Comas 1

Analysis of the Relevance of HER2-Positive Breast Cancer and the Intervention

for its Effective Eradication
I. Background: The amplification and overexpression of the human epidermal growth factor receptor
2, also known as HER2, both greatly influence the role of tumor generation and growth in breast
cancer. The gene, also known as the tumor marker, is recognized as a promoter in the drastic
development of cancer cells in an aggressive variation of breast cancer, called HER2-positive breast
cancer. The presence of this detrimental gene is relatively common since it currently affects 25% to
30% of all breast cancer patients. Also, it targets the youthful population, since younger women are
more likely to be HER2-positive than older women (4). The mechanism that leads to HER2-positive
breast cancer commences with HER2 amplification, caused by the malfunction of the HER2 gene,
which consequently leads to the production of excessive copies of such gene. The disproportionate
assembly of HER2 genes induces the production of excessive HER2 protein receptors, an
occurrence called HER2 protein overexpression, which as an ultimate result makes breast cells grow
in overpowering and overwhelming patterns (1).
HER2-positive breast cancer tends to be more aggressive than other variations of the
condition, meaning that it disperses easily, grows faster, and is less responsive to hormone therapies
that are usually utilized for treating the disease. Not to mention, a study conducted by colleagues
from the University of Texas M.D. Anderson Cancer Center (MDACC) reported that patients who
tested positive for HER2 at an early stage of the disease had an increased risk of recurrence when
compared to HER2-negative cancer (4). A primordial factor that influences tumor recurrence and
aggressiveness is an elevated nuclear grade, which is specifically characteristic of HER2 tumors. In
addition, these tumors are more likely to show positive margins during breast surgery, which as a
result leads to more tissue being removed during mastectomy and harsher treatments afterwards.
There are currently two main drugs that effectively target and eradicate HER2 cancer cells:
Trastuzumab (Herceptin) and Lapatinib (Tykerb), which have experienced modest success. The
Herceptin medication attaches itself to the HER2 protein receptor on breast cancer cells and blocks
them from receiving growth signals, thus slowing down the progression of breast cancer. Similarly,
the Tykerb medication interferes with certain proteins that cause the cell to grow and divide irrationally
(1). Yet, there are two aspects that make the aforementioned treatments ineffective in targeting and
destroying the highly tumorigenic oncogene HER2. First, the tumors unlimited self-renewal

Comas 2
capabilities lead to its relapse; sometimes into a size bigger than it previously was. According to The
New England Journal of Medicine (NEJM), total remission for this disease only occurs in around 7%
to 8% of patients (4). Most importantly, these medications are known to cause detrimental and
painful side effects, which include but are not limited to: fevers, allergic reactions, chills, muscle
aches, rashes, and loose stools. Herceptin, along with the aforementioned flu-like symptoms, can
damage the hearts ability to pump blood and obstructs breathing (2). Similarly, Tykerb has also been
found to cause mild heart damage, bone thinning, and bone and joint pain (5). These side effects,
along with other health effects caused by breast cancer treatments such as chemotherapy and
radiation, seriously affect how the patient continues on with the overall disease management.
II. Objective: I am looking to completely knock out, or delete, the HER2 gene and its manifestation by
identifying HER2 overexpression in a human tumor xenograft in wild-type mice. Then, I will perform
CRISPR/CAS9 mediated gene deletion. My central hypothesis states that the knock down of
oncogene HER2 via CRISPR/CAS9 gene deletion will eliminate the proliferation of lethal tumors while
discarding the chance of painful side effects from drugs. Thus, my central hypothesis will meet the
objective of this grant proposal with the following specific aims:
Aim 1: To target overexpressing HER2 human xenograph introduced into wild-type mice.
Aim 2: To obtain mediated gene deletion of HER2 in targeted cancer cells.
Aim 3: To analyze the results from the gene deletion.
III. Approach
Aim 1: To target overexpressing HER2 human xenograph in wild-type mice, I will perform
xenotransplantation with plasmid DNA.
Live human HER2 dissociated tumor will be introduced into immunodeficient mice via the
xenotransplantation procedure. This will result in the presence of HER2 overexpression in the human
xenograph. The HER2 gene overexpression will be subsequently targeted using homologous
recombination with plasmid DNA from cultured cells, which will then be used to knock down.

Aim 2: To obtain mediated gene deletion of HER2 in targeted cancer cells, I will perform
CRISPR/CAS9.

Comas 3
Genome engineering will be used to delete the gene. The targeted HER2 gene will be knocked out from
the human xenograph via transformation by electroporation with a gene sequence that contains CRISPR
gRNA.

Aim 3: To analyze the results from the gene deletion, I will perform histological and molecular
analysis of targeted cancer cells.
The cells will be sectioned and stained, then observed under a light microscope. This will be done to verify
if other oncogenes are turned off, like for example GRB7, which is co-amplified with the HER2 gene. Also,
the outcome measures will be analyzed, which will be the size of the grafts. Finally, cell proliferation will be
examined, to notice if it decreased or if it completely terminated in the cancer cells.

IV. Relevance: This proposed research is groundbreaking because it is the first study that is looking
to completely inactivate and delete the HER2 gene and its progression in breast tumors, instead of
blocking or interfering with its overall activity. It will halt the risk of full human testing, using mice as
the main subjects until it is definitely confirmed that the proposed alternative is entirely functional and
produces the desired outcomes. The suggested procedure will facilitate the knockout of the HER2
gene, which can later be translated to human subjects in subsequent trials.
My innovative method is the adequate approach to solve this problem because it completely
eliminates the possibility of the harmful side effects that human patients have been exposed to with
drugs such as Trastuzumab (Herceptin) and Lapatinib (Tykerb). Moreover, current drug therapies are
also not fully successful in targeting the HER2 oncogene, as evidenced by the low rate of total
remission in patients. According to the American Cancer Society, it is estimated that about 231,840
new cases of invasive breast cancer will be reported alone in 2015, and it is an unfortunate fact that
about 25% to 30% of these cases will be HER2-positive breast cancer (3). Research in the use of
CRISPR/CAS9, which is a recent revolutionary method in genome engineering, in HER2 gene
deletion will greatly benefit the widely affected female population, and lead to improvements in the
progress of eradicating HER2-positive breast cancer.
The expected outcome of this research will reduce patient distress from the detrimental side
effects and completely eliminate breast cancer tumors and its possibility of relapse. These results will
have a positive impact on the medical field, and will contribute to its advancement by clearing this
hurdle, since it will remove the two main obstacles that are preventing successful remission of HER2positive breast cancer.

Comas 4
Works Cited
1. HER2 Status (2015, March 10). In BreastCancer.Org. Retrieved May 20, 2015, from
http://www.breastcancer.org/symptoms/diagnosis/her2
2. Herceptin Side Effects (2015, January 10). In BreastCancer.Org. Retrieved May 23, 2015, from
http://www.breastcancer.org/treatment/targeted_therapies/herceptin/side_effects
3. How many women get breast cancer? (2014, September). In American Cancer Society. Retrieved
May 20,

2015,

overview-

key-

from

http://www.cancer.org/cancer/breastcancer/overviewguide/breast-cancer-

statistics

4. Madell, R. (2014, October). HER2-Positive Breast Cancer Survival Rates and Other Statistics. In
Healthline. Retrieved May 20, 2015, from http://www.healthline.com/health/breastcancer/her2- positive-survival-

rates-statistics#Genes1

5. Tykerb Side Effects (2015, May 10). In BreastCancer.Org. Retrieved May 20, 2015, from
http://www.breastcancer.org/treatment/targeted_therapies/herceptin/side_effects