A new model for raf kinase inhibitory protein induced chemotherapeutic resistance

Abstract

Therapeutic resistance remains the most challenging aspect of treating cancer. Raf kinase inhibitory protein (RKIP) emerged as a molecule capable of sensitizing cancerous cells to radio- and chemotherapy. Moreover, this small evolutionary conserved molecule, endows significant resistance to cancer therapy when its expression is reduced or lost. RKIP has been shown to inhibit the Raf-MEK-ERK, NFκB, GRK and activate the GSK3β signaling pathways. Inhibition of Raf-MEK-ERK and NFκB remains the most prominent pathways implicated in the sensitization of cells to therapeutic drugs. Our purpose was to identify a possible link between RKIP-KEAP 1-NRF2 and drug resistance. To that end, RKIP-KEAP 1 association was tested in human colorectal cancer tissues using immunohistochemistry. RKIP miRNA silencing and its inducible overexpression were employed in HEK-293 immortalized cells, HT29 and HCT116 colon cancer cell lines to further investigate our aim. We show that RKIP enhanced Kelch-like ECH-associated protein1 (KEAP 1) stability in colorectal cancer tissues and HT29 CRC cell line. RKIP silencing in immortalized HEK-293 cells (termed HEK-499) correlated significantly with KEAP 1 protein degradation and subsequent NRF2 addiction in these cells. Moreover, RKIP depletion in HEK-499, compared to control cells, bestowed resistance to supra physiological levels of H(2)O(2) and Cisplatin possibly by upregulating NF-E2-related nuclear factor 2 (NRF2) responsive genes. Similarly, we observed a direct correlation between the extent of apoptosis, after treatment with Adriamycin, and the expression levels of RKIP/KEAP 1 in HT29 but not in HCT116 CRC cells. Our data illuminate, for the first time, the NRF2-KEAP 1 pathway as a possible target for personalized therapeutic intervention in RKIP depleted cancers.

Figure 1. RKIP level modulation influences KEAP 1 protein expression and degradation.

A, Western blotting (lower panel) and β-actin normalized densitometric measurements (upper panel) for KEAP 1 in HEK-499 and control cells. B, Western blotting and β-actin normalized densitometric measurement (bar charts) for KEAP 1 in RKIP induced cells (Doxy+/RKIP++ are Flp-In T-Rex 293 cells stimulated with Doxycycline and Doxy−/RKIP+ are not treated/stimulated). C, Western blot and densitometric measurements of KEAP 1 protein in HT29 CRC cells transfected with empty vector (EVC) or pBP vector that overexpresses flagged RKIP (fRKIP). D, Quantitative RT-PCR of normalized KEAP 1 mRNA fold changes in HEK-293 control and HEK-499 RKIP depleted cells. E, GAPDH (left) β-actin (right) normalized KEAP 1 fold changes in mRNA expression in HT29 control (KEAP1-1 pBP) and flagged RKIP (KEAP 1-1 RKIP) transfected cells. F, HEK-293 control and HEK-499 RKIP depleted cells exposed to cycloheximide (CHX; 35 µM) for the indicated times. KEAP 1 and GAPDH protein levels were analyzed by Western blotting. G, Western blotting for RKIP, KEAP 1 and tubulin in HT29 CRC cells transfected with empty vector (EVC) or pBP vector that overexpresses flagged RKIP (fRKIP) exposed to cycloheximide (CHX; 35 µM) for the indicated times. All western experiments were performed at least in duplicates and repeated twice. Asterisks indicate statistical significance (p<0.05 compared to equivalent controls).

Figure 2. Antioxidant treatments rescue KEAP 1 in RKIP –depleted HEK-499 cells.

A, Upper panels show confocal images of HEK-293 and 499 stained with dihydro-ethidium bromide (DHE) as an indicator of ROS before and after treatments with 5 mM N-Acetylcysteine (N-Ac). The lower bar chart represents the quantification of cell-number-normalized DHE intensities in depicted cells. B, Upper panel represents western blotting for KEAP 1 and β-actin loading control in HEK-499 cells before and after treatment of cells with the antioxidants N-Ac and/or ALA. The lower western panel depicts KEAP1 and RKIP protein levels in HEK-499 and HEK-293 after the indicated treatment. C, Densitometric measurements of β-actin normalized KEAP 1 expression in an additional independent experiment after treatment of cells with 5 mM of n-Ac for 16 hours, showing the rescue of KEAP 1 protein and concomitant reduction of total NRF2 in the treated HEK-499 cells. Western experiments were performed at least in duplicates and repeated twice. Asterisks indicate statistical significance (p<0.05 compared to equivalent controls).

Figure 3. Immunohistochemical correlation between the expression of KEAP 1 and RKIP proteins in colorectal cancer tissues.

Magnification is at 40×.

Figure 4. RKIP silencing induces nuclear translocation of NRF2.

A, Shows western blotting and densitometric measurements (upper panel) for NRF2, RKIP and β-actin loading control from total protein extracts of depicted cells. B, Shows western blots on nuclear fractions of HEK-control and HEK-499 cells in duplicates with Aurora B as loading control. C, Immunofluorescent confocal microscopy for NRF2 in the depicted cells showing increased nuclear localization of NRF2 in RKIP depleted HEK-499 cells. Western experiments were performed in duplicates and repeated twice.

Figure 5. RKIP modulates NRF2-ARE containing genes.

A, mRNA expression levels by RT-PCR of RKIP and 8 NRF2-inducible genes showing overexpression of genes involved in cellular protection from oxidative stress and chemotherapy in HEK-499 cells with silenced RKIP normalized to HEK-293 control cells. B, Shows fold changes in mRNA expression of the NQO1 gene in HEK-499 cells normalized to their equivalent control. C, mRNA expression levels of RKIP and the same 8 NRF2-inducible genes in Doxycycline induced Flip-in T-Rex 293 cells normalized to uninduced control cells. D, mRNA expression fold change of NQ1 gene in Doxycycline treated Flip-in T-Rex 293 cells normalized to untreated control cells. The gene names are shown on the X-axis. The fold change was calculated using ΔΔCT normalized threshold method. Error bars represent ±S.D. Dashed lines represent 1-fold change in expression. C, Repeat western blot and densitometric analysis of RKIP protein expression levels in the depicted cells confirming the RKIP-mRNA data shown in panel A. Asterisks indicate statistical significance (p<0.05 compared to equivalent controls).

Figure 6. RKIP depletion induces hydrogen peroxide and chemotherapeutic resistance.

A, WST assay showing enhanced survival of RKIP depleted HEK-499 compared to control cells with increasing hydrogen peroxide concentration. B, MTT assay/dose response curve depicting the chemosensitivity of HEK-293 and HEK-499 after treatment with various Cisplatin concentrations for 16 hours and (C) for 60 hours. D, Early apoptosis estimation using Anexin V of HEK-293 and 499 cells after treatment with 10 µg/ml Cisplatin for 16 hours demonstrating significant resistance of HEK-499 cells to apoptotic death. Asterisks indicate statistical significance (p<0.05 compared to equivalent controls).

Figure 7. RKIP level modulations alters HT29 but not HCT116 CRC cells' sensitivity to chemotherapeutic agents.

A, HT29 and B, HCT116 cells were stably transfected with different retroviral expression vectors as indicated. Transfected cells were treated with doxorubicin for 48 h before harvesting. Extracts were prepared for western immunoblot analysis with the specific indicated antibodies. C, HCTT116 cells were stably transfected with different retroviral expression vectors as indicated. Transfected cells were treated with 5-FU for 48 h. Extracts were prepared for cytometric analysis to examine binding to 7-ADD and annexin V.

Figure 8. A proposed model for RKIP-induced drug resistance.

In unstressed normal cells and in the presence of abundant RKIP, NRF2 is kept in the cytoplasm by KEAP 1 binding and shuttled outside of the nucleus by GSK3β, phosphorylated FYN protein. Upon loss or the reduction of RKIP in a cell , inactivation/ubiquitination of KEAP1 ensues. In addition, RKIP loss/depletion causes the inactivation of GSK3β through phosphorylation at its T390 residue by p38 . Both events, ? which may be partly oxidative stress driven, culminate in the stabilization of NRF2 , , . This consequently, enhances the transcription of antioxidants and phase II detoxification genes containing ARE-cis acting element culminating in the induction of drug resistance.