Role of Autophagy in Cisplatin Resistance in Ovarian Cancer Cells

Cisplatin-based treatment is the first line chemotherapy for several cancers including ovarian cancer. The development of cisplatin resistance results in treatment failure, but the underlying mechanisms are not fully understood. Here we show that the induction of autophagy plays an important role in cisplatin resistance in ovarian cancer cells. Specifically, we show that cisplatin resistance is correlated with autophagy induction in a panel of ovarian cancer cells but not in immortalized human ovarian surface epithelial cells. Mechanistically, cisplatin treatment activates ERK and subsequently promotes autophagy. The inhibition of ERK activation with MEK inhibitors or knockdown of ERK expression with siRNA decreases cisplatin-induced autophagy and subsequently sensitizes ovarian cancer cells to cisplatin-induced apoptosis. In ovarian cancer cells that have developed acquired cisplatin resistance, both ERK activation and autophagy induction are increased. Importantly, knockdown of ERK or inhibition of autophagy promotes cisplatin-induced apoptosis in acquired cisplatin-resistant cells. Collectively, our data indicate that ERK-mediated autophagy can lead to cisplatin resistance and suggest that cisplatin resistance can be overcome by inhibition of autophagy in ovarian cancer cells.     

Cloning and Spatio-Temporal Expression of Porcine CDK5 and CDK5R1(p35) Genes

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase homologue attributed to the mitotic cyclin-dependent kinase family. Both the kinase activity and the biological effects of CDK5 in central nervous system are mainly dependent on association with its regulatory subunit 1 known as CDK5R1 (p35). In the present study, the full-length coding regions of CDK5 and CDK5R1 were cloned from pigs. Radiation hybrid mapping localized porcine CDK5 to chromosome 18q12-13, whereas CDK5R1 was electro-localized to chromosome 12q12. Real-time quantitative RT-PCR (qRT-PCR) showed that CDK5 mRNA is ubiquitously present in all porcine tissues examined, with relatively high levels in cerebral cortex, cerebellum, testicle and lung. We also examined the expression profile of porcine CDK5/CDK5R1 in various tissues at different developmental stages. The results indicated that CDK5 mRNA reaches the highest level in cerebral cortex at two months of age and in cerebellum and liver at 4 months of age, respectively, whereas the peak level of CDK5R1 was observed in both cerebral cortex and cerebellum at two months of age, indicating the pivotal role of CDK5/CDK5R1 during the development of porcine brain.     

Parkin Enhances the Expression of Cyclin-dependent Kinase 6 and Negatively Regulates the Proliferation of Breast Cancer Cells

Although mutations in the parkin gene are frequently associated with familial Parkinsonism, emerging evidence suggests that parkin also plays a role in cancers as a putative tumor suppressor. Supporting this, we show here that parkin expression is dramatically reduced in several breast cancer-derived cell lines as well as in primary breast cancer tissues. Importantly, we found that ectopic parkin expression in parkin-deficient breast cancer cells mitigates their proliferation rate both in vitro and in vivo, as well as reduces the capacity of these cells to migrate. Cell cycle analysis revealed the arrestment of a significant percentage of parkin-expressing breast cancer cells at the G1-phase. However, we did not observe significant changes in the levels of the G1-associated cyclin D1 and E. On the other hand, the level of cyclin-dependent kinase 6 (CDK6) is dramatically and selectively elevated in parkin-expressing breast cancer cells, the extent of which correlates well with the expression of parkin. Interestingly, a recent study demonstrated that CDK6 restrains the proliferation of breast cancer cells. Taken together, our results support a negative role for parkin in tumorigenesis and provide a potential mechanism by which parkin exerts its suppressing effects on breast cancer cell proliferation.     

Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation,Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.     

Sphingosine Kinase Mediates Resistance to the Synthetic Retinoid N-(4-Hydroxyphenyl)retinamide in Human Ovarian Cancer Cells

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors.These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.     

Protein-tyrosine Phosphatase 1B Antagonized Signaling by Insulin-like Growth Factor-1 Receptor and Kinase BRK/PTK6 in Ovarian Cancer Cells

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R β-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R β-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rβ signaling in ovarian cancer cells.     

Cyclin K inhibits HIV-1 gene expression and replication by interfering with cyclin-dependent kinase 9 (CDK9)-cyclin T1 interaction in Nef-dependent manner

Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising cyclin-dependent kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.     

MicroRNA-372 is down-regulated and targets cyclin-dependent kinase 2 (CDK2) and cyclin A1 in human cervical cancer, which may contribute to tumorigenesis

MicroRNAs are a class of noncoding RNAs that are ∼22 nucleotides in length. MicroRNAs have been shown to play important roles in cell differentiation and in cancer. Recently, studies have shown that miR-372 is tumorigenic in human reproductive system cancers. However, we provide evidence that miR-372 acts as a tumor suppressor gene in cervical carcinoma. miR-372 was found down-regulated in cervical carcinoma tissues as compared with adjacent normal cervical tissues. Growth curve and FACS assays indicated that ectopic expression of miR-372 suppressed cell growth and induced arrest in the S/G₂ phases of cell cycle in HeLa cells. We used bioinformatic predictions to determine that CDK2 and cyclin A1 were possible targets of miR-372 and confirmed this prediction using a fluorescent reporter assay. Taken together, these findings indicate that an anti-oncogenic role of miR-372 may be through control of cell growth and cell cycle progression by down-regulating the cell cycle genes CDK2 and cyclin A1.     

Mitotic Regulation of SEPT9 Protein by Cyclin-dependent Kinase 1 (Cdk1) and Pin1 Protein Is Important for the Completion of Cytokinesis

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.     

Multisite phosphorylation disrupts arginine-glutamate salt bridge networks required for binding of cytoplasmic linker-associated protein 2 (CLASP2) to end-binding protein 1 (EB1)

A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic "molecular Velcro." Molecular dynamics simulations and (31)P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.     

Inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.     

Elevated Protein Kinase D3 (PKD3) Expression Supports Proliferation of Triple-negative Breast Cancer Cells and Contributes to mTORC1-S6K1 Pathway Activation

Here, we show that the expression of the Golgi-localized serine-threonine kinase protein kinase D3 (PKD3) is elevated in triple-negative breast cancer (TNBC). Using an antibody array, we identified PKD3 to trigger the activation of S6 kinase 1 (S6K1), a main downstream target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Accordingly, PKD3 knockdown in TNBC cells led to reduced S6K1 phosphorylation, which was associated with impaired activation of mTORC1 at endolysosomal membranes, the accumulation of the mannose 6-phosphate receptor in and the recruitment of the autophagy marker light chain 3 to enlarged acidic vesicles. We further show that PKD3 depletion strongly inhibited cell spreading and proliferation of TNBC cells, identifying this kinase as a potential novel molecular therapeutic target in TNBC. Together, our data suggest that PKD3 in TNBC cells provides a molecular connection between the Golgi and endolysosomal compartments to enhance proliferative mTORC1-S6K1 signaling.     

Successive phosphorylation of p27(KIP1) protein at serine-10 and C terminus crucially controls its potency to inactivate Cdk2

During the G(1)-S transition, the activity of Cdk2 is regulated by its association with p27(KIP1), which in rodent fibroblasts undergoes phosphorylation mainly at serine 10, threonine 187, and C-terminal threonine 197 by KIS, Cdk2, and Pim or ROCK, respectively. Recently Cdc6 the AAA+ ATPase, identified initially to assemble pre-replicative complexes on origins of replication and later to activate p21(CIP1)-inactivated Cdk2, was found also to activate p27-bound Cdk2 but only after the bound p27 is C-terminally phosphorylated. On the other hand, the biological significance of the serine 10 phosphorylation remains elusive aside from its involvement in the stability of p27 itself. We report here that serine 10 phosphorylation is required for efficient C-terminal phosphorylation of its own by PIM and ROCK kinases and critically controls the potency of p27 as a Cdk2 inhibitor. In vitro, PIM1 and active ROCK1 efficiently phosphorylated free as well as Cdk2-bound p27 but only when the p27 was phosphorylated at Ser-10 in advance. Consistently, a Ser-10 nonphosphorylatable mutant p27 protein was not phosphorylated at the C terminus in vivo. Furthermore, when double-phosphorylated, free p27 was no longer a potent inhibitor of Cdk2, and Cdk2-bound p27 could be removed by Cdc6 to reactivate the Cdk2. Thus, phosphorylation at these two sites crucially controls the potency of this CDK inhibitor in two distinct modes.