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Reduces Cell Invasion and Motility through Inactivation of RhoA and RhoC GTPases in Head and Neck Squamous Cell Carcinoma
1 Division of Hematology and Oncology, Department of Internal Medicine, 2 Department of Otolaryngology, and 3 Comprehensive Cancer Center, University of Michigan Health System, Ann Arbor, Michigan
Requests for reprints: Quintin Pan, Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan Medical School, 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-647-3408; Fax: 734-615-2719; E-mail: qpan{at}med.umich.edu.
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Abstract |
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is a transforming oncogene and may play a role in HNSCC progression. In this study, we determine the downstream signaling pathway mediated by PKC to promote an aggressive HNSCC phenotype. RNA interference knockdown of PKC in UMSCC11A and UMSCC36, two highly invasive and motile HNSCC cell lines with elevated endogenous PKC levels, resulted in cells that were significantly less invasive and motile than the small interfering RNA–scrambled control transfectants; 51 ± 5% (P < 0.006) and 49 ± 3% (P < 0.010) inhibition in invasion and 69 ± 1% (P < 0.0005) and 66 ± 3% (P < 0.0001) inhibition in motility, respectively. PKC-deficient UMSCC11A clones had reduced levels of active and serine-phosphorylated RhoA and RhoC. Moreover, constitutive active RhoA completely rescued the invasion and motility defect, whereas constitutive active RhoC completely rescued the invasion and partially rescued the motility defect of PKC-deficient UMSCC11A clones. These results indicate that RhoA and RhoC are downstream of PKC and critical for PKC-mediated cell invasion and motility. Our study shows, for the first time, that PKC is involved in a coordinated regulation of RhoA and RhoC activation, possibly through direct post-translational phosphorylation. (Cancer Res 2006; 66(19): 9379-84) |
Introduction |
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50% of all malignancies in some developing nations. Surgery and radiotherapy are highly effective in the treatment of stage I and II disease; however, >70% of patients present with locoregionally advanced stage III and IV disease. In spite of initial aggressive therapy for HNSCC patients with advanced disease, local recurrence rates are upwards of 60% and metastatic disease develops in 15% to 25% of patients, causing a major decline in quality and length of life (1). Fewer than 30% of HNSCC patients are free of disease after 3 years and the 5-year survival rates have remained largely unchanged in the past three decades (2). Thus, critical issues in HNSCC are disease recurrence and metastasis, accounting for the high incidence of morbidity and mortality.
Protein kinase C (PKC) is a family of serine/threonine kinases known to play critical roles in the signal transduction pathways involved in proliferation, differentiation, apoptosis, and migration (3). Decades of work on PKCs have shown that PKC isoforms play heterologous, sometimes paradoxically antagonistic roles in cancer initiation and progression. Thus, it is necessary to understand the role of each individual PKC isoform in oncogenesis. Overexpression of PKC in normal fibroblasts resulted in malignant transformation with changes in morphology, serum- and anchorage-dependent growth, cell cycle progression, and the ability to form tumors in experimental animals (4, 5). Epidermis-specific PKC transgenic mice developed highly malignant and metastatic squamous cell carcinomas in response to 12-O-tetradecanoylphorbol-13-acetate stimulation (6). PKC was shown to regulate hepatocyte growth factor/c-Met signaling, a pathway implicated in angiogenesis, tumorigenesis, and metastasis in HNSCC (7–9). Our laboratory reported that elevated PKC is prognostic of lower overall and disease-free survival in patients with invasive breast cancer (10). Moreover, higher levels of PKC were found to correlate with an increase in disease recurrence and a decrease in overall survival in HNSCC (11). In this study, we report that specific inhibition of PKC is sufficient to dampen the invasive and motile phenotype of aggressive HNSCC. Our results show that targeted disruption of PKC leads to inactivation of RhoA and RhoC, indicating that the PKC-Rho GTPase signaling axis is critical for promoting an invasive and motile tumor cell phenotype in HNSCC.
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Materials and Methods |
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Generation of stable small interfering RNA-PKC UMSCC11A and UMSCC36 clones. Double-stranded oligonucleotides, 5'-GATCGATCCAAGTCAGCAC-3' of PKC were synthesized (Invitrogen, Carlsbad, CA) and cloned into pSilencer2.1-U6 hygro expression vector (Ambion, Austin, TX) and named small interfering RNA (siRNA)-PKC. A 19-bp scrambled sequence with no significant sequence homology to any known human gene sequences (silencer-negative control 1; Ambion) was cloned into pSilencer2.1-U6 hygro expression vector and named siRNA scrambled. Sequencing of siRNA-PKC and siRNA-scrambled expression vectors was done by the University of Michigan DNA Sequencing Core and verified. UMSCC11A and UMSCC36 cells were transfected with siRNA scrambled or siRNA-PKC using electroporation (Nucleofector device, Amaxa Biosystems, Gaithersburg, MD). Single clones were established by culturing transfected cells in the described medium supplemented with 100 µg/mL hygromycin (Invitrogen) for 21 days. Protein levels of PKC were determined by Western blot analysis.
Generation of PKC-deficient/G14V-RhoA and PKC-deficient/G14V-RhoC UMSCC11A clones. NH2-terminal 3x hemagglutinin (HA)–tagged constitutive active (G14V mutant) RhoA and RhoC were obtained from Gutherie cDNA Resource center (Sayre, PA). 3x HA-tagged G14V-RhoA, 3x-HA-tagged G14V-RhoC, or empty vector (pcDNA3.1) was transfected into siRNA-PKC UMSCC11A clones using electroporation. Polyclonal cell populations were established by culturing transfected cells in the described medium supplemented with 100 µg/mL hygromycin and 300 µg/mL G418 for 21 to 28 days. Protein levels of PKC, HA-tagged RhoA, and HA-tagged RhoC were determined by Western blot analysis.
Western blot analysis. Whole-cell lysates (50 µg) were mixed with Laemelli buffer, heat denatured for 3 minutes, separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membrane. Nonspecific binding was blocked by overnight incubation with 2% bovine serum albumin in TBS with 0.05% Tween 20. Immobilized proteins were probed using antibodies specific for PKC (Upstate Biotechnology, Charlottesville, VA), RhoA (Cytoskeleton, Denver, CO), RhoC (Santa Cruz Biotechnology, Santa Cruz, CA), HA (Covance, Princeton, NJ), or actin (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
Cell invasion and random motility assays. Cell invasion was determined as described from the cell invasion assay kit (Chemicon International, Temecula, CA). Cells were harvested and resuspended in serum-free medium. An aliquot (1 x 105 cells) of the prepared cell suspension was added into the chamber and incubated for 24 hours at 37°C in a 10% CO2 tissue culture incubator. Noninvading cells were gently removed from the interior of the inserts with a cotton-tipped swab. Invasive cells were stained and quantified by colorimetric reading at 560 nm. Random cell motility was determined as described from the motility assay kit (Cellomics, Pittsburgh, PA). Cells were harvested, suspended in serum-free medium, and plated on top of a field of microscopic fluorescent beads. After a 16-hour incubation period, cells were fixed and areas of clearing in the fluorescent bead field corresponding to phagokinetic cell tracks were quantified using NIH ScionImager.
Rho GTPase activation assay. Cells were lysed in 300 µL of 50 mmol/L Tris (pH 7.4), 10 mmol/L MgCl2, 500 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, and protease inhibitors. Lysates (1-2 mg) were cleared at 16,000 x g for 5 minutes, and the supernatants were rotated for 2 hours at 4°C with 60 µg glutathione S-transferase (GST)–Rho-binding domain (RBD; GST fusion protein containing the RBD of rhotekin) bound to glutathione-Sepharose beads. Samples were washed in 50 mmol/L Tris (pH 7.4), 10 mmol/L MgCl2, 150 mmol/L NaCl, 1% Triton X-100, and protease inhibitors. Western blot analyses were done on GST-RBD pull-downs with antibodies specific to RhoA, RhoC, or HA.
Statistical analysis. Data are presented as mean ± SE and analyzed using Student's t test. P < 0.05 was considered statistically significant.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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is a predictive biomarker of survival in invasive breast cancer patients and specific disruption of PKC resulted in significant inhibition in tumorigenesis and metastasis in an orthotopic model of breast cancer (10). To date, there is only one publication of note focusing on the role of PKCs in HNSCC. In this study, PKC
, PKCß, PKC, PKC
, and PKC
protein levels were shown to be elevated in the primary tumor tissue of oral cavity patients; however, only PKC was found to be a prognostic marker in this small cohort of 29 patients, even better than the traditional gold standard of tumor-node-metastasis staging (11). Elevated PKC was reported to be significantly associated with an increase in disease recurrence (P < 0.04) and a decrease in overall survival (P < 0.02). These results provide evidence that PKC promotes an aggressive cancer phenotype and that further studies on the role of PKC in HNSCC are warranted.
In our initial experiment, we determined PKC protein levels in a panel of HNSCC cell lines. PKC levels were dramatically elevated in all of the HNSCC cell lines, with the exception of UMSCC38, compared with E6/E7 immortalized oral epithelial cells (NOE; Fig. 1A
). We decided to focus our work on UMSCC11A and UMSCC36 to further examine the role of PKC in promoting an invasive and motile phenotype in HNSCC. As shown in Fig. 1B, UMSCC11A and UMSCC36 cells were significantly more motile than NOE cells; 471 ± 15% and 602 ± 26%, respectively (n = 3; P < 0.001). Moreover, UMSCC11A and UMSCC36 cells were 2-fold more invasive than NOE cells; 116 ± 6% and 110 ± 5%, respectively (n = 5; P < 0.001; Fig. 1C). These results clearly show that UMSCC11A and UMSCC36 cells, two HNSCC cell lines with elevated PKC levels, are significantly more motile and invasive than oral epithelial cells.
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would be sufficient to dampen the invasive and motile phenotype of UMSCC11A and UMSCC36 cells. Stable PKC-deficient UMSCC11A and UMSCC36 clones were generated using a H1 RNA polymerase III promoter-PKC targeting siRNA expression vector. As shown in Fig. 2A
, PKC protein levels were significantly lower in the siRNA-PKC UMSCC11A and UMSCC36 clones than in untransfected or siRNA-scrambled control cells. PKC-deficient UMSCC11A and UMSCC36 clones were significantly less invasive and motile than the parental or siRNA-scrambled control cells (Fig. 2B and C). Cell invasion was decreased by 42% to 59% (n = 3; P < 0.006) and cell motility was suppressed by 68% to 70% (n = 3; P < 0.0005) for siRNA-PKC UMSCC11A clones compared with siRNA-scrambled UMSCC11A cells. Moreover, cell invasion and cell motility for siRNA-PKC UMSCC36 clones were inhibited by 34% to 57% (n = 3; P < 0.01) and 62% to 75% (n = 3; P < 0.0001) relative to siRNA-scrambled UMSCC36 cells, respectively.
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to promote an invasive and motile phenotype is still not completely understood. Our laboratory reported that RNA interference (RNAi) knockdown of PKC in MDA-MB-231 cells, a highly metastatic breast cancer cell line with elevated endogenous PKC levels, resulted in a significant reduction in the levels of activate RhoC compared with siRNA-scrambled control cells (10). RhoC shares significant sequence homology, 82% nucleotide identity and 91% amino acid identity, with RhoA, so we determined if RhoA and RhoC were modulated through a PKC-dependent mechanism in HNSCC. PKC-deficient UMSCC11A clones had significantly lower amounts of active RhoA and RhoC levels compared with siRNA-scrambled control cells (Fig. 3A
). In silico prediction of phosphorylation sites identified multiple serine and threonine residues as putative PKC phosphorylation sites on RhoA and RhoC, suggesting that phosphorylation of RhoA and RhoC through PKC may be a possibility. So, we determined if the levels of serine- and threonine-phosphorylated RhoA and RhoC were modulated in our PKC-deficient cells. As shown in Fig. 3B, PKC-deficient UMSCC11A clones had reduced levels of serine-phosphorylated RhoA and RhoC compared with siRNA-scrambled control cells. Threonine-phosphorylated RhoA and RhoC were not detected for siRNA-scrambled control cells or PKC-deficient clones (data not shown). These results suggest that PKC-mediated regulation of RhoA and RhoC may be at the post-translational level, most likely through serine phosphorylation. Ongoing research in the laboratory is to use mass spectrometry to identify the serine residues on RhoA and RhoC that are phosphorylated by PKC.
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-mediated cell invasion and motility, constitutive active RhoA or RhoC (G14V-RhoA or G14V-RhoC) was overexpressed in PKC-deficient UMSCC11A clones to determine if restoring active RhoA or RhoC will be sufficient to rescue the PKC knockdown loss-of-function phenotype. PKC-deficient UMSCC11A clones that were generated previously were transfected with a 3x HA-tagged G14V-RhoA or G14V-RhoC neomycin-resistant expression vector and grown in selection antibiotics for 14 days, and stable polyclonal cell populations were isolated. The double-transfected PKC-deficient/G14V-RhoA-overexpressing or PKC-deficient/G14V-RhoC-overexpressing UMSCC11A cells had the proper genetic alterations and thus had elevated HA-tagged RhoA or RhoC protein levels in a PKC-deficient background (Fig. 4A
). Moreover, PKC-deficient/G14V-RhoA or PKC-deficient/G14V-RhoC cells had increased levels of active HA-tagged RhoA and RhoC, respectively. siRNA-scrambled control UMSCC11A cells are PKC positive and used as the benchmark for our phenotype rescue experiments. Empty vector transfection of PKC-deficient clones (siRNA-PKC clones 1-3) had minimal effect on cell phenotype as these cells maintained their PKC-deficient loss-of-function invasion and motility defect compared with siRNA-scrambled control (PKC positive) cells. Importantly, ectopic overexpression of constitutive active RhoA or RhoC in PKC-deficient UMSCC11A clones resulted in a significant increase in cell invasion and motility compared with PKC-deficient/empty vector control cells (P < 0.006 for invasion; P < 0.0004 for motility). As shown in Fig. 4C and D, overexpression of active RhoA was able to completely restore the invasion and motility defect of PKC-deficient UMSCC11A cells to levels comparable with PKC positive, siRNA-scrambled UMSCC11A cells; no significant difference in cell invasion (P > 0.09) and motility (P > 0.15) was determined between PKC-deficient C1-3/G14V-RhoA UMSCC11A cells and siRNA-scrambled UMSCC11A cells. Constitutive active RhoC (PKC-deficient/G14V-RhoC) was able to completely rescue the invasion defect but only was able to partially (44%) rescue the motility defect of PKC-deficient UMSCC11A cells. These results reveal that RhoA and RhoC activation are downstream of the PKC signaling cascade and required for PKC-mediated cell invasion and motility. Active RhoA or RhoC was able to completely rescue the invasion defect, suggesting that RhoA and RhoC may have overlapping roles in promoting cell invasion. Interestingly, active RhoA was more effective than active RhoC in rescuing the motility defect, suggesting that RhoA may play a more involved role than RhoC in driving the cell motility phenotype. In any event, our work indicates that inactivation of one Rho GTPase, either RhoA or RhoC, may not be adequate to reduce the incidence of tumor metastasis because RhoA and RhoC seem to have redundant functions in regulating cell invasion and motility. It is likely that a coordinated inactivation of RhoA and RhoC may be necessary to dampen the metastatic potential of aggressive HNSCC.
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It is unclear at this time how PKC modulates the activation of RhoA and RhoC in HNSCC. Several plausible explanations can be drawn from our results. Apparently, phosphorylation of RhoA and RhoC may enhance their interaction with their downstream Rho effectors through an increase in binding affinity or a decrease in binding dissociation leading to an extended Rho activation signal. Additionally, the binding affinities of the negative GDP/GTP cycle regulators, RhoGAPs and RhoGDIs, may be decreased and/or the binding affinities of the positive GDP/GTP cycle regulators, RhoGEFs, may be enhanced to phosphorylated RhoA and RhoC resulting in higher levels of RhoA and RhoC that is GTP bound. Another possibility is that PKC-mediated phosphorylation of RhoA and RhoC may enhance their protein stability through inhibition of protein degradation mechanisms resulting in an increase in the amount of total protein available for activation. This hypothesis is supported by a report showing that cyclic GMP-dependent kinase-mediated phosphorylation of RhoA protected RhoA, particularly the GTP-bound active form, from ubiquitin/proteasome-mediated degradation (20). A logical assumption is that total and active levels of Rho GTPases are concordant; however, there is no clear consensus in the literature to support this notion. Rac3 activation was found to be elevated, whereas Rac3 protein levels were unchanged in human breast cancer cell lines and tumor tissues (21). Additionally, our laboratory showed that RhoC activation levels are independent of total RhoC protein levels in HNSCC (19). Alternatively, we propose that the reduced levels of active RhoA and RhoC observed for PKC-deficient cells may be due, at least in part, to the inability of the regulatory proteins in the GDP/GDP cycle to be stimulated and/or inactivated through PKC-mediated phosphorylation. This possibility is supported by numerous studies showing that PKCs are able to regulate the activities of p115RhoGEF and RhoGDI and modulate the localization of p190 RhoGAP through direct phosphorylation (22–24). Additional work will be necessary to thoroughly examine these possibilities to better understand the mechanism of RhoA and RhoC regulation by PKC.
In summary, specific disruption of PKC was found to inhibit cell invasion and motility in aggressive HNSCC. This study shows, for the first time, that PKC is involved in a coordinated regulation of RhoA and RhoC activation; moreover, the PKC-RhoA/RhoC signaling axis may be indispensable for driving PKC-mediated cell invasion and motility.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7/17/06. Revised 8/ 2/06. Accepted 8/24/06.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts. Oncogene 1993;8:2095–104.[Medline]
and - in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J Biol Chem 1993;268:6090–6. transgenic mice: a unique model for metastatic squamous cell carcinoma. Cancer Res 2001;61:808–12. is a predictive biomarker of aggressive breast cancer and a validated target for RNA interference anti-cancer therapy. Cancer Res 2005;65:8366–71.PKC, a novel prognostic factor for relapse and survival. Head Neck 1995;17:516–25.[Medline] signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem 2001;25:22614–20.-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement. J Biol Chem 2003;278:28793–8.This article has been cited by other articles:
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  L. Xiao, M. Eto, and M. G. Kazanietz ROCK Mediates Phorbol Ester-induced Apoptosis in Prostate Cancer Cells via p21Cip1 Up-regulation and JNK J. Biol. Chem., October 23, 2009; 284(43): 29365 - 29375. [Abstract] [Full Text] [PDF]
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 L. Bao, M. A. Gorin, M. Zhang, A. C. Ventura, W. C. Pomerantz, S. D. Merajver, T. N. Teknos, A. K. Mapp, and Q. Pan Preclinical Development of a Bifunctional Cancer Cell Homing, PKC{varepsilon} Inhibitory Peptide for the Treatment of Head and Neck Cancer Cancer Res., July 15, 2009; 69(14): 5829 - 5834. [Abstract] [Full Text] [PDF]
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N. K. Basu, L. Kole, M. Basu, K. Chakraborty, P. S. Mitra, and I. S. Owens The Major Chemical-detoxifying System of UDP-glucuronosyltransferases Requires Regulated Phosphorylation Supported by Protein Kinase C J. Biol. Chem., August 22, 2008; 283(34): 23048 - 23061. [Abstract] [Full Text] [PDF]
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K.-M. Bae, H. Wang, G. Jiang, M. G. Chen, L. Lu, and L. Xiao Protein Kinase C{varepsilon} Is Overexpressed in Primary Human Non-Small Cell Lung Cancers and Functionally Required for Proliferation of Non-Small Cell Lung Cancer Cells in a p21/Cip1-Dependent Manner Cancer Res., July 1, 2007; 67(13): 6053 - 6063. [Abstract] [Full Text] [PDF]
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