugs. A dicationic pyrazole complex deprotonated to give monocationic pyrazole-pyrazolate complexes, cis-[Ru(bpy)2(R-pz-)(R-pzH)]+, in an aqueous solution with pKa values of 9.5 and 7.2 for R = H and R = Cl, respectively. Upon deprotonation, relative quantum yields of photosubstitution decreased while lipophilicity of the complexes increased according to the measurements of water-octanol coefficients. The ruthenium complex with 4-chloropyrazole ligands displayed high cytotoxicity upon light irradiation (IC50 = 0.060 ± 0.016 µM) toward lung cancer cells, which was 7 times higher than that in the dark (IC50 = 0.44 ± 0.07 µM). Additional experiments for the ruthenium R-pyrazole complexes indicated that (1) selective photodissociation of the 4-chloropyrazole ligand occurs from cis-[Ru(bpy)2(4-Clpz-)(4-ClpzH)]+, (2) photoinduced ligand dissociation is dominant rather than photoinduced generation of singlet oxygen (1O2), and (3) induction of cell death occurs via the intrinsic pathway of apoptosis.

An ionic liquid-assisted sample preparation method for sensitive integral-membrane proteome analysis.

Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.

Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies.

Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination.

An Ionic Liquid-Based Sample Preparation Method for Next-Stage Aggregate Proteomic Analysis.

A wide variety of proteomic methods have been applied for protein profiling of insoluble aggregates or inclusion bodies deposited in various cells or tissues. However, these are essentially optimized or modified classical protein chemistry techniques using conventional denaturing agents such as formic acid, urea, and sodium dodecyl sulfate (SDS). The use of these denaturants has several shortcomings, including limited solubilization, contamination, and restrictions on absolute sample quantity and throughput. Here, we describe an alternative proteomic sample preparation platform for widespread aggregation analysis. This approach combines two techniques, (1) the use of ionic liquid for protein solubilization and (2) the recently published microbead-based and organic-media-assisted proteolysis strategy (BOPs), into a single-tube workflow. We demonstrate that the combined approach (iBOPs) enabled the successful solubilization of heat-aggregated hen egg whites within 10 min and supported sensitive mass spectrometry (MS) analysis. The performance of the iBOPs system surpassed those of conventional detergents and chaotropes. Moreover, this technology enabled ultrasensitive proteomic characterization of protein aggregates deposited in individual Caenorhabditis elegans nematodes. We identified ubiquitin and other molecules as candidate stochastic factors whose accumulation levels varied among aging nematode individuals. The sensitivity and applicability of the present iBOPs make it especially attractive for next-stage aggregate proteomic analysis of various biological processes.

Homophilic complex formation of CDCP1 via the extracellular CUB2 domain facilitates SFK activation and promotes cancer cell migration.

CUB domain­containing protein 1 (CDCP1) is phosphorylated by Src family kinases (SFK), and is thought to serve an important role in tumor metastasis through downstream signaling subsequent to its interaction with protein kinase C δ. The present study investigated the mechanisms of activation for CDCP1 signaling, and demonstrated that CDCP1 is able to activate SFK via a homophilic complex of the extracellular complement C1r/C1s, urchin embryonic growth factor, bone morphogenetic protein 1 (CUB) 2 domain. Deletion of the extracellular CDCP1 region abolished homophilic complex formation of CDCP1 and the ability to promote cancer cell migration. When the culture medium was supplemented with recombinant CUB2 domain protein fused with maltose binding protein (rMBP­CUB2), CDCP1 homophilic complex formation was effectively inhibited. rMBP­CUB2 also inhibited SFK activation and the migratory capacity of invasive human lung adenocarcinoma A549 cells, and human pancreatic BxPC3 cells. These findings demonstrated a novel function for the extracellular CUB2 domain of CDCP1, promoting cancer cell migration via SFK activation on the plasma membrane. It was also indicated that the region blocking the homophilic binding site may be a potential therapeutic target against CDCP1­dependent tumor invasion.

A Sensitive Microbead-Based Organic Media-Assisted Method for Proteomics Sample Preparation from Dilute and Denaturing Solutions.

We developed a robust and sensitive sample preparation method for proteomics termed microbead-based and organic-media-assisted proteolysis strategy (BOPs). BOPs combines two advantages of current techniques, (1) unbiased binding of reversed-phase polymeric microbeads to any type of protein and (2) enhanced trypsin digestion efficiency in CH3CN-aqueous solvent systems, into a single-tube workflow. Compared with conventional techniques, this method effectively concentrates proteins and improves proteolytic digestion, and can be used with submicromolar protein samples in dilute or denaturing solutions, such as 70% formic acid, 8 M urea, or 7 M guanidine hydrochloride without any sample pretreatment. Proteome analysis of single Caenorhabditis elegans organisms demonstrates that BOPs has the sensitivity, reproducibility, and unbiasedness required to characterize worm proteins at a single organism level. We also show that, by simply incorporating an acetone washing step for detergent removal, BOPs is applicable to low concentration samples contaminated with a variety of detergents, including sodium dodecyl sulfate, with negligible protein loss. Moreover, the utility of this modification has also been demonstrated through proteomic characterization of 2000 human (HEK293T) cells lysed using 1% Triton X-100. The simplicity and availability of the present BOPs make it especially attractive for next-stage proteomics of rare and sample-limited systems.

Novel small molecule inhibiting CDCP1-PKCδ pathway reduces tumor metastasis and proliferation.

CUB domain-containing protein-1 (CDCP1) is a trans-membrane protein predominantly expressed in various cancer cells and involved in tumor progression. CDCP1 is phosphorylated at tyrosine residues in the intracellular domain by Src family kinases and recruits PKCδ to the plasma membrane through tyrosine phosphorylation-dependent association with the C2 domain of PKCδ, which in turn induces a survival signal in an anchorage-independent condition. In this study, we used our cell-free screening system to identify a small compound, glycoconjugated palladium complex (Pd-Oqn), which significantly inhibited the interaction between the C2 domain of PKCδ and phosphorylated CDCP1. Immunoprecipitation assays demonstrated that Pd-Oqn hindered the intercellular interaction of phosphorylated CDCP1 with PKCδ and also suppressed the phosphorylation of PKCδ but not that of ERK or AKT. In addition, Pd-Oqn inhibited the colony formation of gastric adenocarcinoma 44As3 cells in soft agar as well as their invasion. In mouse models, Pd-Oqn markedly reduced the peritoneal dissemination of gastric adenocarcinoma cells and the tumor growth of pancreatic cancer orthotopic xenografts. These results suggest that the novel compound Pd-Oqn reduces tumor metastasis and growth by inhibiting the association between CDCP1 and PKCδ, thus potentially representing a promising candidate among therapeutic reagents targeting protein-protein interaction.

TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells.

The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions.

Augmentation of invadopodia formation in temozolomide-resistant or adopted glioma is regulated by c-Jun terminal kinase-paxillin axis.

Temozolomide (TMZ) is one of the few effective anticancer agents against gliomas. However, acquisition of TMZ resistance or adaptation by gliomas is currently a crucial problem, especially increased invasiveness which is critical for the determination of clinical prognosis. This study investigated the molecular regulatory mechanisms of TMZ resistance in gliomas involved in invasiveness, particularly invadopodia formation, a molecular complex formed at the invasive front to cause extracellular matrix degradation during cellular local invasion. The TMZ-resistant clone of the U343 MG human glioma cell line (U343-R cells) was established. U343-R cells demonstrated higher invadopodia formation compared with U343 cells without TMZ resistance (U343-Con cells). Immunoblot analysis of DNA damage-related mitogen-activated protein kinase signals found increased phosphorylation of c-Jun terminal kinase (JNK) and higher activation of its downstream signaling in U343-R cells compared with U343-Con cells. Treatment of U343-R cells with specific inhibitors of JNK or siRNA targeting JNK suppressed up-regulation of invadopodia formation. In addition, paxillin, one of the known JNK effectors which is phosphorylated and affects cell migration, was phosphorylated at serine 178 in JNK activity-dependent manner. Expression of paxillin with mutation of the serine 178 phosphorylation site in U343-R cells blocked invadopodia formation. The present findings suggest that increased formation of invadopodia in U343-R cells is mediated by hyperactivation of JNK-paxillin signaling, and both JNK and paxillin might become targets of novel therapies against TMZ-resistant gliomas.

Oncogenic Ras/ERK signaling activates CDCP1 to promote tumor invasion and metastasis.

UNLABELLED: Involvement of Ras in cancer initiation is known, but recent evidence indicates a role in cancer progression, including metastasis and invasion; however, the mechanism is still unknown. In this study, it was determined that human lung cancer cells with Ras mutations, among other popular mutations, showed significantly higher expression of CUB domain-containing protein 1 (CDCP1) than those without. Furthermore, activated Ras clearly induced CDCP1, whereas CDCP1 knockdown or inhibition of CDCP1 phosphorylation by Src-directed therapy abrogated anoikis resistance, migration, and invasion induced by activated-Ras. Activation of MMP2 and secretion of MMP9, in a model of Ras-induced invasion, was found to be regulated through induction of phosphorylated CDCP1. Thus, CDCP1 is required for the functional link between Ras and Src signaling during the multistage development of human malignant tumors, highlighting CDCP1 as a potent target for treatment in the broad spectrum of human cancers associated with these oncogenes. IMPLICATIONS: CDCP1 protein induced by oncogenic Ras/Erk signaling is essential for Ras-mediated metastatic potential of cancer cells.

Flotillin-1 regulates oncogenic signaling in neuroblastoma cells by regulating ALK membrane association.

Neuroblastomas harbor mutations in the nonreceptor anaplastic lymphoma kinase (ALK) in 8% to 9% of cases where they serve as oncogenic drivers. Strategies to reduce ALK activity offer clinical interest based on initial findings with ALK kinase inhibitors. In this study, we characterized phosphotyrosine-containing proteins associated with ALK to gain mechanistic insights in this setting. Flotillin-1 (FLOT1), a plasma membrane protein involved in endocytosis, was identified as a binding partner of ALK. RNAi-mediated attenuation of FLOT1 expression in neuroblastoma cells caused ALK dissociation from endosomes along with membrane accumulation of ALK, thereby triggering activation of ALK and downstream effector signals. These features enhanced the malignant properties of neuroblastoma cells in vitro and in vivo. Conversely, oncogenic ALK mutants showed less binding affinity to FLOT1 than wild-type ALK. Clinically, lower expression levels of FLOT1 were documented in highly malignant subgroups of human neuroblastoma specimens. Taken together, our findings suggest that attenuation of FLOT1-ALK binding drives malignant phenotypes of neuroblastoma by activating ALK signaling.

Suppression of autophagy by CUB domain-containing protein 1 signaling is essential for anchorage-independent survival of lung cancer cells.

CUB (C1r/C1s, urchin embryonic growth factor, BMP1) domain-containing protein 1 (CDCP1) has been implicated in promoting metastasis of cancer cells through several mechanisms, including the inhibition of anoikis, which is cell death triggered by the loss of extracellular matrix interactions. However, the mechanism inhibiting cell death regulated by CDCP1 remains elusive. Inhibition of CDCP1 expression using small interfering RNA (siRNA) induced the cell death of suspended cancer cells without cleaving caspase-3, a marker of apoptosis; cell death was not inhibited by a general caspase inhibitor, suggesting that the loss of CDCP1 induces caspase-independent cell death. In contrast, knockdown of CDCP1 as well as protein kinase Cδ (PKCδ), a downstream effector of CDCP1, in a suspension culture of lung cancer cells resulted in marked induction of membranous microtubule-associated protein 1 light chain 3 (LC3)-II protein, a hallmark of autophagy, and caused the formation of an autophagosome structure visualized using green fluorescent protein-tagged LC3-II. Expression and phosphorylation of exogenous CDCP1 by Fyn kinase reduced the formation of autophagosomes and inhibited phosphorylation of CDCP1 by PP2, a Src kinase inhibitor or inhibited PKCδ by rottlerin, stimulating autophagosome formation. Moreover, death of suspended lung cancer cells induced by CDCP1 siRNA or by PKCδ siRNA was reduced by the autophagy inhibitor 3-methyladenine. These results indicate that CDCP1-PKCδ signaling plays a critical role in inhibiting autophagy, which is responsible for anoikis resistance of lung cancer cells.

CDCP1 regulates the function of MT1-MMP and invadopodia-mediated invasion of cancer cells.

Complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing protein 1 (CDCP1) is a transmembrane protein that regulates anchorage-independent growth and cancer cell migration and invasion. Expression of CDCP1 is detected in a number of cancer cell lines and tissues and is closely correlated with poor prognosis. Invadopodia are actin-based protrusions on the surface of invasive cancer cells that promote the degradation of the extracellular matrix (ECM) via localized proteolysis, which is mainly mediated by membrane type 1 matrix metalloproteinase (MT1-MMP). MT1-MMP is accumulated at invadopodia by targeted delivery via membrane trafficking. The present study shows that CDCP1 is required for ECM degradation by invadopodia in human breast cancer and melanoma cells. CDCP1 localized to caveolin-1-containing vesicular structures and lipid rafts and was detected in close proximity to invadopodia. Further biochemical analysis revealed that substantial amounts of CDCP1 existed in the Triton X-100 insoluble lipid raft fraction. CDCP1 was coimmunoprecipitated with MT1-MMP and colocalized with MT1-MMP at the vesicular structures. The siRNA-mediated knockdown of the CDCP1 expression markedly inhibited MT1-MMP-dependent ECM degradation and Matrigel invasion and reduced the accumulation of MT1-MMP at invadopodia, as shown by immunofluorescence analysis. These results indicate that CDCP1 is an essential regulator of the trafficking and function of MT1-MMP- and invadopodia-mediated invasion of cancer cells.

Roles of CUB domain-containing protein 1 signaling in cancer invasion and metastasis.

Tumor metastasis is a complex multistep process by which cells from the primary tumor invade tissues, move through the vasculature, settle at distant sites and eventually grow to form secondary tumors. Altered tyrosine phosphorylation signals in cancer cells contribute to a number of aberrant characteristics involved in tumor invasion and metastasis. CUB domain-containing protein 1 (CDCP1) is a substrate of Src family kinases and has been shown to regulate anoikis resistance, migration and matrix degradation during tumor invasion and metastasis in a tyrosine phosphorylation-dependent manner. Knockdown of CDCP1 blocks tumor metastasis or peritoneal dissemination in vivo, without significantly affecting cell proliferation. Moreover, expression levels of CDCP1 are of prognostic value in several cancers. Here, we summarize the studies on CDCP1, focusing on structure and signal transduction, to gain insight into its role in cancer progression. Understanding the signaling pathways regulated by CDCP1 could help establish novel therapeutic strategies against the progression of cancer.

CUB domain-containing protein 1, a prognostic factor for human pancreatic cancers, promotes cell migration and extracellular matrix degradation.

CUB domain-containing protein 1 (CDCP1) is a membrane protein that is highly expressed in several solid cancers. We reported previously that CDCP1 regulates anoikis resistance as well as cancer cell migration and invasion, although the underlying mechanisms have not been elucidated. In this study, we found that expression of CDCP1 in pancreatic cancer tissue was significantly correlated with overall survival and that CDCP1 expression in pancreatic cancer cell lines was relatively high among solid tumor cell lines. Reduction of CDCP1 expression in these cells suppressed extracellular matrix (ECM) degradation by inhibiting matrix metalloproteinase-9 secretion. Using the Y734F mutant of CDCP1, which lacks the tyrosine phosphorylation site, we showed that CDCP1 regulates cell migration, invasion, and ECM degradation in a tyrosine phosphorylation-dependent manner and that these CDCP1-associated characteristics were inhibited by blocking the association of CDCP1 and protein kinase Cdelta (PKCdelta). CDCP1 modulates the enzymatic activity of PKCdelta through the tyrosine phosphorylation of PKCdelta by recruiting PKCdelta to Src family kinases. Cortactin, which was detected as a CDCP1-dependent binding partner of PKCdelta, played a significant role in migration and invasion but not in ECM degradation of pancreatic cells. These results suggest that CDCP1 expression might play a crucial role in poor outcome of pancreatic cancer through promotion of invasion and metastasis and that molecules blocking the expression, phosphorylation, or the PKCdelta-binding site of CDCP1 are potential therapeutic candidates.

Expression of CUB domain containing protein (CDCP1) is correlated with prognosis and survival of patients with adenocarcinoma of lung.

CUB domain containing protein (CDCP1), a transmembrane protein with intracellular tyrosine residues which are phosphorylated upon activation, is supposed to be engaged in proliferative activities and resistance to apoptosis of cancer cells. Expression level of CDCP1 was examined in lung adenocarcinoma, and its clinical implications were evaluated. CDCP1 expression was immunohistochemically examined in lung adenocarcinoma from 200 patients. Staining intensity of cancer cells was categorized as low and high in cases with tumor cells showing no or weak and strong membrane staining, respectively. MIB-1 labeling index was also examined. There were 113 males and 87 females with median age of 63 years. Stage of disease was stage I in 144 cases (72.0%), II in 19 (9.5%), and III in 37 (18.5%). Sixty of 200 cases (30.0%) were categorized as CDCP1-high, and the remaining as CDCP1-low. Significant positive correlation was observed between CDCP1-high expression and relapse rate (P < 0.0001), poor prognosis (P < 0.0001), MIB-1 labeling index (P < 0.0001), and occurrence of lymph node metastasis (P = 0.0086). There was a statistically significant difference in disease-free survival (DFS) (P < 0.0001) and overall survival (OS) rates (P < 0.0001) between patients with CDCP1-high and CDCP1-low tumors. Univariate analysis showed that lymph node status, tumor stage, and CDCP1 expression were significant factors for both OS and DFS. Multivariate analysis revealed that only CDCP1 expression was an independent prognostic factor for both OS and DFS. CDCP1 expression level is a useful marker for prediction of patients with lung adenocarcinoma

CUB-domain-containing protein 1 regulates peritoneal dissemination of gastric scirrhous carcinoma.

CUB-domain-containing protein 1 (CDCP1) is a type-I transmembrane protein that is highly expressed in colon, breast, and lung cancers. We recently revealed that CDCP1 is associated with and phosphorylated by Src family kinases and is involved in the regulation of anchorage independence of certain lung cancer cell lines. In this study, we examined whether CDCP1 is involved in the regulation of tumor progression of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Expression and phosphorylation levels of CDCP1 correlated with the invasive potential of scirrhous gastric cancers. Reduction of CDCP1 expression by siRNA suppressed migration, invasion, and anchorage independence without affecting the proliferation of highly invasive scirrhous gastric cancer cells. However, CDCP1 overexpression promoted gastric cancer cell migration with low potential of invasion. Loss of CDCP1 suppressed invasion and dissemination of cancer cells that were orthotopically implanted in the gastric wall of nude mice. Expression and phosphorylation of CDCP1 were also detected in cancer cells of surgically resected tissues of human scirrhous gastric cancer by immunohistochemical analysis. Our results suggest that CDCP1 promotes invasion and peritoneal dissemination of cancer cells through the regulation of cell migration and anchorage independence. Therefore, it is both a potential prognostic and therapeutic target in certain types of gastrointestinal cancers, and suppression of its phosphorylation might be a useful strategy for modulating cancer metastasis.

Hyperphosphorylated cortactin in cancer cells plays an inhibitory role in cell motility.

Cortactin is frequently overexpressed in cancer cells, and changes of the levels of its tyrosine phosphorylation have been observed in several cancer cells. However, how the expression level and phosphorylation state of cortactin would influence the ultimate cellular function of cancer cells is unknown. In this study, we analyzed the role of cortactin in gastric and breast cancer cell lines using RNA interference technique and found that knockdown of cortactin inhibited cell migration in a subset of gastric cancer cells with a lower level of its tyrosine phosphorylation, whereas it greatly enhanced cell migration and increased tyrosine phosphorylation of p130Cas in other subsets of cells with hyperphosphorylated cortactin. Consistent results were obtained when hyperphosphorylation of cortactin was induced in MCF7 breast cancer cells by expressing Fyn tyrosine kinase. Additionally, immunostaining analysis showed that knockdown of hyperphosphorylated cortactin resulted in the recruitment of p130Cas to focal adhesions. These results suggest that cortactin hyperphosphorylation suppresses cell migration possibly through the inhibition of membrane localization and tyrosine phosphorylation of p130Cas.

CUB domain-containing protein 1 is a novel regulator of anoikis resistance in lung adenocarcinoma.

Malignant tumor cells frequently achieve resistance to anoikis, a form of apoptosis induced by detachment from the basement membrane, which results in the anchorage-independent growth of these cells. Although the involvement of Src family kinases (SFKs) in this alteration has been reported, little is known about the signaling pathways involved in the regulation of anoikis under the control of SFKs. In this study, we identified a membrane protein, CUB-domain-containing protein 1 (CDCP1), as an SFK-binding phosphoprotein associated with the anchorage independence of human lung adenocarcinoma. Using RNA interference suppression and overexpression of CDCP1 mutants in lung cancer cells, we found that tyrosine-phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-related molecule, protein kinase Cdelta, was found to be phosphorylated by the CDCP1-SFK complex and was essential for anoikis resistance downstream of CDCP1. Loss of CDCP1 also inhibited the metastatic potential of the A549 cells in vivo. Our findings indicate that CDCP1 is a novel target for treating cancer-specific disorders, such as metastasis, by regulating anoikis in lung adenocarcinoma.