The Role of Protein Kinase N in Gastric Cancer

Abstract

Gastric cancer (GC) is one of the most common types of cancer in the Western World and accounts for over 700,000 deaths every year worldwide. The prognosis is dismal, with an average 5-year survival rate of less than 20%, mainly because of late diagnosis, due to the early stages are clinically silent. The cause of GC is multifactorial, as infectious agents, enviromental or/and genetic factors. Based on Lauren’s histologic classification, there are 2 types of GC: intestinal (IGC) and diffuse (DGC). Diffuse carcinoma cells lacks cohesion and invade tissues independently or in small clusters, is more common in young patients and behaves more aggressively than the intestinal type. Recent findings published in Nature and Nature Genetics identified frequent hotspots (14-24%) mutations in the small GTPase RHOA in the diffuse type of gastric tumors. To investigate the mechanism that underlies downstream of RHOA we analyzed the capacity of the mutations more commonly found in gastric tumors to bind to different effectors. We observed that the mutations found in gastric tumors specifically affect the capacity of RHOA to bind to PKN effector family. Therefore, in this thesis we study and characterized the role of PKN1 in GC. We demonstrated that the downregulation of PKN1 with shRNA or the deletion mediated by CRISPR/Cas9 results in the increase of proliferation in the diffuse gastric cell lines “in vitro” and “in vivo”. Moreover, the opposite effect is observed when we overexpress the constitutively active form of PKN1 in diffuse gastric cell lines with moderate or low levels of PKN1. In addition, we use a novel mouse model with conditional expression in the gastric mucosa of the RHOA-Y42C mutation (the most frequent mutation found in DGC) to investigate the role of RHOA in gastric tumorogenesis initiated by either N-methyl-N-nitrosourea (MNU) or Apc mutations. The mice with expression of the RHOA- Y42C have a significant increase in the number of tumors indicating that RhoA-Y42C is important for progression of the gastric tumors. Finally, we performed a preclinical testing of a new therapeutic approach, such as dietary supplements of arachidonic acid, a well- established activator of PKN1. The work carried out in this thesis will shed light on the newly identified deregulation of RHOA-PKN signaling in gastric cancer and provide a solid rationale for therapeutic targeting of this pathway.