The role of the antileukoprotease SLPI in smoking‐induced human papillomavirus‐independent head and neck squamous cell carcinomas

Abstract

Recently, we showed that increased SLPI levels prevent human papillomavirus (HPV) infections and metastasis in smokinginduced, non-HPV-driven head and neck squamous cell carcinoma (HNSCC). Here, we focus on the role of SLPI in non-HPVdriven HNSCC, investigating tumor tissue and non-neoplastic mucosa from the same patients and from non-HNSCC patients. Gene and protein expression of SLPI and gene expression of annexin 2 (a SLPI receptor), nicotine receptor (a7AChR) and arylhydrocarbon receptor (AhR) were analyzed in HNSCC patients (20 smokers; 16 nonsmokers). SLPI-results were correlated with the patients' HPV status. Non-neoplastic mucosa of HNSCC patients and normal mucosa from non-HNSCC individuals (18 smokers; 20 nonsmokers) was analyzed for the same parameters. Tissue of the inferior turbinate (n 5 10) was incubated with nicotine for analysis of the same genes. SLPI gene expression in tumor tissue was 109.26 6 23.08 times higher in smokers versus nonsmokers. Non-neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49 6 1.89-fold non-HNSCC; 18.02 6 3.93-fold HNSCC patients). Annexin 2 gene expression was also increased in smokers. SLPI data were corroborated by immunohistochemistry. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r 2 5 0.15, p < 0.001) was shown ex vivo. Nicotine and smoking increased a7AChR and AhR gene expression. Five patients, showing no/ low SLPI expression, were HPV16-positive. A significant correlation between smoking and SLPI expression in tumors and to our knowledge for the first time in mucosa of HNSCC and non-HNSCC patients was established. Together with the finding that all patients with HPV infection showed no/low SLPI expression, these data support our intriguing hypothesis that smoking induced upregulated SLPI prevents HPV infections.Head and neck squamous cell carcinoma (HNSCC) represent 7% of all malignant diseases. They are the sixth most common malignancy in humans and account for 70,000 new cases in the United States and Europe, and 10,000 deaths annually. 1,2 With regard to pathogenesis and clinical behavior, the existence of two distinct HNSCC entities is generally accepted one being predominantly associated with tobacco and alcohol consumption, 3 the other being predominantly associated with human papillomavirus (HPV) infection. 4-7The high-risk HPV genotype 16 plays a pivotal role in the pathogenesis of a subset of HNSCC, specifically of tonsillar cancers. 5,8 Despite constant improvements in tumor surgery as well as radio-and chemotherapy, survival rates of HNSCC patients remained largely unchanged over the last decades, with 5-year overall survival rates of 50%.9 This might partly be due to the fact that there is still no clear strategy of individualization of the conventional therapy regimens, for instance based on the biological properties of the tumor.

Salivary secretory leukocyte protease inhibitor (SLPI) and head and neck cancer: The Cancer Prevention Study II Nutrition Cohort

“…Our finding that SLPI may be differentially associated with HNSCC risk depending on smoking status is consistent with published findings on the effect of cigarette smoking on SLPI production [11, 22, 23]. Nicotine stimulates SLPI expression in the nasal and oral mucosa, and also promotes the proliferation of cancer cells [24].…”

“…Nicotine stimulates SLPI expression in the nasal and oral mucosa, and also promotes the proliferation of cancer cells [24]. In a study of non-HPV driven HNSCC, SLPI gene and protein expression (i.e., intracellular SLPI) was more than 100-fold higher among smokers than non-smokers when evaluated in HNSCC tumor tissue, 20-fold higher among smokers when measured in non-neoplastic tissue of HNSCC patients, and 10-fold higher among smokers when measured in non-HNSCC patients [23]. Similarly, in our study of HNSCC we found current smokers to have significantly higher levels of SLPI than never smokers.…”

SLPI and AnxA2 expression in neoplasm-free palatine tonsils is associated with smoking habit of individuals

“…As shown previously (1)(2)(3), both the secretory leucocyte protease inhibitor (SLPI; an antiproteinase) and the membrane bound receptor Annexin A2 (AnxA2) showed higher expression levels in smokers in comparison to non-smokers. However, SLPI showed a clear surplus compared to AnxA2 in smokers vs. non-smokers whereas AnxA2 vs. SLPI showed a significant surplus in cases tested HPV-positive when compared to HPV-negative cases (1)(2)(3). Previous ex vivo data showed that SLPI expression in clinically healthy mucosa of the lower turbinates can be increased by incubation with nicotine (2).…”

“…However, SLPI showed a clear surplus compared to AnxA2 in smokers vs. non-smokers whereas AnxA2 vs. SLPI showed a significant surplus in cases tested HPV-positive when compared to HPV-negative cases (1)(2)(3). Previous ex vivo data showed that SLPI expression in clinically healthy mucosa of the lower turbinates can be increased by incubation with nicotine (2). Additionally, in vitro studies showed that the cell entry of HPV is hindered when AnxA2 is blocked by SLPI in a cell culture model utilizing a cervix cancer cell line (4).…”

“…qPCR was performed as described previously (10). Primers for SLPI and AnxA2 were designed and used as described elsewhere (2). Primers for the housekeeping genes 18S rRNA, β-actin and b-2-microglobulin (B2M) were purchased from Promolgene (Berlin, Germany) and used according to the manufacturer's protocol.…”