Parkin, an E3 ubiquitin ligase well known for its role in the pathogenesis of juvenile Parkinson disease, has been considered as a candidate tumor suppressor in certain types of cancer. It remains unknown whether parkin is involved in the development of pancreatic cancer, the fourth leading cause of cancer-related deaths worldwide. Herein, we demonstrate the downregulation and copy number loss of the parkin gene in human pancreatic cancer specimens. The expression of parkin negatively correlates with clinicopathological parameters indicating the malignancy of pancreatic cancer. In addition, knockdown of parkin expression promotes the proliferation and tumorigenic properties of pancreatic cancer cells both in vitro and in mice. We further find that parkin deficiency increases the proportion of cells with spindle multipolarity and multinucleation. Parkin-depleted cells also show a significant increase in spindle misorientation. These findings indicate crucial involvement of parkin deficiency in the pathogenesis of pancreatic cancer.
Orientation and positioning of the mitotic spindle are involved in dictating cell division axis and cleavage site, and play important roles in cell fate determination and tissue morphogenesis. However, how spindle movement is controlled to achieve a defined alignment within the dividing cell is not fully understood. Here, we describe an unexpected role for apoptosis signal-regulating kinase 1 (ASK1) in regulating spindle behavior. We find that ASK1 is required for proper mitotic progression and daughter cell adhesion to the substratum. ASK1 interacts with end-binding protein 1 (EB1) and phosphorylates EB1 at serine 40, threonine 154 and threonine 206, enhancing its binding to the plus ends of astral microtubules. Consequently, astral microtubules are stabilized and therefore capable of mediating spindle interaction with the cell cortex, a requirement for spindle movement. These findings reveal a previously undiscovered function of ASK1 in cell division by regulating spindle orientation and positioning, and point to the importance of protein phosphorylation in the regulation of spindle behavior.
Aim:The mitotic kinesin Eg5 plays a critical role in bipolar spindle assembly, and its inhibitors have shown impressive anticancer activity in preclinical studies. This study was undertaken to investigate the effect of dimethylenastron, a specific inhibitor of Eg5, on the migration and invasion of pancreatic cancer cells. Methods: Human pancreatic cancer cell lines PANC1, EPP85, BxPC3, CFPAC1, and AsPAC1 were used. Eg5 expression was examined using immunofluorescence microscopy. Cell migration and invasion were analyzed with wound healing and transwell assays. Cell proliferation was examined using sulforhodamine B and MTT assays. The binding of dimethylenastron to Eg5 was analyzed with a molecular modeling study, and the ADP release rate was examined with the MANT-ADP reagent. Results: Eg5 expression was 9-16-fold up-regulated in the 5 pancreatic cancer cell lines. Treatment of PANC1 pancreatic cancer cells with dimethylenastron (3 and 10 μmol/L) for 24 h suppressed the migratory ability of the cancer cells in a concentration-dependent manner. The invasion ability of the cancer cells was also reduced by the treatment. However, treatment of PANC1 cells with dimethylenastron (3 and 10 μmol/L) for 24 h had no detectable effect on their proliferation, which was inhibited when the cancer cells were treated with the drug for 72 h. Molecular modeling study showed that dimethylenastron could allosterically inhibit the motor domain ATPase of Eg5 by decreasing the rate of ADP release. Conclusion: Dimethylenastron inhibits the migration and invasion of PANC1 pancreatic cancer cells, independent of suppressing the cell proliferation. The findings provide a novel insight into the mechanisms of targeting Eg5 for pancreatic cancer chemotherapy.
Cell migration, a complex process critical for tumor progression and metastasis, requires a dynamic crosstalk between microtubules (MTs) and focal adhesions (FAs). However, the molecular mechanisms underlying this event remain elusive. Herein we identify the proto-oncogenic protein Src as an important player in the regulation of the MT-FA crosstalk. Src interacts with and phosphorylates end-binding protein 1 (EB1), a member of MT plus end-tracking proteins (+TIPs), both in cells and in vitro. Systematic mutagenesis reveals that tyrosine-247 (Y247) is the primary residue of EB1 phosphorylated by Src. Interestingly, both constitutively activated Src and Y247-phosphorylated EB1 localize to the centrosome and FAs. Src-mediated EB1 phosphorylation diminishes its interactions with other +TIPs, including adenomatous polyposis coli (APC) and mitotic centromere associated kinesin (MCAK). In addition, EB1 phosphorylation at Y247 enhances the rate of MT catastrophe and significantly stimulates cell migration. These findings thus demonstrate that the Src-EB1 axis plays a crucial role in regulating the crosstalk between MTs and FAs to promote cell migration.
Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.
Phosphorylation of end-binding protein 1 (EB1), a key member of microtubule plus end-tracking proteins (+TIPs), by apoptosis signal-regulating kinase 1 (ASK1) has been demonstrated to promote the stability of astral microtubules during mitosis by stimulating the binding of EB1 to microtubule plus ends. However, the roles of other members of the +TIPs family in ASK1/EB1-mediated regulation of astral microtubules are unknown. Herein, we show that ASK1-mediated phosphorylation of EB1 enhances the localization of cytoplasmic linker protein 170 (CLIP-170) and p150glued to the plus ends of astral microtubules. Depletion of ASK1 or expression of phospho-deficient or phospho-mimetic EB1 mutants results in changes in the levels of plus-end localized CLIP-170 or p150glued. Mechanistic studies reveal that EB1 phosphorylation promotes its interactions with CLIP-170 and p150glued, thereby recruiting these +TIPs to microtubules. Structural analysis suggests that serine-40 is the primary phosphorylation site on EB1 that exerts these effects. Together, these findings provide novel insight into the molecular mechanisms that regulate the interactions of EB1 with other +TIPs.
Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.
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