Gastrin-releasing peptide receptor: a promising new biomarker to identify cervical precursor lesions and cancer

Abstract

Objective: This study aimed to verify the relation between gastrin-releasing peptide receptor (GRPR), oncogenic Human Papillomavirus (HPV) and cervical lesions severity.

Methods: GRPR mRNA levels were evaluated in cervical cancer-derived cell lines and in primary keratinocytes expressing HPV16 oncogenes by RT-PCR. GRPR protein expression was assessed by immunohistochemistry in organotypic cell cultures derived from keratinocytes transduced with HPV16 oncogenes and in 208 cervical samples, including 59 non-neoplastic tissue, 28 cervical intraepithelial neoplasia grade 3 (CIN3), 44 squamous cell carcinomas (SCC) and 77 adenocarcinomas (ADC). Generic primers (GP5+/GP6+) were used to identify HPV infection in tissue samples. Experiments involving cell lines were analyzed through non-parametric tests (Kruskal Wallis), and Fisher's Exact Test for human tissues samples. All statistical tests were considered significant at p <0.05. Immunohistochemical evaluation was conducted independently and blindly by two observers (AD- LO). Any discordant findings were resolved through discussion to reach a consensus score.

Results: GRPR mRNA levels were not increased in cells expressing HPV16 or HPV18 oncogenes. However, at the protein level, GRPR was upregulated in organotypic cell cultures containing HPV oncogenes. Besides, it was identified an association between GRPR expression and cervical lesion severity (p < 0.0001). The detection rate of high-risk HPV DNA was directly correlated with cervical disease. Nonetheless, HPV infection was not directly associated with GRPR in cervical samples.

Conclusion: GRPR expression is highly predictive of cervical lesion severity, irrespective of HPV infection and might contribute to improving patient's therapeutic management as well as being used a marker of disease progression.

Keywords: Adenocarcinoma; Carcinoma, squamous cell; Gastrin-releasing peptide receptor; Human papillomavirus; Oncogenes; Papillomavirus infections; Uterine cervical dysplasia; Uterine cervical neoplasms.

Figure 1. Relative expression of GRPR mRNA by real time PCR: Relative expression of GRPR mRNA in (A) normal keratinocytes (PHK) and cervical cancer-derived cell lines (C33, SiHa and HeLa) and (B) keratinocytes transduced with the empty vector (pLXSN Ø) or with the vector containing HPV16 oncogenes E6 and/or E7

Figure 2. GRPR protein expression in organotypic cell cultures. GRPR protein expression was evaluated by immunohistochemistry in organotypic cell cultures sections derived from (A) normal keratinocytes; (B) keratinocytes transduced with the empty vector pLXSN (pLXSN Ø); (C) keratinocytes transduced with HPV16 E6 oncogene; (D) keratinocytes transduced with HPV16 E7 oncogene; (E) keratinocytes transduced with HPV16 E6 and E7 oncogenes. (F) Negative control: normal keratinocytes without primary antibody

Figure 3. GRPR protein expression in cervical samples. Representative GRPR protein expression determined by immunohistochemistry in (A) transition area to a high-grade lesion; (B) squamous cell carcinoma and (C) adenocarcinoma. Images in-set: cervicitis (A) and normal glandular tissue (C). Graphic representation of GRPR expression in cervical tissue samples according to the histopathological diagnosis. Samples were analyzed according to GRPR staining intensity and grouped into negative-low or strong groups. The percentage of samples allocated in each group according to histological classification is shown (D). *p < 0.0001 indicates a significantly different distribution between the analyzed variables (GRPR, cervicitis, NIC3, SCC and ADC) (Fisher's exact test)

Figure 4. HPV DNA prevalence in cervical tissue samples according to the histopathological diagnosis. Samples were analyzed according to HPV detection and grouped into negative or positive HPV groups (A). The classification into low and high-risk HPV was also performed (B). The percentage of samples allocated in each group, according to histological classification, is shown. *p < 0.0001 indicates a significantly different distribution between the analyzed variables (HPV, cervicitis, NIC3, SCC and ADC) (Fisher's exact test)

Figure 5. Correspondent Hematoxylin and Eosin (H&E) images of pictures included in figure 3. (A) transition area to a high-grade lesion; (B) squamous cell carcinoma and (C) adenocarcinoma. Images in-set: cervicitis (A) and normal glandular tissue (C)