EGFR-Targeted TRAIL and a Smac Mimetic Synergize to Overcome Apoptosis Resistance in KRAS Mutant Colorectal Cancer Cells

Abstract

TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed DbαEGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of DbαEGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward DbαEGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of DbαEGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing DbαEGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects DbαEGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-RasG12V. In the presence of doxycycline, these cells showed increased resistance to DbαEGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the DbαEGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between DbαEGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that DbαEGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.

Inhibitory effects of tetrandrine on epidermal growth factor-induced invasion and migration in HT29 human colorectal adenocarcinoma cells

“…In addition, the expression levels of EGFR, HER2 and HER3 were determined in primary tumors of colorectal cancer cells, and corresponded with lymph node metastases and liver metastases (42,43). The dysregulation of human EGFR pathways by the overexpression or constitutive activation promote s tumor processes, angiogenesis and metastasis in several types of human cancer (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Previous studies have demonstrated that tetrandrine inhibits cell metastasis in 4T1 breast cancer cells (18) and CT26 colorectal cancer cells in vivo (25).…”

“…The HT29 cells (1x10 5 ) were seeded into a 12-well plate and were exposed to EGF (100 ng/ml) and various concentrations of tetrandrine (0, 0.5, 1 or 2 µM) for 48 h at 37˚C. The conditioned media were collected, and the samples were separated by electrophoresis on an 8% SDS-polyacrylamide gel with 0.1% gelatin (Sigma-Aldrich).…”

“…Abnormal protein activity and/or expression levels of EGFR have been correlated with the etiology of several types of human cancer, including colorectal cancer (4,5), non-small cell lung cancer (6,7), breast cancer (8,9), head and neck squamous cell carcinoma (10,11), pancreatic cancer (12) and brain cancer (13). EGFR-targeted therapy has been validated in human colorectal cancer (14), and the chemotherapeutics include pharmacological agents, including cetuximab (Erbitux), which is an EGFR inhibitor (15).…”

“…Tetrandrine is a bisbenzylisoquinoline alkaloid, which is isolated from the dried root of Stephania tetrandra of the Inhibitory effects of tetrandrine on epidermal growth factor-induced invasion and migration in HT29 human colorectal adenocarcinoma cells CHI-TING HORNG 1,2* , JAI-SING YANG 3* , JO-HUA CHIANG 4 , CHI-CHENG LU 5 , CHIU-FANG LEE 6 , NI-NA CHIANG 6 and FU-AN CHEN Menispermaceae family (18,19). Tetrandrine has been shown to have broad pharmacological actions (20)(21)(22).…”

The effect of model rescaling and normalization on sensitivity analysis on an example of a MAPK pathway model

“…In a first step, signals are normalized to a loading control, in order to minimize artifacts that are due to different loading amounts. Second, since raw signals depend on the specialties of the antibodies, the particular membranes and chemicals in use, they are usually additionally normalized to a reference condition (for examples see [2,3]). This second normalization is required for a comparison of data from different replicates.…”