Receptor-Tyrosine-Kinase-Targeted Therapies for Head and Neck Cancer

Molecular therapeutics for treating epidermal growth factor receptor-(EGFR-) expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics. However, the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment. Yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross-talk between receptor pathways with a key finding of common downstream signaling pathways. Such cross talk may represent a key mechanism for resistance to EGFR-directed therapy. Here we review the interplay between EGFR and Met and the type 1 insulin-like growth factor receptor (IGF-1R) tyrosine kinases, as well as their contribution to anti-EGFR therapeutic resistance in the context of squamous cell cancer of the head and neck, a tumor known to be primarily driven by EGFR-related oncogenic signals

Carcinoma Matrix Controls Resistance to Cisplatin through Talin Regulation of NF-kB

Extracellular matrix factors within the tumor microenvironment that control resistance to chemotherapeutics are poorly understood. This study focused on understanding matrix adhesion pathways that control the oral carcinoma response to cisplatin. Our studies revealed that adhesion of HN12 and JHU012 oral carcinomas to carcinoma matrix supported tumor cell proliferation in response to treatment with cisplatin. Proliferation in response to 30 µM cisplatin was not observed in HN12 cells adherent to other purified extracellular matrices such as Matrigel, collagen I, fibronectin or laminin I. Integrin β1 was important for adhesion to carcinoma matrix to trigger proliferation after treatment with cisplatin. Disruption of talin expression in HN12 cells adherent to carcinoma matrix increased cisplatin induced proliferation. Pharmacological inhibitors were used to determine signaling events required for talin deficiency to regulate cisplatin induced proliferation. Pharmacological inhibition of NF-kB reduced proliferation of talin-deficient HN12 cells treated with 30 µM cisplatin. Nuclear NF-kB activity was assayed in HN12 cells using a luciferase reporter of NF-kB transcriptional activity. Nuclear NF-kB activity was similar in HN12 cells adherent to carcinoma matrix and collagen I when treated with vehicle DMSO. Following treatment with 30 µM cisplatin, NF-kB activity is maintained in cells adherent to carcinoma matrix whereas NF-kB activity is reduced in collagen I adherent cells. Expression of talin was sufficient to trigger proliferation of HN12 cells adherent to collagen I following treatment with 1 and 30 µM cisplatin. Talin overexpression was sufficient to trigger NF-kB activity following treatment with cisplatin in carcinoma matrix adherent HN12 cells in a process disrupted by FAK siRNA. Thus, adhesions within the carcinoma matrix create a matrix environment in which exposure to cisplatin induces proliferation through the function of integrin β1, talin and FAK pathways that regulate NF-kB nuclear activity

Dynamic biochemical tissue analysis detects functional selectin ligands on human cancer tissues

© 2019, The Author(s). Cell adhesion mediated by selectins (expressed by activated endothelium, activated platelets, and leukocytes) binding to their resepective selectin ligands (expressed by cancer cells) may be involved in metastasis. Therefore, methods of characterizing selectin ligands expressed on human tissue may serve as valuable assays. Presented herein is an innovative method for detecting functional selectin ligands expressed on human tissue that uses a dynamic approach, which allows for control over the force applied to the bonds between the probe and target molecules. This new method of tissue interrogation, known as dynamic biochemical tissue analysis (DBTA), involves the perfusion of molecular probe-coated microspheres over tissues. DBTA using selectin-coated probes is able to detect functional selectin ligands expressed on tissue from multiple cancer types at both primary and metastatic sites

Schematic of oral carcinoma response to cisplatin when adherent to the carcinoma matrix.

<p>Cisplatin treatment of oral carcinoma adherent to carcinoma matrix induces proliferation through integrin β₁ dependent pathways. Treatment with cisplatin regulates NF-kB activity of talin overexpressing oral carcinoma via a FAK-dependent mechanism.</p

Role of integrin β₁ in cisplatin induced oral carcinoma proliferation.

<p>Proliferation of JHU012 and JHU012 integrin β₁ deficient cells adherent to A. carcinoma matrix and B. collagen I (10 µg/mL). Cells were treated for 48 hours with DMSO or cisplatin (0.05, 1, 30 µM) as indicated. Shown in the bar graph is the mean proliferation and error bars that indicate standard error. The horizontal line indicates the proliferation of control JHU012 cells after treatment with 30 µM cisplatin. Statistical comparisons for each substratum, a = JHU012/DMSO, b = JHU012/0.05 µM, c = JHU012/1 µM, d = JHU012/30 µM. C. Analysis of surface expression levels of integrin β₁, α₂ and α₆ subunits in JHU012 and JHU012 β₁ deficient cells by flow cytometry. Shown are overlayed histograms, grey is the isotype control and black is the indicated integrin subunit. MFI: Mean Fluorescence Intensity. D. Proliferation of HN12 cells adherent to carcinoma matrix. Control and integrin β₁ siRNA transfected cells were treated for 48 hours with DMSO or 1 µM cisplatin as indicated. Shown in the bar graph is mean proliferation and error bars indicate standard error. Student's t-test <i>p</i> values, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Talin and p130Cas control cisplatin induced proliferation of oral carcinoma.

<p>HN12 cell transfectants (plasmids containing cDNA expression vectors either talin, talin432G, p130Cas, p130CasDM (D416E, D748E) and cDNA3.1) were seeded onto A. carcinoma matrix and B. collagen I. Proliferation was measured following treatment with DMSO or cisplatin (1 and 30 µM) for 48 hours. The mean proliferation is shown with error bars indicating standard error. The data was normalized to cDNA 3.1 control transfectants treated with DMSO on each substratum. This experiment was performed 4 times. Statistical comparisons a = control/DMSO, b = control/1 µM, c = control/30 µM. Student's t-test <i>p</i> values, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Adhesion to carcinoma matrix and talin overexpression regulate NF-kB activity after treatment with cisplatin.

<p>A. HN12 cell transfectants ( (NF-kB)₄ firefly luciferase together with renilla luciferase) adherent to carcinoma matrix or collagen I were treated for 24 hours with 30 µM cisplatin or vehicle DMSO. Shown in the bar graph is the mean renilla normalized firefly luciferase activity in arbitrary light units with error bars indicating standard deviation. B. HN12 cell transfectants (firefly and renilla luciferase constructs with plasmids containing cDNA expression vectors either cDNA3.1, talin or talin432G) adherent to carcinoma matrix and treated with DMSO for 24 hours. The mean renilla normalized firefly luciferase activity in arbitrary light units with error bars indicating standard deviation is shown in the bar graph. C. HN12 cells transfected with talin cDNA expression vector together with (NF-kB)₄ firefly luciferase and renilla luciferase were plated onto carcinoma matrix or collagen. Adherent cells were treated with DMSO or 30 µM cisplatin for 24 hrs and renilla normalized firefly luciferase activity is shown in the bar graph with error bars indicating standard error. D. HN12 cells transfected with talin cDNA expression vector together with tk-luciferase (lacking the NF-kB regulatory element) and renilla luciferase adherent to carcinoma matrix were treated with DMSO and 30 µM cisplatin. Renilla normalized firefly luciferase activity is shown in the bar graph with error bars indicating standard error. E. HN12 cells transfected with (NF-kB)₄ firefly luciferase, renilla luciferase and talin cDNA in combination with either control or FAK siRNA. Carcinoma matrix adherent cells were treated with DMSO or 1 µM cisplatin and renilla normalized firefly luciferase activity is shown in the bar graph with error bars indicating standard error. Student's t-test <i>p</i> values *<i>p</i><0.05, **<i>p</i><0.01.</p

Pharmacological inhibition of oral carcinoma proliferation.

<p>A. Proliferation of HN12 cells transfected with talin, Src or control siRNA adherent to carcinoma matrix following treatment with vehicle DMSO or cisplatin (1 and 30 µM) for 48 hours is shown in the bar graph. B. Western blots to analyze the levels of talin and Src in siRNA-treated HN12 cells. C. and D. HN12 cells transfected with talin or control siRNA were assayed for proliferation 48 hours after treatment with C. vehicle DMSO and the indicated inhibitor or D. 30 µM cisplatin and the indicated inhibitor. The mean proliferation with error bars indicating standard error are shown in the bar graph. The data was normalized in C. to control siRNA/DMSO or in D. to control siRNA/30 µM cisplatin. Cells were treated with MG-132 20 µM, LY294002 20 µM, ZVAD-FMK 10 µM and PD98059 20 µM. E. Proliferation of HN12 cells adherent to carcinoma matrix following treatment with 30 µM cisplatin combined with IKK scrambled peptide (67 µM), IKKβ inhibitor (67 µM) or NLS 360–369 NF-kB p50 (100 µg/ml). F. Proliferation of control siRNA transfected HN12 cells adherent to carcinoma matrix following treatment with DMSO alone or DMSO with IKKβ inhibitor (67 µM). Talin siRNA transfected HN12 cells adherent to carcinoma matrix following treatment with DMSO alone or DMSO with IKKβ inhibitor (67 µM), 1 µM cisplatin alone or 1 µM cisplatin with IKKβ inhibitor (67 µM). Student's t-test <i>p</i> values *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005. Error bars on the mean proliferation indicate standard deviations. Experiments were performed twice in triplicate.</p