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ShRNA targeting Notch1 sensitizes breast cancer stem cell to paclitaxel
The International Journal of Biochemistry & Cell Biology 45 (2013) 1064–1073
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
ShRNA targeting Notch1 sensitizes breast cancer stem cell to paclitaxel Jun Mao a,b,1 , Bo Song a,1 , Yu Shi c , Bo Wang a , Shujun Fan a,b , Xiaotang Yu a,b , Jianwu Tang a,∗∗ , Lianhong Li a,b,∗ a
Department of Pathology, Dalian Medical University, Dalian 116044, PR China The Key Laboratory of Tumor Stem Cell Research of Liaoning Province, Dalian Medical University, Dalian 116044, PR China c Department of Pathology, Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA b
a r t i c l e
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Article history: Received 3 December 2012 Received in revised form 22 February 2013 Accepted 24 February 2013 Available online 14 March 2013 Keywords: Notch1 Breast cancer stem cell Paclitaxel Drug-resistance ABCG2
a b s t r a c t Breast cancer is currently the most lethal gynecologic malignancy in many countries, and paclitaxel is a cornerstone in the treatment of this malignancy. Unfortunately, the efficacy of paclitaxel is limited due to the development of drug resistance. Evidence has suggested that cancer stem cells (CSCs) are involved in resistance to various forms of therapies, including chemotherapy. However, the interaction between paclitaxel resistance and CSCs and its underlying mechanisms have not been previously explored. In this study, we confirmed that paclitaxel enriched breast CSCs (CD44+/CD24−) in a dose-dependent manner in MCF-7 human breast cancer cell line. We then demonstrated that Notch1 was overexpressed in breast CSCs isolated from paclitaxel-treated MCF-7 cells compared to non-CSCs. The short hairpin RNA (shRNA) mediated knock-down of Notch1 inhibited MCF-7 cell proliferation and induced cell apoptosis. The antiapoptosis protein NF-B was decreased significantly when treated with shRNA-Notch1, and this effect was sharply improved by combination with paclitaxel. Paclitaxel decreased CD44+/CD24− cell population in MCF-7 cells and reduced the size and number of primary mammospheres after down-regulating the Notch1. Furthermore, shRNA-Notch1 inhibited the growth of tumor xenografts in nude mice noticeably. RT-PCR and Western blotting analysis showed that the expressions of ALDH1, NICD, Hes-1 and the drug transporter ABCG2 were decreased both in vitro and in vivo. These results suggest that Notch1 might play a critical role in the resistance to paclitaxel, and targeting Notch1 may have important clinical applications in cancer therapy. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Breast cancer is currently the leading cause of death from gynecologic malignancies in many countries. Paclitaxel, originally isolated from Taxus brevifolia (the Pacific yew), is a microtubule stabilizing chemotherapeutic drug used in the treatment of breast cancer, as well as other tumor histologies, such as breast (Hatschek et al., 2012), ovarian (Karlan et al., 2012) and non-small cell lung cancers (Zhu et al., 2012). However, resistance to paclitaxel and other chemotherapeutic agents is still one of the major reasons for the failure of breast cancer treatment. Cancer stem cells (CSC) accounting for a rare fraction of a certain tumor exhibit a self-renewal, low rate of division and proliferation
∗ Corresponding author. Tel.: +86 411 86110050. ∗∗ Corresponding author. Tel.: +86 411 86110002. E-mail addresses: [email protected] (J. Tang), [email protected] (L. Li). 1 These authors contributed equally to this work. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.02.022
that allow them to be more resistant to chemotherapy and radiotherapy (Dean et al., 2005), both of which preferentially target rapidly dividing cells, making CSCs an important reason for the failure of chemotherapy (Diehn and Clarke, 2006; Song et al., 2010). Breast CSCs were first isolated and identified by Al-Hajj et al. (2003) using cell surface phenotype of CD44+/CD24−/low /Lineage− in 2003, and the tumorigenicity of CD44+/CD24−/low /Lineage− cells has been further proved by other studies (Ponti et al., 2005; Patrawala et al., 2006). Ginestier et al. (2007) confirmed that ALDH1 (aldehyde dehydrogenase 1) is a marker of normal and malignant breast stem cell. Several reports have suggested that chemotherapeutic drugs such as epirubicin, cisplatin, docetaxel or doxorubicin, and cyclophosphamide can enrich the putative CSCs in the breast cancer model both in vitro and in vivo, and increased CSCs in turn induce the chemoresistance (Yu et al., 2007; Shafee et al., 2008; Li et al., 2008). However, how CSCs contribute to paclitaxel resistance of breast cancer cells is still not fully understood and further exploration is required. Notch signaling pathway in mammals contains four Notch receptors (Notch1, 2, 3 and 4) and five ligands (Delta-like-1 or
J. Mao et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1064–1073
DLL1, 3, 4 and Jagged1, 2) (Fiuza and Arias, 2007; Yin et al., 2010; Andersen et al., 2012). Notch signaling is initiated by ligand binding, which is followed by intramembranous proteolytic cleavage of the Notch receptor to generate NICD. The NICD subsequently translocates to the nucleus to activate the transcription of Notch target genes, such as Hes/Hey family and nuclear factor-B (NF-B) (Yin et al., 2010; Andersen et al., 2012). And NICD are therefore to be considered as the active form of Notch receptor. Accumulating evidence suggests that deregulation of Notch1 has been implicated in the development, progression and survival of breast cancer (Kovall, 2007; Wu et al., 2007; Zang et al., 2007; Yin et al., 2010). One study showed that Notch1 signaling activation confers chemoresistance to rapamycin in lung cancer cells through inhibiting apoptosis via PI3K-Akt/PKB pathway (Mungamuri et al., 2006). Zang et al. recently reported that knock-down of Notch1 induces breast cancer cell apoptosis through the inactivation of NF-B, and additionally Notch1 down-regulation increases the chemosensitivity to doxorubicin and docetaxel (Zang et al., 2010). Notch signaling pathway also plays important roles in regulating stem cell proliferation, differentiation, self-renewal and apoptosis (Purow et al., 2005; Dang, 2012), which are closely related to tumor genesis. Phillips et al. (2006) reported that fractionated doses of irradiation increases the activation of Notch1 and the percentage of CD44+/CD24−/low breast CSCs. However, the effects and mechanisms of Notch1 in the chemoresistance to paclitaxel in the breast cancer need further investigation. ABCG2 is a member of ATP binding cassette (ABC) transporters, and its overexpression has been implicated in resistance to the chemotherapeutic agents including paclitaxel (Cheng et al., 2012; Seamon et al., 2006). ABCG2 is also widely expressed in a variety of CSC and can be recognized as a universal marker of CSC (Patrawala et al., 2005). Importantly, ABCG2 is verified to be the direct target of Notch signaling, and double immunocytochemical analysis has revealed that Notch1 and ABCG2 are co-localized in the E18 retinal progenitors (Bhattacharya et al., 2007). We reasoned that Notch1 may play an important role in paclitaxel resistance through influencing the proliferation of CSC and the expression of ABCG2 in breast cancer. In this study, we first confirmed that paclitaxel can enrich breast CSC (CD44+/CD24−) in a dose-dependent manner in MCF-7 breast cancer cell line. Then we demonstrated that Notch1 is overexpressed in breast CSCs isolated from paclitaxel-treated MCF-7 cell line. The short hairpin RNA (shRNA) mediated knock-down of Notch1 inhibits MCF-7 cell proliferation and induces cell apoptosis at least partly through the induction of NF-B, and this effect is sharply improved by combination with paclitaxel. Furthermore, knock-down of Notch1 inhibits the proliferative potential of breast CSC and confers breast CSC sensitive to paclitaxel in vitro and suppresses breast cancer tumor growth in vivo. Consistently, ALDH1, NICD, Hes-1, and ABCG2 were found to be decreased in shRNANotch1 transfected cells and in the mouse xenograft tumor tissues. Taken together, shRNA targeting Notch1 inhibits breast CSC proliferation and suppresses ABCG2 expression, thereby conferring sensitivity to paclitaxel. To the best of our knowledge, this work, for the first time, investigated the effects of Notch1 on paclitaxel resistance of breast CSC.
2. Materials and methods 2.1. Cell culture and reagents The human breast cancer cell line MCF-7 was purchased from Shanghai Culture Collection, and was maintained in DMEM/F-12 medium (Gibco Laboratories, Gran Island, NY) at 37 ◦ C in a humidified incubator with 5% CO2 . The culture medium was supplemented
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with 10% fetal bovine serum (Gibco Laboratories, Gran Island, NY), 2 mmol/L glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA). Paclitaxel was purchased from Sigma (St. Louis, MO).
2.2. Cell transfection The short hairpin RNA (shRNA) expression plasmids, pGPU6/GFP/Neo, were obtained from GenePharma (Shanghai, China). The construction of RNAi expression vectors for human Notch1 was as follows, oligo1 encoding human shRNA-Notch1-1 DNA oligomers (shRNA-Notch1forward 5 -GGAGCATGTGTAACATCAACA-3 , reverse 1): 5 -CCTCGTACACATTGT AGTTGT-3 ; oligo2 encoding human Notch1 shRNA-Notch1-2 DNA oligomers (shRNA-Notch1-2): forward 5 -GCAGCTGCACTTCATGTACGT-3 , reverse 5 -CGTCGACGT GAAGTACATGCA-3 . Scrambling nucleotide sequence of Notch1 (GenePharma, Shanghai, China) was used as negative control (shRNA-Control). shRNA-Notch1-1, -2 and shRNA-Control were transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions at a final concentration of 60 nM for 24 h, 48 h, and 72 h. Transfection rates were monitored with fluorescence microscopy. The cells without transfection were used as a blank control. The successfully transfecting cells were selected by additional 350 mmol/L of G418 (Gibco Laboratories, Gran Island, NY). At day 21, cells were collected for RT-PCR and Western blot analysis.
2.3. Paclitaxel treatment Stock solutions of paclitaxel were diluted in DMSO at four different doses (2, 4, 8 and 16 M) for in vitro experiments (Park et al., 2008) and were diluted according to the instructions for in vivo experiments (Kim et al., 2006).
2.4. Mammosphere formation assay Different groups of MCF-7 breast cancer cells were treated with shRNA against Notch1 alone, or combination with paclitaxel for mammosphere culture. Single cells then were plated in ultra-low attachment plates (Corning, Acton, MA) at a density of 2 × 104 viable cells/ml in primary culture, and 5 × 103 cells/ml in subsequent passages with serum-free medium, supplemented with 2 mmol/L L-glutamine (ATCC, Manassas, VA), 2% B27 supplement (Invitrogen, Carlsbad, CA), 20 ng/mL human recombinant epidermal growth factor (EGF) (Prepro Tech, Rocky Hill, NJ) and 20 ng/mL basic fibroblast growth factor (b-FGF) (Prepro Tech, Rocky Hill, NJ), 4 g/mL heparin and 5 g/mL insulin (Sigma, St. Louis, MO). Mammospheres were cultured for 7 days and were counted and collected by centrifugation at 1000 rpm.
2.5. Flow cytometry analysis MCF-7 cells were sorted with multiparametric flow cytometry using BD FACS Aria cell sorter (BD, Franklin Lakes, NJ) under sterile conditions. Cells at a density of 106 were collected and labeled with conjugated anti-human CD44-FITC (Invitrogen, Carlsbad, CA) and CD24-PE (Invitrogen, Carlsbad, CA). Antibodies were diluted in MACS buffer containing 5% BSA, 1 mM EDTA and 15–20% blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) to inhibit nonspecific binding to non-target cells. After 30 min incubation at 4 ◦ C, staining cells were washed, resuspended in 500 l of MACS buffer, and sorted.
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2.6. Isolation of breast cancer stem cell by microbeads Mammospheres were collected and enzymatically dissociated into single-cell suspensions. After counting with a hematocytometer, 1 × 107 cells were incubated with CD44 and CD24 primary mouse IgG antibodies (Gibco Laboratories, Gran Island, NY) for 5 min at 4 ◦ C. Unbounded primary antibodies were removed by PBS following by centrifuge at 300 × g for 10 min. The cells were then resuspended in 80 l of buffer with addition of 20 l of Goat Anti-Mouse IgG MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and incubated for 15 min at 4 ◦ C. Cells were washed again and preceded to magnetic separation. 2.7. Colony formation assay Cells were trypsinized, counted, and seeded for colony formation assay in 6-well plates at 300 cells per well. During colony growth, the culture medium was replaced every 3 days. At 7 days after seeding, the colonies were stained with 0.02% crystal for 1 h and then were counted in 5 random chosen fields under an inverted phase-contrast microscope (Olympus IX71). The colony was counted only if it contains more than 50 cells. Each treatment was carried out in triplicate. 2.8. CCK8 assay Cells were plated in 96-well plates at a density of 3 × 104 per well. At 24, 48, 72 and 96 h post-plating, 10 l Cell Counting Assay Kit-8 solution (Sigma, St. Louis, MO) was added to each well and incubated for 2 h, and the absorbance at 490 and 650 nm was measured using a microplate reader. Addition of DMEM/F12 alone was used as black control group. All experiments were done three times independently. 2.9. Staining with Hoechst 33342 Cells were washed twice in PBS and fixed in PBS containing 1% (wt/vol) paraformaldehyde (Fisher Scientific, Pittsburgh, PA). After rinsing with water, cells were stained with Hoechst 33342 (Sigma, St. Louis, MO) at final concentration of 10 g/ml for 10 min. The morphologic aspect of nuclei was observed under a fluorescence microscope using a 320–350 nm filter. This experiment was repeated three times.
GC-3 (forward) and 5 -CAC CTT CAC CGT TCC AGT TT-3 (reverse). The GAPDH GAPDH primers were 5 -GGTCGGAGTCAAAGGAT-3 (forward) and 5 -ATGAGCCCCAGCCTTCTCCAT-3 (reverse). Above primers were designed by TaKaRa (Dalian, China). Relative quantitation of mRNA expression levels was determined using the relative standard curve method according to the manufacturer’s instructions. 2.12. Western blot analysis Cells were lysed in 1× RIPA buffer (Sigma, St. Louis, MO). The protein concentration was determined by Bradford method with BSA (Sigma, St. Louis, MO) as the standard. Equal amount of protein extract (50 g) were subjected to 12% SDS-PAGE gels and transferred to nitrocellulose membrane (Millipore, Billerica, MA). The primary antibodies of Notch1 (1:500), Hes-1 (1:200), NFB (1:500), ALDH1 (1:200) and ABCG2 (1:200) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-NICD (1:200) was from Millipore (Billerica, MA). GAPDH (1:000) was used as an internal control which was from Santa Cruz Biotechnology (Santa Cruz, CA). 2.13. Tumor growth and morphologic analysis in vivo All studies involving mice were approved by Animal Care and Use Committee of Dalian Medical University. Twenty 6–8-week-old female nude mice were purchased from Experimental Animal Center of Dalian Medical University. Mice were randomly divided into two groups, group 1 was injected with shRNA-Control transfected cells, and group 2 was injected with shRNA-Notch1 transfected cells. To each group, 1 × 106 MCF-7 cells were resuspended in 100 l PBS mixed with matrigel (1:1) and injected into the mouse mammary fat pad. After 5 days, 5 mice were chosen from these two groups respectively and treated with 50 mg/kg paclitaxel every 2 days (Kim et al., 2006). The tumor mass was monitored using caliper. Tumor volumes (V) were calculated by the formula V = L × W2 × 0.5. On day 25 postinoculation, mice were weighed and sacrificed. The transplanted tumors were excised, weighed and frozen at −80 ◦ C until processing for RNA and protein isolation. For histological study, portions of tumors were fixed in 10% neutralbuffered formalin, paraffin-embedded and 4-m sections were stained for HE. For immunohistologic assay, Notch1 and ALDH1 were detected by anti-Notch1 and anti-ALDH1 primary antibodies respectively (Santa Cruz Biotechnology, Santa Cruz, CA).
2.10. Cell apoptosis analysis 2.14. Statistical analysis Cells were harvested and resuspended in 500 l of binding buffer and stained with 5 l of FITC-Annexin-V (BD Biosciences, San Jose, CA) and 5 l of propidium iodide (Sigma, St. Louis, MO) for 15 min in the dark at 4 ◦ C. After washing, cells were analyzed by flow cytometry. This experiment was repeated three times.
The results were expressed as the mean ± SD. Analysis was performed using a two-way Student’s t-test. Statistical significance was set as a P < 0.05. 3. Results
2.11. RNA extraction and RT-PCR assay of Notch1 and ALDH1A1 Total RNA was isolated from MCF-7 cells and mice transplanted tumor tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The concentration and purity of RNA were determined at 260/280 nm using a Nanodrop spectrophotometer (Thermo Scientific, Rockford, IL). Then cDNA was synthesized from approximately 5 ng RNA per 20 L using a cDNA reverse transcription kit (TaKaRa, Dalian, China). The Notch1 primers were 5 -GCA GTT GTG CTC CTG AAG AA-3 (forward) and 5 -CGG GCG GCC AGA AAC-3 (reverse). The ALDH1 primers were 5 -AAT GGC ATG ATT CAG TGA GTG GC-3 (forward) and 5 -GAG GAG TTT GCT CTG CTG GTT TG-3 (reverse). The internal control -actin primers were 5 -GAT CAT TGC TCC TCC TGA
3.1. CD44+/CD24− breast CSCs resistant to paclitaxel treatment overexpress Notch1 at both the mRNA and protein levels To determine whether paclitaxel enriches breast CSC, we treated MCF-7 cells with 2, 4, 8 and 16 M of paclitaxel for 7 days. After sorting by FACS, we found that the percentage of cells with CSC-markers (CD44+/CD24−) increased 1.22 ± 0.08, 1.55 ± 0.11, 2.63 ± 0.07, and 3.19 ± 0.07-fold respectively (Fig. 1A). Next, after treating MCF-7 cells with 8 M of paclitaxel for 7 days, we collected and cultured the surviving cells in low-attachment condition with serum-free medium, as described in Section 2. MCF-7 cells without paclitaxel treatment were used as the negative control. The floating mammospheres generated by small number of cells were passed by
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Fig. 1. Notch1 is highly expressed in CD44+/CD24− breast CSCs. (A) Paclitaxel significantly enriched CD44+/CD24− breast CSCs. MCF-7 cells were treated with various dose of paclitaxel (2, 4, 8, and 16 M) for 7 days and were detected by flow cytometry with CSC-markers (CD44+/CD24−). MCF-7 cells without paclitaxel treatment were used as the negative control. (B) Representative images of mammospheres on day 7. MCF-7 cells were treated with 8 M of paclitaxel for 7 days, and the surviving cells were collected and cultured in low-attachment condition with serum-free medium for 7 days (a, ×200; b, ×400, scale bar = 50 m). (C) CD44+/CD24− cells highly expressed ALDH1 which is a marker of breast CSC. The mammospheres were collected and sorted with CSC-markers by microbeads. The expression of ALDH1 was evaluated by immunologic assay. CSC represents CD44+/CD24− breast CSCs, non-CSCs represents non-CD44+/CD24− breast cancer cells, and un-separated represents the cells before sorting (scale bar = 50 m). (D) Notch1 expression was dramatically increased in breast CSCs compared to non-CSCs at both the mRNA (left) and protein (right) levels, and these differences are even prominent after paclitaxel treatment (8 M). RNA and protein were harvested, and Notch1 expression was determined by RT-PCR and Western blot. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (lower), and -actin and GAPDH were used for normalization respectively. *P < 0.05, **P < 0.01, Student’s t-test.
mechanical and enzymatic dissociation and shown in Fig. 1B. After 11 days of culture, we collected the spherical colonies and isolated the cancer stem cells with CSC-markers by microbeads. To further verify the stem cell nature of CD44+/CD24− cells, we then examined the expression of ALDH1, another confirmed marker of breast CSC (Ginestier et al., 2007) by cellular immunologic assay. The results showed that ALDH1 was expressed in the cytoplasm of more than 70% of CD44+/CD24− cells, whereas almost no ALDH1 expression was found in non-CD44+/CD24− cells, only a few un-separated cells expressed ALDH1 protein (Fig. 1C). These results indicated that CD44+/CD24− can be considered as the effective breast CSC markers. Paclitaxel treatment dramatically enriches CD44+/CD24− breast CSCs, and in turn breast CSCs are more resistant to paclitaxel. We then compared Notch1 expression levels between CD44+/CD24− breast CSCs and non-CD44+/CD24− breast cancer cells using RT-PCR and Western blot. As shown in Fig. 1D, the expression of Notch1 was significantly increased in breast CSCs
compared to non-CSCs at both the mRNA and protein levels, and these differences were even more profound after treating by paclitaxel, suggesting that Notch1 may play important roles in the resistance to paclitaxel possibly through stimulating the development of breast CSC. 3.2. Down-regulation of Notch1 inhibits colony-forming capacity and cell growth potential, and sensitizes MCF-7 breast cancer cells to paclitaxel To investigate the effect of Notch1 in paclitaxel resistance, we first compared two shRNAs specific against Notch1 (shRNANotch1-1, and -2) in silencing the expression of Notch1 in MCF-7 breast cancer cell, scrambling nucleotide sequence of Notch1 (shRNA-Control) was used as the negative control. We found transfection of shRNAs against Notch1 for 48 h had the highest transfection rate than that at any other time points when evaluated
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Fig. 2. Down-regulation of Notch1 inhibits colony-forming capacity and cell growth potential, and sensitizes MCF-7 breast cancer cells to paclitaxel. (A) Comparison of two shRNAs in silencing Notch1 expression at both the mRNA (left) and protein (right) levels. MCF-7 cells were transfected with two shRNAs (shRNA-Notch1-1 and shRNANotch1-2) respectively for 48 h. MCF-7 without treated (control) and scrambling nucleotide sequence of Notch1 (shRNA-Control) were the negative control. Then, cells were cultured under antibiotic pressure for 11 days and collected for RT-PCR and Western blot analysis of Notch1 and GAPDH. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software, and GAPDH was used as a negative control. (B and C) ShRNA-Notch1 alone or combination with paclitaxel reduced the number of colonies in MCF-7 cells. The number of colonies was counted after 7 days. (D) ShRNA-Notch1 alone or combination with paclitaxel inhibited the proliferation rate of MCF-7 cells. CCK8 assay was used to evaluate the cell growth ability. *P < 0.05, **P < 0.01, Student’s t-test. Each condition was repeated 3 times and error bars represent standard deviations.
by fluorescence microscopy (data not shown). The cells then were cultured under antibiotic selection pressure for 21 days and harvested to examine the Notch1 levels by RT-PCR and Western blot. Both the shRNA-Notch1-1 and shRNA-Notch1-2 significantly reduced the expression of, Notch1 compared to shRNA-Control, however, the shRNA-Notch1-2 showed slightly higher efficiency (Fig. 2A). Thus we selected shRNA-Notch1-2 (shRNA-Notch1) to perform the subsequent experiments. Next, colony formation and proliferation assays were performed in MCF-7 breast cancer cells to investigate the potential impact
of Notch1 on cell proliferation. Colonies containing at least 50 cells were counted on day 7 after plating. Our results showed that shRNA-Notch1 significantly suppressed the colony-forming capacity when combined with paclitaxel, indicating that Notch1 silencing sensitizes breast cancer cell line to paclitaxel treatment (Fig. 2B and C). Furthermore, we evaluated the proliferation rate in different groups by CCK8 assay, as Fig. 2D showed, down-regulation of Notch1 suppressed cell growth compared to its negative control, and shRNA-Notch1 combination with paclitaxel dramatically decreased the viability of MCF-7 cells. These results demonstrated
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Fig. 3. ShRNA-Notch1 enhances paclitaxel-induced cell apoptosis in MCF-7 cells partly through down-regulation of NF-B. (A) The apoptotic cells labeled with Hoechst 33342 increased when Notch1 is down-regulated, especially when combined with paclitaxel treatment. MCF-7 cells were transfected with 60 nM of shRNA-Notch1 and treated with 4 M of paclitaxel for 24 h, and then were stained with Hoechst 33342. The apoptotic cells emit strong blue fluorescence (red arrow, scale bar = 50 m). (B) Down-regulation of Notch1 increased the percentage of early period apoptotic cells labeled with Annexin V-FITC/PI. Early apoptosis in the lower right quadrant was taken into account because it is more representative. These experiments were repeated three times, and similar results were obtained. Representative scatter grams from flow cytometry profile represent Annexin V-FITC staining in the x axis and PI in the y axis. (C) The expression of NF-B was determined by Western blot (left). The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right), and GAPDH was used as a negative control. **P < 0.01, Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
that Notch1 down-regulation inhibits colony-forming capacity and cell growth potential of breast cancer cell line, and MCF-7 breast cancer cells with Notch1 down-regulation are more sensitive to paclitaxel. 3.3. Down-regulation of Notch1 enhances paclitaxel-induced cell apoptosis in MCF-7 breast cancer cells To further detect the possible mechanisms by which Notch1 down-regulation sensitizes breast cancer cells to paclitaxel, MCF-7 cells were transfected with shRNA-Notch1 or shRNA-Control and treated with 4 M of paclitaxel for 24 h, cell apoptosis progression was assessed using Hoechst 33342 and Annexin V-FITC/PI staining by flow cytometry. The apoptotic cells were deeply stained, shrunken and fragmented (Fig. 3A). The apoptotic cells labeled with Hoechst 33342 increased in the shRNA-Notch1 group compared to shRNA-Control group, especially when combined with paclitaxel treatment (Fig. 3A). Meanwhile, the percentage of early period apoptotic cells labeled with Annexin V-FITC/PI increased from 2.88% in shRNA-Control group to 8.12% in shRNA-Notch1
group, and the percentage of apoptotic rate in the combination of paclitaxel treatment groups were 11.34% and 19.61% respectively (Fig. 3B). These results suggested that Notch1 down-regulation sensitizes breast cancer cells to paclitaxel, at least partly through inducing apoptosis. To understand the underlying mechanisms, we further examined the levels of NF-B, which is a target gene of Notch1 to inhibit cell apoptosis (Mayo et al., 2001; Yin et al., 2010), by Western blot in different groups. NF-B expression was decreased significantly when treated with shRNA-Notch1, and this effect was sharply improved by combining shRNA-Notch1 with paclitaxel (Fig. 3C). These results indicated that Notch1 down-regulation sensitizes breast cancer cells to paclitaxel treatment through inducing cell apoptosis. This is achieved in part, by the induction of NF-B. 3.4. Down-regulation of Notch1 inhibits the proliferation of breast CSC, and confers breast CSC sensitive to paclitaxel Previously we found paclitaxel treatment can enrich breast CSC, and Notch1 is over-expressed in the breast CSCs compared to
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Fig. 4. Down-regulation of Notch1 inhibits the proliferation of breast CSC and confers breast CSC sensitive to paclitaxel. (A) Notch1 silencing alone or combination with paclitaxel resulted in the decreased volume (left) and number (right) of mammospheres. MCF-7 cells were treated with shRNA-Notch1 alone, or combination with paclitaxel, and then were cultured in ultra-low attachment plates with serum-free medium for 7 days. Mammospheres were collected and evaluated (scale bar = 100 m). (B) The percentage of CD44+/CD24− breast CSC was reduced by Notch1 down-regulation alone or combination with paclitaxel. The phenotype of CD44+/CD24− cells was measured by flow cytometry. Similar results were obtained in three independent experiments, and the representative microscopic picture and flow cytometry pattern were shown. (C) ABCG2, NICD and Hes-1 were down-regulated as Notch1 silencing or combination with paclitaxel (left). The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right), and GAPDH was used as a protein loading control. **P < 0.01, Student’s t-test.
the non-CSCs at both the mRNA and protein levels. It has been reported that mammary stem/progenitor cells are enriched in nonadherent spherical clusters of cells, termed mammospheres (Dontu and Wicha, 2005). Therefore mammosphere formation assay was carried out to determine whether Notch1 interference can suppress the propagation of breast CSC. MCF-7 cells were treated with shRNA-Notch1 alone, or combination with paclitaxel, and then were cultured in ultra-low attachment plates with serum-free medium for 7 days. As Fig. 4A shown, the size of the mammospheres was reduced dramatically, and the number of mammospheres was also reduced by about 45% by shRNA-Notch1 compared to shRNAControl. Furthermore, mammosphere-forming ability was reduced by about 72.4 ± 6.1% in the shRNA-Notch1 transfected cells combination with paclitaxel treatment, and only a small number of cells established mammospheres (Fig. 4A). We then quantified the number of cells with CSC-markers and found that the percentage of CD44+/CD24− was reduced by about 36.3 ± 3.7% and 67.2 ± 5.1% in the shRNA-Notch1 transfected cells and shRNANotch1 combination with paclitaxel treated cells (Fig. 4B). These
results further supported that Notch1 regulates the proliferation of breast CSC, and Notch1 knock-down can improve the chemosensitivity to paclitaxel through inhibiting the proliferation of breast CSC. Meanwhile, we noticed that there was no difference in the size and number of mammospheres between shRNA-Control group and shRNA-Control combination with paclitaxel group (shRNAControl + paclitaxel) (Fig. 4A), indicating that paclitaxel alone is able to inhibit cell proliferation only in breast cancer cell, not in breast CSC. We next performed Western blot to examine the expression of ABCG2 (Patrawala et al., 2005), in shRNA-Notch1 transfected MCF-7 and control cells. As Fig. 4C shown, ABCG2 was dramatically reduced in shRNA-Notch1 transfected cells relative to shRNAControl cells, suggesting that Notch1 down-regulation decreases cell population with ABCG2 positive in MCF-7 breast cancer cell. On the other hand, ABCG2 as a drug transporter was decreased by knock-down of Notch1 (Fig. 4C), thereby confers breast CSC sensitive to paclitaxel. Since ABCG2 is the direct target of Notch signaling (Bhattacharya et al., 2007), these results implied that
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Notch1 down-regulation induces breast CSC sensitive to paclitaxel possibly through inhibiting the proliferative potential and directly suppressing ABCG2 expression of breast CSC. The active form of Notch1, NICD and the downstream target of Notch1, Hes-1 were expressed in lower levels when Notch1 was down-regulated. 3.5. Inhibition of breast cancer growth by Notch1 down-regulation in vivo Given the observed inhibition effects of shRNA-Notch1 and paclitaxel on breast cancer cell proliferation in vitro, we next determined whether shRNA-Notch1 inhibits tumor growth and increases the sensitivity to paclitaxel treatment in vivo. We inoculated 1 × 106 MCF-7 cells stably transfected with shRNA-Control or shRNA-Notch1 into the mouse mammary fat pad for 3 days, and treated them with 50 mg/kg of paclitaxel every 2 days. On day 25 post-inoculation, mice were sacrificed. We found that at all time points, tumors in shRNA-Notch1 group were remarkably smaller than shRNA-Control group, and shRNA-Notch1 combination with paclitaxel treatment significantly inhibited the volume of tumor (Fig. 5A). Tumor weight had the same trend (Fig. 5A). No significant difference was found between shRNA-Notch1 group and shRNA-Control combination with paclitaxel group. By HE staining, shRNA-Notch1 group had more extensive necrosis compared to shRNA-Control group (Fig. 5B). Next we assessed the capacity of shRNA-Notch1 to inhibit breast cancer stem cell proliferation in vivo. We used immunohistochemistry to detect the expression of Notch1 and ALDH1 in the mouse xenograft tumor tissues. As Fig. 5B shown, Notch1 and ALDH1 were highly expressed in shRNA-Control group and shRNA-Control combination with paclitaxel group, whereas shRNA-Notch1 in the absence or presence of paclitaxel induced significant downregulation of ALDH1 protein. We further measured the levels of ALDH1 and ABCG2 by RT-PCR and Western blot in the mouse tumor tissues. We found Notch1 down-regulation significantly suppressed ALDH1 and ABCG2 expression both at mRNA and protein levels (Fig. 5C). The expressions of NICD and Hes-1 were decreased when Notch1 was down-regulated (Fig. 5D). These results indicated that Notch1 down-regulation inhibits the growth of breast cancer cell in vivo, decreases the ALDH1-positive cell population in the xenograft tumors, and confers sensitivity to paclitaxel possibly through suppressing the expression of ABCG2. 4. Discussion In the present study, we tested the role of Notch1 in chemoresistance to paclitaxel in breast cancer. Our data showed that shRNA targeting Notch1 markedly increased paclitaxel-induced cell apoptosis and inhibited cell proliferation in MCF-7 breast cancer cell line, indicating that Notch1 plays a critical role in chemoresistance to paclitaxel. It was reported that a nuclear transcription factor, NF-B, is most commonly involved in inhibiting apoptosis through transactivating anti-apoptotic genes (Mitsiades et al., 2002; Kucharczak et al., 2003). We also found that the expression of NF-B was decreased when treated with shRNA-Notch1 alone or combination with paclitaxel in MCF-7 cells. Notch1 has been reported to cross-talk with NF-B, and down-regulation of Notch1 results in lower activity of NF-B (Vilimas et al., 2006). Since apoptosis is a major manner by which paclitaxel kills cancer cells, these above results implied that Notch1 down-regulation confers breast cancer cells sensitive to paclitaxel through inducing apoptosis via inhibiting anti-apoptotic factor NF-B. Our results were consistent with another recent study (Zang et al., 2010). It showed that down-regulation of Notch1 increases the chemosensitivity of breast cancer cells to doxorubicin and docetaxel, and this effect
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may be caused by cell apoptosis induction through the inactivation of NF-B. Additionally, Notch1 down-regulation also contributes to apoptosis, possibly due to decreased Bcl-2 and Bcl-X(L) protein expression in pancreatic cancer cell, or mediated via inactivation of Akt and mTOR pathways in prostate cancer cell (Wang et al., 2006, 2010). Cancer stem cells (CSC) or tumor-initiating cells have been thought to play a major role in tumor initiation, and chemoand radiation-therapy resistance (Donnenberg and Donnenberg, 2005). To date, several studies have shown that epirubicin, cisplatin, docetaxel or doxorubicin, and cyclophosphamide can enrich the putative CSCs in the breast cancer model both in vitro and in vivo, and increased CSCs in turn induce the chemoresistance to these drugs (Donnenberg and Donnenberg, 2005). We found that paclitaxel can remarkably enrich breast CSCs in a dose-dependent manner in MCF-7 cells, implying that the enrichment of CSCs might be another mechanism for paclitaxel resistance in breast cancer. Many CSCs have deregulated signaling pathways which are involved in normal mammary stem cell regulation, such as Wnt, Hedgehog, Notch and estrogen (Reya and Clevers, 2005; Pannuti et al., 2010; Dontu et al., 2004). In this study, Notch1 was observed to be expressed at high levels in CD44+/CD24− breast CSCs sorted from paclitaxel-treated MCF-7 cell line at both the mRNA and protein levels. Knock-down of Notch1 dramatically reduced the size and number of mammospheres, and this inhibit effect was even more profound after combination with paclitaxel treatment. In line with these findings, the percentage of CD44+/CD24− cells was significantly reduced in shRNA-Notch1 transfected cells and combination with paclitaxel treated cells. We also noticed that paclitaxel alone could not influence the mammosphere-forming ability, further confirming the important role of breast CSC in the resistance to paclitaxel. In vivo, knock-down of Notch1 significantly inhibited the volume and weight of tumor compared to shRNA-Control, and this trend was even more remarkable in the presence of paclitaxel. Current views favor the model that cancer stem cells are innately resistant to chemotherapy through their relative quiescence, their enhanced capacity for DNA repair, decreased entry into apoptosis, and ABC transporter expression (Cheng et al., 2012; Donnenberg and Donnenberg, 2005). Many tumors in patients suffering from relapse to therapy are resistant to multiple drugs (multidrug resistant, MDR) (Gottesman et al., 2009). The P-glycoprotein encoded by the ABCB1 gene was the first ABC transporter identified to be amplified and/or overexpressed in MDR tumor cell lines (Chen et al., 1986). There are two other ABC proteins widely overexpressed in tumor cells, ABCC1/MRP1 and ABCG2 (Haimeur et al., 2004). The conservation of ABCG2 expression in Side Population Progenitor cells (SP cells), enriched from a wide range of tissues including blood, lung and intestine (Patrawala et al., 2005), suggesting that ABCG2 has an important role in the chemoresistance. We found that knock-down of Notch1 remarkably reduced the expression of ALDH1 and ABCG2 in the mammospheres. In vivo, although the tumor was inhibited by paclitaxel a little bit more than by shRNA-Notch1, however, the expression of ABCG2 was down-regulated only by shRNA-Notch1 but not paclitaxel. These data implied that paclitaxel could not eliminate the CSC possibly due to the high level of ABCG2 in CSCs, and this might cause incomplete eradication of CSCs. It is considerable to see that the breast CSC decreased conspicuous in the group treated with shRNANotch1 combination with paclitaxel both in vitro and in vivo and the expressions of NICD, Hes-1 and ABCG2 were reduced much more than paclitaxel treated only. Since ABCG2 was reported to be the direct target of Notch signaling (Bhattacharya et al., 2007), these results implied that Notch1 down-regulation induces breast CSC sensitive to paclitaxel possibly through inhibiting the proliferative potential and directly suppressing ABCG2 expression of breast CSC.
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Fig. 5. Inhibition of shRNA-Notch1 on tumor growth in vivo. (A) ShRNA-Notch1 alone or combination with paclitaxel remarkably reduced the tumor volume and weight. 1 × 106 MCF-7 cells stably transfected with shRNA-Control or shRNA-Notch1 were inoculated into the mouse mammary fat pad for 3 days, and then were treated with 50 mg/kg of paclitaxel every 2 days. On day 25 post-inoculation, mice were sacrificed. The volume of the peripheral tumor was measured every 3 days post-inoculation. Error bars represent standard deviations, n = 5. **P < 0.01, Student’s t-test (scale bar = 1 cm). (B) HE and immunohistochemistry staining with Notch1 and ALDH1 antibodies. a–d: HE staining; c–l: immunohistochemistry staining for Notch1 and ALDH1 (brown color in cytoplasm), ×200 (scale bar = 100 m). (C) RT-PCR (left) and Western blot (right) analyses of ALDH1 in the mouse tumor tissues. (D) Protein levels of Notch1, NICD, Hes-1 and ABCG2 were measured by Western blot in mice tumors (left). As an internal control, GAPDH was used for normalization. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right). Data are presented as mean ± SD, n = 5, **P < 0.01, Student’s t-test.
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In summary, we have shown that shRNA targeting Notch1 could suppress ALDH1 and ABCG2 expression, thereby inhibiting breast CSC proliferation and conferring sensitivity to paclitaxel. These results suggest that Notch1 might play a critical role in the resistance to paclitaxel, and may potentially serve as a therapeutic target for overcoming paclitaxel resistance in human breast cancer. These findings strongly suggest that specific therapeutics targeted stem cell signaling pathways could provide an efficient approach for cancer therapy. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (81272430) and Tumor Stem cell Research Key Laboratory of Liaoning Province. References Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences 2003;100:3983–8. Andersen P, Uosaki H, Shenje LT, Kwon C. Non-canonical Notch signaling: emerging role and mechanism. Trends in Cell Biology 2012;22:257–65. Bhattacharya S, Das A, Mallya K, Ahmad I. Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling. Journal of Cell Science 2007;120:2652–62. Chen C, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman M, Roninson I. Internal duplication and homology with bacterial transport proteins in the mdr1 (Pglycoprotein). Cell 1986;47:381–9. Cheng C, Liu Z, Zhang H, Xie J, Chen X, Zhao X, et al. Enhancing chemosensitivity in ABCB1-and ABCG2-overexpressing cells and cancer stem-like cells by an aurora kinase inhibitor CCT129202. Molecular Pharmaceutics 2012;9:1971–82. Dang TP. Notch, apoptosis and cancer. Advances in Experimental Medicine and Biology 2012;727:199–209. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nature Reviews Cancer 2005;5:275–84. Diehn M, Clarke MF. Cancer stem cells and radiotherapy: new insights into tumor radioresistance. Journal of the National Cancer Institute 2006;98:1755–7. Donnenberg VS, Donnenberg AD. Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis. Journal of Clinical Pharmacology 2005;45:872–7. Dontu G, El-Ashry D, Wicha MS. Breast cancer, stem/progenitor cells and the estrogen receptor. Trends in Endocrinology and Metabolism: TEM 2004;15:193–7. Dontu G, Wicha MS. Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. Journal of Mammary Gland Biology and Neoplasia 2005;10:75–86. Fiuza UM, Arias AM. Cell and molecular biology of Notch. Journal of Endocrinology 2007;194:459–74. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007;1:555–67. Gottesman MM, LudwigJ. Xia D, Szakacs G. Defeating drug resistance in cancer. Discovery Medicine 2009;6:18–23. Haimeur A, Conseil G, Deeley R, Cole S. The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation. Current Drug Metabolism 2004;5:21–53. Hatschek T, Carlsson L, Einbeigi Z, Lidbrink E, Linderholm B, Lindh B, et al. Individually tailored treatment with epirubicin and paclitaxel with or without capecitabine as first-line chemotherapy in metastatic breast cancer: a randomized multicenter trial. Breast Cancer Research and Treatment 2012;131:939–47. Karlan BY, Oza AM, Richardson GE, Provencher DM, Hansen VL, Buck M, et al. Randomized, double-blind, placebo-controlled phase II study of AMG 386 combined with weekly paclitaxel in patients with recurrent ovarian cancer. Journal of Clinical Oncology 2012;30:362–71. Kim JH, Kim YS, Kim SW, Park JH, Kim K, Choi KW, et al. Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. Journal of Controlled Release 2006;111:228–34. Kovall RA. Structures of CSL, Notch and mastermind proteins: piecing together an active transcription complex. Current Opinion in Structural Biology 2007;17:117–27.
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
ShRNA targeting Notch1 sensitizes breast cancer stem cell to paclitaxel Jun Mao a,b,1 , Bo Song a,1 , Yu Shi c , Bo Wang a , Shujun Fan a,b , Xiaotang Yu a,b , Jianwu Tang a,∗∗ , Lianhong Li a,b,∗ a
Department of Pathology, Dalian Medical University, Dalian 116044, PR China The Key Laboratory of Tumor Stem Cell Research of Liaoning Province, Dalian Medical University, Dalian 116044, PR China c Department of Pathology, Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA b
a r t i c l e
i n f o
Article history: Received 3 December 2012 Received in revised form 22 February 2013 Accepted 24 February 2013 Available online 14 March 2013 Keywords: Notch1 Breast cancer stem cell Paclitaxel Drug-resistance ABCG2
a b s t r a c t Breast cancer is currently the most lethal gynecologic malignancy in many countries, and paclitaxel is a cornerstone in the treatment of this malignancy. Unfortunately, the efficacy of paclitaxel is limited due to the development of drug resistance. Evidence has suggested that cancer stem cells (CSCs) are involved in resistance to various forms of therapies, including chemotherapy. However, the interaction between paclitaxel resistance and CSCs and its underlying mechanisms have not been previously explored. In this study, we confirmed that paclitaxel enriched breast CSCs (CD44+/CD24−) in a dose-dependent manner in MCF-7 human breast cancer cell line. We then demonstrated that Notch1 was overexpressed in breast CSCs isolated from paclitaxel-treated MCF-7 cells compared to non-CSCs. The short hairpin RNA (shRNA) mediated knock-down of Notch1 inhibited MCF-7 cell proliferation and induced cell apoptosis. The antiapoptosis protein NF-B was decreased significantly when treated with shRNA-Notch1, and this effect was sharply improved by combination with paclitaxel. Paclitaxel decreased CD44+/CD24− cell population in MCF-7 cells and reduced the size and number of primary mammospheres after down-regulating the Notch1. Furthermore, shRNA-Notch1 inhibited the growth of tumor xenografts in nude mice noticeably. RT-PCR and Western blotting analysis showed that the expressions of ALDH1, NICD, Hes-1 and the drug transporter ABCG2 were decreased both in vitro and in vivo. These results suggest that Notch1 might play a critical role in the resistance to paclitaxel, and targeting Notch1 may have important clinical applications in cancer therapy. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Breast cancer is currently the leading cause of death from gynecologic malignancies in many countries. Paclitaxel, originally isolated from Taxus brevifolia (the Pacific yew), is a microtubule stabilizing chemotherapeutic drug used in the treatment of breast cancer, as well as other tumor histologies, such as breast (Hatschek et al., 2012), ovarian (Karlan et al., 2012) and non-small cell lung cancers (Zhu et al., 2012). However, resistance to paclitaxel and other chemotherapeutic agents is still one of the major reasons for the failure of breast cancer treatment. Cancer stem cells (CSC) accounting for a rare fraction of a certain tumor exhibit a self-renewal, low rate of division and proliferation
∗ Corresponding author. Tel.: +86 411 86110050. ∗∗ Corresponding author. Tel.: +86 411 86110002. E-mail addresses: [email protected] (J. Tang), [email protected] (L. Li). 1 These authors contributed equally to this work. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.02.022
that allow them to be more resistant to chemotherapy and radiotherapy (Dean et al., 2005), both of which preferentially target rapidly dividing cells, making CSCs an important reason for the failure of chemotherapy (Diehn and Clarke, 2006; Song et al., 2010). Breast CSCs were first isolated and identified by Al-Hajj et al. (2003) using cell surface phenotype of CD44+/CD24−/low /Lineage− in 2003, and the tumorigenicity of CD44+/CD24−/low /Lineage− cells has been further proved by other studies (Ponti et al., 2005; Patrawala et al., 2006). Ginestier et al. (2007) confirmed that ALDH1 (aldehyde dehydrogenase 1) is a marker of normal and malignant breast stem cell. Several reports have suggested that chemotherapeutic drugs such as epirubicin, cisplatin, docetaxel or doxorubicin, and cyclophosphamide can enrich the putative CSCs in the breast cancer model both in vitro and in vivo, and increased CSCs in turn induce the chemoresistance (Yu et al., 2007; Shafee et al., 2008; Li et al., 2008). However, how CSCs contribute to paclitaxel resistance of breast cancer cells is still not fully understood and further exploration is required. Notch signaling pathway in mammals contains four Notch receptors (Notch1, 2, 3 and 4) and five ligands (Delta-like-1 or
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DLL1, 3, 4 and Jagged1, 2) (Fiuza and Arias, 2007; Yin et al., 2010; Andersen et al., 2012). Notch signaling is initiated by ligand binding, which is followed by intramembranous proteolytic cleavage of the Notch receptor to generate NICD. The NICD subsequently translocates to the nucleus to activate the transcription of Notch target genes, such as Hes/Hey family and nuclear factor-B (NF-B) (Yin et al., 2010; Andersen et al., 2012). And NICD are therefore to be considered as the active form of Notch receptor. Accumulating evidence suggests that deregulation of Notch1 has been implicated in the development, progression and survival of breast cancer (Kovall, 2007; Wu et al., 2007; Zang et al., 2007; Yin et al., 2010). One study showed that Notch1 signaling activation confers chemoresistance to rapamycin in lung cancer cells through inhibiting apoptosis via PI3K-Akt/PKB pathway (Mungamuri et al., 2006). Zang et al. recently reported that knock-down of Notch1 induces breast cancer cell apoptosis through the inactivation of NF-B, and additionally Notch1 down-regulation increases the chemosensitivity to doxorubicin and docetaxel (Zang et al., 2010). Notch signaling pathway also plays important roles in regulating stem cell proliferation, differentiation, self-renewal and apoptosis (Purow et al., 2005; Dang, 2012), which are closely related to tumor genesis. Phillips et al. (2006) reported that fractionated doses of irradiation increases the activation of Notch1 and the percentage of CD44+/CD24−/low breast CSCs. However, the effects and mechanisms of Notch1 in the chemoresistance to paclitaxel in the breast cancer need further investigation. ABCG2 is a member of ATP binding cassette (ABC) transporters, and its overexpression has been implicated in resistance to the chemotherapeutic agents including paclitaxel (Cheng et al., 2012; Seamon et al., 2006). ABCG2 is also widely expressed in a variety of CSC and can be recognized as a universal marker of CSC (Patrawala et al., 2005). Importantly, ABCG2 is verified to be the direct target of Notch signaling, and double immunocytochemical analysis has revealed that Notch1 and ABCG2 are co-localized in the E18 retinal progenitors (Bhattacharya et al., 2007). We reasoned that Notch1 may play an important role in paclitaxel resistance through influencing the proliferation of CSC and the expression of ABCG2 in breast cancer. In this study, we first confirmed that paclitaxel can enrich breast CSC (CD44+/CD24−) in a dose-dependent manner in MCF-7 breast cancer cell line. Then we demonstrated that Notch1 is overexpressed in breast CSCs isolated from paclitaxel-treated MCF-7 cell line. The short hairpin RNA (shRNA) mediated knock-down of Notch1 inhibits MCF-7 cell proliferation and induces cell apoptosis at least partly through the induction of NF-B, and this effect is sharply improved by combination with paclitaxel. Furthermore, knock-down of Notch1 inhibits the proliferative potential of breast CSC and confers breast CSC sensitive to paclitaxel in vitro and suppresses breast cancer tumor growth in vivo. Consistently, ALDH1, NICD, Hes-1, and ABCG2 were found to be decreased in shRNANotch1 transfected cells and in the mouse xenograft tumor tissues. Taken together, shRNA targeting Notch1 inhibits breast CSC proliferation and suppresses ABCG2 expression, thereby conferring sensitivity to paclitaxel. To the best of our knowledge, this work, for the first time, investigated the effects of Notch1 on paclitaxel resistance of breast CSC.
2. Materials and methods 2.1. Cell culture and reagents The human breast cancer cell line MCF-7 was purchased from Shanghai Culture Collection, and was maintained in DMEM/F-12 medium (Gibco Laboratories, Gran Island, NY) at 37 ◦ C in a humidified incubator with 5% CO2 . The culture medium was supplemented
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with 10% fetal bovine serum (Gibco Laboratories, Gran Island, NY), 2 mmol/L glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA). Paclitaxel was purchased from Sigma (St. Louis, MO).
2.2. Cell transfection The short hairpin RNA (shRNA) expression plasmids, pGPU6/GFP/Neo, were obtained from GenePharma (Shanghai, China). The construction of RNAi expression vectors for human Notch1 was as follows, oligo1 encoding human shRNA-Notch1-1 DNA oligomers (shRNA-Notch1forward 5 -GGAGCATGTGTAACATCAACA-3 , reverse 1): 5 -CCTCGTACACATTGT AGTTGT-3 ; oligo2 encoding human Notch1 shRNA-Notch1-2 DNA oligomers (shRNA-Notch1-2): forward 5 -GCAGCTGCACTTCATGTACGT-3 , reverse 5 -CGTCGACGT GAAGTACATGCA-3 . Scrambling nucleotide sequence of Notch1 (GenePharma, Shanghai, China) was used as negative control (shRNA-Control). shRNA-Notch1-1, -2 and shRNA-Control were transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions at a final concentration of 60 nM for 24 h, 48 h, and 72 h. Transfection rates were monitored with fluorescence microscopy. The cells without transfection were used as a blank control. The successfully transfecting cells were selected by additional 350 mmol/L of G418 (Gibco Laboratories, Gran Island, NY). At day 21, cells were collected for RT-PCR and Western blot analysis.
2.3. Paclitaxel treatment Stock solutions of paclitaxel were diluted in DMSO at four different doses (2, 4, 8 and 16 M) for in vitro experiments (Park et al., 2008) and were diluted according to the instructions for in vivo experiments (Kim et al., 2006).
2.4. Mammosphere formation assay Different groups of MCF-7 breast cancer cells were treated with shRNA against Notch1 alone, or combination with paclitaxel for mammosphere culture. Single cells then were plated in ultra-low attachment plates (Corning, Acton, MA) at a density of 2 × 104 viable cells/ml in primary culture, and 5 × 103 cells/ml in subsequent passages with serum-free medium, supplemented with 2 mmol/L L-glutamine (ATCC, Manassas, VA), 2% B27 supplement (Invitrogen, Carlsbad, CA), 20 ng/mL human recombinant epidermal growth factor (EGF) (Prepro Tech, Rocky Hill, NJ) and 20 ng/mL basic fibroblast growth factor (b-FGF) (Prepro Tech, Rocky Hill, NJ), 4 g/mL heparin and 5 g/mL insulin (Sigma, St. Louis, MO). Mammospheres were cultured for 7 days and were counted and collected by centrifugation at 1000 rpm.
2.5. Flow cytometry analysis MCF-7 cells were sorted with multiparametric flow cytometry using BD FACS Aria cell sorter (BD, Franklin Lakes, NJ) under sterile conditions. Cells at a density of 106 were collected and labeled with conjugated anti-human CD44-FITC (Invitrogen, Carlsbad, CA) and CD24-PE (Invitrogen, Carlsbad, CA). Antibodies were diluted in MACS buffer containing 5% BSA, 1 mM EDTA and 15–20% blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) to inhibit nonspecific binding to non-target cells. After 30 min incubation at 4 ◦ C, staining cells were washed, resuspended in 500 l of MACS buffer, and sorted.
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2.6. Isolation of breast cancer stem cell by microbeads Mammospheres were collected and enzymatically dissociated into single-cell suspensions. After counting with a hematocytometer, 1 × 107 cells were incubated with CD44 and CD24 primary mouse IgG antibodies (Gibco Laboratories, Gran Island, NY) for 5 min at 4 ◦ C. Unbounded primary antibodies were removed by PBS following by centrifuge at 300 × g for 10 min. The cells were then resuspended in 80 l of buffer with addition of 20 l of Goat Anti-Mouse IgG MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and incubated for 15 min at 4 ◦ C. Cells were washed again and preceded to magnetic separation. 2.7. Colony formation assay Cells were trypsinized, counted, and seeded for colony formation assay in 6-well plates at 300 cells per well. During colony growth, the culture medium was replaced every 3 days. At 7 days after seeding, the colonies were stained with 0.02% crystal for 1 h and then were counted in 5 random chosen fields under an inverted phase-contrast microscope (Olympus IX71). The colony was counted only if it contains more than 50 cells. Each treatment was carried out in triplicate. 2.8. CCK8 assay Cells were plated in 96-well plates at a density of 3 × 104 per well. At 24, 48, 72 and 96 h post-plating, 10 l Cell Counting Assay Kit-8 solution (Sigma, St. Louis, MO) was added to each well and incubated for 2 h, and the absorbance at 490 and 650 nm was measured using a microplate reader. Addition of DMEM/F12 alone was used as black control group. All experiments were done three times independently. 2.9. Staining with Hoechst 33342 Cells were washed twice in PBS and fixed in PBS containing 1% (wt/vol) paraformaldehyde (Fisher Scientific, Pittsburgh, PA). After rinsing with water, cells were stained with Hoechst 33342 (Sigma, St. Louis, MO) at final concentration of 10 g/ml for 10 min. The morphologic aspect of nuclei was observed under a fluorescence microscope using a 320–350 nm filter. This experiment was repeated three times.
GC-3 (forward) and 5 -CAC CTT CAC CGT TCC AGT TT-3 (reverse). The GAPDH GAPDH primers were 5 -GGTCGGAGTCAAAGGAT-3 (forward) and 5 -ATGAGCCCCAGCCTTCTCCAT-3 (reverse). Above primers were designed by TaKaRa (Dalian, China). Relative quantitation of mRNA expression levels was determined using the relative standard curve method according to the manufacturer’s instructions. 2.12. Western blot analysis Cells were lysed in 1× RIPA buffer (Sigma, St. Louis, MO). The protein concentration was determined by Bradford method with BSA (Sigma, St. Louis, MO) as the standard. Equal amount of protein extract (50 g) were subjected to 12% SDS-PAGE gels and transferred to nitrocellulose membrane (Millipore, Billerica, MA). The primary antibodies of Notch1 (1:500), Hes-1 (1:200), NFB (1:500), ALDH1 (1:200) and ABCG2 (1:200) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-NICD (1:200) was from Millipore (Billerica, MA). GAPDH (1:000) was used as an internal control which was from Santa Cruz Biotechnology (Santa Cruz, CA). 2.13. Tumor growth and morphologic analysis in vivo All studies involving mice were approved by Animal Care and Use Committee of Dalian Medical University. Twenty 6–8-week-old female nude mice were purchased from Experimental Animal Center of Dalian Medical University. Mice were randomly divided into two groups, group 1 was injected with shRNA-Control transfected cells, and group 2 was injected with shRNA-Notch1 transfected cells. To each group, 1 × 106 MCF-7 cells were resuspended in 100 l PBS mixed with matrigel (1:1) and injected into the mouse mammary fat pad. After 5 days, 5 mice were chosen from these two groups respectively and treated with 50 mg/kg paclitaxel every 2 days (Kim et al., 2006). The tumor mass was monitored using caliper. Tumor volumes (V) were calculated by the formula V = L × W2 × 0.5. On day 25 postinoculation, mice were weighed and sacrificed. The transplanted tumors were excised, weighed and frozen at −80 ◦ C until processing for RNA and protein isolation. For histological study, portions of tumors were fixed in 10% neutralbuffered formalin, paraffin-embedded and 4-m sections were stained for HE. For immunohistologic assay, Notch1 and ALDH1 were detected by anti-Notch1 and anti-ALDH1 primary antibodies respectively (Santa Cruz Biotechnology, Santa Cruz, CA).
2.10. Cell apoptosis analysis 2.14. Statistical analysis Cells were harvested and resuspended in 500 l of binding buffer and stained with 5 l of FITC-Annexin-V (BD Biosciences, San Jose, CA) and 5 l of propidium iodide (Sigma, St. Louis, MO) for 15 min in the dark at 4 ◦ C. After washing, cells were analyzed by flow cytometry. This experiment was repeated three times.
The results were expressed as the mean ± SD. Analysis was performed using a two-way Student’s t-test. Statistical significance was set as a P < 0.05. 3. Results
2.11. RNA extraction and RT-PCR assay of Notch1 and ALDH1A1 Total RNA was isolated from MCF-7 cells and mice transplanted tumor tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The concentration and purity of RNA were determined at 260/280 nm using a Nanodrop spectrophotometer (Thermo Scientific, Rockford, IL). Then cDNA was synthesized from approximately 5 ng RNA per 20 L using a cDNA reverse transcription kit (TaKaRa, Dalian, China). The Notch1 primers were 5 -GCA GTT GTG CTC CTG AAG AA-3 (forward) and 5 -CGG GCG GCC AGA AAC-3 (reverse). The ALDH1 primers were 5 -AAT GGC ATG ATT CAG TGA GTG GC-3 (forward) and 5 -GAG GAG TTT GCT CTG CTG GTT TG-3 (reverse). The internal control -actin primers were 5 -GAT CAT TGC TCC TCC TGA
3.1. CD44+/CD24− breast CSCs resistant to paclitaxel treatment overexpress Notch1 at both the mRNA and protein levels To determine whether paclitaxel enriches breast CSC, we treated MCF-7 cells with 2, 4, 8 and 16 M of paclitaxel for 7 days. After sorting by FACS, we found that the percentage of cells with CSC-markers (CD44+/CD24−) increased 1.22 ± 0.08, 1.55 ± 0.11, 2.63 ± 0.07, and 3.19 ± 0.07-fold respectively (Fig. 1A). Next, after treating MCF-7 cells with 8 M of paclitaxel for 7 days, we collected and cultured the surviving cells in low-attachment condition with serum-free medium, as described in Section 2. MCF-7 cells without paclitaxel treatment were used as the negative control. The floating mammospheres generated by small number of cells were passed by
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Fig. 1. Notch1 is highly expressed in CD44+/CD24− breast CSCs. (A) Paclitaxel significantly enriched CD44+/CD24− breast CSCs. MCF-7 cells were treated with various dose of paclitaxel (2, 4, 8, and 16 M) for 7 days and were detected by flow cytometry with CSC-markers (CD44+/CD24−). MCF-7 cells without paclitaxel treatment were used as the negative control. (B) Representative images of mammospheres on day 7. MCF-7 cells were treated with 8 M of paclitaxel for 7 days, and the surviving cells were collected and cultured in low-attachment condition with serum-free medium for 7 days (a, ×200; b, ×400, scale bar = 50 m). (C) CD44+/CD24− cells highly expressed ALDH1 which is a marker of breast CSC. The mammospheres were collected and sorted with CSC-markers by microbeads. The expression of ALDH1 was evaluated by immunologic assay. CSC represents CD44+/CD24− breast CSCs, non-CSCs represents non-CD44+/CD24− breast cancer cells, and un-separated represents the cells before sorting (scale bar = 50 m). (D) Notch1 expression was dramatically increased in breast CSCs compared to non-CSCs at both the mRNA (left) and protein (right) levels, and these differences are even prominent after paclitaxel treatment (8 M). RNA and protein were harvested, and Notch1 expression was determined by RT-PCR and Western blot. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (lower), and -actin and GAPDH were used for normalization respectively. *P < 0.05, **P < 0.01, Student’s t-test.
mechanical and enzymatic dissociation and shown in Fig. 1B. After 11 days of culture, we collected the spherical colonies and isolated the cancer stem cells with CSC-markers by microbeads. To further verify the stem cell nature of CD44+/CD24− cells, we then examined the expression of ALDH1, another confirmed marker of breast CSC (Ginestier et al., 2007) by cellular immunologic assay. The results showed that ALDH1 was expressed in the cytoplasm of more than 70% of CD44+/CD24− cells, whereas almost no ALDH1 expression was found in non-CD44+/CD24− cells, only a few un-separated cells expressed ALDH1 protein (Fig. 1C). These results indicated that CD44+/CD24− can be considered as the effective breast CSC markers. Paclitaxel treatment dramatically enriches CD44+/CD24− breast CSCs, and in turn breast CSCs are more resistant to paclitaxel. We then compared Notch1 expression levels between CD44+/CD24− breast CSCs and non-CD44+/CD24− breast cancer cells using RT-PCR and Western blot. As shown in Fig. 1D, the expression of Notch1 was significantly increased in breast CSCs
compared to non-CSCs at both the mRNA and protein levels, and these differences were even more profound after treating by paclitaxel, suggesting that Notch1 may play important roles in the resistance to paclitaxel possibly through stimulating the development of breast CSC. 3.2. Down-regulation of Notch1 inhibits colony-forming capacity and cell growth potential, and sensitizes MCF-7 breast cancer cells to paclitaxel To investigate the effect of Notch1 in paclitaxel resistance, we first compared two shRNAs specific against Notch1 (shRNANotch1-1, and -2) in silencing the expression of Notch1 in MCF-7 breast cancer cell, scrambling nucleotide sequence of Notch1 (shRNA-Control) was used as the negative control. We found transfection of shRNAs against Notch1 for 48 h had the highest transfection rate than that at any other time points when evaluated
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Fig. 2. Down-regulation of Notch1 inhibits colony-forming capacity and cell growth potential, and sensitizes MCF-7 breast cancer cells to paclitaxel. (A) Comparison of two shRNAs in silencing Notch1 expression at both the mRNA (left) and protein (right) levels. MCF-7 cells were transfected with two shRNAs (shRNA-Notch1-1 and shRNANotch1-2) respectively for 48 h. MCF-7 without treated (control) and scrambling nucleotide sequence of Notch1 (shRNA-Control) were the negative control. Then, cells were cultured under antibiotic pressure for 11 days and collected for RT-PCR and Western blot analysis of Notch1 and GAPDH. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software, and GAPDH was used as a negative control. (B and C) ShRNA-Notch1 alone or combination with paclitaxel reduced the number of colonies in MCF-7 cells. The number of colonies was counted after 7 days. (D) ShRNA-Notch1 alone or combination with paclitaxel inhibited the proliferation rate of MCF-7 cells. CCK8 assay was used to evaluate the cell growth ability. *P < 0.05, **P < 0.01, Student’s t-test. Each condition was repeated 3 times and error bars represent standard deviations.
by fluorescence microscopy (data not shown). The cells then were cultured under antibiotic selection pressure for 21 days and harvested to examine the Notch1 levels by RT-PCR and Western blot. Both the shRNA-Notch1-1 and shRNA-Notch1-2 significantly reduced the expression of, Notch1 compared to shRNA-Control, however, the shRNA-Notch1-2 showed slightly higher efficiency (Fig. 2A). Thus we selected shRNA-Notch1-2 (shRNA-Notch1) to perform the subsequent experiments. Next, colony formation and proliferation assays were performed in MCF-7 breast cancer cells to investigate the potential impact
of Notch1 on cell proliferation. Colonies containing at least 50 cells were counted on day 7 after plating. Our results showed that shRNA-Notch1 significantly suppressed the colony-forming capacity when combined with paclitaxel, indicating that Notch1 silencing sensitizes breast cancer cell line to paclitaxel treatment (Fig. 2B and C). Furthermore, we evaluated the proliferation rate in different groups by CCK8 assay, as Fig. 2D showed, down-regulation of Notch1 suppressed cell growth compared to its negative control, and shRNA-Notch1 combination with paclitaxel dramatically decreased the viability of MCF-7 cells. These results demonstrated
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Fig. 3. ShRNA-Notch1 enhances paclitaxel-induced cell apoptosis in MCF-7 cells partly through down-regulation of NF-B. (A) The apoptotic cells labeled with Hoechst 33342 increased when Notch1 is down-regulated, especially when combined with paclitaxel treatment. MCF-7 cells were transfected with 60 nM of shRNA-Notch1 and treated with 4 M of paclitaxel for 24 h, and then were stained with Hoechst 33342. The apoptotic cells emit strong blue fluorescence (red arrow, scale bar = 50 m). (B) Down-regulation of Notch1 increased the percentage of early period apoptotic cells labeled with Annexin V-FITC/PI. Early apoptosis in the lower right quadrant was taken into account because it is more representative. These experiments were repeated three times, and similar results were obtained. Representative scatter grams from flow cytometry profile represent Annexin V-FITC staining in the x axis and PI in the y axis. (C) The expression of NF-B was determined by Western blot (left). The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right), and GAPDH was used as a negative control. **P < 0.01, Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
that Notch1 down-regulation inhibits colony-forming capacity and cell growth potential of breast cancer cell line, and MCF-7 breast cancer cells with Notch1 down-regulation are more sensitive to paclitaxel. 3.3. Down-regulation of Notch1 enhances paclitaxel-induced cell apoptosis in MCF-7 breast cancer cells To further detect the possible mechanisms by which Notch1 down-regulation sensitizes breast cancer cells to paclitaxel, MCF-7 cells were transfected with shRNA-Notch1 or shRNA-Control and treated with 4 M of paclitaxel for 24 h, cell apoptosis progression was assessed using Hoechst 33342 and Annexin V-FITC/PI staining by flow cytometry. The apoptotic cells were deeply stained, shrunken and fragmented (Fig. 3A). The apoptotic cells labeled with Hoechst 33342 increased in the shRNA-Notch1 group compared to shRNA-Control group, especially when combined with paclitaxel treatment (Fig. 3A). Meanwhile, the percentage of early period apoptotic cells labeled with Annexin V-FITC/PI increased from 2.88% in shRNA-Control group to 8.12% in shRNA-Notch1
group, and the percentage of apoptotic rate in the combination of paclitaxel treatment groups were 11.34% and 19.61% respectively (Fig. 3B). These results suggested that Notch1 down-regulation sensitizes breast cancer cells to paclitaxel, at least partly through inducing apoptosis. To understand the underlying mechanisms, we further examined the levels of NF-B, which is a target gene of Notch1 to inhibit cell apoptosis (Mayo et al., 2001; Yin et al., 2010), by Western blot in different groups. NF-B expression was decreased significantly when treated with shRNA-Notch1, and this effect was sharply improved by combining shRNA-Notch1 with paclitaxel (Fig. 3C). These results indicated that Notch1 down-regulation sensitizes breast cancer cells to paclitaxel treatment through inducing cell apoptosis. This is achieved in part, by the induction of NF-B. 3.4. Down-regulation of Notch1 inhibits the proliferation of breast CSC, and confers breast CSC sensitive to paclitaxel Previously we found paclitaxel treatment can enrich breast CSC, and Notch1 is over-expressed in the breast CSCs compared to
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Fig. 4. Down-regulation of Notch1 inhibits the proliferation of breast CSC and confers breast CSC sensitive to paclitaxel. (A) Notch1 silencing alone or combination with paclitaxel resulted in the decreased volume (left) and number (right) of mammospheres. MCF-7 cells were treated with shRNA-Notch1 alone, or combination with paclitaxel, and then were cultured in ultra-low attachment plates with serum-free medium for 7 days. Mammospheres were collected and evaluated (scale bar = 100 m). (B) The percentage of CD44+/CD24− breast CSC was reduced by Notch1 down-regulation alone or combination with paclitaxel. The phenotype of CD44+/CD24− cells was measured by flow cytometry. Similar results were obtained in three independent experiments, and the representative microscopic picture and flow cytometry pattern were shown. (C) ABCG2, NICD and Hes-1 were down-regulated as Notch1 silencing or combination with paclitaxel (left). The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right), and GAPDH was used as a protein loading control. **P < 0.01, Student’s t-test.
the non-CSCs at both the mRNA and protein levels. It has been reported that mammary stem/progenitor cells are enriched in nonadherent spherical clusters of cells, termed mammospheres (Dontu and Wicha, 2005). Therefore mammosphere formation assay was carried out to determine whether Notch1 interference can suppress the propagation of breast CSC. MCF-7 cells were treated with shRNA-Notch1 alone, or combination with paclitaxel, and then were cultured in ultra-low attachment plates with serum-free medium for 7 days. As Fig. 4A shown, the size of the mammospheres was reduced dramatically, and the number of mammospheres was also reduced by about 45% by shRNA-Notch1 compared to shRNAControl. Furthermore, mammosphere-forming ability was reduced by about 72.4 ± 6.1% in the shRNA-Notch1 transfected cells combination with paclitaxel treatment, and only a small number of cells established mammospheres (Fig. 4A). We then quantified the number of cells with CSC-markers and found that the percentage of CD44+/CD24− was reduced by about 36.3 ± 3.7% and 67.2 ± 5.1% in the shRNA-Notch1 transfected cells and shRNANotch1 combination with paclitaxel treated cells (Fig. 4B). These
results further supported that Notch1 regulates the proliferation of breast CSC, and Notch1 knock-down can improve the chemosensitivity to paclitaxel through inhibiting the proliferation of breast CSC. Meanwhile, we noticed that there was no difference in the size and number of mammospheres between shRNA-Control group and shRNA-Control combination with paclitaxel group (shRNAControl + paclitaxel) (Fig. 4A), indicating that paclitaxel alone is able to inhibit cell proliferation only in breast cancer cell, not in breast CSC. We next performed Western blot to examine the expression of ABCG2 (Patrawala et al., 2005), in shRNA-Notch1 transfected MCF-7 and control cells. As Fig. 4C shown, ABCG2 was dramatically reduced in shRNA-Notch1 transfected cells relative to shRNAControl cells, suggesting that Notch1 down-regulation decreases cell population with ABCG2 positive in MCF-7 breast cancer cell. On the other hand, ABCG2 as a drug transporter was decreased by knock-down of Notch1 (Fig. 4C), thereby confers breast CSC sensitive to paclitaxel. Since ABCG2 is the direct target of Notch signaling (Bhattacharya et al., 2007), these results implied that
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Notch1 down-regulation induces breast CSC sensitive to paclitaxel possibly through inhibiting the proliferative potential and directly suppressing ABCG2 expression of breast CSC. The active form of Notch1, NICD and the downstream target of Notch1, Hes-1 were expressed in lower levels when Notch1 was down-regulated. 3.5. Inhibition of breast cancer growth by Notch1 down-regulation in vivo Given the observed inhibition effects of shRNA-Notch1 and paclitaxel on breast cancer cell proliferation in vitro, we next determined whether shRNA-Notch1 inhibits tumor growth and increases the sensitivity to paclitaxel treatment in vivo. We inoculated 1 × 106 MCF-7 cells stably transfected with shRNA-Control or shRNA-Notch1 into the mouse mammary fat pad for 3 days, and treated them with 50 mg/kg of paclitaxel every 2 days. On day 25 post-inoculation, mice were sacrificed. We found that at all time points, tumors in shRNA-Notch1 group were remarkably smaller than shRNA-Control group, and shRNA-Notch1 combination with paclitaxel treatment significantly inhibited the volume of tumor (Fig. 5A). Tumor weight had the same trend (Fig. 5A). No significant difference was found between shRNA-Notch1 group and shRNA-Control combination with paclitaxel group. By HE staining, shRNA-Notch1 group had more extensive necrosis compared to shRNA-Control group (Fig. 5B). Next we assessed the capacity of shRNA-Notch1 to inhibit breast cancer stem cell proliferation in vivo. We used immunohistochemistry to detect the expression of Notch1 and ALDH1 in the mouse xenograft tumor tissues. As Fig. 5B shown, Notch1 and ALDH1 were highly expressed in shRNA-Control group and shRNA-Control combination with paclitaxel group, whereas shRNA-Notch1 in the absence or presence of paclitaxel induced significant downregulation of ALDH1 protein. We further measured the levels of ALDH1 and ABCG2 by RT-PCR and Western blot in the mouse tumor tissues. We found Notch1 down-regulation significantly suppressed ALDH1 and ABCG2 expression both at mRNA and protein levels (Fig. 5C). The expressions of NICD and Hes-1 were decreased when Notch1 was down-regulated (Fig. 5D). These results indicated that Notch1 down-regulation inhibits the growth of breast cancer cell in vivo, decreases the ALDH1-positive cell population in the xenograft tumors, and confers sensitivity to paclitaxel possibly through suppressing the expression of ABCG2. 4. Discussion In the present study, we tested the role of Notch1 in chemoresistance to paclitaxel in breast cancer. Our data showed that shRNA targeting Notch1 markedly increased paclitaxel-induced cell apoptosis and inhibited cell proliferation in MCF-7 breast cancer cell line, indicating that Notch1 plays a critical role in chemoresistance to paclitaxel. It was reported that a nuclear transcription factor, NF-B, is most commonly involved in inhibiting apoptosis through transactivating anti-apoptotic genes (Mitsiades et al., 2002; Kucharczak et al., 2003). We also found that the expression of NF-B was decreased when treated with shRNA-Notch1 alone or combination with paclitaxel in MCF-7 cells. Notch1 has been reported to cross-talk with NF-B, and down-regulation of Notch1 results in lower activity of NF-B (Vilimas et al., 2006). Since apoptosis is a major manner by which paclitaxel kills cancer cells, these above results implied that Notch1 down-regulation confers breast cancer cells sensitive to paclitaxel through inducing apoptosis via inhibiting anti-apoptotic factor NF-B. Our results were consistent with another recent study (Zang et al., 2010). It showed that down-regulation of Notch1 increases the chemosensitivity of breast cancer cells to doxorubicin and docetaxel, and this effect
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may be caused by cell apoptosis induction through the inactivation of NF-B. Additionally, Notch1 down-regulation also contributes to apoptosis, possibly due to decreased Bcl-2 and Bcl-X(L) protein expression in pancreatic cancer cell, or mediated via inactivation of Akt and mTOR pathways in prostate cancer cell (Wang et al., 2006, 2010). Cancer stem cells (CSC) or tumor-initiating cells have been thought to play a major role in tumor initiation, and chemoand radiation-therapy resistance (Donnenberg and Donnenberg, 2005). To date, several studies have shown that epirubicin, cisplatin, docetaxel or doxorubicin, and cyclophosphamide can enrich the putative CSCs in the breast cancer model both in vitro and in vivo, and increased CSCs in turn induce the chemoresistance to these drugs (Donnenberg and Donnenberg, 2005). We found that paclitaxel can remarkably enrich breast CSCs in a dose-dependent manner in MCF-7 cells, implying that the enrichment of CSCs might be another mechanism for paclitaxel resistance in breast cancer. Many CSCs have deregulated signaling pathways which are involved in normal mammary stem cell regulation, such as Wnt, Hedgehog, Notch and estrogen (Reya and Clevers, 2005; Pannuti et al., 2010; Dontu et al., 2004). In this study, Notch1 was observed to be expressed at high levels in CD44+/CD24− breast CSCs sorted from paclitaxel-treated MCF-7 cell line at both the mRNA and protein levels. Knock-down of Notch1 dramatically reduced the size and number of mammospheres, and this inhibit effect was even more profound after combination with paclitaxel treatment. In line with these findings, the percentage of CD44+/CD24− cells was significantly reduced in shRNA-Notch1 transfected cells and combination with paclitaxel treated cells. We also noticed that paclitaxel alone could not influence the mammosphere-forming ability, further confirming the important role of breast CSC in the resistance to paclitaxel. In vivo, knock-down of Notch1 significantly inhibited the volume and weight of tumor compared to shRNA-Control, and this trend was even more remarkable in the presence of paclitaxel. Current views favor the model that cancer stem cells are innately resistant to chemotherapy through their relative quiescence, their enhanced capacity for DNA repair, decreased entry into apoptosis, and ABC transporter expression (Cheng et al., 2012; Donnenberg and Donnenberg, 2005). Many tumors in patients suffering from relapse to therapy are resistant to multiple drugs (multidrug resistant, MDR) (Gottesman et al., 2009). The P-glycoprotein encoded by the ABCB1 gene was the first ABC transporter identified to be amplified and/or overexpressed in MDR tumor cell lines (Chen et al., 1986). There are two other ABC proteins widely overexpressed in tumor cells, ABCC1/MRP1 and ABCG2 (Haimeur et al., 2004). The conservation of ABCG2 expression in Side Population Progenitor cells (SP cells), enriched from a wide range of tissues including blood, lung and intestine (Patrawala et al., 2005), suggesting that ABCG2 has an important role in the chemoresistance. We found that knock-down of Notch1 remarkably reduced the expression of ALDH1 and ABCG2 in the mammospheres. In vivo, although the tumor was inhibited by paclitaxel a little bit more than by shRNA-Notch1, however, the expression of ABCG2 was down-regulated only by shRNA-Notch1 but not paclitaxel. These data implied that paclitaxel could not eliminate the CSC possibly due to the high level of ABCG2 in CSCs, and this might cause incomplete eradication of CSCs. It is considerable to see that the breast CSC decreased conspicuous in the group treated with shRNANotch1 combination with paclitaxel both in vitro and in vivo and the expressions of NICD, Hes-1 and ABCG2 were reduced much more than paclitaxel treated only. Since ABCG2 was reported to be the direct target of Notch signaling (Bhattacharya et al., 2007), these results implied that Notch1 down-regulation induces breast CSC sensitive to paclitaxel possibly through inhibiting the proliferative potential and directly suppressing ABCG2 expression of breast CSC.
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Fig. 5. Inhibition of shRNA-Notch1 on tumor growth in vivo. (A) ShRNA-Notch1 alone or combination with paclitaxel remarkably reduced the tumor volume and weight. 1 × 106 MCF-7 cells stably transfected with shRNA-Control or shRNA-Notch1 were inoculated into the mouse mammary fat pad for 3 days, and then were treated with 50 mg/kg of paclitaxel every 2 days. On day 25 post-inoculation, mice were sacrificed. The volume of the peripheral tumor was measured every 3 days post-inoculation. Error bars represent standard deviations, n = 5. **P < 0.01, Student’s t-test (scale bar = 1 cm). (B) HE and immunohistochemistry staining with Notch1 and ALDH1 antibodies. a–d: HE staining; c–l: immunohistochemistry staining for Notch1 and ALDH1 (brown color in cytoplasm), ×200 (scale bar = 100 m). (C) RT-PCR (left) and Western blot (right) analyses of ALDH1 in the mouse tumor tissues. (D) Protein levels of Notch1, NICD, Hes-1 and ABCG2 were measured by Western blot in mice tumors (left). As an internal control, GAPDH was used for normalization. The quantitative results were analyzed by Gel-Pro Analyzer 4.0 software (right). Data are presented as mean ± SD, n = 5, **P < 0.01, Student’s t-test.
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