Accumulation of extracellular hyaluronan by hyaluronan synthase 3 promotes tumor growth and modulates the pancreatic cancer microenvironment

Abstract

Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.

Figure 1

HAS3 overexpression is associated with high extracellular hyaluronan accumulation and HAS2 with increased extracellular and intracellular accumulation of hyaluronan. Amount of secreted, cell surface, and intracellular hyaluronan was analyzed in BxPC-3, BxPC-3 HAS2, and BxPC-3 HAS3 cells with a hyaluronan assay, and results were presented as extracellular hyaluronan showing combined amount of secreted and cell surface hyaluronan (a) and intracellular hyaluronan (b). The results were expressed as % of hyaluronan content in BxPC-3 cells, and data represent mean ± S.D. of three independent experiments. To visualize intracellular hyaluronan, subconfluent cultures of BxPC-3 (c), BxPC-3 HAS2 (d), and BxPC-3 HAS3 cells (e) were stained for intracellular hyaluronan (green) and CD44 (red) using bHABP and anti-CD44s-antibody, respectively. Nuclei were visualized with Prolong Antifade with DAPI. Scale bar in (c–e) is 50 μm. Statistical differences between the groups in (a) and (b) were tested using one-way ANOVA and Tukey's post hoc test (**P < 0.01; ***P < 0.001).

Figure 2

Localization and amount of hyaluronan in BxPC-3, BxPC-3 HAS2, and BxPC-3 HAS3 tumors and response to PEGPH20. Mice carrying BxPC-3, BxPC-3 HAS2, and BxPC-3 HAS3 peritibial xenograft tumors were treated twice with vehicle or 4,500 μg/kg PEGPH20, and tumors were collected 6 h after the last dose. Tumor sections were stained with H&E to visualize morphology of the tumors (a–f) and with bHABP for hyaluronan (g–l). Scale bar in (a–f) and (g–l) is 500 μm. Hyaluronan concentration in the tumors after two weekly treatments of vehicle, 37.5 μg/kg or 1,000 μg/kg of PEGPH20 (n ≥ 3/group) for three weeks was analyzed by hyaluronan assay (m). Statistical differences between the groups shown in figure (m) were tested using two-way ANOVA and Bonferroni's post hoc test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

Pancreatic cancer xenograft tumors overexpressing HAS3 grow faster and respond better to hyaluronan removal than HAS2 or parental tumors. To compare in vivo growth, BxPC-3, BxPC-3 HAS2, and BxPC-3 HAS3 tumor cells were inoculated adjacent to the tibial periosteum in the hind limb of nu/nu mice (n = 7/group), and tumor growth was monitored using ultrasound imaging. Once average tumor size reached 500 mm³, mice were treated twice a week with an i.v. injection of vehicle or PEGPH20 (4,500 μg/kg) (a–c). Statistical difference was tested using repeated measured two-way ANOVA and Bonferroni's post hoc test.

Figure 4

HAS overexpression induces loss of plasma membrane E-cadherin and accumulation of cytoplasmic β-catenin, and removal of hyaluronan translocates them to the plasma membrane. Vehicle- and PEGPH20- (2 doses; 4,500 μg/kg; n = 3/group) treated tumors were stained for E-cadherin (a) and β-catenin (b). E-cadherin was also localized in pancreatic tumors from a KPC mouse model after 0, 8, 24, and 72 h treatment with PEGPH20 (c; n = 3/group) and in human NSCLC biopsies before and after PEGPH20 therapy (d). Scale bar in (a–d) is 250 μm. Insets in (a–d) represent 3x magnification of the original micrograph.

Figure 5

Removal of extracellular hyaluronan decreases HIF-1α and Snail signaling. Vehicle- and PEGPH20- (2 doses; 4,500 μg/kg) treated BxPC-3 and BxPC-3 HAS3 tumors were homogenized and nuclear extracts were analyzed with Western blot using HIF-1α (a) and (b), Snail (d) and (e), β-actin (a) and (b), and Histone 3 (c) and (d) antibodies. In (a) and (b), the blot is a representative of three experiments, and each lane represents a piece of the same vehicle-treated or PEGPH20-treated tumor. The HIF-1α band is indicated with an arrow. In (d) and (e), lysates from the same vehicle-treated or PEGPH20-treated tumor were combined and each lane represents Snail level of one tumor. Intensities of the bands in the cropped blots were quantified using Image-Pro Analyzer 7.0 software and normalized to the intensity of the housekeeping protein ((c) and (f)). Data were plotted as mean ± S.D., and statistical difference between the groups was tested with t test (*P < 0.05; **P < 0.01; and ***P < 0.001).

Figure 6

Effect of HAS2 and HAS3 overexpression and hyaluronan removal on apoptosis and proliferation in BxPC-3, BxPC-3 HAS2, and BxPC-3 HAS3 tumors. Vehicle-treated and PEGPH20-treated (2 doses; 4,500 μg/kg; n = 3/group) BxPC-3, BxPC-3 HAS2, and BxPC-3 HAS3 xenograft tumor sections were stained for CC3 and PH3 to visualize apoptotic and proliferative cells, respectively. Number of positive cells per tumor section was analyzed using Aperio Positive Pixel v9 and Nuclear staining algorithms, respectively, ((a) and (b)). Data were plotted as mean ± S.D., and statistical difference between the groups was tested with two-way ANOVA and Bonferroni's post hoc test (**P < 0.01).