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Overcoming Tamoxifen Resistance of Human Breast Cancer by Targeted Gene Silencing Using Multifunctional pRNA Nanoparticles - PubMed

  • ️Sun Jan 01 2017

Overcoming Tamoxifen Resistance of Human Breast Cancer by Targeted Gene Silencing Using Multifunctional pRNA Nanoparticles

Yijuan Zhang et al. ACS Nano. 2017.

Abstract

Most breast cancers express estrogen receptor (ER) α, and the antiestrogen drug tamoxifen has been widely used for their treatment. Unfortunately, up to half of all ERα-positive tumors have intrinsic or acquired endocrine therapy resistance. Our recent studies revealed that the ER coactivator Mediator Subunit 1 (MED1) plays a critical role in tamoxifen resistance through cross-talk with HER2. Herein, we assembled a three-way junction (3-WJ) pRNA-HER2apt-siMED1 nanoparticle to target HER2-overexpressing human breast cancer via an HER2 RNA aptamer to silence MED1 expression. We found that these ultracompact RNA nanoparticles are very stable under RNase A, serum, and 8 M urea conditions. These nanoparticles specifically bound to HER2-overexpressing breast cancer cells, efficiently depleted MED1 expression, and significantly decreased ERα-mediated gene transcription, whereas point mutations of the HER2 RNA aptamer on these nanoparticles abolished such functions. The RNA nanoparticles not only reduced the growth, metastasis, and mammosphere formation of the HER2-overexpressing breast cancer cells but also sensitized them to tamoxifen treatment. These biosafe nanoparticles efficiently targeted and penetrated into HER2-overexpressing tumors after systemic administration in orthotopic xenograft mouse models. In addition to their ability to greatly inhibit tumor growth and metastasis, these nanoparticles also led to a dramatic reduction in the stem cell content of breast tumors when combined with tamoxifen treatment in vivo. Overall, we have generated multifunctional RNA nanoparticles that specifically targeted HER2-overexpressing human breast cancer, silenced MED1, and overcame tamoxifen resistance.

Keywords: HER2 RNA aptamer; MED1; breast cancer; pRNA of phi29 DNA packaging motor; tamoxifen resistance.

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Figures

Figure 1
Figure 1

Construction and characterization of pRNA–HER2apt–siMED1 nanoparticles. (A) Scheme of the pRNA–HER2apt–siMED1 (p-HER2-siMED1) structure. (B) p1 and p2 strands of pRNA–HER2apt–siMED1were transcribed using an in vitro RNA transcription system and separated in 8% denatured PAGE gel. (C) pRNA–HER2apt–siMED1 nanoparticles were generated by annealing equal molar of strands p1 and p2 and subjected to 8% native PAGE gel electrophoresis. (D) DLS assay of hydrodynamic size of pRNA–HER2apt–siMED1 nanoparticle. (E) Tm value of pRNA–HER2apt–siMED1 nanoparticle determined by TGGE assay. (F) Atomic force microscopy (AFM) images of pRNA–HER2apt–siMED1 nanoparticles. (G) Stability of control unmodified and 2′-F-modified pRNA nanoparticles was examined by 8% native PAGE gel electrophoresis after RNase A, 10% FBS-supplemented DMEM medium, and 8 M urea treatments for the indicated time at 37 °C.

Figure 2
Figure 2

pRNA–HER2apt–siMED1 nanoparticles specifically targeted BT474 cells in vitro and in vivo. (A) Confocal microscopy analyses of the internalization of AF647-labeled control and pRNA–HER2apt–siMED1 nanoparticles by BT474 cells. Scale bar: 10 μm. (B) Flow cytometry assays of the cellular uptake of AF647-labeled control and pRNA–HER2apt–siMED1 nanoparticles by BT474 cells. (C) IVIS Lumina live imaging of BT474 orthotopic xenograft mice 24 h after i.v. injection of indicated AF647-labeled pRNA nanoparticles (10 mg/kg). (D) Major organs and tumors of above mice were excised and imaged for AF647 fluorescence. (E) Frozen tumor sections were examined for localization of AF647-labeled pRNA nanoparticles (red) using confocal microscopy. The blood vessels were stained with anti-CD31 primary antibody and Alexa488-conjugated secondary antibody (green). The nuclei were stained with DAPI (blue). Scale bar: 50 μm. (F) The fluorescence intensity of AF647-labeled pRNA nanoparticles (red) in frozen tumor sections was quantified with Image-pro Plus software.

Figure 3
Figure 3

pRNA–HER2apt–siMED1 nanoparticles silenced MED1 expression and inhibited the cell growth and metastatic capabilities of HER2-positive breast cancer cells in vitro. (A) BT474 cells were incubated with 10 μg/mL control and pRNA–HER2apt–siMED1 nanoparticles for 48 h, and the MED1 mRNA level was determined by real-time PCR. (B) BT474 cells were incubated directly with (as indicated by –) or transfected with indicated pRNAs using lipofectamine 2000. At 48 h post treatment, MED1 protein levels were determined by Western blotting. (C–E) BT474 cells were treated with 10 μg/mL pRNA nanoparticles for 48 h and assayed for cell viability by MTT assay (C). Cells were treated as above and seeded for migration (D) and invasion (E) transwell assays. Scale bar: 50 μm. (F–H) After pRNA treatment for 48 h, the mRNA levels of ERα target genes TFF-1 (F), c-Myc (G), and cyclin D1 (H) in BT474 cells were determined by realtime PCR.

Figure 4
Figure 4

pRNA–HER2apt–siMED1 nanoparticles sensitized HER2-overexpressing BT474 cells to tamoxifen treatment. (A–C) BT474 cells were treated with pRNA nanoparticles in combination with vehicle or 1 μM 4-hydroxytamoxifen (4-OHT) as indicated for 48 h and assayed for cell viability by MTT assay (A). Cells were treated as above, and the migration (B) and invasion (C) capabilities were examined. (D–F) Cells were treated as above and assayed for in vitro mammosphere formation (D). The number of mammospheres was counted (E), and the mammosphere size was calculated (F). Scale bar: 10 μm.

Figure 5
Figure 5

pRNA–HER2apt–siMED1 nanoparticles inhibited HER2-overexpressing breast tumor growth in vivo. (A) BT474 orthotopic xenograft mouse models were treated with control pRNA–HER2apt–siScram or pRNA–HER2apt–siMED1 (4 mg/kg) once a week, in combination with vehicle or tamoxifen (TAM, 0.5 mg/mice/day) 5 days per week. Tumor sizes were measured every 3 days. (B) After 3 weeks, mice were i.p. injected with

d

-luciferin, and representative in vivo images of BT474 tumors were recorded using IVIS Lumina imaging system. (C and D) Average weight of the tumors excised at the end of the treatment (C) and the representative photos of tumors (D). (E-G) MED1 expression in the BT474 tumors was examined using IHC staining (E and F) and immunoblotting (G). (H and I) The expression of Ki-67 in tumor tissues was analyzed using IHC staining (H), and the percentage of Ki-67 positive cells was counted (I). Scale bar: 100 μm.

Figure 6
Figure 6

pRNA–HER2apt–siMED1 in combination with tamoxifen greatly impaired breast cancer lung metastasis, stem cell formation, and associated gene expression in vivo. (A and B) Whole lung tissues were fixed, embedded, and sectioned for H&E staining (A), and metastasis foci in the whole lung tissues were then counted (B). Red arrow indicated the metastasis foci. Scale bar: 100 μm. (C and D) After digesting tumor tissues with trypsin and 0.1% collagenase, tumor cells were stained for CD44 and CD24 and analyzed for CD44+CD24−/low stem cell population using flow cytometry. (E−H) Total RNA in the tumor tissues was extracted using TRIZOL reagent, and the expression of TFF-1 (E), c-Myc (F), cyclin D1 (G), and MMP-9 (H) was determined by real-time PCR.

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