Targeting DNA Repair Pathways in Triple Negative Breast Cancer

Summary

My project on targeting DNA repair pathways in triple negative breast cancer (TNBC) has two objectives: compare the extent of damage between two ethnicities and evaluate their response to targeted treatment 

 I did a literature search on biomarkers present in DNA damage and focused on six of the most common types that indicate DNA damage.  

Two triple negative breast cancer cell lines were used, MDA-MB 231 [from a Caucasian (CA) woman] and MDA-MB 468 [ from an African American (AA) woman]. Western blot analysis was used to determine if these cell lines expressed these biomarkers.  

I tested the effects of two Proliferating cell nuclear antigen (PCNA) on the cancer cell lines. PCNA, a checkpoint in cell replication, is defective in TNBC cells. A cytotoxicity assay was conducted with two PCNA inhibiting drugs that our lab created to evaluate its effect the cell lines. Flow Cytometry was conducted to determine cell cycle distribution of cells when treated with/without the drugs. The David analysis database was used to determine pathways the cancerous cell used for survival. 

The TNBC cells in the AA woman had more damage. The PCNA drugs blocked two DNA damage pathways. Although the CA cell line responded best, AA and those with similar phenotypes will benefit from the treatments. An important question that could be explored is why the AA breast cancer phenotype is more aggressive. Although this project only compared two specific cell lines, results indicate the study could be expanded to include cell lines of other ethnicities.  

 

Question / Proposal

My project on targeting DNA repair pathways in triple negative breast cancer (TNBC) has two objectives: to compare the extent of damage between two ethnicities and to evaluate their response to targeted treatment. TNBC is an aggressive subtype of breast cancer with all three hormone receptors (estrogen, progesterone, and HER-2) not over-expressed in the cancer tumor.  Due to the lack of these targets, traditional treatments like hormone therapy and chemotherapy are ineffective. Of those diagnosed with breast cancer, about 10%-20% of them will be triple negative breast cancer patients, and diagnosis most often occurs prior to age 50. Unfortunately, certain ethnicities are more at risk for this particular cancer sub-type than others.  More specifically, occurrence in TNBC is likely to be found in African Americans, Hispanics, and those with a BRCA1 gene mutation (National Breast Cancer Foundation,). It was hypothesized that triple negative breast cancer cells have an increased dependence on DNA damage for survival. By targeting specific DNA damage response proteins, it could potentially improve treatment of this breast cancer sub-type.

Research

 

Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with all three hormone receptors (estrogen, progesterone, and HER-2) not over-expressed in the cancer tumor.  Due to lack of these targets, traditional treatments like hormone therapy and chemotherapy are ineffective.  Of those diagnosed with breast cancer, about 10%-20% of them will be TNBC cancer patients, and diagnosis most often occurs prior to age 50.  Unfortunately, certain ethnicities are more at risk for this particular cancer sub-type than others- more specifically, occurrence in TNBC is likely to be found in African Americans, Hispanics, and those with a BRCA1 gene mutation (National Breast Cancer Foundation,).  

Despite these lack of effective treatment options, other methods which have been used include chemotherapy, radiation therapy, and targeted treatment, all of which are not the most effective. The laboratory that I worked in created two PCNA inhibitory drugs, a molecule and peptide, to target specific PCNA binding site and the homologous pathways in cancer. With Indepth research, targeting specific pathways in triple negative breast cancer could be the be a potential step to help treat this hard to cure breast cancer subtype. 

With the above-mentioned information, it was hypothesized that Triple Negative Breast cancer cells have an increased dependence on DNA damage for survival. By targeting specific DNA damage response proteins, it could potentially improve treatment of this breast cancer sub-type. This summer I carried out an investigative study with two cell lines: MDA MB 231 derived from a Caucasian woman and MDA MB 468 derived from an African American woman. I first wanted to find out the extent of DNA damage in the two cell lines and then subsequently, their response to treatment. I had two objects when carrying out this project: compare the extent of damage between two ethnicities and evaluate their response to targeted treatment.

                                                          

Method / Testing and Redesign

Western Blot Analysis with SDS PAGE 

 For each of the cell lines (231 and 468), we first harvested and exponentially growing cells of a culture dish. After making cell extracts from each cell pellet, SDS, an anionic detergent, was used to denature the proteins we were probing for (Chk1, P-Chk1, Chk2, P-Chk2, H2AX, and P-H2AX) prior to loading onto Polyacrylamide gel. When an electric current is applied, the denatured proteins will then separate and migrate down the gel according to their molecular weight. Separated proteins were then transferred to a nitrocellulose membrane and then probed with antibodies for specific DNA damage response proteins.  

Cell Cytotoxicity Assay 

A cytotoxicity assay (CellTiter Glo) was used to determine the effect that the two drugs (PCNA inhibiting drugs that target the ATM and ATR pathways) had on both cell lines. 96-well plates were prepared with 100 µl of media per well for both cell lines. The two drugs were administered to each cell line in increasing concentrations, and were incubated for 48 hours. The CellTiter Glo Assay was performed according to manufacturer recommendations. Briefly, to carry out the assay, CellTiter Glo reagent was added to each well. The contents were mixed on an orbital shaker for two minutes and the well plates were incubated at room temperature for ten minutes to stabilize the luminescent signals. A luminometer was used and a dose response curve was created to compare the outcome of the treatment response. Responses were then analyzed using GraphPad Prism. 

Flow Cytometry 

Flow cytometry analysis was conducted on both cell lines utilizing the BD Pharmingen FITC BrdU Flo Kit according to manufacturer recommendations. Both cells were then treated with the PCNA inbiting drugs. Cells were then pulse-labeled with BrDU for 60 minutes prior to harvesting.  Cells were fixed and permeabilized using Cytofix/Cytoperm, which is a reagent that serves to preserve cell morphology, fix cellular proteins, and permeabilize cells for subsequent immunofluorescent staining of intracellular proteins. After the cells were fixed and permeabilized with Cytofix/Cytoperm, the cells were incubated with BD Cytoperm Permeabilization buffer, which is used as a staining enhancer and secondary permeabilization reagent. The cells were then re-fixed, treated with DNase to expose the incorporated BrdU, and labeled with FITC. 7AAD was then added to determine the DNA content. Stained cells were then analyzed by flow cytometry using BD Fortessa 2. 

Results

   Figure 1  

 

From the blot shown in Figure1, it can be seen that the MDA-MB 468 cell line had an increase in DNA damage response compares to the MDA-MB 231 cell line. More specifically, there was an upregulation of Chk1, P-Chk1, Chk2, and P-Chk2 in the MDA MB 468 cell line compared to the MDA-MB 231 cell line. There was slightly more of an upregulation of H2AX in the MDA MB 468 cell line compared to the MDA MB 231 cell line. From this blot analysis, it was hypothesized that the MDA MD 468 cell line had more damage present. 

Based on the preliminary results, it was hypothesized that the MDA-MB 468 cell lines would be more sensitive to the Malkas laboratory’s novel therapeutics. Next, a cytotoxicity assay was conducted to determine the effects the two PCNA inhibiting drugs will have on the cell lines. As shown in Figure’s 2 and 3, the 468 cells were more responsive to both treatments compared to the 231 cells.            

                              

                              Figure 2                                                               Figure 3    

Finally, a flow cytometry analysis was conducted on both cell lines. For the untreated cells, we can see that for the MDA-MB 231 cell line (Figure 4A), 3.81% of the cells were in the sub G0 phase, 36.2% were in the G0/G1 phase, 34.9% were in the S-phase, and 19.6% were in the G2+M phase. The 468 cell line (Figure 4B) had 19.7% of its cells in the sub G0 phase, 46.7% in the G0/G1 phase, 10.3% in the S-phase, and 19.9%in the G2+M phase. From the data points, we can see a significant difference between S-phase among both cell lines. Specifically, more 231 cells were synthesizing DNA whereas more of the 468 cells were still in the G1 phase. When treated with the PCNA Inhibiting peptide and the PCNA Inhibiting molecule, as shown in Figure 4C, 5.37% of the MDA-MB 231 cells were in the G0 phase, 36.4% were in the G0/G1 phase, 37.5% were in the S-phase, and 17.5% of the cells were in the G2+M phase. In comparison, 7.71% of the 468 cells (Figure 4D) were in the sub G0 phase, 47% were in the G0/G1 phase, 18.9% were in the S-phase, and 21% were in the G2+M phase. In the same fashion, when AOH 1996 was administered to both cell lines, the effect of this drug caused a substantial impact. As depicted in Figure 4E. 42% of the 231 cell lines were in the sub G0 phase, 25.5% were in the G0/G1 phase, 9.49% were in the S-phase, and 10.7% were in the G2+M phase. In comparison, 21.2% of the 468 cells (Figure 4F) were in the sub-G0 phase, 5.76% were in the G0/G1 phase, 2.55% were in the S-phase, 44% of the cells were in the G2+M phase.  

      Figure 4 

                  A                                                   B                                      C                   

   

                    D                                     E                                           F            

                                

Conclusion

 I investigated the pathways through which proteins were upregulated due to DNA damage. Through Western Blot analysis, we were able to show upregulation of a number of these DNA damage proteins (Chk1, P-Chk1, Chk2, and P-Chk2) were prevalent in the 468 cell line compared to the 231 cell line.  The difference in upregulation of these proteins was more so visually prevalent in Chk1 and Chk2, which activated the ATR and ATM pathways respectively. The ATM (Ataxia-Telangiectasia-Mutated) pathway is activated by double strand breaks and the ATR (Ataxia Telangiectasia and Rad3-related) pathway is typically activated in the presence of single stranded DNA and occasionally, double strand breaks.  

  An analysis of Chk1 over expression in breast cancer patients was by using the TCGA database and from this, it can be concluded that patients who had TNBC expressed higher levels of Chk1 compared to Non-TNBC patients (Figure8). Furthermore, Patients whose tumor's expressed Chk1 had a lower survival rate compared to patients whose tumors did not      

Based on the data, targeting these DNA damage repair pathways could be useful in treating TNBC.  Proliferating cell nuclear antigen (PCNA) is an important scaffold for DNA repair proteins, DNA replication, repair, cell-cycle control, and chromatin remodeling. The Malkas laboratory where I executed this project, has demonstrated the ability to block PCNA-protein interactions that are unique to cancer with a PCNA inhibiting peptide and small molecule. Both drugs have the ability to block key PCNA-protein interactions in the homologous recombination DNA repair pathway. (Lingeman, Robert G, et al, 2018) and (Gu, Long, et al., 2014). We tested these treatments on both cell lines and the 468 cells proved to be more sensitive to the treatments.      

Through flow cytometry, it can be seen that the sensitivity of the MDA MB 468 cell line to the treatments is most likely due to the cell cycle distribution of the cells within each phase. Another contributing factor as to why the 468 cell lines were more sensitive to the treatment could be due to the specific pathways in each cell cycle phase. The drugs could have the ability to block a certain pathway within a certain phase of the cell cycle.  

Even though this project only compared two specific cell lines, the results indicate that this study could be expanded to a more comprehensive project. The MDA-MB 231 cell line and the MDA-MB 468 cell line had been selected to determine if there was a possible difference between the two based on ethnicity. As our data shows, the African American cell line (MDA-MB 468) exhibited more DNA damage compared to the Caucasian cell line (MDA-MB 231). In future work, this study could be expanded to include more cell lines that are representative of each ethnicity. If the comprehensive study showed a similar trend that the African American populations have an increase in DNA damage, it can then be proposed that this might be linked to health disparities. We could then begin to explore these disparities that are both genetic and epigenetic. 

 

About me

Although I have had a passion for the sciences since middle school, my passion for research was sparked freshman year when I learned about the high rates of lung cancer in the area of West Virginia where I live. This prompted me to search for cancer research opportunities throughout high school. During my junior year, I was selected to conduct triple-negative breast cancer research at the Eugene and Roberts Research Program in California.  This was surreal because my dreams of doing research since my freshman year came true.

Apart from the research, I enjoy participating in math and science competitions at my school. I am a part of my school’s science bowl team, a participant in TEAMS (Tests of Engineering Aptitude, Mathematics, and Science), a Science Olympiad competitor, Key Club officeholder, and a volunteer at the Camden Clark medical center. Additionally, I co-founded a math club at my high school.  In my free time, I love playing my violin, meditating, and hanging out with my friends.

I have participated in a science fair before and it was a wonderful opportunity.  I interacted with students from different parts of West Virginia and was able to learn from their amazing projects across multiple disciplines of science. Winning the Google Science Fair will give me the opportunity to share my project and my love of science with others across the world.

 

Health & Safety

Throughout this Project, antiseptic techniques were used. The culture hood was always sterile before the cells were cultured. My mentor for this project was Dr. Shanna Smith and her email address is shsmith@coh.org . The PI for the laboratory I worked for is Dr. Linda Malkas and her email address is lmalkas@coh.org .

Bibliography, references, and acknowledgements

Acknowledgements

I did this research project at the Eugene and Ruth Roberts Academy at the City of Hope. I would like to thank my mentor for this project, Dr. Shanna Smith, for helping me to execute my project and help me along the way. I would like to thank the PI (Private Investigator) of the lab I worked in, Dr. Linda Malkas, for giving me the opputunity to execute my project at her lab. In addition, I would like to thank the Eugene and Roberts Academy for giving me the oppurtunity to attend this summer research internship. 

Ren, L, et al. “Potential Biomarkers of DNA Replication Stress in Cancer.” Advances in Pediatrics., U.S. National Library of Medicine, 6 June 2017, www.ncbi.nlm.nih.gov/pubmed/28445142. 

Zhang, Jun, et al. “Targeting DNA Replication Stress for Cancer Therapy.” Advances in Pediatrics., U.S. National Library of Medicine, Aug. 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC4999839/ 

Conroy, S M, et al. “Racial/Ethnic Differences in the Impact of Neighborhood Social and Built Environment on Breast Cancer Risk: The Neighborhoods and Breast Cancer Study.” Advances in Pediatrics., U.S. National Library of Medicine, Apr. 2017, www.ncbi.nlm.nih.gov/pubmed/28196846. 

Ashing, Kimlin Tam, et al. “Nurturing Advocacy Inclusion to Bring Health Equity in Breast Cancer among African American Women.” Advances in Pediatrics., U.S. National Library of Medicine, 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4762369/. 

Gu, Long, et al. “A PCNA-Derived Cell Permeable Peptide Selectively Inhibits Neuroblastoma Cell Growth.” Advances in Pediatrics., U.S. National Library of Medicine, 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC3984256/. 

Sancar, A, et al. “Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints.” Advances in Pediatrics., U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/pubmed/15189136. 

Wright, N, et al. “Targeted Drugs and Diagnostic Assays: Companions in the Race to Combat Ethnic Disparity.” Advances in Pediatrics., U.S. National Library of Medicine, 1 Jan. 2017, www.ncbi.nlm.nih.gov/pubmed/27814611. 

Dietlein, F, et al. “Cancer-Specific Defects in DNA Repair Pathways as Targets for Personalized Therapeutic Approaches.” Advances in Pediatrics., U.S. National Library of Medicine, Aug. 2014, www.ncbi.nlm.nih.gov/pubmed/25017190. 

Houtgraaf, J H, et al. “A Concise Review of DNA Damage Checkpoints and Repair in Mammalian Cells.” Advances in Pediatrics., U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/pubmed/16945824. 

Tian, H, et al. “DNA Damage Response--a Double-Edged Sword in Cancer Prevention and Cancer Therapy.” Advances in Pediatrics., U.S. National Library of Medicine, 1 Mar. 2015, www.ncbi.nlm.nih.gov/pubmed/25528631.Network, The  

Cancer Genome Atlas. “Comprehensive Molecular Portraits of Human Breast Tumours.” Nature, U.S. National Library of Medicine, 4 Oct. 2012, www.ncbi.nlm.nih.gov/pmc/articles/PMC3465532/. 

“Gene Profiles in Drug Design - PDF Free Download.” Epdf.tips, EPDF.TIPS, epdf.tips/gene-profiles-in-drug-design.html. 

“DNA Replication: Mammalian.” Encyclopedia of Life Sciences, www.els.net/WileyCDA/ElsArticle/refId-a0001041.html. 

Lingeman, Robert G, et al. “The Anti-Cancer Activity of a First-in-Class Small Molecule Targeting PCNA.” Clinical Cancer Research, American Association for Cancer Research, 1 Jan. 2018, clincancerres.aacrjournals.org/content/early/2018/06/30/1078-0432.CCR-18-0592. 

Nbcf. “Triple Negative Breast Cancer :: The National Breast Cancer Foundation.” Www.nationalbreastcancer.org, www.nationalbreastcancer.org/triple-negative-breast-cancer.