High hyaluronan binding and RHAMM expression identify an invasive and metastatic subpopulation in androgen-resistant prostate cancer cells

Hyaluronan (HA) metabolism in prostate cancer associates with androgen resistance and metastasis. We showed that binding of low molecular weight HA (≤250 kDa) to castration-resistant prostate cancer cells was heterogeneous with most cells binding low amounts of HA (HA^low) while a minor subset bound higher amounts of this polysaccharide (HA^high). HA^high subsets, which were separated by FACS, were stably more metastatic in vivo than HA^low comparators. Multiplexed flow cytometry analyses indicated that both subsets displayed similar expression of the HA receptor CD44 while an elevated RHAMM cell surface display was unique to HA^high subsets. Genomic deletion of RHAMM using CRISPR-Cas9 editing reduced the detection of HA^high subsets by 6mer but not 250 kDa HA fluorescent probes, and phenocopied the lower aggressive properties of HA^low tumor cells. Few differences in the mutation landscape of RHAMM^+/+ vs. RHAMM^-/- tumor cells were detected but pathway analyses of differentially expressed genes predicted RHAMM-loss altered extracellular matrix signaling. Transcriptomic analyses revealed that HA^high subsets and RHAMM^+/+ PC3MLN4 cells shared high expression of follistatin (FST), an activin member of the TGF-β family that is clinically linked to metastases in PCA patients. A causal role for FST in RHAMM^+/+ tumor cell aggression was assessed using motility as a surrogate marker of invasive capability. FST antibodies blocked RHAMM^+/+ PC3MLN4 cell migration while conversely, recombinant FST protein rescued the migration deficit of RHAMM^-/- comparators. These results define a novel form of prostate cancer cell heterogeneity, identify a method for detecting and isolating highly metastatic subsets and highlight a novel RHAMM-regulated pathway that may be targeted to improve patient management by limiting metastasis.