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Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab
GENE-40837; No. of pages: 6; 4C: Gene xxx (2015) xxx–xxx
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab Elham Abu Sharieh a,b, Abdulla S. Awidi b,c, Mamoun Ahram d, Malek A. Zihlif a,⁎ a
Department of Pharmacology, Faculty of Medicine, The University of Jordan, Amman, 11942, Jordan Stem Cell Therapy Center, Faculty of Medicine, The University of Jordan, Amman, 11942, Jordan Department of Internal Medicine Hematology and Oncology Unit, University of Jordan, Amman 11942, Jordan d Department of Physiology and Biochemistry, Faculty of Medicine, The University of Jordan, Amman, 11942, Jordan b c
a r t i c l e
i n f o
Article history: Received 29 March 2015 Received in revised form 23 June 2015 Accepted 7 September 2015 Available online xxxx Keywords: Trastuzumab resistance Epithelial-to mesenchymal transition
a b s t r a c t Development of resistance against cancer therapeutic agents is a common problem in cancer management. Trastuzumab resistance is one of the challenges in management of HER-2-positive breast cancer patients resulting in breast cancer progression, metastasis, and patient poor outcome. The aim of this study is to determine the alteration in gene expression in response to Trastuzumab resistance after long-term exposure to Trastuzumab. The Trastuzumab-resistant MDA-MB-453 (MDA-MB-453/TR) cell line was developed by exposing cells to 10 μM Trastuzumab continuously for 6 months. Sensitivity toward Trastuzumab was tested using cell viability assays. The acquisition of an epithelial-to mesenchymal transition (EMT) phenotype was also observed in parallel with the development of resistance. Based on the real-time-based PCR array technology, several genes were altered affecting multiple networks. The most up-regulated genes were TGF-β1 and EGF, and IGFBP-3. These genes are known to have a critical role in Trastuzumab resistance in breast cancer cell lines and/or in the acquisition of EMT. They are also recognized for their role in cancer progression and metastasis. These alterations indicate that the development of Trastuzumab resistance is multifactorial and involves a development of a mesenchymal like phenotype. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Breast cancer is the most commonly occurring malignancy in women worldwide (Vranic et al., 2011) representing 23% of all cancer cases (Goto et al., 2014). The disease is commonly classified based on gene expression profiles into four main subtypes. The luminal A subtype is characterized by high expression of estrogen receptor alpha (ESR-A), whereas the luminal B subtype is characterized with mixed expression of ESR-A, progesterone (PR) and/or human epidermal receptor 2 (HER-2) (Babayan et al., 2013). A third type is named HER2 enriched subtype, which is characterized by over-expression of the HER2 gene. Lastly, a subtype of breast cancer that lacks expression of any of the
Abbreviations: (ESR-A), estrogen receptor alpha; (PR), progesterone; (HER-2), human epidermal receptor 2; PCR, POLYMERASE chain reaction; (FDA), Food and Drug Administration; (ATCC, USA), American Type Culture Collection; (FBS), fetal bovine serum; (MTT), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; (IC50), the inhibitory concentrations; (ACTB), actin beta; (GAPDH), beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase; (HPRT1), hypoxanthine phosphoribosyltransferase 1; (RPLP0), ribosomal protein large, P0; (DAVID), database for Annotation, Visualization and Integrated Discovery; (IGFBP-3), the insulin-like growth factor binding protein3; (TGFβ1), the transforming growth factor beta 1; (EMT), epithelial-to mesenchymal transition. ⁎ Corresponding author. E-mail address: [email protected] (M.A. Zihlif).
aforementioned receptor genes is known as triple-negative breast cancer. The HER2-positive breast cancer accounts for 20–30% of all breast cancers and has the second poorest prognosis among breast cancer subtypes (Vu and Claret, 2012). Over-activation of HER-2 receptors leads to uncontrolled cellular growth and proliferation, tumor metastasis, and prevention of apoptosis in malignant cells. The production of monoclonal antibodies (mAbs) that are specific against molecular targets of cancer cells or within the tumor microenvironment have shown success in cancer management (Strome et al., 2007). Among these mAbs is Trastuzumab, which is one of the success stories of biologically targeted breast cancer therapy that has revolutionized the management of HER2 positive breast cancer (Damiano et al., 2009). This humanized mAb belongs to the immunoglobulin subclass 1 (IgG1) that was the first targeted therapy approved by Food and Drug Administration (FDA) for management of HER-2 positive breast cancer (Romond et al., 2005). The mAb targets the extracellular domain of HER-2 receptor blocking its function via acting through antibody-dependent cellular cytotoxicity, stimulating receptor internalization and degradation, and, consequently, arresting cancer cells at the G1 phase of the cell cycle (Nahta and Esteva, 2006). Breast cancer patients having positive diagnosis for HER2 and, hence, treated with this mAb have shown reduced tumor growth, decreased tumor size, and increased 5-year survival.
http://dx.doi.org/10.1016/j.gene.2015.09.019 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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Among the most common challenges in treating HER2-positive breast cancers is the development of resistance within the first year of treatment. Trastuzumab resistance can either be primary (de novo) when the Trastuzumab is ineffective for the treatment of breast cancer patients despite tumor expression of HER2 leading to no response to treatment, or secondary (acquired) when patients who initially respond to Trastuzumab treatment, but experience a Trastuzumab-refractory relapse leading to loss of clinical benefits.(Nahta and Esteva, 2003; Nahta and Esteva, 2006; Nahta et al., 2006; Wilken and Maihle, 2010). This phenomenon necessitates identification of changes in gene expression associated with development of Trastuzumab resistance that can lead to better understanding of the perturbations in molecular network. In this study, we utilized the breast cancer MDA-MB453 cell line that over-expresses HER2 to establish resistant line via exposing the cells to a long-term, continuous dose of Trastuzumab. Alterations in gene expression were determined using the PCR array technology that contains most commonly altered human breast cancer genes. 2. Material and methods 2.1. Cell culture growth conditions All cell culture reagents were obtained from Thermo Scientific Inc. (USA). The MDA-MB-453 cell line was originally obtained from the American Type Culture Collection (USA). The cells were maintained as an attached monolayer culture in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM 100 u/mL L-glutamine, and 100 μg/mL penicillin-streptomycin. The cells were incubated at 37 °C in a 90% humidified atmosphere of 5% CO2. 2.2. Development of Trastuzumab resistant MDA-MB-453 cell line Trastuzumab (Herceptin; F. Hoffmann-La Roche, Basel, Switzerland) was utilized in this study. To develop resistant cell line termed MDAMB-453/TR. Two approaches were attempted. The first approach was based on treating the cells with an initial concentration of 2 μM of Trastuzumab and increasing the dose gradually. In the second approach, the cells were exposed to a continuous, steady concentration of 10 μM of Trastuzumab. Both approaches were previously utilized to develop Trastuzumab-resistant cells.(Wang et al., 2011; Wu et al., 2012).
2.4. Gene expression profiling Total RNA was isolated using an RNeasy® Mini kit and the RNasefree DNase set (Qiagen, Germany) according to the manufacturer's instructions. Aliquots containing 1 μg of total RNA were used from each sample to synthesize complementary DNA strands using RT2 First Strand kit (Qiagen, Germany). The Human Breast Cancer PCR Array (Qiagen, USA) was used to determine alterations in gene expression resulting from developing Trastuzumab resistance. The array contains 84 breast cancer-specific genes involved in tumor classification, signal transduction and other commonly affected pathways such as angiogenesis, adhesion, proteolysis, cell cycle, and apoptosis. A diluted cDNA aliquot, equivalent to 1 μg total RNA for each plate, was mixed with the RT2 SYBR® green master mix (Qiagen, USA) according to the manufacturer's instructions, and loaded onto the 96-well array. The PCR cycle was performed using the iCycler (Bio-Rad, USA). Each cell sample was performed in duplicates. The cycle threshold (Ct) values for each sample were given automatically by the iCycler thermal cycler (BioRad, USA) according to the amplification curves of both MDA-MB-453/Control and MDA-MB-453/TR. The threshold value was manually set on 0.01. Changes in gene expression were calculated using the ΔΔCt method utilizing data analysis tool of the SABiosciences Company (Qiagen, USA). The data were normalized, across all plates, to the following housekeeping genes: Actin beta (ACTB), Beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and ribosomal protein large, P0 (RPLP0). 2.5. Gene functional annotation The genes whose expression levels were altered by ≥4-fold were further examined using the web–based, gene annotation, bioinformatics tool, the Database for Annotation, Visualization and Integrated Discovery (DAVID) website (http://david.abcc.ncifcrf.gov/), which provides biological classification of the genes. Results are displayed in comparative tables that describe the most affected biological function by genes changes. 3. Results 3.1. Development and characterization of the MDA-MB-453/TR cells
2.3. Cell proliferation assays
In order to establish Trastuzumab-resistant MDA-MB-453 cells, two approaches were carried out. In the first approach, the cells were initially grown in the presence of low concentration of Trastuzumab (2 μM) and increasing the dose gradually with every cell passage. However,
The anti-proliferative effects of the parental MDA-MB-453 cells and MDA-MB-453/TR cells were evaluated by the Cell Titer Non-Radioactive Cell Proliferation Assay Kit® (Promega, USA) according to manufacturer's instructions. This assay is a colorimetric test that is based on the reduction of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) converting it from a yellow tetrazole to a purple formazan, a process that occurs in the mitochondria of viable cells. The cells were seeded onto 96-well plates (Greiner, Germany) at a concentration of 14 × 103 cells/well and incubated overnight. Horizontal dilutions of the drug ranging from 0.1–100 μM were added. Each concentration was added in triplicates, and every plate contained a control of cells in plain media. Optical density (OD) at 570 nm wavelength was recorded. The inhibitory concentrations (IC50) values were calculated from the logarithmic trend line of the cytotoxicity graphs using the Graph Pad PRISM® 5.0 software (Graph Pad Software, Inc., city, country). The degree of resistance was evaluated in terms of resistance index (R), which was calculated according to the equation: R = IC50 resistant cells/IC50 sensitive cells.
Fig. 1. The MDA-MB-453 cells viability assay curves A. The MDA-MB-453/control cells viability assay curve, the viability assay is done for MDA-MB-453 breast cancer cell line not exposed to Trastuzumab at passage number (P80), the concentration at which (0.5) of cells were viable is approximately 15.84 μM. B. The MDA-MB-453/TR cells viability assay curve, the viability assay is done for Trastuzumab resistant MDA-MB-453 breast cancer cell line (exposed to 10 μM Trastuzumab for 6 months) passage number (P53), the concentration at which (0.5) of cells were viable is approximately 32.28 μM.
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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this approach was not successful as cells became senescent at a concentration of 20 μm and died at a concentration of 36 μM. The second approach was based on exposing the cells to a continuous steady concentration of 10 μM of Trastuzumab for approximately six months for a total of 33 doses. The 10-μM concentration was selected according to the first measured inhibitory concentration of 50% (IC50) value for untreated MDA-MB-453 cells, which was approximately 10 μM. This approach was successful and resistant cells (MDA-MB-453/TR) that could survive and replicate at 36 μM of Trastuzumab were developed. The proliferative capacity and the viability of the parental MDA-MB453 and the MDA-MB-453/TR cells were determined at different concentrations of Trastuzumab. As illustrated in Fig. 1, whereas MDAMB-453/Control had an IC50 of 15.84 ± 1.168 μM (mean ± STD), the IC50 for MDA-MB-453/TR was 32.28 ± 3 μM (mean ± STD). According to the R-value, the MDA-MB-453/TR cells was approximately 2-fold more resistant to Trastuzumab than the parental cell line.
3.1.1. Morphological features Upon developing a resistance phenotype, gradual changes in the cell morphology was observed. The parental cells existed as clusters
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of rounded cells. Continuing treatments and passaging of the Trastuzumab-treated cells induced loose cell connectivity with cells acquiring a spindle-shape and elongated morphology. This morphological change became more pronounced with longer periods of treatments (Fig. 2).
3.2. Alterations in gene expression in association with resistance development A gene expression profile analysis was performed using the Human Breast Cancer PCR array. A standard 4-fold change in expression was used as an arbitrary cut-off. The Scatter plot shown in Fig. 3 illustrates the differences in expression levels of the tested genes between the resistant and parent cell line. Out of the 84 genes, the expression level of 13 genes was altered by more than 4 folds. Four of these genes were up-regulated including the insulin-like growth factor binding protein 3 (IGFBP-3) and the transforming growth factor beta 1 (TGFβ1), both of which showed the highest level of up-regulation of 10 and 8 folds, respectively. Two genes, PRDM2 and EGF, were up-regulated to a lesser degree. On the other hand, nine genes were down-regulated and
Fig. 2. Morphology of MDA-MB-453/TR cells treated with 10 μM Trastuzumab (A) compared to untreated MDA-MB-453/control (B) at different passage numbers (P).
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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Fig. 3. Scatter plots of human breast cancer genes expression in MDA-MB-453/TR cells compared to MDA-MB-453/Control cells. A standard 4-fold change in expression was used as an arbitrary cut-off, the up-regulated genes are marked with red stars and the down-regulated genes are marked with green stars, the unchanged genes are marked with black stars. Abbreviations: IGFBP3: insulin-like growth factor binding protein 3, TGFβ1: transforming growth factor βa1, PRDM2: PR domain containing 2 with ZNF domain, EGF: Epidermal growth factor, IGF1: Insulin-like growth factor 1 (somatomedin C), HIC1: Hyper ethylated in cancer 1, CST6: Cystatin E/M, SERPINE1: Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1, IL-6: interlukin 6, KRT5: Keratin 5, TFF3: Trefoil factor 3 (intestinal), ESR1: Estrogen receptor 1, MUC1: Mucin 1, cell surface associated, GLI1: GLI family zinc finger 1.
included the GLI family zinc finger 1 (GLI1) and Trefoil factor 3 (intestinal) (TFF3), which had a down regulation of 20.5 and 17.5 folds, respectively. The up and down-regulated human breast cancer genes in MDA-MB-453/TR breast cancer cells are illustrated in Table 1. 3.2.1. Gene ontology The DAVID gene annotation website was used to categorize the altered genes into their specified gene ontology groups. The genes that were up-regulated by ≥ 4-fold were functionally clustered into six main categories: regulation of protein modification process, phosphorylation, and regulation of signal transduction, regulation of cell cycle
Table 1 Up and Down-regulated human breast cancer genes in MDA-MB-453/TR cells, relative to MDA-MB-453/Control cells. A standard 4-fold change in expression was used as an arbitrary cut-off. Gene symbol
Fold of regulation
Up-regulated human breast cancer genes IGFBP3 TGFB1 PRDM2 EGF
10 fold 8 fold 5.5 fold 4 fold
Down-regulated human breast cancer genes GLI1 TFF3 KRT5 ESR1 CST6 HIC1 IL6 MUC1 IGF1 SERPINE1
−20.5 folds −17 folds −14.5 folds −12.5 folds −11.5 folds −6 folds −5.5 folds −5 folds −4.5 folds −4 folds
process, regulation of cell migration and regulation of cell motion. The genes that were down-regulated by ≥ 4-fold were functionally clustered into seven main categories: regulation of RNA metabolic process, regulation of transcription DNA dependent, regulation of granule cell precursor proliferation, regulation of epithelial cell proliferation, regulation of signal transduction, regulation of cell proliferation, regulation of cell communication. Main gene ontology categories for upregulated genes and down regulated genes are listed in Tables 2 and 3, respectively. 4. Discussion In this study, we attempted to identify the alterations in gene expression that are associated with Trastuzumab resistance. Wilken and Maihle (2010) have previously illustrated that cancer cells exposed for long-term Trastuzumab have altered phenotype and gene expression (Wilken and Maihle, 2010). In support of the previous finding, we have found that Trastuzumab resistance correlates with relevant changes in gene expression in association with changes in cell morphology. Importantly, these morphological changes resemble cells going through an epithelial-to-mesenchymal transition (EMT). During the EMT process, cells undergo remodeling of the cell cytoskeleton through losing
Table 2 The gene ontology groups for up-regulated genes (≥4-fold) in MDA-MB-453/TR cells according to the database for annotation, visualization and integrated discovery (DAVID) gene annotation website, http://david.abcc.ncifcrf.gov/ Category
Genes
Regulation of protein modification process, Phosphorylation, and signal transduction Regulation of cell cycle process Regulation of cell migration and motility
IGFBP3, TGFB1, EGF IGFBP3, TGFB1, EGF TGFB1, EGF IGFBP3, TGFB1
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
E.A. Sharieh et al. / Gene xxx (2015) xxx–xxx Table 3 The gene ontology groups of down-regulated genes (≥4-fold) in MDA-MB-453/TR cells according to the database for Annotation, Visualization and Integrated Discovery (DAVID) gene annotation website, http://david.abcc.ncifcrf.gov/ Category
Genes
Regulation of RNA metabolic process and transcription Regulation of cell proliferation Regulation of epithelial cell proliferation Regulation of cell communication and signal transduction
ESR1, GLI1, IGF1 GLI1, IGF1 IGF1, KRT5 ESR1, IGF1
their polarity and contact, which are also characterized by loss of their intercellular adhesion, decrease in expression of epithelial markers, an increase in expression of mesenchymal markers, and acquisition of spindle-shape, and single-cell migration (Huber et al., 2005; Thiery and Sleeman, 2006). The EMT process is linked to increased invasiveness and metastatic abilities of cancer cells and appears to play a critical role in drug resistance (Sethi et al., 2011). The cells acquire molecular alterations that may be responsible for the EMT phenotype such as up-regulation of a number of genes including TGF-β1 and EGF and down-regulation of other genes such as GLI1 expression. TGF-β1 was found to reduce the function of natural killer cell and such effect has been proposed to contribute in the development of Trastuzumab resistance. Additionally, TGF-β1 has the ability to promote tumorigenesis through induction of EMT (Brown et al., 2000). The elevation in the expression of TGF-β1 gene increases the activation of TGF-β1 receptors, which causes dissociation of tight junction and inhibition of cell adhesion. Induction of EMT transition through TGFβ1 is thought to occur through the Ras-dependent and the Smaddependent pathways (Christiansen and Rajasekaran, 2006). Ito et al. (2010) found that TGF-β/Smad signaling was inhibited by ESR1 (Ito et al., 2010). Interestingly, our gene expression profiling shows a profound down-regulation of the ESR1 gene suggesting a negative control on the TGF-β/Smad signaling upon acquisition of the Trastuzumab resistance. Another important gene that has been up-regulated in MDA-MB453/TR is that encoding EGF. Over-production of endogenous ligands such as EGF has been shown to over-activate networks involved in the carcinogenesis process (Lichtenstein, 2008). In addition, EGF may also contribute to the EMT process. Lo et al. (2007) illustrated that some cell lines undergo EMT in response to EGF stimulation (Lo et al., 2007). Motoyama, et al. (2002) reported that EGF has the ability to activate MAPK and PI3K pathway in the presence of the anti-proliferative effect of the mAb 4D5, an un-humanized version of Trastuzumab, in the breast cancer BT474 cells (Motoyama et al., 2002). EGF also promotes HER-2 receptor internalization in the form of heterodimers reducing the surface HER-2 receptor in Trastuzumab-exposed cells, which would indeed limit Trastuzumab efficacy and result in disease progression (Hurrell and Outhoff, 2013). Taken together, We can surmise that the up-regulation of TGF-β1 gene and EGF gene together may have a critical role in developing EMT and acquisition of Trastuzumab resistance phenotype that end with invasion and metastasis of such cells. Other altered genes are also involved in the drug resistance process. One of these genes is IGFBP-3. This gene was reported to enhance the growth of cells in vitro and was found to increase proliferation of prostate cancer (LNCaP) cells in the absence of IGFs (Sheen-Chen et al., 2009). Different studies have illustrated a relationship between the tissue levels of IGFBP-3 and unfavorable prognostic factors of breast cancer such as large tumor size, elevated S-phase fraction, and elevated DNA aneuploidy. It is related to the low levels of ER or progesterone receptors (Shao et al., 1992; Vestey et al., 2005). Furthermore, O'Han, et al. (2009) illustrated that high expression of IGFBP-3 in breast cancer Hs578T cells was associated with rapid growth and poor outcome (O'Han et al., 2009). High levels of IGFBP-3 were also associated with Trastuzumab sensitivity in two breast cancer cell lines (Dokmanovic et al., 2011), who also showed that the Trastuzumab-resistant cells have a low level
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of IGFBP-3. The observed up-regulation of IGFBP-3 may also affect the expression level of other proteins like the free IGFs (Ren et al., 2007). This function may explain the observed 4.5 folds down-regulation in the Insulin-like growth factor 1 (somatomedin C) expression levels in the MDA-MB-453/TR gene expression data. PRDM2 gene, which encodes two proteins, the RIZ1 and RIZ2, was found to be up-regulated. RIZ1 acts as a tumor suppressor at the tumor initiation stage through inducing apoptosis and cell cycle arrest and tumor and a tumor promoter at the malignant stage enhancing metastasis (Sun et al., 2011). Furthermore, the previous study illustrated that changes in the RIZ1 and RIZ2 level may have a role in cancer development. Importantly, RIZ1 up-regulates insulin-like growth factorbinding protein 2 (IGFBP-2) and secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine), and may enhance the expression of nuclear factor of activated T cell 1 (NFATc1) that promotes breast cancer cell invasion via cyclooxygenase-2 (Sun et al., 2011). A number of genes found to be down-regulated upon developing Trastuzumab resistance are linked to cell motility. An example is GLI1, which was the most down-regulated gene in the MDA-MB-453/TR cells, may also have a role in the EMT process. This down-regulation was proposed by Joost et al. (2012) to have a link with the loss of some epithelial markers. Such loss leads to gain of a mesenchymal morphology and increasing cell motility in pancreatic ductal adenocarcinoma (Joost et al., 2012). Another gene is the hyper-methylated in cancer 1 (HIC1), which is a tumor suppressor gene that was reported to be down-regulated in different cancers (Pan et al., 2013) and may participate in developing resistance toward Trastuzumab through increasing the expression of EphA2 receptors. The latter molecules are involved in different processes including angiogenesis and cell migration (Foveau et al., 2012). The down-regulation of the Cystatin E/M (CST6) gene is also a common phenomenon in metastatic malignant cells (Ko et al., 2010). ESR1, TFF3, MUC1 are three more genes that have been downregulated considerably. ESR1 is involved in cell proliferation, survival, and cancer progression. It has an important regulatory role in controlling the expression level of different important genes such as TFF3 and MUC1. This explains the observed down-regulation seen in the expression of TFF3 and MUC1 despite their reported role in breast cancer metastasis and progression. The elevated expression of MUC1 human breast cancer correlates with increased metastasis rate (Do et al., 2013) and may contribute in Trastuzumab resistance (Wilken and Maihle, 2010), while TFF3 is considered to be a pro-survival, proinvasive, and pro-angiogenic factor in common human solid tumors. 5. Conclusion In conclusion, Trastuzumab resistance in MDA-MB-453 appears to be a multifactorial process that involves the regulation of many genes and acquisition of a mesenchymal phenotype. The most up-regulated genes were IGFBP-3, TGFβ1, and EGF, which are known to have an important role in developing drug resistance and, more specifically, Trastuzumab resistance in breast cancer. TGF-β1 and EGF have also been recognized for their role in the acquisition of the morphological changes of the cell during EMT, a phenomenon that was observed in this study. On the other hand, the down-regulation of genes known for their role in cancer drug resistance such as GLI1, TFF3, CST6, and ESR1, has also been noted. References Babayan, A., Hannemann, J., Spotter, J., Muller, V., Pantel, K., Joosse, S.A., 2013. Heterogeneity of estrogen receptor expression in circulating tumor cells from metastatic breast cancer patients. PLoS One 8, e75038. Brown, R.E., Bernath, A.M., Lewis, G.O., 2000. HER-2/neu protein-receptor-positive breast carcinoma: an immunologic perspective. Ann. Clin. Lab. Sci. 30, 249–258. Christiansen, J.J., Rajasekaran, A.K., 2006. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res. 66, 8319–8326.
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Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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Research paper
Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab Elham Abu Sharieh a,b, Abdulla S. Awidi b,c, Mamoun Ahram d, Malek A. Zihlif a,⁎ a
Department of Pharmacology, Faculty of Medicine, The University of Jordan, Amman, 11942, Jordan Stem Cell Therapy Center, Faculty of Medicine, The University of Jordan, Amman, 11942, Jordan Department of Internal Medicine Hematology and Oncology Unit, University of Jordan, Amman 11942, Jordan d Department of Physiology and Biochemistry, Faculty of Medicine, The University of Jordan, Amman, 11942, Jordan b c
a r t i c l e
i n f o
Article history: Received 29 March 2015 Received in revised form 23 June 2015 Accepted 7 September 2015 Available online xxxx Keywords: Trastuzumab resistance Epithelial-to mesenchymal transition
a b s t r a c t Development of resistance against cancer therapeutic agents is a common problem in cancer management. Trastuzumab resistance is one of the challenges in management of HER-2-positive breast cancer patients resulting in breast cancer progression, metastasis, and patient poor outcome. The aim of this study is to determine the alteration in gene expression in response to Trastuzumab resistance after long-term exposure to Trastuzumab. The Trastuzumab-resistant MDA-MB-453 (MDA-MB-453/TR) cell line was developed by exposing cells to 10 μM Trastuzumab continuously for 6 months. Sensitivity toward Trastuzumab was tested using cell viability assays. The acquisition of an epithelial-to mesenchymal transition (EMT) phenotype was also observed in parallel with the development of resistance. Based on the real-time-based PCR array technology, several genes were altered affecting multiple networks. The most up-regulated genes were TGF-β1 and EGF, and IGFBP-3. These genes are known to have a critical role in Trastuzumab resistance in breast cancer cell lines and/or in the acquisition of EMT. They are also recognized for their role in cancer progression and metastasis. These alterations indicate that the development of Trastuzumab resistance is multifactorial and involves a development of a mesenchymal like phenotype. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Breast cancer is the most commonly occurring malignancy in women worldwide (Vranic et al., 2011) representing 23% of all cancer cases (Goto et al., 2014). The disease is commonly classified based on gene expression profiles into four main subtypes. The luminal A subtype is characterized by high expression of estrogen receptor alpha (ESR-A), whereas the luminal B subtype is characterized with mixed expression of ESR-A, progesterone (PR) and/or human epidermal receptor 2 (HER-2) (Babayan et al., 2013). A third type is named HER2 enriched subtype, which is characterized by over-expression of the HER2 gene. Lastly, a subtype of breast cancer that lacks expression of any of the
Abbreviations: (ESR-A), estrogen receptor alpha; (PR), progesterone; (HER-2), human epidermal receptor 2; PCR, POLYMERASE chain reaction; (FDA), Food and Drug Administration; (ATCC, USA), American Type Culture Collection; (FBS), fetal bovine serum; (MTT), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; (IC50), the inhibitory concentrations; (ACTB), actin beta; (GAPDH), beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase; (HPRT1), hypoxanthine phosphoribosyltransferase 1; (RPLP0), ribosomal protein large, P0; (DAVID), database for Annotation, Visualization and Integrated Discovery; (IGFBP-3), the insulin-like growth factor binding protein3; (TGFβ1), the transforming growth factor beta 1; (EMT), epithelial-to mesenchymal transition. ⁎ Corresponding author. E-mail address: [email protected] (M.A. Zihlif).
aforementioned receptor genes is known as triple-negative breast cancer. The HER2-positive breast cancer accounts for 20–30% of all breast cancers and has the second poorest prognosis among breast cancer subtypes (Vu and Claret, 2012). Over-activation of HER-2 receptors leads to uncontrolled cellular growth and proliferation, tumor metastasis, and prevention of apoptosis in malignant cells. The production of monoclonal antibodies (mAbs) that are specific against molecular targets of cancer cells or within the tumor microenvironment have shown success in cancer management (Strome et al., 2007). Among these mAbs is Trastuzumab, which is one of the success stories of biologically targeted breast cancer therapy that has revolutionized the management of HER2 positive breast cancer (Damiano et al., 2009). This humanized mAb belongs to the immunoglobulin subclass 1 (IgG1) that was the first targeted therapy approved by Food and Drug Administration (FDA) for management of HER-2 positive breast cancer (Romond et al., 2005). The mAb targets the extracellular domain of HER-2 receptor blocking its function via acting through antibody-dependent cellular cytotoxicity, stimulating receptor internalization and degradation, and, consequently, arresting cancer cells at the G1 phase of the cell cycle (Nahta and Esteva, 2006). Breast cancer patients having positive diagnosis for HER2 and, hence, treated with this mAb have shown reduced tumor growth, decreased tumor size, and increased 5-year survival.
http://dx.doi.org/10.1016/j.gene.2015.09.019 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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Among the most common challenges in treating HER2-positive breast cancers is the development of resistance within the first year of treatment. Trastuzumab resistance can either be primary (de novo) when the Trastuzumab is ineffective for the treatment of breast cancer patients despite tumor expression of HER2 leading to no response to treatment, or secondary (acquired) when patients who initially respond to Trastuzumab treatment, but experience a Trastuzumab-refractory relapse leading to loss of clinical benefits.(Nahta and Esteva, 2003; Nahta and Esteva, 2006; Nahta et al., 2006; Wilken and Maihle, 2010). This phenomenon necessitates identification of changes in gene expression associated with development of Trastuzumab resistance that can lead to better understanding of the perturbations in molecular network. In this study, we utilized the breast cancer MDA-MB453 cell line that over-expresses HER2 to establish resistant line via exposing the cells to a long-term, continuous dose of Trastuzumab. Alterations in gene expression were determined using the PCR array technology that contains most commonly altered human breast cancer genes. 2. Material and methods 2.1. Cell culture growth conditions All cell culture reagents were obtained from Thermo Scientific Inc. (USA). The MDA-MB-453 cell line was originally obtained from the American Type Culture Collection (USA). The cells were maintained as an attached monolayer culture in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM 100 u/mL L-glutamine, and 100 μg/mL penicillin-streptomycin. The cells were incubated at 37 °C in a 90% humidified atmosphere of 5% CO2. 2.2. Development of Trastuzumab resistant MDA-MB-453 cell line Trastuzumab (Herceptin; F. Hoffmann-La Roche, Basel, Switzerland) was utilized in this study. To develop resistant cell line termed MDAMB-453/TR. Two approaches were attempted. The first approach was based on treating the cells with an initial concentration of 2 μM of Trastuzumab and increasing the dose gradually. In the second approach, the cells were exposed to a continuous, steady concentration of 10 μM of Trastuzumab. Both approaches were previously utilized to develop Trastuzumab-resistant cells.(Wang et al., 2011; Wu et al., 2012).
2.4. Gene expression profiling Total RNA was isolated using an RNeasy® Mini kit and the RNasefree DNase set (Qiagen, Germany) according to the manufacturer's instructions. Aliquots containing 1 μg of total RNA were used from each sample to synthesize complementary DNA strands using RT2 First Strand kit (Qiagen, Germany). The Human Breast Cancer PCR Array (Qiagen, USA) was used to determine alterations in gene expression resulting from developing Trastuzumab resistance. The array contains 84 breast cancer-specific genes involved in tumor classification, signal transduction and other commonly affected pathways such as angiogenesis, adhesion, proteolysis, cell cycle, and apoptosis. A diluted cDNA aliquot, equivalent to 1 μg total RNA for each plate, was mixed with the RT2 SYBR® green master mix (Qiagen, USA) according to the manufacturer's instructions, and loaded onto the 96-well array. The PCR cycle was performed using the iCycler (Bio-Rad, USA). Each cell sample was performed in duplicates. The cycle threshold (Ct) values for each sample were given automatically by the iCycler thermal cycler (BioRad, USA) according to the amplification curves of both MDA-MB-453/Control and MDA-MB-453/TR. The threshold value was manually set on 0.01. Changes in gene expression were calculated using the ΔΔCt method utilizing data analysis tool of the SABiosciences Company (Qiagen, USA). The data were normalized, across all plates, to the following housekeeping genes: Actin beta (ACTB), Beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and ribosomal protein large, P0 (RPLP0). 2.5. Gene functional annotation The genes whose expression levels were altered by ≥4-fold were further examined using the web–based, gene annotation, bioinformatics tool, the Database for Annotation, Visualization and Integrated Discovery (DAVID) website (http://david.abcc.ncifcrf.gov/), which provides biological classification of the genes. Results are displayed in comparative tables that describe the most affected biological function by genes changes. 3. Results 3.1. Development and characterization of the MDA-MB-453/TR cells
2.3. Cell proliferation assays
In order to establish Trastuzumab-resistant MDA-MB-453 cells, two approaches were carried out. In the first approach, the cells were initially grown in the presence of low concentration of Trastuzumab (2 μM) and increasing the dose gradually with every cell passage. However,
The anti-proliferative effects of the parental MDA-MB-453 cells and MDA-MB-453/TR cells were evaluated by the Cell Titer Non-Radioactive Cell Proliferation Assay Kit® (Promega, USA) according to manufacturer's instructions. This assay is a colorimetric test that is based on the reduction of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) converting it from a yellow tetrazole to a purple formazan, a process that occurs in the mitochondria of viable cells. The cells were seeded onto 96-well plates (Greiner, Germany) at a concentration of 14 × 103 cells/well and incubated overnight. Horizontal dilutions of the drug ranging from 0.1–100 μM were added. Each concentration was added in triplicates, and every plate contained a control of cells in plain media. Optical density (OD) at 570 nm wavelength was recorded. The inhibitory concentrations (IC50) values were calculated from the logarithmic trend line of the cytotoxicity graphs using the Graph Pad PRISM® 5.0 software (Graph Pad Software, Inc., city, country). The degree of resistance was evaluated in terms of resistance index (R), which was calculated according to the equation: R = IC50 resistant cells/IC50 sensitive cells.
Fig. 1. The MDA-MB-453 cells viability assay curves A. The MDA-MB-453/control cells viability assay curve, the viability assay is done for MDA-MB-453 breast cancer cell line not exposed to Trastuzumab at passage number (P80), the concentration at which (0.5) of cells were viable is approximately 15.84 μM. B. The MDA-MB-453/TR cells viability assay curve, the viability assay is done for Trastuzumab resistant MDA-MB-453 breast cancer cell line (exposed to 10 μM Trastuzumab for 6 months) passage number (P53), the concentration at which (0.5) of cells were viable is approximately 32.28 μM.
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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this approach was not successful as cells became senescent at a concentration of 20 μm and died at a concentration of 36 μM. The second approach was based on exposing the cells to a continuous steady concentration of 10 μM of Trastuzumab for approximately six months for a total of 33 doses. The 10-μM concentration was selected according to the first measured inhibitory concentration of 50% (IC50) value for untreated MDA-MB-453 cells, which was approximately 10 μM. This approach was successful and resistant cells (MDA-MB-453/TR) that could survive and replicate at 36 μM of Trastuzumab were developed. The proliferative capacity and the viability of the parental MDA-MB453 and the MDA-MB-453/TR cells were determined at different concentrations of Trastuzumab. As illustrated in Fig. 1, whereas MDAMB-453/Control had an IC50 of 15.84 ± 1.168 μM (mean ± STD), the IC50 for MDA-MB-453/TR was 32.28 ± 3 μM (mean ± STD). According to the R-value, the MDA-MB-453/TR cells was approximately 2-fold more resistant to Trastuzumab than the parental cell line.
3.1.1. Morphological features Upon developing a resistance phenotype, gradual changes in the cell morphology was observed. The parental cells existed as clusters
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of rounded cells. Continuing treatments and passaging of the Trastuzumab-treated cells induced loose cell connectivity with cells acquiring a spindle-shape and elongated morphology. This morphological change became more pronounced with longer periods of treatments (Fig. 2).
3.2. Alterations in gene expression in association with resistance development A gene expression profile analysis was performed using the Human Breast Cancer PCR array. A standard 4-fold change in expression was used as an arbitrary cut-off. The Scatter plot shown in Fig. 3 illustrates the differences in expression levels of the tested genes between the resistant and parent cell line. Out of the 84 genes, the expression level of 13 genes was altered by more than 4 folds. Four of these genes were up-regulated including the insulin-like growth factor binding protein 3 (IGFBP-3) and the transforming growth factor beta 1 (TGFβ1), both of which showed the highest level of up-regulation of 10 and 8 folds, respectively. Two genes, PRDM2 and EGF, were up-regulated to a lesser degree. On the other hand, nine genes were down-regulated and
Fig. 2. Morphology of MDA-MB-453/TR cells treated with 10 μM Trastuzumab (A) compared to untreated MDA-MB-453/control (B) at different passage numbers (P).
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
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Fig. 3. Scatter plots of human breast cancer genes expression in MDA-MB-453/TR cells compared to MDA-MB-453/Control cells. A standard 4-fold change in expression was used as an arbitrary cut-off, the up-regulated genes are marked with red stars and the down-regulated genes are marked with green stars, the unchanged genes are marked with black stars. Abbreviations: IGFBP3: insulin-like growth factor binding protein 3, TGFβ1: transforming growth factor βa1, PRDM2: PR domain containing 2 with ZNF domain, EGF: Epidermal growth factor, IGF1: Insulin-like growth factor 1 (somatomedin C), HIC1: Hyper ethylated in cancer 1, CST6: Cystatin E/M, SERPINE1: Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1, IL-6: interlukin 6, KRT5: Keratin 5, TFF3: Trefoil factor 3 (intestinal), ESR1: Estrogen receptor 1, MUC1: Mucin 1, cell surface associated, GLI1: GLI family zinc finger 1.
included the GLI family zinc finger 1 (GLI1) and Trefoil factor 3 (intestinal) (TFF3), which had a down regulation of 20.5 and 17.5 folds, respectively. The up and down-regulated human breast cancer genes in MDA-MB-453/TR breast cancer cells are illustrated in Table 1. 3.2.1. Gene ontology The DAVID gene annotation website was used to categorize the altered genes into their specified gene ontology groups. The genes that were up-regulated by ≥ 4-fold were functionally clustered into six main categories: regulation of protein modification process, phosphorylation, and regulation of signal transduction, regulation of cell cycle
Table 1 Up and Down-regulated human breast cancer genes in MDA-MB-453/TR cells, relative to MDA-MB-453/Control cells. A standard 4-fold change in expression was used as an arbitrary cut-off. Gene symbol
Fold of regulation
Up-regulated human breast cancer genes IGFBP3 TGFB1 PRDM2 EGF
10 fold 8 fold 5.5 fold 4 fold
Down-regulated human breast cancer genes GLI1 TFF3 KRT5 ESR1 CST6 HIC1 IL6 MUC1 IGF1 SERPINE1
−20.5 folds −17 folds −14.5 folds −12.5 folds −11.5 folds −6 folds −5.5 folds −5 folds −4.5 folds −4 folds
process, regulation of cell migration and regulation of cell motion. The genes that were down-regulated by ≥ 4-fold were functionally clustered into seven main categories: regulation of RNA metabolic process, regulation of transcription DNA dependent, regulation of granule cell precursor proliferation, regulation of epithelial cell proliferation, regulation of signal transduction, regulation of cell proliferation, regulation of cell communication. Main gene ontology categories for upregulated genes and down regulated genes are listed in Tables 2 and 3, respectively. 4. Discussion In this study, we attempted to identify the alterations in gene expression that are associated with Trastuzumab resistance. Wilken and Maihle (2010) have previously illustrated that cancer cells exposed for long-term Trastuzumab have altered phenotype and gene expression (Wilken and Maihle, 2010). In support of the previous finding, we have found that Trastuzumab resistance correlates with relevant changes in gene expression in association with changes in cell morphology. Importantly, these morphological changes resemble cells going through an epithelial-to-mesenchymal transition (EMT). During the EMT process, cells undergo remodeling of the cell cytoskeleton through losing
Table 2 The gene ontology groups for up-regulated genes (≥4-fold) in MDA-MB-453/TR cells according to the database for annotation, visualization and integrated discovery (DAVID) gene annotation website, http://david.abcc.ncifcrf.gov/ Category
Genes
Regulation of protein modification process, Phosphorylation, and signal transduction Regulation of cell cycle process Regulation of cell migration and motility
IGFBP3, TGFB1, EGF IGFBP3, TGFB1, EGF TGFB1, EGF IGFBP3, TGFB1
Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019
E.A. Sharieh et al. / Gene xxx (2015) xxx–xxx Table 3 The gene ontology groups of down-regulated genes (≥4-fold) in MDA-MB-453/TR cells according to the database for Annotation, Visualization and Integrated Discovery (DAVID) gene annotation website, http://david.abcc.ncifcrf.gov/ Category
Genes
Regulation of RNA metabolic process and transcription Regulation of cell proliferation Regulation of epithelial cell proliferation Regulation of cell communication and signal transduction
ESR1, GLI1, IGF1 GLI1, IGF1 IGF1, KRT5 ESR1, IGF1
their polarity and contact, which are also characterized by loss of their intercellular adhesion, decrease in expression of epithelial markers, an increase in expression of mesenchymal markers, and acquisition of spindle-shape, and single-cell migration (Huber et al., 2005; Thiery and Sleeman, 2006). The EMT process is linked to increased invasiveness and metastatic abilities of cancer cells and appears to play a critical role in drug resistance (Sethi et al., 2011). The cells acquire molecular alterations that may be responsible for the EMT phenotype such as up-regulation of a number of genes including TGF-β1 and EGF and down-regulation of other genes such as GLI1 expression. TGF-β1 was found to reduce the function of natural killer cell and such effect has been proposed to contribute in the development of Trastuzumab resistance. Additionally, TGF-β1 has the ability to promote tumorigenesis through induction of EMT (Brown et al., 2000). The elevation in the expression of TGF-β1 gene increases the activation of TGF-β1 receptors, which causes dissociation of tight junction and inhibition of cell adhesion. Induction of EMT transition through TGFβ1 is thought to occur through the Ras-dependent and the Smaddependent pathways (Christiansen and Rajasekaran, 2006). Ito et al. (2010) found that TGF-β/Smad signaling was inhibited by ESR1 (Ito et al., 2010). Interestingly, our gene expression profiling shows a profound down-regulation of the ESR1 gene suggesting a negative control on the TGF-β/Smad signaling upon acquisition of the Trastuzumab resistance. Another important gene that has been up-regulated in MDA-MB453/TR is that encoding EGF. Over-production of endogenous ligands such as EGF has been shown to over-activate networks involved in the carcinogenesis process (Lichtenstein, 2008). In addition, EGF may also contribute to the EMT process. Lo et al. (2007) illustrated that some cell lines undergo EMT in response to EGF stimulation (Lo et al., 2007). Motoyama, et al. (2002) reported that EGF has the ability to activate MAPK and PI3K pathway in the presence of the anti-proliferative effect of the mAb 4D5, an un-humanized version of Trastuzumab, in the breast cancer BT474 cells (Motoyama et al., 2002). EGF also promotes HER-2 receptor internalization in the form of heterodimers reducing the surface HER-2 receptor in Trastuzumab-exposed cells, which would indeed limit Trastuzumab efficacy and result in disease progression (Hurrell and Outhoff, 2013). Taken together, We can surmise that the up-regulation of TGF-β1 gene and EGF gene together may have a critical role in developing EMT and acquisition of Trastuzumab resistance phenotype that end with invasion and metastasis of such cells. Other altered genes are also involved in the drug resistance process. One of these genes is IGFBP-3. This gene was reported to enhance the growth of cells in vitro and was found to increase proliferation of prostate cancer (LNCaP) cells in the absence of IGFs (Sheen-Chen et al., 2009). Different studies have illustrated a relationship between the tissue levels of IGFBP-3 and unfavorable prognostic factors of breast cancer such as large tumor size, elevated S-phase fraction, and elevated DNA aneuploidy. It is related to the low levels of ER or progesterone receptors (Shao et al., 1992; Vestey et al., 2005). Furthermore, O'Han, et al. (2009) illustrated that high expression of IGFBP-3 in breast cancer Hs578T cells was associated with rapid growth and poor outcome (O'Han et al., 2009). High levels of IGFBP-3 were also associated with Trastuzumab sensitivity in two breast cancer cell lines (Dokmanovic et al., 2011), who also showed that the Trastuzumab-resistant cells have a low level
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of IGFBP-3. The observed up-regulation of IGFBP-3 may also affect the expression level of other proteins like the free IGFs (Ren et al., 2007). This function may explain the observed 4.5 folds down-regulation in the Insulin-like growth factor 1 (somatomedin C) expression levels in the MDA-MB-453/TR gene expression data. PRDM2 gene, which encodes two proteins, the RIZ1 and RIZ2, was found to be up-regulated. RIZ1 acts as a tumor suppressor at the tumor initiation stage through inducing apoptosis and cell cycle arrest and tumor and a tumor promoter at the malignant stage enhancing metastasis (Sun et al., 2011). Furthermore, the previous study illustrated that changes in the RIZ1 and RIZ2 level may have a role in cancer development. Importantly, RIZ1 up-regulates insulin-like growth factorbinding protein 2 (IGFBP-2) and secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine), and may enhance the expression of nuclear factor of activated T cell 1 (NFATc1) that promotes breast cancer cell invasion via cyclooxygenase-2 (Sun et al., 2011). A number of genes found to be down-regulated upon developing Trastuzumab resistance are linked to cell motility. An example is GLI1, which was the most down-regulated gene in the MDA-MB-453/TR cells, may also have a role in the EMT process. This down-regulation was proposed by Joost et al. (2012) to have a link with the loss of some epithelial markers. Such loss leads to gain of a mesenchymal morphology and increasing cell motility in pancreatic ductal adenocarcinoma (Joost et al., 2012). Another gene is the hyper-methylated in cancer 1 (HIC1), which is a tumor suppressor gene that was reported to be down-regulated in different cancers (Pan et al., 2013) and may participate in developing resistance toward Trastuzumab through increasing the expression of EphA2 receptors. The latter molecules are involved in different processes including angiogenesis and cell migration (Foveau et al., 2012). The down-regulation of the Cystatin E/M (CST6) gene is also a common phenomenon in metastatic malignant cells (Ko et al., 2010). ESR1, TFF3, MUC1 are three more genes that have been downregulated considerably. ESR1 is involved in cell proliferation, survival, and cancer progression. It has an important regulatory role in controlling the expression level of different important genes such as TFF3 and MUC1. This explains the observed down-regulation seen in the expression of TFF3 and MUC1 despite their reported role in breast cancer metastasis and progression. The elevated expression of MUC1 human breast cancer correlates with increased metastasis rate (Do et al., 2013) and may contribute in Trastuzumab resistance (Wilken and Maihle, 2010), while TFF3 is considered to be a pro-survival, proinvasive, and pro-angiogenic factor in common human solid tumors. 5. Conclusion In conclusion, Trastuzumab resistance in MDA-MB-453 appears to be a multifactorial process that involves the regulation of many genes and acquisition of a mesenchymal phenotype. The most up-regulated genes were IGFBP-3, TGFβ1, and EGF, which are known to have an important role in developing drug resistance and, more specifically, Trastuzumab resistance in breast cancer. TGF-β1 and EGF have also been recognized for their role in the acquisition of the morphological changes of the cell during EMT, a phenomenon that was observed in this study. On the other hand, the down-regulation of genes known for their role in cancer drug resistance such as GLI1, TFF3, CST6, and ESR1, has also been noted. References Babayan, A., Hannemann, J., Spotter, J., Muller, V., Pantel, K., Joosse, S.A., 2013. Heterogeneity of estrogen receptor expression in circulating tumor cells from metastatic breast cancer patients. PLoS One 8, e75038. Brown, R.E., Bernath, A.M., Lewis, G.O., 2000. HER-2/neu protein-receptor-positive breast carcinoma: an immunologic perspective. Ann. Clin. Lab. Sci. 30, 249–258. Christiansen, J.J., Rajasekaran, A.K., 2006. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res. 66, 8319–8326.
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Please cite this article as: Sharieh, E.A., et al., Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.019