MUPP1 Expression in Breast Cancer Cells




Faaria Fasih

Introduction

     Breast cancer usually begins in the breast lobules, where milk is produced, or in the breast ducts, which transfer the milk from the lobules. Breast cancer metastasis occurs when a cancer cell leaves its tissue of origin, which in this case is the breast tissue, and migrates to a different tissue type. Metastasis is possible when the integrity of the structures holding the cells in place is compromised.

     One of these types of structure are the tight junctions. Tight junctions are a collection of proteins that seal adjacent cells together at the apical surface. Tight junctions hold the cells together and restrict movement of substances in the space between two adjoined cells. In cancer metastasis, tight junction function is disturbed so the metastatic cell can escape its tissue of origin (Musch et al. 2006, Martin and Wiang 2008).

     The protein MUPP1 interacts with the tight junction proteins which are also associated with the cytoskeleton (Hamazaki et al. 2002, Lanaspa et al. 2007). MUPP1 levels are known to decrease in patients with metastatic breast cancer (Martin et al. 2004). The MUPP1 gene is 7454 base pairs and it codes for 2054 amino acids, including 13 PDZ domains. A PDZ domain is a structure of about 70 to 90 amino acids that are characterized by the formation of 6 beta-helices and 2 alpha-helices in a specific orientation (Ranganathan and Ross 1997). The structure of PDZ domains allow them to interact with cytoplasmic proteins as well as anchor to the cytoskeleton.

     The purpose of this study was to determine MUPP1 expression and alternative splicing in various breast ductal carcinoma epithelial cell lines.

Materials and Methods

RNA from 6 cell lines:
     Cervical cancer (HeLa)
     Breast ductal epithelial carcinoma (MDA-MB-231, MDA-MB-435S, MCF-7, MDA-MB-468, and TD-47)
Regions near the beginning (exon 2 to 3), middle (exon 19 to 20), and end (exon 43 to 44) of the MUPP1 transcript were amplified by RT- PCR to examine expression levels
Regions with possible alternative splicing activity amplified by RT-PCR:
     •Exon 17 to 19
     •Exon 34 to 36
     •Exon 25 to 28
Products were confirmed to be regions of MUPP1 transcript by sequencing.

     RNA from the HeLa cell line and the breast ductal epithelial carcinoma cell lines, MDA-MB-231, MDA-MB-435S, MCF-7, MDA-MB-468, and TD-47 were all tested for the presence of the MUPP1 mRNA using RT-PCR. Three primer pairs were designed to amplify three areas in the gene near the beginning, middle, and end of the MUPP1 transcript. After RT-PCR, amplified products were purified and sent for sequencing. Then, to look for possible splicing in the MUPP1 gene, primers were designed from exon 17 to 19, exon 34 to 36, and exon 25 to 28. After RT-PCR, amplified products were gel purified and sent for sequencing.

Results

     There was no clear differential expression of MUPP1 between the MDA-MB-231, MDA-MB-435S, MCF-7, MDA-MB-468, TD-47, and the HeLa cell lines (Figure 1). However, the transcript was spliced differently in the various cell lines (Figure 2). Size variation was also seen at the protein level in a western blot (Figure 3).

Discussion

Interestingly, none of the alternatively spliced exons caused a frame-shift in the amino acid sequence.
     • Exon 18 is 117 base pairs (39 amino acids)
     • Exon 35 is 87 base pairs (29 amino acids)
     • Exon 26 is 111 base pairs (37 amino acids)
     • Exon 27 is 99 base pairs (33 amino acids)
     • Exons 26 and 27 together are 210 base pairs (70 amino acids)

      Even though some amino acids are missing in some transcripts, the amino acids coded for after the alternatively spliced exon are not changed. None of the spliced exons coded for any of MUPP1’s PDZ domains. Therefore, the alternative proteins should still have the PDZ domains to anchor to the cytoskeleton and interact with the tight junctions.

     A western blot of MUPP1 published previously by Abcam shows different sized bands (Figure 3), which is expected since the transcript is alternatively spliced. An implication of the splicing is that protein recognizing compounds such as antibodies need to be designed so they recognize an conserved epitope on MUPP1. Further MUPP1 protein analysis should determine the biological significance of differential splicing events in the metastatic process.

References

Abcam. 2014. Anti-MUPP1 antibody. http://www.abcam.com/mupp1-antibody-ab101276.html. Accessed May 5, 2014.

Hamazaki Y., Masahiko I., Hiroyuki S., Mikio F., Shoichiro T. 2002. Multi-PDZ Domain Protein 1 (MUPP1) Is Concentrated at Tight Junctions through Its Possible Interaction with Claudin-1 and Junctional Adhesion Molecule. The Journal of Biological Chemistry 277: 455-461.

Lanaspa M. A., Almeida N. E., Andres-Hernando A., Rivard C.J., Capasso J. M., and Berl T. 2007. The tight junction protein, MUPP1, is up-regulated by hypertonicity and is important in the osmotic stress response in kidney cells. Proceedings of the National Academy of Sciences USA. 104: 13672-12677.

Martin T. and Wiang J. (2008). Loss of tight junction barrier function and its role in cancer metastasis. BBA Biomembranes 4:872-891.

Musch M.W., Walsh-Reitz M.M., Chang E.B. 2006. Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. American Journal of Physiology. Gastrointestinal and liver Physiology 290: G222-31.

Ranganathan R and Ross E. 1997. PDZ domain proteins: scaffolds for signaling complexes. Current Biology 7: R770–R773.

Figures


Figure 1- A) A depiction of the primer placements in the MUPP1 transcript. The arrows represent the direction and approximate location of each primer. B) Gel electrophoresis of the RT-PCR products are visualized on a 1% agarose gel. Each set of bands shows the amplified product from one primer pair in each cell line. The GAPDH gene was used as a positive control.


Figure 2-A) The three different primer pairs and the possible products RT-PCR products they can generate. B) Gel electrophoresis of the RT-PCR products on a 1% agarose gel. Each band corresponds to a possible product depicted in Figure 3A. More preferential splicing of exon 18 can be seen in the MDA-MB-435S, MCF-7, and MDA-MB-468 cell lines than the other cell lines.


Figure 3-A western blot of MUPP1 done by Abcam using .01 ug/ml anti-MUPP1 antibody. There is 50 ug HeLa cell lysate in lane 1, 15 ug in lane 2, and 5 ug in lane 3. The blot shows different sized bands of MUPP1.



Abstract

     Cancer cells become metastatic when tight junction function is compromised. MUPP1 is a protein that is associated with tight junctions and is reported to have decreased expression in metastatic breast cancer (Martin and Wiang 2008). In this project, MUPP1 gene expression levels were examined by RT-PCR in various breast cancer cells. The expression levels were found to be relatively constant between the cell lines. Previously in the literature, alternatively spliced forms of MUPP1 have been detected. The presence of alternatively spliced forms in these cells lines was examined and it was found that they showed varying alternatively spliced patterns. Interesting, none of the splicing events caused a frameshift in the encoded protein. None of the alternatively spliced exons disrupted any of the 13 PDZ domains in MUPP1. An implication of the alternative splicing is that antibodies need to designed so they recognize a conservatively spliced region of the transcript. The role of differentially spliced MUPP1 transcript in the metastatic process needs to be further examined.

Full Paper

Acknowledgments

     I thank Dr. Rubin for helping me with the project, making it possible in the first place, and always steering me in the right direction. I am deeply grateful for all of Catharine Grubaugh’s and Kate Reid's help every step of the way. I thank Dr. Sylvia Anderson for helping me purify RNA. Also, I greatly appreciate Dr. Wei for helping me design the project and for providing the RNA and cells. In addition, I thank Dr. Wu immensely for preparing the cells and helping me with the RNA. Lastly, I thank all of my classmates for their support throughout the project.


This document was last modified 05/16/2014.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home