Exploring IKKβ as an anti-metastatic therapeutic target in KRAS-induced lung câncer

Author(s)

Vanessa Silva Miranda

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2019-02-07

Examining board members

Daniela Sanchez Bassères; Fábio Luís Forti; Vanessa Morais Freitas; Tiago Góss dos Santos

• Advisor: Daniela Sanchez Bassères

Abstract

Lung cancer is the leading cause of cancer deaths worldwide. The most frequent genetic changes found in lung cancer are driver mutations in the KRAS proto-oncogene. Even though KRAS mutations have been causally linked to the oncogenic process, therapies targeted to oncogenic RAS have failed in clinical trials. One of the main characteristics in oncogenesis is the ability of tumors to acquire metastatic capability. The objective of this project is to identify therapeutic targets that reduce KRASinduced lung cancer metastasis. Based on previous reports that oncogenic KRAS, drives not only tumor initiation, but also promotes a metastatic phenotype, the hypothesis of this project is that (1) the acquisition of metastatic ability induced by KRAS in the lung is potentiated by the IKK kinase; and (2) that IKKβ inhibition will reduce KRAS-induced cell invasive properties and KRAS-induced tumor metastasis. This hypothesis has been formulated on the basis of previous studies showing that the main IKKβ substrate, the transcription factor NF-κB, is activated by KRAS in lung tumors in situ in an IKKβ-dependent manner, that NF-κB is known to promote metastasis in different tumor models, and that pharmacological IKKβ inhibition in a KRAS-induced lung cancer mouse model reduces tumor growth and progression to higher histological tumor grades. Our goal was evaluate how inhibition of IKKβ affects migration and invasion of KRAS-positive lung cells in vitro and whether inhibition of IKKβ is capable of affecting the metatactic capacity of these cells in vivo. First, we evaluated the expression of enzymes involved in the metastatic phenotype, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and also a molecule involved in the integrinmediated adhesion, FAK (focal adhesion kinase), we targeted IKKβ by a highly specific IKK inhibitor (Compound A) or with RNA interference in A549 and H358 cells. We also used colorimetric Matrix Biotrak Activity Assay System to measure the activity of MMPs with RNA interference for KRAS (siKRAS) and IKKβ (IKKβ) and we have seen that IKKβ appears to modulate the expression or activity of MMP-9 and decreases the expression of FAK. The expression of MMP-2 did not change. Then we evaluated migration in A549 cell and invasion in A549 and H358 cells with inhibition of IKK by RNA interference or with Compound A treatment in Transwell assays, and observed a significantly reduced cell migration and invasion in vitro. We then generated cell lines to express luciferase, the A549 pLUC and H358 pLUC lines. A549 pLUC B4 and H358 pLUC F1 cells with RNA interference for KRAS and IKKβ were injected in the tail vein in nude (balb/c) mice and metastases were monitored by in vivo imaging. There were metastases in 20% of IKKβ animals in the anatomical region of the mouth. Animals that received siControl and siKRAS had no visible metastasis in the live imaging, but micrometastases were observed in the histological analyzes of the lungs. The result of this experiment is unexpected, not only due to the fact that it occurs in the IKKβ experimental group, but also due to the anatomical site of the tumor, and a further investigation of the role of IKKβ in this process, can be a random result. When evaluated together, our results suggest that the IKKβ kinase plays an important role in the migratory and invasive phenotype of in KRAS positive lung cancer cells, contributing to metastatic capacity. (AU)

• FAPESP's process: 16/10404-0 - Exploring IKKbeta as an anti-metastatic therapeutic target in KRAS-induced lung câncer
• Grantee: Vanessa Silva Miranda
• Support Opportunities: Scholarships in Brazil - Master