Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 17;8(9):e75681.
doi: 10.1371/journal.pone.0075681. eCollection 2013.

The impact of the receptor of hyaluronan-mediated motility (RHAMM) on human urothelial transitional cell cancer of the bladder

Christian Niedworok  1 , Inga Kretschmer, Katharina Röck, Frank Vom Dorp, Tibor Szarvas, Jochen Heß, Till Freudenberger, Ariane Melchior-Becker, Herbert Rübben, Jens W Fischer
Affiliations

The impact of the receptor of hyaluronan-mediated motility (RHAMM) on human urothelial transitional cell cancer of the bladder

Christian Niedworok et al. PLoS One. .

Abstract

Hyaluronan (HA) is a carbohydrate of the extracellular matrix with tumor promoting effects in a variety of cancers. The present study addressed the role of HA matrix for progression and prognosis of human bladder cancer by studying the expression and function of HA-related genes.

Methods: Tissue samples of 120 patients with different stages of transitional cell bladder cancer, who underwent surgical treatment for bladder cancer at the University Hospital of Essen were analysed. mRNA-expression levels of HA synthases (HAS1-3) and HA-receptors (RHAMM and CD44) were evaluated by real time RT-PCR in comparison to healthy bladder tissue as control. In uni- and multivariate cox proportional hazard survival regression analysis, the impact of the gene expression levels on survival was assessed. In vitro knock-down of RHAMM, CD44 and HAS isoenzymes was achieved by siRNA and lentiviral shRNA in J82 bladder cancer cells. Transfected cells were analysed in vitro with regard to proliferation, cell cycle and apoptosis. J82 cells after knock-down of RHAMM were xenografted into male nu/nu athymic mice to monitor tumor progression in vivo.

Results: In invasive tumor stages RHAMM-, HAS1 and HAS2 mRNA-expression levels were elevated whereas HAS3v1 was reduced as compared to non-invasive tumors. Subsequently, Kaplan-Meier analysis revealed reduced bladder cancer specific survival in patients with high RHAMM mRNA and low HAS3v1 expression. Elevated RHAMM in invasive tumors was confirmed by RHAMM immunohistochemistry. Furthermore, multivariate analysis revealed that only RHAMM expression was associated with poor prognosis independent from other survival factors (HR=2.389, 95% CI 1.227-4.651, p=0.01). Lentiviral RHAMM knock-down revealed reduced J82 cell proliferation in vitro and reduced xenograft tumor growth in vivo.

Conclusion: The data suggest that RHAMM plays a crucial role in mediating progression of muscle-invasive bladder cancer and recommends RHAMM for further evaluation as a prognostic marker or therapeutic target in bladder cancer therapy.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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
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
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
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
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
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
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ploeg M, Aben KK, Kiemeney LA (2009) The present and future burden of urinary bladder cancer in the world. World J Urol 27: 289-293. doi:10.1007/s00345-009-0383-3. PubMed: 19219610. - DOI - PMC - PubMed
    1. Millán-Rodríguez F, Chéchile-Toniolo G, Salvador-Bayarri J, Palou J, Algaba F et al. (2000) Primary superficial bladder cancer risk groups according to progression, mortality and recurrence. J Urol 164: 680-684. doi:10.1016/S0022-5347(05)67280-1. PubMed: 10954628. - DOI - PubMed
    1. Liberman D, Alasker A, Sun M, Ismail S, Lughezzani G et al. (2011) Radical cystectomy for patients with pT4 urothelial carcinoma in a large population-based study. BJU Int 107: 905-911. doi:10.1111/j.1464-410X.2010.09590.x. PubMed: 20860649. - DOI - PubMed
    1. Toole BP (2004) Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 4: 528-539. doi:10.1038/nrc1391. PubMed: 15229478. - DOI - PubMed
    1. de la Motte CA, Hascall VC, Drazba J, Bandyopadhyay SK, Strong SA (2003) Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function. Am J Pathol 163: 121-133. doi:10.1016/S0002-9440(10)63636-X. PubMed: 12819017. - DOI - PMC - PubMed

Publication types

MeSH terms

Grants and funding

The study was supported by DFG Fi682/4-1 and the IFORES Program of the University of Essen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.