Could LxxLL be a key to a New Site for Target Drugs?

We have mapped the entire human genome. We have discovered specific site targeting drugs that have been a huge success in killing cancer. What is next? Of course to further embark on this newly discovered path and target other genes for success! 

How do these targeting drugs work? Lets look at the human papillomavirus (HPV) vaccine. This vaccine is preventing strains 16 and 18 from infecting women.These strains are the cause of at least 70% of cervical cancer cases. When HPV infect someone, it acts as a retrovirus. It copies into human DNA. Then, it makes an E6 protein. This protein is not equivalent to the human one, but from HPV. This E6 protein floats around cells and attaches with ubiquitin ligase, a protein that degrades genes from doing their job. There is a domain on this E6 protein that start with Zinc C domain and terminates with a Zinc N domain. In between is a linker helix domain that is very hydrophobic. This makes up the LxxLL sequence with L being acidic Leucine and x being other hydrophobic amino acids. This hydrophobic pocket then binds with extreme ease to proteins like p53 and RB. When they are in contact, ubiquitin ligase then deregulates the protein E6 is bound to. In turn, this then causes no tumor suppression or cell death and cancer develops. Because HPV is sexually transmitted almost all of these cases result in cervical cancer. 

  Figure 1: E6 protein structure when bound with ubiquitin ligase.
The top image shows the structure of the folding of the E6 protein, in yellow is the first Zinc domain, in grey is the linker helix domain and in purple is the terminal Zinc domain. The bottom images shows the hydrophobic LxxLL pocket. 

A crucial point to notice in this process is that the LxxLL pocket can bind with a plethora of different proteins. Targeted therapies do just this. If we could make a drug/ structure that has a pocket like LxxLL that could go in and bind to proteins that are mutated, we could used this process to treat cancer. This is especially important because proteins like RB and p53 are in almost all cases of cancer and there has not been much research done on these oncoproteins. Targeting drugs have proven in the past to be very successful in curing targeted cancer. This specific pocket could be useful for further research in this field.

Even though this started with the HPV vaccine which does not cure cancer, it prevents it, if science found the technology to to build a protein that when inserted into a cancer cell could latch onto mutated proteins and regulate them again, we could help cure many forms of cancer. Evidence shows through the HPV that it really does work. Our next step is to instead of using it as a prevention, we use this technology as a cure. By knowing how HPV attacks proteins we can do the same with an artificial structure. If we went after proteins as common in mutations as p53 and RB we could change many lives. I would love to see further research on this LxxLL pocket. I feel that having a technology break through would come best with furthering our knowledge on targeting mutations on proteins that influence all cancers. This pocket could be the start of a new era in cancer research. 



Work Cited:

Zanier, K., S. Charbonnier, A. O. M. O. Sidi, A. G. Mcewen, M. G. Ferrario, P. Poussin-Courmontagne, V. Cura, N. Brimer, K. O. Babah, T. Ansari, I. Muller, R. H. Stote, J. Cavarelli, S. Vande Pol, and G. Trave. "Structural Basis for Hijacking of Cellular LxxLL Motifs by Papillomavirus E6 Oncoproteins." Science 339.6120 (2013): 694-98. Web. 12 May 2014. <http://www.sciencemag.org/content/339/6120/694.full>.

Epithelial-Mesenchymal Transition on CTCs

In my previous blog post I mainly addressed the issue of factors that affect circulating tumor cell count in blood samples. As previously mentioned, this could lead to an under or overrepresentation of the cumulative CTC total within the patient. As such, it is important to account for these factors during quantification and ultimate prognosis using CTCs. In my previous post I focused more on the factors of clustering and filtration. The factors that followed, such as cloaking had to do with molecules from within the circulatory system masking CTC presence and as a result prevented accurate analyses to the point where it was deemed another factor in distorting CTC measurement. However one of the factors that was in the article mentioned, but was never given a greater insight to is G in the image below labeled “(partial) EMT”. 

Mechanisms For Trastuzumab Resistance

My cancer project looked at a possible mechanism for Trastuzumab resistance, this being AMF causing HER2 phosphorylation leading to intracellular signaling. While this idea is interesting it is fairly new so I wanted to do more research on what is widely accepted today as the mechanism for Trastuzumab resistance. Trastuzumab is a monoclonal antibody that specifically targets the HER2 receptor. A monoclonal antibody is a synthetic molecule whose primary role is to attack a certain defect in a cancer cell. These antibodies also cause cancer cells to become more visible to the immune system, thereby causing an immunogenic response. Trastuzumab binds to HER2 extracellularly and this causes HER2 to become deactivated. This ceases all signal transduction, which leads to inhibition of both the MAPK pathway and the pi3k pathway. However, the majority of patients with metastatic breast cancer develop a resistance to Trastuzumab within a year. How does this happen?

Cholesterol and Cancer Inhibition

For our project, Matt and I focused on cholesterol and its metabolite’s promoting effects on breast cancer.  However, as Matt mentioned in our presentation, there is a tumor suppressing metabolite of cholesterol.  It is still very much in the works, but research seems to support the suppression of this metabolite, called Dendrogenin A (DDA).  This molecule is an amino-oxysterol metabolite that “arises from cholesterol-5,6-epoxides and histamine in mammals” (Silvente-Poirot).  Very little is actually known about this molecule and its inhibitive abilities, and a Google search turned up this study and one other that identified DDA as a product of cholesterol.  It has recently been discovered to be a natural metabolite occurring in mammals, having been previously synthesized synthetically.  The fact that this molecule may be a successful inhibitor of cancer makes it very exciting.  The fact that it is naturally occurring makes it even better.

We Are All Mutant

Mel Greaves' article "Does everyone develop covert cancer?" discusses that while around 1 in 3 individuals will develop overt cancer, there is increasing evidence that most, if not all, people in their lifetimes develop covert cancer. Greaves begins by defining covert cancer as cancer that is "hidden and subclinical" but that can be "uncovered by more incisive interrogation," as opposed to overt cancer, or full-blown, malignant growths or tumors. The evidence for these covert cancers is primarily gathered from autopsies of individuals who died for non-cancerous reasons. Most autopsy reports show a substantially higher frequency of pre-malignant lesions, or carcinoma in situ (CIS), than of clinical cancer frequencies or cumulative life-time risk of developing overt cancer. This suggests a much higher frequency of CIS than is commonly believed or recorded.

Panitumumab versus Cetuximab - Treatment in Metastatic Colorectal Cancer

Throughout all our research about anti-EGFR treatment in metastatic colorectal cancer, my partner Tien Lu and I often discovered panitumumab and cetuximab as main-line therapies (More in our wiki). Both of these drugs are anti-EGFR treatments. EGFR is the epidermal growth factor receptor, which contains many pathways that lead to cell proliferation and survival. Therefore, it is no surprise EGFR as been important in the study of cancer, as malfunction in its pathways can lead to uncontrollable cell growth, differentiation, and migration. Typically, EGFR ligands bind to the EGFR cell surface receptor to induce cell signaling down the pathway, leading to cell replication. Monoclonal antibodies are anti-EGFR treatment, and bind to the EGFR receptor instead, blocking the downstream signaling and stopping cell proliferation.

Five or More Blistering Sunburns Before 20 Increases Melanoma Risk by 80%

I thought this post would be particularly fitting with summer right around the corner.

I was doing some reading and came across this article, which summarized a study that examined the relationship between sun exposure (along with some other risk factors) and the risk of different types of skin cancers. In this study, researchers were able to follow 108,916 US women over 20 years and determined that having five or more blistering sunburns between ages 15 and 20 resulted in an 80% increased risk for melanoma. Moreover, women in the same age range had a 68% increased risk for basal cell carcinoma and squamous cell carcinoma after having five or more blistering sunburns.

Cancer and Personalized Medicine

If you recall we spent week five of the course discussing the different treatments available for those who have cancer. During the week, we discussed the three most common methods of treatment, those being: surgical oncology, radiation therapy, and medical oncology. Surgical oncology mainly takes place as a treatment in the form of a resection of a solid tumor with wide margins to ensure complete removal. Radiation oncology uses x-rays and carbon ions to directly damage DNA of the cancer cells in hopes the damage will prevent continued growth. Lastly, we discussed medical oncology, wherein we discussed chemotherapy. When we discuss personalized medicine we are generally referring to changing the way in which we use medical oncology.

Will This Man Cure Cancer?

While in the passenger seats of one of my housemates car, I picked up a Forbes magazine from the dashboard published in May 2014.  The large block lettering on the cover read "WILL THIS MAN CURE CANCER?" below a picture of a professional looking man in a full suit.  After spending ten weeks studying many different topics regarding cancer research,  I wondered what this business/financial news based magazine was to say about curing cancer.  The man on the cover is Joseph Jimenez, the CEO of Novartis, a Swiss pharmaceutical company.

  Jimenez plans to "double down" on cancer related drugs by spending the majority of Novartis' budget on cancer therapies.  According to Forbes, Jimenez is a man that should be listened to when it comes to making money.  He transformed Gleevec(tyrosine-kinase inhibitor) from a 400 million dollar annual profit to a 4.5 billion dollar annual profit.  The six page article begins by discussing the case of a five year old girl diagnosed with acute lymphoblastic leukemia(ALL).  Today 85% of ALL patients are cured with FDA approved chemotherapeutic treatment.  This particular young girl is part of the remaining 15% uncured patients.  Her cancer returned while she was waiting for a bone marrow transplant.  Oncologists decided to try a treatment in which they removed blood from the patient and passed it through a machine which removes white blood cells.  They used a modified HIV virus to genetically reprogram those white bloods cells so they would attack cancer cells.  I decided to further research this process of immunotherapy described without much detail in Forbes magazine.

AAC-11 Newly Linked to Cervical Cancer

Introduction
Researchers have found a new genetic link to the development of metastatic cervical cancer. This article discusses how the overexpression of the gene AAC-11 leads to the production of other proteins that induce anti-apoptosis effects in the cell. The researchers found that levels of expression of AAC-11 were low in normal cervical tissue as well as cervical tissue that is in the early stages of becoming cancerous. The levels of AAC-11 increased by 200 to 400% in cervical cancer that had invaded regional lymph nodes or became metastatic (1). This indicates that AAC-11 is not the primary mutation that leads to the development of cervical cancer; rather, it is a mutation that occurs after the cancer develops but aids the progression by inhibiting apoptosis.

Telomerase Peptide Vaccine Tested in Pancreatic Cancer Patients

Telomerase is expressed in 80-95% of cases of pancreatic cancer making it a viable target for cancer therapies.  It follows that a Norwegian study explored the creation of a telomerase peptide vaccine for research subjects with non-resectable (inoperable) pancreatic cancer.  This study was a combined phase I and phase II trial aimed at discovering the proper dosage of the vaccine and its efficacy by measuring its safety, tolerability, and immunogenicity, as well as patient survival.  The peptide chosen for this vaccine, GV1001 is a "promiscuous HLA (human leukocyte antigen) class II epitope", meaning that it is the part of an antigen that the immune system recognizes and binds in a nonspecific manner to HLA molecules.

Further Hindrance in CTC measurement

     My partner’s previous post illustrated several issues which might change the number of detected CTCs in the bloodstream. While the study of CTCs holds many possibilities, my partner made a fair point to discuss that they have a fair number of limitations. One such limitation that he described was entrapment, in which CTCs sometimes get clogged within the capillaries, causing them to be underrepresented in blood samples. In my research I also stumbled upon similar phenomena in which the rate of CTC flow seemed to fluctuate. One particular study looked into the rate of CTC flow in both large and small blood vessels for both melanoma and breast cancer. The figures below display the results for these tests.



TA-65: An Anti-Aging Drug or a Cancer-Facilitating Drug?

There have always been people who have feared growing old and the decline in health associated with aging. However, people with these fears may no longer have to worry thanks to TA-65, an anti-aging drug. Isolated from various species of the Astragalus plant, TA-65 (Cycloastragenol) helps prevent aging by activating telomerase, which results in increased telomere length and ensures that cells stay healthy and live on. People have been taking the drug for a while now, and apparently it works pretty well for them. You can listen to some of the testimonials of people taking TA-65 here. But for all the good TA-65 has done for people already, others are very concerned that TA-65 theoretically increases the risk of oncogene-mediated cancer.

27HC, CYP27A1, and Breast Cancer

Myself and Erik Christensen are investigating the relationship between breast cancer and cholesterol for our Cancer Project. In our presentation we are going to give you a brief overview about what 27HC was, but I wanted to blog a little more in-depth about it. In addition, I wanted to introduce CYP27A1 and its effect on 27HC and breast cancer as we won't be discussing that aspect of our topic in our presentation.

The Cholesterol Metabolite 27-hydroxycholestorol (27HC) promotes cell proliferation. Specifically, in this case we are looking at its promotion of ER positive breast tumors. What makes 27HC different is the fact that it is an abundant primary metabolite of cholesterol. Because of its relationship with cholesterol it can attach to and actually stimulate estrogen receptors in the body. Since 27HC is a cholesterol metabolite transported in the same lipoprotein particles as cholesterol, there was a positive association between lipoprotein particles and cholesterol. Patients with ER+ breast cancer have higher amounts of 27HC in breast tissue than women who have not been specifically diagnosed with ER+ breast cancer. Below you can see the process of cholesterol synthesis and where 27HC stands in comparison to cholesterol.

The precedent behind 27HC that we wanted to try and see was if it really did have an effect on breast cancer. In order to determine if 27HC was indeed a promoter of ER positive breast cancer study was conducted with mice. The goal was to determine if 27HC promoted MCF-7 cell growth (Nelson, et. al. 2013). As we can see in Figure 1A, 27HC promotes MCF-7 cell growth. Due to unesterified plasma levels approximating to 10−8M, the threshold concentration for activation of MCF-7 cell proliferation was 10−8M. This can be seen in figure 1B.

Figure 1. 27HC promotes MCF-7 cell and Ishikawa cell proliferation, and in vivo 27HC stimulates MCF-7 cell xenograft growth and a uterotrophic response. A–D. Cell proliferation was evaluated by quantifying BrdU (A) or 3H-thymidine incorporation (B–D), n= 4–8. A. Growth responses of MCF-7 cells to E2 (10−8M) or 27HC treatment (10−8 to 10−6M) for 24h were compared. B. The dose-response of MCF-7 cells to 27HC (10−9 to 10−6M, for 24h) was determined. C. The requirement for ERα in the growth response of MCF-7 cells to E2 (10−8M) or 27HC (10−6M) was evaluated in cells treated with methyl-piperidino-pyrazole (MPP, 10uM) for 24h. D.

So with this information it would be wise to see the potential impacts of 27HC on ER+ breast cancer. Since 27HC is a cholesterol metabolite transported in the same lipoprotein particles as cholesterol, there was a predictable positive association between serum 27HC and cholesterol in both controls and cancer patients. We can see that this was eventually proven to be true in Figure 2C and 2D. In addition, compared with controls, there was a 3 fold greater 27HC concentration in normal breast tissue from cancer patients than controls. 27HC levels were 2.3 fold higher in the breast tumor itself than in the breast tissue.

27HC content is increased in normal breast tissue and tumors from ER+ breast cancer patients, and it is locally modulated. A,B. Serum 27HC (A) and total cholesterol concentration (B) in control and breast cancer patients (n= 17 and 58, respectively). Values for 10 cancer patients with serum 27HC greater than 2SD above the mean value for controls are shown in red. C,D. Relationship of serum 27HC to serum cholesterol concentration in controls (C, n=17) and cancer patients (D, n=58). E. 27HC content in normal breast tissue from controls (n=17) and cancer patients (n=48), and in tumors (n=32). *p<0.05 vs control, †p<0.05 vs cancer patient normal breast. F,G. Relationship of normal breast 27HC content to serum 27HC in controls (F, n=17) and cancer patients (G, n=40). H,I. Relationship of tumor 27HC content to serum 27HC (H) or normal breast 27HC content (I) in cancer patients (n=27).

Along the same lines, CYP27A1 is a gene sometimes referred as sterol 27-hydroxylase. Generally, it is expressed in the macrophages of the cell. We are interested in it because it is required for the conversion of Cholesterol into the metabolite 27HC. Interestingly enough, no matter whether macrophages of the breast tissue were malignant or benign, they consistently stained positively and strongly for the CYP27A1 protein. With an already established connection between macrophage infiltration and breast cancer (Wu, et al. 2013), we clamored to try and find other data and studies that incorporated this connection between the macrophage and breast cancer.

There were a couple of important findings from the next set of data we found research. The first was that (bone derived) macrophages were unable to support MCF7 cell proliferation when treated with CYP27A1 inhibitors. More importantly, introducing 27HC could reverse the effects of the CYP27A1 inhibitors. With the reversal process so effective, this indicated that local production of 27HC by macrophages in the tumor has a high impact on tumor pathology. The second observation was that CYP27A1 was expressed in the cancer cells themselves. From the data, the determination can be made that the expression was based on the size of the tumor. This can be seen below.

Table 1. Overexpression of CYP27A1 increases the likelihood of a higher tumor grade. Results of immunohistochemical analysis of CYP27A1 expression in human breast cancer tissue microarrays are shown. CYP27A1 expression was determined to be low or high and correlated with tumor grade. A Fisher’s exact test was used to determine P values for the likelihood of association. Ordinal logistic regression was used to estimate the odds ratio. N, sample number; N/A, not applicable (because sample number is too small).

The second main area studied was CYP27A1’s relation to a high cholesterol diet. In order to do this, researchers performed a study on mice where a group of mice was fed a high cholesterol diet from birth and another group on a control diet from birth. In addition to the different diet, both CYP27A1 positive and negative mice were tested on either one diet or another. The results show that CYP27A1 positive mice on a high cholesterol diet developed larger tumors faster and earlier than the mice on the control diet. Furthermore, the tumors were tested and those mice with the high cholesterol diet were found to high much higher levels of 27HC in the tumor than those mice that ate a controlled diet. Most surprisingly, the CYP27A1 negative mice had undetectable levels of 27HC in tumors and tumors formed much later and grew lower. Lastly, if the CYP27A1 negative mice that were fed the control diet were injected with 27HC, they started to produce the same exact results as the CYP27A1 positive mice on the high cholesterol diet. 

Genetic or pharmacological inhibition of 27HC production attenuates hypercholesterolemia- promoted tumor growth in mice. The latency and growth of tumors in the MMTV-PyMT mouse model of breast cancer were evaluated in mice in which the conversion of cholesterol into 27HC was inhibited by disruption of the CYP27A1 gene (CYP27A1−/−). For this study, MMTV-PyMT mice were bred onto a CYP27A1+/+ or a CYP27A1−/− background. (A) Tumor latency and (B) tumor growth were measured in mice on a control diet (CD) or a high-cholesterol diet (HCD) from weaning. Note that in the tumor growth studies, daily injection of 27HC overcame the inhibitory effect of CYP27A1 deletion. Significance between curves is indicated by a connecting black line and an asterisk (P < 0.05, n = 9 to 25).

In my opinion, this is the study’s most significant finding because it indicates that 27HC is the direct breast tumor promoter. In addition, the d at a hints that cholesterol metabolite 27HC stimulates MCF-7 cell growth in mice and lastly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to cancer-free controls, and tumor 27HC abundance is further increased.

References:

1. Nelson, E. R., S. E. Wardell, J. S. Jasper, S. Park, S. Suchindran, M. K. Howe, N. J. Carver, R. V. Pillai, P. M. Sullivan, V. Sondhi, M. Umetani, J. Geradts, and D. P. Mcdonnell. "27-Hydroxycholesterol Links Hypercholesterolemia and Breast Cancer Pathophysiology." Science 342.6162 (2013): 1094-098. Web.
2. Wu, Qian, Tomonori Ishikawa, Rosa Sirianni, Hao Tang, Jeffrey G. Mcdonald, Ivan S. Yuhanna, Bonne Thompson, Luc Girard, Chieko Mineo, Rolf A. Brekken, Michihisa Umetani, David M. Euhus, Yang Xie, and Philip W. Shaul. "27-Hydroxycholesterol Promotes Cell-Autonomous, ER-Positive Breast Cancer Growth." Cell Reports 5.3 (2013): 637-45. Web.
3. Umetani, Michihisa, and Philip W. Shaul. "27-Hydroxycholesterol: The First Identified Endogenous SERM." NIH (2011): 1-10. National Institute of Health. Web.
4. Bianchi F, Kaaks R, Viano H. Overweight, obesity, and cancer risk. Lancet Oncol. 2002; 3:565-574. Web

Clinical Trials to Support KRAS Mutations and Cetuximab Resistance

To conclude my series of blog posts, I will focus on the last important piece of the paper that I have been dissecting. My other posts focused on the data of the cellular models used to explain the molecular basis of the secondary acquired resistance. Through two different models, the authors concluded that KRAS amplification and/or mutation had an effect on resistance to cetuximab. To determine if these conclusions are clinically relevant, the researchers examined tumor biopsies from colorectal cancer patients.