Hierarchically arranged lists of pathways differentially targeted by AMP-18 in SCC-61 and HaCaT cells.

<p>SCC-61 and HaCaT cells were treated with AMP-18 for 2 h, total RNA was purified and subjected to RNA microarray analysis. Differentially expressed pathways were identified as stated in the Methods with no AMP-18 treatment as a control.</p

Effect of AMP peptide and radiation treatment in an orthotopic model of HNC cancer in the tongue assessed using IVIS.

<p>An orthotopic model of HNC cancer was established by inoculating bioluminescent SCC-25-LUC cells into the anterior tongue of nude mice. Tumor size in different groups was assessed and compared on Days 5 and 8 as well as Days 10 and 14 using IVIS after injection of D-luciferin. Mice in Group 1 and Group 3 were not irradiated, whereas animals in Groups 2 and 4 received 30 Gy on Day 0 of the study. Treatment with PBS (Group 2) or AMP peptide (Group 4) was administered by injection s. c. once daily. In the irradiated mice, treatment with AMP peptide was associated with much lower IVIS radiance compared to treatment with PBS, indicating that AMP peptide reduced tumor growth. Representative IVIS images of tongue tumors from 32 mice are shown.</p

Mucositis Scoring Scale.

<p>Representative images of radiation-induced mucositis used for scoring are shown. See details in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152995#sec002" target="_blank">Methods</a>.</p

Differential effects of AMP-18 on cleavage of caspase 3 in HaCaT and SCC-25 cells.

<p>Nontransformed human keratinocyte (HaCaT cells) and human HNC (SCC-25) cell cultures were each exposed to TNF-α for 6 h to induce apoptosis displayed as increased cleavage of caspase 3; 8.1-fold in HaCaT, and 2.8-fold in SCC-25 cells. Treatment with rhAMP-18 appeared to block TNF-α –induced cleavage of caspase 3 in HaCaT (50–75%, * P = 0.02) but not in HNC cells. Each immunoblot represents one of three experiments.</p

A Novel Peptide for Simultaneously Enhanced Treatment of Head and Neck Cancer and Mitigation of Oral Mucositis

<div><p>We have characterized a novel 21 amino acid-peptide derived from Antrum Mucosal Protein (AMP)-18 that mediates growth promotion of cultured normal epithelial cells and mitigates radiation-induced oral mucositis in animal models, while suppressing <i>in vitro</i> function of cancer cells. The objective of this study was to evaluate these dual potential therapeutic effects of AMP peptide in a clinically relevant animal model of head and neck cancer (HNC) by simultaneously assessing its effect on tumor growth and radiation-induced oral mucositis in an orthotopic model of HNC. Bioluminescent SCC-25 HNC cells were injected into the anterior tongue and tumors that formed were then subjected to focal radiation treatment. Tumor size was assessed using an <i>in vivo</i> imaging system, and the extent of oral mucositis was compared between animals treated with AMP peptide or vehicle (controls). Synergism between AMP peptide and radiation therapy was suggested by the finding that tumors in the AMP peptide/radiation therapy cohort demonstrated inhibited growth vs. radiation therapy-only treated tumors, while AMP peptide-treatment delayed the onset and reduced the severity of radiation therapy-induced oral mucositis. A differential effect on apoptosis appears to be one mechanism by which AMP-18 can stimulate growth and repair of injured mucosal epithelial cells while inhibiting proliferation of HNC cells. RNA microarray analysis identified pathways that are differentially targeted by AMP-18 in HNC vs. nontransformed cells. These observations confirm the notion that normal cells and tumor cells may respond differently to common biological stimuli, and that leveraging this finding in the case of AMP-18 may provide a clinically relevant opportunity.</p></div

Effect of AMP peptide and radiation treatment of HNC tumors in the anterior tongue of nude mice with time assessed by measuring tumor IVIS radiance.

<p>Irradiated animals were treated with AMP peptide or PBS, and IVIS radiance was measured on Days 5 and 8, and then on Days 10 and 14. Means ± SE were compared. In mice given PBS, IVIS radiance appeared to increase between Days 5 and 8 and 10 and 14, whereas radiance in animals treated with AMP peptide was unchanged between Days 5 to 14. On Days 10 and 14, treatment with AMP peptide significantly reduced tumor size compared to treatment with PBS (* P = 0.03). In non-irradiated mice, the orthotopic tumors in the tongue grew progressively; administration of AMP peptide did not change tumor size significantly (not shown).</p

Inhibitory effects of AMP peptide and AMP-18 on HNC cells.

<p>SCC-25 (<b>A</b>) or SCC-61 (<b>B</b>) cells were left untreated, exposed to AMP-18 (2 μg/ml), cisplatin (2 μM for SCC-25 cells, 10 μM for SCC-61 cells) only, cisplatin in the presence of 2 μg/ml AMP peptide, or rhAMP-18 for 48 h. Cell viability was assayed by CellTiter-Blue reagent. Experiments were performed in quadruplicate. Values are mean ± SE. Adding either AMP peptide or rhAMP-18 amplified the cytotoxicity of cisplatin by inhibiting growth of SCC-25 cells by >30% (AMP peptide, P = 0.013; rhAMP-18, P = 0.03), and SCC-61 cells by 65% (P<0.05) and 75% (P<0.02), respectively.</p

p62/SQSTM1 Accumulation in Squamous Cell Carcinoma of Head and Neck Predicts Sensitivity to Phosphatidylinositol 3-Kinase Pathway Inhibitors

<div><p>The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of <i>ATG7</i> in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN.</p></div

Overexpression of p62/SQSTM1 increases drug resistance in both SCCHN and breast cancer cells.

<p><b>A</b>) MCF-7 cells were transiently transfected with p62/SQSTM1 expressing vector and SCC61 cells were stably transduced with lentivirus carrying the same p62/SQSTM1 vector. Western blotting shows the ectopically expressed p62/SQSTM1-HA protein detected with HA antibody (3F10). The same blot was reprobed with p62/SQSTM1 antibody to show the total p62/SQSTM1 protein expression in both cell types. <b>B</b>) Cell viability assays demonstrate that p62/SQSTM1 expression increases drug resistance in both MCF-7 and SCC61 cell lines.</p

Effects of down regulation of p62/SQSTM1 in SCC35 cells and association of p62/SQSTM1protein level with drug resistance in both SCCHN and breast cancer cells.

<p><b>A</b>) Western blotting shows knockdown of p62/SQSTM1 in SCC35-p62KD cell line compared to SCC35-control (left). The p62/SQSTM1 bands were quantified and shown graphically on the right. <b>B</b>) Dose-response curves of SCC35-p62KD, SCC35-control, parental cells (SCC35), and SCC61 cells to MK-2206. <b>C</b>) Detection of p62/SQSTM1 protein expression in panels of SCCHN (Top) and breast cancer cell lines (Middle) by western blotting as described in Methods. Dose-responsive curves of the panel of breast cancer cell lines treated with MK-2206 (Bottom). <b>D and E</b>) Reduced expression of Nrf2 (<b>D</b>) and NQO1 (<b>E</b>) in SCC35-p62KD and SCC35-control cells. Western blotting shows duplicate samples from each derived cell line. Graph represents quantified fold change normalized to control.</p