Breast cancer progression correlates with altered hyaluronan metabolism, including increased deposition of hyaluronan in the nests of carcinoma cells, and especially in the stromal tissue in the invading edges of breast carcinomas.1, 2 Stromal fibroblasts activated by the breast cancer cells most likely contribute to the enrichment of hyaluronan in the immediate peritumoral stroma.3In vitro studies revealed that the most aggressive breast carcinoma cell lines both synthesize high amounts of hyaluronan and express the cell surface hyaluronan receptors, CD44 and RHAMM, unlike the less aggressive cell lines.4, 5, 6
Hyaluronan is synthesized by hyaluronan synthases, which exist in 3 isoforms (HAS1, HAS2 and HAS3), and is degraded by hyaluronidases (HYAL1, HYAL2 and HYAL3, and PH-20).7, 8 Although each of the HAS isoforms is capable of hyaluronan synthesis, they synthesize hyaluronan of different lengths. The HAS2 isoform synthesizes hyaluronan molecules larger than 3.9 × 106, HAS3 synthesizes polydisperse hyaluronan (Mw of 0.12–1 × 106) and HAS1 synthesizes much smaller chains (Mw of 0.12 × 106). Moreover, the HAS isoforms exhibit different catalytic activities; HAS3 is catalytically more active than HAS2, which in turn is more active than HAS1.9 Disruption of the HAS2 gene causes embryonic lethality in mice, whereas deletion of HAS1 or HAS3 do not.10 Hyaluronan fragmentation is catalyzed by HYAL1 and HYAL2, which are widely distributed in tissues at low concentrations. Both hyaluronidases are active at an acidic pH, and HYAL1 exhibits high specific activity whereas HYAL2 is less active.11, 12, 13
Several studies have demonstrated a close correlation between abnormal production of hyaluronan and the promotion of the malignant phenotype of tumors. In a colon carcinoma model, we showed that expression of HAS2 enhances tumor growth whereas expression of HYAL1 delays tumor development, and the transplantable tumor cells gave rise to tumors of lower frequency.14 Recently, studies using antisense inhibition of HAS2 and/or HAS3 genes in aggressive prostate and breast carcinoma experimental models further highlight the importance of HAS2 and HAS3 in initiation and progression of the tumors.15, 16 Although these approaches using stable transfection of the cancer cell lines offered an initial promise, these studies achieved partial inhibition of the messenger RNA. To investigate further the importance of hyaluronan and hyaluronan synthesizing and/or degrading enzymes for breast cancer cell invasiveness, we determined the hyaluronan production and expression of HAS1, HAS2 and HAS3, as well as HYAL1 and 2 mRNA, in a panel of highly aggressive and less aggressive breast cancer cells. The analyses revealed that the invasive Hs578T cells both synthesized the largest amounts of hyaluronan and predominantly expressed the HAS2 gene. RNAi technology is currently providing new promise in the experimental and therapeutic silencing of genes.17, 18 We explored the applicability of this technique to the silencing of the HAS2 gene, with the aim of characterizing the importance of this enzyme in the maintenance of the invasive and malignant phenotype of the Hs578T breast cancer cell line.
FBS, fetal bovine serum; HAS, hyaluronan synthase; HYAL, hyaluronidase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline without Ca2+ and Mg2+; PDGF β-receptor, platelet derived growth factor β-receptor; SDS, sodium dodecylsulfate; siRNA, small interfering RNA.
Material and methods
Cell culture and quantification of hyaluronan synthesis
Human breast cancer cell lines (4.5 × 104 cells/well in 12-well plates) expressing high levels of progesterone and estrogen receptors (ZR-75-1, MCF-719, 20) or low receptor levels (MDA-MB-231, Hs578T21, 22, 23), as well as high progesterone but low estrogen receptor levels (HTB-12224), were cultured in Dulbecco's modified Eagles medium (DMEM, Veterinary Institute, Uppsala, Sweden) containing 10% fetal bovine serum (FBS), 4 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (complete medium); the cell lines were generously provided by Professor J. Bergh (Karolinska institute, Stockholm, Sweden). Twenty four hours after plating, the conditioned media were collected, and the hyaluronan content quantified using a microtiter-based assay taking advantage of the formation of a complex between hyaluronan and the hyaluronan binding protein (HABP) domain of aggrecan, essentially as described previously.14 Briefly, MaxiSorp 96-well Nunc-Immuno Plates (Nunc 439454) were precoated overnight with 1 μg HABP/ml. Then, samples of conditioned media at appropriate dilutions or hyaluronan standards (0–100 ng/ml) were applied onto the plates. After 1 hr of incubation at 37°C, plates were washed, 100 μl of biotin labeled HABP (b-HABP; 1 μg/ml) was added and the plates were incubated for an additional 1 hr. After washing, the b-HABP specifically bound to the immobilized hyaluronan was detected with streptavidin biotinylated horseradish peroxidase (Amersham) followed by the addition of 100 μl 3,3′,5,5′ tetramethylbenzindine substrate solution (Sigma T4444) for 15 min. Following addition of 50 μl of 2 M H2SO4, the absorbance was measured at a wavelength of 450 nm, and the sample hyaluronan content was calculated from a standard curve.
siRNA-mediated silencing of HAS2 expression and HAS2 overexpression in Hs578T breast cancer cells
Four siRNA duplexes designed with symetric 3′TT overhangs to target different nucleotide sequences (no 1, 1043–1061; no 2, 1402–1420; no 3, 1617–1637; no 4, 1671–1691) of the human HAS2 gene (Genbank accession number U54804) were obtained from Qiagen, UK. Subconfluent Hs578T cells (about 50–60% confluent) grown in complete medium were transfected separately with each 1 of the 4 siRNA duplexes or with a control (non-silencing) siRNA (sense, r(UUC UCC GAA CGU GUC ACG U)dTdT; antisense: r(ACG UGA CAC GUU CGG AGA A)dTdT) at different concentrations (50, 100 and 200 nM) using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. The suppression efficiency of each 1 of the 4 siRNA duplexes was examined both by measuring the hyaluronan content in the conditioned culture media after 24, 48, 72 and 96 hr of transfection, and the HAS2 mRNA expression levels using real-time PCR, 48 hr after transfection. The HAS2 siRNA [sense: r(GCG AUU AUC ACU GGA UUC U)dTdT; antisense: r(AGA AUC CAG UGA UAA UCG C)dTdT] corresponding to nucleotide sequences 1671–1691 of the HAS2 cDNA (AAGCGATTATCACTGGATTCT) (no 4) was used for all experiments; in some of the experiments the effects of HAS2 siRNA duplex [sence: r(CACGUAACGCAAUUGGUCU)dTdT; antisense: r(AGACCAAUUGCGUUACGUG)dTdT] corresponding to target region 1043–1061 (AACACGTAACGCAATTGGTCT) (no 2) were also studied. Furthermore, hyaluronan at different concentrations (molecular mass 2.5 × 106) was included into the culture media in some of the transfection experiments, 6 hr after the start.
Subconfluent cultures of Hs578T were transfected with 2 μg/ml of pCIneo vector containing the open reading frame for HAS225 using LipofectAMINE 2000, according to the manufacturer's instructions. Then, cultures were screened for hyaluronan content in the conditioned media, as described above.
Quantification of mRNA expression levels for hyaluronan synthesizing and degrading enzymes
Real time RT-PCR was used to quantify the relative mRNA levels of HAS1, HAS2 and HAS3, as well as HYAL1 and 2 in wild-type breast cancer cell lines, and in control siRNA- and HAS2 siRNA-transfected Hs578T cells. Total RNA was purified using a RNAqueous™-4PCR kit for isolation of DNA-free RNA (Ambion), according to the manufacturer's instruction. Purified total RNAs (3 μg for each sample) were reverse transcribed (SuperScript™ II RNase H− Reverse Transcriptase Kit, Invitrogen) according to the manufacturer's instruction. Then, 3 μl of each reverse transcribed cDNA was subjected to real-time PCR (qPCR™ Core kit for SYBR™ Green. Eurogenetic) using the ABI Prism 7700 Detection system (version 1.6 software; Applied Biosystem, Weiterstadt, Germany) according to the manufacturer's protocol. The specific primers were designed based on cDNA sequences deposited in the GenBank data base (accession numbers: HAS1, U59269; HAS2, U54804; HAS3, AF232772; HYAL1, U03056; HYAL2, AJ000099 and GAPDH, NM_002046) using a Primer Express™ software (Version 1.5, Applied Biosystems) (Table I). The conditions for real-time PCR were as follows: One cycle at 95°C for 10 min, 40 cycles at 95°C for 15 sec, 60°C for 1 min and one cycle at 25°C for 2 min. Confirmation of all primer pairs specificity was documented by performing melting curve analysis (data not shown). Standard curves for each transcript were generated by serial dilutions of input total RNAs from 0–100 ng; the slope values of each standard curve were approximately equal, indicating a similar amplification efficiency for each target (within 10% from the theoretical value which should be −3.3).26 Hence, quantification of each target gene relative to an endogenous reference gene, GAPDH, was determined by using the formula, target gene/GAPDH = 2−DCT; DCT values corresponds to difference between the threshold cycle (CT) values of each target gene and GAPDH (i.e. number of cycles to see amplification of the target gene). The expression level for each target gene relative to control siRNA (arbitrarily set to 1) was quantified using the relative standard curve method (Applied Biosystems, User Bulletin 2).
Table I. Primer Sequences Used for the Quantification of Gene Expression in Breast Cancer Cell Line
Genebank accession number
For 5′ GGAATAACCTCTTGCAGCAGTTTC 3′ Rev 5′ GCCGGTCATCCCCAAAAG 3′
For 5′ TCGCAACACGTAACGCAAT 3′ Rev 5′ ACTTCTCTTTTTCCACCCCATTT 3′
For 5′ AACAAGTACGACTCATGGATTTCCT 3′ Rev 5′ GCCCGCTCCACGTTGA 3′
For 5′ GATGTCAGTGTCTTCGATGTGGTA 3′ Rev 5′ GGGAGCTATAGAAAATTGTCATGTCA 3′
For 5′ CTAATGAGGGTTTTGTGAACCAGAATAT 3′ Rev 5′ GCAGAATCGAAGCGTGGATAC 3′
For 5′ CCCATGTTCGTATGGGTGT 3′ Rev 5′ TGGTCATGATCCTTCCACGATA 3′
Determination of the molecular mass and cell-localization of hyaluronan synthesized by control siRNA- and HAS2 siRNA-transfected Hs578T cells
The molecular mass of de novo synthesized hyaluronan by Hs578T cells (2 × 105 cells/well in a 6-well plate), transiently transfected for 48 hr with control siRNA or HAS2 siRNA, was determined after incubation for an additional 24 hr in 1 ml complete medium containing 10 μCi D-[6-3H]glucosamine hydrochloride, a precursor of both hyaluronan and sulfated glycosaminoglycans. Conditioned media were collected and dialyzed (MW cut-off of 3500; Spectrum Laboratories, USA) for 48 hr against 0.1 M sodium acetate buffer, pH 5.5, supplemented with 5 mM EDTA. The dialysate was then divided into 2 equal aliquots. One aliquot was treated overnight with 10 U/ml Streptomyces hyaluronidase at 37°C (hyaluronidase-sensitive radioactivity was considered to represent 3H-hyaluronan), whereas the other aliquot was treated in a similar way but in the absence of the hyaluronidase, as a control. Then, the enzyme was heat-inactivated and each sample was subjected to gel chromatography using a Sephacryl-HR column (100 × 1 cm), composed of 3 layers of Sephacryl-HR with different porosities27 and equilibrated with 0.25 M NaCl. Fractions of 0.82 ml were collected and the radioactivity was measured in a Pharmacia LKB Wallac scintillation counter.
Hs578T cells (1 × 105 cells/well in 4-well chamber slides; Lab/Tek, Falcon) transfected with 100 nM of control siRNA or HAS2 siRNA were cultured for 48 hr. Then, untreated or Streptomyces hyaluronidase-treated cells (2 U/ml for 10 min to remove pericellular hyaluronan, Seikagaku, Japan) were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, washed, permeabilized in 0.15% Triton X-100 in PBS for 10 min, and washed again in PBS-D. To detect hyaluronan in situ, cultures were incubated with b-HABP (5 μg/ml in PBS-1% BSA) for 1 hr. After washing, cells were incubated with Alexa Fluor 488 conjugated streptavidin (1 μg/ml; Molecular Probes) for 1 hr at 37°C. The specificity of hyaluronan staining was tested by preincubating the cells with 10 U/ml Streptomyces hyaluronidase for 2 hr at room temperature.
Immunoblotting and hyaluronan binding assay
Cells (1 × 106 cells in 6-well plates) were transfected with control or HAS2 siRNAs and incubated in the absence or presence of exogenously added hyaluronan (100 μg/ml) for 48 hr, whereafter they were solubilized with ice-cold RIPA buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 0.65 mM MgSO4, 1 mM CaCl2, 0.5% deoxycholate) containing proteinase inhibitors (10 turbidity units/ml aprotinin, 1 μg/ml leupeptin, 0.1 mM pefabloc, 1 μM pepstatin) for 2 hr at 4°C, on a rocking platform. Lysates were centrifuged at 13,000 rpm for 15 min at 4°C, and the protein content in the supernatants was determined by BCA kit (Pierce). Then, samples (20 μg of protein) were subjected to SDS-PAGE using 10% polyacrylamide gels and the protein was electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden) using semi-dry transfer system (Bio-Rad). Membranes were blocked over-night at 4°C with 5% defatted milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, 137 mM NaCl, pH 7.6), supplemented with 0.1% Tween-20, and probed with primary antibodies for 3 hr at room temperature (2 μg/ml of poly-Ab against p16 or 2 μg/ml mAb against p21, cyclin A, cyclin B or cdc2 p34, Santa Cruz, UK, or 5 μg/ml Hermes-1 a mAb against CD44; an ammonium sulphate precipitate from serum free supernatant, generously provided by Professor S. Jalkanen, Turku, Finland, was used). After 3 washes in TBS-T, the membranes were incubated with appropriate secondary antibodies for 1 hr, and immunocomplexes were detected by enhanced chemiluminescence (Amersham Bioscience, Uppsala, Sweden) according to the manufacturer's instructions. The blots were quantified using a scanner and associated software (Aida Image Analyzer).
The hyaluronan binding capacity of HAS2 siRNA- and control siRNA-treated Hs578T cells was investigated essentially as described previously.14, 28 Briefly, Hs578T cells expressing or deprived of HAS2 mRNA were incubated in starvation medium overnight and then treated with testicular hyaluronidase (100 U/ml, for 1 hr at 37°C) to ensure that the cell surface receptors for hyaluronan were not occupied by endogenously produced hyaluronan. Following extensive washing, 0.25 μg [3H]hyaluronan/ml (prepared from conditioned equine synovial cells, as described by Fraser et al.29) was added, and the cells were incubated for 1 hr at room temperature in the absence or presence of exogenously added nonlabeled hyaluronan (100 μg/ml, Mw 1.2 × 106, Q-Med, Uppsala, Sweden). Some of the cell monolayers were preincubated for 30 min at 4°C with Hermes-1 antibody which specifically inhibits the binding of [3H]hyaluronan to cell surface CD44.30, 31 After washing, cell layers were solubilized in 0.3 M NaOH, 1% SDS (w/v) for 30 min, followed by neutralization with 2 M HCl and scintillation counting.
Soft agar growth assay
The soft agar assay was conducted essentially as described previously.32 Control siRNA- or HAS2 siRNA-transfected Hs578T cells (0.5 × 105 cells/well in 6-well plate) were suspended in 3 ml DMEM containing 10% FBS (pre-warmed to 37°C), and 300 μl of 3% agarose in PBS (prewarmed to 60°C) was added. Agar-suspended cells (1 ml/well in 6-well plates) were plated out in dishes coated with 1 ml of agar-coated dishes (0.6% agarose in DMEM). After solidification at room temperature for 20 min, 3 ml complete medium was added to each well and cells were incubated at 37°C in a humidified atmosphere of 5% CO2 (v/v) in air for 3 weeks. After this period, 20 fields were randomly selected, and the number of colonies comprising greater than 6 arbitrary units were counted under a Nikon microscope. The area of the colonies was measured using an NIH image program.
Cells (3 × 104 cells/well in 24-well plates) were transfected either with control siRNA or each one of the HAS2 siRNAs (no 2 or no 4), as described above. Differences in the proliferative capacity between HAS2 siRNA- and control siRNA-transfected cells were examined in the absence or presence of exogenous hyaluronan (Mw 2.5 × 106, Q-Med, Uppsala, Sweden), by counting cell numbers up to 8 days after transfection. At the indicated time points, cells were detached using trypsin-EDTA and the cell numbers were determined using a Particle Coulter counter (Beckman Instruments AB).
Cell cycle analysis
Hs578T cells (2 × 105 cells/well in 12-well plates) transfected with HAS2 siRNA or control siRNA were cultured in complete medium, for various periods of time after transfection. Cells were then washed in PBS, trypsinized and pelleted by centrifugation. For DNA analysis, the cell pellets were resuspended in 450 μl prechilled 70% ethanol and stored at −70°C for at least 5 min. After fixation, the cells were pelleted by centrifugation, washed in PBS and resuspended in 300 μl PBS containing 0.1% Triton X-100 and 180 U/ml RNase A (USB, USA). The cell suspensions were then incubated at 37°C for 20 min followed by addition of 250 μl propidium iodide (to a final concentration of 50 μg/ml, Sigma) and 10 min incubation at 4°C. The DNA content of cell samples and cell cycle kinetics were detected by using a CellQuest software in a Becton Dickinson FACStar plus flow cytometer.
Wild type Hs578T cells (4 × 105 cells/well in 6-well plates) and cells transfected with HAS2 siRNA or control siRNA were grown for 48 hr until confluency. Two horizontal and one longitudinal cell-free areas were made across the dish with a 200 μl pipette tip. Detached cells were removed and fresh complete medium was added. In some cases, to investigate the effect of exogenously added hyaluronan or CD44 on cell migration, cells were cultured in complete media supplemented with hyaluronan at different concentrations (1–50 μg/ml) or with blocking CD44 antibodies (Hermes-1; 50 μg/ml). The cell-free areas were photographed immediately after wounding; the closure of the wounds by migrating cells were determined after 14 hr and 28 hr using an Olympus CK2 inverted phase contrast microscope with a Nikon camera and quantified by NIH Image Software. The data was analyzed by student t test.
Matrigel invasion assay
The invasive behavior of Hs578T cells lacking HAS2 expression compared to HAS2 expressing cells was performed essentially as described before.33 Polycarbonate membrane inserts (6.5 mm, 8 μm pores) in Transwell 24 permeable supports (Costar, Corning) were layered with Matrigel matrix (basement membrane extract growth factor reduced, 7–11 μg protein/mm2; R&D Systems). The Matrigel-layered inserts were allowed to solidify for 30 min at at 37°C, in humidified 95% air and 5% CO2. Growth medium (0.6 ml/well) was added to the transwell unit, and the 48 hr transfected-cells with 100 nM of control siRNA or HAS2 siRNA were trypsinized, counted and plated on the unlayered or Matrigel-layered membrane inserts (1 × 105 cells per 100 μl growth medium); cell transfection, migration or invasion was performed in the absence or presence of 100 μg/ml hyaluronan (Mw 2.5 × 106). After 24 hr of incubation in a tissue culture incubator, media were removed and the polycarbonate filters with the migrating and invaded cells were washed once with PBS followed by fixation with 2.5% EM grade glutaraldehyde (Fluka) in PBS for 15 min at room temperature. Matrigel matrix and noninvading cells at the upper surface of the membrane were removed by wiping with a cotton swap. The migrating and invading cells on the underside of the membrane were stained with Giemsa solution for 30 min, rinsed in water and the membranes were removed from the insert by a scalpel blade. After dehydration through graded alcohols, the membranes were mounted onto glass slides. The invading cells were counted in 3 randomly selected fields of triplicate membranes, under the microscope at 200× magnification. The data is expressed as the percent invasion through the Matrigel-layered membrane relative to the migration through the unlayered one.
Quantification of hyaluronan synthesizing capacity, and expression of mRNA for HAS and HYAL isoforms in a panel of breast cancer cell lines
We first assessed the levels of hyaluronan in 24 hr conditioned media derived from the highly invasive cell lines Hs578T, MDA-MB-231 and HTB-122, and the less invasive cell lines MCF-7 and ZR-75-1.34 As shown in Figure 1a, breast cancer cells exhibiting an invasive phenotype produce higher amounts of hyaluronan compared to less aggressive cell lines. To elucidate the contribution of the HAS and HYAL enzymes to the hyaluronan levels in the conditioned media, the gene expression profiles of HAS1, HAS2 and HAS3, as well as HYAL1 and HYAL2, were investigated using real-time PCR. The relative expression levels of HAS and HYAL mRNAs, in comparison to the reference gene GAPDH, are depicted in Figure 1b. ZR-75-1, HTB-122 and MCF-7 cells expressed about 24-fold, 38-fold and 3-fold higher levels of HAS1 mRNA, respectively, compared to HAS1 mRNA transcript levels in Hs578T and MDA-MB-231 cells (mRNA expression level relative to GAPDH = 0.006 × 10−3). Notably, HAS2 mRNA was abundantly expressed in Hs578T, MDA-MB-231, as well as in the HTB-122 cells, at levels about 80-, 8- and 20-fold, respectively, over the levels in MCF-7 cells (mRNA expression level relative to GAPDH = 0.05 × 10−3). The relatively high transcriptional activities of HAS3 (mRNA expression level relative to GAPDH = from 0.6 × 10−3 to 1.7 × 10−3) and HYAL2 (mRNA expression level relative to GAPDH = from 3.2 × 10−3 to 11.4 × 10−3) genes differed only slightly among the breast cancer cells examined, except for the more elevated signals in ZR-75-1 cell cultures. The expression of HYAL1 mRNA, although almost undetectable in Hs578T, MDA-MB-231, HTB-122 and MCF-7, was high in ZR-75-1 cells (mRNA expression level relative to GAPDH = 5.0 × 10−3). Thus, the high hyaluronan content in the conditioned media of Hs578T cells is likely to be dependent primarily by the balanced expression of HAS2, HAS3 and HYAL2.
Silencing of the HAS2 gene in Hs578T cells induces HAS1 and HAS3 and HYAL1 transcripts
Several studies have implicated HAS2 expression in the promotion of tumor cell growth, both in vitro and in vivo.14, 32, 35, 36 On the basis of our finding that the aggressive Hs578T cells, which synthesize the highest amounts of hyaluronan among the 5 studied human breast cancer cell lines, predominantly expressed HAS2 mRNA (Fig. 1b), we investigated the consequences of suppression of HAS2 gene by designing specific HAS2 siRNA duplexes; transfection of cells by 100 nM of siRNA duplexes targeted to different sites of HAS2 cDNA caused about 50% reduction of hyaluronan amount released into the 48 hr conditioned media compared to control siRNA (Fig. 2a). Notably, 100 nM of the HAS2 siRNA led to HAS2 transcript suppression of about 85% (Fig. 2b), and to an about 50% reduction of hyaluronan content (Fig. 2c) compared to control siRNA. Similar results were obtained even after extended incubation, up to 96 hr after transfection (data not shown). The HAS2 siRNA-mediated strong reduction of HAS2 mRNA and its ability as well as the ability of 3 other HAS2 siRNAs (each one targeted to different sequences of HAS2 mRNA sequence) to suppress in a similar manner the production of hyaluronan, confirm the specific nature of the siRNA silencing used.
Notably, using real-time PCR we found that down-regulation of HAS2 mRNA levels led to a robust increase of HAS1 mRNA (about 15-fold). Furthermore, two-fold increases in both HAS3 and HYAL1 transcript levels were detected (Fig. 2c). However, the amount of HYAL2 mRNA did not change. These observations suggest that the invasive breast cancer cell line Hs578T, upon deprivation of HAS2 mRNA, evoked mechanisms to maintain hyaluronan production.
Silencing of HAS2 in Hs575T cells results in hyaluronan synthesis of lower molecular mass
Given the intriguing observation that silencing of the HAS2 mRNA promoted the up-regulation of mRNAs for HAS1 and HAS3, as well as HYAL1, we determined the size of hyaluronan synthesized by HAS2 siRNA-transfected Hs578T breast cancer cells by Sephacryl HR size exclusion chromatography. As shown in Figure 3a, the Hs578T cells transfected with control siRNA synthesized preferentially high molecular weight hyaluronan of an average of 2.9 × 106, whereas silencing of HAS2 in tumor cells resulted in the reduction of both the amount and size of hyaluronan chains released into the conditioned media (about 1 × 106; first peak, Streptomyces hyaluronidase sensitive material). A second peak of a molecular weight lower than 0.46 × 106 was insensitive to Streptomyces hyaluronidase, and thus considered to represent other glycosaminoglycans. The relative proportion of the glycosaminoglycans was only slightly affected by inhibition of the HAS2 mRNA. Thus, silencing of the HAS2 gene lead to reduced synthesis of hyaluronan molecules of apparently smaller size, either because the hyaluronan was synthesized by HAS1 and HAS3, which are known to synthesize hyaluronan of lower molecular weight than HAS2,9 and/or because the amount of hyaluronidase activity was increased.
Furthermore, we investigated whether HAS2 silencing affects the amount and localization of cell-associated hyaluronan. The intensity of hyaluronan staining was stronger in permeabilized Hs578T cells transfected with nonsilencing HAS2, than the nonpermeabilized ones (Fig. 3b). Notably, removal of the cell surface-associated hyaluronan with Streptomyces hyaluronidase prior to permeabilization did not completely abolish the staining for cell-associated hyaluronan, indicating that a part of hyaluronan resides within the tumor cells. As expected, the staining of hyaluronan in permeabilized HAS2 siRNA-transfectants was much less compared to the cells transfected with nonsilencing siRNA; also in these cells, a portion of hyaluronan was detected inside the cells, as ascertained by the weak staining remaining after Streptomyces hyaluronidase treatment. The data suggest that the Hs578T breast cancer cells have both a pericellular and an intracellular pool of hyaluronan, and that the endogenously produced hyaluronan is accumulated predominantly in the cytoplasm with or without HAS2 gene suppression.
We next investigated the consequence of HAS2 gene suppression on the total CD44 expression levels and cell surface CD44 hyaluronan binding capacity (Fig. 4). Western blot analysis revealed a slight decrease of the total CD44 molecules in Hs578T cells deprived of HAS2 mRNA compared to that of tumor cells expressing HAS2 mRNA. The predominant CD44 isoforms expressed had molecular masses of 80–95 kDa (Fig. 4a). Since hyaluronan mediates its signaling effects through binding to cell surface receptors, we next determined the binding capacity of [3H]hyaluronan on cells expressing high versus low HAS2 mRNA levels (Fig. 4b). The control siRNA transfectants exhibited a slightly higher capacity to bind [3H]hyaluronan than the HAS2 siRNA transfectants. To investigate whether cell surface CD44 molecules mediate [3H]hyaluronan binding, Hermes-1 antibodies, which block the binding of hyaluronan to CD44, were used. About 80% inhibition of the specific binding of [3H]hyaluronan was detected in the presence of blocking Hermes-1 antibodies, suggesting that the CD44 molecules on the cell surface are the major hyaluronan binding sites on Hs578T cells.
HAS2 mRNA silencing reduces cellular growth
To investigate further the importance of HAS2 gene silencing, and the subsequent suppression of hyaluronan synthesis, for the tumorigenicity of the Hs578T breast cancer cell line, we studied the growth rates of the nonsilencing siRNA- and HAS2 siRNA-transfectants. We used a soft agar assay to determine the anchorage-independent growth of the cells, which reflects the malignant phenotype of cells in vitro.37 We observed that the Hs578T cells treated with HAS2 siRNA formed about 40% smaller colonies compared to cells transfected with control siRNA, and parental Hs578T cells (Fig. 5a). However, no obvious differences in the number of clones were detected (data not shown). To further assess the role of HAS2 silencing for the proliferative capacity of Hs578T breast cancer cells, we analyzed their cell cycle profile using flow cytometry (Fig. 5b). At 48 hr after silencing of the HAS2 gene, a significantly higher proportion of cells were in G1 phase (52.0% ± 2.3%) compared to the mock-transfected cells (42.6% ± 2%). In contrast, HAS2 siRNA-transfectants had a significantly lower proportion of cells both in S phase (21.4% ± 1.6%) and G2/M phase (26.6% ± 2%) compared to non-silencing siRNA-transfectants (25.3% ± 2.9% in S and 31.0% ± 3% in G2/M phase). These data are consistent with a slower proliferation rate of Hs578T cells expressing low amounts of HAS2 mRNA.
To further evaluate the importance of HAS2 silencing in cell proliferation, the number of cells expressing or lacking HAS2 was investigated at different time points after the start of the transfection (Fig. 5c). No obvious differences between control cells and HAS2 siRNA-treated cells in the growth rate of subconfluent cultures (up to about 50% of confluency, reached at day 2), was observed. However, in confluent Hs758T cultures (at day 4 and after) the suppression of HAS2 gene caused about a 2-fold lower proliferation rate compared to control siRNA-transfectants. Addition of 100 μg/ml of exogenous hyaluronan had no effect on their proliferative capacities (Fig. 5c). The importance of exogenous hyaluronan, in a concentration-dependent manner, to rescue the growth kinetics of tumor cells lacking HAS2, was further investigated using cultures at 6 days after transfection with siRNAs targeted to different sites in HAS2 mRNA (no 2 and no 4) or control siRNA (Fig. 5d). Again, the proliferative capacity of each one of the two HAS2 siRNA-transfectants were similarly lowered in comparison to control cells, and the inclusion into the culture media of low or high amounts of hyaluronan (1–50 μg/ml) had no effect. Notably, the hyaluronan content in 48 hr conditioned media from the 2 different HAS2 siRNA-transfectants was half as much as from control siRNA-transfectants (data not shown). These observations indicate that HAS2 mRNA suppression decreases the malignant phenotype of Hs578T cells and that inclusion of exogenous hyaluronan into the culture medium did not restore their growth kinetics.
The effect of silencing of HAS2 on cell growth prompted us to investigate, by immunoblotting, the relation between HAS2 gene silencing and the expression levels of proteins in the cytoplasm that control cell cycle progression. The analysis revealed that the expressions of cyclin A and B, and cdc2 p34, which are important for cells to complete the cell cycle, were considerably decreased in HAS2 siRNA-transfectants compared to wild-type or nonsilencing siRNA-transfected Hs578T cells (Fig. 6a). No significant changes in the expression levels of negative regulators of the cell cycle (p16 and p21) were observed. These results suggest that the low proliferation rate of HAS2-silenced cells involves suppression of mitotic cyclins A and B, and cdc2 p34 kinase. Then, we investigated whether addition of exogenous hyaluronan could counteract the suppression of the cell cycle regulators caused by silencing the HAS2 gene. As shown in Figure 6a, exogenously added hyaluronan partially restored the expression of the cdc2 p34. Notably, overexpression of HAS2 by transfection led to significant increase in the expression levels of cyclins A and cyclin B (Fig. 6b), suggesting that hyaluronan synthesized by the tumor cells stimulates traverse through the cell cycle. Thus, HAS2-mediated hyaluronan synthesis stimulates the growth of breast tumor cells.
The constitutive expression of cyclins A and B, and cdc2 p34 was high even in wild-type Hs578T cells cultured under starvation condition (0.1% FBS; Fig. 6c). Notably, addition of exogenous hyaluronan or hyaluronan oligosaccharides (a mixture of 12–20 monosaccharide units; Fig. 6c) into the medium of starved wild-type cells strongly increased the expression levels of cyclin B; in addition, hyaluronan fragments promoted the expression of cyclin A, a result not observed after the addition of high molecular weight hyaluronan. Furthermore, neither high nor low molecular weight hyaluronan exerted any effect on the expression of the negative regulators of the cell cycle. The effects of the exogenously added high molecular weight or fragmented hyaluronan were not apparent when cells were cultured in complete medium (data not shown).
Suppression of HAS2 slows down both the migratory and invasive capacities of Hs578T cells
We also compared the motility of Hs578T cells treated with HAS2 siRNA or control siRNA, using a cell culture wounding assay (Fig. 7) and a Transwell permeable support assay (Table II). As shown in Figure 7a, HAS2 siRNA-transfectants exhibited about 50% lower migration capacity than control siRNA-transfectants. To investigate whether CD44 was implicated in the migration, Hermes-1 antibodies that specifically block the binding of hyaluronan to CD44 were added. Hermes-1 suppressed the migratory capacity of control siRNA-transfectants during the wound closure over a 28 hr period by about 27% compared to control siRNA treated cells (Fig. 7b). Thus, hyaluronan-CD44 interactions are partially involved in the regulation of breast cancer cell migration, but most likely also other hyaluronan receptors and other mechanisms are involved in mediating Hs578T cell migration. We then investigated if exogenously added high molecular weight hyaluronan could restore the migratory ability of HAS2-deprived cells. Cells separately transfected with two HAS2 siRNAs, no 2 and no 4, migrated about half as much slower than these tumor cells expressing HAS2, but the inclusion of exogenous hyaluronan at various concentrations into the culture media did not restore cell migration (Fig. 7c). In agreement with the results of this wounding assay, HAS2 expressing Hs578T cells migrated through the unlayered polycarbonate membrane inserts at least 2-fold faster than the HAS2 deprived cells, independently of the presence of exogenously added hyaluronan (Table II).
Table II. Effect of HAS2 Gene Silencing on the Invasive Potential of Hs578T Cells
Unlayered and Matrigel-layered polycarbonate membranes (8 μm pore size) in Transwell 24 units were used for the invasion assay as described in Material and methods section. Values represent the average of triplicates of a representative experiment out of two ± SD.
To investigate whether HAS2 is important for the invasive property of Hs578T cells, we subjected cells treated with control siRNA or HAS2 siRNA to an in vitro invasion assay in which cells invade through a Matrigel matrix layered on top of the 8 μm porous membrane inserts. We found that the HAS2-deprived tumor cells invaded the Matrigel matrix less than half as efficiently as HAS2 expressing cells (Table II). Hyaluronan inclusion into the media had no effect on breast cancer cell invasion. These results indicate that HAS2 is a critical component of Hs578T cells migratory and invasive behavior.
In this study, we show that upon silencing of the HAS2 gene, the invasive and malignant phenotype of the Hs578T breast cancer cell line is significantly decreased. The reduction of the aggressive characteristics of this cell line was manifested by a lower growth rate under both anchorage-dependent and anchorage-independent conditions, and suppression of the migratory and invasive phenotype. Interestingly, tumor Hs578T cells expressing low copy numbers of HAS2 mRNA showed increased expressions of HAS1 and HAS3, and HYAL1 genes, i.e. compensatory mechanisms, which led to a net reduction in hyaluronan production of only about 50%, but the hyaluronan chains produced were considerably smaller than those synthesized by HAS2 mRNA expressing cells. Notably, siRNA-mediated knock-down of HAS2 had no effect on HYAL2 mRNA expression (Fig. 2c), and only slightly decreased the CD44 protein levels, as well as its specific hyaluronan binding capacity (Fig. 4). In contrast, antisense HAS2 inhibition in the highly metastatic MDA-MB-231 cell line led to a significant suppression of CD44 and HYAL2 genes, but had no effect on the other HAS genes.16 Our findings, together with previous reports, suggest that HAS2 gene expression is important for maintenance of the malignant phenotype of breast cancer cells. Moreover, there appears to be a coordinated expression of genes encoding HAS and degrading enzymes, as well as CD44 hyaluronan receptors.
Quantification of HAS and HYAL mRNA in a panel of breast cancer cell lines demonstrated a correlation between HAS2 expression and aggressive breast tumor cell phenotype, whereas HAS1 and HYAL1 expression occurred in tumor cells of low invasive potential (Fig. 1b). Although HAS3 and HYAL2 mRNAs were expressed by all breast cancer cells investigated, independently of their degree of aggressiveness, they were most profoundly expressed by the noninvasive ZR-75-1 cell line. However, the relatively higher expression levels of HAS2 and HYAL2 mRNA, compared to other genes involved in hyaluronan turnover, are consistent with a dominant role of these 2 genes in the advancement of human breast carcinomas. Also Udabage et al.,38 observed that HAS2 and HYAL2 genes are implicated in breast tumor cell proliferation and invasiveness. In the present study, we clearly demonstrated a central role of HAS2 gene expression for the survival and proliferation of Hs578T tumor cells, since its silencing suppressed cellular proliferation, anchorage-independent growth and expression levels of positive regulator proteins of the cell cycle. In contrast, HAS2 overexpression promoted the expression of cell cycle regulators (Fig. 6b). Studies by us and other laboratories revealed that HAS2 is the most highly differentially regulated HAS isoform in response to growth factors.39, 40 A large number of studies have shown that hyaluronan promotes the growth of both normal and malignant cells. In particular, endogenous hyaluronan has been shown to regulate ErbB2 signaling and consequently it can promote embryonic morphogenesis or the malignant properties of tumor cells.10, 41, 42
siRNA-mediated knock-down of HAS2 caused lower hyaluronan production and suppression of cell growth and migration whereas addition of exogenous hyaluronan only partially restored cyclin B expression, and did not restore growth and migration of the Hs578T cells. We consider it unlikely that this lack of rescue is due to off-target effects of the siRNA used, since we obtained the same results using 2 different siRNA molecules targeting sequences from different parts of the HAS2 mRNA sequence. It is likely that exogenous addition of hyaluronan is not equivalent to endogenously produced hyaluronan, which possibly show a different cellular localization because of its interactions with the HAS molecule itself,43 and the intracellular hyaluronan binding proteins cdc3744 and RHAMM,45 interactions that might not be mimicked by exogenously added hyaluronan. Our results are consistent with those of other studies in which exogenously added hyaluronan did not rescue the growth of prostate cancer cells deprived of hyaluronan synthesis.15 Moreover, whereas endogenous hyaluronan has been found to promote the activation of several tyrosine kinase receptors including the PDGF β-receptor,46 we recently found that exogenous hyaluronan inhibits the PDGF β-receptor.47
Ample evidence support that there is an interplay between a tumor and its environment, which is important in tumor progression and aggressiveness. Our finding that HAS2 silencing in Hs578T breast cancer cells suppresses their motility and invasive potential to about half of that of HAS2 expressing cells (Fig. 7a, Table II) indicate that hyaluronan appears to be an integral component of breast carcinoma motility and invesiveness,48 which could be linked to lymph node and hematogenous distant metastases. However, it is possible that hyaluronan in the extracellular matrix function as a linker between CD44 expressing tumor cells and CD44 expressing vessel endothelial cells or lymphatic endothelial cells, which express the hyaluronan receptors CD44 and LYVE1, respectively,49, 50 but it could also function in a cell-surface independent manner by creating a malleable matrix that promote migration. Notably, the noninvasive behavior of ZR-75-1 and MCF-7 breast cancer cell lines tightly correlated with low CD44 expression and low hyaluronan production.5, 38 It should also be noted that siRNA-mediated knock-down of HAS2 caused about a 50% reduction of migration, so there are most likely also nonhyaluronan-mediated mechanisms involved.
Our findings in this study and those of Udabage et al.16, 38 predict that HAS2 has an important role in breast cancer progression, and that HAS2 cooperates with HYAL2 and the hyaluronan receptor CD44, suggesting that these molecules are critical for the aggressive character of breast cancer cells. Further studies are warranted on the molecular mechanisms that regulate the expression of HAS and HYAL isoforms, as well as hyaluronan receptors in breast carcinomas, to explore their usefulness as potential targets for tumor therapy.
The authors thank professor C.-H. Heldin for constructive criticism of this work.