Targeting surface nucleolin with a multivalent pseudopeptide delays development of spontaneous melanoma in RET transgenic mice

Abstract

Background: The importance of cell-surface nucleolin in cancer biology was recently highlighted by studies showing that ligands of nucleolin play critical role in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, we recently reported that HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in the athymic nude mice without apparent toxicity.

Methods: The in vivo antitumoral action of HB-19 treatment was assessed on the spontaneous development of melanoma in the RET transgenic mouse model. Ten days old RET mice were treated with HB-19 in a prophylactic setting that extended 300 days. In parallel, the molecular basis for the action of HB-19 was investigated on a melanoma cell line (called TIII) derived from a cutaneous nodule of a RET mouse.

Results: HB-19 treatment of RET mice caused a significant delay in the onset of cutaneous tumors, several-months delay in the incidence of large tumors, a lower frequency of cutaneous nodules, and a reduction of visceral metastatic nodules while displaying no toxicity to normal tissue. Moreover, microvessel density was significantly reduced in tumors recovered from HB-19 treated mice compared to corresponding controls. Studies on the melanoma-derived tumor cells demonstrated that HB-19 treatment of TIII cells could restore contact inhibition, impair anchorage-independent growth, and reduce their tumorigenic potential in mice. Moreover, HB-19 treatment caused selective down regulation of transcripts coding matrix metalloproteinase 2 and 9, and tumor necrosis factor-alpha in the TIII cells and in melanoma tumors of RET mice.

Conclusions: Although HB-19 treatment failed to prevent the development of spontaneous melanoma in the RET mice, it delayed for several months the onset and frequency of cutaneous tumors, and exerted a significant inhibitory effect on visceral metastasis. Consequently, HB-19 could provide a novel therapeutic agent by itself or as an adjuvant therapy in association with current therapeutic interventions on a virulent cancer like melanoma.

Figure 1

HB-19 inhibits significantly spontaneous melanoma development. RET mice were treated with HB-19 according to the schedule indicated in Methods and diagnosed frequently over a period of 300 days. A/B. HB-19 treatment delays significantly the onset of small cutaneous tumors (≥ 4 mm²) in panel A and of huge cutaneous tumors (> 60 mm²) in panel B (Log-rank Wilcoxon test, p < 0.001 for both graphs). The dotted lines correspond to data from untreated mice (n = 11) and full lines correspond to HB-19 treated mice (n = 9). C/D. HB-19 treatment significantly delays the incidence of facial (C) and dorsal (D) cutaneous nodules in RET mice. The dotted lines correspond to data from untreated mice and full lines correspond to HB-19 treated mice (Log-rank Wilcoxon test, p < 0.001). E/F. Number of facial (E) and dorsal (F) cutaneous nodules during the period of 120 days. The histograms show the progression of the number of nodules (calculated for 10 mice) in untreated (hatched bars) and HB-19 treated (black bars) mice, respectively. HB-19 treated mice displayed less cutaneous nodules than untreated mice (p < 0.05 for both graphs).

Figure 2

Reduced density of blood vessels in tumors recovered from HB-19 treated RET mice. Sections of tumors from control untreated (panel A; localized under the ear) and HB-19 treated (panel B; localized under the eye) RET mice were stained with antibodies against the CD34 endothelial marker and analyzed by fluorescence microscopy (Methods). Representative macroscopic images (magnification 200×) from each group of sections are presented. C. Quantification of angiogenesis was estimated by image analysis of CD34-labeled endothelial cells in tumor sections from control and HB-19 treated RET mice. Statistical significance: * 0.01 < p < 0.1.

Figure 3

Restoration of growth contact-inhibition in HB-19 cultured TIII cells. TIII cells passaged 12-times in the absence or presence of 5 μM of HB-19 were fixed in PFA-Triton, and intracellular actin filaments were stained with FITC-conjugated phalloidin. Immunofluorescence (A) and confocal (B) microscopy was as described in Methods.

Figure 4

HB-19 affects the malignant phenotype of melanoma TIII cells. A. Reduced colony formation in soft agar. TIII cells were cultured for twelve passages in the absence (control, histogram C) or presence of 1, 5 and 10 μM of HB-19 before assay for colony formation in the absence of further HB-19 treatment (***p < 0.001). B. Reduced tumorigenicity in mice. Tumor size was measured fourteen days after transplanting control (open circles, n = 14) or HB-19 precultured (black circles, n = 15) TIII cells. The graph corresponds to the results of two separate experiments. The mice transplanted with HB-19 precultured cells displayed tumors smaller than those treated with control cells (ANOVA, Mann Whitney test, p = 0.0276). C. Reduced lung metastasis in mice. Mice injected in the tail vein with TIII cells were treated (closed circles, n = 8) or not (open circles, n = 10) with HB-19, and the number of black lung macro-metastases was determined (ANOVA, Mann Whitney test, p = 0.0014).

Figure 5

HB-19 treatment of TIII cells results in the reduction of transcripts coding MMP-2, MMP-9 and TNF-α. A. Expression of transcripts 24 hours after HB-19 treatment. Two days after seeding, TIII cells were cultured for 24 hours in the absence (Control) or presence of 10 or 25 μM of HB-19. B. Expression of transcripts in control and HB-19 cultured TIII cells (cultured as described in Figure 3). C. Expression of transcripts in control and HB-19 precultured TIII cells (as in section B) that were further passaged 7-times in the absence of HB-19. The expression of specific mRNAs was investigated by RT-PCR using primers for MMP-2, MMP-9, VEGF-A, TNF-α, STAT-1, MIA and GAPDH.

Figure 6

Reduced expression of transcripts coding MMP-2, MMP-9 and TNF-α in melanoma tumors recovered from HB-19 treated RET mice. The expression of transcripts coding MMP-2, MMP-9, VEGF-A, TNF-α, STAT-1, MIA and GAPDH was investigated RT-PCR in tumors located at the eye, ear and intraperitoneum of control and HB-19 treated RET mice (as in Figure 5). The eye, ear and intraperitoneal tumors are representative for the firstly diagnosed nodule, a cutaneous nodule and a metastatic nodule, respectively. Equivalent tumor masses at similar locations were considered for comparison.