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Comparative Study
. 2009 Aug;119(8):1518-30.
doi: 10.1002/lary.20506.

CD44 variant isoforms in head and neck squamous cell carcinoma progression

Steven J Wang  1 , Gabriel Wong, Anne-Martine de Heer, Weiliang Xia, Lilly Y W Bourguignon
Affiliations
Comparative Study

CD44 variant isoforms in head and neck squamous cell carcinoma progression

Steven J Wang et al. Laryngoscope. 2009 Aug.

Abstract

Objectives/hypothesis: The CD44 family of receptors includes multiple variant isoforms, several of which have been linked to malignant properties including migration, invasion, and metastasis. The objective of this study was to investigate the role of the CD44 v3, v6, and v10 variant isoforms in head and neck squamous cell carcinoma (HNSCC) tumor progression behaviors.

Study design: Laboratory study involving cell cultures and clinical tissue specimens.

Methods: Analysis of the expression of standard CD44s and the CD44 variant isoforms v3, v6, and v10 was carried out in the HNSCC cell line, HSC-3. The role of CD44 isoforms in migration, proliferation, and cisplatin resistance was determined. Immunohistochemical analysis was performed on clinical tissue specimens obtained from a series of 82 HNSCC patients. The expression of standard CD44s and the CD44 v3, v6, and v10 variants in primary tumor specimens (n = 82) and metastatic cervical lymph nodes (n = 24) were analyzed with respect to various clinicopathologic variables.

Results: HSC-3 cells express at least four CD44 isoforms, and these CD44 isoforms mediate migration, proliferation, and cisplatin sensitivity. Compared with primary tumors, a greater proportion of metastatic lymph nodes demonstrated strong expression of CD44 v3 (lymph node 14/24 vs. primary tumor 38/82), CD44 v6 (lymph node 18/24 vs. primary tumor 26/82), and CD44 v10 (lymph node 14/24 vs. primary tumor 16/82), while expression of standard CD44 was not significantly different in metastatic lymph nodes and primary tumors (lymph node 10/24 vs. primary tumor 60/82). Expression of CD44 variant isoforms were associated with advanced T stage (v3 and v6), regional (v3) and distant (v10) metastasis, perineural invasion (v6), and radiation failure (v10). CD44 v6 and CD44 v10 were also significantly associated with shorter disease-free survival.

Conclusions: CD44 isoforms mediate migration, proliferation, and cisplatin sensitivity in HNSCC. Furthermore, expression of certain CD44 variants may be important molecular markers for HNSCC progression and should be investigated as potential therapeutic targets for therapy.

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Figures

FIGURE 1
FIGURE 1. Schematic exon map of human CD44 gene
Up to 10 possible variable exons may be inserted between exon 5 and exon 15 of CD44. Exon 7 contains the CD44 v3 exon; exon 10 contains the v6 exon; exon 14 contains the v10 exon.
FIGURE 2
FIGURE 2. CD44 isoform RNA expression in HSC-3 cells
Quantitative real-time RT-PCR reactions were performed on total RNA extracts from HSC-3 cells using primers specific for CD44s (1), CD44 v3 (2), CD44 v6 (3), and CD44 v10 (4). RNA levels are expressed relative to the expression of 36B4, a human acidic ribosomal phosphoprotein P0 whose expression was unchanged in the tumor cells. Error bars were determined from standard deviations.
FIGURE 3
FIGURE 3. CD44 isoform protein expression in HSC-3 cells
Protein lysates from HSC-3 cells transfected with CD44 siRNA (lane 2) or non-transfected cells (lane 1) were immunoblotted with anti-CD44 antibody (panel a) or anti-actin antibody (panel b) as a loading control.
FIGURE 4
FIGURE 4. In vitro tumor cell migration assays
Using hyaluronan as a chemoattractant, Transwell migration assays of HSC-3 cells transfected with CD44 siRNA (3) or scrambled sequence siRNA (2) were performed as described in Methods. The migration of non-transfected HSC-3 cells (1) served as a reference control (designated 100%). Error bars were determined from standard deviations.
FIGURE 5
FIGURE 5. In vitro tumor cell growth assays
A) HSC-3 cells (untransfected or transfected with CD44siRNA or with non-targeting scrambled siRNA) were cultured for 24 hours followed by treatment with increasing concentration of cisplatin for 48 hours. The in vitro growth of these cells was analyzed by MTT assays. The 50% inhibitory concentration (IC50) was identified as a concentration of drug required to achieve a 50% growth inhibition relative to untreated controls. Error bars were determined from standard deviations. B) Untreated HSC-3 cells (1) and HSC-3 cells that were treated with normal rat immunoglobulin (2) (control for CD44 antibody), anti-CD44 antibody (3), normal rabbit immunoglobulin (4) (control for CD44 v3, v6, and v10 antibodies), anti-CD44v3 antibody (5), anti-CD44v6 antibody (6), or anti-CD44v10 antibody (7) underwent MTT assays to determine in vitro tumor cell growth. Error bars were determined from standard deviations.
FIGURE 5
FIGURE 5. In vitro tumor cell growth assays
A) HSC-3 cells (untransfected or transfected with CD44siRNA or with non-targeting scrambled siRNA) were cultured for 24 hours followed by treatment with increasing concentration of cisplatin for 48 hours. The in vitro growth of these cells was analyzed by MTT assays. The 50% inhibitory concentration (IC50) was identified as a concentration of drug required to achieve a 50% growth inhibition relative to untreated controls. Error bars were determined from standard deviations. B) Untreated HSC-3 cells (1) and HSC-3 cells that were treated with normal rat immunoglobulin (2) (control for CD44 antibody), anti-CD44 antibody (3), normal rabbit immunoglobulin (4) (control for CD44 v3, v6, and v10 antibodies), anti-CD44v3 antibody (5), anti-CD44v6 antibody (6), or anti-CD44v10 antibody (7) underwent MTT assays to determine in vitro tumor cell growth. Error bars were determined from standard deviations.
FIGURE 6
FIGURE 6. CD44 variant isoform expression in head and neck squamous cell carcinoma (HNSCC) primary tumors and lymph nodes
A panel of anti-CD44 variant antibodies (CD44s, CD44v3, CD44v6, and CD44v10) were used to examine CD44 isoform protein expression in HNSCC primary tumors and metastatic lymph nodes. Representative examples of primary tumors and lymph nodes stained for each of the CD44 antibodies are shown (original magnification x200). CD44 expression was scored as “strong,” “weak,” or “none,” as described in Methods. The example shown for CD44v10 expression in a lymph node was classified as weak, while the other examples shown were classified as strong.
FIGURE 7
FIGURE 7. Disease-free survival analysis of study population
A) Disease free survival rates in patients, grouped by clinical stage (p=.0001); B) Disease free survival rates in patients, grouped by primary tumor site (p=.02); C) Disease free survival rates in patients with or without positive cervical lymph nodes (p=.002); D) Disease free survival rates in patients with or without distant metastasis (p<.0001); E) Disease free survival rates in patients with or without primary tumor expression of CD44 v6 (p=.03); F) Disease free survival rates in patients with or without primary tumor expression of CD44 v10 (p=.04).
FIGURE 7
FIGURE 7. Disease-free survival analysis of study population
A) Disease free survival rates in patients, grouped by clinical stage (p=.0001); B) Disease free survival rates in patients, grouped by primary tumor site (p=.02); C) Disease free survival rates in patients with or without positive cervical lymph nodes (p=.002); D) Disease free survival rates in patients with or without distant metastasis (p<.0001); E) Disease free survival rates in patients with or without primary tumor expression of CD44 v6 (p=.03); F) Disease free survival rates in patients with or without primary tumor expression of CD44 v10 (p=.04).
FIGURE 7
FIGURE 7. Disease-free survival analysis of study population
A) Disease free survival rates in patients, grouped by clinical stage (p=.0001); B) Disease free survival rates in patients, grouped by primary tumor site (p=.02); C) Disease free survival rates in patients with or without positive cervical lymph nodes (p=.002); D) Disease free survival rates in patients with or without distant metastasis (p<.0001); E) Disease free survival rates in patients with or without primary tumor expression of CD44 v6 (p=.03); F) Disease free survival rates in patients with or without primary tumor expression of CD44 v10 (p=.04).
FIGURE 7
FIGURE 7. Disease-free survival analysis of study population
A) Disease free survival rates in patients, grouped by clinical stage (p=.0001); B) Disease free survival rates in patients, grouped by primary tumor site (p=.02); C) Disease free survival rates in patients with or without positive cervical lymph nodes (p=.002); D) Disease free survival rates in patients with or without distant metastasis (p<.0001); E) Disease free survival rates in patients with or without primary tumor expression of CD44 v6 (p=.03); F) Disease free survival rates in patients with or without primary tumor expression of CD44 v10 (p=.04).
FIGURE 7
FIGURE 7. Disease-free survival analysis of study population
A) Disease free survival rates in patients, grouped by clinical stage (p=.0001); B) Disease free survival rates in patients, grouped by primary tumor site (p=.02); C) Disease free survival rates in patients with or without positive cervical lymph nodes (p=.002); D) Disease free survival rates in patients with or without distant metastasis (p<.0001); E) Disease free survival rates in patients with or without primary tumor expression of CD44 v6 (p=.03); F) Disease free survival rates in patients with or without primary tumor expression of CD44 v10 (p=.04).
FIGURE 7
FIGURE 7. Disease-free survival analysis of study population
A) Disease free survival rates in patients, grouped by clinical stage (p=.0001); B) Disease free survival rates in patients, grouped by primary tumor site (p=.02); C) Disease free survival rates in patients with or without positive cervical lymph nodes (p=.002); D) Disease free survival rates in patients with or without distant metastasis (p<.0001); E) Disease free survival rates in patients with or without primary tumor expression of CD44 v6 (p=.03); F) Disease free survival rates in patients with or without primary tumor expression of CD44 v10 (p=.04).
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References

    1. Parkin DM, Pisani P, Ferlay J. Global cancer statistics, 2002. Ca Cancer J Clin. 2005;55:74–108. - PubMed
    1. Toole BP, Hascall VC. Hyaluronan and tumor growth. Am J Pathol. 2002;161:745–747. - PMC - PubMed
    1. Turley E, Noble P, Bourguignon L. Signaling properties of hyaluronan receptors. J Biol Chem. 2002;277:4589–92. - PubMed
    1. Bourguignon LY, Singleton PA, Diedrich F, et al. CD44 interaction with Na+-H+ exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion. J Biol Chem. 2004;279:26991–7007. - PubMed
    1. Bourguignon LY, Zhu H, Zhou B, et al. Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185HER2 and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth. J Biol Chem. 2001;276:48679–92. - PubMed

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