Elevated hyaluronan and hyaluronan-mediated motility receptor are associated with biochemical failure in patients with intermediate-grade prostate tumors

Abstract

Background: The clinical course of prostate cancer (PCa) measured by biochemical failure (BF) after prostatectomy remains unpredictable in many patients, particularly in intermediate Gleason score (GS) 7 tumors, suggesting that identification of molecular mechanisms associated with aggressive PCa biology may be exploited for improved prognostication or therapy. Hyaluronan (HA) is a high molecular weight polyanionic carbohydrate produced by synthases (HAS1 through HAS3) and fragmented by oxidative/nitrosative stress and hyaluronidases (HYAL1 through HYAL4, SPAM1) common in PCa microenvironments. HA and HA fragments interact with receptors CD44 and hyaluronan-mediated motility receptor (HMMR), resulting in increased tumor aggressiveness in experimental PCa models. This study evaluated the association of HA-related molecules with BF after prostatectomy in GS7 tumors.

Methods: Tissue microarrays were constructed from a 96-patient cohort. HA histochemistry and HAS2, HYAL1, CD44, CD44v6, and HMMR immunohistochemistry were quantified using digital pathology techniques.

Results: HA in tumor-associated stroma and HMMR in malignant epithelium were significantly and marginally significantly associated with time to BF in univariate analysis, respectively. After adjusting for clinicopathologic features, both HA in tumor-associated stroma and HMMR in malignant epithelium were significantly associated with time to BF. Although not significantly associated with BF, HAS2 and HYAL1 positively correlated with HMMR in malignant epithelium. Cell culture assays demonstrated that HMMR bound native and fragmented HA, promoted HA uptake, and was required for a promigratory response to fragmented HA.

Conclusions: HA and HMMR are factors associated with time to BF in GS7 tumors, suggesting that increased HA synthesis and fragmentation within the tumor microenvironment stimulates aggressive PCa behavior through HA-HMMR signaling.

Keywords: biomarkers; digital pathology; hyaluronan; hyaluronan-mediated motility receptor; prostate cancer.

Figure 1

Representative immunohistochemical staining for HAS2 (A), HA (B), HYAL1 (C), CD44 (D), CD44v6 (E), and HMMR (F) on prostate cancer tissue microarrays. Scale bar represent 50 μm.

Figure 2

(A–F) Prostate cancer tissue microarrays were stained by immunohistochemistry. (G–L) Genie Pattern Recognition software (Aperio) subclassified tumor areas into malignant epithelium (dark blue), stroma (yellow), and glass (light blue). (M–R) DAB staining in malignant epithelium (HAS2, HYAL1, CD44, CD44v6 and HMRR) or stroma (HA) was deconvolved from counterstain using Color Deconvolution (Aperio). (S–X) DAB staining was quantified in these areas and pseudocolored for weak (yellow), moderate (orange), and strong (red) staining. Scale bars represent 50 μm.

Figure 3

Kaplan-Meier curve demonstrating the time to biochemical failure for the sample population.

Figure 4

Representative immunohistochemical staining for HA (A–D) and HMMR (E–H) in biochemical failure (BF) and non-failure (non-BF) groups. Scale bar represent 50 μm.

Figure 5

(A) Western blot analysis confirmed protein expression of HAS2 and HYAL1 in PC3MLN4 cells. (B) Cell migration assays demonstrated increased motility in response to 6.2 and 10 kDa HA (5 μg/mL) and decreased motility in response to 2.1 kDa HA (5 μg/mL) relative to PBS controls. (C) HA pull-down assays were performed with 10 kDa HA coupled to Sepharose beads. Western blot analysis revealed that full-length (FL) and truncated (TR) HMMR expressed by PC3MLN4 cells bound efficiently to 10 kDa HA at similar levels to HMMR-transfected 10T1/2 cells (positive control). (D) PC3MLN4 cells bound and internalized Alexa 647-labeled HA fragments (top) while addition of anti-HMMR antibody blocked binding and uptake of Alexa 647-labeled HA fragments (bottom) detected by fluorescent microscopy. Binding was blocked by excess unlabeled HA and confirmed specificity of labeled HA (data not shown). (E) Cell migration increased in response to 10 kDa HA fragments (5 μg/mL) and was inhibited by HMMR mimetic peptide P15 (20 μg/mL), but not by its scrambled peptide control. Error bars, SEM of triplicate determinations. Statistically significant differences were observed (p<0.01) as determined by Student’s t-test.