Regulation of Oncogenic Stress Response in Gastric Cancer
Department
Biological Sciences
Faculty Advisor
Alexander Agoulnik
Start Date
29-9-2020 10:00 AM
End Date
29-9-2020 11:00 AM
Abstract
Gastric cancer remains a major worldwide health problem and is considered the second most common cause of cancer death in the world. Despite numerous multimodal treatment options (i.e. chemotherapy, radiation therapy, targeted surgery, etc.), this malignancy is considered to be poorly treatable and carry significant morbidity and mortality rates. In order to better treat and prevent this disease, it is extremely important to thoroughly understand the factors involved in gastric cancer development. A fundamental, innate response to oncogenic transformation is enhanced cellular stress (i.e. metabolic, ER stress, oxidative, DNA damage). Oncogenic stress response is a powerful and persistent antiproliferative response brought on by oncogenic signaling due to the mutation of an oncogene, or the inactivation of a tumor- suppressor gene (i.e. p53). In order to accurately understand the specific metabolic pathways affected and proteins involved in gastric tumorigenesis, a model system was created. Through the cloning process, the TetOne-kRas vector grants a perfect opportunity to understand how oncogenic stress response functions. In order to observe oncogenic stress response, a model system was created through the standard cloning procedure. The TetOne vector was chosen because it contains all the components needed to induce mutated kRas expression (i.e. Tet-On 3G Transactivator Protein, PTRE3GS Inducible Promoter, etc.). Previous research studies have reported that mutated kRas cause tumor initiation, which is the perfect reason as to why it was selected as our insert. In order to properly insert kRas into the TetOne vector, the restriction sites were located and selected: Not1-HF and BamHI. In order to prepare kRas for ligation, PCR, PCR purification, restriction digest, gel, and gel purification were performed. In order to prepare TetOne vector for ligation, restriction digest, gel, and gel purification were performed. Once the vector/insert ratio was calculated (1:2), the ligation was conducted. The ligation mix was then streaked onto experimental and control LB agar-ampicillin plates. After several attempts, the colonies grew and eight individual colonies were selected for plasmid isolation. After isolation, gel electrophoresis was performed and analyzed to ensure that the insert and vector were present. From the eight total samples, three were selected for sequencing in order to guarantee no mutations. Since the model system was successfully created utilizing the pBlueScript vector, the insert, kRas, will be transferred to the TetOne vector. For future directions, this model system will be utilized for transfection in mammalian cells and investigated for oncogenic stress response function. With the creation of this model system, it will now be possible to easily detect which metabolic pathways are affected and what key proteins are involved in the regulation of oncogenic stress. Future studies will be conducted with this model system to understand gastric tumorigenesis and help identify potential targets in the prevention of gastric cancer. Overall, the main goal of this research is to prevent and cure one of the most lethal cancers in the world, gastric cancer.
File Type
Event
Regulation of Oncogenic Stress Response in Gastric Cancer
Gastric cancer remains a major worldwide health problem and is considered the second most common cause of cancer death in the world. Despite numerous multimodal treatment options (i.e. chemotherapy, radiation therapy, targeted surgery, etc.), this malignancy is considered to be poorly treatable and carry significant morbidity and mortality rates. In order to better treat and prevent this disease, it is extremely important to thoroughly understand the factors involved in gastric cancer development. A fundamental, innate response to oncogenic transformation is enhanced cellular stress (i.e. metabolic, ER stress, oxidative, DNA damage). Oncogenic stress response is a powerful and persistent antiproliferative response brought on by oncogenic signaling due to the mutation of an oncogene, or the inactivation of a tumor- suppressor gene (i.e. p53). In order to accurately understand the specific metabolic pathways affected and proteins involved in gastric tumorigenesis, a model system was created. Through the cloning process, the TetOne-kRas vector grants a perfect opportunity to understand how oncogenic stress response functions. In order to observe oncogenic stress response, a model system was created through the standard cloning procedure. The TetOne vector was chosen because it contains all the components needed to induce mutated kRas expression (i.e. Tet-On 3G Transactivator Protein, PTRE3GS Inducible Promoter, etc.). Previous research studies have reported that mutated kRas cause tumor initiation, which is the perfect reason as to why it was selected as our insert. In order to properly insert kRas into the TetOne vector, the restriction sites were located and selected: Not1-HF and BamHI. In order to prepare kRas for ligation, PCR, PCR purification, restriction digest, gel, and gel purification were performed. In order to prepare TetOne vector for ligation, restriction digest, gel, and gel purification were performed. Once the vector/insert ratio was calculated (1:2), the ligation was conducted. The ligation mix was then streaked onto experimental and control LB agar-ampicillin plates. After several attempts, the colonies grew and eight individual colonies were selected for plasmid isolation. After isolation, gel electrophoresis was performed and analyzed to ensure that the insert and vector were present. From the eight total samples, three were selected for sequencing in order to guarantee no mutations. Since the model system was successfully created utilizing the pBlueScript vector, the insert, kRas, will be transferred to the TetOne vector. For future directions, this model system will be utilized for transfection in mammalian cells and investigated for oncogenic stress response function. With the creation of this model system, it will now be possible to easily detect which metabolic pathways are affected and what key proteins are involved in the regulation of oncogenic stress. Future studies will be conducted with this model system to understand gastric tumorigenesis and help identify potential targets in the prevention of gastric cancer. Overall, the main goal of this research is to prevent and cure one of the most lethal cancers in the world, gastric cancer.