Reversal of endocrine resistance in breast cancer
Ma Ran
Degree project inapplied biotechnology, Master ofScience (2years), 2010 Examensarbete itillämpad bioteknik 45 hp tillmasterexamen, 2010
Biology Education Centre, Uppsala University, and Dept ofOncology and Pathology, Karolinska Institutet
Supervisor: Johan Hartman, MD, PhD
Reversal of endocrine resistance in breast cancer Ma Ran
Summary
Breast cancer is the most common female cancer worldwide. In Sweden, each year around 7300 women are affected and about 1500 women die of it.
Following development of early detection, diagnose and therapies have been reported marked progress in patients and survivals. Normally, premenopausal women are the main group getting breast cancer.
The majority of breast cancers in premenopausal women are initially dependent on estrogens for growth. Therefore, those patients can be effectively treated with adjuvant endocrine therapy such as tamoxifen or aromatase inhibitors, resulting in reduced tumor growth and increased cancer cell-death. This is by far the most effective therapeutic strategy and is associated with few side-effects. However, breast cancer cells which are not completely eradicated by the initial therapy will finally gain resistance to endocrine therapy. Thus patients with recurrent, endocrine resistant breast cancers are not possible to cure. At this stage, treatment is focused on palliative care and prolonged survival with cytotoxic drugs.
For this reason, it is important to increase the knowledge of endocrine resistance in breast cancer and to further develop new targeted therapies to reverse this process.
The mechanism behind endocrine resistance involves both estrogen receptors and tyrosine kinase signaling cascades. In some endocrine resistant breast cancers, expression of estrogen receptor (ER) is lost, and the cell can use other factors than estrogen to grow. Nevertheless, in many cases, the tumor contains ER but the receptor is inactivated by post-translational modifications.
These modifications comprise phosphorylations of certain amino acid residues.
To identify compounds with the ability to reverse endocrine resistance in vitro study, we performed cell proliferation assays, real-time PCR, apoptosis assay and wetern-blotting. However, our study results didn’t show obvious increasing expression of ERs in the results of RT-PCR, but apoptosis was induced and p44/42 MAPK (Erk1/2) signaling pathway was activated by treatments with compounds we used in this project and thus implied additional mechanisms behind how these compounds effect on cells other than reversal of ERs expression.
Introduction
Breast cancer subtypes and their molecular profiling
Molecular profiling, e.g., global scale comparative studies of mRNA or protein, is a very helpful scientific approach to provide biological evidence for identifying breast cancer intrinsic subtypes and heterogeneity (49).
Through years of estrogen receptor (ER) -related gene expression studies by using DNA microarray, as well as the evidence from earlier epidemiologic and marker studies, clinical breast cancer tumor samples are reported biologically different in ER positive and ER negative diseases. Breast cancer intrinsic subtypes have prognostic implications in diagnostics. Generally, two main categories of subtypes exist in breast cancer. The ER positive subtypes include luminal A and B, the ER negative subtype characterized as HER2+/ER- (Human Epidermal growth factor Receptor 2) in table 1(47, 49).
Breast cancer intrinsic subtype Gene expression status
Luminal A ER or PR+/HER2-
Luminal B ER or PR+/HER2+
HER2+/ER- ER-/PR-/HER2+
Table 1: breast cancer subtypes and related gene status
ERα and ERβ
Estrogen as a dominant regulator of growth and differentiation mediates estrogen signaling in many physiological processes, including cell growth, organ development specific gene regulation in central nerve and skeleton systems(1, 6). It is also known to influence several pathological processes of hormone dependent diseases such as breast cancer, endometrial cancer, ovarian cancer as well as osteroporosis (2). Estrogen biological activities are controlled by binding to one of two specific estrogen receptors; ERα and ERβ, which were identified in turn by Elwood Jensen and colleagues in 1962 and George G. Kuiper in 1996. Both of them belong to the nuclear receptor superfamily, which is a family of ligand regulated transcription factors (1, 4).
Ligand-binding induces conformational change of the ER, secondary dimerization and recruitment of coregulators. Finally the ER complex regulates gene transcription by binding to estrogen response elements (ERE) or interacts with other proteins (4). Former studies demonstrated that both ERα and ERβ exposure to estrogen can modulate expression and for example increase the activities of promoter regions.(7-9)
Both ERα and ERβ are expressed throughout the human body. ERα is expressed mainly in the liver, heart, uterus and kidney whereas ERβ is expressed in the bladder, lung, prostate, ovary, hematopoietic, gastrointestinal tract and central nervous systems. Apparently, they display sometimes distinctly in a variety of tissues (2, 3). But distinct expression of one or the other receptor sometimes occur in various tissues such as epididymis, adrenal, thyroid, bone, mammary gland and some regions of brain.(1).
Structural and functional domains of ERα and ERβ are evolutionarily conserved and typical as other nuclear receptor family members in their five distinguishable domains named A/B, C, D, E and F, including DNA-binding domains, dimerization domains, ligand binding domains and transcriptional activation which are high-lightened as colored bars in the figure 1 (5). ERs have less than 20% amino acids identity in the N-terminal A/B domain, since this domain is the most variable region, it may work as to distinguish ER subtype specific actions on target genes. (10) However, ERs share common structural characteristics and 95% identity in their central C-domain, which is the DNA-binding domain involved in specific DNA binding and receptor dimerization (6). The D-domain located between DNA-binding domain and ligand-binding domain is very flexible and thus referred to as a hinge domain (6). Even though this domain has only 30% identity in ERα and ERβ, it contains a nuclear localization signal and so implied to be important for nuclear translocation (11). There are two separate transcription activation functions (AFs) mediating the transactivating functions of ERα and ERβ, AF-1 is harbored in A/B domain at the N-terminal whereas AF-2 is located within the ligand-binding domain at C-terminal (7). AF-2 is hormone-dependent; it allows the receptors to stimulate estrogen-regulated genes transcription by ligand binding and dimerization (10). AF-1 is ligand-independent and contributes to specificities in cell and promoter activities (6). Interestingly, the three-demensional structures of LBDs in ERs are quite similar but the amino acids forming binding cavities are different at two sites (12, 13) and thus can complex with agonists or antagonists. The functions of the F-domain in two ER subtypes remain undefined.
Fig.1: Schematic Structures of ERα and ERβ including five regions, DNA binding domain(DBD), ligand binding domain(LBD) and two functions(AF-1/2)
Cancer Therapy Vol 6, 149-162, 2008 Molecular mechanisms of estrogen receptor function
Estrogen activities in target tissues are carried out via binding to ERα or ERβ.
The relative estrogen-dependent transcriptional function is regulated by cooperation between AF-1 in A/B region and AF-2 in ligand-binding domain.
Some studies show that unlike ERα, the AF-1 function in ERβ seems to be weaker and thus might depend more on AF-2, the ligand-dependent function to exert its transcriptional activation (14).
Two models describing the mechanisms of ERs actions have been proposes, where estrogen participate in several distinct pathways and biological processes. In the classic model, activated ERs will specifically bind to DNA at their estrogen-responsive elements (EREs) as homo-dimers or as hetero-dimers (1, 6). A second model explains an additional mechanism where estrogen can also modulate gene expression via tethering to other transcription factors such as stimulation protein 1(SP1) and activating protein-1 (AP-1) through a transcription factor cross-talk process (15, 17)
After ERs binding to DNA-response elements, transcription will be regulated through recruitment of co-activators or co-repressors. Those co-regulatory proteins are functionally distinguished by their types of ligand, promoter structure and receptor subtype. (20, 21). The steroid receptor coactivator (SRC) family is one of the main groups of co-activators affecting nuclear receptor function. Members in this family share similar structures and are indicated to be important mediators for both ERs through interaction process (1, 21). One member AIB1 (or SRC-3/RAC3) in the SRC family of co-activators
has been found overexpressed in more than 50% and gene-amplified in 5-10%
of breast cancers (18, 22, 23, 24,). Mice models proved that SRC-3/AIB1/RAC3 is necessary for normal development of mammary glands, but overexpression of this coactivator in mammary epithelial cells will lead to malignant mammary tumors. (18, 25). Another member SRC-2/TIF2/RAC2 along with SRC-3/AIB1/RAC3 can be recruited to and enhance both ER subtypes to mediate transcription with the same affinity (1, 26).
Tamoxifen and resistance
Tamoxifen is an antagonist of the estrogen receptor in breast tissue. It can bind and block estrogen receptors on the surface of cells, preventing estrogens from binding and activating the cell. It is used in patients for treating and preventing breast cancer, especially efficient for those with ERα-positive breast cancer. But recently, resistance to tamoxifen therapy has been noticed, which has become an important clinical problem.
The mechanisms behind endocrine resistance may be summarized by several reasons. In some endocrine resistant breast cancers, expression of ER is lost, and the cell is dependent on other factors than estrogen to grow. Nevertheless, research has shown that non-steroidal stimuli such as growth factors or the signaling molecule cAMP (18, 27, 28), which by elevating intracellular kinase activities, can induce ER-dependent transcription even in the case of absence of ligand-binding or presence of tamoxifen. Released signals then activate cellular kinase cascades and further phosphorylate ERs and their coregularoty proteins. The AF-1 domain of ERs is most influenced and activated by phosphorylation at many sites, involving multiple signaling kinases including Akt, MAPKs, c-Src as well. Phosphorylation of coactivators will elevate activity of genomic ERs action because of their increased subcellular nuclear localization, therefore potentially reinforced to recruit other more transcriptional co-regulators and interact with ERs forming pre-complex (18, 29).
Phosphorylation of corepressors results in nuclear export to prevent them approach to ERs transcriptional complexed in nucleus (30).
ERs related breast cancer in women
In the growth of the mammary glands, estrogen plays an essential role and has been related to breast cancer promotion. As mentioned before, ERs are located in different cell types. In normal or malignant breast tissue, ERα is mainly found in the lobular epithelial and ductal cells but less in stroma, whereas ERβ is observed in all these three cell types and is important for breast cancer cell invasion and proliferation (34,35), but ERβ is as the predominant subtype in the normal breast (32). In further investigation and studies of ERβ expression in normal and breast cancer specimens, the role in
response to endocrine treatment and the relations with other clinical features have been extensively examined at both mRNA and protein levels. ERβ expression is decreased in most of breast tumors and a possible reason behind might be promoter methylation (32, 33). Since this phenomenon is frequently observed in other types of cancers such as gene promoter methylation in colorectal cancer and bladder cancer, it implies a protective role of ERβ as a potential tumor suppressor gene in breast cancer development.
But results from immunochemical staining showed ERβ expressed in 50%-70% of clinical breast cancer biopsies as well as the percentage of ERα expression in breast tumor, the assumption about the role ERβ played in breast cancer needs further investigation.
Cell proliferation assays
Cell proliferation is the increase in cell number as a result of cell division.
Accurate detection of cell proliferation is a fundamental method for assessing cell health, and evaluating anticancer drugs. The measurement of cell proliferation and viability has become a key technology in the research of life science. The need for reliable, sensitive, easy and fast methods has led to the development of several standard assays. The most accurate method utilizes direct measurement of new DNA synthesis. Generally, it is performed by interfusing bromodeoxyuridine (BrdU) with DNA, then followed by detection with an anti-BrdU antibody. Although effective, this method requires DNA denaturation to expose the BrdU to the antibody. Other colorimetric assays are based on the cleavage of tetrazolium salts, such as MTT, that are added to the culture medium. These assays do not require either washing or harvesting of cells. The complete assay can be performed in the same microplate.
Cell Proliferation Reagent WST-1 has several advantages compared to other popular compounds. Compared to MTT, which is cleaved to water-insoluble formazan crystal and therefore has to be solubilized after cleavage, WST-1 yields water-soluble cleavage products which can be measured without an additional solubilization step. Futhermore, WST-1 is more stable and can be used as a ready-to-use solution and can be stored at 2°C to 8°C for several weeks without significant degradation. It also has a wider linear range and shows accelerated color development.
Aim of the study
The aim of this study was to identify compounds with the ability to reverse endocrine resistance of breast cancer cells. This was achieved by in vitro studies by using different concentrations of small molecules combined with tamoxifen on several breast cancer cell lines. Cell proliferation assays were first performed with 300 commercial natural compounds from KaroBio and
another 2 compounds 107,108 from Axentra which have effects in colon cancers and needed to be tested in breast cancer as well. Primary results from proliferation assays showed strong effects of 107 and 108 on inhibiting cell growth in all breast cancer cell lines but less convinced evidence from the other 300 compounds. Thus, we mainly focused on the studies of 107 and 108.
In order to investigate detailed mechanisms behind their effects, RT-PCR, western blotting and apoptosis assay were thus performed to analyze if cell growth decrease was induced because of endocrine resistance.
Results
In order to find general effects of compounds at different concentrations, cell proliferation assay was compared by estimating cell numbers from photo- -graphs. Real-time PCR, apoptosis assay and western bolting were then performed to discover possible mechanisms about why the treatments inhibited cell growth and how they worked behind.
Two compounds 107 and 108, which are marked as A and B in the following text, combined with tamoxifen(4-OHT) were used in all the tests. Another 300 natural commercial compounds were tested together through cell proliferation assay at the beginning but results are not shown, due to the weak corresponding evidences in all the cell lines, we thus only focused on 107 and 108 in the following tests.
Cell proliferation assay
Cell proliferation assays were made as a first test to observe if the compounds could influence cell growth in vitro. Treated concentration of compounds A (compound 107) and B (compound 108) were 10uM and 50uM (marked as 10A, 10B, 50A and 50B in the test) diluted in DMSO. Photos were collected through microscope before and after incubation, and both compounds showed notable impact on blocking cell proliferation under observation.
Control 10A 10B 50A 50B
Fig.2: MCF7 cells incubated with compounds for 5 days
Control 10A 10B 50A 50B
Fig.3: MDA231 cells incubated with compounds for 5 days
Primary results suggested under the treatments at 10uM and 50uM, both A and B had negative effects on cell proliferation in both MCF7 and MDA231 cell lines, with fewer cell numbers compared to control group (figure2 and figure 3).
Related cell number then measured in cell proliferation assay by using WST-1 reagent.
Fig.4: MCF7 cells + compounds for 120h, analyzed with WST-1 proliferation assay
Around 600 cells/well were seeded into 96-well plate as five groups with different treatments. Compounds A and B were diluted into DMSO and tamoxifen was diluted into ethanol. Medium RPMI1640 with 0.1% DMSO and 0.1% ethanol was used as a control in order to avoid the deviation from solvents DMSO and ethanol. Relative cell number was calculated according to the absorbance value of WST-1 cell proliferation assay. Group of MCF-7 cells showed decreased cell number when cultured with compounds, however, treated cells co-cultured with tamoxifen (4OHT in figure 4) appeared slightly increased compared to the ones cultured without. For compound A, 10uM final concentration had a weaker effect on MCF-7 cells compared to 50uM, and compound B had a stronger effect on cell number than A (figure 4).
Fig.5: MDA231 cells + compounds for 120h, analyzed with WST-1 proliferation assay
For cell proliferation test with MDA-231 cells, around 800cells/well were seeded and treated as described above. The proliferation of treated MDA-231 cells co-cultured with or without tamoxifen (4-OHT in figure 5) was in all cases weakened by compounds A and B. Still interestingly, groups cultured with tamoxifen showed slightly higher cell numbers.
FACS analysis
Fig.6: MCF7 cells + compounds for 72h, apoptosis was checked by FACS
Aimed at finding the possible relations between ERs and apoptosis, MCF-7(ER+) cell line was analyzed with flow cytometry. Cells were seeded into 6-well plates with groups of treatments. After 3 days of incubation, both supernatant and adherent cells were collected into tubes for characterization of apoptosis and necrosis by Annexin-V and PI. Results from figure 6 showed that MCF-7 cells cultured with tamoxifen had higher apoptotic numbers;
however, three of the compounds treated groups co-cultured with tamoxifen showed lower apoptotic numbers than the control group. Compound B had a more severe effect on induced apoptosis.
Expression of ERα and ERβ
Fig.7: mRNA levels of ER/PS2 in MCF7 cells after 24h treatment
To examine if the expression of ERα was inhibited and ERβ expression was stimulated by both of the compounds, as well as PS2 expression which is positively related to ERα expression, all of them were detected together by real-time PCR. Columns showed a general trend of reducing expression for ERα(ER1), ERβ(ER2) and PS2 except abrupt rise in 50B group ( in figure7), but same as control group. Normally, ERβ expression was generally lower than ERα expression in MCF-7 cells.
Fig.8: mRNA levels of ER/PS2 in MDA231 cells after 24h treatment
MDA231 cells were analyzed with the same procedure as MCF-7 cells.
Interestingly, ERβ expression was markedly stimulated in MDA231(ERs-) cells with the treatment with 50A (figure8). Due to time limitation, we failed to
provide sufficient data on ERα expression in this experiment in time, new data will be made later.
Western blotting
Actin
MAPK
Control 50A 50B
Fig.9: Effect of 50A and 50B on MAPK expression in MCF-7 cells after 24h of treatment.
Results from previous tests failed to show notable increasing reversal of estrogen expression but strong effects on apoptosis; therefore more information was needed to explain our results. Accordingly, we made western blotting to analyze possible activated signaling pathway such as MAPK which is involved in many cellular programs including cell proliferation and cell death at protein expression level. Developed bands showed induced MAPK expressions in treated groups comparing to the blank control. Actin was also used as internal standard control for normalization.
Discussion
Data collected from this project showed that two compounds 107 and 108 are strong inhibitors of proliferation in breast cancer cells. They can induce notable apoptosis as well. However as described above, the aim of this study was to identify compounds in vitro with the ability to reverse endocrine resistance of breast cancer cells treated with tamoxifen, in this respect, the results were unexpected, because the tumor suppressive function seems to be independent of breast cancer cell types. For example, in the cell proliferation assay, for both MCF-7 and MDA-231 cell lines, treated groups co-cultured with tamoxifen showed higher cell numbers than treated group which without tamoxifen, even though most of them were still lower than the control group.
But compound A at concentration of 10uM enhanced MCF-7 cells growth and the similar result was observed in MDA-231 cells as well. Since the molecular background of MCF-7 and MDA-231 cells is different, MCF-7 is ERα positive and MDA-231 is ERα negative, which determine whether they can respond to estrogen resistance or not. We then performed RT-PCR to obtain more detailed information about ERα and ERβ expression levels. ERs expression of
MCF-7 cells in the group of 10uM compound A treatment was higher than 50uM compound A and 10uM compound B, but ERβ was not fully reversed with treatments because the relevant results in cell proliferation, cells number of 10A was more than 50A and 10B. We’ve mentioned about the importance of ERβ in healthy cells and its potential tumor-suppressive function, theoretically growth of tumor cells should be retarded along with the increased ERβ expression. But ERβ expression in group 50B got the highest value and it corresponded to the lowest number of cells in proliferation experiment. Still, the role ERβ exerted in the process was hard to explain clearly since ERα also worked in the same process and interrelated with some pathways that ERβ participated in. However ERβ seemed reversed a lot in MDA231 with the group of 50A treatment, it was interesting because ERs should have minor trace in MDA231 cells.
Studies of apoptosis also gave us some indications. For example the sum of apoptotic cells in group 10A was less than any other groups and this was also accord with the higher scores from proliferation assay and RT-PCR. Group 50B, even though it got highest expression of ERs in both cell lines, it failed to show strongest induction for cell apoptosis. But group 10B, the lowest expression of ERs in MCF7 made most apoptotic cells.
Considering those results implies other additional mechanisms behind how these compounds affect cells. They inhibited cells proliferation and induced apoptosis through another way instead of working on ERs directly. One of the possible mechanism might be the activation of the p42/p44 MAPK cascades and we thus tested out obvious MAPK(Erk) expression in treated group 50A and 50B compared to control group in western blotting. Mitogen-activated protein kinases (MAPKs) is a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines. Thus, other possible involved pathways such as PI3-K, PKC/PKA or EGFR/HER2 should also be checked in future studies.
Methods and Material
Compounds and reagents
Around 1400 compounds were provided from two companies in different times.
In this project, two compounds 107(mark as A in report) and 108(mark as B in report) were fully examined by series of experiments, another 320 compounds
were checked by first high throughput screening and the rest is still in progress.
Compounds were dissolved into 100% DMSO or 99% ethanol as designed or required concentrations.
Cell lines and culture conditions
Human breast cancer MCF-7(ERα+, ERβ-, Tam+R), MDA-MB-231(ER- EGFR+ Tam-R) and SKBR-3(ER- Her2+ Tam-R) cells are purchased from (ATCC). MCF-7 and MDA-MB-231 were maintained in RPMI1640 (Invitrogen) and SKBR-3 was kept in modified IMEM (Invitrogen) with supplement of 5%
FBS (Invitrogen) and 1% PEST(Invitrogen). Before treatment, cells were seeded into 96-well plates or 6-well plates for treatments, after 3 hours of attachment, compounds and tamoxifen were added as designed format with recommended concentration for further 96 hours under the condition of 40%
CO2, 37℃ and 95% humidity.
RNA extraction
Around 106 cells were seeded into 6-well plates and treated as introduced before. Total RNA was isolated using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. RNA concentration was measured by NanoDrop.
cDNA synthesis
500-800 ng of total RNA was reverse-transcribed for cDNA at 40℃, 40min by using RT-enzyme mix and random primers from NBS first strand system (Nordic Bioservice).
RT-PCR
Real time PCR then was operated with NBS SYBR mastermixed (Nordic Bioservice) and performed in ABI PRISM 7500(Applied Biosysterm) according to recommended conditions as 95℃, 10min hot start; 60℃ 50s in 40 cycles.
Finally SYBR-Green primer pairs were adjusted with melting curve analysis.
Primer pairs were used as follows in the project: 18s (Rev: AGC TAT CAA TCT GTC, Fw: GCT TAA TTT GAC TCA), ERα (Rev: TCT GGC GCT TGT GTT, Fw:
GCT ACG AAG TGG GAA), ERβ (Rev: CAG ATG TTC CAT GCC, Fw: ACT TGC TGA ACG CCG),PS2 (Rev: CTC TGG GAC TAA TCA, Fw: CAT CGA CGT CCC TCC)
Proliferation assay
Cells were pre-cultured in 96-well plates with different numbers basic on their growth curve for 3 hours, plating efficiency were similar among plates and then treated with designed format with or without tamoxifen for another 96-120
hours depending on kinds of compounds. Then WST-1(Roche) was used for cell proliferated detection. Considering its sensitivity to incubation temperature and CO2 concentration, we made some modifications for some steps compared to the original methods in this project. For example, before adding WST-1, cells in plate need to change fresh medium as same volume. The detection time also need to be revised according to cell types and numbers in a well, normally examined at 1.5h, 2h, 3h instead of after 4h directly. Final absorbance of the samples against a background control was measured by using a microplate (ELISA) reader at 440 nm.
Western blotting
Cells were seeded into 6-well plates about 60% confluence in RPMI1640 medium supplemented with 5% fetal bovine serum and 1% penicillin. After 3 hours of attachment, compounds added in to medium for 24 hours of treatment.
At the time point, cells were washed by cold PBS and harvested in lysis buffer at 4℃. Final protein concentrations were measured by the Bradford method to decide the same amount of protein from each treatment. SDS-PAGE then was performed for running and gel was electrotransferred to PVDF membrane.
β-Actin (13E5) Rabbit mAb and P44/42 MAPK (Erk1/2) primary antibodies(Cell Signaling) were used for first incubation( name of production ) to against actin and MAPK and peroxidase-conjugated antibodies (SIGMA) were used for second incubation.
Flow cytometry
Flow cytometry is a technique for counting and analyzing cells and chromosomes in research. Cells will be stained with fluorescence-conjugated antibodies depending on their surface markers. They need to suspend as fluid first and pass through as a stream by an electronic detection apparatus. Finally excited fluorescence can emit signal light at certain wavelengths, which will be picked up by detectors and collected for later analysis. Flow cytometry is routinely used in the diagnosis of health disorders.
Cell apoptosis can also be detected by flow cytomety, since different types of particles such as phosphatidylserine or DNA would be exposed outside membrane during different stages in apoptotic cells. In this project, the analysis on the outer apoptotic cell membranes was performed by using Annexin-V-Fluos (Roche) and propidium iodid (Invitrogen) for cell characterization from necrotic cells or labeling with a cell surface marker.
Acknowledgements
I would like to show my great appreciation to Jonas Bergh’s group in CCK Karolinska. Thanks to Dr Jonas, our respectable group leader, his comprehensive knowledge and generosity leave me strong impression of a successful scientist. Thanks to Tao, Anna-lena, Johanna and Linda, my dear group members, they always gave me lots of help and tolerated my mistakes kindly. And especially, my sincere gratitude to Johan, my best supervisor ever, who has provided me with valuable guidance in every stage of my thesis, gave me enough training of my scientific ability with his trust. His keen and rigorous scholarship influenced me a lot in my study now and future.
I shall also extend my thanks to all the people in CCK, R8:03, who really make me a lovely corridor with their friendliness! Thanks to pink group, I feel so lucky to share cell culture room and crazy stories with them. Thanks to those who I still can not tell the names, but their generous help will be unforgettable.
Finally, I am grateful to my professors and coordinator in Uppsala University, thanks very much for you all to provide us such a resourceful chance of study and encourage us to chase our real interests. Thanks again for your great help!
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