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. 2014 Jan 1;6(217):217ra2.
doi: 10.1126/scitranslmed.3007048.

Silencing HoxA1 by Intraductal Injection of siRNA Lipidoid Nanoparticles Prevents Mammary Tumor Progression in Mice

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Free PMC article

Silencing HoxA1 by Intraductal Injection of siRNA Lipidoid Nanoparticles Prevents Mammary Tumor Progression in Mice

Amy Brock et al. Sci Transl Med. .
Free PMC article

Abstract

With advances in screening, the incidence of detection of premalignant breast lesions has increased in recent decades; however, treatment options remain limited to surveillance or surgical removal by lumpectomy or mastectomy. We hypothesized that disease progression could be blocked by RNA interference (RNAi) therapy and set out to develop a targeted therapeutic delivery strategy. Using computational gene network modeling, we identified HoxA1 as a putative driver of early mammary cancer progression in transgenic C3(1)-SV40TAg mice. Silencing this gene in cultured mouse or human mammary tumor spheroids resulted in increased acinar lumen formation, reduced tumor cell proliferation, and restoration of normal epithelial polarization. When the HoxA1 gene was silenced in vivo via intraductal delivery of nanoparticle-formulated small interfering RNA (siRNA) through the nipple of transgenic mice with early-stage disease, mammary epithelial cell proliferation rates were suppressed, loss of estrogen and progesterone receptor expression was prevented, and tumor incidence was reduced by 75%. This approach that leverages new advances in systems biology and nanotechnology offers a novel noninvasive strategy to block breast cancer progression through targeted silencing of critical genes directly within the mammary epithelium.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Gene network inference pipeline
In the first phase, the MNI algorithm was trained on a compendium of 3000 mouse microarray data sets to construct a basal network connectivity model. Genome-wide expression data from wild type and 8 week-old transgenic mammary glands were then interrogated using the GRN model to pinpoint alterations in gene behavior that were unique to particular stages of tumorigenesis. The significance of the predicted key mediator genes of the disease stage of interest is quantified with a z-score. Two-tailed p-values are calculated based on the z-score value. Genes are ranked according to the p-value, and top-ranked genes are selected as probable key mediators of the disease state.
Figure 2
Figure 2. In vitro silencing of HoxA1 normalizes mouse mammary tumor cell spheroid cultures, restoring polarity and reducing proliferative index
(A) Disease progression in the C3(1)-SV40TAg glands is marked by atypia and hyperplasia, ductal filling and tumor invasion (H&E) with a progressive loss of GM130 (green) and laminin (red) polarity by 12 weeks and basement membrane thinning by 16 weeks. Scale bars, 100 μm. (B) Confocal images through the center of carmine-labeled normal mouse mammary (Eph4) and mouse mammary tumor (M6 and M6C) spheroid cultures reveal filled lumens in tumor cell cultures and a hollow organized lumen morphology when HoxA1 is silenced. Cell polarity was visualized by immunocytochemistry of sectioned spheroids with staining of an apical Golgi marker GM130 and the basal basement membrane protein, laminin. Nuclei are stained with DAPI (blue) and F-actin is labeled with phalloidin (green). Scale bars, 20 μm. Images representative of n = 3 studies, 150–300 spheroids imaged per sample) Downregulation of HoxA1 protein expression was confirmed by immunoblotting in lysates prepared from M6 and M6C cells (n=3). (C and D) The effects of silencing HoxA1 on hollow lumen formation (C) and cell proliferation (D) in collagen-Matrigel spheroid cultures of mouse M6 and M6C tumor cells. Eph4 mouse mammary cells served as normal controls. Data are means +/− SD (n = 3, 150–300 spheroids imaged per sample; *p < 0.05, **p<0.01, Fisher’s exact t-test). (E) Quantitation of apoptosis by TUNEL staining after treatment of M6 and M6C cells with HoxA1 siRNA (n=3, 150–300 spheroids imaged per sample) *p<0.05, Fisher’s exact t-test.
Figure 3
Figure 3. Silencing HoxA1 in human breast cancer cells reduces cell growth and restores lumen organization in tumor cell spheroid cultures
(A) The Oncomine compendium of cancer transcriptome profiles was used for analysis and visualization of HoxA1 expression data in human breast tumors and normal breast tissue. Differential expression analysis identified 10 patient datasets in which HoxA1 was over-expressed in human breast lesions by greater than 2-fold (see references 26–31). The type of tumor represented in the datasets is categorized as D, ductal breast carcinomas; L, lobular breast carcinomas; A; all breast carcinomas. The rank order of HoxA1 in each analysis is displayed (red, increased rank %; blue, decreased rank %). (B) Confocal images through the center of carmine-labeled spheroid cultures of normal human breast epithelial cells (MCF10A) and human breast cancer MD-MBA-468 and HCC1937 cell lines reveal. Breast cancer cells were also treated withsiHoxA1. Scale bar, 20 μm. (C) Hollow lumen formation and (D) proliferation of cancer cells in collagen-Matrigel spheroid cultures. Quantitation of lumen formation in non-tumorigenic human MCF10A breast epithelial cells and cells treated with scrambled siRNA controls are shown for comparison. Data are means +/− SD (n = 3, 150–300 cells per sample) **p<0.01, Fisher’s exact t-test.
Figure 4
Figure 4. Silencing HoxA1 in the mammary epithelium reduces tumor incidence, reduces proliferative index, and prevents the loss of hormone receptor expression
(A) Virgin C3(1)-SV40TAg mammary glands injected in parallel with 20 μl of a fluorescent control siRNA-nanoparticle to visualize the efficiency of its delivery throughout the ductal tree. The entire injected gland was removed and whole mounted for fluorescent imaging. Scale bar, 1 mm. (B) Confocal images of mammary tissue sections show distribution of fluorescent control siRNA (red) in the duct at 2, 7, and 14 days post-injection. Tissues were counterstained with laminin (green) and DAPI (blue). Bar= 100 μm. (C) Tumor incidence measured at 21 weeks in control C3(1)-SV40TAg mice versus mice injected with siHoxA1 bi-weekly for 9 weeks (n=10 siNT, n=8 siHoxA1, n=5 untreated). (D) Timing of onset of tumor formation in untreated, siNT control, and siHoxA1 treated mice. (E) H&E and F) immunohistochemistry for PR, ER, and PCNA in mammary glands. Scale bar, 20 μm. (G, H, I) Percentage of PCNA+, ER+, PR+ cells after 9 weeks of treatment are shown in Data are mean +/− SEM, n=5 animals, 400–550 cells scored per animal, **p<0.01, ***p< 0.001, Pearson’s chi-square test. (For each sample, data).
Figure 5
Figure 5. siHoxA1 reduces mammary tumor cell proliferation in vivo through modulation of p44/42 MAPK signaling
(A) Microarray analysis of MAPK pathway signature genes in untreated transgenic C3(1)-SV40TAg mammary glands (n = 3) at progressive disease stages. (B) The effect of bi-weekly siHoxA1 injections on protein expression of MAPK components, ERK1/2, SRC, IER3, KRAS, PCNA and GRB2, in mammary glands of 21-week old transgenic animals (n = 3) compared with injection of non-targeting control siRNA (siNT) (n = 3).

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