GENE EXPRESSION OF MYPT1 AND ROCK1 IN ENDOMETRIAL CANCER

Master Degree Project in Biomedicine One year 30 ECTS Spring term 2015

Student Name: Arisa Ziou Email address: [email protected]

Supervisor: Neha Singh

Examiner: Afrouz Behboudi Email address: [email protected] Abstract Endometrial adenocarcinoma is a cancer that develops from the endometrium, from cells that form the glands in the endometrium. Animal models such as the BDII rat have the characteristic property that they are prone to cancers, characterized by rapid tumor growth, that resemble human cancers. Such rat models are therefore used to study expression and signal pathways in cancer. Cancer cells have the ability to invade surrounding tissues through lymphatic and/or vascular circulation to produce secondary tumors at new sites. The Rho/ROCK pathway orchestrates cell motility thereby contributing in significantly to metastasis. ROCK, through phosphorylation of myosin phosphatase target subunit 1 (MYPT1), inhibits MYPT1 and induces actomyosin contraction which contributes to many cellular processes. In this study, we were interested in investigating the expression of MYPT1 and ROCK1 in malignant and non-malignant endometrial cell lines in BDII rat model, in human endometrial cancer cells represented by the Ishikawa cells line, and in human embryonic kidney 293 (HEK293) human non-malignant control cells. Although statistical significance was not reached due to the small sample size, a difference in the levels in malignant and non-malignant cells was observed. The results from the present study showed a higher expression of MYPT1 and ROCK1 genes in cancer cells (rat and human) compared to non-cancer cells, and we can conclude that this implicates the significance of the MYPT1/ROCK pathway in endometrial cancer and its deregulation. Popular scientific summary Endometrial cancer is the most common gynecological malignancy. It can affect women in pre-, peri- and post-menopausal phase, in the majority of endometrial cancer cases appear in women after the age of 50, whereas 25% of the cases are diagnosed in pre- or peri- menopausal women. Treatment of endometrial cancer, depending on the stage of endometrial cancer, includes hysterectomy, removal of fallopian tubes and ovaries, radiotherapy and chemotherapy. These treatments cause many side effects such as nausea, temporary hair loss, night sweats, vaginal dryness. Moreover, the cost of treatment and support of cancer patients has significant impact on the economy of a country, because treatment of cancer demands not only the equipment and adjuvants but also a wide range of experts, like medical oncologists and social workers. Furthermore, the psychological effect of treatments can cause the patients to experience disorders such as depression and anxiety and disturb the communication between patient, family and friends, expanding to society. In order to reduce such negative impacts, scientists are continuously trying to develop new therapies with reduced side-effects and cheaper. Development of new therapeutic strategies mostly aim in inhibition of cellular processes which enhance the development of endometrial cancer, and therefore demands a better understanding of the molecular basis of endometrial cancer.

In the present study, we were interested in investigating the expression of two genes involved in cell movement in an animal model of the human endometrial cancer. We also wanted to investigate the expression of these genes in a human endometrial cancer cell line. The two genes which we investigated regulate cell movement which is mediated in processes during development such as tissue regeneration and embryological development, and also in the development of cancer. Our results showed that these two genes were expressed more in the endometrial cancer cells thereby implicating these genes in cancer cell movement. Designing agents which inhibit cell movement can lead to better endometrial cancer therapies with reduced side effects as compared to other treatment options such as chemotherapy and radiotherapy, thereby contributing positively to a better quality of life in cancer patients. Contents Abstract...... Popular scientific summary...... Contents...... Abbreviations...... 1. Introduction...... 1 1.1. Endometrial cancer...... 1 1.2. Cancer and animal models...... 1 1.3. Rho-ROCK pathway and cell motility...... 2 1.4. Myosin phosphatase target subunit 1 biological role and implication in diseases and cancer...... 3 1.5. Aim of the study...... 4 2. Materials and Methods...... 5 2.1. Animal crosses and tumor material...... 5 2.1. Cell culture...... 6 2.2. RNA isolation...... 7 2.3. Reverse Transcription...... 7 2.4. Quantitative-PCR...... 7 2.5. Statistical analysis...... 8 3. Results...... 8

4. Discussion...... 12 5. Ethical aspects and impact of the research on the society...... 16 6. Conclusion...... 17 7. References...... 18 8. Appendices...... 23 8.1. Table 2: Components and volumes of reverse transcription reaction...... 23 8.2. Table 3: Concentration, ratio A260/280 and A260/230 measured by nanodrop...... 23 8.3. Table 4: The IDs of TaqMan probes used in the study...... 23 8.4. Table 5:...... 24 8.5. Table 6:...... 24 8.6. Table 7:...... 25 Abbreviations Cq Quantitation Cycle DMEM Dulbecco's Modified Eagle's medium DMSO Dimethyl sulfoxide EMT Epithelial-Mesenchymal Transition FBS Foetal Bovine Serum FGFR2 Fibroblast Growth Factor Receptor 2 GLUT4 Glucose Transporter Type 4 IRS1 Insulin Receptor Substrate 1 ILK Integrin-Linked kinase MAT Mesenchymal-Amoeboid Transition MEM Modified Eagle's Medium MLC Myosin light chain MP Myosin Phosphatase MYPT1 Myosin Phosphatase Target Subunit 1 NEAA Non-Essential Amino Acids NUT N1 Uterine Tumour PDK1 Phosphoinositide-Dependent kinase 1 PP1cδ Protein Phosphatase Type 1 δ isoform PPP1R12A Regulatory Subunit 12A REF Rat Embryo Fibroblast RPMI Roswell Park Memorial Institute 1. Introduction

1.1. Endometrial cancer

Endometrium is the inner mucous membrane of uterus and it is highly influenced by hormones changes of hypothalamic-pituitary system (Behera, 2013). Endometrial adenocarcinoma is developed in the endometrium from cells that form the glands in the endometrium. In 2011, the National Board of Health and Welfare of Sweden reported 1.431 cases of uterine cancer representing 5.2% of the tumors reported in the females of Sweden, the majority of which are estimated to be endometrial adenocarcinomas (National Board of Health and Welfare, 2011). Endometrial adenocarcinomas are classified into two types – type I and II (Bansal et al 2009) adenocarcinomas. Type 1 endometrial adenocarcinomas which consist 70%-80% of endometrial cancers are estrogen-dependent low-grade carcinomas that have favorable prognosis, and affect women in pre- or peri-menopausal phase. Type II endometrial adenocarcinomas are non-estrogen-dependent high-grade carcinomas, that have a poor prognosis, and affect post-menopausal women (Bansal et al 2009; Bokhman, 1983).

1.2. Cancer and animal models

Cancer is a complex disease, and its complexity can be observed in the heterogeneity of cells that can be found in a single biopsy. Τhe first report of the heterogeneity of cancer was by Gerlinger et al. who demonstrated that two biopsies from different sites in patients with kidney cancer had the majority of cells having different mutations and traits (Gerlinger et al., 2012). The rapid growth and division of cells results to damage of DNA which leads to defects of the cell function and multistep process of transformation of normal cells to malignant cells (Hanahan-Weinberg, 2000). Animal models contribute to the study of cancer and biochemical pathways in cancer cells, and also in evaluation of the efficacy of new therapeutic drugs. Animal models such as the BDII rat have the characteristic property that they are prone to cancers, characterized by rapid tumor growth, that resemble human cancers. Such rat models are therefore used to study gene expression and signal pathways in cancer (Cekanova- Rathore, 2014).

1.3. Rho-ROCK pathway and cell motility

Cancer cells have the ability to invade surrounding tissues through lymphatic and/or vascular circulation to produce secondary tumors at new sites (Hanahan D and Weinberg R.A, 2011).

1 Disruption of extracellular matrix, change of cell shape and cell motility through the reorganization of actin cytoskeleton are some critical processes of cancer invasion and metastasis (Matsuoka-Yashiro, 2014; Hanahan D and Weinberg R.A, 2011; Yamazaki et al, 2005). Actin reorganization in all the above steps is a major contributor and is regulated by Rho-GTPases (Matsuoka-Yashiro, 2014; Yamazaki et al, 2005). The Rho/ROCK pathway orchestrates cell motility thereby contributing in a significant way to metastasis (Matsuoka- Yashiro, 2014). Apart from cell motility, the Rho/ROCK pathway mediates cytoskeletal tension which is produced by actin cytoskeleton and plays a fundamental role in gene expression, cell proliferation and differentiation (Bhadriraju et al, 2007). Rho-kinases ROCK1/ROKβ/Rho-kinaseβ and ROCK2/ROKα/Rho-kinaseα are downstream effectors of Rho-GTPases (Matsuoka-Yashiro, 2014; Amano et al 2010; Olson and Sahai, 2009).Rho- associated, coiled-coil containing protein kinase 1 and 2 (ROCK1 and ROCK2) mediate many cellular processes such as motility during metastatic process of cells (Rath-Olson, 2012; Itoh et al, 1999), and systems of the human body such as the nervous and the cardiovascular system (Schmandke et al 2007; Ohtsu et al, 2005).

The human ROCK1 gene located on 18 (18q11.1) encodes a protein serine/threonine kinase and it is the binding of the GTP-bound form of Rho which activates the kinase (Figure 2) (National Center of Biotechnology Information, 2015). ROCK1 through phosphorylation of different substrates like myosin phosphatase target subunit 1 (MYPT1), myosin light chain (MLC) and insulin receptor substrate (IRS), catalyze a plethora of processes which participate in morphological changes that occur during metastasis (Akagi et al 2014; Geetha et al, 2012; Amano et al, 2010). Processes such as structural rearrangements, adhesion, transformation, cytokinesis, migration, proliferation and changes in cellular polarity occur in morphological changes (Majid, et al 2012; Amano et al, 2010). ROCK1 possesses oncogenic activity and depending on the cell type, participates in proapoptotic responses creating a complex balance between them (Akagi et al 2014). In a previous study, three somatic mutations were found in ROCK1 gene which lead to increased ROCK1 and subsequently drove enhanced motility and decreased adhesion, which are features of cancer progression, mediated by actin cytoskeleton rearrangements (Lochhead et al, 2010). ROCK functions through phosphorylation of myosin phosphatase target subunit 1 (MYPT1) which inhibits MYPT1, and also increase the phosphorylated-myosin light chain (p-MLC) which induces actin contraction (Figure 2) (Matsuoka-Yashiro, 2014; Amano et al, 2010). In other previous studies, ROCK1 was found to induce the destabilization of actin cytoskeleton via Myosin light chain 2 (MLC2) phosphorylation, and ROCK2 mediated stabilization of actin cytoskeleton via cofilin contributing to cell contraction (Matsuoka-Yashiro, 2014; Amano et al, 2010). Additionally, activation of ROCK results in rounded shape of cells due to increased

2 MLC phosphorylation through actomyosin contractility which is driven by Rho/ROCK pathway. The rounded shape assists in invasion and metastasis (Rath-Olson, 2012).

1.4. Myosin phosphatase target subunit 1 biological role and implication in diseases and cancer

MYPT1 also called protein phosphatase 1 regulatory subunit 12A (PPP1R12A), is one of the three subunits of myosin phosphatase (MP) and regulates the activity of actomyosin (National Center of Biotechnology Information, 2015; Iwasaki et al, 2013). MYPT1 is activated by many receptors such as tyrosine kinases receptors, lipoproteins receptors and insulin receptors, and participates in many cellular processes (Schmandke et al, 2007; Geetha et al, 2012). MYPT1 influences MLC phosphorylation and subsequently influences cytokinesis and chromosome stability (Iwasaki et al, 2013; Wu et al, 2010). Moreover, MYPT1 forms a complex with δ isoform type 1 protein phosphatase (PP1cδ), IRS1 and p85 subunit of PI3K, and activates phosphoinositide-dependent kinase 1 (PDK1) which activates AKT and which in turn activates mTOR/Raptor. Activation of mTOR/Raptor leads to cell survival, cell differentiation and protein synthesis (Geetha et al, 2012).

By activation of MYPT1 from kinases like ZIPK and activated MYPT1, MYPT1 contributes to apoptosis through down-regulation of MP and activates myosin II which plays a role in cell migration, apoptosis and cytokinesis (Iwasaki et al, 2013). MYPT1 also mediates activation or degradation of YAP-TAZ oncogene through dephosphorylation of MYPT1 by integrin- linked kinase (ILK), which consequently leads to degradation of YAP-TAZ and inhibition of tumor growth (Serrano et al, 2013).

3 Figure 1. The Rho/ROCK pathway contributes to actomyosin contraction. Activated receptors stimulate guanine exchange factors (GEFs), which subsequently activate RhoA. ROCK1 and 2 are activated by RhoA and promote different phosphorylation. In particular, ROCK1 mediates phosphorylation of MLC and LIMK mediates phosphorylation of LIMK. The phosphorylation promotes actomyosin contraction, actomyosin filament bundling and actin filament stabilization, which participates in several cellular processes such as motility, adhesion, proliferation and apoptosis (Rath-Olson, 2012).

1.5. Aim of the study

The overall aim of this study was to investigate the expression levels of MYPT1 and ROCK1 genes in malignant and non-malignant endometrial cell lines in BDII rat model, in human endometrial cancer cells represented by the Ishikawa cells line, and in human embryonic kidney 293 (HEK293) cells used as human non-malignant control cells from another tissue other than the endometrium.

4 2. Materials and Methods

2.1. Animal crosses and tumor material

The tumor material used in this study was obtained by crossing female BDII rats with low- incidence endometrial adenocarcinoma rat strains (BN or SPRD) to produce F1 progeny. The F1 progeny were then crossed to produce F2, and the F1 also backcrossed with the BDII females (Deerberg & Kaspareit, 1987; Roshani et al, 2005), to produce N1 progeny. N1 uterine tumor (NUT) cells which develop endometrial adenocarcinoma and are estrogen- dependent were then derived from the N1 offspring (Deerberg & Kaspareit, 1987). A total of 8 Malignant and non-malignant NUT cells (Table 1) were used in this study to compare the gene expression of MYPT1 and ROCK1 in rat cells. The rat cell lines were normalized by GAPDH housekeeping gene, which plays the role of endogenous control and normalizes differences in quality and quantity between samples. Additionally, rat embryo fibroblast (REF) was used as an exogenous control to normalize the differences arising from inadequate RNA isolation or reverse transcription, and to ensure that quantification is optimal.

Table 1. An overview of the cell lines used in the study

CELLS TISSUE ORIGIN

NUT cells: Malignant NUT 6 Endometrium

NUT 43 NUT 128 NUT 97 non-malignant NUT 129 NUT 75 NUT 110 NUT 118 Ishikawa Malignant Endometrium sample

HEK293 Non-Malignant Kidney sample

5 Figure 2. A schematic overview of the study. The BDII rat cell lines described in Table 1 were cultured in appropriate growth media. Cell cultures were examined every day and trypsinization when reached to 80% confluence were sub cultured. When the desired concentration of cells was reached, total RNA for all cell lines was isolated. Total RNA was reverse transcribed to cDNA and cDNA was relative quantified using TaqMan probes for all cell lines. Cq values were obtained, rat cells were normalized by endogenous and exogenous controls, and human cell lines were normalized by an endogenous control. After normalization, log 2 values were calculated.

2.1. Cell culture

The human embryonic kidney 293 (HEK293) cell line, and human endometrial cancer cell line Ishikawa were also used in this study. The Ishikawa cell line is derived from well- differentiated, estrogen receptor positive adenocarcinomas of the endometrium (Vollmer,

2003). The HEK293 cell line is derived from a transformed kidney and possesses the desired non- or low tumorigenic qualities appropriate for testing oncogenic properties of cancer- associated genes. Primary cell lines cultures obtained from Ishikawa and HEK293 cell lines both purchased from Sigma-Aldrich (Sigma-Aldrich, Missouri, United States) were cultured in RPMI 1640 media growth supplemented with 10 mM Hepes, 1 mM Sodium pyruvate, 2mM L-glut, 10% FBS and 1X PEST (also from Sigma-Aldrich, Missouri, United States). The NUT cell line was propagated in DMEM media growth supplemented with 1X MEM Vitamins, 2mM L- glut, 1X MEM AA (amino acid), 1X MEM NEAA, 10% FBS and 1X PEST (Sigma-Aldrich, Missouri, United States).

All cells were grown at 37°C in an atmosphere of 95% humidity and 5% CO2. Cells were observed every day under microscope with x10 magnification and harvested through trypsinization when the confluence reached to 80%. The harvested cells were then counted and subcultured. The human cell lines (the Ishikawa cell line and HEK293 cells) were normalized by GAPDH.

2.2. RNA isolation

Cell pellets from NUT cells, Ishikawa cells and HEK293 cells were obtained after cell culture and RNA isolation was performed using mirVana™ PARIS™ RNAkit (Applied Biosystems, California, United States), in an RNAase-free environment and following manufacturer's instructions manual.

6 The eluted solution from RNA isolation was collected, the concentration and purity of every sample was measured through Nanodrop ND-1000 (Nanodrop technologies, USA) and stored at -20°C.

2.3. Reverse Transcription

Reverse Transcription was performed by using High-Capacity RNA-to-cDNA kit (Applied Biosystems, California, United States). The reverse transcription reactions were prepared by following the manufacturer's instructions manual (Appendix A) in a final volume of 20μl, and total RNA samples of 2μg per 20 μl per reaction were introduced in the reactions l. The thermocycler was programmed for the following cycles: step 1 for 60 min at 37°C, step 2 for 5min at 95° C, and step 3 for ∞ at 4° C.

2.4. Quantitative-PCR

TaqMan® Universal PCR Master Mix was used for relative quantification (Applied Biosystems, California, United States) and different TaqMan probes (Applied Biosystems, California, United States, Appendix B) were used for rat and human cells. cDNA samples were diluted 1/100 before using cDNA samples for quantitative PCR. Each sample was loaded as triplicate in a 96-well plate, and 9μl of cDNA samples, 1μl of probe and 10μl of master mix were loaded to the reaction tube to a final volume of 20μl of each reaction. No template controls were used for each different probe. Rat samples were normalized by GAPDH (endogenous control) and Rat Embryo Fibroblast (REF) (exogenous control), while human cells were normalized by GAPDH. The reactions were ran by the thermocycler 7300 (Applied Biosystems, California, United States) for 40 cycles and in the following cycles: step 1 for 2min at 50°C, step 2 for 10min at 95°C, step 3 for 15sec at 95°C, and step 4 for 1min at 60 °C.

2.5. Statistical analysis

Cq values were obtained from the quantification plots, normalized by the GAPDH values, and log2 values were then calculated using the values of REF (Appendix D) and through the Livak method for the rat cell lines. This method is used to calculate relative changes in gene expression and is derived from quantitative PCR and calculated as follows (Yilmaz et al 2012):

- (Ct -Ct ) test sample - (Ct -Ct ) calibrator 2 target gene reference gene target gene reference gene

7 A simpler formula for the Livak method - the ΔCq method - was used for calculating the fold changes in human cell lines as follows (Appendix D):

- (Ct -Ct ) test sample 2 target gene reference gene

The log2 values were introduced to SPSS (IBM SPSS statistics 22). Mean and standard deviation were calculated for each sample and p-values were obtained using a confidence interval of 95%. Statistical significance was defined as P<0.05.

3. Results In this study, we were interested in investigating the expression of MYPT1 and ROCK1 genes in malignant and non-malignant endometrial cell lines in BDII rat model, in human endometrial cancer cells represented by the Ishikawa cells line, and in human embryonic kidney 293 (HEK293) human non-malignant control cells. Therefore, as indicated in Table 1, 8 NUT cell lines, Ishikawa cells and HEK293 cells were quantified for the expression of MYPT1 and ROCK1 genes. As seen in figures 3 - 6, a general pattern of higher expression of these genes in malignant cell lines was observed compared to the non-malignant cell lines.

A general view of the figures 3 and 4 below shows a higher expression of MYPT1 in malignant rat cells compared to non-malignant rat cells. Significant differences were indicated for values with P values < 0.05. The exception among the rat non-malignant cells was NUT 129 which displayed a higher expression of MYPT1 than the other non-malignant cells. A possible explanation for the higher expression of MYPT1 in NUT129 non-malignant cell could be that the cell line did exceed the passages that a normal cell line divides and became immortalized using the same mechanisms employed by cancer cells. Similar to cancer cells, during the cell culturing, this cell line exhibited an aggressive proliferation in comparison with the other non-malignant cells and it reached 80% confluence within shorter incubation period than the other cell lines. Among human cells, Ishikawa cells showed a higher expression of the MYPT1 gene compared to HEK293 cells as seen in figure 4.

8 Figure 3. Relative quantification of MYPT1 gene expression in NUT 75, NUT 110, NUT118, NUT 129, NUT6, NUT 43, NUT 128, NUT 97. Values of rat cells were normalized by GAPDH and REF was used as an exogenous control. Results showed that MYPT1 was expressed more in malignant cell lines than in non-malignant rat cells based on the fold change. Statistical significance was defined as P<0.05.

Figure 4. Relative quantification of MYPT1 expression in HEK293 and Ishikawa cells. Values of human cells were normalized by GAPDH which was also used as an endogenous control. Results showed a higher expression of MYPT1 in Ishikawa cells as compared to HEK293 cells. Statistical significance was defined as P<0.05.

9 As seen in Figures 5 and 6 below, there was a higher expression of ROCK1 in the endometrial cancer cells compared to the non-malignant endometrial cells. Similar to what was observed earlier in the expression of MYPT1 gene, an over-expression of ROCK1 was also observed in the non-malignant NUT 129 cell line thus strengthening our hypothesis that this cell line did indeed progress to a malignant stage. For the human cells, Ishikawa cells displayed a higher expression of ROCK1 compared to the HEK293 cells as seen in figure 6.

Figure 5. Relative quantification of ROCK1 in NUT 75, NUT 110, NUT118, NUT 129, NUT6, NUT 43, NUT 128, NUT 97. Values were normalized by GAPDH and REF was used as an exogenous control. Results showed a higher expression of MYPT1 in malignant cell lines than in non-malignant rat cells based on fold change. Statistical significance was defined as P<0.05.

10 Figure 6. Relative expression of ROCK1 in HEK293 and Ishikawa cells. Values of human cells were normalized by GAPDH which was also used as an endogenous control. Results showed a higher expression of ROCK1 in Ishikawa cells as compared to HEK293 cells.

4. Discussion Endometrial adenocarcinoma is the sixth most common cancer in women all over the world, and accounts for 4.8% of all cancers diagnosed in women (Ferlay et al, 2013). With a cumulative risk of 1% by age of 75 years and a death risk of 0.2%, it is the most common malignancy of the female reproductive tract (Van Nyen, Moiola, Colas, Annibali, & Amant, 2018). Endometrial adenocarcinoma is influenced by the balance of estrogen and progesterone and several factors such as estrogen therapy, birth control pill and obesity, have been reported to affect this balance therefore contributing to endometrial cancer development (American Cancer Society, 2015; Kaaks et al 2002). This is the reason why it is crucial to investigate the pathways mediated in endometrial adenocarcinoma.

Since the Rho/ROCK pathway, and especially ROCK, is involved in a wide spectrum of cellular processes, it is important to understand that malfunction of the protein and its effectors can contribute to the development of diseases and cancer (Matsuoka-Yashiro, 2014; Cheng et al,2013). ROCK has been reported to contribute in many types of cancer. In 1999, Itoh and his colleagues utilizing rat MM1 hepatoma cells proposed that ROCK participates fundamentally in tumor cell invasion (Itoh et al, 1999). Additionally, Sahai et al. in 1999 suggested that ROCK mediates the transformation of the NIH3T3 fibroblasts cells (Sahai et al, 1999). Further, Lochhead et al (2010) studied ROCK1 activity in NIH 3T3 mouse fibroblast cells and showed that the enhanced activity of ROCK1 in cancer cells is a direct consequence of three somatic mutations (Lochhead et al, 2010). In 2003, Kamai and his colleagues, investigating the protein expression of RhoA, RhoC and ROCK in tumor and non- tumor bladder cells as well as metastatic and uninvolved lymph nodes, showed overexpression of the mentioned proteins and correlation of this overexpression with poor tumor differentiation and lymph node metastasis (Kamai et al, 2003).

The Rho/ROCK pathway and its downstream effectors which play a pivotal role in many cellular functions have been investigated in many studies. The Rho/ROCK pathway is involved in the modulation of smooth muscle tone, stress fiber formation, maintenance and focal adhesions as reported in the review of Amano et al. in 2010 (Amano et al, 2010). Moreover, the Rho/ROCK pathway contributes to the actomyosin-based contractile ring

11 during cell division by phosphorylation of MLC by ROCK mediated by MYPT1 inhibition, therefore also contributing to Micro-cytokinesis (Amano et al, 2010; Wu et al, 2010). One of the downstream effectors of the Rho/ROCK pathway is the MYPT1. The MYPT gene family is very significant in the development of diseases such as cancer, hypertension, Parkinson’s disease et cetera, and studies have indicated that MYPT1 has important functions in the development and progression of cancers, mediating many cellular processes such as cell motility, apoptosis, cell differentiation and cytokinesis (Lin et al., 2017). Deregulation of MYPT1 can lead to diseases and may also affect cancer cells. For example, in the review by Rath and Olson, it was reported that activation of ROCK and downstream effectors (MLC and MYPT1) was associated with cell survival and cell growth of tyrosine kinases-driven hematological malignancies (Rath-Olson, 2012). Further, Cheng et al. reported that MYPT1 downregulation may contribute to atherosclerosis progression through abnormal contractility and reduce vascular smooth muscle cell migration in cultured human aortic smooth muscle cells (Cheng et al, 2013). In other previous reviews, Hartshorne et al., and Ito et al. stated that the activity of Myosin Phosphatase (MP) is strongly affected by the activity of its subunit MYPT1, and that MP is the only phosphatase that regulates MLC and subsequent processes like smooth muscle contraction (Ito et al., 2004; Hartshorne et al. 1998). Wu et al. showed that the decrease of MLC is associated with failure in cytokinesis and the processes of multinucleation and multipolarity in oral cancer cells. The study also concluded that the deficiency in MLC is due to deficiency of MLCK or due to overexpression of MYPT1 (Wu et al, 2010). MYPT1 has many different binding sites and many subcellular interactions, and resistance or activation of MYPT1 expression may be a therapeutic target for tumors (Given, Ogut, & Brozovich, 2007).

The present study was conducted on the BDII rat endometrial cancer cells and a human endometrial cancer cell line. The aim of the study was to investigate the expression of MYPT1 and ROCK1 genes in endometrial adenocarcinoma. The cell lines used in this study are widely accepted models for investigating the molecular mechanisms of female reproductive tract tumors. The BDII/Han rat cell lines, from whence the N1 uterine tumour (NUT) cells used in this study were derived, are also an excellent model system for studying type I endometrial cancer. They are characterized by the stable expression of estrogen receptors, and are characteristically predisposed to developing spontaneous endometrial adenocarcinoma which is very similar to the human endometrial adenocarcinoma type 1 both in cell biology and in pathogenesis (Deerberg & Kaspareit, 1987). The Ishikawa cell line is an excellent model for studying general and molecular mechanisms in the biology of endometrial cancer (Vollmer, 2003) due to their sensitivity to estrogens and

12 progesterone, as they contain estrogen and progesterone receptors (Kuramoto-Nishida, 2002; Vollmer, 2003; Yang et al, 2001; Lessey et al, 1996;). The cell line was originally established from an endometrial adenocarcinoma from a 39-year-old Japanese patient. The Ishikawa cell line was used in this study due to its comparability with the rat endometrial cancer cells which exhibits large similarities in cell biological aspects in the pathogenesis of endometrial cancers in humans. Due to these desirable properties, the Ishikawa cell line provided the endometrial cancer cell lines in this study. The human embryonic kidney 293 (HEK293) cell line was originally derived from a transformed kidney of an aborted human embryo. This transformation was succeeded by integration of adenoviral genome of Adenovirus 5 in chromosome 19 and produces a protein which inhibits apoptosis. HEK293 cells are used in a wide range of experiments - from signal transduction to rapid small-scale protein expression and biopharmaceutical production. It is a hypotriploid human cell line, with a modal chromosome number of 64 (Yao-Cheng Lin et al, 2014). For their application as non- or low tumorigenic for testing oncogenic properties of cancer-associated genes, HEK293 cells were used as human non-malignant control cells in this study. Although statistical significance was not reached due to the small sample size, a difference in the gene expression levels in malignant and non-malignant cells was observed (figures 3- 6). As seen, rat malignant cells expressed ROCK1 more than rat non-malignant cells. A higher expression of MYPT1 in rat endometrial cancer cells and in human Ishikawa cells was also observed.

It is well known that ROCK is involved in many cellular processes as well as in cancer. Elevated expression of ROCK1 has been reported by previous studies in pancreatic tumors, esophageal squamous cell carcinomas, in testicular germ cell tumors, and in bladder tumors (Kamai 2003, 2002; Zhou 2003; Kaneko 2002). In other previous studies, Wu et al. ascertained that oral cancer cells exhibited increased multinucleation and multipolar spindles defects which derive from defects in MLC (decreased phosphorylation) as a result of overexpression of MYPT1 (Wu et al, 2010). Grindrod et al. estimated the expression of MYPT1 in order to assess MLCP association with prostate cancer progression and found overexpression of MYPT1 in prostatic intraepithelial neoplasia as compared to precancerous prostatic intraepithelial tissues (Grindrod et al, 2011). The present study, where a higher expression of MYPT1 was observed, is in agreement with all the above-mentioned previous studies. From these results, it can be suggested that the difference in expression of MYPT1 and ROCK1 in the cancer cells compared to normal cells of the study could implicate the crucial role of MYPT1/ ROCK pathway in endometrial cancer. This therefore merits further investigation of the role of MYPT1 and ROCK1 genes in

13 endometrial carcinogenesis, as well as regulation of the ROCK/MYPT1 pathway in endometrial cancer in future studies.

Several studies have focused on creating successful inhibitors of ROCK when investigating methods of cancer treatments. For example, in the study by Itoh et al., the ROCK inhibitor Y27632 was shown to block the hepatoma cell line from forming nodules in the peritoneal area (Itoh et al, 1999). Further, several inhibitors of ROCK have been utilized in the therapy of types of cancer like prostate, lung and melanoma (Routhier et al, 2010; Rattan et al, 2006; Somlyo et al, 2000). As ROCK inhibitors succeed in blocking tumour growth, the downstream effectors of ROCK may also contribute in reduction of tumour growth. Therefore, designing an effector for activation of MLC or designing a component for inhibition of MYPT1 may contribute successfully in reduction of cancer cells development. It has also been shown that blocking overexpression of MYPT1 in cancer cells can in turn block multinucleation and multipolarity, which is observed in the early stages of human tumour formation (Wu et al, 2010). Putting these findings and our results into consideration, we suggest that MYPT1 might be a potential biomarker and therapeutic strategy for endometrial cancer.

As cell lines capture only a small part of tumor heterogeneity, an important limitation in this study was the small sample size of the cell lines used. As a result, strong conclusions could not be reached due to lack of statistical significance. Therefore, future studies should be conducted with a higher number of cell lines to enable achievement of statistically significant results and confirm if the pattern and the results observed in this study can be replicated. Such future studies should include paired cell lines to also remedy the issue of tumor heterogeneity.

5. Ethical aspects and impact of the research on the society The use of animals is applied in a wide range of experiments in research for development of new drugs and understanding the mechanisms of development and maintenance of an organism. However, this wide spectrum of animal use is always an issue of debate between the scientists and animal-rights organizations due to the ethical aspects involved. Due to this, legislations and laws have been introduced and implemented in many countries around the world over the last few decades to govern experiments involving animal use. It is an obligation for every researcher to prioritize the welfare of the animals, and to minimize the suffering of animals in any research. Further, alternatives of animal use must be first explored, and proper design of experiments assessing the benefits of the experiments

14 must be conducted. Russell and Burck, in their book "The Principles of Humane Animal Experimental Techniques″, introduced the three main principles – ‘the three Rs'- which a researcher must take into consideration when designing an experiment (Russell and Burck, 1959). The ‘three Rs'- Reduction, Refinement and Replacement- must govern every study conducted. Reduction is targeting the minimization of the number of animals used in an experiment and can be achieved by enhancement of experimental design and use of appropriate statistical analysis, refinement is associated with improvement of animal welfare by minimizing animal pain or distress, and replacement includes complete avoidance or replacement of animals used in a study through the use of other alternatives such as human tissues, established animal cell lines, microorganisms, plants, eggs, reptiles, amphibians and invertebrates, and digital modeling.

Our understanding of endometrial cancer has greatly increased over years as a result of the advanced molecular techniques applied to the different available preclinical models. Significant promise in form of potential biomarkers and therapeutic strategies for endometrial cancer has also been achieved from the many experimental studies done. In this study, we observed a difference in MYPT1 and ROCK1 gene expression levels in malignant and non- malignant cell. These findings directly and significantly contribute to the knowledge acquired so far about the MYPT1/ROCK pathway in endometrial cancer, and strengthen the hypothesis that both MYPT1 and ROCK1 genes may be potential biomarkers for endometrial cancer.

6. Conclusion In summary, the results from the present study showed a higher expression of MYPT1 and ROCK1 genes in cancer cells (rat and human) compared to non-cancer cells, and we can conclude that this implicates the significance of the MYPT1/ROCK pathway in endometrial cancer and its deregulation. From this, further studies with greater sample size to achieve conclusive results with statistical significance are recommended. Further studies should also be conducted to explore the mechanism behind the higher expression of MYPT1 and ROCK1 in clinical samples of endometrial adenocarcinoma.

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20 8. Appendices

8.1. Table 2: Components and volumes of reverse transcription reaction

Component +RT reaction (μl) -RT reaction (μl) 2X RT Buffer 10 10 20X Mix 1 - RNA sample 1.7 1.7

Nuclease-free H2O 7.3 7.3 Total per reaction 20 20

8.2. Table 3: Concentration, ratio A260/280 and A260/230 measured by nanodrop

Cells Concentration (mg/ml) A260/280 A260/230 Ishikawa 83.0 2.17 1.25 HEK293 78.6 2.10 1.92 NUT 6 102.2 2.18 1.98 NUT 43 250.3 2.10 1.69 NUT 128 85.1 2.12 2.16 NUT 97 123.5 2.18 1.54 NUT 75 136.8 2.17 1.79 NUT 110 76.8 2.06 1.89 NUT 118 198.7 2.19 1.80 NUT 129 143.9 2.07 1.98

8.3. Table 4: The IDs of TaqMan probes used in the study

TaqMan probes ID MYPT1 human Hs01552899_m1 ROCK1 human Hs01127699_m1 MYPT1 rat Rn00588037_m1 ROCK1 rat Rn00579490_m1

8.4. Table 5:

Reaction components Volume per reaction (μl) TaqMan® probes 1 TaqMan® Universal PCR Master Mix 10 cDNA 9 Total 20

8.5. Table 6:

CCTE AAVG CCTC AVG ΔCT ΔCT Sample (ROCK (ROCK (GAPD (GAPD (ROCK (GAPD ΔΔCT 2^- LOG2 1) 1) H) H) 1) H) ΔΔCT

21 NUT75 36,22 36,21 27,52 27,48 10,17333 1,446667 8,72666 0,002 - 7 8,72667

NUT75 36,22 27,51

NUT75 36,19 27,41

NUT11 32,76 32,76667 25,09 24,85667 6,73 -1,17667 7,90666 0,004 - 0 7 7,90667

NUT11 32,72 25,01 0

NUT11 32,82 24,47 0

NUT11 31,78 31,74667 23,42 23,49667 5,71 -2,53667 8,24666 0,003 - 8 7 8,24667

NUT11 31,74 23,56 8

NUT11 31,72 23,51 8

NUT12 24,46 24,47333 24,5 24,48333 -1,56333 -1,55 - 1,009 0,01333 9 0,01333 3

NUT12 24,49 24,39 9

NUT12 24,47 24,56 9

NUT6 24,56 24,57 24,54 24,57 -1,46667 -1,46333 - 1,002 0,00333 0,00333 3 ’ NUT6 24,58 24,68

NUT6 24,57 24,49 0 0 NUT43 27,43 27,45333 27,51 27,45 1,416667 1,416667 1,000

NUT43 27,46 27,37

NUT43 27,47 27,47

NUT12 25,76 25,83333 25,78 25,80667 -0,20333 -0,22667 0,02333 0,984 - 8 3 0,02333

NUT12 25,83 25,76 8

NUT12 25,91 25,88 8

NUT97 24,15 24,13333 24,13 24,13 -1,90333 -1,90333 3,55E- 1,000 -3,5E- 15 15

NUT97 24,14 24,09

NUT97 24,11 24,17

REF 26,06 26,03667 26,06 26,03333

22

REF 26,01 26,02

REF 26,04 26,02

8.6. Table 7:

Sample CTE AVG AVG ΔCT ΔCT ΔΔC (2^- LOG2 (MYPT1) (MYPT1) (GAPD (MYP (GAP T ΔΔCT) H) T1) DH) NUT75 36,22 27,480 10,173 1,447 8,727 0,002 -8,727 NUT75 36,22 NUT75 36,19 NUT110 32,76 24,857 6,730 -1,177 7,907 0,004 -7,907 NUT110 32,72 NUT110 32,82 NUT118 31,78 23,497 5,710 -2,537 8,247 0,003 -8,247 NUT118 31,74 NUT118 31,72 NUT129 24,46 24,483 -1,563 -1,550 - 1,009 0,013 0,013 NUT129 24,49 NUT129 24,47 NUT6 24,56 24,570 -1,467 -1,463 - 1,002 0,003 0,003 NUT6 24,58 NUT6 24,57 NUT43 27,43 27,450 1,417 1,417 0,000 1,000 0,000 NUT43 27,46 NUT43 27,47 NUT128 25,76 25,807 -0,203 -0,227 0,023 0,984 -0,023 NUT128 25,83 NUT128 25,91 NUT97 24,15 24,130 -1,903 -1,903 0,000 1,000 0,000 NUT97 24,14 NUT97 24,11 26,06 26,033 REF 26,01 REF

23 26,04 REF

24