Hyperproduction of hyaluronan in neu-induced mammary tumor accelerates angiogenesis through stromal cell recruitment: possible involvement of versican/PG-M

Abstract

Elevated concentrations of hyaluronan are often associated with human breast cancer malignancy. Here, we investigated the roles of hyaluronan in carcinogenesis and cancer progression using the mouse mammary tumor virus (MMTV)-Neu transgenic model of spontaneous breast cancer. Conditional transgenic mice that express murine hyaluronan synthase 2 (Has2) by Cre-mediated recombination were generated and crossed with the MMTV-Neu mice. In expressing Cre recombinase under the control of the MMTV promoter, the bigenic mice bearing Has2 and neu transgenes exhibited a deposition of hyaluronan matrix and aggressive growth of Neu-initiated mammary tumors. Notably, forced expression of Has2 impaired intercellular adhesion machinery and elicited cell survival signals in tumor cells. Concurrent with these alterations of tumor cells, intratumoral stroma and microvessels were markedly induced. To reveal the molecular basis of hyaluronan-mediated neovascularization, various hyaluronan samples were examined for their ability to potentiate in vivo angiogenesis. In Matrigel plug assays, basic fibroblast growth factor-induced neovascularization was elevated in the presence of either hyaluronan oligosaccharides or a hyaluronan aggregate containing versican. Administration of hyaluronan-versican aggregates, but not native hyaluronan alone, promoted stromal cell recruitment concurrently with the infiltration of endothelial cells. Taken together, these results suggest that hyaluronan overproduction accelerates tumor angiogenesis through stromal reaction, notably in the presence of versican.

Figure 1

Generation of Has2 conditional transgenic mice. A: Schematic of the transgenic construct. FLAG-tagged murine Has2 cDNA was positioned downstream of the transgene unit, including CAG promoter (CAG Pro), a loxP sequence, the Neo-resistance gene (Neo), the SV40 poly(A) signal (pA), and a second loxP sequence. On recognition of the loxP site, Cre recombinase deletes the Neo cassette along with one of the loxP sequences and then joins the CAG promoter and Has2 cDNA, leading to expression of Has2 mRNA. White, gray, and black triangles represent CAG promoter, PGK-Neo, and Has2-R7 primers, respectively. B: PCR analysis of Cre-mediated genomic DNA recombination. Genomic DNA samples were isolated from Neu-initiated mammary tumors. PCR screening (#1) with the CAG promoter and PGK-Neo primers gave the anticipated 360-bp DNA product (arrow) for the Has2^+Neo tumor, but not for the Has2^ΔNeo tumor. PCR screening (#2) with the CAG promoter and Has2-R7 primers gave the predicted 1880-bp DNA product (white arrowhead) for the Has2^+Neo tumor and 670-bp product (black arrowhead) for the Has2^ΔNeo tumor. This change in size of the amplified product demonstrated that deletion of the Neo cassette was successfully achieved by Cre-mediated transgene recombination in mammary tumors of Has2^ΔNeo mice. All PCR products were not amplified for the genomic DNA from Cre:Neu and Neu tumors. C: HA matrices of the tumor cells established from tumor tissues of Has2^ΔNeo and Has2^+Neo mice. Tumor cells were surgically isolated from Neu-initiated mammary tumors and cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS. HA matrices surrounding the tumor cells were visualized by the particle exclusion assay as described previously. The HA matrix occupies the clear area (arrowheads) between the fixed erythrocytes and the tumor cells.

Figure 2

Histopathological and immunohistochemical analyses of Neu-initiated mammary tumors. Tumor sections from the indicated genotypes were stained with hematoxylin and eosin. By detection using b-HABP, intense HA staining (red) was observed at the intercellular boundaries of tumor cells and particularly in the tumor stroma of Has2^ΔNeo (arrows). HA-rich matrix was also prominent in the perivascular elastic structure of angiogenic microvessels (arrowheads). An analogous stromal staining was observed for versican (green) as a HA-bound matrix component (arrows). In contrast, few deposition of HA and versican was observed in Has2^+Neo mice.

Figure 3

Decreased organization of intercellular junctions in Has2-overexpressing mammary tumors. Tissue sections from Has2^+Neo and Has2^ΔNeo tumors were immunostained with antibodies against E-cadherin (green) and β-catenin (red), and the cell nucleus were stained with DAPI (blue). The immunolocalization was visualized by fluorescent and confocal microscopy. Staining of E-cadherin and β-catenin at intracellular boundaries was observed in Has2^+Neo tumors (arrowheads).

Figure 4

Growth acceleration of Neu-initiated tumor by Has2 overexpression. A: Growth of mammary tumors in Has2 transgenic mice. After discovery of tumor, the length and width were measured daily with calipers until the tumors reached approximately 1 cm in diameter. The mean tumor volume (length × width²)/2 is shown as a function of elapsed time after first detection of the tumors of seven Has2^ΔNeo mice and eight controls (Has2^+Neo, Cre:Neu, and Neu). Data are presented as mean ± SE (*P < 0.05). B: The percentage of PCNA-positive nuclei was calculated with the formula [(number of PCNA⁺ nuclei)/(total nuclei) × 100]. n = 30 fields (five random fields of six tumors per genotype). The data point for each field is shown by an open circle. Closed circles and bars represent means ± SE (**P < 0.01). C: The percentage of TUNEL-positive nuclei was calculated by using the formula [(number of TUNEL⁺ nuclei)/(total nuclei) × 100]. n = 15 fields (five random fields of three tumors per genotype). The data point for each field is shown by an open circle. Closed circles and bars represent means ± SE (**P < 0.01).

Figure 5

Activation of cell survival signals by HA overproduction. A: Phosphorylation of Akt in Has2-overexpressing tumor. Tissue sections from Has2^+Neo and Has2^ΔNeo tumors were immunostained with anti-phospho-Akt, which recognizes the Ser473-phosphorylated epitope of Akt. HA was counterstained with b-HABP (green). Phosphorylation (red) of Akt was predominantly detected in Has2^ΔNeo tumor cells near the stroma-like structures (arrowhead) that are surrounded by HA-rich matrix. B: Tumor homogenates were analyzed by Western blotting using anti-Akt (non-phospho) and anti-phospho-Akt antibodies, and each band was quantified by densitometric imaging as described in Materials and Methods. Four independent tumors from each group were used for the comparison. Data represent the mean ± SE (*P < 0.05 versus control).

Figure 6

HA-induced recruitment of endothelial and stromal cells. A: CD31 immunostaining of tumor sections showing the vascular density in tumors. Tissue sections from Has2^+Neo and Has2^ΔNeo tumors were stained with a rat antibody against murine CD31. Tumor microvessels of smaller size were more numerous in the Has2^ΔNeo mice compared with Has2^+Neo tumors. Stromal reaction was demonstrated by immunostaining of type I collagen (Col I) and fibronectin (FN). B: Microvascular density in tumor sections from Has2^ΔNeo and control mice. The graph represents average number and the size distribution of the microvessels in five random (×200) fields of three tumors per genotype, showing that tumors had a significant increase in the number of microvessel with less than 1000 μm². Data represent the mean ± SE (*P < 0.05 versus control). C: Relative mRNA expression of angiogenesis-related genes in mammary tumor. Total RNA from Has2^ΔNeo and control tumors was transcribed into cDNA, and the transcriptional levels were analyzed by real-time quantitative PCR. All values are normalized to GAPDH mRNA. Data represent the mean ± SE from more than three different tumors of each group (*P < 0.05, **P < 0.01 versus control).

Figure 7

Effects of various HA samples on vascularization of Matrigel implants. A: Gel filtration chromatography of HA samples isolated from tumor tissues of Has2^ΔNeo and Has2^+Neo mice. HA-containing tumor homogenates were prepared as described in Materials and Methods and then separated by gel filtration chromatography using a Superose 6 HR 10/30 column. HA content was determined in each fraction from the column using the IBA method. Representative elution pattern is shown. B: The degree of angiogenesis in Matrigel plugs containing various HA samples. The Matrigel plug model of in vivo angiogenesis was conducted in C57BL/6 mice with the indicated treatments. The indicated concentration of HA oligosaccharides (M_r 6.8 kd, white column), rooster comb native HA (gray column), Streptococcus HA (hatched column), or human umbilical cord HA (black column) was mixed with the Matrigel and subcutaneously injected into the abdomen of mice. Matrigel plugs were removed 7 days after implantation, and the hemoglobin (Hb) concentration was quantified as described in Materials and Methods. Data represent the mean of two independent experiments. Dot blot detection of human versican in the umbilical cord HA sample was performed using 2B1 antibody specific for human versican (inset). C: Dot blot detection of HA and versican in mammary tumor extract (T) or in fractions (A1–A6) of CsCl density gradient ultracentrifugation. Tumor extracts from three different genotypic mice were fractionated into six fractions by CsCl density gradient ultracentrifugation under associative condition (0.4 mol/L guanidine hydrochloride) and examined for the sedimentation patterns of HA and versican. Significant amounts of HA and versican were recovered in the A1 fraction with lowest buoyant density. D: The degree of angiogenesis in Matrigel plugs containing rooster comb native HA and/or versican. Total RNA samples were isolated from Matrigel plugs supplemented with or without umbilical cord versican (V) and dermal versican (dV). The expression levels of CD31 mRNA were assessed as an index of angiogenesis by real-time quantitative PCR. The mRNA levels were normalized to GAPDH mRNA. Data represent the average from more than five different implants. Data represent the mean ± SE from more than five different implants (*P < 0.05 versus PBS control).

Figure 8

Infiltration of stroma cells into Matrigel plugs supplemented with or without native HA-versican aggregates (HA/V). A: Matrigel plugs were analyzed by hematoxylin and eosin staining or by immunostaining of CD31 of paraffin sections. Scale bars = 100 μm. B: Sections from the center of each embedded Matrigel plugs (three animals per group) were quantified for cellular invasion. Data represent the means ± SE (**P < 0.01). C: The sections of Matrigel plugs were immunostained with anti-human versican (2B1), α-SMA, or vimentin antibody. All specimens were counterstained with b-HABP. Scale bar = 20 μm.