Filopodia play an important role in the trans-mesothelial migration of ovarian cancer cells.

Ovarian cancer cells shed from primary tumors can spread easily to the peritoneum via the peritoneal fluid. To allow further metastasis, the cancer cells must interact with the mesothelial cell layer, which covers the entire surface of the peritoneal organs. Although the clinical importance of this interaction between cancer and mesothelial cells has been increasingly recognized, the molecular mechanisms utilized by cancer cells to adhere to and migrate through the mesothelial cell layer are poorly understood. To investigate the molecular mechanisms of cancer cell trans-mesothelial migration, we set up an in vitro trans-mesothelial migration assay using primary peritoneal mesothelial cells. Using this method, we found that downregulation of filopodial protein fascin-1 or myosin X expression in ES-2 cells significantly inhibited the rate of trans-mesothelial migration of cancer cells, whereas upregulation of fascin-1 in SK-OV-3 cells enhanced this rate. Furthermore, downregulation of N-cadherin or integrin ß1 inhibited the rate of cancer cell trans-mesothelial migration. Conversely, downregulation of cortactin or TKS5 or treatment with the MMP inhibitor GM6001 or the N-WASP inhibitor wiskostatin did not have any effect on cancer cell trans-mesothelial migration. These results suggest that filopodia, but not lamellipodia or invadopodia, play an important role in the trans-mesothelial migration of ovarian cancer cells.

ATAD2 is associated with malignant characteristics of pancreatic cancer cells.

Pancreatic cancer is one of the most aggressive human cancers and is associated with a poor prognosis. To develop a novel strategy for pancreatic cancer treatment, it is essential to elucidate the molecular mechanisms underlying the invasion and proliferation of cancer cells. ATPase family AAA domain containing protein 2 (ATAD2) is a highly conserved protein with an AAA+ domain and a bromodomain. Accumulating studies have demonstrated that ATAD2 is associated with the progression of multiple cancers. The present study demonstrated that ATAD2 depletion suppressed cell invasion and migration. In addition, ATAD2 knockdown suppressed anchorage-independent growth of pancreatic cancer cells. Finally, ATAD2 depletion was demonstrated to sensitize pancreatic cancer cells to gemcitabine. The results of the present study indicate that ATAD2 is involved in the malignant characteristics of pancreatic cancer.

FAM98A is localized to stress granules and associates with multiple stress granule-localized proteins.

Stress granules are evolutionally conserved ribonucleoprotein structures that are formed in response to various stress stimuli. Recent studies have demonstrated that proteins with low complexity (LC) regions play a critical role for the formation of stress granules. In this study, we report that FAM98A, whose biological functions are unknown, is a novel component of stress granules. FAM98A is localized to stress granules, but not to P-bodies, after various stress stimuli. Analysis with deletion mutants revealed that C-terminal region that contains LC region was essential for FAM98A accumulation to stress granules. Depletion of FAM98A using two different siRNAs decreased the number of stress granules formed per cell. Finally, we show that FAM98A associates with stress granule-localized proteins, such as DDX1, ATXN2, ATXN2L, and NUFIP2. Our results show a partial role of FAM98A for the organization of stress granules.

PAI-1 secreted from metastatic ovarian cancer cells triggers the tumor-promoting role of the mesothelium in a feedback loop to accelerate peritoneal dissemination.

The mesothelium, covered by a continuous monolayer of mesothelial cells, is the first protective barrier against metastatic ovarian cancer. However, mesothelial cells release tumor-promoting factors that accelerate the process of peritoneal metastasis. We identified cancer-associated mesothelial cells (CAMs) that had tumor-promoting potential. Here, we found that plasminogen activator inhibitor-1 (PAI-1) induced the formation of CAMs, after which CAMs increasingly secreted the oncogenic factors interleukin-8 (IL-8) and C-X-C motif chemokine ligand 5 (CXCL5), further promoting the metastasis of ovarian cancer cells in a feedback loop. After the formation of CAMs, PAI-1 activated the nuclear factor kappa B (NFκB) pathway in the CAMs, thus transcriptionally upregulating the expression of the downstream NFκB targets IL-8 and CXCL5. Moreover, PAI-1 correlated with peritoneal metastasis in ovarian cancer patients and indicated a poor prognosis. In both ex vivo and in vivo models, after PAI-1 expression was knocked down, the metastasis of ovarian cancer cells decreased significantly. Therefore, targeting PAI-1 may provide a potential target for future therapeutics to prevent the formation of CAMs and alleviate peritoneal metastasis in ovarian cancer patients.

The role of E2F8 in the human placenta.

Recent studies have reported that E2F transcription factor (E2F) 8, an atypical E2F transcription factor, serves a critical role in promoting the growth and development of the murine placenta. However, the function of E2F8 in the human placenta remains unknown. Invasion of extravillous trophoblasts (EVTs) into the maternal decidua is known to be important for the development of the human placenta. To investigate the role of E2F8 in human placental development, E2F8 localisation was examined in the human placenta and E2F8 mRNA expression was detected in primary cultured EVTs. The human EVT cell line, HTR­8/SVneo, was divided into two groups and treated separately, one with retrovirus expressing short hairpin (sh)­RNA against E2F8 (shE2F8 cells) and the other with non­target control shRNA (shControl cells). The cell functions, including cell cycle, proliferation, invasion and adhesion, were compared between the shE2F8 and shControl cells. A histological examination revealed that E2F8 was localised in the decidua cells, EVTs, and cytotrophoblasts in the placenta. E2F8 mRNA was confirmed to be expressed in cultured primary EVTs. No significant difference was observed in the cell cycle, proliferation or adhesion between the shE2F8 and shControl cells. The invasive ability was ~2­fold higher in the shE2F8 cells when compared with the shControl cells (P<0.01). Production of matrix metalloproteinase­1 was significantly increased in the shE2F8 cells when compared with the shControl cells (P<0.05). Taken together, E2F8 is present in the EVTs of the human placenta, but, unlike murine placenta, it may suppress the invasiveness of EVTs. E2F8 was also present in cytotrophoblasts in cell columns, which have no invasive ability and differentiate into EVTs. In conclusion, E2F8 also exists in the human placenta, and its function may be different from that in the murine placenta, although further investigation is required.

Hematopoietic lineage cell-specific protein 1 (HS1), a hidden player in migration, invasion, and tumor formation, is over-expressed in ovarian carcinoma cells.

Hematopoietic lineage cell-specific protein 1 (HS1), which is the hematopoietic homolog of cortactin, is an actin-binding protein and Lyn substrate. It is upregulated in several cancers and its expression level is associated with increased cell migration, metastasis, and poor prognosis. Here we investigated the expression and roles of HS1 in ovarian carcinoma cells. We analyzed the expression of HS1 in 171 ovarian cancer specimens and determined the association between HS1 expression and clinicopathological characteristics, including patient outcomes. In patients with stage II-IV disease, positive HS1 expression was associated with significantly worse overall survival than negative expression (P < 0.05). HS1 was localized in invadopodia in some ovarian cancer cells and was required for invadopodia formation. Migration and invasion of ovarian cancer cells were suppressed by down-regulation of HS1, but increased in cells that over-expressed exogenous HS1. Furthermore, ovarian cancer cells that expressed HS1 shRNA exhibited reduced tumor formation in a mouse xenograft model. Finally, we found that tyrosine phosphorylation of HS1 was essential for cell migration and invasion. These findings show that HS1 is a useful biomarker for the prognosis of patients with ovarian carcinoma and is a critical regulator of cytoskeleton remodeling involved in cell migration and invasion.

KIF20A expression as a prognostic indicator and its possible involvement in the proliferation of ovarian clear­cell carcinoma cells.

Kinesin family member 20A (KIF20A), which is involved in cytokinesis and intracellular transportation, has been recently reported to be upregulated in several malignancies and may contribute to chemotherapeutic resistance. We examined the distribution and expression of KIF20A in clear­cell carcinoma (CCC) of the ovary to elucidate its clinical significance and molecular mechanism. Paraffin sections from ovarian CCC tissues (N=43) were immunostained with KIF20A antibody, and the staining intensities were semi­quantitatively evaluated. Furthermore, we investigated whether silencing of KIF20A contributes to the proliferation­inhibitory potential using CCC cells. During the observational period, 18 patients (41.9%) developed recurrence. The median time to recurrence was 11.5 months. Patients in the high KIF20A expression group showed poorer progression­free survival (PFS) and overall survival (OS) than those in the low expression group (P=0.0443 and P=0.0478, respectively). In multivariable analyses, KIF20A expression was also a significantly independent indicator of PFS and a marginally significant indicator of OS [PFS: HR (high vs. low), 5.488; 95% CI, 1.410­24.772 (P=0.0136); OS: HR, 2.835; 95% CI, 0.854­11.035, (P=0.0897)]. In in vitro studies, the ovarian CCC cell proliferation was significantly decreased by KIF20A silencing or in the presence of KIF20A inhibitor in CCC cells. Cell cycle G2/M arrest and a higher apoptosis­induced fraction were more frequently observed in si­KIF20A­transfected CCC cells than in the control cells. Although the present study was preliminary, these data indicate the possible involvement of KIF20A in the proliferation of CCC, suggesting that targeting this molecule may contribute to reversing the malignant potential consequently affecting the oncologic outcome of CCC patients.

Hematopoietic lineage cell-specific protein 1 immunoreactivity indicates an increased risk of poor overall survival in patients with ovarian carcinoma.

Hematopoietic lineage cell-specific protein 1 (HS1) is a 75-kDa intracellular protein that is expressed primarily in hematopoietic cells. Several previous studies have demonstrated the association between HS1 expression and a poor prognosis in hematopoietic malignancies; however, in solid tumors, no studies not been reported. The present study examined the distribution and expression of HS1 in human epithelial ovarian carcinoma (EOC) to determine its clinical significance. Paraffin sections were obtained from EOC tissues and immunostained with HS1 antibody, and then the staining intensities were evaluated. Overall survival (OS) was determined using the Kaplan-Meier estimator method, and multivariate analysis was performed using the Cox proportional hazards analysis. In total, 195 patients with EOC (median age, 56 years) were enrolled into the present study. HS1 immunoreactivity was categorized based on expression levels: Low (89/195; 45.6%) and high (106/195; 54.4%). Results demonstrated no association between expression level(s) and any clinicopathological parameter including age, International Federation of Gynecology and Obstetrics (FIGO) staging, type of chemotherapy or type of surgery received. The 5-year OS rates of patients who demonstrated low (n=89) and high (n=106) HS1 expression were 90.4 and 66.7%, respectively. The OS times for patients with high HS1 expression were significantly shorter compared with those for patients exhibiting low HS1 expression (P=0.0065). Results obtained from the multivariate analysis demonstrated that the FIGO stage and the amount of HS1 expressed were significant independent prognostic markers for poorer OS (hazard ratio, 3.539; 95% confidence interval, 1.221-12.811; P=0.0187). High HS1 expression levels may serve as a useful biomarker in patients with EOC who are likely to exhibit an unfavorable clinical outcome.

A novel mechanism of neovascularization in peritoneal dissemination via cancer-associated mesothelial cells affected by TGF-ß derived from ovarian cancer.

Epithelial ovarian cancer (EOC) is believed to cause peritoneum dissemination through microenvironmental cell­to-cell communication between the tumor and mesothelium, leading to the further acquisition of progressive and metastatic potentials. In the present study, we aimed to determine the role of cancer-associated mesothelial cells (CAMCs) in the promotion of tumor neovascularization and vascular permeability via enhanced vascular endothelial growth factor (VEGF) production. We examined whether a characteristic morphological change in human peritoneal mesothelial cells (HPMCs) was observed in the presence of malignant ascites and tumor-derived TGF-ß. We focused on the enhanced production of VEGF in CAMCs and its crucial role in endothelial migration and tube formation. Normal HPMCs showed an epithelial morphology with a cobblestone appearance. When HPMCs were co-cultured with malignant ascites from patients with advanced EOC, a dramatic morphologic change was noted from an epithelioid pattern to an α-SMA-positive fibroblastic, mesenchymal pattern. Additionally, we found that EOC-derived TGF-ß induced typical EMT-like morphological alteration in HPMCs, which was associated with CAMCs. We further discovered that CAMCs play a crucial role in the enhanced migration and tube formation of endothelial cells by the promotion of VEGF production. In conclusion, our findings indicate the possible involvement of CAMCs in the neovascularization of EOC and enhancement of vascular permeability, resulting in the formation of malignant ascites. The novel mechanism of CAMCs as a facilitator of EOC progression is displayed by microenvironmental cell-to-cell communication between EOC and the mesothelium.

Inhibition of ZEB1 leads to inversion of metastatic characteristics and restoration of paclitaxel sensitivity of chronic chemoresistant ovarian carcinoma cells.

ZEB1, a member of the zinc-finger E-box binding homeobox family, is considered to play a crucial role in cancer progression and metastasis. In the current study, we investigated the role of ZEB1 in metastasis and chronic chemoresistance of epithelial ovarian carcinoma (EOC) cells. Using several EOC and acquired paclitaxel (PTX)-resistant EOC cell lines, we investigated whether silencing ZEB1 led to a reversal of the chemoresistance and metastatic potential in vitro and in vivo. Subsequently, the expression of ZEB1 in EOC tissues and its association with the oncologic outcome were investigated. According to the immunohistochemical staining of EOC tissues, as the positivity of ZEB1 expression was increased, the overall survival of EOC patients became poorer (P = 0.0022 for trend). Additionally, cell migration and invasion were significantly decreased by ZEB1 silencing in both PTX-sensitive and PTX- resistant cells. Although PTX-sensitivity was not changed by silencing ZEB1 in parental EOC cells, the depletion of ZEB1 made the PTX-resistant EOC cells more sensitive to PTX treatment. In an animal model, mice injected with ZEB1-silencing PTX-resistant cells survived for longer than the control cell-injected mice. Although the intravenous injection of PTX did not affect the tumor weight of shCtrl cells, the tumor weight of shZEB1 cells was significantly reduced by PTX treatment. The current data indicate the possible involvement of ZEB1 in the metastasis and paclitaxel resistance of EOC, and suggest that targeting this molecule may reverse the malignant potential and improve the oncologic outcome for EOC patients.

Impact of positive ZEB1 expression in patients with epithelial ovarian carcinoma as an oncologic outcome-predicting indicator.

Several previous studies have revealed that the expression of zinc finger E-box binding homeobox 1 (ZEB1) in solid malignancies has an important significance on the clinical outcome of patients. However, the association between ZEB1 expression and survival in patients with epithelial ovarian carcinoma (EOC) remains unclear. The objective of the present study was to examine the extent of ZEB1 expression in EOC using immunohistochemical staining and investigate its association with patient outcome. A total of 40 patients with EOC initially treated with cytoreductive surgery and systematic chemotherapy were enrolled. ZEB1 expression was immunohistochemically categorized as negative, weak, moderate and strong according to the size of the staining area, and intensity. Subsequently, the associations between ZEB1 expression and recurrence/progression-free survival (RFS) rate were examined. The median age of patients in the current study was 54 years old (range, 22-72 years old). Among these patients, 15 (37.5%) exhibited International Federation of Gynecology and Obstetrics stage I disease, and 10 (25.0%), 13 (32.5%), and 2 (5%) had stage II, III, and IV disease, respectively. No patients with negative expression of ZEB1 experienced recurrence. In addition, ZEB1 expression was identified to be a significant predictor of a poorer RFS rate compared with negative expression (negative vs. weak, moderate and strong, P=0.0126). Furthermore, multivariate analyses revealed that moderate and strong ZEB1 expression levels were significant prognostic indicators of a poorer RFS rate in patients with EOC (hazard ratio, 2.265; 95% confidence interval, 1.072-8.021; P=0.0349). Confining analysis to patients with the clear-cell/mucinous histological type, those with moderate/strong ZEB1 expression demonstrated a significantly poorer RFS rate (P=0.0025). Positive ZEB1 expression may be an indicator to predict unfavorable RFS in patients with EOC.

Phosphorylation of DEPDC1 at Ser110 is required to maintain centrosome organization during mitosis.

DEPDC1 (DEP domain containing 1) is overexpressed in multiple cancers and is associated with cell cycle progression. In this report, we have investigated the expression, localization, phosphorylation and function of DEPDC1 during mitosis. DEPDC1 has two isoforms (isoform a and isoform b), and both of them are increased in mitosis and degraded once cells exit mitosis. DEPDC1a is localized to the centrosome in metaphase, whereas DEPDC1b is localized to the entire cell cortex during mitosis. DEPDC1a, but not DEPDC1b, was required for the integrity of centrosome and organization of the bipolar spindle. Mass spectrometry and biochemical analyses revealed phosphorylation of DEPDC1 at Ser110. The phosphorylation of Ser110 is essential for localization of DEPDC1a to the centrosome. Consistently, non-phosphorylation mutants of DEPDC1a did not rescue disruption of centrosome organization by depletion of endogenous DEPDC1. Our results show a novel role for DEPDC1 in maintaining centrosome integrity during mitosis for the accurate distribution of chromosomes.

HOXD8 exerts a tumor-suppressing role in colorectal cancer as an apoptotic inducer.

Homeobox (HOX) genes are conserved transcription factors which determine the anterior-posterior body axis patterning. HOXD8 is a member of HOX genes deregulated in several tumors such as lung carcinoma, neuroblastoma, glioma and colorectal cancer (CRC) in a context-dependent manner. In CRC, HOXD8 is downregulated in cancer tissues and metastatic foci as compared to normal tissues. Whether HOXD8 acts as a tumor suppressor of malignant progression and metastasis is still unclear. Also, the underlying mechanism of its function including the downstream targets is totally unknown. Here, we clarified the lower expression of HOXD8 in clinical colorectal cancer vs. normal colon tissues. Also, we showed that stable expression of HOXD8 in colorectal cancer cells significantly reduced the cell proliferation, anchorage-independent growth and invasion. Further, using The Cancer Genome Atlas (TCGA), we identified the genes associated with HOXD8 in order to demonstrate its function as a suppressor or a promoter of colorectal carcinoma. Among inversely related genes, apoptotic inhibitors like STK38 kinase and MYC were shown to be negatively associated with HOXD8. We demonstrated the ability of HOXD8 to upregulate executioner caspases 6 & 7 and cleaved PARP, thus inducing the apoptotic events in colorectal cancer cells.

AMPD3 is associated with the malignant characteristics of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract. It is well known that activating mutations in the receptor tyrosine kinases KIT and platelet-derived growth factor receptor-α have essential roles in the pathogenesis of GISTs. The activation of these receptor protein kinases triggers multiple signaling pathways that promote cell proliferation and survival; however, the exact mechanism by which the activation of these kinases promotes the progression of GISTs remains uncertain. The aim of the present was to search for genes that are associated with the progression of GIST. The present study used reverse transcription-quantitative polymerase chain reaction to demonstrate that adenosine monophosphate deaminase 3 (AMPD3) was highly expressed in GISTs. Furthermore, transfection of GIST-T1 cells with KIT-specific small interfering RNA (siRNA) demonstrated that the expression of AMPD3 was dependent on KIT expression, while the depletion of AMPD3 in human GIST-T1 cells using AMPD3-specific siRNA resulted in the suppression of cell migration and invasion. In addition, AMPD3 depletion sensitized GIST-T1 cells to the tyrosine kinase inhibitor imatinib. The results of the present suggested that the combined inhibition of tyrosine kinases and AMPD3 may be effective for the treatment of GISTs.

Interleukin-1ß activates focal adhesion kinase and Src to induce matrix metalloproteinase-9 production and invasion of MCF-7 breast cancer cells.

Interleukin-1ß (IL-1b) is a pleiotropic cytokine that is important in tumor progression and invasion. Matrix metalloproteinase-9 (MMP-9), which is a secreted matrix-degrading enzyme, is one of the key regulators of tumor invasion and metastasis. The current report indicated that IL-1b promotes MMP-9 production and cell invasion in non-metastatic MCF-7 breast cancer cells. IL-1b activated focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src). Moreover, inhibiting the Src/FAK pathway reduced the IL-1b-induced production of MMP-9 and cell invasion. To investigate the functional role of FAK in MMP-9 production cell lines expressing mutant FAK in FAK knock-out mouse fibroblasts were generated. In wild-type FAK-expressing cells, MMP-9 production was induced by IL-1b stimulation. By contrast, IL-1b-induced MMP-9 production was abrogated in FAK knock-out, FAK Y397F, FAK Y925F, and kinase dead mutant-expressing cells. Therefore the results of the current study indicate that FAK and Src kinases are activated by IL-1b and play a critical role in MMP-9 production and tumor cell invasion.

Role of Caveolin-1 for Blocking the Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is one of the most severe ocular diseases. Fibrotic changes in retinal cells are considered to be involved in the pathogenesis of PVR. Epithelial-mesenchymal transition (EMT) of RPE cells is one of the main concepts in the pathogenesis of fibrovascular membranes (FVMs) in PVR. In this study, we examined the expression of Caveolin-1 in human FVMs from patients with PVR. We also examined the role of Caveolin-1 in the pathogenesis of PVR. Methods: Western blotting, real-time PCR, and immunohistochemistry were performed with human FVMs and mouse eyes with PVR. Cell migration assays were performed to evaluate the involvement of Caveolin-1 in EMT using primary human and mouse RPE cells. Results: Caveolin-1 was expressed in human FVMs and upregulated in the mouse eyes with PVR. The alpha-smooth muscle actin (αSMA) expression and migration ability were increased in RPE cells with knockout or knockdown of Caveolin-1, whereas zonula occludens-1 (ZO-1) immunohistochemistry showed reduced morphology and expression of ZO-1. In addition, migration assays showed that Caveolin-1 reduction increased RPE cell migration abilities. Conclusions: These results indicated that Caveolin-1 in RPE cells prevents PVR by blocking EMT.

FAM98A associates with DDX1-C14orf166-FAM98B in a novel complex involved in colorectal cancer progression.

Protein Arginine Methyl Transferase 1 (PRMT1) is deemed to be a potential oncogenic protein considering its overexpression in several malignancies including colorectal cancer. However, the molecular pathogenesis regarding PRMT1 overexpression and overall poor patient survival involved in this devastating and life threatening cancer remains obscured. In our previous study, we have identified FAM98A as a novel substrate of PRMT1 and also identified its role in ovarian cancer progression. Here, we showed that the two structural homologs FAM98A and FAM98B included in a novel complex with DDX1 and C14orf166 are required for PRMT1 expression. Analysis of the data from The Cancer Genome Atlas (TCGA) database and clinical colorectal cancer specimens also demonstrated a strong positive correlation and co-occurrence of PRMT1, FAM98A and FAM98B. These findings provide a mechanistic insight into how knockdown of FAM98A or FAM98B can suppress the malignant characteristics of cancer cells. Besides, we showed that FAM98A and FAM98B are working in the same axis as knockdown of both proteins together does not cause additional reduction in the cellular proliferation and colony formation of colorectal cancer cells.

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas.

World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69-80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83-90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 µl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.

A regulatory subunit of protein phosphatase 2A, PPP2R5E, regulates the abundance of microtubule crosslinking factor 1.

Dynamic changes in microtubule organization are regulated by numerous microtubule-associating proteins and their post-translational modification. Microtubule crosslinking factor 1 (MTCL1) is a recently identified protein that regulates microtubule organization. To obtain further insight into its functions, we searched for proteins that associate with it using mass spectrometry analysis. We found that PPP2R5E, a regulatory subunit of protein phosphatase 2A, interacted with MTCL1. Depletion of PPP2R5E reduced the abundance of MTCL1 abundance, whereas exogenous expression of PPP2R5E increased endogenous MTCL1. Furthermore, inhibition of phosphatase activity by okadaic acid reduced MTCL1, which was restored by the addition of the protease inhibitor MG132. Finally, we show that cells depleted of PPP2R5E and MTCL1 exhibited defects in microtubule organization. Our results suggest that the PPP2R5E phosphatase may contribute to microtubule organization by stabilizing MTCL1.

Nek2 siRNA therapy using a portal venous port-catheter system for liver metastasis in pancreatic cancer.

Nek2 (NIMA-related kinase 2) is a serine-threonine kinase and human homolog of the mitotic regulator NIMA of Aspergillus nidulan. We reported the efficiency of Nek2 siRNA in several cancer xenograft models using cholangiocarcinoma, breast cancer and colorectal cancer. Pancreatic cancer is difficult to treat due to its rapid progression and resistance to chemotherapy. Novel treatments are urgently required to improve survival in pancreatic cancer, and siRNA are a promising therapeutic option. However, finding an in vivo drug delivery system of siRNA remains a major problem for clinical application. In this study, the overexpression of Nek2 was identified in pancreatic cancer cell lines. Nek2 siRNA inhibited tumor growth in a subcutaneous xenograft mouse model of pancreatic cancer, prolonged the survival time in an intraperitoneal xenograft mouse model and efficiently prevented the progression of liver metastasis using a portal venous port-catheter system. Taken together, Nek2 is an effective therapeutic target in pancreatic cancer. An adequate delivery system is considered important in treating advanced pancreatic cancer, such as peritoneal dissemination and liver metastasis. Further investigations are required on the safety and side effects of the portal venous port-catheter system. We hope that Nek2 siRNA will be a novel therapeutic strategy for pancreatic cancer with liver metastasis and peritoneal dissemination.