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RhoC GTPase Expression as a Potential Marker of Lymph Node Metastasis in Squamous Cell Carcinomas of the Head and Neck

Authors' Affiliations: Departments of ¹ Pathology, ² Biostatistics, ³ Internal Medicine, and ⁴ Otolaryngology-Head and Neck Surgery and ⁵ Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan

Requests for reprints: Theodoros N. Teknos, Department of Otolaryngology-Head and Neck Surgery, University of Michigan, 1904 Taubman Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-936-3172; Fax: 734-936-9625; E-mail: teknos{at}umich.edu.

	Abstract

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Purpose: Survival rates for squamous cell carcinoma of the head and neck (SCCHN) have remained unchanged for several decades due to local tumor recurrences as well as regional and distant metastases. Recent evidence has shown that RhoC GTPase is overexpressed in stages III and IV regionally metastatic SCCHN compared with stages I and II localized disease. This study evaluated the expression of RhoC in head and neck carcinoma and investigated the prognostic use of this marker on a large cohort of previously untreated patients with SCCHN.

Experimental Design: Standard Western blot techniques were used to evaluate RhoC protein expression in nine established head and neck cancer cell lines and in normal oral epithelium. In vivo expression of RhoC in metastatic and nonmetastatic SCCHN was investigated using immunohistochemical analysis on a tissue microarray composed of 113 independent tumor samples. RhoC expression was analyzed as it related to clinical and pathologic variables of interest.

Results: Levels of RhoC protein were increased in the SCCHN cell lines compared with normal oral epithelium. The in vivo expression of RhoC correlated with advanced clinical stage and lymph node metastases for the entire patient cohort as well as in small primary tumors (T₁ and T₂).

Conclusions: This study is the first to examine the expression of RhoC GTPase protein in SCCHN and normal squamous epithelium. It is clear from the results that RhoC is a specific marker of lymph node metastases in patients with this challenging form of carcinoma. RhoC levels seem to identify a subset of patients with early tumor stage primary tumors and high metastatic potential that might benefit from more aggressive therapy. Through continued investigation, blockade of RhoC activity may be a potential target in the development of novel strategies for treating metastases of head and neck cancer.

Many of the factors necessary to convey the metastatic phenotype to cancer cells are controlled by the members of the Ras superfamily of small GTP-binding proteins. RhoC GTPase is a member of the Ras superfamily, and its activation results in the assembly of the actin-myosin contractile filaments into focal adhesion complexes that, ultimately, lead to cell polarity and facilitate motility (1–3). Our laboratory has detected overexpression of RhoC mRNA in advanced breast cancers by in situ hybridization and characterized RhoC as a transforming oncogene for human mammary epithelial cells. In addition, RhoC overexpression results in a highly motile and invasive phenotype that recapitulates the most lethal form of locally advanced breast cancer, inflammatory breast cancer. Further work showed that RhoC is specifically expressed in invasive breast carcinomas capable of metastasizing, and it may be clinically useful in patients with tumors <1 cm to guide treatment (4, 5). Subsequent investigations in other tumor types have shown that RhoC overexpression enhances tumor metastasis in melanoma (6), ovarian carcinoma (7), ductal adenocarcinoma of the pancreas (8), and lung carcinoma (9). To date, no studies have investigated the role of RhoC in the metastatic phenotype of head and neck carcinoma. Abraham et al. (10) did immunohistochemical studies investigating other Ras superfamily motility-related gene expression but did not specifically stain for RhoC. Interestingly, however, they concluded that there was a significant difference in the expression of these proteins between normal epithelium and malignant cells and hypothesized that motility-related genes may prove to be a marker of malignancy and/or aggressiveness in head and neck cancer (10).

Through gene expression profiling studies, we have identified RhoC as being differentially overexpressed in stages III and IV regionally metastatic SCCHN compared with stages I and II localized disease (11). This finding led us to hypothesize that RhoC may be a clinically useful marker of metastatic potential in SCCHN. In this study, we have analyzed RhoC protein expression in normal squamous epithelium as well as in a panel of squamous cell carcinoma cell lines in vitro. We then investigated the prognostic use of detecting RhoC protein in situ by immunohistochemistry on human tissues from a large cohort of previously untreated patients with SCCHN.

	Materials and Methods

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Western blots. UMSCC and normal oral epithelium cell lines were homogenized in 0.2 mol/L Tris-HCl buffer (pH 7.4) containing 1 mmol/L EGTA, 2.5 mmol/L EDTA, 5 mmol/L DTT, 0.3 mol/L sucrose, 10 µg/mL leupeptin, 50 µg/ml aprotinin, 2 mmol/L Na₃VO₄, and 0.1% Triton X-100. The homogenate was centrifuged at 500 x g for 10 minutes at 4°C to remove all cell debris. Total proteins were estimated in clear supernatants by dye-binding method using Bradford reagent. Equal amounts of protein were boiled in Laemmli buffer for 5 minutes and then analyzed by 10% SDS-PAGE. After transferring the separated proteins onto nitrocellulose membranes, the membranes were blocked for 3 hours at room temperature with TBS-0.1% Tween 20 containing 5% nonfat dry milk. The membranes were incubated overnight with polyclonal RhoC antibody (1:1,200) at 4°C with mild shaking. After incubation with primary antibody, the membranes were blotted for 1 hour with a secondary horseradish peroxidase–conjugated antibody (1:5,000). Next, the membranes were washed and the proteins were visualized using enhanced chemiluminescence Western detection system (Amersham, Piscataway, NJ). The analysis of activated RhoC was done using the RhoC Activation Assay Biochem kit (Cytoskeleton, Denver, CO) following manufacturer's protocols. The cells were lysed using lysis buffer, and cell lysates were centrifuged at 10,000 rpm for 5 minutes to remove cell debris. The clear supernatants were taken out and incubated with 60 µL of pretreated beads for 1 hour at 4°C. The beads were separated from the supernatants by centrifuging at 10,000 rpm for 5 minutes at 4°C. The protein-bead complex was washed once with lysis buffer followed by a clear wash with the wash buffer (supplied). A small portion of this complex was used to determine the protein concentration (Bradford assay). The remaining portion was used to load exact amounts of proteins on 12% SDS gels following standard Western blot techniques as described previously above.

Selection of patients and tissue microarray development. In vivo expression of RhoC in metastatic and nonmetastatic SCCHN was investigated using immunohistochemical analysis on a tissue microarray composed of 113 independent tumor samples. All patients for this validation group were presented to the University of Michigan Hospital (Ann Arbor, MI) between 1997 and 2000 with newly diagnosed, previously untreated HNSCC. University of Michigan institutional review board approval and written consent were obtained. Patients underwent clinical and pathologic staging followed by surgical tumor resection and regional lymphadenectomy. A surgical pathologist used H&E staining to evaluate cryotome sections (5 µm) from each paraffin-embedded primary tumor block. Representative areas of primary tumor and normal squamous cell mucosa were marked. A high-density tissue microarray was constructed from the marked areas using three replicate tumor cores (0.6-mm diameter) and one normal mucosa core per patient. By obtaining three cores from different areas of each tumor, we accounted for tumor heterogeneity.

Immunohistochemical studies. Immunohistochemistry was done on the tissue microarray by using a standard biotin-avidin complex technique and a polyclonal antibody against RhoC that was developed and validated in our laboratory by immunoblot analysis and immunohistochemistry (12). RhoC expression was evaluated at least thrice for every tissue microarray element and at least nine times for each tumor. Using this method, the pathologist was blinded to tumor stage and clinical information. The mean value of all measurements from a single individual was used for subsequent analyses.

Because RhoC protein interacts with the contractile cytoskeleton of the cell and is localized to the submembrane space, cytoplasmic stain was expected. Not surprisingly, myoepithelial cells and vascular smooth muscle cells were strongly positive in all cases, serving as consistent internal positive controls. The intensity of cytoplasmic staining was scored using a four-tiered system by comparison to the positive internal controls (score 1 = negative, score 2 = weak, score 3 = moderate, and score 4 = strong staining). This scoring system has been previously validated (12). Based on the biological characterization of RhoC in SCCHN, we defined high RhoC expression when there was moderate or strong cytoplasmic staining (scores 3 and 4) and low RhoC expression when staining was negative or weak (scores 1 and 2). Furthermore, positive controls for staining consisted of head and neck cell lines known to overexpress RhoC and negative controls were done by omitting the primary antibody.

Statistical analysis. The goal of the analysis was to determine if RhoC expression in primary tumor specimen relates to clinical variables of interest. The clinical variables of interest included lymph node metastases, presence of nodal extracapsular spread, tumor stage, clinical stage, and presence of perineural invasion, primary tumor differentiation, disease-free survival, and overall survival. The mean score (rounded to the nearest integer) of all measurements from a single individual was used for analysis. To evaluate the association of RhoC with covariates of interest, generalized linear models were fit to the data using a generalized estimating equation approach to account for both within and between subject variations derived from repeated measurements. A multinomial distribution with cumulative logit link was used for the staining intensity of RhoC. The clinical variables of interest were tested as a main effect in these models. The relationship between RhoC and survival outcomes was explored by the Kaplan-Meier method. The log-rank test was used to compare the homogeneity of survival rate between each scoring category.

For exploratory purposes, a separate analysis was conducted by using only the maximum RhoC staining of each subject. Spearman rank correlation coefficient was used to assess the association between clinical variables and the maximum RhoC staining scores. All statistical analyses were done using SAS version 8.2 (SAS, Carey, NC). A two-tailed P 0.05 was considered to be statistically significant.

	Results

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RhoC protein levels are elevated in SCCHN. To illustrate that RhoC is up-regulated in squamous carcinomas when compared with normal squamous epithelium, we did Western blots on a panel of squamous cell carcinoma cell lines and compared it with that of normal oral epithelium. As illustrated in Fig. 1 , levels of RhoC and activated RhoC were increased in all the cancer cell lines tested compared with normal epithelial cells. This recapitulates and validates the findings of our previous gene array studies.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Western blots illustrating the protein expression levels of RhoC (A) and activated RhoC (B) in a panel of UMSCC cell lines and normal oral epithelium (NOE).

The clinical and pathologic features of the 68 patients in our cohort are summarized in Table 1 . Of the 68 invasive carcinomas, 15 (22.1%) were early stage (stages I and II) and 53 (77.9%) were advanced stage (stages III and IV). As expected, based on our previous studies in breast cancer, RhoC was expressed mainly in the cytoplasm (Fig. 2 ). SCCHN that expressed high levels of RhoC and those that expressed low levels of RhoC were readily identifiable (Fig. 2). In our cohort of 68 tumors, RhoC protein was expressed in 82.35% (56 of 68) of patients and high RhoC protein was detected in 32 (47%) of the carcinomas, whereas low RhoC expression was found in 36 (53%) of the SCCHN tumors.

View larger version (129K): [in this window] [in a new window]   Fig. 2. Typical staining patterns seen with RhoC immunohistochemistry in the head and neck high-density tissue microarray. Low RhoC expression was defined as negative or weak cytoplasmic staining (scores 1 and 2), whereas high RhoC expression was defined as moderate or strong cytoplasmic staining (scores 3 and 4).

	Discussion

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The formation and growth of metastases is the principal cause of death for most cancer patients, particularly those with head and neck cancer (13, 14). An ever-expanding body of evidence is implicating RhoC GTPase as a critical determinant of metastasis for a variety of tumor types (1–9). Ours is the first study to investigate the role of RhoC in the behavior of SCCHN.

Through the in vitro portion of this study, we have illustrated that RhoC protein expression is present in all squamous cell carcinoma cell lines tested and at levels dramatically higher than normal squamous epithelium. This is consistent with our findings in a gene array analysis comparing normal squamous epithelium versus SCCHN, thus validating these data (11). It is also in line with our previous work in breast cancer where we showed that RhoC expression increases once breast cancer cells acquire the ability to invade the host tissues and metastasize (3). Finally, although previous authors did not specifically address RhoC, our findings confirm the work of Abraham et al. (10), who concluded that motility-related proteins are overexpressed in squamous cell carcinoma relative to normal keratinocytes and may prove to be markers of metastasis.

To further evaluate the importance of RhoC in conferring a metastatic phenotype in SCCHN, we correlated in vivo levels of RhoC with known demographic and clinical variables using a large cohort of previously untreated head and neck cancer patients. Collectively, our data indicate that increasing levels of RhoC expression are associated with advanced-stage tumors and with lymph node metastasis even in small primary tumors. Furthermore, there was a trend noted, in which increasing RhoC expression was appreciated in patients with extracapsular spread in their lymph node metastases, indicating aggressive tumor behavior. Interestingly, although RhoC correlated with lymph node metastases and advanced tumor stage, a clear correlation with survival and distant metastases could not be illustrated. This may be due to the relatively short length of clinical follow-up (41 months) and could reach significance as the patient cohort matures. Furthermore, the bias of our institution toward aggressive postoperative treatment of patients with high-risk tumors (i.e., multiple lymph nodes and extracapsular spread) with combined chemoradiation therapy may have minimized the survival difference between the two groups. Regardless, our study clearly illustrates that RhoC expression is correlated with lymph node metastasis and clinical stage in head and neck cancer. Even with small primary tumors, RhoC may identify those small (T₁ and T₂) carcinomas with high metastatic potential and may assist clinicians in selecting patients who would benefit from aggressive surgical and adjuvant therapy. In this pilot study, high RhoC expression was able to identify a metastatic phenotype in small tumors, with a relatively high degree of specificity and positive predictive value.

Our findings in this study are consistent with observations made by others in different tumor types. In melanoma, for instance, genomic analysis of highly metastatic tumors revealed an essential role for RhoC by inducing a highly motile, invasive tumor phenotype, which was abrogated in vitro with the introduction of dominant-negative Rho (6). Similarly, Wang et al. (15) identified RhoC as strong predictor of lymph node metastasis in hepatocellular carcinoma using genomic analysis and tissue microarray immunohistochemistry. In an orthotopic model of non–small cell lung cancer, Ikoma et al. (9) found that metastasis, in vitro migration, and invasion were significantly enhanced by overexpression of RhoC. They further concluded that RhoC does not affect primary tumor growth but enhances the metastatic nature of lung cancer not only by stimulating cell motility but also by up-regulating the expression of certain matrix metalloproteinases. Similar data correlating RhoC expression with lymph node metastasis and tumor progression have been noted in pancreatic and ovarian carcinoma (7, 8).

The emerging role of RhoC in the metastatic process strongly suggests that attenuation of RhoC activity may be a potential target for novel strategies in the treatment of head and neck as well as other carcinomas and melanoma. To that end, Rho proteins are dependent on post-translational isoprenylation for their biological activity (16, 17) RhoC specifically depends on geranylgeranylation, and this can be inhibited by the statin class of drugs, which are commonly used for their cholesterol lowering affect (18). These drugs, by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase, cause dramatic reductions not only in cholesterol but also in its isoprenoid precursors (farnesyl pyrophosphate and geranylgeranyl pyrophosphate). Recently, atorvastatin was found to inhibit RhoC isoprenylation and metastasis in a mouse model of human melanoma (19). Furthermore, clinical trials of statins for heart disease have noted antineoplastic effects of statins in general (20) and in melanoma in particular (21). Ongoing studies in our laboratory are investigating the effect of statin therapy on growth, invasion, and metastasis of head and neck cancer through RhoC inhibition. If the preliminary findings in this study and future investigations indicate efficacy of statin therapy, this may prove to be an important adjunct to existing therapies in head and neck cancer to prevent regional and distant metastasis.

In conclusion, this study is the first in examining the expression of RhoC GTPase protein in SCCHN and normal squamous epithelium. It is clear from the results that RhoC is a specific marker of lymph node metastases in patients with this challenging form of carcinoma. Importantly, although our data are preliminary, RhoC levels seem to identify a subset of patients with early tumor stage primary tumors and high metastatic potential that might benefit from more aggressive therapy. Through continued investigation, blockade of RhoC activity may be a potential target in the development of novel strategies for treating metastases of head and neck cancer.

	Footnotes

 
Grant support: Specialized Programs of Research Excellence grant in Head and Neck NIH/National Institute of Dental and Craniofacial Research/National Cancer Institute 1P50CA/DE97248-01 (T.N. Teknos and S.D. Merajver), Army grants DAMD17-02-1-491 (C.G. Kleer) and DAMD-17-00-1-0345 (S.D. Merajver), and NIH grants K08 CA 090876 (C.G. Kleer), R01CA107469 (C.G. Kleer), RO1CA77612 (S.D. Merajver), and 1 P50-CADE97258 (S.D. Merajver).

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: C.G. Kleer and T.N. Teknos are cofirst authors on this article.

Received 2/20/06; revised 4/ 6/06; accepted 5/16/06.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes with actin stress fibers, lamellipodia, and filopodia. Cell 1995;81:53–62.[CrossRef][Medline]
2. Leung T, Chen XQ, Manser E, Lim L. The p160 RhoA-binding kinase ROK is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 1996;16:5313–27.[Abstract]
3. Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 1996;273:245–8.[Abstract]
4. van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD. RhoC-GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res 2000;60:5832–8.[Abstract/Free Full Text]
5. Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, Merajver SD. Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J of Pathol 2002;160:579–84.
6. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000;406:532–5.[CrossRef][Medline]
7. Horiuchi A, Imai T, Wang C, et al. Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma. Lab Investig 2003;83:861–70.[Medline]
8. Suwa H, Ohshio G, Imamura T, et al. Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 1998;77:147–52.[Medline]
9. Ikoma T, Takahashi T, Nagano S, et al. A definitive role of RhoC in metastasis of orthotopic lung cancer in mice. Clin Cancer Res 2004;10:1192–200.[Abstract/Free Full Text]
10. Abraham MT, Kuriakose MA, Sacks PG, et al. Motility-related proteins as markers for head and neck squamous cell cancer. Laryngoscope 2001;111:1285–9.[CrossRef][Medline]
11. Schmalbach CE, Chepeha DB, Giordano TJ, et al. Molecular profiling and the identification of genes associated with metastatic oral cavity/pharynx squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2004;130:295–302.[Abstract/Free Full Text]
12. Van den Eynden GG, Van der Auwera I, Van Laere S, et al. Validation of a tissue microarray to study differential protein expression in inflammatory and non-inflammatory breast cancer. Breast Cancer Res Treat 2004;85:13–22.[CrossRef][Medline]
13. Lin M, van Golen KL. Rho-regulatory proteins in breast cancer cell motility and invasion. Breast Cancer Res Treat 2004;84:49–60.[CrossRef][Medline]
14. Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty eighth GHA Clowes Memorial Award Lecture. Cancer Res 1990;50:6130–8.[Abstract/Free Full Text]
15. Wang W, Yang LY, Huang GW, et al. Genomic analysis reveals RhoC as a potential marker in hepatocellular carcinoma with poor prognosis. Br J Cancer 2004;90:2349–55.[Medline]
16. Collisson EA, Carranza DC, Chen IY, Kolodney MS. Isoprenylation is necessary for the full invasive potential of RhoA overexpression in melanoma cells. J Invest Dermatol 2002;119:1172–6.[CrossRef][Medline]
17. Allal C, Favre F, Couderc B, et al. Rho prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription. J Biol Chem 2000;275:31001–8.[Abstract/Free Full Text]
18. Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A. Non-lipid related effects of statins. Ann Med 2000;32:164–76.[Medline]
19. Collisson EA, Kleer C, Wu M, et al. Atorvastatin prevents RhoC isoprenylation, invasion, and metastasis in human melanoma cells. Mol Cancer Ther 2003;2:941–8.[Abstract/Free Full Text]
20. Pedersen TR, Wilhelmsen L, Faergeman O, et al. Follow-up study of patients randomized in the Scandinavian simvastatin survival study (4S) of cholesterol lowering. Am J Cardiol 2000;86:257–62.[CrossRef][Medline]
21. Papadakis JA, Mikhailidis DP. Coronary events with lipid-lowering therapy: the AFCAPS/TexCAPS trial. Air Force/Texas Coronary Atherosclerosis Prevention Study. JAMA 1999;281:416–9.[Medline]

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