Evaluating The Function of Genes Implicating Glioblastoma Multiforme (GBM) Formation Using C. Elegans – Final Report

Evaluating The Function of Genes Implicating Glioblastoma Multiforme (GBM) Formation Using C. Elegans

The purpose of this project was to determine if genes implicating Glioblastoma Multiforme (GBM) formation play a role in cell division in Caenorhabditis elegans (C.elegans). To be able to detect genes predisposing to GBM, it is important to understand the role of cell division. Mutations in cell division cause the proliferation of cancerous cells. In order for cell division to produce malignant cell growth, genes responsible for proper cell division have to be altered. Gene regulation is usually affected making some cells do things that they should not be doing, like overproducing proteins or growth factors that may be useful for the destructive cancerous cells. To detect GBM genes, it is important to know what genes have increased variance in GBM affected patients. Genetic variation is important in cancer, because it causes evolutionary genetic susceptibility to cellular malfunctions, such as damage in DNA repair and replication. Using the Backes et al. (2012) study, we used the data published on GBM patients, which yielded multiple genes that had increased or abnormal genetic variance. These genes were not declared as markers of GBM, although with their suspicious presence in the studied GBM patients implicates that those genes may have a role in brain cancer development. There were some genes in the publication’s dataset that had high variance, but those genes were ignored because they have been already declared as cancerous genes and predisposition markers from other research studies.

To understand whether these genes have a role in GBM, we used C.elegans as a model organism to understand the morphological and cellular significance of the suspected genes. C.elegans is a nematode that possesses a nervous system. Its nervous system comprises of 302 neurons and 56 glial cells. (Oikonomou and Shaham, 2011) These cells have an influence on sensory and motor functions. We implicated in this study that if we knockout a suspected GBM gene from C.elegans, and observed changes in glial cell development using behavioral assay and advanced microscopy, then will be able to confirm the suspected genes association with GBM.

Using several online databases, including Wormbase, Ensembl and Ortholist, we were able to find 10 genes that had orthologous genes with C.elegans. We had to research the background of the 10 genes to pinpoint which ortholog had any association with glial cells or the nervous or neuromuscular system in C.elegans. One gene, Lev-9, a levamisole resistant gene, was one of the few genes from the bunch that had a connection to the nervous system in the nematode. Utilizing the services of a company that specializes in knocking out selected genes in organisms, we were able to order two strains of Lev-9: RB1717 and ZZ16. The two strains with similar genotypes allowed us to investigate the effects of the Lev-9 gene knockout.

Figure 1: Above graphs the median progeny count for two strains of Lev-9 C.elegans: RB1717 and ZZ16; and control, N2 (wild-type) strain. Median progeny count is taken from several tests performed using the Lev-9 strains and control. Blue indicated the number of unhatched eggs found on the plate. Orange indicates the amount of early larval stage (L1-L3) organisms inhabit a plate. Purple indicated the total amount of progeny present on the plate when counted after 48 hours of incubation in 20 C.

 

According to the data, it is clear that there is some statistical significance in the results we have collected. To determine significance, we conducted four tests on the N2 control and three tests each for both RB1717 and ZZ16 Lev-9 strain C.elegans. In Figure 1, the total progeny count is substantially lower in ZZ16 than in RB1717 in comparison to the control. We expected ZZ16 and RB1717 to have less progeny, since this would indicate that there are problems with cell division. In each test, ZZ16 still remained in its L4 stage, and unlike the other strains, it appeared that it needed a longer period of incubation time in order to develop into an adult. This suggests that ZZ16 may have developmental issues and a harder time laying eggs at the same pace as a wild-type. On the contrary, RB1717 appeared to develop at an increased rate and yielded an even higher progeny count than N2. This result was surprising, because we expected RB1717 to behave more closely as ZZ16 since they are both mutants from the same gene.

From the data we have collected, we can start discussing how the progeny count is affecting the nematode and whether this data is relevant to glial cells. We predicted that the organisms would have a deteriorated nervous system and that the neural tube would also be affected by the KO of the Lev-9 gene. The organisms did exhibit abnormal behavior and uncoordinated movement when observed under a 10X microscope. Body curling and slowed movements were two of the most obvious observations, when compared to a normal organism (control). This raised question whether glial cells have been affected. To determine whether glia are directly affected, we will need to follow up on our behavioral results with a much more advanced experiment.

Keeping in mind that there is no research adequate enough to understand to what extent GBM is hereditary, and the details of it being linked to cell division, this study has the potential to help provide significant insight for the gap of knowledge in genetic inheritance and variability in GBM. GBM has significant molecular characterization for its category as a tumor, and not all therapies like chemotherapy or radiotherapy have not been found to be completely effective. This study may contribute to biomedical and bioengineering professionals that are working on solutions for cancer development prevention and those who are finding simpler ways for disease prognosis. Additionally, this study may add information to new studies concerning C.elegans and cell reproductive health. It is also greatly anticipated that the contributions from this research can provide more direction for the development of both better drugs and therapies for life-threatening mutations in cell division. it is anticipated that the present and future studies may help reshape the current predisposition testing for cancer. For a bigger picture, it is hoped that the small, but significant information from this research and other studies can redesign treatment options, and revolutionize education on how cancer and may other diseases work. With the help of my mentor, this project has definitely enriched and enhanced my education in the fundamental knowledge of the reproductive biology and cellular biology. I hope that my research experience can be a motivation to others interested in the scientific field and that determination and commitment has the potential to create great things.

 

 

References:

Backes C, Harz C, Fischer U, et al. New insights into the genetics of glioblastoma multiforme by familial exome sequencing. Oncotarget. 2015;6(8):5918-5931.

Oikonomou G, Shaham S. The glia of Caenorhabditis elegans. Glia. 2011 Sep; 59(9):1253-63. Doi:10.1002/glia.21084.

 

Using Caenorhabditis elegans to understand if particular genes have a role in Glioblastoma Multiforme formation

Using Caenorhabditis elegans to understand if particular genes have a role in Glioblastoma Multiforme formation:

The goal of this project was to continue to identify and investigate genes that implicate Glioblastoma Multiforme (GBM) formation using Caenorhabditis elegans (C.elegans). To understand whether these genes have a role in GBM, we used C.elegans as a model organism to understand the morphological and cellular significance of the suspected genes. C.elegans is a nematode that possesses a nervous system. Its nervous system comprises of 302 neurons and 56 glial cells. (Oikonomou and Shaham, 2011) These cells have an influence on sensory and motor functions. We implicated in this study that if we knockout a suspected GBM gene from C.elegans, and observed changes in glial cell development using behavioral assay and advanced microscopy, then will be able to confirm the suspected genes association with GBM.  Using several sequencing platforms, we were able to find 10 genes from the Backes et al study (2015) that had orthologous genes with C.elegans. We had to research the background of the 10 genes to pinpoint which ortholog had any association with glial cells or the nervous or neuromuscular system in C.elegans. One gene, Lev-9, a levamisole resistance gene, was one of the few genes from the bunch that had a connection to the nervous system in the nematode. Utilizing the services of a company that specializes in knocking out selected genes in organisms, we were able to order two strains of Lev-9: RB1717 and ZZ16. The two strains with similar genotypes allowed us to investigate the effects of the Lev-9 gene knockout.

To understand if cell division is affected in the knockout (KO) C.elegans, we carried out several assays in order to detect any problems with reproduction and embryonic development in the organisms. In the beginning of these assays, two young adult (L4 stage) C.elegans were picked onto a fresh NGM agar plate with OP50 bacterial lawns. This step was required three times to separately plate the two Lev-9 strains and N2 strain (wild-type) C.elegans, which would serve as our control. Using a standard protocol on culturing C.elegans, we allowed the young adults to grow at 20 C for 72 hours. This test was repeated several times and the progeny count of on each plate was recorded. The progeny consisted of eggs and any offspring inhabited the agar plate ranging between L1-L3 stage C.elegans. The test yielded inconsistent and unquantifiable results, which suggested that we develop another test to study the reproductive system in the KO strains of C.elegans. The next test consisted of picking an equal amount of L4 C.elegans per strain and the control onto new agar plates. The plates were incubated at 20 C for 24 hours, then the adult mothers would be picked off or terminated using heat over a flame. The plates are then incubated for another 24 hours in 20 C, and thereafter the plates are counted for the amount of eggs laid and the amount of L1-L3 stage C.elegans inhabiting the agar plates. This allowed us to understand the time it took for a ZZ16 and RB1717 strains of Lev-9 C.elegans to hatch and start developing.

            From the data we have collected, we can start discussing how the progeny count is affecting the nematode and whether this data is relevant to glial cells. We predicted that the organisms would have a deteriorated nervous system and that the neural tube would also be affected by the KO of the Lev-9 gene. The organisms did exhibit abnormal behavior and uncoordinated movement when observed under a 10X microscope. Body curling and slowed movements were two of the most obvious observations, when compared to a normal organism (control). This raised questions whether glial cells have been directly affected. To determine whether glia are directly affected, we would need to perform a series of behavioral assays, such as “nose-touch” assay, to confirm their associations. In order to perform this sub-experiment, we will require a strain of C.elegans that would be immune to nose-touch assays, such as Grl-1. There is another prestige university lab that specialized with glia cells in C.elegans and we anticipate on trying to collaborate this their resources to see if the glial in the Lev-9 C.elegans is being affected.

Keeping in mind that there is no research adequate enough to understand what extent GBM is hereditary, and the details of it being linked to cell division, this study has the potential to help provide significant insight for the gap of knowledge in genetic inheritance and variability in GBM. GBM has significant molecular characterization for its category as a tumor, and not all therapies like chemotherapy or radiotherapy have not been found to be completely effective. This study may contribute to biomedical and bioengineering professionals that are working on solutions for cancer development prevention and those who are finding simpler ways for disease prognosis. Additionally, this study may add information to new studies concerning C.elegans and cell reproductive health. It is also greatly anticipated that the contributions from this research can stimulate more direction for the development of both better drugs and therapies for life-threatening mutations in cell division. It is anticipated that the present and future studies may help reshape the current predisposition testing for cancer. For a bigger picture, it is hoped that the small, but significant information from this research and other studies can redesign treatment options, and revolutionize education on how cancer and may other diseases work. This project has definitely enriched and enhanced my education in the fundamental knowledge of the reproductive biology and cellular biology. I hope that my research experience can be a motivation to others interested in the scientific field and that determination and commitment has the potential to create great things.

References:

Backes C, Harz C, Fischer U, et al. New insights into the genetics of glioblastoma multiforme by familial exome sequencing. Oncotarget. 2015;6(8):5918-5931.

Oikonomou G, Shaham S. The glia of Caenorhabditis elegans. Glia. 2011 Sep; 59(9):1253-63. Doi:10.1002/glia.21084.

Blog Post #1

According to the National Brain Tumor Society (NBTS), nearly 700,000 people in the United States are living with a brain tumor that has yet to be diagnosed and treated. Most patients undergo a variety of treatments, as it is very unlikely to treat this disease using only one form of treatment. Patients can opt to undergo specialized therapies: radiation: in the form of radiation and x-rays that attempt to destroy the tumor cells in the body, chemotherapy: in the form of drugs and chemicals that are used to destroy dividing tumor cells, target therapy: which focuses on disrupting or altering certain molecules or pathways that are required for tumor cell growth, and tumor-treating field therapy: wearable device that exerts an electric field that disrupts the cell division via electrical charge inside tumor cells. Very commonly, patients may decide to undergo surgery to remove the tumor. However it is very difficult to completely remove a brain tumor since there is a high risk of developing a recurring tumor due to presence of the residual tumor and cells from the primary tumor. Despite these treatment options, the NBTS reports that over 16,500 people will still die from a malignant brain tumor this year.

Glioblastoma Multiforme (GBM) is the deadliest form of brain cancer. The deadly tumor arises from glial cells, which are star-shaped cells that serve to support and maintain healthy nerve cells located in the brain. The survival rate for glioblastoma patients is very small in comparison to other types of brain cancers, such as lower-stage astrocytoma or oligodendroglioma. According to NBTS, the survival rate percentage of people who lived at least five years after being diagnosed with GBM was 5.1%. According the American Cancer Society (ACS), the five-year survival rate for adults diagnosed with GBM estimates to about 8%. Although the disease is not as common as lung, colorectal or breast cancer, it has the same lethal capacity of destroying one of the most vital and functional organs in the body. Diagnosis and advanced biotechnology can have the capability of helping save lives and prevent people from developing GBM.

Currently, there are several ways of diagnosing potential GBM patients. Standardly, neurological exams are carried out on the patients to detect visible signs of impairments in vision, hearing and movement. Movement coordination and reflexes are examined to determine whether the nervous system is properly functioning, since they are essentially all signals from the brain. Brain imaging technology, such as computer tomography (CT) and magnetic resonance imaging (MRI) are utilized to detect tumor formations in the brain. Positron emission tomography (PET scan) can be used to detect the presence of GBM as well. Aside from imaging technology, biopsies of suspected masses or tissue can be done, and the extraction of cerebrospinal fluid. Both samples can be used to observe unusual and abnormal pathological characteristics. Determining the presence of GBM is helpful in having the premature disease treated and cleared as soon as possible. However, if we are able to pinpoint specific genes in humans that predispose to GBM is there a way we can try to eliminate GBM completely?

This study concentrates on understanding which genes predispose to GBM. According to Urbanska et al. (2014) GBM is often spontaneously developed in patients, although there have been familial cases recorded. Most of these familial cases show a history of GBM in ancestry that is believed to be passed onto their children and grandchildren. For all we may know, a person with familial history of GBM may have genetic predisposition to the disease and not even know about it nor expect it. Understanding these familial cases and the genetic differences in GBM affected patients, we may be able to pinpoint GBM-related genes. Utilizing knowledge and data from several publications on GBM patients, we will be observing whether the presence of variations in certain genes are linked to GBM.

To understand whether these genes truly have a role towards GBM formation, a model organism, Caenorhabditis elegans(C.elegans) will be used to observe the impact of the knock-out of specific genes. C.elegansis a roundworm or nematode that is commonly used for genetic and reproductive research. Using this organism allows us to understand the effect of the gene knockout based on the possible adverse effects it has on the reproductive and general nervous and muscular system of the nematode. C.elegansis practically a microscopic organism with a simple anatomy, which advantageously consists of a nervous system and structure that serves as a brain called the neural tube. If the genes that we observe truly cause physical and reproductive alterations to the organism, then staining microscopy will be used to better understand the cellular and molecular changes of the organism. In this study, we would like to understand what changes are applied to the glial cells that are present in C.elegans.

The genetics behind GBM are understudied and therefore is a lack of effective GBM-specialized target therapy. Understanding and confirming these changes in a model organism will allow us to apply the knowledge from this study to help improve GBM predisposition technology and target therapy that can be used to treat people that are in danger of developing this disease. More people consider undergoing genetic testing to find out whether they are prone to developing a genetically-related disease and adding new information to the screening panel can be very beneficial.

References:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248049/

https://events.braintumor.org/wp-content/uploads/2016/03/BrainTumorsBytheNumbers_12.04.15.pdf

https://www.cancer.org/cancer/brain-spinal-cord-tumors-adults/detection-diagnosis-staging/survival-rates.html

Mid-Year Research Update

For the past months, I have attempted to collect data for my research study. Collecting data was not fairly simple, because it was a retrospective approach to collecting data, meaning that I didn’t get to follow a step by step protocol in order to get preliminary results. It took some time trying to digest all the research papers available on my topic of interest and if any data would be able to provide me with feasible information on genes correlated to C.elegans and Glioblastoma multiforme.

I was able to successfully screen over 30+ human GBM linked genes to see if they were orthologous to C.elegans. I successfully found 10 possible C.elegans genes that could be correlated to GBM in humans. I was able to do this with the help of two high-density genome platforms called Ensembl and Ortholist. It is satisfying to know that I was able to retrieve a few, but significant number of genes, although I feel that I need to look for more genes, and perform more screens on more potential GBM human genes.  This will requite me to look through another vast number of papers which I am willing to do. This must be done, before any RNAi experiments can be performed to knock out any desired genes.

 

Evaluating the function of genes implicated Glioblastoma multiforme (GBM) formation using C. elegans

The title of my project is “Evaluating the function of genes implicated Glioblastoma multiforme (GBM) formation using C. elegans.Caenorhabditis elegans (C.elegans) is an ideal organism for studying genes and proteins in humans on a genetic and molecular level. Humans have many genes in common with C.elegans, some of which may be associated with the development of cancer in humans. C. elegans is a valuble model system that we can study to get insights into how cell division is controlled and to understand how misregulation of cell division can ultimately lead to cancer. The goal of this study is to identify which genes present in C.elegans play a role in cancer development. In particular we are interested in genes that are known to play a role in the development of Glioblastoma multiforme (GBM), which is an aggressive form of brain cancer with very low rates of survival.

There are current and past studies that demonstrate there is a link between genetic variance and tumorigenesis (cancer development) in humans. These studies involved real-life and model cases where gene mutation resulted in the cancer. Furthermore, many studies implicate that the mutation of one specific gene is capable of disrupting other genes in the genome that have linked molecular/cellular interactions with that mutated gene. The changes in the genes may be so small, involving even a few proteins. This project will help elucidate and affirm how changes in genes could affect cell function that, in turn, could ultimately cause cancer.

First, I will identify which genes implicated in GBM development are also present in C. elegans.  Then I will conduct RNA inference (RNAi) experiments in C. elegans, which will allow me to disrupt gene function and determine the function of those genes. After the RNAi experiments I will assess if cell division is disrupted by looking at embryo development. During embryo development, the cell must maintain the proper regulation of the cell cycle if not the embryo will not develop properly, which is similar to the errors that are seen in many cancers. These experiments will allow us to determine mechanistically what parts of the cell cycle are being affected and pinpoint the essential proteins that are linked to these abnormalities.