Keratin Down-Regulation in Vimentin-Positive Cancer Cells Is Reversible by Vimentin RNA Interference, Which Inhibits Growth and Motility

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Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility

Rachel J. Paccione,1,2 Hiroshi Miyazaki,1 with reduced vimentin reexpressed differentiation-specific Vyomesh Patel,4 Ahmad Waseem,5 keratins K13, K14, and K15 as a result of increased gene J. Silvio Gutkind,4 Zendra E. Zehner,2,3 transcription as judged by quantitative PCR and promoter- and W. Andrew Yeudall1,2,3 reporter assays. Furthermore, cells in which vimentin expression was reduced showed a greatly decreased tumorigenic 1Philips Institute, 2Department of Biochemistry and Molecular potential, as tumors developing from these cells were 70% Biology, and 3Massey Cancer Center, Virginia Commonwealth smaller than those from control cells. The data suggest 4 University, Richmond, Virginia; Oral and Pharyngeal Cancer that reversal of the mesenchymal phenotype by inhibiting Branch, National Institute of Dental and Craniofacial Research, Bethesda, Maryland; and 5Centre for Clinical and Diagnostic Oral vimentin expression results in reexpression of epithelial char- Sciences, Barts and The London Queen Mary’s School of acteristics and reduced tumor aggressiveness. [Mol Cancer Medicine and Dentistry, Institute of Cell and Molecular Sciences, Ther 2008;7(9):2894–903] London, United Kingdom Introduction Abstract In cancers at a more advanced stage of tumor progression, At later stages of tumor progression, epithelial carcino- epithelial to mesenchymal transition (EMT) may occur genesis is associated with transition to a mesenchymal and is associated with tumor invasiveness (reviewed in phenotype, which may contribute to the more aggres- ref. 1). For example, many metastatic tumors are charac- sive properties of cancer cells and may be stimulated by terized by partial or complete EMT, in which the epithelial growth factors such as epidermal growth factor and trans- phenotype of tight intercellular junctions and polarity forming growth factor-B. Previously, we found that cells across the epithelial layer is replaced by a more mesenchy- derived from a nodal metastatic squamous cell carcinoma mal phenotype with reduced cell-cell adhesions, altered are highly proliferative and motile in vitro and tumorigenic shape, expression of mesenchymal cellular markers, and in vivo. In the current study, we have investigated the enhanced cell motility (2, 3). Thus, EMT is now recognized role of vimentin in proliferation and motility. Cells derived as a hallmark of tumor progression, characterizing highly from nodal metastasis express high levels of vimentin, invasive and metastatic carcinomas (4). which is undetectable in tumor cells derived from a syn- Intermediate filament (IF) proteins are crucial structural chronous primary lesion of tongue. Vimentin expression elements that are differentially expressed in a cell type- was enhanced by epidermal growth factor and transform- and lineage-dependent manner. There are approximately ing growth factor-B both independently and in combina- 50 different types of IF protein that are categorized into six tion. Use of RNA interference resulted in the generation subtypes (5). Keratins are the major IF proteins expressed of stable cell lines that express constitutively low levels in cells of epithelial origin, whereas vimentin is expressed of vimentin. RNA interference-mediated vimentin knock- in mesenchymal tissues, although its expression is wide- down reduced cellular proliferation, migration, and inva- spread during embryogenesis. However, when tumor cells sion through a basement membrane substitute by 3-fold undergo EMT, the expression of differentiation-specific compared with nontargeting controls. In addition, cells keratins is down-regulated with vimentin becoming the major IF protein in these cells. Thus, it has been suggested that vimentin may be a potential diagnostic marker for the initial progression of cells from a localized epithelial lesion Received 5/8/08; revised 6/17/08; accepted 6/19/08. to become migratory, metastatic tumor cells (6). Grant support: Philip Morris External Research Program UK Medical Although vimentin expression may be useful as a Research Council (COSB1A1R); Philip Morris USA (W.A. Yeudall) and biomarker of EMT, studies have suggested that vimentin Medical Research Council (A. Waseem). contributes to the altered biological properties of tumor The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked cells. For example, vimentin expression in prostate cancer advertisement in accordance with 18 U.S.C. Section 1734 solely to cells has been shown to correlate with the invasive indicate this fact. phenotype in vitro (7), whereas anchorage independence Note: R.J. Paccione and H. Miyazaki contributed equally to this work. of squamous carcinoma cells was associated with vimentin Requests for reprints: W. Andrew Yeudall, Philips Institute of Oral and positivity (8). In contrast, down-regulation of keratins has Craniofacial Molecular Biology, P.O. Box 980566, Richmond, VA 23298-0566. Phone: 804-828-6415; Fax: 804-828-0150. been linked to epithelial tumor progression. Squamous E-mail: [email protected] carcinoma cells expressing low levels of K19 were reported Copyright C 2008 American Association for Cancer Research. to have a higher capacity to invade in vitro, which was doi:10.1158/1535-7163.MCT-08-0450 decreased by ectopic expression of this IF protein (9). In

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addition, down-regulation of K15 has been reported in Quantitative Real-time PCR hyperproliferative conditions such as psoriasis and hyper- Quantitative real-time PCR (qRT-PCR) was done using trophic scarring (10), whereas K14 down-regulation was an ABI 7500 Fast system (Applied Biosystems) and a SYBR documented in a model of cervical carcinoma (11). How- Green-based procedure as described previously (16). ever, the relationship between keratin expression and tumor Oligonucleotide pairs for use as PCR primers (Supplemen- progression remains unclear, as a recent report indicated tary Table S1)7 were designed using the Primerbank Web- that K14 overexpression correlated with high-risk squa- based database (17).8 cDNA for use as template was reverse mous cell carcinoma (SCC; ref. 12). Furthermore, expression transcribed from 1 Ag total cellular RNA using standard of comparable levels of keratin and vimentin was reported procedures (SuperScript II; Invitrogen). Serial dilutions to indicate breast tumors with a poor prognosis (13). were made using previously generated PCR products, In previous studies, we compared gene expression in a assigned arbitrary values corresponding to the dilutions, cell line model derived from synchronous primary and and used to construct relative standard curves for each metastatic head and neck cancer exhibiting epithelial or gene target. mesenchymal characteristics, respectively (14). We found Western Blot Analysis down-regulation of keratin gene expression in the metas- Total protein extracts were prepared from cell lines, tasis-derived cells, together with up-regulated expression essentially as described previously (16). Cleared lysates of vimentin, which was further enhanced by transforming were combined with SDS-PAGE sample buffer, denatured growth factor-h (TGF-h) and epidermal growth factor for 5 min at 100jC, and resolved by electrophoresis in (EGF), both of which enhance motility of the metastasis- 10% (w/v) polyacrylamide-SDS gels. Fractionated proteins derived cells (14, 15). The purpose of the present study, were electroblotted onto polyvinylidene difluoride mem- therefore, was to determine the contribution of vimentin to branes (Immobilon-P; Millipore) overnight, blocked with the biological behavior of advanced stage squamous 4% (w/v) nonfat milk in 0.03% (v/v) Tween-TBS for 1 h at carcinoma cells in vitro and in vivo. room temperature, and incubated with anti-vimentin monoclonal antibody diluted 1:1,000 in blocking buffer Materials and Methods for 1 h at ambient temperature. Membranes were then washed three times with 0.03% Tween-TBS, incubated with Cells horseradish peroxidase–conjugated goat anti-mouse HN4 cells derived from a primary tongue SCC and HN12 monoclonal antibody diluted 1:10,000 in blocking buffer cells derived from a synchronous nodal metastasis were for 1 h at ambient temperature, and then washed four times cultured as described previously (15). Recombinant human h in 0.03% Tween-TBS. The specific antigen-antibody inter- EGF and TGF- 1 were purchased from Austral Biologicals actions were detected using enhanced chemiluminescence and used as described previously (15). (ECL Plus; Amersham Biosciences). Plasmids Immunofluorescence Targeting sequences for vimentin-short hairpin RNA Cells were plated on sterile coverslips and allowed to (shRNA) plasmids were designed using Web-based soft- attach for 24 h. Cells were then serum starved for 48 h, ware (Ambion).6 Sequences of sense/antisense oligonu- 7 washed, fixed with cold methanol for 20 min, washed with cleotides are shown in Supplementary Table S1 together PBS for 5 min, and blocked in 5% bovine serum albumin, with oligonucleotides for nontargeting ‘‘scrambled’’ con- 0.1% (v/v) Triton X-100 in PBS for 1 h at ambient trols. Complementary oligonucleotides were diluted to A j temperature. Cells were incubated with monoclonal anti- 10 mol/L, mixed together, denatured for 5 min at 100 C, vimentin antibody (Sigma) diluted 1:250 in blocking buffer annealed by cooling to ambient temperature, and ligated j Bam Eco overnight at 4 C, washed three times with PBS, and into HI- RI-digested pSIREN-Retro-Q retroviral incubated with a FITC-conjugated anti-mouse antibody vector (BD Biosciences). (1:500 dilution) for 1 h at ambient temperature. Coverslips Generation of Cell LinesThat Stably Express Vimentin were mounted using Vectashield (Vector Labs) and viewed shRNA with a Zeiss Axiovert 200 microscope with excitation at HN12 cells containing vimentin shRNA (shVim) or 488 nm. nontargeting control (NTC) plasmids were prepared Proliferation Assays essentially as described (16). Cells were cultured to 60% A Cells were trypsinized, washed, resuspended in complete confluency and then transfected with 3 g plasmid DNA growth medium containing 1 Ag/mL puromycin, counted, using TransIT Keratinocyte Reagent (Mirus Bio). Forty- and plated at a density of 2 Â 103 per well in 24-well plates. eight hours later, cells were selected in the presence of A Triplicate wells were counted daily for 8 consecutive days 1 g/mL puromycin. Individual puromycin-resistant colo- using a hemocytometer. nies were isolated, propagated, and characterized. Migration and Invasion Assays Cells at 70% confluence were detached from culture plates in the absence of trypsin, washed twice in DMEM/

6 http://www.ambion.com/techlib/misc/siRNA_finder.html 7 Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). 8 http://pga.mgh.harvard.edu/primerbank/index.html

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0.1% bovine serum albumin, and resuspended in DMEM/ invasion (15), we treated HN4 and HN12 cells with these 0.1% bovine serum albumin. Cells (1 Â 105) were added to growth factors and determined levels of vimentin mRNA the upper chamber of an 8 Am pore size Transwell insert and protein by qRT-PCR and Western blot, respectively. (Corning). EGF (2.5 pmol/L) in DMEM/0.1% bovine serum Figure 1C shows that both TGF-h and EGF are able to albumin was added as a chemoattractant to the lower stimulate expression of vimentin in both cell lines, although chamber and cells allowed to migrate for 7 h. Cells were the magnitude of the response is dramatically lower in fixed in 0.05% glutaraldehyde, washed, and stained with HN4. Furthermore, combination treatment with TGF-h and 0.1% crystal violet solution. Nonmigratory cells on the EGF provided a further increase in vimentin levels upper surface of the membrane were removed, the mem- compared with single growth factor treatments. brane was mounted on a microscope slide, and migrated shRNA-Mediated Down-regulation of Vimentin cells were counted in 20 random high-power fields. Expression Invasion assays were carried out in a similar manner but To investigate the contribution of vimentin to the cellular using Matrigel-coated Transwell inserts as described phenotype, we used RNA interference to inhibit expression previously (15). in HN12 cells. Plasmids targeting vimentin, or a NTC, were TumorigenicityAssays introduced into HN12 cells and stable clones selected. Assessment of tumorigenic potential was carried out as Vimentin gene expression was determined first by qRT- described previously (16), in accordance with local Institu- PCR, which indicated up to 95% inhibition of gene expres- tional Animal Care and Use Committee regulations. Briefly, sion (Fig. 2A) compared with controls. Western blotting cells were cultured to 70% confluence, trypsinized, washed, showed that cells harboring NTC plasmids expressed high counted, and resuspended in serum-free medium. Cells levels of vimentin; however, the protein was undetect- (1 Â 106) were injected s.c. on the flanks of 4-week-old able in clones containing the vimentin targeting plasmid nu/nu mice (Charles River Laboratories) using 5 mice per (Fig. 2B). A similar result was obtained by indirect immu- group. Animals were monitored daily for general health nofluorescence (Fig. 2C). Taken together, these data and tumor formation for 4 weeks post-transplantation. indicate that vimentin expression can be inhibited success- Tumors were excised from euthanized animals and tumor fully in HN12 cells, thus providing a suitable model in volume was calculated. Experiments were carried out which to assay cellular functions. using individual NTC and shVim cell lines on three sepa- Vimentin Contributes to the Growth and Motility of rate occasions. Cancer Cells Statistical Analysis Cells at a more advanced stage of tumor progression may Data obtained from migration, invasion, and qRT-PCR exhibit properties such as more rapid proliferation and assays, as well as tumorigenicity experiments, were anal- enhanced motility. Indeed, HN12 cells grow more rapidly yzed by t test using the SPSS version 13 software pack- and migrate faster than HN4 cells in in vitro assays. age (SPSS). P < 0.05 was considered to be statistically Therefore, to determine whether vimentin expression significant. contributes to these properties of HN12 cells, we first carried out proliferation assays of vimentin knockdown and control cells. Cells were seeded in 24-well culture Results plates and triplicate wells were trypsinized and counted Vimentin Is Overexpressed in Metastasis-Derived daily. As shown in Fig. 3A, growth of shVim clones was Head and Neck SCC Cells reduced compared with NTC clones (P < 0.0001). To It has long been recognized that EMT is a common investigate whether vimentin affected cell motility, we feature of malignant progression and that cells at a more carried out Transwell assays. NTC or shVim clones were advanced stage of tumor progression may express vimen- seeded in the upper chambers of Transwell inserts, and the tin, an IF protein characteristic of mesenchymal cells number of cell migrating to the lower chamber after 6 h was (18, 19). Our previous microarray studies comparing gene determined by counting. Figure 3B shows that, whereas expression in cells derived from primary head and neck NTC clones showed similar migratory abilities to parental SCC with that in cells from a lymph node metastasis HN12 cells, the motility of shVim clones was markedly indicated that vimentin expression was elevated at more reduced by over 4-fold (P < 0.0001). Similarly, in vitro advanced stages of tumor progression (14). As shown in invasion assays using Matrigel-coated membranes revealed Fig. 1A, quantitative reverse transcription-PCR experi- that the invasive potential of shVim clones was reduced by ments indicate that metastasis-derived HN12 cells express 3-fold compared with controls (Fig. 3C; P < 0.0001). Taken 5-fold higher levels of vimentin transcripts than HN4 cells, together, these data suggest that elevated expression of which were derived from a synchronous primary lesion. vimentin enhances proliferation, migration, and invasion of However, analysis of protein levels by Western blotting head and neck SCC cells. (Fig. 1B) show that, whereas there is robust expression of Down-regulation of Vimentin Affects Keratin Gene vimentin in HN12 cells, the protein is undetectable in HN4 Expression cells. As our previous data (14) indicated that vimentin As we have successfully inhibited expression of vimentin expression could be modulated by exposure to TGF-h or in HN12 cells, thereby altering biological properties asso- EGF, which are known to enhance cell migration and ciated with malignancy, we sought to determine whether

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Figure 1. Vimentin is overexpressed in advanced head and neck SCC cells and is increased by growth factors. A, HN4 and HN12 cells were cultured to 70% confluence in complete growth medium. Total RNA was prepared, reverse transcribed, and used as template for qRT-PCR as described in Mate- rials and Methods. Relative expression of vimentin is shown, normalized to actin. Bars, 1 SD. B, total protein lysates were Western blotted with the indicated antibodies. C, cells were cultured in the presence or absence of the indicated growth factors; total RNA was extracted and subjected to qRT-PCR (top). Relative expression of vimentin is shown, normalized to actin. Bars, 1 SD. Parallel cultures, similarly treated, were used to prepare protein lysates and Western blotted with the indicated antibodies (bottom).

this occurred at least in part by a reversal of EMT. K14 expression was undetectable. Conversely, K14 was Therefore, we determined the expression of differentia- reexpressed in shVim cells and vimentin expression was tion-specific keratins. Protein lysates from NTC and shVim considerably reduced. Similar results were obtained with clones, as well as the HN4 and HN12 parental cell lines, K13 and K15 (data not shown). were Western blotted with antibodies that recognize K13, To determine whether the increase in keratin expression K14, or K15. As shown in Fig. 4A, expression of these was a result of increased levels of transcription, we used keratins was high in HN4 but low or undetectable in HN12 quantitative PCR and promoter assays. As shown in cells and NTC clones. However, down-regulation of Fig. 5A, HN12/shVim cells expressed 4-fold higher levels vimentin in shVim clones resulted in up-regulation of all of K13 mRNA compared with NTC (P = 0.017). Sim- three keratins and, in the case of K14, to a level similar to ilarly, K14 and K15 expression was up-regulated 3-fold that seen in HN4 cells. Western blots were normalized (P = 0.002) and 2-fold (P = 0.023), respectively (Fig. 5A). using actin as an internal control. We also determined Transfection of a plasmid containing the K13 promoter expression using indirect immunofluorescence (Fig. 4B). upstream of an enhanced green fluorescent protein cDNA Whereas vimentin expression was strong in NTC clones, resulted in readily detectable expression of enhanced green

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fluorescent protein in HN12/shVim cells, whereas en- sion. Our previous microarray studies indicated over- hanced green fluorescent protein expression was almost expression of vimentin in HN12 cells derived from a undetectable in HN12/NTC cells (Fig. 5B). These data lymph node metastasis of a tongue SCC compared with indicate that shRNA-mediated inhibition of vimentin cells derived from the primary tumor (14). In the present results in reexpression of epithelial keratins by increasing study, we have shown that elevated vimentin expression their transcription. contributes to the biological properties of these cells, Targeted Reduction of Vimentin Inhibits Tumor including enhanced proliferation, motility, and tumorigen- Growth In vivo ic potential. In this regard, our results are consistent with To determine whether the reduced growth and motility those of others. For example, a recent study using colon of vimentin knockdown cells observed in culture resulted and breast carcinoma cell lines showed a decrease in cell in altered growth in vivo, we used a standard xenograft adhesion and migration following small interfering RNA- assay. HN12/NTC or HN12/shVim cells were transplanted mediated repression of vimentin (20). Also, it has been to the flanks of athymic mice and tumor growth was reported previously that, in in vitro wound-healing assays, monitored over a 4-week period. Compared with controls, expression of vimentin in MCF-10A breast epithelial cells tumors formed from HN12/shVim cells were 70% smaller was higher at the wound edge and correlated with (Fig. 6A; P = 0.0098). Notably, tumors formed from HN12/ migration speed (21). These authors also documented an shVim cells showed characteristics associated with a well- increase in vimentin expression and motility in the differentiated phenotype (Fig. 6C) compared with those presence of EGF. In addition to stimulating proliferation generated with the control cells (Fig. 6B). Thus, reversal of epithelial cells, EGF is a well-recognized motogenic of the mesenchymal phenotype by inhibiting vimentin factor and has been implicated in the migration of expression reduces tumorigenic potential and enhances squamous carcinoma cells (22, 23) and exposure of HN12 epithelial differentiation. cells to EGF also enhances vimentin expression (14) and motility (15). Using a model system of human papillomavirus-immortalized gingival keratinocytes, Chamulitrat et Discussion al. (24) found that exposure to ethanol, an etiologic factor The acquisition of a mesenchymal phenotype by epithelial for oral carcinogenesis, resulted in a switch to a vimentin- cancer cells is strongly associated with malignant progres- positive cell phenotype and was associated with a spindle

Figure 2. RNA interference-mediated reduction of vimentin expression. HN12 cells were transfected with plasmids encoding shVim or a NTC and clones were isolated as described in Materials and Methods. A, total RNA was prepared from the indicated cells and reverse transcribed and vimentin expression was determined by qRT-PCR, normalized to actin. B, total protein lysates were electrophoresed and Western blotted with the indicated antibodies. C, indicated cells were fixed in acetone/methanol, incubated with vimentin antibody followed by FITC-tagged secondary antibody, and counterstained with 4,6-diamidine-2-phenylindole. Original magnification, Â400.

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recognized as a feature of epithelial tumor progression (25). For example, in breast epithelia, K8 and K18 are expressed in the differentiated compartment and are down-regulated in a high percentage of breast carcinomas, a feature that correlates with a worse prognosis (13, 26, 27). Similarly, micrometastatic cell lines derived from the bone marrow of breast cancer patients showed loss of cytokeratin expression with concomitant up-regulation of vimentin (28). Furthermore, these authors were able to correlate changes in keratin and vimentin expression with more aggressive lesions, indicating clinical significance. In addition, K14 expression has been reported to be dramatically reduced in cervical SCC as well as in cervical epithelial cells transformed by human papillomavirus-16 and activated ras (11), whereas more recent studies using cDNA micro- arrays indicate down-regulation of K13 in head and neck SCC tissues (29). Recent proteomic analysis has also revealed a decrease in differentiation-associated keratins and an increase in vimentin expression in advanced tongue cancer (30). Our own previous studies documented a decrease in expression of multiple keratins, including K13, K14, and K15, in metastasis-derived HN12 cells compared with their primary counterpart, providing further correlation of keratin down-regulation with increased tumor aggressiveness (14). However, the findings of the current study (that keratins associated with differentiation become reexpressed on targeted inhibition of vimentin) were unexpected. At present, the mechanism regulating this is unclear. One possibility is that vimentin regulates expression of a factor(s) that acts to repress keratin expression directly. The promoters of both K14 and K15 have been shown to be repressed by nuclear factor-nB transcription factors (31), which are frequently up-regulated during malignant progression (32). It has been reported previously that vimentin expression is enhanced by nuclear factor-nB (33); whether vimentin enhances NF-nB activity is unknown. There has been recent debate over the involvement of EMT in tumor progression (34–36). Our data support a role for the acquisition of mesenchymal characteristics in more advanced stages of squamous carcinogenesis, with expres- Figure 3. Vimentin knockdown inhibits growth and motility. A, NTC sion of vimentin, repression of keratin expression, and and shVim independent clones were seeded in 24-well plates. Triplicate altered cell morphology together with enhanced cell moti- wells were trypsinized and counted daily for 8 d. B, indicated cells were lity and tumorigenic potential. However, it seems clear seeded in the upper chamber of Transwell inserts and allowed to migrate for 6 h. Migrated cells were fixed, stained, and counted in 20 random high- that this process is reversible, at least to some extent, as power fields. C, indicated cells were seeded in the upper chamber of inhibiting the expression of mesenchymal IF proteins Matrigel-coated Transwell inserts and allowed to migrate for 16 h. results in reemergence of an epithelial IF network and Migrated cells were fixed, stained, and counted in 20 random high-power concomitant reduction in tumor cell aggressiveness in vitro fields. and in vivo. Thus, our studies support the notion of epithelial plasticity during tumor progression (37, 38). morphology and growth in soft agar. Thus, factors In a ras-driven model of breast tumor progression, Huber important for oral tumor progression such as EGF, human et al. showed a central requirement for nuclear factor-nBin papillomavirus, and alcohol can alter the IF profile of the acquisition of a mesenchymal and metastatic pheno- epithelial cells and their biological properties. type (37). Consistent with our observations, these authors Transition from an epithelial to a mesenchymal pheno- reported up-regulation of vimentin and decreased expres- type is not only associated with expression of vimentin but sion of keratin 14, among other targets. It must be recog- also with altered expression of differentiation-specific nized, however, that multiple proteins are involved in EMT keratins. Dysregulation of keratin expression has long been during tumor progression, in addition to vimentin. For

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example, several key transcription factors play direct roles, consistent with our own results indicating roles for including Snail, Slug, Twist, and FoxC2 (39, 40), with the phosphoinositide 3-kinase and multiple mitogen-activated potential to alter expression of multiple transcriptional protein kinase pathways in EGF-and TGF- h-mediated cell targets. In addition, down-regulation of E-cadherin and motility (14, 15). Indeed, the participation of TGF-h in the associated loss of adherens junctions are hallmarks of emergence of mesenchymal characteristics during tumor tumor-associated EMT. Notably, ZEB1 and ZEB2, which progression has been extensively documented in several are repressors of E-cadherin, are also elevated during this systems (43). Moreover, TGF-h has been shown to enhance process (39). Furthermore, ZEB2 has also been shown to vimentin gene expression directly despite the lack of a regulate vimentin expression in breast cancer cells (41). consensus TGF-h response element in the vimentin We found that exposure of cells to EGF and TGF-h promoter (44). Here, Smads were found bound to tandem enhanced the expression of a mesenchymal IF network. In activator protein-1 sites in the vimentin promoter, in addition, treatment with both growth factors elevated complex with activator protein-1 family members c-jun vimentin levels further. Recent studies in a model of and c-fos, together with Sp1/Sp3. An interesting study intestinal carcinogenesis reported synergy between these by Han et al. used a model system of mice transgenic in two growth factors in producing EMT in a ERK and the epidermis for a dominant-negative form of the type II phosphoinositide 3-kinase-dependent manner (42) as well TGF-h receptor and/or TGF-h1 (45). These authors showed as regulation of migration and invasion. These data are a requirement for TGFhRII signaling via Smads in the

Figure 4. Cells with vimentin knockdown reex- press differentiation-specific keratins. A, total protein lysates were prepared from parental, vimentin knockdown and control cells and then electrophoresed and Western blotted with the antibodies indicated. B, vimentin knockdown and control cells were subjected to indirect immunofluorescence with vimentin or keratin 14 antibodies or normal mouse IgG as control. Bound antibody was detected with FITC-conjugated secondary antibody. Cells were counterstained with 4,6-diamidine-2-phenylindole and viewed by fluores- cence microscopy. Original magnification, Â400.

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Figure 5. Keratin reexpression is due to enhanced mRNA levels. A, control and vimentin-knockdown cells were subjected to qRT-PCR analysis for keratin 13, 14, or 15 as indicated. Relative expression is shown, normalized to actin. B, HN12/NTC or HN12/shVIM cells were transiently transfected with a plasmid containing the K13 promoter upstream of an enhanced green fluorescent protein reporter sequence. Forty-eight hours later, total cell lysates were prepared and Western blotted with the indicated antibodies. induction of EMT; however, lack of a functional receptor to manage human cancer. In this regard, our data indicate suppressed EMT but enhanced the development of meta- that targeted suppression of vimentin is sufficient to inhibit static tumors, thus drawing a distinction between the path- migration and invasion in vitro as well as growth in vitro ways required for these two components of advanced-stage and in vivo. We found that RNA interference-mediated tumors. inhibition of vimentin expression resulted in reemergence The ability to reverse the advanced phenotype of meta- of keratin gene expression in cell culture and the produc- static tumors cells may be of use as a therapeutic approach tion of smaller, more differentiated tumors in athymic mice,

Figure 6. Down-regulation of vimentin inhibits tumor growth in vivo. A, vimentin knockdown (shVim) and control cells were transplanted to the flanks of athymic mice and tumor formation was monitored over a 4-wk period as described in Materials and Methods. Tumor volume is indicated F SE. B and C, tumor sections stained with H&E. HN12/NTC (B) and HN12/shVIM (C). Original magnification, Â100.

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suggesting potential for development as a treatment moda- 17. Wang X, Seed B. A PCR primer bank for quantitative gene expression analysis. Nucl Acids Res 2003;31:e154:1 – 8. lity. Pharmacology-based differentiation therapies have 18. Hendrix MJ, Seftor EA, Chu YW, Trevor KT, Seftor RE. Role of been developed by others, including the use of retinoids intermediate filaments in migration, invasion and metastasis. Cancer (46–48) and butyrates (49). Combination therapy of reti- Metastasis Rev 1996;15:507 – 25. noid acid receptor and retinoid X receptor agonists with 19. Kokkinos MI, Wafai R, Wong MK, Newgreen DF, Thompson EW, Waltham M. Vimentin and epithelial-mesenchymal transition in human chemotherapeutic drugs has shown some promise clin- breast cancer-observations in vitro and in vivo. Cells Tissues Organs ically in the treatment of lung cancer (50). In the present 2007;185:191 – 203. study, we achieved a partial response purely by targeting 20. McInroy L, Maatta A. Down-regulation of vimentin expression inhibits vimentin expression. Based on this finding and work by carcinoma cell migration and adhesion. Biochem Biophys Res Commun 2007;360:109 – 14. others, developing a combined therapeutic approach for 21. Gilles C, Polette M, Zahm JM, et al. Vimentin contributes to human SCC is warranted. mammary epithelial cell migration. J Cell Sci 1999;112:4615 – 25. 22. Xie H, Pallero MA, Gupta K, et al. EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of Disclosure of Potential Conflicts of Interest the motility-associated PLCg signaling pathway. J Cell Sci 1998;111: 615 – 24. No potential conflicts of interest were disclosed. 23. Kassis J, Lauffenburger DA, Turner T, Wells A. Tumor invasion as dysregulated cell motility. Semin Cancer Biol 2001;11:105 – 17. References 24. Chamulitrat W, Schmidt R, Chunglok W, Kohl A, Tomakidi P. Epithelium and fibroblast-like phenotypes derived from HPV16 E6/ 1. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. E7-immortalized human gingival keratinocytes following chronic ethanol Nat Rev Cancer 2002;2:442 – 54. treatment. Eur J Cell Biol 2003;82:313 – 22. 2. Ruiz P, Gunthert U. The cellular basis of metastasis. World J Urol 1996; 25. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of 14:141 – 50. human cytokeratins: patterns of expression in normal epithelia, tumors and 3. Boyer B, Valles AM, Edme N. Induction and regulation of epithelial- cultured cells. Cell 1982;31:11 – 24. mesenchymal transitions. Biochem Pharmacol 2000;60:1091 – 9. 26. Schaller G, Fuchs I, Pritze W, et al. Elevated keratin 18 protein 4. Birchmeier C, Birchmeier W, Brand-Saberi B. Epithelial-mesenchymal expression indicates a favorable prognosis in patients with breast cancer. transitions in cancer progression. Acta Anat (Basel) 1996;156:217 – 26. Clin Cancer Res 1996;2:1879 – 85. 5. Kim S, Coulombe PA. Intermediate filament scaffolds fulfill mechanical, 27. Woelfle U, Sauter G, Santjer S, Brakenhoff R, Pantel K. Down- organizational, and signaling functions in the cytoplasm. Genes Dev 2007; regulated expression of cytokeratin 18 promotes progression of human 21:1581 – 97. breast cancer. Clin Cancer Res 2004;10:2670 – 4. 6. Gilles C, Polette M, Piette J, et al. Vimentin expression in cervical 28. Willipinski-Stapelfeldt B, Riethdorf S, Assmann V, et al. Changes in carcinomas: association with invasive and migratory potential. J Pathol cytoskeletal protein composition indicative of an epithelial-mesenchymal 1996;180:175 – 80. transition in human micrometastatic and primary breast carcinoma cells. 7. Singh S, Sadacharan S, Su S, Belldegrun A, Persad S, Singh G. Clin Cancer Res 2005;11:8006 – 14. Overexpression of vimentin: role in the invasive phenotype in an androgen- 29. Kainuma K, Katsuno S, Hashimoto S, et al. Differences in the independent model of prostate cancer. Cancer Res 2003;63:2306 – 11. expression of genes between normal tissue and squamous cell carcinomas 8. Tomson AM, Scholma J, Meijer B, Koning JG, de Jong KM, van der of head and neck using cancer-related gene cDNA microarray. Acta Werf M. Adhesion properties, intermediate filaments and malignant Otolaryngol 2006;126:967 – 74. behaviour of head and neck squamous cell carcinoma cells in vitro. Clin 30. Patel V, Hood BL, Molinolo AA, et al. Proteomic analysis of laser- Exp Metastasis 1996;14:501 – 11. captured paraffin-embedded tissues: a molecular portrait of head and neck 9. Crowe DL, Milo GE, Shuler CF. Keratin 19 downregulation by oral cancer progression. Clin Cancer Res 2008;14:1002 – 14. squamous cell carcinoma lines increases invasive potential. J Dent Res 31. Blumenberg M. Transcriptional regulation of keratin gene expression. 1999;78:1256 – 63. In: Paramio J, editor. Intermediate filaments. New York (NY): Springer; 10. Waseem A, Dogan B, Tidman N, et al. Keratin 15 expression in 2006. p. 93 – 109. stratified epithelia: downregulation in activated keratinocytes. J Invest 32. Karin M, Cao Y, Greten FR, Li ZW. NF-nB in cancer: from innocent Dermatol 1999;112:362 – 9. bystander to major culprit. Nat Rev Cancer 2002;2:301 – 10. 11. Bowden PE, Woodworth CD, Doniger J, DiPaolo JA. Down-regulation 33. Chen JH, Vercamer C, Li Z, Paulin D, Vandenbunder B, Stehelin D. of keratin 14 gene expression after v-Ha-ras transfection of human PEA3 transactivates vimentin promoter in mammary epithelial and tumor papillomavirus-immortalized human cervical epithelial cells. Cancer Res cells. Oncogene 1996;13:1667 – 75. 1992;52:5865 – 71. 34. Tarin D, Thompson EW, Newgreen DF. The fallacy of epithelial 12. Chung CH, Parker JS, Ely K, et al. Gene expression profiles identify mesenchymal transition in neoplasia. Cancer Res 2005;65:5996 – 6001. epithelial-to-mesenchymal transition and activation of nuclear factor-nB 35. Thompson EW, Newgreen DF, Tarin D. Carcinoma invasion and signaling as characteristics of a high-risk head and neck squamous cell metastasis: a role for epithelial-mesenchymal transition? Cancer Res carcinoma. Cancer Res 2006;66:8210 – 8. 2005;65:5991 – 5. 13. Thomas PA, Kirschmann DA, Cerhan JR, et al. Association between 36. Garber K. Epithelial-to-mesenchymal transition is important to keratin and vimentin expression, malignant phenotype, and survival metastasis, but questions remain. J Natl Cancer Inst 2008;100:232 – 9. in postmenopausal breast cancer patients. Clin Cancer Res 1999;5: 2698 – 703. 37. Huber MA, Azoitei N, Baumann B, et al. NF-nB is essential for epithelial-mesenchymal transition and metastasis in a model of breast 14. Miyazaki H, Patel V, Wang H, Ensley JF, Gutkind JS, Yeudall WA. cancer progression. J Clin Invest 2004;114:569 – 81. Growth factor-sensitive molecular targets identified in primary and metastatic head and neck squamous cell carcinoma using microarray 38. Gotzmann J, Mikula M, Eger A, et al. Molecular aspects of epithelial analysis. Oral Oncol 2006;42:240 – 56. cell plasticity: implications for local tumor invasion and metastasis. Mutat Res 2004;566:9 – 20. 15. Yeudall WA, Miyazaki H, Ensley JF, Cardinali M, Gutkind JS, Patel V. Uncoupling of epidermal growth factor-dependent proliferation and 39. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour invasion in a model of squamous carcinoma progression. Oral Oncol progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2005;41:698 – 708. 2007;7:415 – 28. 16. Miyazaki H, Patel V, Wang H, Edmunds RK, Gutkind JS, Yeudall WA. 40. Mani SA, Yang J, Brooks M, et al. Mesenchyme forkhead 1 (FOXC2) Downregulation of CXCL5 inhibits squamous carcinogenesis. Cancer Res plays a key role in metastasis and is associated with aggressive basal-like 2006;66:4279 – 84. breast cancers. Proc Natl Acad Sci US A 2007;104:10069 – 74.

Mol Cancer Ther 2008;7(9). September 2008

41. Bindels S, Mestdagt M, Vandewalle C, et al. Regulation of vimentin 46. Clarke N, Germain P, Altucci L, Gronemeyer H. Retinoids: potential in by SIP1 in human epithelial breast tumor cells. Oncogene 2006;25: cancer prevention and therapy. Expert Rev Mol Med 2004;6:1 – 23. 4975 – 85. 47. Short SC, Suovuori A, Cook G, Vivian G, Harmer C. A phase II study 42. Uttamsingh S, Bao X, Nguyen KT, et al. Synergistic effect between using retinoids as redifferentiation agents to increase iodine uptake in EGF and TGF-h1 in inducing oncogenic properties of intestinal epithelial metastatic thyroid cancer. Clin Oncol (R Coll Radiol) 2004;16:569 – 74. cells. Oncogene 2007;27:2626 – 34. 48. Tighe AP, Talmage DA. Retinoids arrest breast cancer cell prolifera- 43. Zavadil J, Bottinger EP. TGF-h and epithelial-to-mesenchymal tran- tion: retinoic acid selectively reduces the duration of receptor tyrosine sitions. Oncogene 2005;24:5764 – 74. kinase signaling. Exp Cell Res 2004;301:147 – 57. 44. Wu Y, Zhang X, Salmon M, Lin X, Zehner ZE. TGFh1 regulation of 49. Butt AJ, Hague A, Paraskeva C. Butyrate- but not TGFh1-induced vimentin gene expression during differentiation of the C2C12 skeletal apoptosis of colorectal adenoma cells is associated with increased myogenic cell line requires Smads, AP-1 and Sp1 family members. Biochim expression of the differentiation markers E-cadherin and alkaline phospha- Biophys Acta 2007;1773:427 – 39. tase. Cell Death Differ 1997;4:725 – 32. 45. Han G, Lu SL, Li AG, et al. Distinct mechanisms of TGF-h1-mediated 50. Nandan R. Promising results achieved with a combination of epithelial-to-mesenchymal transition and metastasis during skin carcino- chemotherapy and two retinoids in patients with advanced non-small-cell genesis. J Clin Invest 2005;115:1714 – 23. lung cancer. Lung Cancer 2006;51:387 – 8.

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