THE ENDOPLASMIC RETICULUM STRESS-INDEPENDENT AND DEPENDENT ROLES OF INOSITOL-REQUIRING ENZYME 1 IN THE DEVELOPMENT OF SKIN CANCERS
Restricted (Penn State Only)
- Author:
- Mogre, Saie
- Graduate Program:
- Pathobiology (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 20, 2022
- Committee Members:
- Adam Glick, Chair & Dissertation Advisor
Kumble Prabhu, Major Field Member
Anthony Schmitt, Professor in Charge/Director of Graduate Studies
Connie Rogers, Outside Field Member
Santhosh Girirajan, Outside Unit Member - Keywords:
- Unfolded Protein Response
ER Stress
IRE1alpha
PERK
HRas
TGFbeta1
Carcinogenesis
Skin Cancer - Abstract:
- The endoplasmic reticulum (ER) is a highly adaptive intracellular organelle that can respond to the changing cellular environment during cell cycle progression, differentiation, and other intracellular signaling events, as well as counter several cell-intrinsic and environmental stresses. The ER functions by monitoring protein homeostasis, and regulates biogenesis, trafficking, and degradation of secreted and membrane-bound proteins. Interference with the normal functions of the ER can lead to a disturbed state of cellular homeostasis, a condition known as ER stress, causing the activation of the unfolded protein response (UPR) to restore the equilibrium between protein folding and synthesis. The UPR is mediated by three stress-sensing ER transmembrane proteins, Inositol-Requiring Enzyme 1 (IRE1), Protein kinase RNA-like ER Kinase (PERK), and Activating Transcription Factor 6 (ATF6). These sensors can collaborate to upregulate the protein folding capacity of the cells through increased transcription of the ER-resident molecular chaperones and ER expansion, downregulating ER load of mRNAs and proteins, and activating the ER-associated degradation (ERAD) machinery. Not surprisingly, tumor-suppressive and tumor-promoting roles of UPR pathways have been illustrated in many cancer models. In addition, tissue-specific mutations in all three UPR proteins that can potentially modify the UPR network have been identified; however, their biological relevance is poorly understood. Using a multicellular spheroid model of SV40-T immortalized mouse keratinocytes, we show that IRE1a mutations can differentially activate several biological processes, such as proliferation, migration, and EMT, and these roles occur without the classical activation of the UPR pathway. RNA-sequencing analysis from multicellular spheroids expressing wildtype or mutant forms of IRE1a suggested differential activation of the Rho family GTPases, RhoA and Rac1. Increased RhoA activation in keratinocytes expressing IRE1a inactivating mutations promoted cell migration, and this occurred as a result of an escape from the degradation of Angptl4 mRNA due to the dampened IRE1a-RIDD axis in these cells. Pharmacological inhibition of RhoA and siRNA-mediated silencing of Angptl4 destabilized the actin cytoskeleton and significantly lowered cell migration in keratinocytes expressing the RNase-dead mutant. On the other hand, the keratinocytes expressing IRE1a hyperactive mutants were slow migrating due to a visible dysregulation of the actin cytoskeleton, lower Angptl4 expression, and inhibition of RhoA signaling. Genetic and pharmacological inhibition of IRE1a, as well as secondary mRNA structure prediction, also confirmed the likelihood of Angptl4 as a target of the IRE1a-RIDD axis. While this ability of IRE1a to degrade mRNAs that can increase the proliferative and migratory potential appears to be consistent with its role as a potential tumor suppressor, we also demonstrated that keratinocytes expressing IRE1a activating mutations caused elevated levels of active Rac1, which may provide survival benefits under UV-induced apoptosis. Interestingly, in a v-RasHa spheroid model expressing IRE1a activating mutations, we noted the expression of several genes involved in proliferation and migration, including Angptl4, were no longer suppressed, indicating a pro-tumorigenic role of these mutations under these conditions. Our results provide a rationale for the development of targeted therapeutics in cancers based on the IRE1a mutational signatures. Next, we showed that TGFb1, a potent tumor suppressor in the early stages of cancer development, mediates a cross-talk between the UPR proteins, IRE1a and PERK, to control cell fate in keratinocytes expressing v-RasHa. Our previous studies have reported selective activation of IRE1a in keratinocytes expressing oncogenic forms of HRas. Transforming Growth Factor b1 (TGFb1) is a critical regulator of tumor progression in response to HRas. Recently, TGFb1 has been shown to trigger ER stress in many disease models; however, its role in oncogene-induced ER stress is unclear. TGFb1 suppressed IRE1a phosphorylation and activation by HRas in in vitro and in vivo models while simultaneously activating the PERK pathway. However, the increase in ER stress by TGFb1 indicated an uncoupling of ER stress and IRE1a activation. Pharmacological and genetic approaches demonstrated that TGFb1-dependent dephosphorylation of IRE1a was mediated by PERK through RNA Polymerase II Associated Protein 2 (RPAP2), a PERK-dependent IRE1a phosphatase. In addition, TGFb1-mediated growth arrest in oncogenic HRas keratinocytes was partially dependent on PERK-induced IRE1a dephosphorylation and inactivation. Loss of TGFb1 is associated with an increased risk of malignant conversion. Our results propose a role of increased Xbp1 splicing as an important mechanism toward malignant progression to squamous cell carcinoma.