A thermally targeted c-Myc inhibitory polypeptide inhibits breast tumor growth

Abstract

Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. We have developed a thermally responsive polypeptide, based on the sequence of Elastin-like polypeptide (ELP), that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. Following systemic administration, the ELP - delivered c-Myc inhibitory peptide was targeted to tumors using focused hyperthermia, and significantly reduced tumor growth in an orthotopic mouse model of breast cancer. This work provides a new modality for targeted delivery of a specific oncogene inhibitory peptide, and this strategy may be expanded for delivery of other therapeutic peptides or small molecule drugs.

Fig. 1

Heating tumors with infrared light. Tumor temperature (as monitored by a needle thermocouple in the tumor core), body temperature, and skin temperature over the heated site was recorded while illuminating the tumor with 950 nm light from an LED light source. Data represent the mean of three mice bearing 250 mm³ E0771 mammary tumors, bars, s.d.

Fig. 2

Plasma stability, plasma clearance and tumor uptake of Bac-ELP-H1. (A) Stability of the rhodamine label on Bac-ELP-H1-Rho was determined by incubation in fresh mouse plasma at 37 °C, thermal precipitation of the labeled polypeptide, and spectrophotometric detection of released label in the remaining plasma. Bars, s.d. (B) Plasma levels with time following IV or IP injection of Bac-ELP1-H1-Rho. Data represent the mean ± s.d. of 6 animals per group. (C) Representative images of tumor sections 3 h after IV or IP injection of rhodamine-labeled Bac-ELP1-H1. The perfused vasculature was marked by infusion of high molecular weight dextran 1 min prior to euthanasia (top panel), and rhodamine fluorescence was used to follow the localization of the polypeptide within the tumor (middle panel). (D) Bac-ELP1-H1-Rho tumor levels 3 h after IV or IP administration of rhodamine-labeled Bac-ELP1-H1 with and without tumor hyperthermia. Bars, s.e. (E) Bac-ELP1-H1-Rho and Bac-ELP2-H1-Rho tumor levels 3 h after IV administration of polypeptides with or without hyperthermia. Bars, s.e. (F) High magnification images of the tumors in C were obtained using a fluorescence microscope and a 40 × objective. Cell nuclei were stained with Hoechst 33342.

Fig. 3

Biodistribution of Bac-ELP-H1 Following IV or IP Injection. Organ distribution of Bac-ELP1-H1-Rho 3 h after IV or IP administration with or without tumor heating. Bars, s.e. *, Organ levels are statistically different, (one-way ANOVA, p = 0.0006 (liver), p = 0.005, (spleen), post hoc Bonferroni, 95% CI). ** Brain levels were not detectable over background fluorescence following IV injection.

Fig. 4

In vivo tumor deposition of Bac-ELP-H1 with time. (A) Representative fluorescence images 8 h after injection with saline (left panel) or AlexaFluor 750-labeled Bac-ELP1-H1 (right panels). A mouse with no tumor (second panel), an unheated tumor (third panel), and a heated tumor (fourth panel) are shown. The position of the tumor or the corresponding non-tumor bearing mammary fat pad are shown by the ovals. (B) Quantitative analysis of the tumor fluorescence at each time point. Data represent the mean ± s.e. of 5 mice/group. *, The difference between heated and unheated tumors is statistically significant (p = 0.007, student’s t-test).

Fig. 5

Tumor Volume Reduction by Bac-ELP-H1. E0771 Tumor volume (A and C) and corresponding body weight (B and D) after 7 daily treatments (arrows) with saline, saline + hyperthermia, Bac-ELP1, Bac-ELP1-H1, Bac-ELP1-H1 + hyperthermia (A and B), Bac-ELP2-H1, and Bac-ELP2-H1 + hyperthermia (C and D). Data represent the mean ± s.d. of 6 mice/group. *, Tumor volumes are statistically significant from control (one-way ANOVA, p = 0.0017, post hoc Bonferroni, 95% CI). (E) Tumor mass on day 14. *, Tumor mass is statistically significant from control (one-way ANOVA, p = 0.0008, post hoc Bonferroni, 95% CI).