Expression and Signalling in Breast Cancer Cells, Essay Example

Abstract

This paper is based on exploring the role of occluding in HER2 expression and signalling in Cancer cells of Breast cancer. Breast cancer refers to a tumour that is malignant and has been developed in the cells of breast. It is one of the common type of cancer in women globally. HER2 is a (human epidermal growth factor receptor 2) which is found on the breast cells and are protein receptors. One of the major molecules associated with tight junction is occludin. It is an important stability for the barrier functions and tight junction stability. In this research, functional antagonism or occludin knockdown in MCF7-HER2 breast cancer cells is done in 4 individual experiences to figure out the influences of this TJ in levels of HER2. The analysis is done using statistics t-test where the four independent experiments were tested at the significance level when p values <.05. In the results it was figured out that the occludin molecule has no impact on the HER2 expression. The major points in this research is to know that re-probing and stripping for actin results in the protein loss so that’s why in the data and results actin was excluded. In the end of the research, the negative results showed that occludin doesn’t play a crucial role in the breast cancer.

Introduction

Breast cancer refers to malignant tumour that has developed in breast cells. It is the most common type of cancer in women worldwide.  Breast cancer develops when mutations or abnormal changes occur in genes responsible for regulation of cell growth.¹ there are many risk factors that contribute to the development of breast cancer some of which are non-modifiable such as age, gender, family history. However, there are some risk factors that can be modified or controlled such as, weight, physical activity, and exposure to oestrogen.² One of many genes that help regulating cell growth and repair is the HER2 gene (human epidermal growth factor receptor 2). This gene produces HER2 proteins. HER2 protein receptors are found on breast cells. About 25% of breast cancer tests positive for this protein.  Mutations or changes of the gene cause overexpression of HER2 receptors in cancer cells promoting their growth.³ This type tends to be more aggressive than other types of breast cancer. It’s less sensitive to therapy and resistance is a big issue. ⁴

Studies have shown that over expression of Junctional Adhesion Molecule-A (JAM-A) is associated with poor patient outcome. Also, over expression of JAM-A is correlated with over expression of the oncogene HER2. JAM-A family of tight junctions (TJs) is a cell adhesion molecule that is expressed in epithelial and endothelial cells, and also hematopoietic cells, such as leukocytes, platelets, and erythrocytes. JAM-A has an important regularity functions in several cellular adhesive processes. These studies found that silencing of JAM-A resulted in subsequent reduction in the expression of HER2. This suggests that regulation of JAM-A expression might affect tumorigenic signalling of HER2.⁵

One of the first identified TJ associated molecules is occludin. Occludin is important in tight junction stability and barrier function. In this study, occludin knockdown or functional antagonism in MCF7-HER2 breast cancer cells was done in 4 individual experiments to see if it influences the levels of HER2.

Aim of the Research

This project will contribute new knowledge about the signalling pathways that control HER2 tumorigenesis in breast cancer cells.

Materials & methods

There are many materials and method used in conducting this experiment. These comprises of Cell line, plating cells, protein extraction, knock down via siRNA transfection, BCA Assay, Western Blotting, Stripping for actin and statistical analysis. The description of these methods and materials is given below;

Cell line

MCF7 cells transfected for high levels of HER2 (MCF7-HER2). HER2 is a member of epidermal growth factor receptor (EGFR) and plays a major role in cancer progression and metastasis. These cells show high resistance to cytotoxic therapies.⁶ these cells are maintained in a 10ml medium added to each flask which is  made up of minimum essential medium (MEM) 435ml, L-Glutamine 5ml, non-essential amino acid (NEAA) 5ml,penicillin streptomycin 5ml, foetal bovine serum(FBS) 50ml. Cells are kept at 37^oC incubation.

Plating cells

The cells were 80% confluent .The media was discarded and cells were washed with 10 ml of phosphate buffered saline (PBS). In order to detach cells from flask 2 ml of trypsin was added and incubated for 5 minutes. Media (8ml) is then added.  The solution is then transferred into a 15 ml tube and centrifuged at 1000 RCF for 3 minutes.

A 6-well plate was used to seed the cells. In each well a 100000 cells were seeded in 2 ml medium cells were incubated for 24 hours at 37^oC.

Knockdown via siRNA transfection

The first well was for control. The cells in the 5 remaining wells were transfected using short interference RNA -dharmafect  (Dharmafect- siNEG –siOCCLUDIN – siJAM(1) – siOCCLUDIN+siJAM(1) )in antibiotic free media. Following treatment the cells were incubated for 72 hours at 37^oC.

Protein extraction

The solution in each well was aspirated.  PBS was added to each well to wash cells for 5 minutes and then discarded. 200µl of Cell lysis buffer (relax buffer1.4ml, Triton-X100 14 µl, phosphatase inhibitors (2) 14 µl and (3) 14 µl, protease inhibitor14 µl) was added to each well. The protein lysate was then centrifuged at 4C at 1500rcf for 5 minutes. The supernatant was transferred into new tubes.

BCA Assay

In 96-well plate a 10µl of samples and 10µl of standards (5mg BSA in 1ml ddH₂O aliquoted as 5mg/ml, 2.5mg/ml, 1.25mg/ml, .625mg/ml, .3125mg/ml, and 0mg/ml) were added into wells in duplicate.  200 µl of BCA solution was added on top and incubated at 37^oC for 30 minutes covered in foil. The protein concentration was then measured in each sample using multi-label reader (PerkinElmer-Victor X3)

Western blotting

Whole cell lysates extracted from cells 18µg, 8µg, 17µg, 20µg (amount of protein loaded for each experiment respectively) of denatured proteins were electrophoresed on 2 (9%) gels and were transferred into PVDF membranes. membranes were blocked with milk for (JAM-A and Occludin) and with Bovine serum albumin for (HER2). The membranes were then immune blotted with primary antibodies against all three proteins using mouse anti JAM-A (1:1000), mouse anti HER2 (1:1000), rabbit anti occludin (1:1000) and kept on roller overnight in 4^oC room. The following day the membranes were washed 3 times with TBS-T for 5 minutes and then blocked with secondary antibodies(anti mouse(1:5000), anti-rabbit(1:2500)) for an hour and washed again. Finally, the immuno-reactions were visualized on x-ray films using the enhanced chemo-luminescence (ECL) system.

Stripping for actin

The membranes were stripped using 70µg of ?- mercaptoethanol in 10ml stripping buffer for each membrane for 10 minutes in 60^oC heating oven. Followed by X3- 5minutes washing with TBS-T. The membranes were blocked with milk for an hour and then immune blotted with primary antibodies against actin (rabbit anti actin (1:5000)) and left overnight in 4^oC room. The following day the membranes were washed 3 times with TBS-T for 5 minutes and then blocked with secondary antibody (anti-rabbit (1:2500)) for an hour and washed again. Finally, the immuno-reactions were visualized on x-ray films using the enhanced chemo luminescence (ECL) system.

Statistical analysis

All comparisons were made against control. The t-test was used to analyse all data in four independent experiments and were considered significant when p values <.05.

Results

In the results we can see that there are 3 figures. One is he JAM-A, second is HER2 and the third is Occludin. These figures values are divided into six variables. These are Control, Dhamafect, siNEG, siOccludin, siJAM and siBoth. In regards to HER2 signalling effects we can see that it has less Dhamafect and siNEG values as compared to JAM-A and Occludin. In Occludin, there is very less impact of siOccludin which indicates that occluding has less impact on HER2 expression in controlling the breast cancer.

Knockdown of occluding does not affect the levels of HER2 expression

From all four experiments the knockdowns of JAM-A, occludin, and both (JAM-A + Occludin) were successful. The knockdown of JAM-A resulted in a significant (p<0.004) reduction of HER2 expression as anticipated. The knockdown of occludin does not seem to have an effect on HER2 levels (p<0.2). However, the double knockdown (JAM-A + Occludin) reduced the levels of HER2 (p<.006). The reduction in HER2 levels in the double knockdown is therefore regarded to the effect of JAM-A silencing but not the occludin.

Discussion

The results show that the system level and genomic analysis of the breast cancers and the cell lines derived from that signify a diversity that is tremendous. One is that the cells from different compartments or layers are evolved of the epithelium mammary. Second is that the addiction differential to the oncogenic networks and oncogenes. It is established firmly now that the cancer of breast has subtypes which response differently to various treatments. The major component of the HER2 therapy for a positive breast cancer is the selective inhibitors usage like lapatinib. Therefore, this research helps in knowing the cell responses and the control of HER2 for the breast cancer cells.

In this research, the circuitry molecular connecting the signalling pathways of HER2 and the machinery apoptotic involved in the pathways of mitochondrial were explored. The system controlling and the signalling of GHER 2 leads to the BIM accumulation and is the association subsequent to BIM. Moreover, there is a direct association with BAK/BAX to enhance the mitochondrial pores.

Moreover, this study was interested in exploring the role of occludin in HER2 expression in breast cancer. We wanted to know whether other adhesion molecules beside JAM-A influence cell signalling and levels of HER2. We found that silencing of occludin molecule has no effect on the expression of HER2. Interestingly, we found that a slight increase in her2 expression was observed in some of the experiments. Whether this is a true effect or not can be confirmed by setting up new experiments. Key points in this study is to know that stripping and re probing for actin resulted in loss of protein so  we couldn’t get actin blots for all experiments. For that reason when we analysed the data we excluded actin and we used the row data by comparing everything to control.

Conclusion

In conclusion, as we got negative result and we found that occludin does not play a role in this part of cancer it might have other effects that can contribute to new knowledge of cell signalling that can play a role in tumorogenises. Its known that it plays a role in cell premature senescence.⁷  Other adhesion molecules such as cloudin and many others could be tested and see the effect that might open a new therapeutic target for this and many other types of cancer.

Acknowledgment

We thank prof. D Slamon (UCLA) and Dr N.O’Donovan (DCU) for providing us with the mcf7 her2 cells.

References

McSherry EA¹, McGee SF, Jirstrom K, Doyle EM, Brennan DJ, Landberg G, Dervan PA, Hopkins AM, Gallagher WM. JAM-A expression positively correlates with poor prognosis in breast cancer patients. Int J Cancer. 2009 Sep 15; 125(6):1343-51. doi: 10.1002/ijc.24498. http://www.ncbi.nlm.nih.gov/pubmed/19533747

Liang K¹, Lu Y, Jin W, Ang KK, Milas L, Fan Z. Sensitization of breast cancer cells to radiation by trastuzumab. Mol Cancer Ther. 2003 Nov; 2(11):1113-20. http://mct.aacrjournals.org/content/2/11/1113.long

Makoto Osanai,1,3 Masaki Murata,1 Nami Nishikiori,2 Hideki Chiba,1 Takashi Kojima,1 Norimasa Sawada Occludin-mediated premature senescence is a fail-safe mechanism against tumorigenesis in breast carcinoma cells Departments of 1 Pathology and 2 Ophthalmology, Sapporo Medical University School of Medicine.