Bombesin antagonists inhibit growth of MDA-MB-435 estrogen-independent breast cancers and decrease the expression of the ErbB-2/HER-2 oncoprotein and c-jun and c-fos oncogenes

Abstract

Previous studies showed that antagonists of bombesin (BN)/gastrin-releasing peptide (GRP) inhibit the growth of various cancers by interfering with the growth-stimulatory effects of BN-like peptides and down-regulating epidermal growth factor receptors on tumors. Because the overexpression of the human epidermal growth factor receptor-2 (ErbB-2/HER-2/neu) oncogene plays a role in the progression of many breast cancers, we investigated whether BN/GRP antagonists can affect HER-2 in mammary tumors. Female nude mice bearing orthotopic xenografts of MDA-MB-435 human estrogen-independent breast cancers were treated daily with BN/GRP antagonists RC-3095 (20 microg) or RC-3940-II (10 microg) for 6 weeks. The expression of BN/GRP receptors on tumors was analyzed by reverse transcription-PCR and immunoblotting. We also evaluated whether the mRNA expression for the c-jun and c-fos oncogenes is affected by the therapy. Both BN/GRP antagonists significantly inhibited growth of MDA-MB-435 cancers; RC-3095 reduced tumor volume by 40% and RC-3940-II by 65%. The GRP receptors (subtype 1) were detected in MDA-MB-435 tumors, showing that they mediate the inhibitory effect of the antagonists. Tumor inhibition was associated with a substantial reduction in the expression of mRNA and protein levels of the ErbB/HER receptor family as well as with a decrease in the expression of c-jun and c-fos oncogenes. BN/GRP antagonists RC-3940-II and RC-3095 could be considered for endocrine therapy of estrogen-independent breast cancers that express members of the ErbB/HER receptor family and the c-jun and c-fos oncogenes.

Figure 1

Changes in tumor volume of MDA-MB-435 human estrogen-independent breast carcinomas, implanted orthotopically into athymic female nude mice, during treatment with BN/GRP antagonists RC-3095 or RC-3940-II. The vertical bars show SE values. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

Figure 2

(A) Expression of mRNA for BRSs in MDA-MB-435 human breast carcinoma. Lanes 2–4, GRPR; lanes 6–8, NMBR; lanes 10–12, BRS-3; lanes 1, 5, and 9, positive controls for GRPR (PC3 human prostatic cancer cell line), NMBR (MDA-MB-468 human breast cancer cell line), and BRS-3 (H-69 human SCLC cell line), respectively (Upper). All preparations were normalized according to the expression of mRNA for β-actin (Lower). PCRs yielded products of the expected size of 158, 484, 375 and 459 bp for the GRPR, NMBR, BRS-3, and β-actin, respectively. (B) RT-PCR analysis of the expression of mRNA for GRPR and β-actin in MDA-MB-435 human estrogen-independent breast cancers. The lanes represent control tumors and those treated with 20 μg/day of RC-3095 or 10 μg/day of RC-3940-II as indicated. The sizes of the expected products are shown. Lane M, 100-bp molecular DNA marker.

Figure 3

Expression of mRNA for EGFR and HER-2, -3, and -4 in MDA-MB-435 human estrogen-independent breast cancers grown in nude mice after treatment with RC-3095 (20 μg/day) or RC-3940-II (10 μg/day). Total RNA (3 μg) was reverse-transcribed, and cDNA was amplified with specific primers. The PCR products were resolved on a 2% agarose ethidium-bromide gel. mRNA expression for β-actin was used as internal control. M, 100-bp molecular DNA marker. The sizes of the expected products are shown. mRNA levels were standardized according to β-actin mRNA levels and are expressed as a percentage of control value shown in Table 1. Results are the mean of at least two independent experiments.

Figure 4

Effect of BN antagonists RC-3095 (20 μg/day) and RC-3940-II (10 μg/day) on protein levels of EGFR and HER-2, -3, and -4 in MDA-MB-435 human estrogen-independent breast cancer grown in nude mice. Membranes were resolved and immunoblotted by using specific antisera (see Materials and Methods). Actin was used as internal control. The molecular masses of the proteins are indicated. Four representative samples from each group are shown. Protein levels were normalized to actin protein and are expressed as percentages of control values shown in Table 1. The data are the mean of at least three independent experiments.

Figure 5

Expression of mRNA for c-jun and c-fos oncogenes in MDA-MB-435 human estrogen-independent breast cancers grown in nude mice. (A) PCR products after electrophoresis in 2% agarose gel for control and groups treated with RC-3095 and RC-3940-II. The expression of β-actin was used as internal control. M, 100-bp molecular DNA marker. The size of the expected products is also shown. (B) the levels of c-jun and c-fos mRNA were standardized according to the levels of β-actin mRNA and are expressed as percentages of control values. The results are the mean of at least two independent experiments. The vertical bars show ± SE. *, P < 0.05 vs. control; **, P < 0.005 vs. control.