Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma

Abstract

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14 days (p < 0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

The novel capsazepine analog, CIDD‐99, significantly inhibits oral squamous cell carcinoma in vivo through a TRPV1‐independent induction of ER stress, mitochondrial dysfunction, and apoptosis

“…Cell viability was determined using the Cell Titer 96 ® Aqueous Non‐Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) as described . Cells were plated and treated for 24 hours with serum‐free DMEM containing CPZ analogs at the indicated concentrations.…”

“…Mice were injected subcutaneously in the flank with 3 × 10 6 Cal‐27 cells as previously described . When tumors grew to 100 mm 3 , mice were stratified into five experimental groups (n = 5 per group) that received the following treatments via intra‐tumor injection every other day for four weeks: vehicle control (100 μL of 0.25% EtOH diluted in sterile saline), 120 μg of CPZ, CIDD‐24, CIDD‐99, or CIDD‐111 diluted in 100 μL sterile saline (final concentration of 0.25% EtOH).…”

“…Cell lysates (40 μg) were separated using electrophoresis in 10% SDS‐PAGE, separated proteins were transferred to PVDF membrane, and membrane blocked in 5% milk solution. Rabbit polyclonal antibodies (Cell Signaling, Danvers, MA, USA) against cPARP (#5625S; 1:1250), Bip (#3177S; 1:1000), and CHOP (#2895T; 1:1000) and rabbit monoclonal anti‐α/β‐tubulin antibody (Cell Signaling, #CS2148; 1:1000) were diluted in 6 mL diluent (1% milk in PBS‐0.1% Tw‐20) and incubated overnight at 4°C . Membranes were washed with PBS‐Tw‐20, incubated with ECL Plus detection solution (GE Healthcare, South San Francisco, CA, USA) for 1 minutes, and signal was detected by radiographic exposure for 30 seconds.…”

Induction of anaplastic lymphoma kinase (ALK) as a novel mechanism of EGFR inhibitor resistance in head and neck squamous cell carcinoma patient-derived models

“…regulating invasiveness and metastatic progression in HNSCC, 10 and ALK is upregulated in advanced disease compared to early-stage tumors. 11 A recent study reported that cotargeting ALK and EGFR using TAE684 and gefitinib significantly reduces HNSCC cell proliferation in vitro and decreases tumor volumes of a cell line derived xenografts by 30%. 11 However, whether the effectiveness of the combination of gefitinib and TAE684 was due to inhibition of EGFR and ALK was uncertain, since TAE684 has multiple targets other than ALK.…”

Thymol inhibits oral squamous cell carcinoma growth via mitochondria‐mediated apoptosis

“…Cytotoxicity was assessed using the Cell Titer 96 ® Aqueous Non‐Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) according to manufacturer's protocol. Cells were plated and treated for 48 hours with DMEM containing thymol at the indicated concentrations as previously described . Final DMSO concentrations were maintained at 0.1%.…”