Silencing of hyaluronan synthase 2 suppresses the malignant phenotype of invasive breast cancer cells

Abstract

Accumulation of hyaluronan has been demonstrated in the peritumoral breast cancer stroma and nests of tumor cells. In this study, we have quantified the production of hyaluronan and the expression of mRNAs encoding hyaluronan synthesizing (HAS) and hyaluronan degrading (HYAL) enzymes in a panel of breast cancer cell lines. The analysis revealed that highly invasive breast cancer cells produce high amounts of hyaluronan and express preferentially HAS2 mRNA, whereas less invasive breast cancer cells produce low amount of hyaluronan and express HAS1 and HYAL1 mRNAs. We explored the importance of HAS2 expression for breast cancer tumorigenicity, by specifically silencing the HAS2 gene using RNA interference (RNAi)-mediated suppression in the invasive breast cancer cell line Hs578T. This led to a less aggressive phenotype of the breast tumor cells, as assessed by cell growth, both in anchorage-dependent and anchorage-independent cultures. siRNA-mediated knock down of HAS2 in Hs578T breast tumor cells led to an up-regulation of HAS1, HAS3 and HYAL1 mRNAs, resulting in only a 50% decrease in the net hyaluronan production; however, the synthesized hyaluronan was of lower size and more polydisparse compared to control siRNA-treated cells. Interestingly, Hs578T cells deprived of HAS2 migrated only half as efficiently as HAS2 expressing cells through cell-free areas in a culture wounding assay and through Transwell polycarbonate membrane as well as invaded a Matrigel layer. These results imply that alterations in HAS2 expression and endogenously synthesized hyaluronan affect the malignant phenotype of Hs578T breast cancer cells. ' 2007 Wiley-Liss, Inc.Key words: migration; hyaluronidase; hyaluronan synthase; CD44; breast carcinomas; hyaluronan Breast cancer progression correlates with altered hyaluronan metabolism, including increased deposition of hyaluronan in the nests of carcinoma cells, and especially in the stromal tissue in the invading edges of breast carcinomas.1,2 Stromal fibroblasts activated by the breast cancer cells most likely contribute to the enrichment of hyaluronan in the immediate peritumoral stroma. 3In vitro studies revealed that the most aggressive breast carcinoma cell lines both synthesize high amounts of hyaluronan and express the cell surface hyaluronan receptors, CD44 and RHAMM, unlike the less aggressive cell lines. [4][5][6] Hyaluronan is synthesized by hyaluronan synthases, which exist in 3 isoforms (HAS1, HAS2 and HAS3), and is degraded by hyaluronidases (HYAL1, HYAL2 and HYAL3, and PH-20). 7,8 Although each of the HAS isoforms is capable of hyaluronan synthesis, they synthesize hyaluronan of different lengths. The HAS2 isoform synthesizes hyaluronan molecules larger than 3.9 3 10 6 , HAS3 synthesizes polydisperse hyaluronan (M w of 0.12-1 3 10 6 ) and HAS1 synthesizes much smaller chains (M w of 0.12 3 10 6 ). Moreover, the HAS isoforms exhibit different catalytic activities; HAS3 is catalytically more active than HAS2, which in turn is more active than HAS1.9 Disruption of the HAS2 gene ca...

4-Methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells

“…In this study, we showed that 4-MU treatment in CF33 cells resulted in inhibition of cell growth associated with downregulation of HAS2 mRNA expression. Our data are consistent with a recent report showing that HAS2 is related to inhibition of cell proliferation, migration and metastasis in human breast cancer cells (35). Therefore, 4-MU may also serve as a potential therapeutic agent for breast cancer in dogs.…”

“…A possible explanation for the results of our study is that 4-MU causes positive feedback for HAS3 mRNA expression to compensate for the inhibition of HA synthesis. These observations are likely to occur in HA-associated genes (17,35). Our results further showed that the downregulation of HAS2 after treatment with 4-MU induced marked growth retardation and apoptosis in CF33 cells.…”

“…Furthermore, it was recently reported that HAS2 plays an important prometastatic role by generating a favorable microenvironment of cancer stem-like cells (41). In human breast cancer cells, HAS2 is increasingly recognized as a key molecule for maintenance of the malignant phenotype (17,35). In this study, we showed that 4-MU treatment in CF33 cells resulted in inhibition of cell growth associated with downregulation of HAS2 mRNA expression.…”

“…The rate of cell proliferation is often associated with that of HA synthesis (35). Furthermore, it was previously reported that cell proliferation is inhibited by 4-MU in various cancer cells (27,28).…”

Fragmented hyaluronan is an autocrine chemokinetic motility factor supported by the HAS2-HYAL2/CD44 system on the plasma membrane

“…HAS1 and HAS2 produce HA of 200-2000 kDa, whereas HAS3 mainly synthesizes HA of 100-1000 kDa (14). Knockdown of the HAS genes in tumor cells was reported to inhibit their proliferation, invasion and motility in vitro and tumor growth and metastasis in vivo (16)(17)(18)(19)(20)(21)(22). In particular, it has been reported that the levels of HAS2 expression are often correlated with malignant behavior in various cancer cells such as breast cancer, fibrosarcoma and osteosarcoma cells (17,(23)(24)(25).…”