Vav2 oncoprotein, a guanine nucleotide exchange factor (GEF), is known to be overexpressed in human breast cancer, promoting tumor growth as well as metastasis. It is believed that Vav2 GEF, after being phosphorylated by the tyrosine kinases, activates certain proteins named small guanosine triphosphatases (GTPases) of the Rho family. These GTPases act as molecular switches that impact actin cytoskeletal dynamics, which influence cell adhesion and motility. GTPases cycle from an inactive state bound to guanosine diphosphate (GDP) into an active state bound to guanosine triphosphate (GTP). The limiting step is the exchange of GDP for GTP, which is facilitated by GEFs such as Vav2.
Previously our laboratory performed mass spectroscopy analysis to discover proteins that associate with Vav2 purified from a human breast cancer cell line, MCF-7. Peptide-spectrum matches for GTPases, including RhoG, RhoA, and Cdc42 small GTPases have been identified. My study aimed to discover which specific small GTPases are activated by Vav2 GEF as well as observe their impact on cell morphology and transformation. In addition, my research investigated interactions upstream of Vav2 including the effect of IGF-IR (Insulin-like growth factor type I receptor) tyrosine kinase. Understanding molecular pathways of cancer is crucial to develop effective therapeutics. Biochemical signaling and markers can be used to identify patients who are at risk of more aggressive tumors, as well as be a critical target for drug treatments.
To research GTPase function within a breast cancer model, several knockdown (KD), gene deficient, cell lines were established utilizing lentiviral particles. Separate MCF-7 cells that either overexpress (Wild Type, WT) and do not express (Dead Kinase, DK) IGF-IR were infected with shRNA sequences that either target RhoA, RhoG, or Cdc42. Protein expression of each GTPase was measured by western blot to confirm inhibition. Approximately 70% of RhoG and 80% of RhoA expression was knocked down. Cdc42, however, seemed to resist lentiviral infection; new experiments are utilizing new lentiviral sequences and siRNA techniques to achieve inhibition.
KD cells were subsequently transfected to overexpress Vav2 tagged with enhanced green fluorescence protein (EGFP). Successfully transfect cells, the ones that showed green fluorescence, were fixed onto coverslips and imaged for morphological changes.
In addition, transfected cells were used to observe the direct interactions of small GTPases and Vav2-EGFP. Cells were lysed and run through selective EGFP immunoprecipitation that isolated Vav2 and proteins bound to it. The resulting lysates were run through a gel an analyzed by western blot. RhoG was detected on the membrane signifying that it was bound to Vav2. However, RhoA was not present.
Future experiments include measuring RhoA, RhoG, or Cdc42 GTPase activities using specific targets that bind only to the active form of the GTPases. Activity of EGFP-Vav2 transfected cells will be compared to those that only contain endogenous Vav2. Rescue experiments will also be performed. The KD cells will be transfected with plasmid that express the small GTPases they have lost. The cells will be studied to determine if the morphological changes observed can revert to its original phenotype.
Vav2 oncoprotein, a guanine nucleotide exchange factor (GEF), is known to be overexpressed in human breast cancer, promoting tumor growth as well as metastasis. It is believed that Vav2 GEF, after being phosphorylated by the tyrosine kinases, activates certain proteins named small guanosine triphosphatases (GTPases) of the Rho family. These GTPases act as molecular switches that impact actin cytoskeletal dynamics, which influence cell adhesion and motility. GTPases cycle from an inactive state bound to guanosine diphosphate (GDP) into an active state bound to guanosine triphosphate (GTP). The limiting step is the exchange of GDP for GTP, which is facilitated by GEFs such as Vav2.
Previously our laboratory performed mass spectroscopy analysis to discover proteins that associate with Vav2 purified from a human breast cancer cell line, MCF-7. Peptide-spectrum matches for GTPases, including RhoG, RhoA, and Cdc42 small GTPases have been identified. My study aimed to discover which specific small GTPases are activated by Vav2 GEF as well as observe their impact on cell morphology and transformation. In addition, my research investigated interactions upstream of Vav2 including the effect of IGF-IR (Insulin-like growth factor type I receptor) tyrosine kinase. Understanding molecular pathways of cancer is crucial to develop effective therapeutics. Biochemical signaling and markers can be used to identify patients who are at risk of more aggressive tumors, as well as be a critical target for drug treatments.
To research GTPase function within a breast cancer model, several knockdown (KD), gene deficient, cell lines were established utilizing lentiviral particles. Separate MCF-7 cells that either overexpress (Wild Type, WT) and do not express (Dead Kinase, DK) IGF-IR were infected with shRNA sequences that either target RhoA, RhoG, or Cdc42. Protein expression of each GTPase was measured by western blot to confirm inhibition. Approximately 70% of RhoG and 80% of RhoA expression was knocked down. Cdc42, however, seemed to resist lentiviral infection; new experiments are utilizing new lentiviral sequences and siRNA techniques to achieve inhibition.
KD cells were subsequently transfected to overexpress Vav2 tagged with enhanced green fluorescence protein (EGFP). Successfully transfect cells, the ones that showed green fluorescence, were fixed onto coverslips and imaged for morphological changes.
In addition, transfected cells were used to observe the direct interactions of small GTPases and Vav2-EGFP. Cells were lysed and run through selective EGFP immunoprecipitation that isolated Vav2 and proteins bound to it. The resulting lysates were run through a gel an analyzed by western blot. RhoG was detected on the membrane signifying that it was bound to Vav2. However, RhoA was not present.
Future experiments include measuring RhoA, RhoG, or Cdc42 GTPase activities using specific targets that bind only to the active form of the GTPases. Activity of EGFP-Vav2 transfected cells will be compared to those that only contain endogenous Vav2. Rescue experiments will also be performed. The KD cells will be transfected with plasmid that express the small GTPases they have lost. The cells will be studied to determine if the morphological changes observed can revert to its original phenotype.