Characterisation of human kallikrein 6/protease M expression in ovarian cancer

Abstract

Kallikrein 6 (hK6, also known as protease M/zyme/neurosin) is a member of the human kallikrein gene family. We have previously cloned the cDNA for this gene by differential display and shown the overexpression of the mRNA in breast and ovarian primary tumour tissues and cell lines. To thoroughly characterise the expression of this kallikrein in ovarian cancer, we have developed a novel monoclonal antibody specific to hK6 and employed it in immunohistochemistry with a wide range of ovarian tumour samples. The expression was found elevated in 67 of 80 cases of ovarian tumour samples and there was a significant difference in the expression levels between normal and benign ovarian tissues and the borderline and invasive tumours (P<0.001). There was no difference of expression level between different subtypes of tumours. More significantly, high level of kallikrein 6 expression was found in many early-stage and low-grade tumours, and elevated hK6 proteins were found in benign epithelia coexisting with borderline and invasive tissues, suggesting that overexpression of hK6 is an early phenomenon in the development of ovarian cancer. Quantitative real-time reverse transcription-polymerase chain reactions also showed elevated kallikrein 6 mRNA expression in ovarian tumours. Genomic Southern analysis of 19 ovarian tumour samples suggested that gene amplification is one mechanism for the overexpression of hK6 in ovarian cancer.

Figure 1

Use of monoclonal antibody 2D4 in immunostaining and immunohistochemistry. Immunostaining of (A) tetracycline-induced hK6 in SKOV3 cell line; (B) the same cell line without tetracycline induction; (C) OVCA429 ovarian carcinoma cell line; (D) immortalised normal HOSE cells. Immunohistochemical staining for hK6 in (E) normal ovary; (F) endometrioid; (G) mucinous; (H) stage I clear cell; (I) stage I serous; and (J) stage III serous ovarian tumour tissues. Immunopositive cells were stained brown in the cytoplasm. To highlight the tumour cells, the slides were counterstained with haematoxylin (purple).

Figure 2

Elevated expression of hK6 in benign and borderline lesions of mucinous ovarian tumours. Positive 2D4 staining in immunohistochemistry of (A) a mucinous case, showing both single stratified layer of benign (BN) epithelium and borderline (BOT) tumour component with pleomorphism; (B) a mucinous tumour showing positive 2D4 staining in both borderline (BOT) cells and invasive (INV) tumour components.

Figure 3

Genomic Southern analysis and quantitative real-time PCR of tumour DNA. Equal amounts of 19 tumour DNA were restricted with BamHI and electrophoresed on an agarose gel and Southern blot analysis was performed using the KLK6 cDNA probe. The 5- and 3.2-kb bands of the KLK6 gene are marked by arrows. Genomic DNA of a normal ovarian epithelial primary culture (HOSE) was used as control. Results of the quantitative real-time PCR of the tumour samples relative to the normal HOSE cells are shown at the bottom.