Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer.

Chromosome 17q23 amplification occurs in ~11% of human breast cancers. Enriched in HER2+ breast cancers, the 17q23 amplification is significantly correlated with poor clinical outcomes. In addition to the previously identified oncogene WIP1, we uncover an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing 17q23 amplicons. The 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 selectively inhibits the proliferation, survival and tumorigenic potential of the HER2+ breast cancer cells harboring 17q23 amplification. To overcome the resistance of trastuzumab-based therapies in vivo, we develop pH-sensitive nanoparticles for specific co-delivery of the WIP1 and miR-21 inhibitors into HER2+ breast tumors, leading to a profound reduction of tumor growth. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the trastuzumab-resistant HER2+ breast cancers.

Introduction

Genomic studies have identified distinct breast cancer subtypes with differences in survival and response to therapy, including luminal A, luminal B, basal-like, HER2+ and Claudin-low subtypes[1,2]. HER2 overexpression leads to aggressive cancer phenotype and poor patient survival. Trastuzumab, a humanized HER2 antibody, shows considerable clinical efficacy and extends the overall survival of patients with HER2+ breast cancer. However, the overall response rate to trastuzumab-containing therapies needs to be improved[3-5]. Multiple trastuzumab-resistance mechanisms have been identified in preclinical studies, in which constitutive activation of the PI3K pathway owing to PTEN deficiency or PIK3CA mutations seems to be one of the most prevalent events[6-8]. The better understanding of breast cancer biology has been translated into the development of new anti-HER2 agents with varying mechanisms of action[9,10]. The small molecular tyrosine kinase inhibitor lapatinib has demonstrated activity in HER2+ metastatic breast cancer and in the preoperative setting[11,12]. Pertuzumab, a monoclonal antibody with a distinct binding site from trastuzumab, inhibits receptor dimerization[13,14]. The addition of pertuzumab to combination therapy has led to improved progression-free survival in patients with HER2+ metastatic breast cancer and higher response rates in the preoperative setting[14].

To maintain genome stability, eukaryotic cells have evolved with the ability to detect and translate the initial signals of DNA damage to proper cellular responses. The key components of the DNA damage response (DDR) include ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related), and DNA-PKcs (DNA dependent protein kinase catalytic subunit)[15]. These kinases initiate DNA damage signaling cascades and activate cell cycle checkpoints and DNA repair[16,17]. Advanced tumors often show inactivation of DDR markers, suggesting that silencing of the DDR is an important prerequisite for cancer progression. The wildtype p53-induced phosphatase 1 (WIP1, also known as PPM1D) functions as a homeostatic regulator and a master inhibitor of the DDR. It is a type 2C serine/threonine phosphatase that is induced in response to DNA damage in a p53-dependent manner. Previous studies demonstrated that WIP1 dephosphorylates multiple key proteins in the DDR, such as CHK1, CHK2, p53, MDM2, and H2AX[18]. Importantly, WIP1 suppresses p53 by multiple mechanisms, including dephosphorylation of p53 kinases (CHK1, CHK2), p53 itself, and MDM2.

Consistent with its oncogenic functions, the WIP1 gene in the 17q23 chromosome region is amplified and overexpressed in 11–18% of human breast cancer[18,19]. The WIP1-null mice are resistant to spontaneous and oncogene-induced tumors due to enhanced DNA damage and p53 responses[20,21]. While previous studies ruled out the possibility of any protein-coding oncogenes in the WIP1-containing 17q23 amplicon, we identified an oncogenic miRNA gene, MIR21, in almost all the WIP1 amplicons. Moreover, a majority of the WIP1/MIR21-amplified cancer samples have concurrent HER2 amplification. Here we showed that the chromosome 17q23 amplification in the HER2+ breast cancer results in aberrant elevation of WIP1 and miR-21, which not only contributes to breast tumorigenesis, but also causes resistance to anti-HER2 therapy. Therefore, targeted inhibition of WIP1 and miR-21 could be an effective therapeutic approach for trastuzumab-resistant HER2+ breast cancer.

In this study, we propose to develop a therapy for trastuzumab-resistant HER2+ breast cancer with the combined use of a small molecular inhibitor against WIP1 (GSK2830371), anti-miR-21 oligonucleotides (antagomiR21), and trastuzumab. However, GSK2830371 has poor solubility in water with poor bioavailability in vivo[22]. Although antagomiR21 is highly soluble in water, it is relatively unstable in blood. Furthermore, neither GSK2830371 nor antagomiR21 can efficiently enter cells by itself. To address these challenges, we developed a nanoparticle system using biocompatible polymers, to co-encapsulate GSK2830371 and antagomiR21 for targeted co-delivery into HER2+ breast tumor. A major hurdle to nanoparticle-based delivery of RNAs (including antagomiR21) is that the RNAs can be easily degraded by the many RNases in endo/lysosomes after cell uptake of the RNA-laden nanoparticle by endocytosis[23]. This challenge can be overcome with our nanoparticle because it has the nanobomb effect when exposed to the low pH in endo/lysosomes to break up the endo/lysosomes. As a result, the antagomiR21 can escape the endo/lysosomes into the cytosol (where it performs its function) before degradation. We show that the combined treatment with WIP1 and miR-21 inhibitors using the nanoparticle significantly reduces trastuzumab-resistant HER2+ tumor growth, confirming that co-inhibition of WIP1 and miR-21 is a promising therapeutic strategy.

RESULTS

Co-amplification of WIP1 and MIR21 in the 17q23 amplicon

The WIP1-containing 17q23 region is amplified in a subset (~11%) of human breast tumors[24]. Extensive analyses of breast cancer genomics revealed that the 17q23 amplicon can span up to over 10 Mb, including a number of protein-coding and non-coding genes. Early studies identified WIP1 as the only oncogene in the amplicon due to incomplete breast cancer genomic databases and lack of noncoding RNA information[19,24]. To search for other potential driver oncogenes, the 17q23 amplicon was analyzed using TCGA (The Cancer Genomics Atlas) breast cancer databases[25,26]. Our results revealed that the amplicon region focuses on an approximately 2.73 Mb region (Fig. 1a), where 21 protein-coding genes (MED13, INTS2, BRIP1, NACA2, TBX4, C17ORF82, TBX2, BCAS3, APPBP2, C17ORF64, USP32, CA4, HEATR6, RNFT1, RPS6KB1, TUBD1, VMP1, PTRH2, CLTC, DHX40, YPEL2) and 3 miRNA genes (MIR4737, MIR21, MIR4729) showed highly similar amplification profiles to WIP1 (Fig. 1b). Transgenic mice overexpressing WIP1 in mammary glands showed no abnormal overt phenotype and did not develop spontaneous mammary tumors[27]. However, their mammary tumor incidence was accelerated in the presence of the ErbB2 (the mouse homolog of HER2) transgene, suggesting that WIP1 plays an important role in HER2-initiated breast cancer. Interestingly, we found that the WIP1 amplification was enriched in human HER2+ breast cancer (Fig. 1c). While only 10% of all types of breast cancer in the dataset were identified as HER2+, the HER2+ subtype existed in 30% (3-fold more, p = 3.6e−13) of the WIP1-amplified breast cancers, compared to 7% of breast cancers without WIP1 amplification (Fig. 1c). We found that 14 out of 24 genes in the amplicon were significantly (p < 0.001) overexpressed with the gene amplification. WIP1, as well as MIR21, a well characterized oncomiR, were both overexpressed, similar to the aberration of HER2 (ERBB2) (Fig. 1d and Supplementary Fig. 1a–c). Consistently, the expression levels of WIP1 and miR-21 were also significantly upregulated in ErbB2-transgenic mice when compared to their normal littermates, although depletion of WIP1, miR-21 or both only had minimal effect on the ErbB2 expression (Supplementary Fig. 1d–h). Furthermore, we assessed the transformation ability of the 14 genes that are overexpressed in the 17q23-amplified breast cancers (Fig. 1e and Supplementary Fig. 2). Soft-agar colony formation assays were performed by transducing primary mammary epithelial cells from MMTV-ErbB2 transgenic mice with lentivirus expressing each individual gene. These results demonstrated that both WIP1 and MIR21, but not any other genes in the amplicon, induced in vitro transformation, indicating their functional roles in HER2+ breast cancers.

Fig. 1

Association between gene amplification, gene expression and HER2+ breast cancer on chromosome 17. a Heatmap of genomic segment copy-number abnormalities (log-ratio measurements) of human chromosome 17 in 1,080 breast invasive carcinomas. Positive log-ratios indicate degree of copy number gain (red) whereas negative values mark the loss (blue). b Co-amplification of WIP1 and genes from 17q22 to 17q23. c HER2+ subtype significantly enriched in breast cancers harboring WIP1-contating 17q23 amplicon. The TCGA dataset was downloaded from Xena Public Data Hubs (see Methods for details). d Correlation between gene expression aberration and copy number variation for genes, WIP1, MIR21, and HER2 (ERBB2) in breast tumors. e Soft agar colony formation assays with MMTV-ErbB2 mouse mammary epithelial cells transduced with control vector (Ctrl) or lentiviral vector expressing the indicated genes. **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test was used (d, e). Data are presented as mean ± SD and are representative of 3 independent experiments

Depletion of miR-21 and WIP1 inhibits mammary tumorigenesis

Both WIP1−/− and MIR21−/− mice are known to have normal mammary gland development and functions[21,24,28], suggesting that WIP1 and miR-21 are dispensable for developmental and physiological functions. To study their potential oncogenic roles, mammary tumorigenesis of MMTV-ErbB2 transgenic mice were examined in the contexts of WIP1 or MIR21 knockout (Fig. 2a). All the females in the experimental groups were kept virgin during the 20-month observation period. All the MMTV-ErbB2 transgenic female mice (12 out of 12) died of mammary tumors before the end of observation period. We found that knockout of WIP1 impaired the ErbB2-induced mammary tumorigenesis, consistent with previous studies[20,21]. Similar to the WIP1 knockout, depletion of miR-21 rendered the MMTV-ErbB2 mice considerably more resistant to tumor formation. Only 6 out of 13 MIR21-/-;MMTV-ErbB2 females developed mammary tumors (p < 0.001). Overall, WIP1−/−;MMTV-ErbB2 and MIR21−/−;MMTV-ErbB2 females showed a significant increase in lifespan in comparison with their control MMTV-ErbB2 littermates. In line with these mouse studies, analysis of human breast cancer genomics using TCGA databases showed that copy number gains of WIP1 or MIR21 are significantly correlated with poor clinical outcomes in patients with HER2+ breast cancer, but not in patients with luminal A, luminal B or basal-like breast cancer (Fig. 2b). We next assessed whether inhibiting WIP1 or miR-21 impact the proliferation and mammosphere formation of the mouse tumor cell line H605, which was isolated and established from a mouse MMTV-ErbB2 mammary tumor[29]. Either WIP1 or miR-21 knockdown markedly reduced the tumor cell growth rate, whereas knockdown of both had a more profound inhibition of cell proliferation (Fig. 2c and Supplementary Fig. 3a, b). We also found that depletion of WIP1 or miR-21 significantly diminished the number and size of mammospheres formed by H605 cells, and knockdown of both WIP1 and miR-21 further inhibited the mammosphere formation (Fig. 2d). In addition, depletion of WIP1, miR-21 or both promoted G1/S phase arrest and apoptosis (Supplementary Fig. 3c, d). These results validated the oncogenic potential of both WIP1 and MIR21 genes.

Fig. 2

Suppression of miR-21 and WIP1 inhibits proliferation and tumorigenic potential of HER2+ breast cancer cells. a Kaplan–Meier analysis of tumor-free survival in female MMTV-ErbB2 wild type (n = 12), MMTV-ErbB2; WIP1 (n = 12) and MMTV-ErbB2; MIR21 (n = 13) mice. b Amplification of MIR21 and WIP1 is associated with poor overall survival in patients with HER2+ breast cancer, but not with patients with luminal A, luminal B or basal-like breast cancer. c Cell growth curve of H605 cells (MMTV-ErbB2 tumor cells) expressing doxycline (Dox)-inducible control shRNA or specific shRNA targeting WIP1 or mature miR-21. d Mammosphere formation assay in H605 cells expressing Dox-inducible control shRNA, specific shRNA targeting WIP1 or mature miR-21. Right panel demonstrates the quantitative data using Image J software. Scale bar, 500 µm. *p < 0.05; ***p < 0.001**; ***p < 0.001; Log-rank Mantel–Cox test (a, b) and one-way ANOVA followed by Tukey’s t test (c, d) were used. Data are presented as mean ± SD and are representative of 3 independent experiments

Oncogene-induced senescence (OIS) in mammary glands is a protective mechanism against breast tumorigenesis[30]. Accumulating evidence supported an important role of the ATM-p53 signaling pathway in OIS[31]. However, recent study pointed out that TGF-β signaling pathway is responsible for the ATM-independent OIS in mammary glands[32]. While WIP1 is a master inhibitor for the ATM-p53 signaling, we found that miR-21 may suppress the TGF-β signaling. To identify physiologically relevant miR-21 targets, we analyzed genome-wide gene expression profiles in mammary epithelial cells (MMECs) isolated from MIR21-/-;MMTV-ErbB2 female mice and their wildtype littermates. Gene transcripts were identified as potential miR-21 targets in MMECs, which are most significantly increased in the MIR21-/- cells and modulate crucial tumor cell activities (Fig. 3a and Supplementary Fig. 4). There is significant overlap between in silico predicted miR-21 target genes and those genes up-regulated in the MIR21−/− MMECs (Supplementary Fig. 4a). Interestingly, a number of miR-21 targets are associated with the TGF-β signaling pathway, including MAP2K3, BMPR1B, INHBA, MAP2K4, MAPK1, MAPK8, NRAS, SMAD2, SMURF2 and SOS1 (Fig. 3a). TGF-β/SMAD signaling pathways are among the most enriched pathways regulated by miR-21 (Supplementary Fig. 4b). We performed individual gene expression analyses to validate these target genes in MIR21+/+ and MIR21−/− mouse mammary glands. Mouse mammary glands were harvested from 8-week littermate females and the purified mRNAs were subjected to reverse transcription and quantitative PCR analyses (Fig. 3b). While WIP1 plays a primary role in the DDR and DNA repair, no significant overlaps were identified between WIP1 and miR-21 targets, suggesting that these two oncogenes play non-redundant roles in mammary tumorigenesis. In addition, MMECs, isolated from MMTV-ErbB2 transgenic mouse, were passaged and examined for OIS. The MMECs, after three passages, exhibited an increase in cell size, cell spreading, vacuolization, multinucleated morphology and positive staining for senescence-associated β-galactosidase (SA-β-Gal) activity (Fig. 3c). Ectopic expression of WIP1 or miR-21 alone partly rescued MMECs from senescence, indicated by reduced number of cells positive for SA-β-Gal. Co-expression of WIP1 and miR-21 had a profound inhibition on the OIS of the MMECs overexpressing ErbB2, suggesting that amplification of WIP1 and miR-21 may allow MMECs to overcome OIS in oncogenic transformation.

Fig. 3

Overexpression of miR-21 and WIP1 promotes oncogenic transformation of HER2+ breast cancer cells. a The differential expression heat map of miR-21 target genes in the mammary epithelial cells (MMECs) derived from MIR21−/−;MMTV-ErbB2 mice (8 weeks old). b Relative expression levels of the predicted miR-21 targets in primary MMECs isolated from wild-type or MIR21-/- virgin females at the age of 8 weeks. Data represents the mean expression levels normalized to the endogenous snoRNA55 control from three independent experiments. c MMECs derived from MMTV-ErbB2 mice were transduced with lentivirus expressing miR-21 and/or WIP1. Top panel: representative photomicrographs of SA-β-galactosidase (SA-β-Gal) staining observed in bright field. Bottom panel: miR-21 and WIP1 expression levels as determined by q-PCR, and percentages of SA-β-Gal-positive cells were calculated. Scale bar, 10 µm. *p < 0.05; **p < 0.01; unpaired 2-tailed t-test was used (b, c). Data are presented as mean ± SD and are representative of 3 independent experiments

Co-amplified DDX5 facilitates pri-miR-21 maturation

While no miRNA-sequence specificity exists for Drosha and DGCR8, two core components, in the microprocessor, emerging evidence has shown that regulatory RNA-binding proteins in the Drosha complexes may recruit specific primary miRNAs (pri-miRs) for processing[33]. We performed a MS2-TRAP (MS2-tagged RNA affinity purification) assay to identify a pri-miR-21-specific regulatory component in the Drosha microprocessor[34]. The assay is based on the addition of a specific MS2 RNA hairpin loop sequence (from bacteriophage MS2) to pri-miR-21, followed by co-expression of the MS2-tagged RNA together with GST-tagged MS2P that specifically binds the MS2 RNA sequence. In addition to those predicted proteins (Drosha and DGCR8) in the microprocessor complex, DEAD-Box helicase 5 (DDX5) was identified in the pri-miR-21-protein complex (Supplementary Fig. 5a). Interaction of DDX5 with Drosha was further confirmed in the immunoprecipitation and western blotting assay (Fig. 4a). DDX1, identified in our previously study[35], was used as a positive control for the interaction with Drosha. RNA immunoprecipitation (RIP) assays further verified specific interaction between DDX5 and pri-miR-21 (Fig. 4b). As a positive control, pri-miR-16 was reported as a DDX5-interacting RNA[36]. Pri-miR-200a was used as a negative control that had no interaction with DDX5[35]. Knockdown of DDX5 inhibited the processing of pri-miR-21 and resulted in accumulation of unprocessed pri-miR-21 and decreased levels of mature miR-21 in three breast cancer cell lines harboring 17q23 amplicon (Fig. 4c and Supplementary Fig. 5b). Interestingly, genomic analysis of HER2+ breast cancer revealed that DDX5 is adjacent to the WIP1-MIR21 amplicon and is co-amplified in 67% of HER2+ breast cancer with MIR21 amplification. Consistent with the analysis of miR-21 and DDX5 mRNA expression levels using TCGA datasets (Supplementary Fig. 5c), DDX5 protein levels are positively correlated with miR-21 levels, determined by immunohistochemistry staining of HER2+ breast tumor tissue microarray (Fig. 4d and Supplementary Fig. 5d). Using colony formation assay, we found that overexpression of DDX5 significantly promoted anchorage-independent cell growth compared to the control, which was abrogated by silencing of miR-21, but not miR-16 (Fig. 4e). Collectively, DDX5 is potentially an important player that promotes the oncogenic function of the 17q23 amplicon via facilitating miR-21 maturation.

Fig. 4

DDX5 gene is co-amplified with MIR21 and DDX5 facilitates maturation of pri-miR-21. a Immunoprecipitation (IP) and western blotting analyses were performed using indicated antibodies. Normal immunoglobulin G (IgG) was used as a negative control for IP. The RNA-binding protein DDX1 was used as a positive control for the Drosha-binding proteins. b The DDX5-bound pri-miRNAs were immunoprecipitated with DDX5 and analyzed by qRT-PCR. Control IgG was used as a negative control. c Levels of primary or mature forms of miR-21 were analyzed in control and DDX5-knockdown breast cancer cells harboring 17q23 amplicon. d Levels of mature miR-21 and DDX5 were analyzed in breast tumor samples using in situ hybridization and immunohistochemistry. Representative staining images of tissue samples are shown. Scale bar, 100 μm. e Soft agar colony formation assays with MMTV-ErbB2 mouse mammary epithelial cells transduced with control vector or lentiviral vector expressing the indicated genes. Representative images are shown in the middle panel and relative expression levels of mature miR-16 or miR-21 are shown in the right panel. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test (b, c and e). Data are presented as mean ± SD and are representative of 3 independent experiments

Inhibition of WIP1 and miR-21 kills HER2+ breast cancer cells

WIP1 is a major inhibitor for the activity of p53[37]. Thus, inhibiting WIP1 may impact the survival of HER2+ breast cancer cells in a p53-dependent manner. HER18 cells bearing wildtype p53 were highly sensitive to the treatment of the WIP1 inhibitor GSK2830371, which markedly increased the levels of phosphorylated CHK2 and p21 (Fig. 5a). However, both of the HER2+ breast cancer cell lines with mutant p53 (BT474 and MDA-MB453) were insensitive to the inhibition of WIP1 (Fig. 5b, c), although they also have 17q23 amplicon. By contrast, treatment of antagomiR21 significantly reduced the cell proliferation and viability of these HER2+/p53-mutant cells (Fig. 5d, e). Furthermore, inhibition of miR-21 dramatically sensitized HER18 cells to the treatment of the WIP1 inhibitor (Fig. 5d). These results suggest that inhibiting miR-21 is a potentially therapeutic approach to target breast cancers harboring 17q23 amplification regardless of p53 mutations.

Fig. 5

Inhibition of miR-21 and WIP1 kills HER2+ breast cancer cells harboring 17q23 amplicon. a–c HER18 (a), BT-474 (b) or MDA-MB-453 (c) cells were incubated with or without the WIP1 inhibitor GSK2830371 at the indicated concentrations for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. Protein levels are shown at the bottom of each panel by immunoblotting. d, e HER18 (d) or BT-474 (e) cells with or without Dox-induced miR-21 knockdown were incubated indicated concentrations of GSK2830371 for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD (left panels). The cell lysates were subjected to Western blot analyses with the indicated antibodies (middle panels). Knockdown efficiency of miR-21 was measured by luciferase reporter assay (right panels). *p < 0.05; **p < 0.01; unpaired 2-tailed t-test was used (d, e). Data are presented as mean ± SD and are representative of 3 independent experiments

Multiple trastuzumab resistance mechanisms have been identified, in which activation of the PI3K pathway seems to be the most prevalent. In line with the previous report concerning the function of miR-21 in negative regulation of PTEN[38], we also found that miR-21 directly inhibited PTEN in MMECs (Fig. 3a, b). Hence we reasoned that inhibiting miR-21 may sensitize HER2+ breast cancer cells to the treatment of trastuzumab. Trastuzumab-resistant cell lines were generated from parental BT-474 and HER18 lines via the treatment of escalating doses up to 15 µg ml−1 of trastuzumab (Fig. 6a, b). The half maximal inhibitory concentration (IC50) was increased by ~10-fold in both resistant cell lines. Treatment of trastuzumab inhibited cell proliferation (increased p27 levels) and AKT signaling pathway (reduced pAKT levels) in parental BT-474 and HER18 cells, which were not observed in the resistant cells (BT-474R and HER18R) (Fig. 6a, b). Knockdown of miR-21 notably inhibited the AKT signaling as expected and suppressed cell proliferation. Moreover, we observed that miR-21 depletion markedly sensitized both BT-474R and HER18R cells to the treatment of trastuzumab, indicating the p53-independent activity of miR-21 inhibition. By contrast, knockdown of WIP1 had a similar effect on the resistant cells expressing wildtype p53 (HER18R), but not on the p53-mutant BT-474R cells (Fig. 6c, d). To test whether inhibiting WIP1 and miR-21 had synergistic effects, HER18R cells were treated with the WIP1 inhibitor, antagomiR21 or both inhibitors, along with a low concentration of trastuzumab (1 µg ml−1) (Fig. 6e). While they were insensitive to trastuzumab as single agent, HER18R cells were sensitized by miR21 inhibitor or WIP1 inhibitor alone, but the effects were drastically magnified by treatment with both inhibitors.

Fig. 6

Inhibition of miR-21 and WIP1 sensitizes HER2+ breast cancer cells to the treatment of trastuzumab. a, b Parental or trastuzumab-resistant HER2+ breast cancer cells (HER18 or BT-474) were incubated with trastuzumab at the indicated concentrations for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. The cell lysates were subjected to western blot analyses with the indicated antibodies (right panels). c, d HER18R (c) or BT-474R (d) cells with or without Dox-induced miR-21 knockdown were incubated with indicated concentrations of GSK2830371 for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. The cell lysates were subjected to western blot analyses with the indicated antibodies (right panels). e HER18R cells with or without Dox-induced miR-21 knockdown were incubated with GSK2830371 (0.2 µM) and/or trastuzumab (1 µg ml−1) for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. f, g Gross tumor images (f) and tumor growth curves (g) of xenograft tumors derived from orthotopically implanted parental or trastuzumab-resistant HER18 cells expressing Dox-inducible WIP1 or DDX5 shRNA or anti-miR21 oligonucleotides with trastuzumab treatment (5 mg kg−1, twice per week). *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test (c–e) and one-way ANOVA followed by Tukey’s t test (g) were used. Data are presented as mean ± SD and are representative of 3 independent experiments in a–e

To further test the suppressive effect of WIP1 and miR-21 inhibition on breast tumor growth in vivo, HER18R cells expressing doxycycline (Dox)-inducible control, WIP1, miR-21 or DDX5 short-hairpin RNAs (shRNAs) (>70% knockdown efficiency) were injected to mouse mammary fat pads to establish xenograft breast tumor models in female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Knockdown efficiency of WIP1, miR-21, and DDX5 shRNAs was confirmed in the HER18R cells (Supplementary Fig. 6a, b). Once tumors were established at three weeks after transplantation, the tumor-bearing mice were treated with 5 mg kg−1 of trastuzumab. While the HER18R tumors were resistant to the trastuzumab treatment as expected, in comparison with the trastuzumab sensitivity of the HER18 tumors. Depletion of miR-21 or WIP1 markedly decreased the growth of xenograft tumors (Fig. 6f, g and Supplementary Fig. 6c, d), and their dual depletion led to more severe tumor growth inhibition, indicating a synergistic role of miR-21 and WIP1 in the trastuzumab resistance of HER2+ breast cancer. Notably, inhibition of DDX5 also exhibited modest effect on the inhibition of HER18R tumor growth, supporting its role in promoting miR-21 expression and other documented oncogenic functions[39].

Nanoparticles for co-delivery of miR-21 and WIP1 inhibitors

The nanoparticle was synthesized using four biocompatible materials (three polymers and one phospholipid) approved by the U.S. Food and Drug Administration (FDA) for medical use (Fig. 7a): Poly(d,l-lactide-co-glycolide) (PLGA), Pluronic F127 (PF127), chitosan, and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC). To generate the nanobomb effect, chitosan was modified with guanidine group to form chitosan-guanidine (CG)[40]. The guanidine group can react reversibly with carbon dioxide (CO2) to form chitosan-guanidinium (CG-CO2) in a pH-dependent manner (Fig. 7a)[41], which may be utilized to capture/store CO2 at neutral pH and release at acidic pH. To synthesize the nanoparticle, the aqueous solution of CG was bubbled with CO2 gas to form CG-CO2 and then mixed with antagomiR21. The aqueous mixture was emulsified with organic solvent or oil (dichloromethane: tetrahydrofuran 40:1) containing DPPC, PLGA, and GSK2830371 to form a water-in-oil structure, where the CG-CO2 and oligonucleotides were encapsulated in the aqueous core while PLGA, DPPC, and GSK2830371 were in the surrounding oil. Next, the first emulsion was emulsified with the aqueous solution of PF127-Chitosan (P127 modified with chitosan[42], which served as the stabilizer during the second emulsion). The organic solvent was then evaporated to produce the two agents (inhibitors of miR-21 and WIP1)-laden nanoparticles (in-MW@NP).

Fig. 7

Synthesis of nanopariticles for drug delivery. a Nanoparticles encapsulating therapeutic agent(s) were synthesized using a double-emulsion water-in-oil-in-water method. The inner water phase containing anti-miR21 inhibitor and CG-CO2 were encapsulated in the core, and hydrophobic WIP1 inhibitor (GSK2830371) together with PLGA and DPPC dissolved in oil phase was used to form the shell structure. b TEM images of nanoparticles in pH 7.4, 6.0, and 5.0, respectively. Nanoparticles maintained spherical morphology with clear core-shell structure in pH 7.4, and enlarged and broken under low pH (pH 6.0 and pH 5.0) indicated the pH-responsive behavior. c Electrophoretic stability assay of in-MW@NP and free anti-miR21 inhibitor at different incubation time points in serum at 37 °C. The observable signal of in-MW@NP indicated that the nanoparticle encapsulation could protect anti-miR21 inhibitor from degradation in serum up to 36 h. d Typical confocal imaging of cells incubated with Dex-Rho@NP for 1–6 h. The change of the fluorescence overlap of Dex-Rho (red) and Lysotracker (green) shows the CO2-associated pH-responsive endo/lysosomal escape in HER18R cells. Cell nuclei were stained with DAPI (blue), endo/lysosomal vesicles were stained with LysoTracker Green, and DIC represented differential interference contrast. Data are representative of 3 independent experiments

The resulting nanoparticles have a smooth spherical morphology and core-shell structure with diameter of 115 ± 9.7 nm at pH 7.4 (Fig. 7b). At pH 6.0, defect (dark spots) can be seen on the shell of some nanoparticles. Under more acidic pH 4.0, extensive defects can be seen in most of the nanoparticles and they forms large aggregates owing to the low pH-activated nanobomb effect of the nanoparticles (Fig. 7b and Supplementary Fig. 7a, b). More specifically, the CG-CO2 encapsulated in in-MW@NP can generate/release CO2 gas at the reduced pH to break up the nanoparticles, and the severely damaged nanoparticles may tangle together to form large aggregates.

The stability of antagomiR21 encapsulated in the nanoparticles was examined by incubating it in serum at 37 °C (Fig. 7c). The oligonucleotide encapsulated in in-MW@NP was observable even after 36 h of incubation, while the free (non-encapsulated) oligonucleotide started to degrade in a few minutes and disappeared in less than 2 h. By contrast, there was no notable degradation within at least 6 h when the free oligonucleotides were incubated with RNase-free buffer (Supplementary Fig. 7c). A detergent (1% alconox, w/v) was used to treat the NPs as a positive control for the release of antagomiR21. After 6 h incubation, there was a sharp band of free antagomiR21 observed, indicating the release of antagomiR21 from nanoparticles (Supplementary Fig. 7c). These results suggest that the nanoparticle could protect the antagomiR21 from degradation by serum RNases at neutral pH. To study the low pH-activated nanobomb effect of in-MW@NP, Dextran (molecular weight of 10 KDa, similar to that of the antagomiR21) labeled with Rhodamine (Dex-Rho) was used in the nanoparticle (DexRho@NP). After incubating the nanoparticles with trastuzumb-resistant HER18R breast cancer cells for 1–6 h, the intracellular distribution of Rhodamine (red fluorescence) was examined against lysotracker (green fluorescence) that stains the endo/lysosomes. The red and green fluorescence largely overlapped after incubating for 1 and 3 h (Fig. 7d), indicating that the cells take up the nanoparticles mainly by endocytosis. However, their overlap became minimal at 6 h, indicating successful escape of Dex-Rho from endo/lysosomes, probably due to the low pH-activated nanobomb effect to break open the endo/lysosomes. By contrast, cellular uptake of free Dex-Rho was minimal even after 6 h of incubation and it largely localized to endo/lysosomes (Fig. 7d). Collectively, it is clear that the CG-CO2 encapsulated in the nanoparticle can disrupt the endo/lysosomes under low pH by generating CO2, which greatly enhance the cytosolic delivery of antagomiR21.

Anti-tumor activity of co-delivered miR-21 and WIP1 inhibitors

We next examined if the miR-21 and WIP1 inhibitors-laden nanoparticles are effective in killing trastuzumb-resistant breast cancer cells. First, we investigated the anticancer capacity of the drug-laden nanoparticles in vitro (Fig. 8a). HER18R cells were treated with free WIP1/miR-21 inhibitors, blank nanoparticles, and various amounts of in-MW@NP for a total of 72 h and the cell viability was measured. The blank nanoparticles were not detrimental to the cells and did not impact the cell growth, suggesting their minimal cytotoxicity. Although the aggregation of NPs was found in the low pH buffer due to the CO2 generation and NPs disruption (Fig. 7b and Supplementary Fig. 7a), nonspecific effects on cellular functions were not observed because the CO2 generation of the NPs, unlike in the buffer solution, only occurs when they are in the endo/lysosomes. In this case, the NPs aggregation should be minimal in the cell. The drug-laden nanoparticles were significantly more cytotoxic to the trastuzumab-resistant HER18R cells than free WIP1 or miR-21 inhibitors due to the enhanced drug delivery and release of the nanoparticles.

Fig. 8

In vivo efficacy of nanoparticle-encapsulated WIP1 and miR-21 inhibitors in trastuzumab-resistant breast tumor models. a Relative cell viability of trastuzumab-resistant HER18R treated with indicated doses of nanoparticles encapsulating WIP1 or miR-21 inhibitors for 72 h. b In vivo whole animal imaging of ICG fluorescence at 1 h, 6 h, and 24 h, respectively, after intravenous injection of saline, free ICG and NP-ICG nanoparticles. Ex vivo imaging of ICG fluorescence of tumor together with five important organs collected when the mice were sacrificed at 24 h post-injection. The arrows indicate the locations of tumors in mice. c–e Gross tumor images (c), tumor growth curves (d) and tumor weight (e) of xenograft tumors derived from orthotopically implanted trastuzumab-resistant HER18R cells. Once tumors were palpable, mice were randomly divided to 4 groups and then treated with either control, WIP1 (5 mg kg−1) and/or miR-21 (1 mg kg−1) inhibitor nanoparticles (twice per week) intravenously. f, g Quantification of cell proliferation (Ki-67 staining, f) and apoptosis (cleaved caspase-3 staining, g) in the abovementioned xenografted tumor tissues. **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test was used e–g. Data are presented as mean ± SD

We next investigated the tumor-targeting capability of the nanoparticles in mice. Indocyanine green (ICG) was encapsulated in the nanoparticles for in vivo imaging. Highly enhanced fluorescence of ICG was observable in tumor at 6 h after intravenous injection of the nanoparticles, compared to free ICG (Fig. 8b). We further checked the fluorescence of ICG at 24 h, the ICG-laden nanoparticles were exclusively localized in the tumor, while no notable signal was seen for free ICG. Various organs were harvested for ex vivo imaging to check the biodistribution of ICG after sacrificing the mice at 24 h. Indeed, only tumors from the nanoparticles treated groups showed strong fluorescence of ICG, in comparison with the tumors from the saline or free ICG-treated mice. As expected, weak fluorescence was observed in the liver from mice treated with free ICG or ICG-laden nanoparticles probably due to minimal nonspecific uptake and retention. The stronger signal present in the liver in the ex vivo than in vivo probably due to the penetration depth of fluorescence. We further investigated the anti-tumor efficacy and safety of the WIP1 and/or miR-21 inhibitor-laden nanoparticles in vivo by treating orthotopic HER18R or BT-474R mammary tumor-bearing mice. When tumor reached a volume of ∼50 mm3, the tumor-bearing mice were divided randomly into four groups: blank nanoparticles, WIP1 inhibitor-laden nanoparticles, antagomiR21-laden nanoparticles, and WIP1 + miR-21 inhibitors-laden nanoparticles. Mice were treated with antagomiR21 (1.0 mg kg–1) and/or WIP1 inhibitor (5.0 mg kg–1) encapsulated in the nanoparticles via intravenous injection twice weekly. Inhibiting WIP1 or miR-21 by their inhibitor-laden nanoparticles both significantly inhibited the mammary tumor growth with ~60% of reduction in tumor volumes and ~50% of reduction in tumor weights (Fig. 8c–e). For the HER18R tumor bearing wild type p53, dual inhibition of WIP1 and miR-21 exhibited the best anti-tumor capacity with over 95% of tumor growth inhibition when compared with the tumor from the control group (Fig. 8c–e). However, for the BT-474 tumor harboring mutant p53, inhibition of miR-21 significantly impaired mammary tumor growth although WIP1 inhibitor only had a modest effect (Supplementary Fig. 8a–c). In addition, we also analyzed the effect of WIP1 or miR-21 inhibition on tumor metastasis by intravenously injecting HER18R cells into female nude mice. Knockdown of WIP1 or miR-21 alone showed significant reduction in lung metastasis, whereas inhibition of both had a more profound reduction of lung nodules (Supplementary Fig. 8d–f), suggesting that inhibiting WIP1 and miR-21 is a promising therapeutic approach for both primary and metastatic tumors. Inhibiting miR-21 and WIP1 resulted in profound apoptosis in the HER18R tumor cells, as well as significant suppression of cancer cell proliferation (Fig. 8f, g and Supplementary Fig. 8g). Importantly, we did not notice any evident side effect of the drug-laden or blank nanoparticles. Neither death nor significant drop of body weight was noted for the mice treated with saline, blank nanoparticles, and all the three drug formulations (WIP1 inhibitor, miR-21 inhibitor, and WIP1/miR-21 inhibitors) (Supplementary Fig. 8h), suggesting the excellent safety of the nanoparticles for targeted delivery of WIP1 and miR-21 inhibitors in vivo.

Discussion

There is substantial evidence to support the oncogenic properties of WIP1, but much less is known regarding the clinical significance of WIP1 aberrations in human cancers. Although the WIP1 amplification is closely correlated with poor clinical outcome, the WIP1 transgene itself fails to promote mammary tumorigenesis in mice[22,43]. Here, in-depth analysis of the human 17q23 amplicon revealed that MIR21 is the other potential oncogene that may cooperate with WIP1 in mammary tumor initiation and progression. This finding is important because WIP1 and miR-21 deactivate two major tumor suppression pathways: p53 and PTEN pathways, respectively. We also made a clinically significant finding that the WIP1/MIR21 amplification is enriched in HER2+ breast cancer, suggesting that WIP1/miR-21 aberrations cooperate with HER2 in tumors with poor prognosis.

Aberrant expression of WIP1 and miR-21 may promote breast tumorigenesis by inhibiting OIS in mammary epithelial cells. OIS is a key anti-cancer barrier at the early stage of tumorigenesis, which involves the ATM-p53 and TGF-β signaling pathways in mammary glands. While the importance of the ATM-p53 signaling has been extensively studied, recent evidence shows that the TGF-β signaling is responsible for the ATM-independent OIS in mammary glands[32]. Although WIP1 is a master inhibitor in the ATM-p53 signaling, overexpression of WIP1 in transgenic mice does not induce mammary tumorigenesis, suggesting that intact TGF-β signaling needs to be overcome for breast cancer initiation. We found that miR-21 is probably a potent inhibitor of the TGF-β pathway by suppressing a number of key components in the pathway. Our results suggest that WIP1 and miR-21 cooperatively inhibit these two pathways to override OIS and promote mammary tumorigenesis.

Post-transcriptional processing of pri-miRNAs is an essential step in miRNA biogenesis. While Drosha and DGCR8 are the core components in the microprocessor, neither of them has binding specificity for individual pri-miRNAs[44-46]. We identified DDX5 as a miR-21-specific regulator in the Drosha microprocessor. Thus, co-amplification of DDX5 with MIR21 facilitates the efficient processing of pri-miR-21 transcripts and results in elevated levels of miR-21. This hypothesis is also supported by the positive correlation between DDX5 copy numbers and miR-21 levels in mammary tumor tissues. In addition to its role in miRNA processing, DDX5 is an essential gene in the development as DDX5 knockout mice are embryonically lethal[47]. By its interaction with mRNA, DDX5 is also involved in the processing, splicing and degradation of mRNA. Mazurek and colleagues reported that DDX5 directly regulates DNA replication factor expression by promoting the recruitment of RNA polymerase II to E2F-regulated gene promoters[39]. DDX5 was suggested as a promising candidate for targeted therapy of breast tumors with DDX5 amplification. In line with their study, we showed that inhibiting DDX5 suppressed the growth of HER2+ tumors in vivo, suggesting that DDX5 is likely one of the important regulators for mammary tumors induced by 17q23 amplification.

Although RNA interference (RNAi) has shown as a promising therapeutic strategy for cancer, few RNAi-based therapies have passed/entered Phase II/III clinical trial[48-50]. This is partly because naked RNAs have poor stability in blood, do not enter cells, and are instable in the endo/lysosome inside cells[23]. In addition, the small molecule drugs for conventional chemotherapy is either insoluble in water or can diffuse to both normal tissue and tumor, which may induce significant side effects[51]. Nanotechnology have demonstrated great potential for overcoming the challenges facing conventional chemo/RNAi therapy[51-53]. In this study, we designed and synthesized a unique nanoparticle to encapsulate both WIP1 and miR-21 inhibitors for combination therapy. It not only can improve the solubility and bioavailability of GSK2830371 for inhibiting WIP1 but also achieve cytosolic delivery of antagomiR21. The nanoparticle could stabilize antagomiR21 by preventing it from the enzymatic degradation during circulation and preferentially targets tumor. After entering tumor, it could enhance cellular uptake of the encapsulated agents. More importantly, after being taken up by cancer cells via endocytosis, the nanoparticle could generate carbon dioxide gas to break open endo/lysosomes. Moreover, our nanoparticle-based approach ensures that the dose ratio of the two agents in the tumor can be maintained to be the same as that at injection, while the dose ratio of the two agents in tumor may be very different from that at injection due to vast difference in bioavailability of the hydrophobic GSK2830371 and hydrophilic antagomiR21. This well-designed nanoparticle is an excellent vehicle for delivering the antagomiR21 (and other RNAs) to overcome both the extracellular and intracellular barrier to the use of RNAs for cancer therapy.

In summary, we identified that co-amplification of MIR21 and WIP1 in HER2+ breast cancer harboring 17q23 amplicon generates therapeutic vulnerabilities and provides an effective treatment strategy for breast cancers containing such genomic events. We demonstrated that miR-21 and WIP1 functionally cooperate with the HER2 gene in breast tumorigenesis, and that inhibiting them circumvents resistance to anti-HER2 therapies. It is anticipated that development of nanoparticles encapsulating miR21 and WIP1 inhibitors will facilitate this promising therapeutic approach in treating trastuzumab-resistant breast cancers.

Methods

TCGA analysis

The TCGA breast cancer data were downloaded from the website: https://xenabrowser.net/datapages/?cohort = TCGA%20Breast%20Cancer%20(BRCA), which include copy number variation (CNV) at segment level in log-ratio, CNV at gene level estimated by using the GISTIC2 algorithm, miRNA mature strand expression data in logarithm (base-2), and patient information about HER2 positive or negative. To study the amplification (Amp) similarity between other genes and WIP1, we focused on only patients with WIP1 amplification. The ratio of WIP1-Amp patients with Amp on the gene as well was calculated to represent such similarity between the gene and WIP1.

Tissue culture

MCF-7, MDA-MB453, BT474, HMC18, MDA-MB231, MCF10A cell lines were purchased from the American Type Culture Collection. HER18 cells (stably overexpress HER2 and were derived from MCF-7) were a gift from Dr. Mong-Hong Lee (MD Anderson Cancer Center). These cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS at 37°C in 5% CO2. MCF10A cells were maintained in DMEM/F12 supplemented with 5% Horse Serum, epidermal growth factor (20 ng ml−1), Hydrocortisone (0.5 mg ml−1), Cholera Toxin (100 ng ml−1) and Insulin (10 μg ml−1). BT474 and HER18 cells were grown and selected in 10 ug ml−1 Trastuzumab for several weeks and defined as trastuzumab-resistant cells (HER18R, BT474R). Cell lines were banked in multiple aliquots on receipt to reduce risk of phenotypic drift and were tested negative for mycoplasma contamination using the Mycoplasma detection kit (Lonza). Cell identity was confirmed by validating the STR DNA fingerprinting using the AmpFLSTR Identifiler Kit according to the manufacturer’s instructions (Applied Biosystems).

Antibodies and reagents

Anti-WIP1 antibody (A300-664A, 1:500), anti-DDX1 (A300-521A, 1:2000), anti-Drosha (A301-886A, 1:2000) and anti-DDX5 (A300-523A, 1:2000) were purchased from Bethyl Laboratories. Anti-HER2 antibody (18299-1-AP, 1:2000) was purchased from Proteintech. Anti-p21 (sc-397, 1:1000), anti-WIP1 (sc-376257, 1:500), anti-GAPDH (sc-20357, 1:5000), anti-Actin (sc-1616, 1:5000), HRP-anti-goat IgG (#2020, 1:5000), HRP-anti-rabbit IgG (#2054, 1:5000) and HRP-anti-mouse IgG (#2055, 1:5000) antibodies were purchased from Santa Cruz. Anti-AKT (#4691, 1:2000), anti-phospho-AKT (S473, #9018, 1:2000), anti-Chk2 (#2662 S, 1:2000), anti-phospho-Chk2 (#2661 S, 1:2000), anti-Ki-67 (#9449, 1:500) and anti-cleaved caspase3 (#9661 S, 1:200) were purchased from Cell Signaling. Trastuzumab (Herceptin, Genentech) and GSK-2830371 (Active Biochem) were used to target HER2 and WIP1 in vitro and in vivo. Antisense miR-21 miRZip (System Bioscience) expressing constructs were used to inhibit mature miR-21 function in vitro and in vivo (Dox-induced stable knockdown as shown in Fig. 6). To evaluate and measure mature miR-21 activity in the cells, pmirGLO Dual Luciferase miR-21 vector was used in vitro. mirVana miR-21 inhibitor (Ambion, Life Technology) was used for in vivo miR-21 inhibition with nanoparticles. PLGA (lactide: glycolide = 75: 25, MW: 4,000-15,000), PF127, and organic solvents were purchased from Sigma (St. Louis, MO, USA). Agarose, ethidium bromide and loading buffer were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). DPPC was purchased Anatrace (Maumee, OH, USA). Chitosan oligosaccharide of pharmaceutical grade (MW: 1.2 KDa, 95% deacetylation) was purchased from Zhejiang Golden Schell Biochemical Co. Ltd (Zhejiang, China). Methyl aminomethanimidothioate hydroiodide was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Generation of doxycycline-inducible knocked-down cell lines

Lentiviral pGIPZ vector expressing non-specific silencing shRNA control, WIP1 and DDX5 shRNAs were obtained from the MD Anderson shRNA and ORFome Core Facility (originally from Open Biosystems) and sub-cloned to pTRIPZ vector according to the manufacturer’s instructions. To generate Dox-inducible anti-sense miR-21 miRZip expressing cell lines, anti-miR21 expression cassette was sub-cloned to pLKO-Tet-On vector. Cells were infected with lentiviruses in the presence of polybrene (8 μg ml−1). To establish stable knockdown cell line, lentiviral shRNA-transduced cells were selected with puromycin (2 μg ml−1) 48 h post-infection and individual colonies were propagated and validated for expression by Western blotting (protein) and qRT-PCR (mRNA).

Genomic DNA isolation and copy number validation

Total genomic DNA was extracted from human cell lines using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s purification instructions. The copy number validation for HER2, WIP1, and MIR21 were determined by quantitative PCR assays using iTaq Universal SYBR Green Supermix (Bio-Rad) on an Applied Biosystems 7900HT Sequence Detection System.

Immunoblotting

Immunoblotting was performed as described previously[54]. Briefly, total cell lysates were solubilized in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 5 mM sodium vanadate, 1 μg each of aprotinin, leupeptin, and pepstatin per ml). Proteins were resolved by SDS-PAGE gels and then proteins were transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk for 1 h at room temperature prior to incubation with indicated primary antibodies. Subsequently membranes were washed and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Following several washes, chemiluminescent images of immunodetected bands on the membranes were recorded on X-ray films using the enhanced chemiluminescence (ECL) system (Perkinelmer) according to the manufacturer’s instructions. Uncropped scans of the western blots are presented in the Supplementary Fig. 9.

Cell viability assay

Equal numbers of cells were plated in 12 well plates in triplicate. After incubation with indicated reagents for 3–5 days, cell viability was quantified using Vi-Cell cell viability analyzer (Beckman Coulter).

Soft agar colony formation assay

Two thousand cells per well mixed in a 0.35% agarose/complete media suspension were seeded onto 0.7% agarose/complete media bottom layer in 12-well plates. Cells grew at 37 °C for 3 weeks until the colonies were visible to the naked eye. Colonies were then stained with p-iodonitrotetrazolium violet (1 mg ml−1, Sigma) overnight and then photographed. The colonies were counted by Image J software, and the data were plotted. Each experiment was performed in triplicate.

Mammosphere culture

Mammary tumor cells were plated onto ultralow attachment plates (Corning) at a density of 20,000 viable cells ml−1 (to obtain primary mammospheres) in a serum-free DMEM-F12 (Invitrogen) supplemented with 5 μg ml−1 insulin, 20 ng ml−1 epidermal growth factor and 20 ng ml−1 basic fibroblast growth factor (Sigma), and 0.4% bovine serum albumin (Sigma). After 10 days, number and size of mammospheres were estimated.

Immunoprecipitation

Cells were lysed on ice for 30 min in IP buffer (1% NP-40, 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA) containing protease inhibitor cocktail. Cell lysates (700 μg) were incubated overnight with 3 µg of antibodies or normal IgG at 4 °C with rotary agitation. Protein A-sepharose beads were added to the lysates and incubated for additional 4 h. Beads will be washed three times with IP buffer and boiled for 10 min in 3% SDS sample buffer. Total cell lysates and immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting.

RNA immunoprecipitation (RIP) assay

Cell were crosslinked for 20 min with 1% formaldehyde, and cell pellets were resuspended in buffer B (1% SDS, 10 mM EDTA, 50 mM Tris–HCl (pH 8.1), 1×protease inhibitor, 50 U ml−1 RNase inhibitor). Incubated 10 min in ice, the pellets were disrupted by sonication, and the lysates were subjected to immunoprecipitation with control IgG or anti-DDX1 or anti-Drosha antibody, followed by stringent washing, elution, and reversal of crosslinking. The RNA was resuspended in 20 μl of TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 50 U ml−1 RNase inhibitor) and incubated with DNase I for 30 min at 37 °C to remove any remaining DNA. After extraction with phenol:chloroform:isoamyl alcohol (25:24:1), RNA was precipitated with ethanol and dissolved in 20 μl of DEPC-treated water. RNA (5 μl) was used for the cDNA synthesis reaction. Quantitative PCRs were then performed on real-time PCR machine.

RNA isolation, qRT–PCR, and miRNA PCR array

Total RNA was isolated using TRIzol reagent (Life Technologies) and then reverse-transcribed using iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was used for qPCR using iTaq Universal SYBR Green Supermix (Bio-Rad) with gene-specific primers and these results were normalized to β-actin control. To analyze miRNAs, total RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Life Technologies) and was then reverse transcribed with a Universal cDNA Synthesis Kit II (Exiqon). cDNA was used for qPCR using SYBR Green master mix (Exiqon) on ABI 7900 real-time PCR cycler. miRNA LNA PCR primer sets (Exiqon) and gene specific primers were used for detecting miRNA and mRNA levels and data was normalized to internal control, U6 (miRNA) or GAPDH (mRNA). RT-PCR primers are shown in Supplementary Table 1. For deep sequencing, primary mouse mammary epithelial cells (8 weeks old) were isolated as previously reported[55,56] and then total mRNA was extracted using Direct-zol RNA extraction kit (Zymo Research) and then submitted for deep sequencing. Both raw and processed data have been deposited in the GEO database (Accession Number GSE119210).

miR-21 in situ hybridization

Breast cancer tissue array (BR10010) was purchased from Biomax.us, containing 50 cases of breast carcinoma. As described previously[57], the tissue slides were first digested with 15 μg ml−1 proteinase K for 10 min at room temperature, and then hybridized with the double-DIG-labeled mercury LNA microRNA probe (Exiqon) for 2 h at 50 °C on Ventana Discovery Ultra (Ventana Medical Systems). The digoxigenins were then detected with a polyclonal anti-DIG antibody and alkaline phosphatase-conjugated second antibody using NBT-BCIP as the substrate. Raw images were captured with the same exposure and gain settings from all slides and saved as TIF files, and were analyzed using intensity measurement tools of Image-Pro Plus software.

Synthesis of nanoparticles

The nanoparticles were synthesized using a double emulsion method with a slight modification[58]. First, Chitosan was modified with guanidine group according to the literature[59] to form chitosan-guanidine (CG). To capture carbon dioxide (CO2), CG aqueous solution was bubbled with CO2 for 1 h to form CG-CO2. PLGA (20 mg) and DPPC (10 mg) were dissolved in 2 ml dichloromethane (DCM) and 50 µl GSK2830371 in tetrahydrofuran (THF) solution (40 mg ml−1) was then added, the above mixture together with 400 µl of DI water containing 500 µg ml−1 anti-miR21 oligonucleotide and 500 µg ml−1 CG-CO2 were transferred into a 50 ml centrifuge tube. Then the immiscible solutions were emulsified by sonication for 1 min using a Bransan 450 sonifier. Next, this first emulsion and 8 ml of chitosan-PF127 solution (in DI water) were emulsified by sonication for 1 min. After rotary evaporation to remove the organic solvent, the nanoparticles were collected by centrifugation at 10,000×g for 10 min at room temperature and washed twice with DI water.

Characterization of nanoparticles

The nanoparticle was characterized using both transmission electron microscopy (TEM) and dynamic light scattering (DLS). First, nanoparticles were soaking in soaking in Phosphate Buffer (pH 7.4, pH 6.0) or Acetate Buffer (pH 5.0), respectively, for 6 h. For TEM study, the nanoparticles were negatively stained with uranyl acetate solution (2%, w/w) and examined using an FEI (Moorestown, NJ, USA) Tecnai G2 Spirit transmission electron microscope. The nanoparticle size was determined using a Brookhaven (Holtsville, NY, USA) 90 Plus/BI-MAS dynamic light scattering instrument.

Electrophoretic gel assay

Free anti-miR-21 oligonucleotide and in-MW@NP (in PBS or serum) were mixed with loading buffer, and then loaded into 2% agarose gel with 0.5 μg ml−1 ethidium bromide. Electrophoresis was conducted in 1× TEA buffer at 80 V for 10 min. The result gels were analyzed using a UV illuminator (FluorChemTM E System, CA, USA) to show its location of anti-miR21 oligonucleotide.

Cell uptake and intracellular distribution of nanoparticles

Dex-Rho was encapsulated inside the nanoparticles using the same method described above. To study cellular uptake and subcellular localization of nanoparticles, HER8R cells were treated with Dex-Rho@NP or free Dex-Rho for 1–6 h at 37 °C. The cells were further treated with medium containing 90 nM LysoTracker Green and 50 nm DAPI. Then, the cells were further examination using an Olympus FluoViewTM FV1000 confocal microscope.

Mouse lines

The MMTV-ErbB2 transgenic mice[60] and miR-21 knockout mice[28] were obtained from The Jackson Laboratory, Bar Harbor, ME. Details of the WIP1 deficient mice have been previously described[61]. Mice were interbred and maintained on FVB/C57BL6 hybrid background in pathogen-free conditions at M.D. Anderson Cancer Center and used in accordance with Institutional Animal Care and Use Committee-approved protocols. Virgin MMTV-ErbB2 and bitransgenic MMTV-ErbB2; MIR21 and MMTV-ErbB2; WIP1 female mice were palpated weekly to detect the presence of mammary tumors starting at 90 days of age. When tumor reachs 10 mm in diameter, tumor and normal, non-tumourigenic mammary tissue was harvested from the same animal at the time of tumor harvest.

Breast tumor xenograft mouse model

Female NOD/SCID mice (6–8 weeks old) were purchased from Jackson Laboratories. All studies were approved and supervised by the Institutional Animal Care and Use Committee at the MD Anderson Cancer Center and Indiana University School of Medicine. For the breast cancr orthotopic xenograft model, nude mice were injected with 5 × 106 HER18 (mixed with matrigel in 100 µl PBS) human breast cancer cells in the mammary fat pad as discribed previously[62,63]. The nude mice were supplementary with weekly subcutaneous estradiol cypionate injections (3 mg kg−1 per week), starting 1 week prior to injection of tumor cells. After initial establishment of tumor (50 mm3), mice were randomly grouped and treated with or without 1 µg ml−1 Dox in drinking water for 4 weeks. The Dox water was changed every other day. The adminstration of trastuzumab (5 mg kg−1) was performed biweekly for 4 consecutive weeks by intravenous injection.

For xenograft tumor studies using nanoparticles, mice bearing trastuzumab-resistant HER18 or BT-474 tumors were randomized to four groups and received the following treatments: (1) control nanoparticles; (2) WIP1 inhibitor nanoparticles (5 mg kg−1); (3) miR-21 inhibitor nanoparticles (1 mg kg−1); and (4) WIP1 inhibitor + miR-21 inhibitor nanoparticles. Nanoparticles were administered twice weekly by intravenous injection, and tumor size and body weight changes were monitored biweekly. Tumor size was measured using a caliper, and tumor volume was calculated using the standard formula: 0.5 × L × W2, where L is the longest diameter and W is the shortest diameter. For the tumor metastasis model, female NOD/SCID mice were injected with trastuzumab-resistant HER18-luc cells via tail vein injection. Lung metastasis was monitored by the IVIS bioluminescent imaging sysem. Mice were euthanized when they met the institutional euthanasia criteria for tumor size and overall health condition. Tumors were removed, photographed and weighed. The freshly dissected tumor tissues were fixed in 10% buffered formalin overnight, transferred to 70% ethanol, embedded in paraffin, sectioned and stained with hematoxylin and eosin and indicated antibodies.

Immunohistochemistry

The tumors were fixed in 10% neutral buffered formalin and embedded in paraffin and 5-µm tissue sections were serially cut and mounted on slides. The sections were de-paraffinized in xylene, re-hydrated, and boiled for 10 min in antigen retrieval buffer. After retrieval, the sections were washed with distilled water and endogenous peroxidase activity was blocked using 3% H2O2 in TBS for 15 min and then blocked with blocking solution (1% bovine serum albumin, 10% normal serum in 1× TBS). Samples were incubated with primary antibodies overnight at 4 °C, washed three times with TBST buffer, and then incubated with biotinylated goat anti-rabbit or anti-mouse IgG (GR608H, BioCare Medical). A streptavidin–biotin peroxidase detection system with 3, 3′-diaminobenzidine as substrate was used according to the manufacturer’s instructions (DAB Peroxidase Substrate Kit, Vector Laboratory). Sections were counterstained with haematoxylin. For immunocytochemistry, cells on chamber slide were fixed in 3.7% paraformaldehyde for 10 min, and permeabilzed by 0.2% Triton X-100 for 5 min and then blocked with blocking solution. After incubation with primary antibodies, cells were washed with PBS three times and incubated with Alexa fluor 488 or 594 conjugated antibodies (Life Technologies) and counter stained with Hoechst 33258. After mounting, signals were observed under microscope. Breast cancer tissue array (BR10010) was purchased from Biomax.us.

Statistical analysis

Each experiment was repeated three times or more. Unless otherwise noted, data are presented as mean ± SD, and Student’s t-test (unpaired, two-tailed) was used to compare two groups of independent samples, whereas one-way ANOVA followed by Tukey’s t-test was conducted to compare three or more groups of independent samples. Survival analysis was done using the Kaplan–Meier method, as assessed using a log-rank Mantel-Cox test. p < 0.05 was considered statistically significant.

Supplementary information