Siah-1 is associated with expression of hypoxia-inducible factor-1α in oral squamous cell carcinoma

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Siah-1 is associated with expression of hypoxia-inducible factor-1a in oral squamous cell carcinoma Mitsuharu Aga, Satoru Kondo, Naohiro Wakisaka, Makiko Moriyama-Kita, Kazuhira Endo, Yosuke Nakanishi, Shigeyuki Murono, Hisashi Sugimoto, Takayoshi Ueno, Tomokazu Yoshizaki * Division of Otolaryngology, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 December 2015 Accepted 10 June 2016 Available online xxx

Objective: Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of the HIF-1a and HIF-1b subunits. HIF-1 is a central regulator of responses to hypoxia; it enhances metastasis-related factors such as matrix metalloproteinases and vascular endothelial growth factor (VEGF). We have reported critical roles for HIF-1a in tumor microenvironments, and oncogenic properties of HIF-1a have been suggested in malignancies. Seven in absentia homologue (Siah) appeared to upregulate HIF-1 production, which prompted us to investigate the Siah association with HIF-1a expression in oral squamous cell carcinoma (OSCC). Methods: Samples from fifty-five patients with OSCC were evaluated by immunohistochemistry for the protein expressions of Siah-1 and -2, HIF-1a, and VEGF. The expression levels of each protein and clinicopathological data were statistically analyzed. Results: Siah-1 and, Siah-2, HIF-1a, and VEGF were immunolocalized on the cell membranes and cytoplasm of the tumor cells. The expression of Siah-1 showed a linear dependence on the expression of HIF-1a (r = 0.627, p < 0.001). In 17 cases of the large tumor size category (T3 and 4), the mean Siah-1 expression score was significantly higher than in 41 cases of the small tumor size category (T1 and 2; p = 0.001). In addition, in 16 cases of the lymph node metastasis-positive category (N1–3), the mean Siah-1 expression score was significantly higher than that in 42 cases of the lymph node metastasis-negative category (N0, p = 0.001). Conclusion: These results suggested that the expressions of Siah-1 and HIF-1a were clearly correlated in OSCC. Moreover, Siah-1 appears to be correlated with clinicopathological data, particularly tumor size. ß 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Siah-1 HIF-1a Oral squamous cell carcinoma

1. Introduction Cancers have a specific potential for rapid and unlimited growth; hypoxia is a common feature of many cancers and contributes to local and systemic cancer progression [1]. We have been investigating metastasis-related factors such as

* Corresponding author. Fax: +81 76 234 4265. E-mail address: [email protected] (T. Yoshizaki).

matrix metalloproteinases (MMPs), syndecans, and vascular endothelial growth factor (VEGF), which predict the treatment outcome of patients with oral squamous cell carcinomas (OSCC [2–5]). Recently, interleukin (IL)-33, a member of the IL-1 family, has been shown to be associated with microvessels and mast cell densities in stroma [6]. Hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. HIF-1, the principal oxygensensing transcription factor, is composed of two subunits, HIF1a and HIF-1b [7]. The level of HIF-1a increases

http://dx.doi.org/10.1016/j.anl.2016.06.007 0385-8146/ß 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Aga M, et al. Siah-1 is associated with expression of hypoxia-induciblefactor-1a in oral squamous cell carcinoma. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.06.007

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exponentially in response to decreases in cellular oxygen concentration, whereas HIF-1b is not regulated by oxygen tension [7,8]. The specific binding of the HIF-1a and HIF-1b heterodimers to the hypoxic response element (HRE) activates the transcription of genes whose products are required for metabolism, erythropoiesis, vascularization, and tumor progression [7,9]. Under normoxic conditions, HIF-1a is degraded via a proteasome-degradation pathway [10]. Recently, we reported that HIF-1a is transported to the tumor microenvironment via extracellular vesicles, called exosomes, and functions as a transcriptional factor [11]. The Siah-family proteins are homologues of Drosophila seven in absentia (Sina) protein, which is essential for the formation of R7 photoreceptor cells during development. Mice and rats have three unlinked Siah homologue genes (Siah1a, Siah1b, and Siah2), whereas human beings have single SIAH1 and 2 genes [12,13]. Recently, a new pathway for controlling the levels of HIF-1a has been proposed; murine Siah stabilizes HIF-1a in mouse embryonic fibroblasts [14]. Blocking Siah function could be a promising approach to impairing angiogenesis and proliferation in carcinomas. Overexpression of HIF-1a has been demonstrated in a variety of cancers including renal, bladder, colorectal, breast, ovarian, endometrial, cervical, and head and neck cancers [15–32]. In some of these cancers, expression of HIF-1a has been found to be significantly associated with the histologic grade, lymph node metastasis, clinical stage, pathologic stage, survival rate, and cancer prognosis [16–22,25–27,29,32]. For years, our group revealed the function of HIF-1a and Siah-1 in latent membrane protein 1 (LMP1) positive nasopharyngeal carcinoma (NPC). In terms of molecular biology, we have shown that the Epstein–Barr virus oncogenic protein, LMP1, stabilizes HIF-1a [33] and it is via upregulation of Siah-1 [34]. Moreover, HIF-1a induced by LMP1 is transferred to LMP1-negative cell using exosomal cargo [11]. From the clinical view point, we evaluated the pattern and clinical significance of Siah-1 and HIF-1a expression in NPC and found that there was a remarkable correlation between the expression of Siah-1 and LMP1, and a weak correlation between the expression of Siah-1 and HIF1a. These findings indicate that Siah-mediated HIF-1a stabilization is an important phenomenon in cancer biology of NPC [35]. In oral squamous cell carcinoma (OSCC), the overexpression of HIF-1a has been detected and significantly correlated with T and N statuses, and clinical stages of OSCC [36]. Moreover, the presence of hypoxia in oral cancers is partially responsible for the upregulation of VEGF. The elevation of VEGF expression in OSCC tissue correlates with increased microvessel density and severity of the disease [37]. Moreover, our group demonstrated expressions of VEGF in regional lymph nodes of pathological N0 oral squamous cell carcinoma associated with intranodal lymphangiogenesis [5]. Thus, it is tempting to speculate whether Siah-1 is associated with expression of HIF-1a in OSCC. In addition, the association of Siah-1 with clinical status of OSCC was examined. The aim of this study is investigating the involvement of Siah-1 in clinical status of OSCC.

2. Materials and methods 2.1. Patient characteristics This study included 58 patients (37 men and 21 women) with OSCC who were initially diagnosed and underwent surgery between 1988 and 2005 in the Division of Otolaryngology, Kanazawa University Hospital. The age of the patients ranged from 25 to 93 years (mean, 60.7 years). Each patient’s chart was retrospectively reviewed for age, sex, and stage. According to the fifth TNM classification system of the Union for International Cancer Control in 1997, histologically, there were 20 (34%) stage I, 15 (26%) stage II, 11 (19%) stage III, and 12 (21%) stage IV. Pathological data of the patients are summarized in Table 1. 2.2. Immunohistochemical staining and evaluation Formalin-fixed, paraffin-embedded blocks were sectioned (3-mm in thickness) and used for hematoxylin–eosin (H&E) staining. H&E slides were used to reevaluate the histologic characteristics. The paraffin-embedded tissue sections were also used for immunohistochemical staining. Briefly, 3-mm sections were de-waxed in xylene and hydrated with graded ethanol. Afterwards, sections were incubated with a prediluted primary antibody. Primary antibodies against Siah-1 and Siah-2 (Trans Genic), HIF-1a (H-206, Santa Cruz), and VEGF (A-20, Santa Cruz) were used. Immunoreactions were visualized with the diaminobenzidine chromogen, and the sections were counterstained using Meyer’s hematoxylin and mounted in Entellan. The sections were exposed to secondary antibody with the EnVision kit (DAKO, Glostrup, Denmark). Stained samples were reviewed using light microscopy. Three areas that showed high densities of stained cells in a 40 field were selected, then the number of stained cells from a group of 200 cells was carefully counted in the three areas in a 200 field without any previous clinicopathological knowledge of the patients. The percentage of positive cell numbers obtained as an Table 1 Patients’ pathological data. Variables Sex Male Female T stage pT1 pT2 pT3 pT4 Nodal status pN0 pN1 pN2 pN3 Clinical stage I II III IV

Total number 37 21 22 20 9 7 42 7 7 2 20 15 11 12

Please cite this article in press as: Aga M, et al. Siah-1 is associated with expression of hypoxia-induciblefactor-1a in oral squamous cell carcinoma. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.06.007

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average of three counts was used as the expression score. For the purposes of clinical correlation, expression scores of these proteins were arbitrarily divided into two categories consisting of high (10% of tumor cells) and low (<10%).

were visualized as dark brown staining. Siah-1 and HIF-1a were expressed in the cytoplasm and cell membranes, and partially in the nucleus (Fig. 1A and C). Siah-2 and VEGF were located in the cytoplasm and cell membranes (Fig. 1B and D).

2.3. Statistical analysis

3.2. Association of HIF-1a with expression of Siah-1, Siah-2, and VEGF

The relationship between the expression score of Siah-1, Siah-2, HIF-1a, and VEGF was examined using the Spearman rank correlation coefficient. The correlation among each protein expression and clinicopathological data were analyzed with the Spearman correlation and Mann–Whitney U test. Multivariate analyses were carried out with logistic regression model. All of the analyses were performed using Dr. SPSS version 11.0.1 (SPSS, Chicago, IL). The significance level was set at p < 0.05. 3. Results 3.1. Immunostaining for Siah-1, Siah-2, HIF-1a, and VEGF In recent studies, the expression of HIF-1a and VEGF has been detected in OSCC [35,36], and Siah contributes to stabilization of HIF-1a [34]. Therefore, we wondered whether Siah-1, Siah-2, HIF-1a, and VEGF were overexpressed in OSCC. To evaluate this, the expression of Siah-1, Siah-2, HIF1a, and VEGF was examined by immunohistochemical staining on surgically resected OSCCs. Of 58 tumors, Siah-1 expression was observed in 24 cases (41.4%), Siah-2 expression was observed in 17 cases (29.3%), HIF-1a expression was observed in 26 cases (44.8%), and VEGF expression was observed in 39 cases (67.2%). These proteins

Because Siah proteins demonstrate involvement in hypoxia signaling via regulation of HIF-1a, which induces expression of VEGF, the correlation of HIF-1a and Siah-1, and/or Siah-2 or VEGF was examined [34,36]. A strong correlation was found between the expressions of HIF1a and Siah-1 (r = 0.627, p < 0.001 [Fig. 2]) or that of VEGF (r = 0.620, p < 0.001 [Fig. 3]). There was also strong correlation between Siah-1 and VEGF (r = 0.5759, p < 0.001 [Fig. 4]). There was a weak correlation between the expressions of HIF-1a and Siah-2 (r = 0.309). 3.3. Relationship of clinicopathological features with expression of Siah-1, Siah-2, HIF-1a, and VEGF It has been reported that HIF-1a and VEGF expression in OSCC correlates with severity of the disease [35,36]. Thus, we tested the relationship between clinicopathological features and the expression of Siah-1, Siah-2, HIF-1a, and VEGF. T classification was significantly associated with the expression of Siah-1 (p = 0.001), HIF-1a (p = 0.01), and VEGF (p = 0.03). No statistically significant association was observed with those of Siah-2. N classification was significantly associated with the expression of Siah-1 (p = 0.001), HIF-1a (p = 0.001), and VEGF (p = 0.001). No statistically significant association was

Fig. 1. Immunohistochemical staining of Siah-1, Siah-2, HIF-1a, and VEGF in OSCC sections. (A) Immunostaining for Siah-1. Siah-1 signals are detected in the cytoplasm and cell membranes, and partially in the nucleus, of the tumor cells. (B) Immunostaining for Siah-2. Siah-2 signals are detected in cytoplasm and cell membrane of the tumor cells. (C) Immunostaining for HIF-1a. HIF-1a signals are detected in the cytoplasm and cell membranes, and partially in the nucleus, of the tumor cells. (D) Immunostaining for VEGF. VEGF signals are detected in cytoplasm and cell membranes of the tumor cells.

Please cite this article in press as: Aga M, et al. Siah-1 is associated with expression of hypoxia-induciblefactor-1a in oral squamous cell carcinoma. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.06.007

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VEGF (Expression score) (%)

60 50 40 30 20 10 0

r = 0.5759, p < 0.001 - 10 - 10

0

10

20

30

40

50

60

Siah1 (Expression score) (%) Fig. 2. Correlation of Siah-1 expression with HIF-1a. The Siah-1 and HIF-1a expression scores for 58 OSCC cases are plotted. Siah-1 expression demonstrated a significant correlation with HIF-1a expression according to the Spearman correlation coefficient (r = 0.627 and p < 0.001).

observed with that of Siah-2 (Table 2), sex, age, smoking and alcohol intake. The clinical significance of the expression of Siah-1, HIF-1a, and VEGF was therefore assessed by multivariate analysis in order to adjust the other parameters significantly associated with clinical significance. This analysis showed that only Siah-1 was significantly associated with T (p = 0.001) and N (p = 0.008) classification (Table 3). 4. Discussion Hypoxia is commonly seen in many of the pathological conditions in which neovascular growth is observed. A local decrease in oxygen levels has been considered a major stimulus for the induction of angiogenic factors [38]. Previous studies revealed that Siah proteins have played an important role in stabilizing HIF-1a [39] and that HIF-1a had induced the expression of VEGF [37]. In this study, we found that Siah-1,

Fig. 3. Correlation of VEGF expression with HIF-1a. The VEGF and HIF-1a expression scores for 58 OSCC cases are plotted. VEGF expression demonstrated a significant correlation with HIF-1a expression according to the Spearman correlation coefficient (r = 0.620 and p < 0.001).

Fig. 4. Correlation of VEGF expression with Siah-1. The VEGF and Siah-1 expression scores for 58 OSCC cases are plotted. VEGF expression demonstrated a significant correlation with Siah-1 expression according to the Spearman correlation coefficient (r = 0.5759 and p < 0.001).

Siah-2, HIF-1a, and VEGF were expressed in OSCC, and that there was a correlation between the expression of Siah-1 and that of HIF-1a or VEGF in OSCC. We believe that this mechanism is essential for tumor growth and lymph node metastasis induced by hypoxic conditions (Fig. 5). A recent study demonstrated that Siah-1 plays a role in proteasomedependent degradation of HIF-1a [39]. In addition, there is good evidence of an association of HIF-1a and VEGF in tumor angiogenesis [36] and our group demonstrated expressions of VEGF in regional lymph nodes of pathological N0 oral squamous cell carcinoma associated with intranodal lymphangiogenesis [5]. Loss of HIF-1a inhibits a number of important parameters of endothelial cell behavior during angiogenesis, including proliferation, chemotaxis, extracellular matrix penetration, and wound healing [40]. It has been reported that the expression of HIF-1a and VEGF is significantly correlated with the severity of OSCC [36]. In this study, the expression of HIF-1a, VEGF, and Siah-1 correlated with both the T and N factors with univariate analysis. However, multivariate analysis showed that only Siah1 was significantly associated with T and N factors. As rapid tumor growth results in large size tumors with some hypoxic areas, and hypoxia is a strong inductor of HIF-1a, this may be the major reason of increased HIF-1a expression in larger OSCCs [41,42]. In addition, HIF-1a can control the expression of genes encoding VEGF [42,43]. Because VEGF can stimulate the proliferation and migration of endothelial cells, resulting in the promotion of tumor angiogenesis, hypoxia-induced elevated production of HIF-1a may subsequently enhance oral cancer progression through the promotion of tumor angiogenesis [44,45]. HIF-1a can stimulate Matrigel invasion by human colon carcinoma cells in vitro [46]. VEGF-C is considered to induce both lymphatic invasion and lymph node metastasis in various types of cancer associated with lymphangiogenesis [47]. Hypoxia can increase VEGF-C expression in venous endothelial cells and human seminoma cells [48]. Katsuta et al. demonstrated a significant correlation of HIF-1a expression

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Table 2 Relationship between clinicopathological features and the expression of Siah-1, Siah2, HIF-1a, and VEGF. Variable

T stage T1, 2 T3, 4 Nodal status N0 N1, 2, 3

Total number

Siah1 (+)

Siah2 (+)

HIF1a (+)

VEGF (+)

Expression score (%)

p

Expression score (%)

p

Expression score (%)

p

Expression score (%)

p

41 17

10.27  11.77 35.53  13.70

0.001*

10.90  16.09 15.94  20.78

0.324

12.15  13.48 24.71  15.22

0.01*

12.15  13.48 32.00  13.06

0.03*

42 16

11.95  13.56 32.69  15.71

0.001*

10.07  16.77 18.44  18.68

0.11

12.21  13.75 25.31  14.46

0.001*

17.50  15.10 31.94  12.95

0.001*

p-Values were generated by comparisons between the two groups. Tumor (T), node (N) classification, and overall stage were classified on the basis of the UICC classification, 1997. * Significant.

Table 3 Multivariate analyses for relationship between clinicopathological features and the expression of Siah-1, HIF-1a and VEGF. Multivariate analysis T (T1,2 vs. T3,4)

HIF-1a expression Siah1 expression VEGF expression *

N (N0 vs. N1-3)

Hazard ratio

95% confidence interval

p

Hazard ratio

95% confidence interval

p

0.978 1.114 1.042

0.917–1.043 1.048–1.184 0.978–1.110

0.489 0.001* 0.201

1.001 1.071 1.033

0.945–1.061 1.018–1.126 0.975–1.094

0.96 0.008* 0.273

Significant.

with VEGF-C expression and lymphatic invasion in esophageal squamous cell carcinomas [47]. HIF-1a-regulated effector proteins such as VEGF-C, VEGF-F, Epo, or MMP2 can increase both cancer cell and lymphatic invasion, lymphangiogenesis, and tumor angiogenesis [43–48]. Siah proteins are implicated in a variety of cellular processes, such as apoptosis and tumor suppression. However, the biological significance of Siah-dependent b-catenin ubiquitination and its regulation in normal and cancer cells is largely unknown [49,50]. Recently, Brauckhoff et al. reported that nuclear accumulation of the E3 ubiquitin ligase Siah-1 supports different protumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis

[51]. In addition, the multivariate analysis of present study revealed that only Siah-1 was significantly associated with T and N classification. Therefore, blocking Siah-1 function could be a promising approach to impairing angiogenesis and proliferation. Interfering with HIF-1a upregulation through Siah inhibition is appealing, as the impact on the tumor’s adaptive responses to hypoxia is more significant than inhibition of the individual downstream HIF-1a effectors, such as VEGF. In conclusion, the present study suggested that the expressions of Siah-1 and HIF-1a clearly correlated in OSCC. Moreover, Siah-1 appears to be correlated with clinicopathological data, particularly tumor size. Conflicts of interest statement None declared. Acknowledgment This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 16K20235. References

Fig. 5. Schematic representation of the mechanism by which hypoxia induces tumor growth and lymph node metastasis.

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Please cite this article in press as: Aga M, et al. Siah-1 is associated with expression of hypoxia-induciblefactor-1a in oral squamous cell carcinoma. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.06.007