Loss of p12CDK2-AP1 Expression in Human Oral Squamous Cell Carcinoma with Disrupted Transforming Growth Factor-β-Smad Signaling Pathway

RESEARCH ARTICLE

Neoplasia . Vol. 8, No. 12, December 2006, pp. 1028 – 1036

1028

www.neoplasia.com

Loss of p12CDK2-AP1 Expression in Human Oral Squamous Cell Carcinoma with Disrupted Transforming Growth Factor-B–Smad Signaling Pathway1 Hui Peng *,2, Satoru Shintani y,z,2, Yong Kim * and David T. Wong *,§,b,#,**

*Dental Research Institute, School of Dentistry, University of California-Los Angeles, Los Angeles, CA, USA; Department of Oral and Maxillofacial Surgery, Showa University School of Dentistry, Tokyo, Japan; zDepartment of Oral and Maxillofacial Surgery, Ehime University School of Medicine, Ehime, Japan; §Division of Head and Neck Surgery/Otolaryngology, David Geffen School of Medicine, Los Angeles, CA, USA; b Henry Samueli School of Engineering, University of California-Los Angeles, Los Angeles, CA, USA; #Molecular Biology Institute, University of California-Los Angeles, Los Angeles, CA, USA; **Jonsson Comprehensive Cancer Center, University of California-Los Angeles, Los Angeles, CA, USA y

Abstract We examined correlations between TGF-B1, TBR-I and TBR-II, p12CDK2-AP1, p21WAF1, p27KIP1, Smad2, and pSmad2 in 125 cases of human oral squamous cell carcinoma (OSCC) to test the hypothesis that resistance to TGF-B1 – induced growth suppression is due to the disruption of its signaling pathway as a consequence of reduced or lost p12CDK2-AP1. Immunoreactivity for TBR-II decreased in OSCC with increasing disease aggressiveness; however, no differences were observed for TBR-I and TGF-B1. The expression of TBR-II significantly correlated with p12CDK2-AP1 and p27KIP1 (P < .001 and P < .01, respectively). Furthermore, there was a significant relationship between TBR-II expression and p-Smad2 (P < .001). The in vivo correlation of the levels of TBR-II, p12CDK2-AP1, and p27KIP1 was confirmed in normal and OSCC cell lines. Additionally, in vitro analysis of TGF-B1 – treated cells showed that TGF-B1 treatment of normal keratinocytes suppressed cell growth with upregulation of p-Smad2, p12CDK2-AP1, and p21WAF1 expression, whereas there was no effect on OSCC cell lines. These results provide evidence of a link between a disrupted TGF-B – Smad signaling pathway and loss of induction of cell cycle – inhibitory proteins, especially p12CDK2-AP1 in OSCC, which may lead to the resistance of TGF-B1 growth-inhibitory effect on OSCC. Neoplasia (2006) 8, 1028 – 1036 Keywords: Head and neck squamous cell carcinoma, transforming growth factor-b1, transforming growth factor-b receptor, p12CDK2-AP1, p27KIP1.

Introduction Transforming growth factor-b1 (TGF-b1) is a potent growth inhibitor of various cell types, including epithelial, endothe-

lial, and hematopoietic cells [1 – 5]. TGF-b1 signaling is mediated mainly by two serine threonine kinase receptors, TGF-b receptor I (TbR-I) and TGF-b receptor II (TbR-II), which activate Smad2/3 and Smad4 transcription factors. The phosphorylation and activation of these proteins are followed by the formation of the Smad2/3 – Smad4 complex, which translocates to the nucleus regulating transcriptional responses to TGF-b1 [6 – 8]. Previous studies have demonstrated that loss of function or decreased expression of TbR-II is associated with tumor progression [9,10]. The mechanism of TGF-b1 growth inhibition in epithelial cells is mediated, in part, by induction of p21WAF1 expression [11]. Recently, TGF-b1 has been found to induce the expression of a novel cyclin-dependent kinase (CDK) 2 inhibitor, p12 CDK2 – associating protein 1 (p12CDK2-AP1), in normal epithelial cells [12]. Mechanistically, p12 plays a role in TGF-b1 – mediated growth suppression by modulating CDK2 activities and pRb phosphorylation [12]. Head and neck cancer affects more than half a million patients worldwide yearly; despite improvements in surgery, chemotherapy, and radiation therapy, overall survival has not improved over the past 30 years [13]. Molecular alterations frequently associated with head and neck cancer include cyclin D1 amplification or overexpression, decreased expression of p53 and pRb, and decreased expression of CDK inhibitors p27KIP1, p21WAF1, and p16INK4A [14,15]. More recently, decreased expression of p12CDK2-AP1 has been associated with Abbreviations: TGF-b, transforming growth factor-b; TbR-I, TGF-b receptor I; TbR-II, TGF-b receptor II; p12CDK2-AP1, p12 CDK2 – associating protein 1; OSCC, oral squamous cell carcinoma Address all correspondence to: Dr. David T. Wong, Dental Research Institute, School of Dentistry, University of California-Los Angeles, 10833 Le Conte Avenue, 73-017 CHS, Los Angeles, CA 90095. E-mail: [email protected] 1 This work was supported by Public Health Service (PHS) grant R01 DE14857 to D.T.W. 2 Hui Peng and Satoru Shintani contributed equally to this work. Received 22 August 2006; Revised 5 October 2006; Accepted 9 October 2006. Copyright D 2006 Neoplasia Press, Inc. All rights reserved 1522-8002/06/$25.00 DOI 10.1593/neo.06580

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

the development of oral squamous cell carcinoma (OSCC) [16,17]. p12CDK2-AP1 is an S-phase – associated growth suppressor that binds with DNA polymerase A/primase and/or CDK2 [18,19]. Studies have shown that p12 is differentially expressed in normal and tumor oral mucosa, with a reduced expression in 14.3% of oral dysplasias and in 64% to 72% of oral cancers, suggesting a potential role for p12CDK2-AP1 as a tumor suppressor in oral keratinocytes [16,17]. A defective TGF-b – Smad signaling pathway, which leads to loss of response to the proliferation-inhibitory effect of TGF-b1, has been found in several human cancers, such as hepatocellular carcinoma, pancreatic cancer, colon carcinoma, and glioblastoma [20 – 23]. Recently, mutations of Smad2 and Smad4 have been found in human head and neck squamous cell carcinoma cell lines; however, the expression of TGF-b – Smad signaling proteins in OSCC has not been reported. In addition, the biologic effects of TGF-b1 on OSCC remain unknown [24]. In the present study, we tested the hypothesis that resistance to TGF-b1 growth suppression in OSCC is due to the preferential reduction or loss of p12 expression. We investigated the expression of TGFb1, its receptors, and the intracellular TGF-b – Smad signaling pathway in human OSCC. We also examined whether TGFb1 is biologically functional in oral cancer.

Materials and Methods Tissue Samples Tissue samples were obtained from 125 previously untreated OSCC patients at the Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine (Ehime, Japan) from 1991 to 2003. The median age of the OSCC patients was 68.2 years (SD = 14.1). Twenty-eight patients were in stage I, 41 patients were in stage II, 23 patients were in stage III, and 33 patients were in stage IV, according to tumor – node – metastasis classification [25]. The grade of tumor differentiation was determined according to the criteria proposed by the World Health Organization [26]. Histologic mode of invasion was classified according to the classification of Anneroth et al. [27]. Immunohistochemical Analysis Tissues were fixed in 10% buffered formalin and embedded in paraffin. Paraffin blocks were cut at 4 mm thickness. Sections were deparaffinized with xylene and rehydrated in graded alcohol. Three percent hydrogen peroxide was then applied to block endogenous peroxidase activity. The sections were incubated overnight at 4jC with anti-human TGFb1 (diluted 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), TbR-I (diluted 1:100; Santa Cruz Biotechnology), TbR-II (diluted 1:100; Upstate Biotech, Inc., Lake Placid, NY), Smad2 (diluted 1:100; Chemicon, Inc., Temecula, CA), pSmad2 (diluted 1:100; Santa Cruz Biotechnology), p21WAF1 (diluted 1:50; DAKO, Carpinteria, CA), p27KIP1 (diluted 1:50; DAKO), and Ki67 (diluted 1:100; Abcam, Inc., Cambridge, MA). Immunostaining was performed with the Envision system (DAKO, Carpinteria, CA) in accordance with the manu-

Neoplasia . Vol. 8, No. 12, 2006

Peng et al.

1029

facturer’s instructions. Peroxidase activity was visualized by applying diaminobenzidine chromogen containing 0.05% hydrogen peroxidase. The sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted. Negative control staining was carried out by substituting nonimmune goat or mouse serum for primary antibodies. The immunoreactivity of antibodies to TGF-b1 and its receptors was assessed on a visual analogue scale by semiquantifying cytoplasmic staining. Immunoreactivity was scored as () absent, (1+) low (< 25% of positive tumor cells), (2+) moderate (26– 75% of positive tumor cells), or (3+) diffuse (> 75% of positive tumor cells) [27,28]. As for Smad2, p-Smad2, p21WAF1, p27KIP1, p12CDK2-AP1, and Ki67, nuclear staining of these antibodies was evaluated by estimating the percentage of positive nuclei. OSCCs were assessed based on at least 10 randomly selected fields. All slides were interpreted by two investigators. Cell Culture The human immortalized normal oral epithelium cell lines OKF6 tert-1 and OKF6 tert-2 were gifts from Dr. Jim Rhienwald (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA). They were maintained in a serum-free keratinocyte growth medium (Cell Applications, Inc., San Diego, CA) containing glutamine, apo-transferrin, bovine pituitary extract, insulin, human epidermal growth factor, and hydrocortisone. The human malignant oral epithelium cell lines SCC4, SCC9, SCC66, UM1, and 1483 were maintained in DMEM/F12 supplemented with 10% fetal bovine serum and 1% PSF. TGF-b1 was obtained from Sigma (St. Louis, MO). Cells were seeded in six-well plates, TGFb1 was added when cells reached 50% confluence, and the rate of cell duplication was determined by counting cells for three consecutive days after TGF-b1 treatment. Protein Preparation and Western Blot Analysis Subconfluent cells were harvested by trypsinization and lysed with lysis buffer (50 mM Tris – HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM PMSF, 5 mg/ml leupeptin, 0.2% vol/vol Triton X-100, and 0.5% vol/vol Nonidet P-40). Lysates were incubated for 30 minutes at 4jC and were then centrifuged for 15 minutes at 4jC. Protein concentrations of lysate were determined using the Bio-Rad DC protein assay system (Bio-Rad, Hercules, CA), with bovine serum albumin as standard. Equal amounts of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (8 – 15%) and transferred to PVDF membranes. Membranes were incubated overnight with antibodies specific for p12 (pAb86 rabbit polyclonal antibody; Strategic Biosciences, Newark, DE), p27, p21, TbR-I, TbR-II, Smad2, p-Smad2/3 (Santa Cruz Biotechnology), and b-actin (Sigma), washed, and incubated with secondary antibody against mouse or rabbit IgG conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) for 60 minutes. After washing, visualization was performed by ECL (Amersham Biosciences) according to the manufacturer’s recommendations. Band densities

1030

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

Peng et al.

were measured with UN-SCAN-IT software (Silk Scientific, Orem, UT). Statistical Analysis The correlation between clinicopathological parameters (i.e., surgical staging) and immunohistochemical analysis results was assessed, as was the correlation between TGF-b1, its receptors, and cell cycle – related proteins, and that between these proteins and the Ki67 labeling index. Comparative data were analyzed using Mann-Whitney U test. For all statistical analyses, P < .05 was considered statistically significant.

Results Abrogated Expression of T R-II, p-Smad2, and p12CDK2-AP1 in Human OSCC In Vivo TGF-b1 signaling is mediated mainly by two serine threonine kinase receptors, TbR-I and TbR-II. Loss of function or decreased expression of TbR-II has been reported in several tumors; however, the level of TbR-I, TbR-II, and TGF-b1, and their association with OSCC carcinogenesis and tumor progression have not been fully described. In this study, we first examined the expression levels of TGF-b1, TbR-I, and TbR-II in 125 cases of human OSCC by immunohistochemistry. Table 1 shows the characteristics of tissue samples used for the immunohistochemical study. Oral squamous epithelium obtained in surgical procedures not associated with squamous neoplasia has consistently revealed moderate to intense homogenous cytoplasmic expression of TGF-b1 and TGF-b receptors I and II. Homogeneous, moderate, intense cytoplasmic expression of TGF-b1 and TbR-I was also shown in OSCC tissues; however, there was significant weak expression or no expression of TbR-II proteins shown in OSCCs (Figure 1). According to the progression of clinical stage, the expression of TbR-II decreased. The intensity of TGF-b1, TbR-I, and TbR-II expression in different clinical tumor stages is summarized in Table 2. There was no correlation between the expression of TGF-b1, TbR-I, or TbR-II and clinicopathological parameters such as differentiation and mode of invasion (data not shown). These results suggest that, in

Table 1. Characteristics of Tissue Samples Used for Immunohistochemical Analysis. Age (years) Gender Male Female T classification T1 T2 T3 T4 N classification N0 N1 N2

23 – 94 70 55 29 64 9 23 83 27 15

OSCC, there is a significant progressive loss of expression of TbR-II, which associates with highly aggressive lesions. There is no significant loss of expression of TbR-I in OSCC; TbR-I expression is not directly related to the degree of tumor differentiation or to local aggressiveness. Smad2 and Smad4 transcription factors are mediators of TGF-b1 signaling [6 – 8]. Phosphorylation activation of Smad2 is a key step in TGF-b1 signaling for the induction expression of downstream molecules, such as cell cycle inhibitors p21 and p27 [11,29,30]. In our study, Smad2 and p-Smad2 were shown in the nuclei of the normal epithelium (Figure 2). Although Smad2 expression was similarly shown in the nuclei of OSCC, p-Smad2 expression was significantly decreased compared to that in the normal epithelium (Figure 2). Statistical analysis showed that there was a significant correlation between the expressions of TbR-II and p-Smad2 in OSCC (P < .001), whereas there was no correlation between the expressions of TGF-b1, TbR-I, and p-Smad2 in OSCC (Table 3). Cell cycle – regulatory proteins are downstream molecules of TGF-b – Smad signaling. The growth-inhibitory effect of TGF-b1 is mediated by inducing the expression of important cell cycle – inhibitory proteins such as p21WAF1, p12CDK2-AP1, and p27KIP1 [11,12,29]. Our immunohistochemistry study showed that there was loss of expression of p12CDK2-AP1 and p21WAF1 in OSCC (Table 4). The mean values of the labeling indices for p12CDK2-AP1, p21WAF1, p27KIP1, Ki67, Smad2, and p-Smad2 in OSCC are summarized in Table 4. A decrease in TbR-II immunoreactivity had a statistically significant correlation with decreased p12CDK2-AP1 and p27KIP1 expression (P < .001 and P < .01, respectively) (Table 5). No correlation between other TGF-b receptors and p21WAF1 was found. In addition, no correlation between TGF-b1, TbR-I, and cell cycle – related proteins was observed. Our immunohistochemistry results showing the loss of expression of TbR-II, p-Smad2, p21WAF1, and p12CDK2-AP1 in OSCC provide evidence of aberrant TGF-b – Smad pathway in this human cancer and also indicate the important role of p21WAF1 and p12CDK2-AP1, potentially inhibiting tumor formation or progression. Decreased Expression of T R-II, p12, Smad2, and p-Smad2/3 in Human OSCC Lines In Vitro The role of the TGF-b – Smad signaling pathway in the carcinogenesis of head and neck cancer has not been fully evaluated. To examine this signaling pathway in human OSCC, we first examined the basal expression levels of TbR-I, TbR-II, Smad-2, and p-Smad2/3 in human OSCC cell lines by immunoblotting. Compared with normal keratinocytes, an increasing level of TbR-I in OSCC was detected (Figure 3A); however, the expression of TbR-II was decreased in OSCC (Figure 3A). We also detected decreasing levels of Smad2 and p-Smad2/3 in OSCC (Figure 3B). p27KIP1, p21WAF1, and p12CDK2-AP1 are negative cell cycle regulators and growth inhibitors, and p21WAF1 and p12CDK2-AP1 are also implicated to be induced transcriptionally by TGF-b1 in epithelial cells [11,12]. To study the correlation between TGFb – Smad signaling and these growth regulators, we also

Neoplasia . Vol. 8, No. 12, 2006

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

Peng et al.

1031

Figure 1. Decreased expression of T R-II in human OSCC tissues. The levels of TGF- 1 and its associated receptors (T R-I and T R-II) in human OSCC tissues were compared to those in normal tissues, as described in the Materials and Methods section. The levels of TGF- 1 and T R-I did not show any significant alterations in OSCC, but there was significant reduction of T R-II in OSCCs compared to that in the normal epithelium.

examined the basal expression levels of p27KIP1, p21WAF1, and p12CDK2-AP1. Compared with normal oral keratinocytes, p12CDK2-AP1 expression decreased in most OSCC cell lines (Figure 3A); however, decreased expression of p27KIP1 and p21WAF1 was not consistently found in OSCC cell lines. These results indicate that a defective TGF-b –Smad signaling pathway exists in human OSCC and provides a possible link between loss of p12CDK2-AP1 expression and defective TGFb –Smad signaling in OSCC. Loss of the Growth-Inhibitory Effect of TGF- 1 in Human OSCC TGF-b1 is a potent growth inhibitor of epithelia. To test the response of human OSCC with a disrupted TGF-b – Smad pathway to TGF-b1, we compared the growth pattern of OSCC with that of normal oral keratinocytes after TGF-b1 treatment. Growth suppression was observed in normal human oral keratinocyte after 24 hours of TGF-b1 exposure (Figure 4A); however, five malignant SCC cell lines keep growing even after 72 hours of TGF-b1 treatment (Figure 4,

Neoplasia . Vol. 8, No. 12, 2006

B – F ). Among the five tumor cell lines, only UM1 showed partial growth inhibition by TGF-b1 (Figure 4F ). The result confirmed that human OSCC has significant resistance to TGF-b1 growth-inhibitory effect. The key step of TGF-b – Smad signaling is mediated by the phosphorylation activation of p-Smad2/3 and the induction Table 2. Immunohistochemical Analysis of TGF-b1 and Its Receptors in OSCC Tissues. Samples

TGF-b1 0 to 1+ 2+ to 3+ TbR-I 0 to 1+ 2+ to 3+ TbR-II 0 to 1+ 2+ to 3+

OSCC [n (%)] I (n = 28)

II (n = 41)

III (n = 23)

IV (n = 33)

2 (7.1) 26 (92.9)

9 (21.9) 32 (78.1)

3 (13.0) 20 (87.0)

7 (21.2) 26 (78.8)

4 (14.3) 24 (85.7)

10 (24.4) 31 (75.6)

7 (30.4) 16 (69.6)

11 (33.3) 22 (66.7)

13 (46.6) 15 (53.6)

28 (68.3) 13 (31.7)

18 (78.3) 5 (21.7)

24 (72.7) 9 (27.3)

1032

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

Peng et al.

Figure 2. Significant reduction of p-Smad2 in human OSCC tissues. The levels of Smad2 and p-Smad2, known regulators in TGF- 1 – mediated growth suppression, were analyzed in human OSCC tissues. Immunohistochemical analysis showed nuclear staining of Smad2 and p-Smad2. There was significant reduction of p-Smad2 in OSCC, but there was no significant alteration in Smad2.

expression of downstream cell cycle inhibitors, such as p21WAF1 and p12CDK2-AP1, in epithelial cells. To understand TGF-b1 resistance in OSCC, we further examined the induction levels of p-Smad2/3, p21WAF1, and p12CDK2-AP1, as well as the components of the TGF-b – Smad pathway in OSCC compared with normal oral keratinocytes. Increased levels of p-Smad2/3, as well as p21WAF1 and p12CDK2-AP1, were found in normal keratinocytes after 24 hours of TGF-b1 exposure; however, among the five OSCC lines, p21WAF1 could only be induced in UM1, and p12CDK2-AP1 induction was found only in SCC66 (Figure 5, A – D). Besides, a complete failure of p-Smad2/3 induction was found in all OSCCs examined (Figure 5, A and B). TbR-I, Smad2, and p27KIP1 levels have no consistent changes among the five OSCC cell lines. These results strongly suggest that the failure of induction of

p12CDK2-AP1 or p21WAF1 expression is an important event in the TGF-b1 resistance observed in human OSCC.

Discussion TGF-b receptors mediate the biologic effects of TGF-b1. The direct involvement of TbR-I and TbR-II in TGF-b signal transduction suggests that changes in the expression of these two receptors may lead to loss of TGF-b sensitivity and may ultimately negate the autocrine growth-regulatory mechanism imposed by TGF-b [31]. TGF-b is a potent regulator of proliferation, growth, and differentiation in normal squamous epithelium. TGF-b exerts its antiproliferative effect through TbR-II. Loss of function or decreased expression of TbR-II has been reported in several tumors, and it has been described as having an association with carcinogenesis and tumor progression [9,10,32,33]. Recently, Grady et al. [34]

Table 3. Statistical Analysis of the Correlation of TGF-b1 and Its Receptors with Smad2 and p-Smad2 in OSCC. Samples TGF-bI 0 to 1+ (n = 21) 2+ to 3+ (n = 104) TbR-I 0 to 1+ (n = 32) 2+ to 3+ (n = 93) TbR-II 0 to 1+ (n = 83) 2+ to 3+ (n = 42) NS, not significant.

Smad2

P

p-Smad2

P

Table 4. Levels of Known Cell Cycle Regulators and Signaling Molecules in the TGF-b1 – Mediated Pathway in OSCC.

31.7 ± 3.6 33.9 ± 1.6

NS

16.6 ± 1.7 16.8 ± 2.6

NS

Samples

30.5 ± 2.9 34.6 ± 1.7

NS

15.3 ± 2.1 17.1 ± 1.2

NS

22.3 ± 1.8 36.1 ± 2.5

NS

12.3 ± 1.1 25.2 ± 1.6

< .001

Ki67 p12CDK2-AP1 p21WAF1 p27KIP1 Smad2 p-Smad2

Normal (n = 28) 6.3 31.3 25.8 30.1 41.2 32.8

± ± ± ± ± ±

1.9 1.9 2.3 3.2 3.3 4.1

OSCCs I (n = 28)

II (n = 41)

III (n = 23)

IV (n = 33)

26.6 17.5 8.0 24.3 40.0 24.5

28.9 13.7 8.9 25.6 32.1 17.0

34.2 13.6 7.0 20.0 30.3 13.5

36.0 14.3 6.2 23.1 32.0 12.0

± ± ± ± ± ±

1.8 1.8 1.3 2.3 3.1 2.1

± ± ± ± ± ±

1.5 1.5 1.0 1.9 2.6 1.7

± ± ± ± ± ±

1.9 1.9 1.4 2.6 3.4 2.3

± ± ± ± ± ±

1.7 1.6 1.2 2.1 2.8 1.9

Neoplasia . Vol. 8, No. 12, 2006

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC Table 5. Statistical Analysis of the Correlation of TGF-b1 and Its Receptors with Cell Cycle Inhibitors in OSCC. Samples TGF-b1 0 to 1+ (n = 21) 2+ to 3+ (n = 104) TbR-I 0 to 1+ (n = 32) 2+ to 3+ (n = 93) TbR-II 0 to 1+ (n = 83) 2+ to 3+ (n = 42)

p12CDK-AP1 P

p21CIP/WAF1 P

p27KIP1

13.9 ± 2.0 14.7 ± 0.9

NS

5.7 ± 1.4 7.8 ± 0.6

NS 23.1 ± 1.2 NS 26.5 ± 2.7

13.4 ± 1.7 15.0 ± 1.0

NS

8.0 ± 0.7 5.5 ± 1.1

NS 23.6 ± 1.3 NS 23.8 ± 2.2

10.7 ± 0.8 22.2 ± 1.2

< .001 8.7 ± 1.0 6.7 ± 0.7

P

NS 20.2 ± 1.9 < .01 25.3 ± 1.3

NS, not significant.

reported that mutations in TbR-II coincide with the transformation of benign adenomas to malignant carcinomas in colon cancers with microsatellite instability. These obser-

Peng et al.

1033

vations have led to the hypothesis that escape from TGFb – mediated negative growth control is an important aspect of malignant phenotype in many epithelial neoplasms. In the present study, we have analyzed the expression of TGF-b1 and the direct involvement of TbR-I and TbR-II in OSCC. Our results indicate that expression of TbR-II inversely correlates with clinical stages and suggest that aberrant TbR-II expression is a contributing factor to OSCC development. To investigate whether anomalies in TbR-II expression are specific for this tumor, we have also analyzed the expression of TbR-I, another component of the TGF-b signal transduction pathway. The normal squamous epithelium had moderate to high levels of both receptors. Furthermore, TbR-I was consistently expressed in all tumors analyzed and in all areas within the tumors. In contrast to TbR-I, the distribution of TbR-II expression was markedly

Figure 3. Basal expression level of TGF- – Smad signaling proteins in OSCC. (A) Western blot analysis of T R-I, T R-II, p12 CDK2-AP1, and p27 KIP1 in the normal oral keratinocytes OKF6 tert1 and OKF6 tert2, as well as in OSCC. Expression of T R-I increased in OSCC compared with that in normal keratinocytes. Expression of T R-II and p12CDK2-AP1 decreased in OSCC. Multiple bands of T R-II represent different isoforms. (B) Western blot analysis of Smad2 and pSmad2/3 in OKF6 tert1 and OSCC shows decreased Smad2 and p-Smad2/3 expression in OSCC. Forty micrograms of total proteins was used for Western blot analysis. The level of actin is shown as loading control. Protein levels were quantitated by scanning and analysis with UN-SCAN-IT software. Values indicate total pixel values of a particular protein/actin. The same findings were made from three independent experiments.

Neoplasia . Vol. 8, No. 12, 2006

1034

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

Peng et al.

Figure 4. Loss of the growth-inhibitory effect of TGF- 1 on OSCC. Cells were seeded in six-well plates, 1 ng/ml TGF- 1 was added when cells reached 50% confluence, and the rate of cell duplication was determined by counting cells for three consecutive days after TGF- 1 treatment. Values represent cell number, and data are expressed as the mean and SD of triplicate experiments. (A) Complete growth suppression by 24 hours of TGF- 1 exposure in normal keratinocyte OKF6 tert1. (B – E) Loss of growth suppression even by 72 hours of TGF- 1 exposure in OSCC. (F) OSCC UM1 exhibited partial growth inhibition after 48 hours of TGF- 1 exposure.

different. In the majority of OSCCs, particularly in infiltrating regions, TbR-II expression was either markedly reduced or completely absent. The mechanisms of resistance to the antiproliferative effects of TGF-b are unknown. However, a decrease in the number of TbR-II – binding sites has been associated with loss of responsiveness to the TGF-b1 cell line in the present study. Similar findings were observed in a number of studies [35 – 39]. This decrease in TbR-II expression correlates with tumorigenicity in many cell lines. Moreover, an abnormal TGF-b signal transduction pathway could contribute to progressive loss of differentiation in carcinomas. Previous studies revealed cell type – dependent variations in the effect of TGF-b1 on cell cycle regulators. p27KIP1 and p21WAF1, both CDK inhibitors, have also been reported to have an essential role in cell cycle inhibition

induced by TGF-b1 [29,40 – 42]. Recently, we have suggested that p12CDK2-AP1 may play a role in TGF-b1 – mediated growth suppression [12]. This study revealed that TbR-II, p12CDK2-AP1, p21WAF1, p27KIP1, and p-Smad2 were concordantly decreased, supporting the view that TbR-II and cell cycle control proteins may play an important role in the process of cell progression in OSCC. In addition, this result may provide a link between the effects of growth factor and cell cycle control in this tumor. Smad proteins play a critical role in transmitting TGF-b superfamily signals from the cell surface to the nucleus [43]. By forming a heteromeric complex composed of Smad2/3/4, Smad proteins transduce TGF-b or activin signals [30]. In the current study, immunohistochemistry and Western blot analysis showed the correlation between the abrogated expression of TbR-II, p-Smad2,

Neoplasia . Vol. 8, No. 12, 2006

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

Peng et al.

1035

Figure 5. Induction level of TGF- – Smad signaling proteins by TGF- 1 in normal oral keratinocytes and OSCCs. Cells exposed to 1 ng/ml TGF- 1 for 24 hours were used for Western blot analysis. Forty micrograms of total proteins was loaded. The level of actin is shown as loading control. Protein levels were quantitated by scanning and analysis with UN-SCAN-IT software. Values indicate the total pixel values of a particular protein/actin. (A) Western blot analysis of T R-I, Smad2, p-Smad2/3, p12CDK2-AP1, p21WAF1, and p27KIP1 in normal oral keratinocyte OKF6 tert1 and OSCC with or without TGF- 1 exposure. (B) Increased p-Smad2/3 expression in normal oral keratinocyte OKF6 tert1 under TGF- 1, but complete loss of p-Smad2/3 induction was found in all OSCCs. (C) Induction of p12CDK2-AP1 in OKF6 tert1 under TGF- 1; however, p12CDK2-AP1 was induced only in SCC66. (D) Induction of p21WAF1 in OKF6 tert1 under TGF- 1; however, in OSCC, this TGF- 1 effect was found only in UM1. The results were confirmed in three independent experiments.

and TbR-II, and cell cycle control proteins such as p27KIP1, p21WAF1, and p12CDK2-AP1. Our data provide evidence of a disrupted TGF-b – Smad signaling pathway; consequently, abrogated expression of cell cycle inhibitors such as p12CDK2-AP1 may lead to loss of response to TGF-b1 growthsuppressive effects on OSCC.

Acknowledgement The authors are grateful to Steven Mok for technical support.

References [1] Tucker RF, Shipley GD, Moses HL, and Holley RW (1984). Growth inhibitor from BSC-1 cells is closely related to the platelet type b transforming growth factor. Science 226, 705 – 707. [2] Shipley GD, Pittelkow MR, Wille JJ Jr, Scott RE, and Moses HL (1986). Reversible inhibition of normal human prokeratinocyte proliferation by type b transforming growth factor-growth inhibitor in serum-free medium. Cancer Res 46, 2068 – 2071.

Neoplasia . Vol. 8, No. 12, 2006

[3] Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, and Sporn MB (1985). Type-beta transforming growth factor: a bifunctional regulator of cellular growth. Proc Natl Acad Sci USA 82, 119 – 123. [4] Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, Sporn MB, and Fauci AS (1986). Transforming growth factor-beta is an important immunomodulatory protein for human B lymphocytes. J Immunol 137, 3855 – 3860. [5] Knabbe C, Lippman ME, Wakefield LM, Flanders KC, and Kasid A (1987). Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells. Cell 48, 417 – 428. [6] Nakao A, Roijer E, Imamura T, Souchelnytskyi S, Stenman G, Heldin CH, and ten Dijke P (1997). Identification of Smad2, a human Madrelated protein in the transforming growth factor beta signaling pathway. J Biol Chem 272, 2896 – 2900. [7] Macias-Silva M, Abdollah S, Hoodless PA, Pirone R, Attisano L, and Wrana JL (1996). MADR2 is a substrate of the TGFb receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87, 1215 – 1224. [8] Yingling JM, Das P, Savage C, Zhang M, Padgett RW, and Wang XF (1996). Mammalian dwarfins are phosphorylated in response to TGF-b and are implicated in control of cell growth. Proc Natl Acad Sci USA 93, 8940 – 8944. [9] Paterson IC, Matthews JB, and Huntley S (2001). Decreased expression

1036

[10]

[11]

[12]

[13] [14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

p12CDK2-AP1 and Resistance to TGF-B1 in Human OSCC

Peng et al.

of TGF-b cell surface receptors during progression of human oral squamous cell carcinoma. J Pathol 193, 458 – 467. Williams RH, Stapleton AMF, and Yang G (1996). Reduced levels of transforming growth factor-b receptor type II in human prostate cancer: an immunohistochemical study. Clin Cancer Res 2, 635 – 640. Pardali K, Kurisaki A, Moren A, Dijke PT, Kardassis D, and Moustakas A (2000). Role of Smad proteins and transcription factor Sp1 in p21WAF/ Cip regulation by transforming growth factor-beta. J Biol Chem 275, 29244 – 29256. Hu MG, Hu G-F, Kim Y, Tsuji T, McBride J, Hinds P, and Wong DTW (2004). Role of p12CDK2-AP1 in transforming growth factor-b1 – mediated growth suppression. Cancer Res 64, 490 – 499. American Cancer Society (2004). Cancer Facts and Figures. Atlanta, GA: American Cancer Society. Le QT and Giaccia AJ (2003). Therapeutic exploitation of the physiological and molecular genetic alterations in head and neck cancer. Clin Cancer Res 9, 4287 – 4295. Forastiere A, Koch W, Trotti A, and Sidransky D (2001). Head and neck cancer. N Engl J Med 345, 1890 – 1900. Shintani S, Mihara M, Terakado N, Nakahara Y, Matsumura T, Kohno Y, Ohyama H, McBride J, Kent R, Todd R, et al. (2001). Reduction of p12DOC-1 expression is a negative prognostic indicator in patients with surgically resected oral squamous cell carcinoma. Clin Cancer Res 7, 2776 – 2782. Shintani S, Mihara M, Nakahara Y, Kiyota A, Ueyama Y, Matsumura T, and Wong DT (2002). Expression of cell cycle control proteins in normal epithelium premalignant and malignant lesions of oral cavity. Oral Oncol 38, 235 – 243. Shintani S, Ohyama H, Zhang X, McBride J, Matsuo K, Tsuji T, Hu MG, Hu G, Kohno Y, Lerman M, et al. (2000). p12(DOC-1) is a novel cyclin-dependent kinase 2 – associated protein. Mol Cell Biol 20, 6300 – 6307. Matsuo K, Shintani S, Tsuji T, Nagata E, Lerman M, McBride J, Nakahara Y, Ohyama H, Todd R, and Wong DT (2000). p12 (DOC-1), a growth suppressor, associates with DNA polymerase a/primase. FASEB J 14, 1318 – 1324. Ji GZ, Wang XH, Miao L, Liu Z, Zhang P, Zhang FM, and Yang JB (2006). Role of transforming growth factor-beta1 – smad signal transduction pathway in patients with hepatocellular carcinoma. World J Gastroenterol 12, 644 – 648. Li M, Becnel LS, Li W, Fisher WE, Chen C, and Yao QZ (2005). Signal transduction in human pancreatic cancer: roles of transforming growth factor beta, somatostatin receptors, and other signal intermediates. Arch Immunol Ther Exp 53, 381 – 387. Hsu S, Huang F, Hafez M, Winawer S, and Friedman E (1994). Colon carcinoma cell switch their response to transforming growth factor-b1 with tumor progression. Cell Growth Differ 5, 267 – 275. Jennings MT, Maciunas RJ, Carver R, Bascom CC, Juneau P, Misulis K, and Moses HL (1991). TGF-b1 and TGF-b2 are potential growth regulators for low-grade and malignant gliomas in vitro: evidence in support of an autocrine hypothesis. Int J Cancer 49, 129 – 139. Qiu W, Schonleben F, Li X, and Su GH (2006). Disruption of transforming growth factor beta – Smad signaling pathway in head and neck squamous cell carcinoma as evidenced by mutations of SMAD2 and SMAD4. Cancer Lett 12 [Epub ahead of print]. Sobin LH and Fleming ID (1997). TNM Classification of Malignant

[26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41] [42]

[43]

Tumors, Fifth Edition. Union International Contre le Cancer and the American Joint Committee on Cancer. Cancer 80, 1803 – 1804. Wasi PN, Cohen B, Luthra UK, and Torloni H (1971). Histological typing of oral and oropharyngeal tumors. International Histological Classification of Tumor. Geneva, WHO, pp 17 – 18. Anneroth G, Batsakis J, and Lunba M (1987). Review of the literature and a recommended system of malignancy grading in oral squamous cell carcinomas. Scand J Dent Res 95, 229 – 249. Gobbi H, Dupont WD, and Simpson JF (1999). Transforming growth factor-beta and breast cancer risk in women with mammary epithelial hyperplasia. J Natl Cancer Inst 91, 2096 – 2101. Polyak K, Kato M, and Solomon MJ (1998). p27KIP1, a cyclin-Cdk inhibitor, links transforming growth factor-b and contact inhibition to cell cycle arrest. Genes Dev 160, 770 – 777. Massague J (1998). TGF-b signal transduction. Annu Rev Biochem 67, 753 – 791. Franchi A, Arganini L, and Baroni G (1998). Expression of transforming growth factor beta isoforms in osteosarcoma variants: association of TGF-b1 with high-grade osteosarcomas. J Pathol 185, 284 – 289. Laiho M, Weis FM, and Boyd FT (1991). Responsiveness to transforming growth factor-beta (TGF-b) restored by genetic complementation between cells defective in TGF-b receptors I and II. J Biol Chem 266, 9108 – 9112. Guo Y, Jacobs SC, and Kyprianou N (1997). Down-regulation of protein and mRNA expression for transforming growth factor-beta (TGF-b1) type I and type II receptors in human prostate cancer. Int J Cancer 71, 573 – 579. Grady WM, Rajput A, Myeroff L, Liu DF, Kwon K, Willis J, and Markowitz S (1998). Mutation of the type II transforming growth factor-b receptor is coincident with the transformation of human colon adenomas to malignant carcinomas. Cancer Res 58, 3103 – 3104. Lagna G, Hata A, and Massague J (1996). Partnership between DPC4 and SMAD protein in TGF-b signaling pathway. Nature 383, 832 – 836. Guo YP, Jacobs SC, and Kypriano N (1996). Decreased expression of TGFb1 type I and type II receptors in human prostate cancer. Proc Am Assoc Cancer Res 37, 245. Jakolew SB, Mathias A, Chung P, and Moody TW (1995). Expression of transforming growth factor b ligand and receptor messenger RNA in lung cancer lines. Cell Growth Differ 6, 465 – 476. Wang D, Song H, Evans JA, Lang JC, Schuller DE, and Weghorst CM (1997). Mutation and down-regulation of the transforming growth factor b type II receptor gene in primary squamous cell carcinomas of the head and neck. Carcinogenesis (London) 18, 2285 – 2290. Garrique-Antar L, Souza RL, Vellucci VF, Meltzer SJ, and Reiss M (1996). Loss of transforming growth factor-b type II receptor gene expression in primary human esophageal cancer. Lab Invest 75, 263 – 272. Polyak K, Lee MH, and Erdjument-Bromage J (1994). Cloning of p27KIP1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78, 59 – 66. Ewen ME, Sluss HK, and Whitehouse LL (1993). TGF-b inhibition of cdk4 synthesis is linked to cell cycle arrest. Cell 74, 1009 – 1020. Datto MB, Li Y, and Panus JF (1995). Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53independent mechanism. Proc Natl Acad Sci USA 92, 5545 – 5549. Derynck R, Zhang Y, and Feng XH (1998). Smads: transcriptional activators of TGF-b responses. Cell 95, 737 – 740.

Neoplasia . Vol. 8, No. 12, 2006