Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling

Abstract

Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and β-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell-cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.

Figure 1. Re-entry to S phase following synchronization is significantly accelerated by Hyal1 expression and delayed by HA overproduction

(A) 22Rv1 cells stably expressing GFP (vector control), Hyal1 or HAS3 were synchronized in serum free medium with 0.2 mM nocodazole for 24h, then released by addition of serum (0h). At the indicated times, cells were trypsinized, fixed, stained with propidium iodide and analyzed by flow cytometry. Mean percentage of cells in S phase is plotted ± SEM. * p<0.05 (B) A model for the interplay of cell cycle regulators in proliferation illustrates the prominent roles of ERK activation, cyclin D1 stabilization and cyclin dependent kinase inhibitors p21 and p27 in maintaining appropriate timing of S phase entry and exit.

Figure 2. Expression of Cyclins and cyclin dependent kinase inhibitors is altered in unsynchronized prostate tumor cells producing excess HA

Equal amounts of protein from serum replete asynchronous cultures of 22Rv1 stable transfectants constitutively expressing GFP (control), Hyal1, or HAS3 were analyzed by western blot for cyclin D1, cyclin A1, p21, p27, and tubulin (A). Expression of each cell cycle regulator was quantified by densitometric analysis of the blots normalized to the respective tubulin band. The blot is representative of three independent experiments, for which the mean ± SEM is plotted (B). Equal protein from vehicle or dox-treated (2 μg/ml) 22Rv1 Tet-ON cells transfected with pTRE-Tight GFP (control), Hyal1, or HAS3 constructs was immunoblotted and densitometrically quantified for cyclin D1 (C), p21 (D) or p27 (E). Mean ± SEM is plotted for three experiments. In panels B–E, * p < 0.05.

Figure 3. Transient activation of ERK by EGF and bFGF is unaffected by altered HA metabolism

22Rv1 cells stably expressing GFP (control), Hyal1, or HAS3 were serum starved for 24 hours, then treated with growth factor cocktail (GF, 20 ng/ml of EGF and 10 ng/ml of bFGF) for 20 min. Vehicle (−) and GF treated (+) cells were harvested and lysed in RIPA buffer with protease and phosphatase inhibitors. Cell lysates were analyzed by western blot for phosphorylated and total ERK1/2, with tubulin as a loading control. The blot is representative of three independent experiments.

Figure 4. Hyal1 overexpression reduces sustained ERK activation without altering cyclin D1

Cell populations in serum containing (asynchronous, AS) or serum free (SFM) media for 24h were treated with growth factors for 3h (GF 3h), and 12h (GF 12h). (A) At each time, cells were harvested, lysed in RIPA buffer with protease and phosphatase inhibitors, and analyzed by western blot probed with the indicated antibodies. Representative data from a single experiment are shown, along with densitometric quantification of p-ERK2 to t-ERK2 ratio (B) and cyclin D1 to tubulin ratio (C). Mean ± SEM of three independent experiments is plotted; * p < 0.05.

Figure 5. Expression of cyclin dependent kinase inhibitors and growth factor mediated ERK activation inversely reflect S phase entry kinetics in response to HA

GFP, Hyal1 and HAS3 transfectants were synchronized with nocodazole for 24h (Noc Arr), then released by the application of GF containing media. (A) The cells were harvested at 3h, 12h and 24h post release and assessed by western blot probed for total and phosphorylated ERK1/2, cyclin D1, p21 and p27 in comparison to synchronized cells. Representative data from one experiment are shown. (B) ERK phosphorylation was quantified by densitometry and plotted as a ratio to total ERK, normalized to tubulin. Similar quantification analysis is shown for p27 (C) and p21 (D). The mean ± SEM of three independent experiments is plotted for arrested cells and cells that have been arrested and released for 12h; * p < 0.05 compared to GFP in each condition.

Figure 6. ERK activation in prostate tumor cells is dependent on MEK

Subconfluent cultures of 22Rv1 stable transfectants were treated with vehicle (DMSO), 10 μM U0126 (MEK inhibitor) and 100 nM Wortmannin (PI3K inhibitor) for 2 hours. The cells were harvested and equivalent protein was assessed by western blot for pERK, tERK and Tubulin.

Figure 7. Differential effects of Hyal1 and HA on ERK phosphorylation and cell cycle progression are not coincident with Rac or FAK activation upon integrin ligation

Single cell suspensions of 22Rv1 GFP, Hyal1 and HAS3 transfectants were allowed to adhere to microwell plates coated with either BSA or fibronectin (FN). After 30 min, plates were centrifuged for 5 min to pellet all cells. Cell lysates were prepared and analyzed by western blot for total Rac expression, phosphorylated and total FAK, and tubulin. Activated Rac (GTP bound form) was specifically immunoprecipitated for comparison among transfectants.

Figure 8. 22Rv1 prostate tumor cells express both E-cadherin and N-cadherin but only N-cadherin expression is suppressed by HAS3 and HA synthesis

(A) Total RNA was isolated from stable 22Rv1 transfectants, reverse transcribed and individual transcripts for E-cadherin, N-cadherin and GAPDH (housekeeping control) quantified by PCR as indicated. On the right, whole cell lysates were analyzed for protein levels of E- and N-cadherin by western blot. (B) Lysates were separated into cytosolic (C) and nuclear (N) fractions and subjected to western analysis using antibodies against β-catenin, lamin B (nuclear marker) or tubulin (cytosol). (C) 22Rv1 Tet-ON cells were transiently transfected with plasmids inducible for GFP, Hyal1, or HAS3 and incubated in the presence (+) or absence (−) of 2 μg/ml doxycycline for 48h. Whole cell lysates were immunoblotted for E- and N-cadherin. (D) Subcellular localization of adherens junction components in 22Rv1 transfected cells. 22Rv1 stable transfectants expressing GFP, Hyal1 or HAS3 were grown on glass coverslips overnight, then fixed and immunostained using primary antibodies specific for E-cadherin, N-cadherin, and β-catenin, as indicated, followed by secondary detection with Cy3 conjugate.