J Cancer Prev 2020; 25(4): 234-243
Published online December 30, 2020
© Korean Society of Cancer Prevention
1Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, 2Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, 3College of Pharmacy, Seoul National University, 4Cancer Research Institute, Seoul National University, Seoul, Korea
Aberrant activation of Ras has been implicated in aggressiveness of breast cancer. Among Ras isoforms (H-, K-, and N-), H-Ras has been known to be primarily responsible for invasion and metastasis of breast cancer cells. Phosphorylation of serine (Ser) or threonine (Thr) is a key regulatory mechanism responsible for controlling activities and functions of various proteins involved in intracellular signal transduction. Peptidyl-prolyl
Keywords: Breast neoplasm, Drug resistance, H-Ras, Peptidyl-prolyl
Genetic inhibition of Pin1 in H-
Apoptosis acts as a natural barrier to prevent initiated cells with DNA damage from being converted to (pre)malignant cells [15,16]. However, cancer cells can escape from apoptosis by activating anti-apoptotic signals, thereby acquiring the survival advantage under cytotoxic stress. Pin1 inhibits pro-apoptotic signals while activating anti-apoptotic signals in cancer cells. The activation of the proapoptotic Bax and its mitochondrial localization were significantly increased after Pin1 blockade . Pin1 is highly overexpressed in malignant human gliomas and inhibits D cellular apoptosis mediated by the death-associated protein Daxx . Pin1 also potentiates the anti-apoptotic function of Bcl-2 and myeloid cell leukemia-1 [19,20]. In addition, Pin1 upregulates LC-3 expression to induce protective autophagy, which consequently confers the resistance of breast cancer cells to tamoxifen . The depletion of Pin1 significantly inhibits tumorigenesis in mice with mutated p53 , over-activated HER2/Ras  or constitutively overexpressed c-Myc .
Acquired resistance to chemotherapy is a major obstacle to successful cancer treatment. Proteins of the ABCC/MRP transporter family play pivotal roles in drug resistance by actively exporting chemotherapeutic drugs in various cancers . In experimental models, manifestation of the MDR phenotype is often accompanied by the expression of the MDR1 gene (also known as
Here we report that Pin1 interacts with GTP-H-Ras and upregulates drug resistance genes, conferring survival advantage of transformed or cancerous mammary epithelial cells under chemotherapy.
Dulbecco’s Modified Eagle medium (DMEM) Nutrient mixture F-12 (Ham), penicillin/streptomycin mixtures and FBS were obtained from Gibco BRL (Grand Island, NY, USA). TRIzol® reagent and StealthTM RNAi negative control duplexes were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA, USA). Primary antibody for Pin1 was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibody for GTP-H-Ras was supplied by Abcam (Cambridge, UK). Secondary antibodies were purchased from Zymed Laboratories Inc. (San Francisco, CA, USA). Western blot detection kit (Absignal) was obtained from Abclon (Seoul, South Korea). Control and
MCF10A and H-Ras MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 20 ng/mL EGF, 0.1 μg/mL cholera enterotoxin, 100 units/mL penicillin-streptomycin, 2 mM L-glutamine, and 0.5 μg/mL amphotericin. Cells were maintained in a humidified atmosphere with 95% air and 5% CO2 at 37°C. The human breast cancer (MDA-MB-231) cell line obtained from American type culture collection was maintained in DMEM containing 5% FBS at 37°C in a 5% CO2/95% air incubator.
Lentiviruses were produced by transfecting HEK293T cells using lentiviral vectors. In brief, HEK293T cells transfected with Pin1 short hairpin RNA (shRNA; shPin1) lentiviral vector were re-transfected with VSV-G-, pLP1- and pLP2-expressing plasmids, and lentiviral supernatants were collected at 48-hour and 72-hour post-transfection. H-Ras MCF10A cells were infected with shPin1 or control virus with 5 μg/mL polybrene, and stable clones were selected using 1 μg/mL puromycin.
MDA-MB-231 cells were plated in 6-well plates at a density of 150 cells per well. Cells were treated with 25 μM ATRA, a pharmacologic inhibitor pf Pin-1. The DMEM medium was changed every other day. After one week of incubation, the colonies were fixed in cold methanol and stained by 0.5% crystal violet for 4 hours. The stained colonies were washed with PBS to remove the excess dye. For experiments with shPin1 H-Ras MCF10A cells, cells were plated in 6-well plates at a density of 150 cells per well. Cells were treated with 10 nM paclitacxel. The DMEM/F12 medium was changed every other day. After one week of incubation, the colonies were fixed in cold methanol and stained by 0.5% crystal violet for 4 hours. The stained colonies were washed with PBS to remove the excess dye. Quantitative changes in clonogenicity were determined by extracting stained dye with 10% acetic acid, and the absorbance at 570 nm was measured.
The proximity ligation assay (PLA) was carried out using the DUOLinkTM kit (OLINK, Uppsala, Sweden) according to the manufacturer’s instructions. In brief, MDA-MB-231 cells on glass coverslips were fixed, permeabilized, and blocked with blocking solution (0.1% Triton in PBS containing 5% bovine serum albumin) and incubated with the antibodies against Pin1 (1 : 10) and GTP-H-Ras (1 : 5) overnight at 4°C. PLA plus and minus affinity probes were then added and incubated for 1 hour at room temperature. The probes were hybridized using a ligase to be a closed circle. The DNA was then amplified (a rolling-circle amplification) and detected by fluorescence microscopy.
MCF10A, H-Ras MCF10A and MDA-MB-231 cells were lysed in lysis buffer (250 mM sucrose, 50 mM Tris-HCl [pH 8.0], 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 2 mM NaF, 2 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) for 1 hour on ice followed by centrifugation at 18,000 ×
Cells were lysed in 250 mM sucrose, 50 mM Tris-HCl (pH 8.0), 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 2 mM NaF, 2 mM sodium orthovanadate, and 1 mM PMSF. Total protein (500 μg) was subjected to immunoprecipitation by shaking with Pin1 and GST-H-Ras primary antibodies followed by the addition of protein A/G-agarose bead suspension and additional shaking at 4°C for 12 hours. After centrifugation at 1,000 ×
Tumor tissues were fixed in 3.7% paraformaldehyde for 24 hours. Paraffin-embedded tissues were serially sectioned in 4 μm and mounted on a slide. For antigen retrieval, citrate buffer (DakoCytomation; Glostrup, Denmark) was used. The tissue sections were treated with primary antibodies for anti-Pin1 (1 : 100), and anti-GST-H-Ras (1 : 200) at 4°C, overnight. The secondary antibodies were treated at room temperature for 1 hour; biotinylated anti-rabbit (1 : 500), and anti-mouse Alexa568 (1 : 200). MCf10A, H-Ras MCF10A, and MDA-MB-231 cells were fixed with 4% paraformaldehyde solution for 15 minutes at room temperature, incubated with blocking agents (0.1% Triton in PBS containing 10% bovine serum albumin), washed with PBS and co-incubated overnight with GST-H-Ras (1 : 200) and Pin1 (1 : 100) primary antibodies at 4°C. After washing with PBS, samples were co-incubated with diluted (1 : 5,000) FITC-conjugated anti-rabbit and anti-mouse immunoglobulin G secondary antibodies for additional 1 hour at room temperature. The cells were then examined under a fluorescence microscope (Nikon; Tokyo, Japan) or a confocal microscope (Leica; Berlin, Germany).
siRNA specifically targeting
Total RNA was isolated using Trizol® reagent (Invitrogen). The RNA quality was assessed by Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies; Amstelveen, The Netherlands), and RNA quantification was performed using ND-2000 Spectrophotometer (Thermo Inc.; Wilmington, DE, USA).
For control and test RNAs, the construction of the library was performed using QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen Inc.; Vienna, Austria) according to the manufacturer’s instructions. In brief, each total RNA was prepared and an oligo-dT primer containing an Illumina-compatible sequence at its 5’ end was hybridized to the RNA and reverse transcription was performed. After the degradation of the RNA template, second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5’ end. The double-stranded library was purified by using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library was purified from PCR components. High-throughput sequencing was performed as single-end 75 sequencings using NextSeq 500 (Illumina, Inc.; San Diego, CA, USA).
QuantSeq 3’ mRNA-Seq reads were aligned using Bowtie2. Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for aligning to the genome and transcriptome. The alignment file was used for assembling transcripts, estimating their abundances and detecting differential expression of genes. Differentially expressed gene was determined based on counts from unique and multiple alignments using coverage in Bedtools. The Read Count data were processed based on the quantile normalization method using EdgeR within R using Bioconductor. Gene classification was based on searches done by DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (http://www.ncbi.nlm.nih.gov/). The results were presented as mean ± SD. To determine the statistical significance, the Student’s unpaired
All data are presented as the mean ± SD. Experiments were repeated at least three times. Two-tailed unpaired Student’s
To investigate the role of Pin1 in breast cancer progression, we compared the expression of Pin1 in normal and tumor tissues from the different subtypes of breast cancer patients. As illustrated in Figure 1A, expression of Pin1 was upregulated in the triple negative breast cancer (TNBC) and luminal type breast tumor tissues, compared with that in the adjacent normal tissues. Next, we investigated the expression of Pin1 in human breast cancer cells. As shown in Figure 1B, the level of Pin1 protein was significantly higher in breast cancer cells than in the non-transformed MCF10A normal cells.
In order to explore the cooperative role of Pin1 and GTP-H-Ras in breast cancer development and progression, we examined their co-localization in human breast tumor specimens. While normal tissues exhibited relatively low immunofluorescence signals detected by antibodies recognizing Pin1 and GTP-H-Ras, the tumor tissues showed highly enhanced intensities and co-localization of both proteins (Fig. 1C). There was a significant correlation between Pin1 and GTP-H-Ras in TNBC tissues (Fig. 1D). The subcellular distribution of Pin1 and GTP-H-Ras in MCF10A, H-Ras MCF10A and MDA-MB-231 cells was examined by immunofluorescence analysis. We found that H-Ras MCF10A and MDA-MB-231 cells express Pin1 and GTP-H-Ras to a greater extent than the MCF10A parental cells (Fig. 2A).
The co-localization of Pin1 and GTP-H-Ras prompted us to examine whether both proteins could physically interact with each other. The data from human protein reference database indicate the presence of WW motifs of GTP-H-Ras suitable for binding to Pin1 (Fig. 2B). An immunoprecipitation assay revealed that there was a pronounced interaction between Pin1 and GTP-H-Ras in MDA-MB-231 cells (Fig. 2C). The direct interaction between Pin1 and GTP-H-Ras was verified by the PLA assay, which detects an enhanced fluorescent signal when two proteins are localized in proximity (Fig. 2D).
The regulation of MDA-MB-231 cell growth and proliferation by Pin1 was evidenced by marked reduction in colony formation (Fig. 3A) following treatment with the Pin1 inhibitor, ATRA.
In another experiment, treatment of H-Ras MCF10A cells with administration of paclitaxel (PTX), significantly reduced the number of colonies in shPin1 stable cells compared with that in shControl group (Fig. 3B). PTX treatment induced elevated production of the cleaved forms of Caspase 3 and PARP which are hallmarks of apoptotic cell death, and this was more prominent in
The expression of GTP-H-Ras was not different between
Breast cancer has been estimated as one of the most common causes of cancer-related death among women. Although
It has been reported that the hypervariable region (HVR) of H-Ras is important for its activation [27-29]. HVR, consisting of amino acids 166 to 189 of H-Ras, is essential for manifestation of the invasive phenotype of human breast epithelial cells . The single point mutation at the amino acid residue 12 (Gly to Asp) of H-Ras is more often found in mammary carcinoma, whereas the same mutation of N-Ras is detected in teratocarcinoma and leukemia .
ERK activation was found to be required for H-Ras-mediated migration and invasiveness of human breast epithelial MCF10A cells . Previous studies have shown that p38 lies downstream of the Ras-related GTP-binding proteins Rac and Cdc42, and is directly activated by MKK3, MKK6, and MKK4 [30-33]. It appears that the p38- and ERK-mediated signaling pathways are independent of each other, and both pathways cooperate in H-Ras-mediated migrative and invasive responses in MCF10A cells.
Ras proteins are also subjected to ubiquitination and proteasomal degradation. In addition to GDP/GTP exchange, activities of Ras molecules are regulated by site-specific ubiquitination, which occurs on multiple lysine residues. This modification has been shown to alter the Ras function by altering its subcellular localization and protein–protein interactions as well as promoting its degradation . Glycogen synthase kinase 3β, a negative regulator of the Wnt/β-catenin pathway, contributes to the phosphorylation of H-Ras and the subsequent recruitment of β-TrCP-E3 ligase to phosphorylated H-Ras, leading to the degradation of H-Ras, but not other Ras isoforms [35,36]. Another study has revealed that LZTR1 facilitates the polyubiquitination and degradation of Ras proteins, leading to the inhibition of the Ras-MAPK signaling .
Pin1 cooperates with activated JNK or H-Ras in increasing the transcriptional activity of phosphorylated c-Jun bound to activate the cyclin D1 promoter [38,39]. Consequently, overexpressed cyclin D1 contributes to cell transformation . In the HVR of H-Ras, there are three Pro residues at positions 173, 174, and 179, whereas N-Ras harbours none of these residues. It is noticeable that Pro173 and Pro174 are extensively conserved among species (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/pubmed/), indicative of their importance for the H-Ras function . Pro-rich sequences, often found in the hinge regions, are known to restrict the conformation of proteins and play an indispensable role in the regulation of various cellular responses [10,40].
In this study, we have examined the expression of Pin1 in breast cancer tissues and in some breast cancer cells. The protein levels of Pin1 in breast cancer tissues and cell lines are much higher than those in normal tissues and non-transformed mammary epithelial cells, respectively. Further, expression levels of Pin1 and H-Ras were significantly correlated in TNBC tissues, and H-Ras-transformed MCF10A, and MDA-MB-231 cells, suggesting the Pin1 and H-Ras complex as a potential marker for breast cancer. GTP-H-Ras harbours three WW binding motifs responsible for interaction with Pin1 (Fig. 2B). Immunoprecipitation and PLA assays indicated the strong interaction between GTP-H-Ras and Pin1 in MDA-MB-231 cells. We speculate that H-Ras protein bound to Pin1 is stabilized by avoiding ubiquitination and proteasomal degradation (Fig. 5).
The inhibition of Pin1 activity by ATRA significantly decreased the clonogenicity of MDA-MB-231 cells, indicating the oncogenic role of Pin1. We have also demonstrated that Pin1 silencing sensitized H-Ras transformed MCF10A cells to PTX-induced apoptosis. Pin1 silencing increased the cleavage of Caspase-3 and PARP, each of which is a hallmark of apoptosis. As knock down of Pin1 triggers tumor cells to undergo apoptosis, overexpressed Pin1 may represent an anti-cancer target.
MRP4 has been implicated in the high proliferative growth of some tumors including prostate tumors and neuroblastoma [41,42]. In addition to its drug (and drug metabolite) transporting function, MRP4 mediates the cellular efflux of several endogenous metabolites that play critical roles in signalling pathways involved in such processes as differentiation, pain perception and inflammation . Tumor chemotherapy not only acts on cancer cells, but also exerts toxicity on normal cells, and often results in rapid development of acquired drug resistance in tumor cells, particularly through upregulation of MDR transporters . It is believed that MDR is the most important self-protection mechanism of tumor cells which contributes to the failure of chemotherapy. So far, three MDR isoforms have been identified. Overexpression of MDR1 is widely considered to account for resistance to chemotherapy in women with breast tumor . We found that MRP4 as well as MDR1 was highly overexpressed in H-Ras transformed MCF10A cells compared with that in non-oncogenic MCF10A cells. Conversely, the expression of aforementioned drug resistance genes was significantly reduced in Pin1 silenced stable cells.
Taken all together, the results of the present study indicate interplay between Pin1 and H-Ras which may play a significant role in the growth and progression of breast cancer.
The authors acknowledge the supply of human breast cancer specimens by Prof. Wonshik Han of Seoul National University Hospital. This study was supported by the Global Core Research Center (GCRC) grant (No. 2011-0030001) from the National Research Foundation, Republic of Korea.
No potential conflicts of interest were disclosed.
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