Chondroitin sulfate proteoglycan 4, a targetable oncoantigen that promotes ovarian cancer growth, invasion, cisplatin resistance and spheroid formation.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease encompassing several distinct molecular subtypes and clinical entities. Despite the initial success of surgical debulking and adjuvant chemotherapy, recurrence with chemotherapy resistant tumors is common in patients with EOC and leads to poor overall survival. The extensive genetic and phenotypic heterogeneity associated with ovarian cancers has hindered the identification of effective prognostic and predictive biomarkers in EOC patients. In the current studies, we identify a tumor cell surface oncoantigen, chondroitin sulfate proteoglycan 4 (CSPG4), as an independent risk factor for decreased survival of patients with EOC. Our results show that CSPG4 promotes EOC cell invasion, cisplatin resistance and spheroid formation in vitro and tumor expansion in vivo. Mechanistically, spheroid formation and tumor cell invasion are due to CSPG4-stimulated expression of the mesenchymal transcription factor ZEB1. Furthermore, we have developed a novel monoclonal anti-CSGP4 antibody against the juxtamembrane domain of the core protein that limits CSPG4-stimulated ZEB1 expression, tumor cell invasion and promotes EOC apoptosis within spheroid cultures. We therefore propose that CSPG4 expression drives phenotypic heterogeneity and malignant progression in EOC tumors. These studies further demonstrate that CSPG4 expression levels are a potential diagnostic biomarker in EOC and indicate that targeting cells which express this oncoantigen could limit recurrence and improve outcomes in patients with EOC.

Identification, design and synthesis of tubulin-derived peptides as novel hyaluronan mimetic ligands for the receptor for hyaluronan-mediated motility (RHAMM/HMMR).

Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2, 4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease.

Oral administration of hyaluronan reduces bone turnover in ovariectomized rats.

The effect of oral hyaluronan (HA) on bone loss in ovariectomized (OVX) 3-month-old rats was measured using serum markers of bone turnover and bone mineral density. OVX rats were administered 1 mg/kg HA (OVX + HA) or phosphate-buffered saline (PBS) (OVX + PBS) by oral gavage (5 days/week for 54 days). Additional controls included sham ovariectomy with PBS gavage (Sham + PBS) and no treatment. Oral administration of HA resulted in approximately 50% (p < 0.05) increases in serum HA. Gel filtration analyses showed this was high molecular weight HA (300-500 kDa). Osteopenia was mild due to the young age of the animals. Thus, ovariectomy resulted in a 30% increase in serum collagen N-terminal telopeptides (p < 0.001), a 20% increase in serum nitrate/nitrite levels (p = 0.05), and a 5-6% decrease in femur bone mineral density/content (p < 0.05). HA gavage blunted the development of osteopenia in this model as determined by preventing the 30% increase in serum collagen N-terminal telopeptide levels (p < 0.001) and by reducing bone mineral content loss from 6 to 4%. These results show that oral supplements of HA (gavage solution, 0.12% solution) significantly reduce bone turnover associated with mild osteopenia in rats.