Abstract
Our recent studies have shown that Fibroblast Growth Factor-2 (FGF2) and Syndecan-1 (SDC1) expressions are upregulated, by 45- and 24-fold respectively, in CD30+ cells from tissues of relapsed versus relapsed-free Hodgkin lymphoma (HL) patients (p<0.005; Fig. 1A). Normal control and relapsed-free groups showed similar FGF2 and SDC1 expression profiles in tissue samples. The circulating HL cells in peripheral blood lymphocytes (PBL) have demonstrated that FGF2 and SDC1 mRNA were upregulated, by nearly 7000- and 9000-fold respectively, in relapsed versus relapsed-free HL patient samples (p<0.0001; Fig. 1B). Preliminary Kaplan-Meier analyses indicate that the HL patient group (n=4) with elevated co-expression of FGF2 and SDC1 had shortened survival by six-fold when compared to the majority of patients (n=11) with significantly lowered expression of FGF2/SDC1 (p < 0.05). This suggests that high levels of FGF2/SDC1 contribute to poor prognosis in HL patients. Western blot analyses showed that shed SDC1 regulate the HL cell division rate by a negative feedback mechanism. However, secretion of FGF2 and an abundance of four FGF receptor (FGFR) proteins were not associated with the cell division rate.
Among eleven HL cell lines examined, HD-MyZ represented the best model for relapsed/refractory HL due to its highest cell proliferation rate (35 hour doubling time) and overexpression of FGF2 and SDC1, by 60- and 150-fold respectively, when compared with the B lymphoblast cell line, C1RB7. Immunofluorescence and co-immunoprecipitation studies in normal fibroblasts and HL cells demonstrate that FGF2 and SDC1 are co-localized on the cell membrane and bind together as ligand and receptor. Additionally, FGF2 binds to FGFRs in HL cells with lower affinity than in normal fibroblasts with predominant binding to FGFR1, suggesting that FGF2 signaling largely involves both SDC1 and FGFR1. Fluorescence microscopy studies showed that FGFR1 is constitutively phosphorylated in HL cells, and it stimulates cell proliferation by an autocrine pathway. Treatment with exogenous FGF2 in the absence of serum resulted in phosphorylation of both AKT and ERK, thereby activating FGF2-mediated cell proliferation. FGF2 induction also upregulated mRNA expressions of SDC1 (12-fold), proliferation marker- CCND1 (242-fold) and metastatic marker- MMP9 (67-fold) compared to unstimulated cells.
To examine the molecular mechanism, whereby SDC1 is involved in the proliferation and metastasis of HL cells, SDC1 knockdown (KD) was achieved near 100% with 1nM siRNA after 48 hours of transfection. The SDC1 KD condition did not affect cell morphology but significantly attenuated cell proliferation by two-fold (p < 0.05; Fig. 2A) by downregulating BCL2 (87%), CCND1 (74%), and CDK4 (64%), indicating that SDC1 has pivotal functional roles in proliferation of HL cells. Transwell chamber assays showed that SDC1 KD reduced migration of HL cells by two-fold (p < 0.05; Fig. 2B) and invasion by three-fold (p < 0.005; Fig. 2C) by downregulating metastatic markers MMP9, ADAM9, ITGB3, and VEGF . The flow cytometry data demonstrated that 50% of HL cells expressed residual SDC1 on the cell membrane after SDC1 KD for 48 hours, which potentially maintains the normal HL cells morphology and cellular functions. However, four days of prolonged SDC1 KD caused an induction of apoptosis by upregulating CASP3, and continuous KD for 17 days caused near eradication of surviving HL cells.
In conclusion, our results provide evidence that increased FGF2/SDC1 expression and signaling may drive and maintain the hyperproliferation of HL cells, thereby contributing to disease progression and poor prognosis in HL patients. Therefore, a novel method to inhibit altered FGF2/SDC1 signaling offers an excellent potential for treating high-risk and refractory HL patient populations.
Goy: Genentech: Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics / J&J: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Feldman: Janssen: Speakers Bureau; Celgene: Speakers Bureau; AbbVie: Speakers Bureau; Bristol-Myers Squibb: Consultancy; Kite Pharma: Speakers Bureau; Seattle Genetics: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Speakers Bureau. Leslie: KITE pharma: Speakers Bureau; celgene: Speakers Bureau; seattle genetics: Speakers Bureau. Pecora: COTA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Caladrius Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
