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Research Articles: Therapeutics, Targets, and Development

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

Rachel J. Paccione, Hiroshi Miyazaki, Vyomesh Patel, Ahmad Waseem, J. Silvio Gutkind, Zendra E. Zehner and W. Andrew Yeudall
DOI: 10.1158/1535-7163.MCT-08-0450 Published September 2008

Abstract

At later stages of tumor progression, epithelial carcinogenesis is associated with transition to a mesenchymal phenotype, which may contribute to the more aggressive properties of cancer cells and may be stimulated by growth factors such as epidermal growth factor and transforming growth factor-β. Previously, we found that cells derived from a nodal metastatic squamous cell carcinoma are highly proliferative and motile in vitro and tumorigenic in vivo. In the current study, we have investigated the role of vimentin in proliferation and motility. Cells derived from nodal metastasis express high levels of vimentin, which is undetectable in tumor cells derived from a synchronous primary lesion of tongue. Vimentin expression was enhanced by epidermal growth factor and transforming growth factor-β both independently and in combination. Use of RNA interference resulted in the generation of stable cell lines that express constitutively low levels of vimentin. RNA interference-mediated vimentin knockdown reduced cellular proliferation, migration, and invasion through a basement membrane substitute by 3-fold compared with nontargeting controls. In addition, cells with reduced vimentin reexpressed differentiation-specific keratins K13, K14, and K15 as a result of increased gene transcription as judged by quantitative PCR and promoter-reporter assays. Furthermore, cells in which vimentin expression was reduced showed a greatly decreased tumorigenic potential, as tumors developing from these cells were 70% smaller than those from control cells. The data suggest that reversal of the mesenchymal phenotype by inhibiting vimentin expression results in reexpression of epithelial characteristics and reduced tumor aggressiveness. [Mol Cancer Ther 2008;7(9):2894–903]

Keywords:
  • EMT
  • growth factor
  • migration
  • invasion
  • oral cancer
  • gene regulation

Introduction

In cancers at a more advanced stage of tumor progression, epithelial to mesenchymal transition (EMT) may occur and is associated with tumor invasiveness (reviewed in ref. 1). For example, many metastatic tumors are characterized by partial or complete EMT, in which the epithelial phenotype of tight intercellular junctions and polarity across the epithelial layer is replaced by a more mesenchymal phenotype with reduced cell-cell adhesions, altered shape, expression of mesenchymal cellular markers, and enhanced cell motility (2, 3). Thus, EMT is now recognized as a hallmark of tumor progression, characterizing highly invasive and metastatic carcinomas (4).

Intermediate filament (IF) proteins are crucial structural elements that are differentially expressed in a cell type- and lineage-dependent manner. There are approximately 50 different types of IF protein that are categorized into six subtypes (5). Keratins are the major IF proteins expressed in cells of epithelial origin, whereas vimentin is expressed in mesenchymal tissues, although its expression is widespread during embryogenesis. However, when tumor cells undergo EMT, the expression of differentiation-specific 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 to become migratory, metastatic tumor cells (6).

Although vimentin expression may be useful as a biomarker of EMT, studies have suggested that vimentin contributes to the altered biological properties of tumor cells. For example, vimentin expression in prostate cancer cells has been shown to correlate with the invasive phenotype in vitro (7), whereas anchorage independence of squamous carcinoma cells was associated with vimentin positivity (8). In contrast, down-regulation of keratins has been linked to epithelial tumor progression. Squamous carcinoma cells expressing low levels of K19 were reported to have a higher capacity to invade in vitro, which was decreased by ectopic expression of this IF protein (9). In addition, down-regulation of K15 has been reported in hyperproliferative conditions such as psoriasis and hypertrophic scarring (10), whereas K14 down-regulation was documented in a model of cervical carcinoma (11). However, the relationship between keratin expression and tumor progression remains unclear, as a recent report indicated that K14 overexpression correlated with high-risk squamous cell carcinoma (SCC; ref. 12). Furthermore, expression of comparable levels of keratin and vimentin was reported to indicate breast tumors with a poor prognosis (13).

In previous studies, we compared gene expression in a cell line model derived from synchronous primary and metastatic head and neck cancer exhibiting epithelial or mesenchymal characteristics, respectively (14). We found down-regulation of keratin gene expression in the metastasis-derived cells, together with up-regulated expression of vimentin, which was further enhanced by transforming growth factor-β (TGF-β) and epidermal growth factor (EGF), both of which enhance motility of the metastasis-derived cells (14, 15). The purpose of the present study, therefore, was to determine the contribution of vimentin to the biological behavior of advanced stage squamous carcinoma cells in vitro and in vivo.

Materials and Methods

Cells

HN4 cells derived from a primary tongue SCC and HN12 cells derived from a synchronous nodal metastasis were cultured as described previously (15). Recombinant human EGF and TGF-β1 were purchased from Austral Biologicals and used as described previously (15).

Plasmids

Targeting sequences for vimentin-short hairpin RNA (shRNA) plasmids were designed using Web-based software (Ambion).6 Sequences of sense/antisense oligonucleotides are shown in Supplementary Table S17 together with oligonucleotides for nontargeting “scrambled” controls. Complementary oligonucleotides were diluted to 10 μmol/L, mixed together, denatured for 5 min at 100°C, annealed by cooling to ambient temperature, and ligated into BamHI-EcoRI-digested pSIREN-Retro-Q retroviral vector (BD Biosciences).

Generation of Cell Lines That Stably Express Vimentin shRNA

HN12 cells containing vimentin shRNA (shVim) or nontargeting control (NTC) plasmids were prepared essentially as described (16). Cells were cultured to 60% confluency and then transfected with 3 μg plasmid DNA using TransIT Keratinocyte Reagent (Mirus Bio). Forty-eight hours later, cells were selected in the presence of 1 μg/mL puromycin. Individual puromycin-resistant colonies were isolated, propagated, and characterized.

Quantitative Real-time PCR

Quantitative real-time PCR (qRT-PCR) was done using an ABI 7500 Fast system (Applied Biosystems) and a SYBR Green-based procedure as described previously (16). Oligonucleotide pairs for use as PCR primers (Supplementary Table S1)7 were designed using the Primerbank Web-based database (17).8 cDNA for use as template was reverse transcribed from 1 μg total cellular RNA using standard procedures (SuperScript II; Invitrogen). Serial dilutions were made using previously generated PCR products, assigned arbitrary values corresponding to the dilutions, and used to construct relative standard curves for each gene target.

Western Blot Analysis

Total protein extracts were prepared from cell lines, essentially as described previously (16). Cleared lysates were combined with SDS-PAGE sample buffer, denatured for 5 min at 100°C, and resolved by electrophoresis in 10% (w/v) polyacrylamide-SDS gels. Fractionated proteins were electroblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore) overnight, blocked with 4% (w/v) nonfat milk in 0.03% (v/v) Tween-TBS for 1 h at room temperature, and incubated with anti-vimentin monoclonal antibody diluted 1:1,000 in blocking buffer for 1 h at ambient temperature. Membranes were then washed three times with 0.03% Tween-TBS, incubated with horseradish peroxidase–conjugated goat anti-mouse monoclonal antibody diluted 1:10,000 in blocking buffer for 1 h at ambient temperature, and then washed four times in 0.03% Tween-TBS. The specific antigen-antibody interactions were detected using enhanced chemiluminescence (ECL Plus; Amersham Biosciences).

Immunofluorescence

Cells were plated on sterile coverslips and allowed to attach for 24 h. Cells were then serum starved for 48 h, washed, fixed with cold methanol for 20 min, washed with PBS for 5 min, and blocked in 5% bovine serum albumin, 0.1% (v/v) Triton X-100 in PBS for 1 h at ambient temperature. Cells were incubated with monoclonal anti-vimentin antibody (Sigma) diluted 1:250 in blocking buffer overnight at 4°C, washed three times with PBS, and incubated with a FITC-conjugated anti-mouse antibody (1:500 dilution) for 1 h at ambient temperature. Coverslips were mounted using Vectashield (Vector Labs) and viewed with a Zeiss Axiovert 200 microscope with excitation at 488 nm.

Proliferation Assays

Cells were trypsinized, washed, resuspended in complete growth medium containing 1 μg/mL puromycin, counted, and plated at a density of 2 × 103 per well in 24-well plates. Triplicate wells were counted daily for 8 consecutive days using a hemocytometer.

Migration and Invasion Assays

Cells at 70% confluence were detached from culture plates in the absence of trypsin, washed twice in DMEM/0.1% bovine serum albumin, and resuspended in DMEM/0.1% bovine serum albumin. Cells (1 × 105) were added to the upper chamber of an 8 μm pore size Transwell insert (Corning). EGF (2.5 pmol/L) in DMEM/0.1% bovine serum albumin was added as a chemoattractant to the lower chamber and cells allowed to migrate for 7 h. Cells were fixed in 0.05% glutaraldehyde, washed, and stained with 0.1% crystal violet solution. Nonmigratory cells on the upper surface of the membrane were removed, the membrane was mounted on a microscope slide, and migrated cells were counted in 20 random high-power fields. Invasion assays were carried out in a similar manner but using Matrigel-coated Transwell inserts as described previously (15).

Tumorigenicity Assays

Assessment of tumorigenic potential was carried out as described previously (16), in accordance with local Institutional Animal Care and Use Committee regulations. Briefly, cells were cultured to 70% confluence, trypsinized, washed, counted, and resuspended in serum-free medium. Cells (1 × 106) were injected s.c. on the flanks of 4-week-old nu/nu mice (Charles River Laboratories) using 5 mice per group. Animals were monitored daily for general health and tumor formation for 4 weeks post-transplantation. Tumors were excised from euthanized animals and tumor volume was calculated. Experiments were carried out using individual NTC and shVim cell lines on three separate occasions.

Statistical Analysis

Data obtained from migration, invasion, and qRT-PCR assays, as well as tumorigenicity experiments, were analyzed by t test using the SPSS version 13 software package (SPSS). P < 0.05 was considered to be statistically significant.

Results

Vimentin Is Overexpressed in Metastasis-Derived Head and Neck SCC Cells

It has long been recognized that EMT is a common feature of malignant progression and that cells at a more advanced stage of tumor progression may express vimentin, an IF protein characteristic of mesenchymal cells (18, 19). Our previous microarray studies comparing gene expression in cells derived from primary head and neck SCC with that in cells from a lymph node metastasis indicated that vimentin expression was elevated at more advanced stages of tumor progression (14). As shown in Fig. 1A , quantitative reverse transcription-PCR experiments indicate that metastasis-derived HN12 cells express 5-fold higher levels of vimentin transcripts than HN4 cells, which were derived from a synchronous primary lesion. However, analysis of protein levels by Western blotting (Fig. 1B) show that, whereas there is robust expression of vimentin in HN12 cells, the protein is undetectable in HN4 cells. As our previous data (14) indicated that vimentin expression could be modulated by exposure to TGF-β or EGF, which are known to enhance cell migration and invasion (15), we treated HN4 and HN12 cells with these growth factors and determined levels of vimentin mRNA and protein by qRT-PCR and Western blot, respectively. Figure 1C shows that both TGF-β and EGF are able to stimulate expression of vimentin in both cell lines, although the magnitude of the response is dramatically lower in HN4. Furthermore, combination treatment with TGF-β and EGF provided a further increase in vimentin levels compared with single growth factor treatments.

Figure 1.
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 Materials 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).

shRNA-Mediated Down-regulation of Vimentin Expression

To investigate the contribution of vimentin to the cellular phenotype, we used RNA interference to inhibit expression in HN12 cells. Plasmids targeting vimentin, or a NTC, were introduced into HN12 cells and stable clones selected. Vimentin gene expression was determined first by qRT-PCR, which indicated up to 95% inhibition of gene expression (Fig. 2A ) compared with controls. Western blotting showed that cells harboring NTC plasmids expressed high levels of vimentin; however, the protein was undetectable in clones containing the vimentin targeting plasmid (Fig. 2B). A similar result was obtained by indirect immunofluorescence (Fig. 2C). Taken together, these data indicate that vimentin expression can be inhibited successfully in HN12 cells, thus providing a suitable model in which to assay cellular functions.

Figure 2.
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, ×400.

Vimentin Contributes to the Growth and Motility of Cancer Cells

Cells at a more advanced stage of tumor progression may exhibit properties such as more rapid proliferation and enhanced motility. Indeed, HN12 cells grow more rapidly and migrate faster than HN4 cells in in vitro assays. Therefore, to determine whether vimentin expression 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 plates and triplicate wells were trypsinized and counted daily. As shown in Fig. 3A , growth of shVim clones was reduced compared with NTC clones (P < 0.0001). To investigate whether vimentin affected cell motility, we carried out Transwell assays. NTC or shVim clones were seeded in the upper chambers of Transwell inserts, and the number of cell migrating to the lower chamber after 6 h was determined by counting. Figure 3B shows that, whereas NTC clones showed similar migratory abilities to parental HN12 cells, the motility of shVim clones was markedly reduced by over 4-fold (P < 0.0001). Similarly, in vitro invasion assays using Matrigel-coated membranes revealed that the invasive potential of shVim clones was reduced by 3-fold compared with controls (Fig. 3C; P < 0.0001). Taken together, these data suggest that elevated expression of vimentin enhances proliferation, migration, and invasion of head and neck SCC cells.

Figure 3.
Figure 3.

Vimentin knockdown inhibits growth and motility. A, NTC and shVim independent clones were seeded in 24-well plates. Triplicate wells were trypsinized and counted daily for 8 d. B, indicated cells were 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-power fields. C, indicated cells were seeded in the upper chamber of Matrigel-coated Transwell inserts and allowed to migrate for 16 h. Migrated cells were fixed, stained, and counted in 20 random high-power fields.

Down-regulation of Vimentin Affects Keratin Gene Expression

As we have successfully inhibited expression of vimentin in HN12 cells, thereby altering biological properties associated with malignancy, we sought to determine whether this occurred at least in part by a reversal of EMT. Therefore, we determined the expression of differentiation-specific keratins. Protein lysates from NTC and shVim clones, as well as the HN4 and HN12 parental cell lines, were Western blotted with antibodies that recognize K13, K14, or K15. As shown in Fig. 4A , expression of these keratins was high in HN4 but low or undetectable in HN12 cells and NTC clones. However, down-regulation of vimentin in shVim clones resulted in up-regulation of all three keratins and, in the case of K14, to a level similar to that seen in HN4 cells. Western blots were normalized using actin as an internal control. We also determined expression using indirect immunofluorescence (Fig. 4B). Whereas vimentin expression was strong in NTC clones, K14 expression was undetectable. Conversely, K14 was reexpressed in shVim cells and vimentin expression was considerably reduced. Similar results were obtained with K13 and K15 (data not shown).

Figure 4.
Figure 4.

Cells with vimentin knockdown reexpress 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 fluorescence microscopy. Original magnification, ×400.

To determine whether the increase in keratin expression was a result of increased levels of transcription, we used quantitative PCR and promoter assays. As shown in Fig. 5A , HN12/shVim cells expressed 4-fold higher levels of K13 mRNA compared with NTC (P = 0.017). Similarly, K14 and K15 expression was up-regulated 3-fold (P = 0.002) and 2-fold (P = 0.023), respectively (Fig. 5A). Transfection of a plasmid containing the K13 promoter upstream of an enhanced green fluorescent protein cDNA resulted in readily detectable expression of enhanced green fluorescent protein in HN12/shVim cells, whereas enhanced green fluorescent protein expression was almost undetectable in HN12/NTC cells (Fig. 5B). These data indicate that shRNA-mediated inhibition of vimentin results in reexpression of epithelial keratins by increasing their transcription.

Figure 5.
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.

Targeted Reduction of Vimentin Inhibits Tumor Growth In vivo

To determine whether the reduced growth and motility of vimentin knockdown cells observed in culture resulted in altered growth in vivo, we used a standard xenograft assay. HN12/NTC or HN12/shVim cells were transplanted to the flanks of athymic mice and tumor growth was monitored over a 4-week period. Compared with controls, tumors formed from HN12/shVim cells were 70% smaller (Fig. 6A ; P = 0.0098). Notably, tumors formed from HN12/shVim cells showed characteristics associated with a well-differentiated phenotype (Fig. 6C) compared with those generated with the control cells (Fig. 6B). Thus, reversal of the mesenchymal phenotype by inhibiting vimentin expression reduces tumorigenic potential and enhances epithelial differentiation.

Figure 6.
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 ± SE. B and C, tumor sections stained with H&E. HN12/NTC (B) and HN12/shVIM (C). Original magnification, ×100.

Discussion

The acquisition of a mesenchymal phenotype by epithelial cancer cells is strongly associated with malignant progression. Our previous microarray studies indicated overexpression of vimentin in HN12 cells derived from a lymph node metastasis of a tongue SCC compared with cells derived from the primary tumor (14). In the present study, we have shown that elevated vimentin expression contributes to the biological properties of these cells, including enhanced proliferation, motility, and tumorigenic potential. In this regard, our results are consistent with those of others. For example, a recent study using colon and breast carcinoma cell lines showed a decrease in cell adhesion and migration following small interfering RNA-mediated repression of vimentin (20). Also, it has been reported previously that, in in vitro wound-healing assays, expression of vimentin in MCF-10A breast epithelial cells was higher at the wound edge and correlated with migration speed (21). These authors also documented an increase in vimentin expression and motility in the presence of EGF. In addition to stimulating proliferation of epithelial cells, EGF is a well-recognized motogenic factor and has been implicated in the migration of squamous carcinoma cells (22, 23) and exposure of HN12 cells to EGF also enhances vimentin expression (14) and motility (15). Using a model system of human papillomavirus-immortalized gingival keratinocytes, Chamulitrat et al. (24) found that exposure to ethanol, an etiologic factor for oral carcinogenesis, resulted in a switch to a vimentin-positive cell phenotype and was associated with a spindle morphology and growth in soft agar. Thus, factors important for oral tumor progression such as EGF, human papillomavirus, and alcohol can alter the IF profile of epithelial cells and their biological properties.

Transition from an epithelial to a mesenchymal phenotype is not only associated with expression of vimentin but also with altered expression of differentiation-specific keratins. Dysregulation of keratin expression has long been 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 microarrays 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-κB 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-κB (33); whether vimentin enhances NF-κB 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 expression of vimentin, repression of keratin expression, and altered cell morphology together with enhanced cell motility and tumorigenic potential. However, it seems clear that this process is reversible, at least to some extent, as inhibiting the expression of mesenchymal IF proteins results in reemergence of an epithelial IF network and concomitant reduction in tumor cell aggressiveness in vitro and in vivo. Thus, our studies support the notion of epithelial plasticity during tumor progression (37, 38). In a ras-driven model of breast tumor progression, Huber et al. showed a central requirement for nuclear factor-κB in the acquisition of a mesenchymal and metastatic phenotype (37). Consistent with our observations, these authors reported up-regulation of vimentin and decreased expression of keratin 14, among other targets. It must be recognized, however, that multiple proteins are involved in EMT during tumor progression, in addition to vimentin. For example, several key transcription factors play direct roles, including Snail, Slug, Twist, and FoxC2 (39, 40), with the potential to alter expression of multiple transcriptional targets. In addition, down-regulation of E-cadherin and associated loss of adherens junctions are hallmarks of tumor-associated EMT. Notably, ZEB1 and ZEB2, which are repressors of E-cadherin, are also elevated during this process (39). Furthermore, ZEB2 has also been shown to regulate vimentin expression in breast cancer cells (41).

We found that exposure of cells to EGF and TGF-β enhanced the expression of a mesenchymal IF network. In addition, treatment with both growth factors elevated vimentin levels further. Recent studies in a model of intestinal carcinogenesis reported synergy between these two growth factors in producing EMT in a ERK and phosphoinositide 3-kinase-dependent manner (42) as well as regulation of migration and invasion. These data are consistent with our own results indicating roles for phosphoinositide 3-kinase and multiple mitogen-activated protein kinase pathways in EGF- and TGF-β-mediated cell motility (14, 15). Indeed, the participation of TGF-β in the emergence of mesenchymal characteristics during tumor progression has been extensively documented in several systems (43). Moreover, TGF-β has been shown to enhance vimentin gene expression directly despite the lack of a consensus TGF-β response element in the vimentin promoter (44). Here, Smads were found bound to tandem activator protein-1 sites in the vimentin promoter, in complex with activator protein-1 family members c-jun and c-fos, together with Sp1/Sp3. An interesting study by Han et al. used a model system of mice transgenic in the epidermis for a dominant-negative form of the type II TGF-β receptor and/or TGF-β1 (45). These authors showed a requirement for TGFβRII signaling via Smads in the induction of EMT; however, lack of a functional receptor suppressed EMT but enhanced the development of metastatic tumors, thus drawing a distinction between the pathways required for these two components of advanced-stage tumors.

The ability to reverse the advanced phenotype of metastatic tumors cells may be of use as a therapeutic approach to manage human cancer. In this regard, our data indicate that targeted suppression of vimentin is sufficient to inhibit migration and invasion in vitro as well as growth in vitro and in vivo. We found that RNA interference-mediated inhibition of vimentin expression resulted in reemergence of keratin gene expression in cell culture and the production of smaller, more differentiated tumors in athymic mice, suggesting potential for development as a treatment modality. Pharmacology-based differentiation therapies have been developed by others, including the use of retinoids (46–48) and butyrates (49). Combination therapy of retinoid acid receptor and retinoid X receptor agonists with chemotherapeutic drugs has shown some promise clinically in the treatment of lung cancer (50). In the present study, we achieved a partial response purely by targeting vimentin expression. Based on this finding and work by others, developing a combined therapeutic approach for SCC is warranted.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

  • ↵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

  • Grant support: Philip Morris External Research Program UK Medical Research Council (COSB1A1R); Philip Morris USA (W.A. Yeudall) and Medical Research Council (A. Waseem).

  • 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.

  • Note: R.J. Paccione and H. Miyazaki contributed equally to this work.

    • Accepted June 19, 2008.
    • Received May 8, 2008.
    • Revision received June 17, 2008.

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Molecular Cancer Therapeutics: 7 (9)
September 2008
Volume 7, Issue 9

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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, Hiroshi Miyazaki, Vyomesh Patel, Ahmad Waseem, J. Silvio Gutkind, Zendra E. Zehner and W. Andrew Yeudall
Mol Cancer Ther September 1 2008 (7) (9) 2894-2903; DOI: 10.1158/1535-7163.MCT-08-0450
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