Dual Inhibition of Focal Adhesion Kinase and Epidermal Growth Factor Receptor Pathways Cooperatively Induces Death Receptor-mediated Apoptosis in Human Breast Cancer Cells

The focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) are protein-tyrosine kinases that are overexpressed and activated in human breast cancer. To determine the role of EGFR and FAK survival signaling in breast cancer, EGFR was stably overexpressed in BT474 breast cancer cells, and each signaling pathway was specifically targeted for inhibition. FAK and EGFR constitutively co-immunoprecipitated in EGFR-overexpressing BT474 cells. In low EGFR-expressing BT474-pcDNA3 vector control cells, inhibition of FAK by the FAK C-terminal domain caused detachment and apoptosis via pathways involving activation of caspase-3 and -8, cleavage of poly(ADP-ribose) polymerase, and caspase-3-dependent degradation of AKT. This apoptosis could be rescued by the dominant-negative Fas-associated death domain, indicating involvement of the death receptor pathway. EGFR overexpression did not inhibit detachment induced by the FAK C-terminal domain, but did suppress apoptosis, activating AKT and ERK1/2 survival pathways and inhibiting cleavage of FAK, caspase-3 and -8, and poly(ADP-ribose) polymerase. Furthermore, this protective effect of EGFR signaling was reversed by EGFR kinase inhibition with AG1478. In addition, inhibition of FAK and EGFR in another breast cancer cell line (BT20) endogenously overexpressing these kinases also induced apoptosis via the same mechanism as in the EGFR-overexpressing BT474 cells. The results of this study indicate that dual inhibition of FAK and EGFR signaling pathways can cooperatively enhance apoptosis in breast cancers

HER4 D-Box Sequences Regulate Mitotic Progression and Degradation of the Nuclear HER4 Cleavage Product s80HER4

Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80HER4, containing nuclear localization sequences) and results in G2/M delay by unknown signaling mechanisms. We report herein that s80HER4 contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by APC/C, a multisubunit ubiquitin ligase. s80HER4 ubiquitination and ptoteosomal degradation occurred during mitosis but not during S-phase. Inhibition of an APC subunit (APC2) using siRNA knock-down impaired s80HER4 degradation. Mutation of the s80HER4 D-box sequence stabilized s80HER4 during mitosis, and s80HER4-dependent growth inhibition via G2/M delay was significantly greater with the D-box mutant. Polyomvirus middle-T antigen-transformed HC11 cells expressing s80HER4 resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80HER4 or the empty vector. Cells expressing s80HER4 with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80HER4 limits its growth inhibitory action, and stabilization of s80HER4 enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

<div><p>The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, <i>NGScopy</i>, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.</p></div

SNV Calling in Lung Cancer Specimens Using the UNCseq Assay for SNV Listed in the OncoMap System (‘Conservative’ SNV).

<p>Percentage, actual number of significantly mutated genes, and particular SNV types, nonsynonymous (nonsense, missense) and synonymous, are shown for each tumor sample in relation to its tumor histology and tumor purity. Abbreviations: SqCC: Squamous Cell Carcinoma; SmCC: Small Cell Carcinoma; ADC/BAC: Adenocarcinoma or Bronchio-alveolar Carcinoma; LCC: Large Cell Carcinoma; AD-SqC: Adenosquamous Carcinoma or Combined/Mixed; Carcinoid/NSmCC: Carcinoid-Atypical, Carcinoid-Typical, or Non-small cell carcinoma.</p

SNV Calling of <i>KRAS</i> Hotspots Using First- and Next-Generation Sequencing.

<p><b>(A)</b> Sequencing chromatograms (Finch TV trace viewer v1.4.0) obtained from two tumor tissue examples showing concordance (sample 24) or discordance (sample 38) in <i>KRAS</i> SNV calling. <b>(B)</b> SNV calling at hot-spot loci in <i>KRAS</i> codon 12 and 13 for all 16 tumors using either of the two sequencing strategies. Calls by Sanger and NGS are colored in orange and blue, respectively. Calls by both platforms are colored in half orange and half blue. NGS coverage depth, purity, and MAF are also shown. <b>(C)</b> Boxplots of MAF, tumor purity, and coverage depth between discordant and concordant SNV calls are shown (<i>p</i>-value = 0.0006, two-sided Wilcoxon test).</p

The UNCseq project.

<p><b>(A)</b> The UNCseq project is an initiative that involves clinicians and patients interested to participate in a non-therapeutic clinical trial conducted through the Lineberger Comprehensive Cancer Center (IRB-approved protocol 11–1115), as well as a multidisciplinary team that involves clinical and research faculty (medical oncologists, pathologists, bioinformaticians, and molecular biologists) who generate, critically assess, and discuss NGS data in relation to patients’ clinical history and review previously identified genetic aberrations to determine which are potentially clinically actionable and targeted for downstream validation using validated methods in a CLIA-certified laboratory. <b>(B)</b> Following consent to 11–1115, tumor tissues and peripheral blood are collected from cancer patients. Hematoxylin and eosin (H&E)-stained representative tissue sections from tumor samples (SF or FFPE) are assessed by a certified pathologist for the percentage of viable tumor/stroma content and presence/absence of necrosis (sample QC). Extracted DNA from tumor samples is processed through various steps (fragmentation, DNA library preparation, in-solution capture of DNA fragments of interest, small-scale amplification of captured DNA fragments) prior to Illumina NGS. Data generated are discussed in a multidisciplinary Molecular Tumor Board meeting. Following validation in a CLIA-certified laboratory, these genetic aberrations are reported in patients’ personal electronic medical records.</p

DNA Copy Number Analysis Using Affymetrix Human SNP Array 6.0 Microarray (Panel A) and UNCseq (Panel B, C) or Both (Panel D) of Lung Cancer Samples.

<p><b>(A)</b> Copy number gains (red) and losses (blue) are plotted along the normal genome per each chromosome for each of the 60 completed tumor samples in relation to tumor histology and tumor purity. (SqCC: Squamous Cell Carcinoma; SmCC: Small Cell Carcinoma; ADC/BAC: Adenocarcinoma or Bronchio-alveolar Carcinoma; LCC: Large Cell Carcinoma; AD-SqC: Adenosquamous Carcinoma or Combined/Mixed; Carcinoid/NSmCC: Carcinoid-Atypical, Carcinoid-Typical, or Non-small cell carcinoma) <b>(B)</b> Examples of chromosome-level CNV in various chromosomes (6, 14, and 19) using UNCseq in two tumor samples (27 and 90). Black dots represent the per nucleotide relative copy number ratios (CNRs) in log₂. Segmentation-derived regions of equal copy number are indicated in red lines. A red triangle at 10.6 Kbp position of chromosome 19 in the sample (ID: 90) indicates the zoomed regions in panel C. <b>(C)</b> Example of small (gene-level) structural variations across exons (from 5’ to 3’) of the <i>KEAP1</i> gene (RefGene ID: <i>NM_203500</i>) for all (black) but one (red) tumor samples. Markers from the Genome-Wide Human SNP Array 6.0 corresponding to the chromosome area where <i>KEAP1</i> gene is located are highlighted in red triangles. <b>(D)</b> Boxplot analysis illustrating the SNP array signals at these two markers in C. Signals of tumor sample 90 are in red.</p