Elsevier

Oral Oncology

Volume 48, Issue 5, May 2012, Pages 417-423
Oral Oncology

Tumor-associated carbonic anhydrase XII is linked to the growth of primary oral squamous cell carcinoma and its poor prognosis

https://doi.org/10.1016/j.oraloncology.2011.11.015Get rights and content

Summary

The pattern of protein expression in tumors is under the influence of nutrient stress, hypoxia, and low pH, which determines the survival of neoplastic cells and the development of tumors. Carbonic anhydrase (CA) XII is a transmembrane enzyme that catalyzes the reversible hydration of cell-generated carbon dioxide into protons and bicarbonate. Hypoxic conditions activate its transcription and translation, and enhanced expression is often present in several types of tumors. However, CA XII expression in oral squamous cell carcinoma (OSCC) and its correlation with patients’ prognosis have not been investigated so far. In this study, we detected the expression of CA XII in 264 patients with OSCC using tissue microarrays (TMAs), and evaluated its correlation with clinicopathologic factors and disease prognosis. CA XII expression was present in 185/264 (70%) cases and was associated with more-advanced clinical stages (p = 0.003), a larger tumor size (p < 0.001), and postoperative recurrence (p = 0.047), but was not associated with positive lymph node metastasis or distal metastasis. Importantly, CA XII expression was correlated with a poorer patient prognosis in a univariate (p = 0.034, log-rank test) survival analysis. According to our results, the expression of CA XII in OSCC samples can predict the progression of OSCC and survival of OSCC patients.

Introduction

Oral cancer is a fatal disease, accounting for the fourth highest incidence of malignancy in males and the seventh in females in Taiwan. Oral squamous cell carcinoma (OSCC) accounts for >95% of all oral malignancies. The relatively high prevalence of oral cancer in Taiwan is mainly because there is a high-risk group of 2.5 million people with the habits of smoking and betel nut chewing. Oral mucosal diseases such as leukoplakia, oral submucosal fibrosis, oral precancerous lesions, and oral cancer are strongly associated with the use of betel quid.[1], [2] Unfortunately, in Taiwan, around 50% of new cases diagnosed with oral cancer present with advanced TNM stage III or IV lesions and have a shorter than 5-year survival after treatment.[1], [3], [4] Thus, identifying new tissue-based biomarkers that can predict the risk of progression and recurrence is warranted and urgently needed to improve the control of this deadly form of cancer.

As hypoxia-regulated molecular pathways were analyzed in recent years, hypoxia-inducible genes are becoming interesting candidates as predictors of disease outcomes.5 More than 100 genes involved in pH regulation, tumor metabolism, angiogenesis, migration, and invasion are currently known to be regulated by hypoxia-inducible factor (HIF)-1, a key mediator of the cellular response to hypoxia.6 Among the corresponding gene products, isoenzymes of the carbonic anhydrase (CA) family, CA IX and CA XII, are induced under hypoxic conditions in a variety of tumors and cultured tumor cells, and their expressions are downregulated after a return to normoxia.[7], [8], [9], [10] CA XI and CA XII are transmembrane zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to form bicarbonate (H2O + CO2  H+ + HCO3-). The enzymatic activity of these CAs is likely involved in modulating a variety of physiological processes including the transport of carbon dioxide and solutes, and acidification of the microenvironment that can modulate a tumor’s malignant phenotype.[11], [12] Previous studies described two mechanisms that control the expressions of CA XII and CA IX. Regulation through hypoxia plays a major role, as CA9 is directly upregulated via the binding of HIF to a hypoxia-response element (HRE) within the basal promoter of CA9.[5], [10] The other regulatory mechanism involves the protein product of the von Hippel-Lindau tumor suppressor gene (pVHL), which downregulates the expressions of CA IX and CA XII.8 However, it is noteworthy that neither the expression nor tissue distribution of the CA XII protein is correlated with the expression of CA IX.8 CA XII was originally identified as a protein overexpressed in renal cancer cells,9 but it is also known to be overexpressed in different human cancers, such as diffuse astrocytomas and colorectal, gastrointestinal, breast, pancreatic, ovarian, and renal carcinomas.[13], [14], [15], [16], [17], [18] In vitro and in vivo studies revealed that both CA IX and CA XII are functionally connected to neoplastic processes, and promote tumor cell survival and growth by counteracting acidosis through regulating intracellular pH levels.[19], [20], [21] Moreover, recent studies showed that the combined silencing of CA IX and CA XII can reduce the rate of growth of colon xenograft tumors.19

Whereas tumor expressions of HIF-1 and CA IX were shown to be correlated with poor survival in OSCC patients,[22], [23] the significance of the expression of CA XII, which lacks the N-terminal proteoglycan domain of CA IX, that is implicated in cell adhesion,24 has not been examined in OSCC. In addition, the potential role of tumor CA XII expression as a prognostic biomarker in OSCC has not been investigated.

In this study, we examined the expression of CA XII in a large collection of OSCC tissue samples to assess if CA XII might serve as a predictor of outcomes. Specifically, we attempted to assess associations between CA XII expression and clinicopathological parameters of OSCC patients and the relationship to outcomes.

Section snippets

Patients and tissue microarrays

We constructed formalin-fixed, paraffin-embedded tissue microarrays composed of 264 OSCC tissue cores. Diagnosis of OSCC was based on histological examination of hematoxylin and eosin-stained tissue sections. Patients with clinical N0 stage were treated with primary excision and supraomohyoid neck dissection. Wide excision of the primary tumor and radical neck dissection or modified radical neck dissection were performed in patients with metastasis to a regional lymph node. Adjuvant radiation

Clinicopathologic characteristics of patients with OSCC

As shown in Table 1 and 249 patients were male and 15 were female. Patient ages ranged 32–88 (mean, 55.33 ± 10.93) years. The histologic type of all 264 tumors was SCC. According to the American Joint Committee on Cancer (AJCC) system, tumors were classified into stages I (n = 45), II (n = 44), III (n = 22), and IV (n = 153). Metastasis to cervical lymph nodes was detected in 96 cases (36.4%). Disease recurrence was observed in six tumors (2.3%), and death occurred in 109 cases (41.3%).

IHC analysis of CA XII expression on TMAs

CA XII expression

Discussion

Hypoxia increases cancer radio- and chemoresistance and is therefore regarded as an adverse prognostic factor for survival and local control.25 Head and neck cancers (HNCs) including OSCC are tumor types that are particularly resistant to chemo- and radiotherapy, and hypoxia and concomitant expression of HIF-1α and downstream target genes (e.g., vascular endothelial growth factor (VEGF) and CA IX) are common.[26], [27], [28] While expressions of HIF-1α, VEGF, and CA IX are well investigated in

Conflict of interest statement

We wish to confirm that there are no known conflicts of interest associated with this publication, and there was no significant financial support for this work that could have influenced its outcome.

Acknowledgements

This study was supported by a Grant from Taipei Medical University (TMU99-AE1-B05 to Dr. Chien) and NSC-96-2628-B-040-021-MY3 to Dr. Yang.

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