Graduation Date

Summer 8-11-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Michel Ouellette

Second Advisor

Surinder Batra

Third Advisor

Ying Yan

Abstract

Deregulation of the KRas (Kirsten rat sarcoma virus) GTPase is one of the early hallmarks of Pancreatic Cancer (PC). The most common genetic alteration found in PC are mutations in the KRas protein that block its ability to hydrolyze GTP to GDP and resulting in higher levels of GTP-bound KRas, its active form. Pancreatic tumors driven by oncogenic mutants of KRas tend to be addicted to the oncogene, to the extent that its repression leads to the induction of cell death. This addiction to the KRAS oncogene makes the KRas protein an ideal target for cancer therapy. However, the globular structure of this small GTPase with smooth surfaces, except for one binding pocket that binds the GDP/GTP with high affinity, made it difficult to design inhibitors against the oncogenic KRas protein (1). Currently, there are no inhibitors available to block the function of KRas(G12D), the most common mutant form of KRas found in PC. Hence, it is the need of the hour to identify new vulnerabilities in KRas signaling that can be exploited to eradicate Ras-addicted PC cells in the clinics. With that aim, here in chapter 4, we have identified a new pathway that regulates the levels of KRas proteins in PC cells. The GSK3 kinase is overexpressed and aberrantly activated in PC cells. In recent work, we have discovered that the inhibition of GSK3 induces the proteasomal degradation of Ras family proteins, including the oncogenic KRas proteins. We have observed these effects in many different PC cell lines, using both pharmacological 3 and physiological inhibitors of GSK3 kinase activity and the transfection of siRNA against the two GSK3 isoforms, GSK3α and GSK3β. In PC cells addicted to oncogenic KRAS, this loss of Ras proteins leads to caspase 3 activation and apoptosis. We have further identified the LZTR1 protein as being required for the degradation of Ras proteins induced by the silencing or inhibition of GSK3. The LZTR1 protein is a substrate receptor for an E3 ubiquitin ligase (LZTR1-Cul3-Rbx1) that targets Ras proteins for proteasomal degradation, including KRas, HRas, NRas, and others (2-4). LZTR1 uses BTB-BACK domains to associate with Cul3 and a series of Kelch repeats (K1-K6) to interact with Ras proteins. Our results show that the knockdown of LZTR1 expression blocks the degradation of Ras proteins induced by the silencing or inhibition of GSK3. Both, in vitro and in mice implanted with pancreatic tumor xenografts, we have exploited this pathway to reduce Ras proteins in Ras-addicted PC cells for inducing apoptosis and inhibiting tumor growth. In addition, in chapter 5, we have identified yet another vulnerability downstream of KRas, in the Rac1 signaling pathway. The Rac1 GTPase is a member of Rho family of small GTPases and an important downstream effector of oncogenic KRas, one involved in promoting migration and therapeutic resistance. In mouse models of Ras-driven PC, the pancreas-specific knockout of Rac1 prevents the progression of the disease (5). In a previous article, the Ouellette lab had shown that blocking Rac1 made PC cells, but not normal pancreatic cells, more sensitive to the effects of ionizing radiation (IR), a DNA-damaging agent. In Chapter 5, we show that Rac1 regulates the radioresponse of PC cells, at least in part, by regulating the stability of Claspin, an adaptor protein required for ATR/Chk1 signaling. The ATR/Chk1 cascade is an important component of the DNA damage response (DDR), and the Claspin protein is required for the phosphorylation of Chk1 by ATR. Here, we have discovered that Claspin is destabilized in PC cells exposed to Rac1 inhibitors or when these cells are transfected with siRNA against Rac1. This destabilization of Claspin was determined 4 to require the 26S proteasome and the SCFβTRCP E3 ubiquitin ligase. Compared to normal cells, PC cells express much higher levels of active GTP-bound Rac1 and hence higher levels of Claspin. Most significantly, PC cells were found to be more reliant on Claspin for their survival in the face of DNA damage. In PC cells, but not normal cells, Rac1 inhibition or the knockdown of Claspin could both synergize with IR to induce apoptosis. Therefore, the Rac1-regulated Claspin/ATR/Chk1 cascade protects the PC cells from irradiation-induced DNA damage. Collectively, these studies point to novel vulnerabilities in the Ras/Rac pathway that could eventually be exploited to selectively sensitize PC cells to DNA damaging drugs, such as those used in conventional pancreatic cancer therapies.

Comments

2023 Copyright, the authors

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