The+Effect+of+Autocrine+Motility+Factor+on+HER2+Phosphorylation

=Our Project=

For our project, we decided to study a new ligand which is said to induce phosphorylation in HER2 positive breast cancer. First, we plan on introducing the molecular mechanisms and signal transduction pathways that compose HER2 positive breast cancer. Next, we will introduce Autocrine Motility Factor,explaining its many roles in the human body and how it relates to cancer. Using what information we have, the next section will be dedicated to explaining the research paper we chose. toc

=HER2 Breast Cancer=

A Brief Overview
Human epidermal growth factor receptor 2 (HER2) is a gene that can play a role in aggressive breast cancers. The gene encodes for the HER2 receptor, located on the surface of a cell. In some breast cancers, this gene is mutated, creating many copies of itself, leading to overexpression of the receptor. In a normal healthy breast cell, there are about 20,000 HER2 receptors. In the 25% of people that have the HER2 mutation, there are about 2,000,000 HER2 receptors. This mutation is specific to the cancer cell only, and cannot be inherited by a parent. The overexpression of the HER2 receptor leads to uncontrolled growth and division. When breast cancer contains the HER2 mutation, it is said to be HER2-positive. However since the target of choice is a receptor, there are numerous targeted therapies available to patients.

HER2 Signal Transduction
In order to understand our research paper, it is important that the mechanism of HER2 signal transduction be elucidated. HER2, also called ErbB2, is part of a family of receptor tyrosine kinases, the ErbB family. This family of receptors triggers signaling pathways responsible for cell division, cell death, cell motility, and adhesion. The ErbB family is composed of four members: HER1 (also called EGFR), HER2, HER3, and HER4. Each receptor has an extracelllar domain, transmembrane domain, and an intracellular domain (Yardin, et al. 2001). In order to activate HER2, it must dimerize with another HER2 receptor (homodimerization) or any other members of the ErbB family (heterodimerization). All members of the ErbB family, except HER2, remain in a closed conformation. When bound to a ligand, these receptors undergo a conformational change enabling these receptors to dimerize and start signal transduction. HER2 exists naturally in an open conformation rendering it ready to dimerize at any time. As stated above, the large amount of HER2 receptors (2,000,000) on the surface of the cell can lead to unregulated growth and division. Upon dimerization, the members of the ErbB family initiate a phosphorylation cascade along the transmembrane domain, leading to activation of proliferation and survival pathways (Yardin, et al. 2001).

MAPK Signaling
The MAPK signaling pathway plays a vital role in the regulation of gene expression, cellular growth, and survival. MAPK signaling is initiated by receptor tyrosine kinases upon their activation by growth factors, which take place in the extracellular space. There are adaptor molecules that interact directly with the intracellular section. These molecules mediate the recruitment and activation of signaling molecules in the pathway including: Ras, Raf, MEK, and ERK. Abnormal MAPK signaling leads to uncontrolled cell proliferation,resistance to apoptosis, and resistance to chemotherapy.

Pi3K Signaling
Pi3K, Akt, and mammalian target of rapamycin(mTOR) comprise of the 3 main checkpoints of the pi3k signaling pathway. The Pi3K signaling cascade are activated by signaling from upstream receptor tyrosine kinases.The Pi3K pathway leads to the Akt pathway as well as the mTOR pathway. The Akt leads to apoptosis, while mTOR leads to cell growth.

=Current Targeted Therapies for HER2=

As more research is being done to understand the different changes genes undergo that lead to cancer, researchers are able to develop drugs that can target these changes. These drugs have less severe side effects than other chemotherapy drugs.

Trastuzumab (Herceptin)
This drug is known as a monoclonal antibody. A monoclonal antibody is a synthetic version of a very specific immune system protein. Herceptin binds to HER2, slows the growth of the cancer cell, and also helps to trigger an immune response to attack the cancer cell. Trastuzumab is usually used as an adjuvant therapy for HER2 positive cancer to reduce the risk of remission. IT has been shown that treatments that combine Trastuzumab with other chemo lead to better results than chemo alone. If a patient is taking a combination of Trastuzumab and chemo and the patient’s condition worsens often the chemo is changed. In terms of side effects they are mild compared to other chemotherapies. The side effects are also less frequent.(Genentech Inc. 1998).

Ado-trastuzumab emtansine (Kadcyla)


Ado-trastuzumab emtansine(TDM-1) is known as an antibody –drug conjugate. It consists of the same monoclonal antibody found in Trastuzumab, which is attached to the chemo drug DM-1. In this kind of drug, the antibody acts as a homing device, which is then able to direct the chemo drug right into the cancer drug. This drug is given to treat advanced breast cancer, and is administered by itself. This drug leads to more serious side effects including severe allergic reactions, liver and heart damage, and lung problems.(Genentech Inc. 2013).

Pertuzumab(Perjeta)
Pertuzumab, like Trastuzumab, is also a monoclonal antibody that attaches to the HER2 protein. However, this drug seems to target a different section of the protein than Tastusumab does. Pertuzumab is used along side Trastuzumab and Taxotere to treat advanced breast cancer. This unique three drug cocktail is often used as a neoadjuvant(pre-surgery) therapy to treat earlier stages of breast cancer. This combination of drugs has side effects including hair loss, nausea, low white blood cell count, diarrhea, fatigue, and rash. Similar to Trastuzumab patients with weaker hearts cannot be administered Pertuzumab because this drug also weakens the heart.(Genentech Inc. 2013).

Everolimus(Afinitor)
Everolimus is a targeted therapy that inhibits mTOR, a protein that promotes cell growth and division. By inhibiting mTOR Everolimus helps to stop cancer cells from growing. Another affect is that Everolimus is able to stop angiogenesis, the development of new blood vessels, which in turn limits the growth of these cancer cells, and also can inhibit metastasis. This is because as we learned angiogenesis is a key feature of cancer that helps spread the cancer cells throughout the body. This drug is mainly used to treat advanced hormone receptor positive, HER2 negative, breast cancer in women who have gone through menopause. This drug is usually administered alongside exemestane in women whose cancers have grown while taking only anastrozole. A recent study showed that taking Everolimus alongside exemestane was more effective than exemestane alone in shrinking tumors and stoping their growth in post-menopausal women with hormone receptor positive, HER2 negative breast cancer. Side effects include mouth sores, diarrhea, nausea, fatigue, low blood counts, and even an increase in cholesterol and triglycerides. Due to the last side effects doctors periodically check your blood to make sure everything is stable. The risk of infection is also very high while on Everolimus so the doctors must watch the patient closely for any signs of infection.(Novartis Corp. 2012).

Bevacizumab(Avastin)
New blood vessels are vital fro tumors to not only grow but to spread. This is the reason why angiogenesis is a major characteristic of cancer. Therefore, Bevacizumab is very efficient because it's main function is to hinder and destroy these new blood vessels. Bevacizumab is also a monoclonal antibody that is primarily used in patients with metastatic breast cancer. The antibody targets VEGF(vascular endothelial growth factor) which is the main protein that creates these new blood vessels.The side effects for this drug can be quite sever. In some cases holes can form in the colon resulting in immediate surgery. More common side effects include blood clots, high blood pressure, and low white blood cell counts. This drug is very controversial because the FDA first approved this drug in 2008 due to a study that showed women who took Bevacizumab and Taxol had a longer metastatic free time period than women who took Taxol alone. However, a new study in July 2010 was brought to the FDA;s attention. The study proved that though Bevacizumab did slow down the progression of breast cancer in some women for a short period of time, it did not help those women live longer. On November 18, 2011 the FDA having decided that the risks outweigh the benefits, withdrew this drug from the list of drugs used for metastatic breast cancer. However, this drug is still used and is effective for other kinds of cancer.(Genentech Inc, 2013).

Now let us look closer to Trastuzumab and how it interacts and effects the HER2 receptors found on the surface of the cancerous breast cell.

=Trastuzumab(Herceptin) Mechanism=

To help visualize this mechanism this pink structure will represent the monoclonal anitbody that is Herceptin. This is a HER2 positive tumor cell found in the breast tissue. As you can see the amount of HER2 receptors have become amplified. Like stated above in a normal cell there are only 20,000 HER2 receptors on the surface of the cell. The effect of an increased amount of HER2 receptors is a tremendous increase in receptor mediated intracellular signaling. This causes the cell to proliferate uncontrollably. This leads to a form of breast cancer that progresses very quickly. Herceptin has an extended half-life that allows for constant interaction with tumor cells so this allows for continuous inhibition of HER2 activity. Herceptin also affects both the extracellular and intracellular domains of the HER2 receptor. Herceptin blocks all growth factors from binding to the extracellular domain of the HER2 receptor. Herceptin is the only approved therapy that can bind to the extracellular domain of the HER2 receptor, and flag them for distraction by the immune system. In addition, bound Herceptin prevents HER2 receptor activity by blocking intracellular HER2 signaling and ultimately stopping cell proliferation.

=Autocrine Motility Factor=

Our topic is based on a paper that was published in 2013, and for this reason the way that AMF is portrayed is different than how it was previously know. While in the paper AMF is reported as having ligand qualities in previous literature, AMF was known as a cytokine that promoted cancer cell growth and motility. AMF is part of a group of cytokines that stimulates tumor cell motility in an autocrine fashion. A cytokine is a general term for proteins that are important for cell signaling. This protein factor is expressed and secreted by cancer cells via an autocrine route. This action has been known to stimulate the motility of cancer cells. Malignant tumors can often be characterized by their unregulated growth, invasion into local areas, and their eventual growth into distant organs. One can conclude that for any of these goals to be accomplished the tumor needs to have an efficient cell migration mechanism. AMF was distinguished among the cytokines due to its ability to stimulate directional and random motility of AMF producing cells.(Raz, et al. 2006).

AMF is identical in structure to glucose 6-phosphate isomerase, which is the second enzyme in glycolysis, and neuroleukin which is a neurotrophic factor for spinal and sensory neurons. Though AMF and the others, GPI and NLK, have differing functions they all come from the same gene. AMF monomers are made of two domains, both these domains are in a alpha-beta-alpha conformation. The smaller domain contains a five-stranded beta-sheet surrounded by alpha helices. The larger domain has a six-stranded beta sheet. The functional AMF dimer is composed of two identical monomers. The active site of each monomer is created by a cleft between the to domains and the dimer interface.(Watanabe, et al. 1996).

In patients who suffer from breast cancer usually the cause of death is metastasis. The stages of metastasis involve complex processes such as cell-cell dissociation at the primary site, matrix degradation and invasion, and the resettling in a new site where cells will re-develop as metastatic tumors. This complicated process requires cell motility for the metastatic process to be successful. Some prominent growth factors include HGF, autotoxin, MSF, and AMF.

In a recent immunohistochemical staining of AMF test the scientists found evidence that AMF and AMFR levels had a direct link to the growth of tumors. They found that AMF's relationship to metastasis and angiogenesis proved this link. Researchers found that there were high levels of AMF in recently metastasized tumor cell. They also found high levels of AMF and AMFR in vascular tissue cells. This was prominent because these new blood vessels had been created through angiogenesis.

Recent trials conducted by the forerunners of AMF research found that AMF and its receptor AMFR have a direct link with breast cancer. High levels of AMF and AMFR were found in patients who died of breast cancer, and also in those who had reoccurrences. Also a significant relationship was found between high levels of AMF and long-term survival. The researchers concluded that AMF and AMFR are over-expressed in breast cancer and have a negative association with the patients long term outcome. This confirms their belief that the AMF-AMFR complex has a significant role in the progression and severity of breast cancer. The figure below shows the researchers findings from a ten year clinical study on AMF. As we can see those patients who died from breast cancer and those who had remissions had higher levels of AMF and AMFR than those who who remained disease free. The researchers concluded that AMF had significant effects on the overall severity of breast cancer. Not only did high levels of AMF seem to cause recurrences, but it also seemed to be connected with patient health and metastasis.

Along with these characteristics AMF also protects cancer cells from undergoing apoptosis and is involved in up-regulation of cyclin dependent kinase activity, which we learned about in the beginning of this quarter. Now that we have learned about the research that has been done thus far about AMF let us next look at how AMF and HER2 interact.

=HER2/Autocrine Motility Factor Connection=

Introduction
Now what is the relationship between HER2, a receptor tyrosine kinase, and autocrine motility factor, a protein with many different aliases and functions? In 2013, a paper (Kho, et al. 2013) published in the journal Cancer Research studied the connection with these two. The authors seem to have discovered something novel: a possible mechanism of Trastuzumab resistance. The authors also seem to have discovered the fact that autocrine motility factor, herein referred to as AMF unless otherwise noted, induces HER2 phosphorylation, leading to downstream signaling. Here, we take a look at data sets that support their conclusion, albeit with a couple caveats.

AMF Induces HER2 Shedding
One of the first findings this research paper uncovered was the fact that AMF induces HER2 shedding. HER2 shedding occurs when the extracellular domain of the HER2 receptor is clipped, leaving an intact, 95kD transmembrane and intracellular domain portion. The resulting HER2 protein still embedded in the membrane is constitutively active, or always firing. This provides the cell with ligand independent growth and survival signals (Wang, et al 2013). The shedding of the extracellular domain, coupled with the massive overexpression of the HER2 receptor on the surface of the cell, make disrupting HER2 shedding relatively important. In this figure, T47D breast cancer cells were treated with AMF and analyzed for total HER2 phosphorylation and HER2 shedding. Looking at the total phospho-HER2 blot, at time zero, there is an immediate phosphorylation of HER2. The signal peaks 2 minutes after AMF treatment, then immediately drops off after. We suspect that the authors used a phospho-HER2 antibody that detects phosphorylation on the extracellular domain, since there is a noticeable drop off in signal as time progresses. The 185kD band is the full size HER2 protein. At time zero, with no AMF treatment, there is still a strong signal at 185kD. 2 minutes after treatment, a signal at the 95kD band begins to appear, indicating that shedding is occurring. As time goes on, the signal at the 185kD band decreases, while the signal at the 95kD band increases. 30 minutes after AMF treatment, there is almost no signal at the 185kD band and a very strong signal at the 95kD band when compared to time zero. According to this data, it seems that AMF does indeed induce phosphorylation of HER2, and induces shedding of the extracellular domain fragment.

AMF Induces HER2 Phosphorylation
We chose to analyze this data set because it is a strong indicator that AMF does induce HER2 phosphorylation. However upon further review of their experimental technique, we thought it was peculiar for one reason: rather than using breast cancer cells that express HER2, the authors used human embryonic kidney cells (HEK293) transfected with a HER2 gene. We are not sure why the authors did this, but it is worthy to note. However, the data seems to support their claim. The negative portion of the blot indicates that the cells were not transfected with the HER2 gene; the positive portion of the blot indicates that the cells were transfected with the gene. Looking at the figure, you can see that there is a strong signal at the 185kD band for both positive regions, indicating that there is a large amout of HER2 protein present. The control was treated with bovine serum albumin, an inert protein. When looking at the total phospho-HER2 blot, as expected, there is little to no reaction. What little signal appears can be due to background noise. Looking at the AMF treated portion of the phospho-HER2 data, there is a strong signal. When compared with the control, it seems that AMF does indeed induce phosphorylation in HER2. However, since the cells used in this study were not breast cancer cells, these results are questionable. We would like to see this experiment repeated, with different breast cancer cell lines. If they produce the same result, the data from this set could be trusted.

AMF Interacts With HER2
The authors made it clear that there was an interaction between AMF and HER2. However the only proof that they were able to provide of this interaction was this computer generated image as seen on the left. We also thought this was peculiar; if the authors really wanted to prove the interaction between AMF and HER2, we thought that they should have used more advanced techniques rather than seemingly pasting the 3-D structures of these two molecules together. They could have quantified the binding of AMF and HER2 by running an ELISA. They could have mapped the binding sites to prove that the binding is real. The authors could have performed x-ray crystallography in order to show a real picture of HER2/AMF binding. All of these possible experiments are much better proof than what they chose to display in the research article.

=Conclusion=

Upon further review of outside literature, it seems that there are very few studies that correlate AMF and HER2. Though the data seems to be promising, it looks as if these authors massaged the data to prove their points, as seen in the "AMF induces HER2 Phosphorylation" section above. If they really wanted to prove AMF induces HER2 phosphorylation, why didn't they use breast cancer cell lines instead of using HEK293 cells transfected with the HER2 gene. Another figure that is problematic is the computer generated diagram of the HER2/AMF interaction. Rather than the computer generated diagram, they could have used real scientific methods to assess the binding interaction, if it is real. There are many problems with published research, one of the main ones being reproducibility. As we learned in class, a large majority of published papers are not reproducible. From firsthand experience, many of these experiments were not reproducible. The data seems promising, but we would like to see other experimenters, from outside labs with no connection to the authors, try to reproduce these experiments and create the same results. If these results hold, AMF could possibly be a new drug target for those who have developed resistance to Herceptin, a monumental discovery. In terms of other drug therapies we did some creative thinking about other targets to attack, besides the HER2 receptor. We thought of therapies, which target the actual signaling pathways, MAPK and Pi3k, because these are what actually lead to the cell proliferation and cell survival. The fact remains that cancer is an ever-mutating and always adapting disease. So we believe that we need to start looking into therapies, which can also adapt and mutate so as to stay ahead of the disease. These creative methods, such as the topic we analyzed for our project, are vital if we wish to truly make advances in cancer treatment research.

=References=


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