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

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 ATG7 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

Ex Vivo Antibody-Dependent Cellular Cytotoxicity Inducibility Predicts Efficacy of Cetuximab

We conducted in vitro studies and a clinical trial for patients with squamous cell carcinoma of the head and neck (SCCHN) to study the relationship between FcγRIIIa polymorphisms and antibody-dependent cellular cytotoxicity (ADCC). In vitro, FcγRIIIa genotype was correlated with ADCC and innate cytotoxicity using natural killer (NK) cells harvested from healthy donors. In the phase II study, patients with recurrent or metastatic SCCHN were treated with cetuximab (500 mg/m(2) i.v. every 2 weeks) and lenalidomide (25 mg daily). FcγRIIIa genotype and ex vivo ADCC were correlated with clinical response, progression-free survival (PFS), and overall survival (OS). In vitro, healthy donors with a FcγRIIIa 158-V allele demonstrated more effective ADCC against two colon cancer cell lines HT29 and SW480, mean cytotoxicity: FF 16.1%, VF/VV 24.3% (P = 0.015) and FF 11.7%, VF/VV 21.0% (P = 0.008), respectively. We observed a linear relationship between ADCC response and innate cytotoxicity. In the phase II trial, 40 patients received cetuximab and lenalidomide with median PFS of 7.2 weeks and OS of 16.4 weeks. Thirty-six patients had FcγRIIIa genotype: VV (2), VF (20), and FF (14), and 25 patients had sufficient NK-cell yield to perform ex vivo ADCC. FcγRIIIa genotype was not associated with any clinical outcomes. Patients mounting ex vivo ADCC response had a higher likelihood of stable disease (P = 0.01) and showed a trend toward increased PFS: 14 weeks versus 6.8 weeks, respectively (P = 0.13). Enhanced ex vivo ADCC and innate immunity responses were more predictive of clinical response than FcγRIIIa and may offer a functional assay to select patients suitable for cetuximab therapy

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

Autophagy is induced in SCCHN cell lines by MK-2206 and SAR245408 and autophagy competence positively correlates with drug sensitivity.

<p><b>A</b>) Dose-response curve of SCCHN cell lines to MK-2206 and SAR245408. Cells were treated with the inhibitors for 72 hours and cell viability were assayed using Cell-Titer Blue reagent as described in Methods. <b>B</b>) SCCHN cell lines show variable autophagic activity defined by the ratio of LC3-II/LC3-I. The intensity of LC3-I and LC3-II bands were quantified with a Li-Cor imaging scanner. <b>C</b>) Western blots show PI3K/mTOR inhibitors induce autophagy in several SCCHN cell lines but not in SCC35 cells. <b>D</b>) Effect of MK-2206 and bafilomycin A1 on LC3-II protein in SCCHN cell lines. Cells were treated for 24 hours and bafilomycin A1 (final concentration 100 nM) was added 4 hours before harvesting. Western blotting was probed with LC3 antibody. LC3-II was quantified and normalized to tubulin. <b>E</b>) Drug sensitivity to MK-2206 and SAR245408 correlated with intrinsic autophagy competence.</p

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

SCC35 cell line is deficient in ATG7 expression and is macro-autophagy deficient.

<p>Time course of autophagy induction by MK-2206 in SCC61 (<b>A</b>) and SCC35 (<b>B</b>) cells. Western blots show LC3-II and p62/SQSTM1 levels from cells treated with MK-2206 (5 µM) at the time points indicated. Fold change was calculated by the ratio of the MK-2206 treated to untreated controls (C). <b>C</b>) Formation of ATG5-ATG12 conjugates in SCCHN cell lines. Western blotting was probed sequentially with ATG12 and ATG7 antibodies (top). The same lysates were analyzed with an ATG5 antibody (bottom). <b>D</b>) ATG7 protein expression in a panel of SCCHN cell lines by western blotting. A non-specific band was detected below the ATG7 protein. <b>E</b>) Detection of <i>ATG7</i> mRNA by Real-time PCR in both untreated (control) and MK-2206 treated SCC35 and SCC61 cells. <b>F</b>) Methylation-specific PCR amplification of the 5′ untranslated region of <i>ATG7</i> gene in SCC35 and SCC61 cells. Untreated and bisulfite treated SCC35 and SCC61 genomic DNAs were used as templates for PCR amplification. The PCR products were analyzed on a 1.2% agrose gel. The unmodified genomic DNA was used with the wild type primers and bisulfite treated genomic DNAs were used for both unmethylated and methylated specific primers. The primer region contains 2 CpG sites as listed in Methods.</p

Induction of autophagy by MK-2206 and SAR245408 in SCC61 cells, not in SCC35 cells, is shown by transmission electron microscopy.

<p>Cells were treated with the inhibitors for 24(AP) and autophagolysosomes (AL) are indicated by arrows.</p

Expression of phospho-AKT and p62/SQSTM1 in tissue microarrays of normal, dysplastic, and malignant oral mucosa.

<p><b>A</b>) Expression of phospho-AKT. Tissue microarrays were stained with phospho-AKT specific antibodies and scored as described in Methods. A total of 362 individual samples were analyzed which included 76 normal, 87 dysplasia, and 199 cancer samples. Histograms show score distribution from cancer samples. <b>B</b>) Expression of p62/SQSTM1. The same tissue microarrays were stained with p62/SQSTM1 antibody and scored as described in Methods. Histograms show score distribution from cancer samples. <b>C</b>) Images (20× magnification) of representative immunohistochemical staining in normal, dysplasia, and cancer tissues obtained from the same patient.</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

Regulation of ATG7 modulates drug sensitivity in SCC61.

<p><b>A</b>) Depletion of ATG7 protein expression in SCC61 cells reduced LC3-II. SCC61 cells were transfected with control siRNA or ATG7 siRNA and analyzed by western blotting. LC3-II protein was quantified and the results shown graphically. <b>B</b>) Generation of conditional knockdown of ATG7 in SCC61-hAtg7shRNA cells. Doxycycline (1 µg/ml) was added for a week to knockdown ATG7 expression. Western blotting for ATG7 expression and graphic representation of quantified ATG7 are shown (top panel). The SCC61-hAtg7shRNA cells were treated with MK-2206 and Bafilomycin as described in Methods. Western blots show ATG7, ATG5, ATG5-ATG12 conjugates, and p62/SQSTM1 protein. p62/SQSTM1 quantification was normalized to tubulin and fold change was compared to untreated control (first lane). <b>C</b>) ATG7 depleted SCC61 cells are significantly more resistant to MK-2206. Cell viability assays were preformed in the presence of MK-2206 (5 µM) or the vehicle control (DMSO).</p