Figure 1.
RHAMM mRNA expression is elevated in muscle-invasive, high grade tumor stages compared to non-invasive low grade tumor stages.
RHAMM, CD44, HAS1, HAS2 and HAS3v1 mRNA expression in human BC tissue, shown as boxplot-diagrams in 25% to 75% intervals with minimum to maximum whiskers. Real-time-RT-PCR (total patients n=120, non-invasive tumors n=55, invasive tumors n=65; Ta n=31, T1 n=22, T2 n=21, T3 n=25, T4 n=19; low grade n=63, high grade n=57).
Figure 2.
Increased RHAMM staining during tumor progression.
(A-C) representative immunohistological staining of RHAMM in paraffin-embedded human BC tissue. (A) in nonmuscle-invasive tumor stages T1 the bladder structure is still preserved. (B) in tissue of high stage tumors the malignant cells start to proliferate and form independent cell clusters surrounded by stromal cells (pT2). (C) in non-organ confined tumor stages (pT4), the bladder structure is severely disturbed by infiltrating tumor cells. (D) the area fraction of RHAMM positive cells is elevated in highly muscle-invasive tumor stages as determined by image analysis (non-invasive, n=8; invasive n=12; *p=0.03, t-test). (E) correlation of RHAMM positive area fraction with higher tumor stages (n=4; *p=0.0129, Pearson-correlation); mean ± SEM.
Figure 3.
High and intermediate RHAMM as well as low HAS3v1 mRNA expression levels are associated with reduced cancer-specific survival.
The patient cohort was split into three subgroups consisting of high, intermediate and low expression of either RHAMM, CD44, HAS1, -2 or -3 mRNA (A-E). Kaplan-Meier survival probability test was performed showing significant increase in the risk of cancer-related death for the subgroups of high and intermediate RHAMM mRNA expression levels compared to low RHAMM mRNA expression (A). Transcript levels of CD44 (B), HAS1 (C) and HAS2 (D) were associated with significant changes of disease-specific survival. Notably, high HAS3v1 mRNA expression was significantly associated with lower risk of cancer-specific death compared to the subgroups with low and intermediate HAS3v1 mRNA expression (E). Data from a total of n=120 patients; analysis was performed in three groups (n=40 each) of high, intermediate and low mRNA expression of the respective genes.
Figure 4.
RHAMM knock-down reduces tumor growth in vivo.
(A-C) xenograft tumor growth in athymic mice is reduced when human J82 BC cells were transduced with shRHAMM prior to injection. (A) control group that received J82 cells transduced with scrambled shRNA; (B), tumors derived from injection of J82 cells transduced with shRHAMM. (C) 45 days after cell application tumor volume was significantly reduced by shRHAMM. (D-F) immunostaining of RHAMM and Ki67 (G-I). Data represent mean ± SEM, n=7 for scrambled and shRHAMM each, *p < 0.05).
Figure 5.
Inhibition of HA synthesis and RHAMM knock-down reduce cell proliferation in vitro.
(A) HAS1-, HAS2- and HAS3 mRNA expression in J82 BC cells as determined by RT-PCR. (B-D) proliferation of J82 cells 4 days after transfection with either HAS1-, HAS2-, HAS3- and RHAMM siRNA in 10% FCS or after 2 days of treatment with 4-MU (300 µM). (E) representative bright field images of J82 cells 4 days after knock-down of HAS3 and RHAMM plusminus 100 µg HA/ml (Healon 5). (F) proliferation expressed as J82 cells/well treated either with siRNA against HAS3 or RHAMM or 4-MU for 2 days and the respective controls. HA (100 µg/ml) was added to test whether the effects could be rescued by exogenous HA; mean ± SEM, *, p<0.05.
Figure 6.
Knock-down of HAS3 causes apoptosis in bladder carcinoma cells in vitro.
Analysis of apoptosis and cell cycle progression were performed 4 days after transfection with siRNA targeting HAS3 and RHAMM as detailed also in Figure 5. (A) immunoblotting of PARP and cleaved PARP after knock-down of HAS3 and RHAMM by siRNA plusminus 100 µg/ml HA. (B) apoptotic activity was expressed as the ratio of cleaved and uncleaved PARP; experiments were performed as described in A. (C), cell cycle analysis by FACS; black line, scrambled; green line, siRHAMM; red line; siHAS3; representative FACS histograms. (D) quantitative analysis of FACS analysis with respect to G0/G1 phase representing cell cycle arrest; G2 phase representing premitotic cells and sub G1 indicating apoptotic cells; mean ± SEM, *, p<0.05.
Figure 7.
Knock-down of CD44 inhibits proliferation of bladder carcinoma cells in vitro.
(A) representative bright field images of J82 cells 4 days after knock-down of RHAMM, CD44 and RHAMM plus CD44 plusminus 100 µg HA/ml (Healon 5) in 10% FCS. (B) proliferation of J82 cells after knock-down of RHAMM, CD44 and CD44 plus RHAMM in J82 cells 4 days after transfection plusminus exogenous HA (100 µg/ml). (C) analysis of apoptosis was performed 4 days after transfection with siRNA as in A and B by immunoblotting of PARP and cleaved PARP. (D) PARP cleavage was quantified as the ratio of cleaved and uncleaved PARP; mean ± SEM, *, p<0.05.