Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma

Abstract

Background: Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.

Methods: A total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry.

Results: The mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).

Conclusion: The results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.

Figure 1

HAS 2-3 mRNA expression in human endometrium and its lesions. Relative mRNA levels of A) HAS2 and B) HAS3 in different tissues. The boxes show the ranges between 25th and 75th percentiles, with a horizontal line at the median value. The whiskers extend to the 10th and 90th percentiles. The open circles represent the outlier values. NE = normal pre-menopausal endometrium, PME = post-menopausal proliferative endometrium, CAH = complex atypical hyperplasia, G1 = grade 1 endometrioid endometrial adenocarcinoma, G2 + 3 = grade 2 + 3 endometrioid endometrial adenocarcinoma.

Figure 2

HYAL 1-2 mRNA expression in human endometrium and its lesions. Relative mRNA levels of A) HYAL1 and B) HYAL2 in different tissues. The boxes show the ranges between 25th and 75th percentiles, with a horizontal line at the median value. The whiskers extend to the 10th and 90th percentiles. NE = normal pre-menopausal endometrium, PME = post-menopausal proliferative endometrium, CAH = complex atypical hyperplasia, G1 = grade 1 endometrioid endometrial adenocarcinoma, G2 + 3 = grade 2 + 3 endometrioid endometrial adenocarcinoma.

Figure 3

HAS 1-3 immunoreactivity and HA-staining in normal and neoplastic human endometrium. Immunostaining of HAS1 (A, B), HAS2 (C, D), HAS3 (E, F) and HA (G, H) in normal human endometrium (A, C, E, G) and in grade 2 endometrioid endometrial carcinoma tissue sections (B, D, F, H). The brown color (DAB) indicates HAS (A-F) or HA (G, H), and blue color (haematoxylin) indicates nuclei. A-F: 200× original magnification. G, H: 100× original magnification.