- Open Access
Coordination of glioblastoma cell motility by PKCι
© Baldwin et al; licensee BioMed Central Ltd. 2010
Received: 22 February 2010
Accepted: 3 September 2010
Published: 3 September 2010
Glioblastoma is one of the deadliest forms of cancer, in part because of its highly invasive nature. The tumor suppressor PTEN is frequently mutated in glioblastoma and is known to contribute to the invasive phenotype. However the downstream events that promote invasion are not fully understood. PTEN loss leads to activation of the atypical protein kinase C, PKCι. We have previously shown that PKCι is required for glioblastoma cell invasion, primarily by enhancing cell motility. Here we have used time-lapse videomicroscopy to more precisely define the role of PKCι in glioblastoma.
Glioblastoma cells in which PKCι was either depleted by shRNA or inhibited pharmacologically were unable to coordinate the formation of a single leading edge lamellipod. Instead, some cells generated multiple small, short-lived protrusions while others generated a diffuse leading edge that formed around the entire circumference of the cell. Confocal microscopy showed that this behavior was associated with altered behavior of the cytoskeletal protein Lgl, which is known to be inactivated by PKCι phosphorylation. Lgl in control cells localized to the lamellipod leading edge and did not associate with its binding partner non-muscle myosin II, consistent with it being in an inactive state. In PKCι-depleted cells, Lgl was concentrated at multiple sites at the periphery of the cell and remained in association with non-muscle myosin II. Videomicroscopy also identified a novel role for PKCι in the cell cycle. Cells in which PKCι was either depleted by shRNA or inhibited pharmacologically entered mitosis normally, but showed marked delays in completing mitosis.
PKCι promotes glioblastoma motility by coordinating the formation of a single leading edge lamellipod and has a role in remodeling the cytoskeleton at the lamellipod leading edge, promoting the dissociation of Lgl from non-muscle myosin II. In addition PKCι is required for the transition of glioblastoma cells through mitosis. PKCι therefore has a role in both glioblastoma invasion and proliferation, two key aspects in the malignant nature of this disease.
Glioblastoma multiforme is a primary brain tumor with a very poor prognosis. Despite the use of aggressive therapeutic approaches combining surgery, radiation and chemotherapy, the median survival time for patients is only 12-14 months . The highly invasive nature of glioblastoma cells blurs tumor margins, making complete surgical resection impossible. Additionally, it is thought that invading cells may be more resistant to radiation and chemotherapy . Inhibition of cell invasion may therefore be an effective strategy to improve the treatment of glioblastoma.
Glioblastoma cell invasion requires that cells have enhanced motility, along with an ability to degrade local tissue barriers. The phosphoinositide 3-kinase (PI 3-kinase) pathway is often constitutively active in glioblastoma as a result of mutations in PTEN, as well as mutation and amplification of the epidermal growth factor receptor . These genetic alterations have been shown to promote motility and invasion of glioblastoma cells [4, 5]. The PI 3-kinase pathway can activate multiple downstream effectors including the atypical protein kinase C family member PKCι [6, 7]. The importance of PKCι as a downstream effector in the PI 3-kinase pathway is emphasized by the fact that PKCι can function as an oncogene in several tumor types [8–10]. On this basis it has been proposed that PKCι is a promising new target for cancer therapy .
The activation of PKCι involves direct phosphorylation by phosphoinositide-dependent kinase 1 and association with Cdc42, a small GTPase that is extensively involved in cell migration [6, 7, 12, 13]. The atypical PKCs (PKCι and PKCζ) have been shown to play a role in the establishment of multiple forms of cell polarity, including asymmetric cell division and apical-basal polarity . They form a conserved polarity complex with the scaffold protein, Par-6, that links the atypical PKCs to other proteins including Cdc42, Par-3 and Lgl .
We have shown previously that PKCι promotes motility and invasion of glioblastoma cells . PKCι has also been shown to promote the invasiveness of lung cancer cells . These studies have given insight into the role of PKCι in cellular motility and invasion; however they have relied on static analyses of invasion, and did not define precisely the role of PKCι in the dynamic process of cancer cell migration. In this study, we have investigated the role that PKCι plays in the regulation of glioblastoma cell motility using time-lapse videomicroscopy. This showed that PKCι has a critical role in coordinating lamellipod leading edge formation, an essential step in glioblastoma invasion. Interestingly, videomicroscopy also revealed a role for PKCι in mitosis, indicating an additional role for PKCι in the malignant phenotype of glioblastoma.
Downregulation of PKCι expression by shRNA
The motility and invasive properties of U87MG cells transduced with pshPKCιA were assessed using Transwell chambers. To examine cell motility, control and PKCι-depleted U87MG cells were seeded at the same density in Transwell chambers and 22 h later the number of cells that crossed through the chamber were counted. Stable depletion of PKCι resulted in a 65% decrease in the number of cells that crossed through the chamber (Figure 1B, top). To assess the effects on invasion, control and PKCι-depleted U87MG cells were seeded at equal densities into Transwell chambers that were coated with a Matrigel layer. PKCι depletion also caused a significant reduction (61%) in the number of cells that were able to pass through the Matrigel-coated chambers (Figure 1B, bottom). The fact that the differences in the number of cells that crossed through the chamber in the presence or absence of Matrigel are similar indicates that PKCι affects the invasion of glioblastoma cells primarily by promoting cell motility. This is the same phenotype that we described previously with transient transfection of two different RNA duplexes targeting PKCι .
Time-lapse videomicroscopy of cell motility in U87MG cells stably depleted of PKCι
Additional file 2: Video 1. Time lapse videomicroscopy of U87MG cells. The video shown is of U87MG cells that were transduced with retrovirus made with pLPCX vector expressing a shRNA to green fluorescent protein (as a control). Time lapse videomicroscopy was performed as described in Materials and Methods. Images were taken at 5 min intervals for 20 h. The video is a representative example of three videos of these cells. (MPEG 1 MB)
Additional file 3: Video 2. Time lapse videomicroscopy of U87MG cells expressing PKCι shRNA. The video shown is of U87MG cells that were transduced with retrovirus expressing PKCι shRNA. Other conditions were as in Additional file 2. The video is a representative example of three videos of these cells. (MPEG 2 MB)
Inhibition of PKCι activity using an atypical PKC specific pseudosubstrate peptide impairs cell motility
Additional file 4: Video 3. Time lapse videomicroscopy of U87MG cells treated with PKCι pseudosubstrate inhibitor peptide. The video shown is of U87MG cells treated with 20 μM pseudosubstrate peptide. Other conditions were as in Additional file 2. The video is a representative example of three videos of these cells. (MPEG 878 KB)
Effects of PKCι on Lgl in migrating glioblastoma cells
Impaired cell division in U87MG cells depleted of PKCι
We previously showed that PKCι promotes glioblastoma cell invasion . This was primarily due to the ability of PKCι to promote cell motility and was linked to repression of RhoB expression by PKCι. To extend these findings, we have used time lapse videomicroscopy to characterize the motility defects in PKCι-depleted glioblastoma cells. PKCι-depleted cells actively extended multiple short protrusions, but have a markedly reduced ability to form a single leading edge lamellipodium, an essential feature of productive cell movement. This is consistent with the established role of the atypical PKCs in generating cell polarity in multiple contexts, including apical/basolateral polarity and asymmetric cell division. Our work is also consistent with the work of Etienne-Manneville et al., which showed the presence of an atypical PKC at the leading edge of migrating astrocytes in association with Cdc42, although in their study this was ascribed to PKCζ rather than PKCι .
Depletion or inhibition of PKCι in glioblastoma cells caused a marked decrease in cell proliferation under normal tissue culture conditions. This is in contrast to findings in other cancer types, where PKCι only affected anchorage-independent proliferation [10, 25] and suggests a unique role for PKCι in glioblastoma. Videomicroscopy showed that the impaired proliferation was due, at least in part, to an impairment in mitosis. This was seen when PKCι levels were depleted by stable expression of a shRNA, or when PKCι activity was reduced using a selective inhibitor. PKCι has not been shown to have a role in mitosis previously. However, Wirtz-Peitz et al. have shown that in Drosophila atypical PKC is activated by Aurora-A kinase during mitosis and linked this activation to the establishment of asymmetric cell division in Drosophila neural precursors . Our work shows a more direct role for atypical PKC in mitosis itself. Glioblastoma cells with reduced PKCι activity appeared to have two mitosis-related defects: (1) a reduction in cells entering mitosis; (2) a delay or failure to progress through mitosis normally. Aurora-A also has roles at multiple points during mitosis . This parallel aspect to the behavior of the two kinases suggests that PKCι may be a downstream mediator of Aurora-A in mitosis. It will be important to determine if Aurora-A is in fact responsible for PKCι activation in this context. Some or all of the effects of PKCι on glioblastoma cell proliferation could be mediated by inactivation of Lgl. In both Drosophila and mice, mutational inactivation of Lgl not only causes polarity defects, but also induces uncontrolled proliferation in neural tissue [28, 29]. A role for Lgl inactivation in glioblastoma proliferation would explain our observation that we could not isolate a stable population of glioblastoma cells expressing a non-phosphorylatable version of Lgl that cannot be inactivated by PKCι.
Our data indicate show a role for PKCι in both glioblastoma cell motility and mitosis. Cell movement and mitosis are mutually exclusive events: cells arrest movement and decrease their matrix attachments prior to mitosis. It is possible that these two processes involve separate intracellular pools of PKCι. Alternatively, a limited pool of PKCι might be co-opted away from motility functions during mitosis, contributing to the uncoupling of these two processes.
PKCι promotes glioblastoma cell invasion by coordinating lamellipod leading edge formation and has a role in remodeling the cytoskeleton at the lamellipod leading edge, promoting the dissociation of Lgl from non-muscle myosin II. In addition PKCι is required for progression through mitosis in glioblastoma cells. The data presented here, along with our previously published data [16, 18], show that PKCι has a role in multiple aspects of glioblastoma cell malignancy. These include the repression of apoptosis in response to DNA damage, aberrant proliferation and metastasis. PKCι is activated by several different oncogenic mutations in glioblastoma, and appears to have a non-redundant role in mediating signaling downstream of these mutations. These features suggest that PKCι is a promising target for glioblastoma therapy that warrants further investigation.
Materials and methods
Chemicals and antibodies
Antibodies to phospho-PKCι T555 and total PKCι were from BD Biosciences (Mississauga, ON, Canada). Mouse anti-Flag M2 antibody and rabbit non-muscle myosin IIA antibody were from Sigma (Oakville, ON, Canada). Antibody to PKC consensus phosphorylation site was from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies Alexa-Fluor 488 chicken anti-rabbit and Alexa Fluor 555 goat anti-mouse were from Invitrogen (Burlington, ON, Canada). Myristoylated atypical PKC pseudosubstrate peptide was from Invitrogen (Burlington, ON, Canada).
The human glioblastoma cell line U87MG was obtained from Dr. W. Cavenee (Ludwig Institute for Cancer Research, La Jolla, CA). A172 cells were from the American Type Culture Collection. Cells were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 10% (v/v) of a 2:1 mixture of donor bovine serum and fetal bovine serum. To make glioblastoma cell lines stably depleted of PKCι, short hairpin DNA target sequences (see Additional file 1, Figure S1A) were designed and ordered from Integrated DNA Technologies (Coralville, IA, USA). Sense and antisense strands were annealed and subcloned into the pSUPER.retro.puro backbone (OligoEngine, Seattle, WA, USA). Replication-incompetent retroviruses containing pshGFP, pshPKCιA or pshPKCιB were made as described previously . Cells were gown in media containing puromycin (1 μg/mL) to select for transductants. Control retroviruses contained empty vector or shRNA to green fluorescent protein. Cells were used within three weeks of selection because of their marked growth impairment, which over time selected for cells with reduced PKCι depletion. To make U87MG cells expressing Flag-tagged Lgl, full-length LLGL1 cDNA (exact match with GenBank accession number NM_004140) was cloned from normal human astrocyte mRNA and subcloned into the retroviral vector pLPCX (Clontech, Palo Alto, CA. USA). Site-directed mutagenesis was then used to add an amino-terminal Flag epitope. Flag-tagged Lgl was expressed in U87MG cells using retroviral transduction followed by puromycin selection. Transient depletion of PKCι by RNA interference was done as described previously ; in some experiments an additional RNA duplex (designated C) was used that has been described previously .
Western blot analysis
Western blotting was performed as described previously . After electrophoretic transfer from the gel, blots were stained with amido black to confirm that equal sample loading and transfer was achieved.
RNA was isolated using the Qiagen's RNeasy Plus Mini Kit and cDNA was generated using the Qiagen Quantitect RT Kit. PCRs were then performed using the following primers: for PUM1 (reference gene), 5'TGAGGTGTGCACCATGAAC 3' and 5' CAGAATGTGCTTGCCATAGGG 3'; for PKCι, 5'GTCCGGGTGAAAGCCTACTAC 3' and 5'ACGGGTCTCCTTCCTCATCT 3'; for PKCζ 5'CCAAGAGCCTCCAGTAGACG 3' and 5'CCATCCATCCCATCGATAAC 3'.
Live cell number was determined using a Vi-Cell XR cell viability analyzer with trypan blue exclusion (Beckman Coulter Canada Inc., Mississauga, ON, Canada).
Cell motility and invasion assays
Chambers (BD Biocoat Matrigel invasion chambers, BD Biosciences, Mississauga, ON, Canada) were rehydrated and equilibrated for 2 h with 500 μL of serum free DMEM medium. After 2 h, the medium in the inserts was aspirated and inserts were placed into the wells containing complete DMEM (10% FBS:DBS). Chambers that were not coated with Matrigel (control inserts) were used to measure motility. Each chamber contains a membrane with 8 μm pores. U87MG stably expressing PKCι short hairpin were counted and resuspended in serum free DMEM medium at 1 × 105 cells/ml. Five hundred μl of cell suspension (50 000 cells) were added to each chamber. The chambers were incubated for 22 h at 37°C in a 5% CO2 atmosphere. The media was then removed and the upper surface of the membrane was scrubbed ten times with a cotton swab. Cells on the lower surface of the scrubbed membranes were fixed in 10% methanol and stained with Diff-Kwik (Dade-Behring, Newark, DE) according to the manufacturer's instructions. Three random fields were counted from each chamber under the light microscope at 40× magnification.
Time lapse videomicroscopy
Control and PKCi depleted or pseudosubstrate treated U87MG cells were plated into a Bioptechs Delta T (Butler, PA) live cell imaging plate in 2 mL of complete DMEM at a density of 103 cells to allow space for migration. Cells were maintained at 37°C in 5% CO2 for the duration of the videos. Videomicroscopy was done using an inverted microscope (Ziess Axiovert 200 M) equipped with phase-contrast microscopy using a 10× objective. Images were acquired with a CCD camera (AxioCam HRm) driven by Zeiss Axiovision 4.5 software. Phase contrast images of the cells were taken at 5 min intervals for 17-20 h and compiled to generate a time-lapse video. To quantify the migration distance per minute, cells from three independent time-lapse imaging experiments of each cell line were analyzed using the Zeiss LSM image browser software. Cell nuclei were tracked to determine migration distance and divided by the travel time. To quantify leading edges, cells from independent movies were assessed for the formation of a single dominant leading edge. Once a cell generated and formed a single leading edge it was counted. A cell was only counted once throughout the duration of the movie (i.e. if it changed direction and generated a new leading edge it was not counted a second time).
Immunofluorescence and confocal microscopy
Cells were grown in 6-well TC dishes containing glass coverslips pre-coated 0.15% Gelatin. They were washed briefly in cold (4°C) PBS and fixed in cold 4% paraformadehyde for 30 minutes. Next, cells were washed in PBS 3 times for 5 minutes each, permeablized for 10 minutes in 0.2% Triton-X 100 (diluted in PBS), and washed in PBS again 3 times for 5 minutes each. Cells were blocked in a solution made of 5% normal goat serum and 5% normal chicken serum in PBS for 30 minutes at room temperature. Cells were then incubated for 1 hour at RT with the primary antibody cocktail which consisted of 1 ug/ml Mouse anti-Flag M2 antibody and 1:200 dilution of rabbit non-muscle myosin IIA diluted in the blocking solution. Cells were washed gently 3 times for 10 minutes each in PBS. This was followed by a 45 minute at RT incubation with the secondary antibody cocktail consisting of 2 ug/ml each of Goat anti-Mouse Alexa Fluor 555 and 2 ug/ml Chicken anti-Rabbit Alexa-Fluor 488 diluted in the blocking solution. Finally the cells were washed with three 10 minutes washes in PBS, mounted on a glass slide with Prolong gold with DAPI (Invitrogen Cat # P-36931) and allowed to air dry overnight at RT in the dark. The following day, coverslips were sealed with permanent mounting media (DAKO Cat. # S3026).
Fluorescent labeling was observed using a Zeiss Observer. Z1 microscope (63X/1.40 oil DIC M27 objective) connected to a Zeiss LSM 510 Meta confocal unit. Alexa Fluor 555 was excited using the He-Ne 543 laser set at 50% power and channeled through an HFT 488/548 main dichroic filter, an NFT 545 secondary dichroic filter and a BP 560-615 IR. Images were captured using Zeiss' ZEN (version 4.5) software for the confocal microscope. AF 488 was excited with the Argon laser set at 20% power and channeled through an HFT 488/548 main dichroic filter and a BP 505-530 filter. DAPI was excited using the He-Ne 405 laser set at 20% power channeled through an HFT 405/488 main dichroic filter and a BP 420-480 filter. Image stacks were collected with the software Pinhole set at 1 Airy unit and a slice interval of 0.41 um.
All results were expressed as the mean ± S.D. Statistical analysis was performed using the Student's t test. P < 0.05 was considered statistically significant and is indicated by the symbol *.
This work was supported by grants to IL from the Canadian Institutes of Health Research. IL holds the J. Adrien and Eileen Leger Chair in Cancer Research at the Ottawa Hospital Research Institute. RMB was a Research Student of The Terry Fox Foundation through an award from the National Cancer Institute of Canada. JAP is the recipient of a Frederick Banting and Charles Best Canada Graduate Scholarship-Master's Award from the Canadian Institutes of Health Research.
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