RPPA SPACE: an R package for normalization and quantitation of Reverse-Phase Protein Array data.

SUMMARY: Reverse-Phase Protein Array (RPPA) is a robust high-throughput, cost-effective platform for quantitatively measuring proteins in biological specimens. However, converting raw RPPA data into normalized, analysis-ready data remains a challenging task. Here, we present the RPPA SPACE (RPPA Superposition Analysis and Concentration Evaluation) R package, a substantially improved successor to SuperCurve, to meet that challenge. SuperCurve has been used to normalize over 170 000 samples to date. RPPA SPACE allows exclusion of poor-quality samples from the normalization process to improve the quality of the remaining samples. It also features a novel quality-control metric, 'noise', that estimates the level of random errors present in each RPPA slide. The noise metric can help to determine the quality and reliability of the data. In addition, RPPA SPACE has simpler input requirements and is more flexible than SuperCurve, it is much faster with greatly improved error reporting. AVAILABILITY AND IMPLEMENTATION: The standalone RPPA SPACE R package, tutorials and sample data are available via https://rppa.space/, CRAN (https://cran.r-project.org/web/packages/RPPASPACE/index.html) and GitHub (https://github.com/MD-Anderson-Bioinformatics/RPPASPACE). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteo-genomic characterization of virus-associated liver cancers reveals potential subtypes and therapeutic targets.

Primary liver cancer is a heterogeneous disease in terms of its etiology, histology, and therapeutic response. Concurrent proteomic and genomic characterization of a large set of clinical liver cancer samples can help elucidate the molecular basis of heterogeneity and thus serve as a valuable resource for personalized liver cancer treatment. In this study, we perform proteomic profiling of ~300 proteins on 259 primary liver cancer tissues with reverse-phase protein arrays, mutational analysis using whole genome sequencing and transcriptional analysis with RNA-Seq. Patients are of Japanese ethnic background and mainly HBV or HCV positive, providing insight into this important liver cancer subtype. Unsupervised classification of tumors based on protein expression profiles reveal three proteomic subclasses R1, R2, and R3. The R1 subclass is immunologically hot and demonstrated a good prognosis. R2 contains advanced proliferative tumor with TP53 mutations, high expression of VEGF receptor 2 and the worst prognosis. R3 is enriched with CTNNB1 mutations and elevated mTOR signaling pathway activity. Twenty-two proteins, including CDK1 and CDKN2A, are identified as potential prognostic markers. The proteomic classification presented in this study can help guide therapeutic decision making for liver cancer treatment.

Frequent post-operative monitoring of colorectal cancer using individualised ctDNA validated by multiregional molecular profiling.

BACKGROUND: Circulating tumour DNA (ctDNA) is known as a tumour-specific personalised biomarker, but the mutation-selection criteria from heterogeneous tumours remain a challenge. METHODS: We conducted multiregional sequencing of 42 specimens from 14 colorectal tumours of 12 patients, including two double-cancer cases, to identify mutational heterogeneity to develop personalised ctDNA assays using 175 plasma samples. RESULTS: "Founder" mutations, defined as a mutation that is present in all regions of the tumour in a binary manner (i.e., present or absent), were identified in 12/14 tumours. In contrast, "truncal" mutations, which are the first mutation that occurs prior to the divergence of branches in the phylogenetic tree using variant allele frequency (VAF) as continuous variables, were identified in 12/14 tumours. Two tumours without founder and truncal mutations were hypermutators. Most founder and truncal mutations exhibited higher VAFs than "non-founder" and "branch" mutations, resulting in a high chance to be detected in ctDNA. In post-operative long-term observation for 10/12 patients, early relapse prediction, treatment efficacy and non-relapse corroboration were achievable from frequent ctDNA monitoring. CONCLUSIONS: A single biopsy is sufficient to develop custom dPCR probes for monitoring tumour burden in most CRC patients. However, it may not be effective for those with hypermutated tumours.

Analysis of mutational and proteomic heterogeneity of gastric cancer suggests an effective pipeline to monitor post-treatment tumor burden using circulating tumor DNA.

Circulating tumor DNA (ctDNA) is released from tumor cells into blood in advanced cancer patients. Although gene mutations in individual tumors can be diverse and heterogenous, ctDNA has the potential to provide comprehensive biomarker information. Here, we performed multi-region sampling (three sites) per resected specimen from 10 gastric cancer patients followed by targeted sequencing and proteomic profiling using reverse-phase protein arrays. A total of 126 non-synonymous mutations were identified from 30 samples from 10 tumors. Of these, 16 (12.7%) were present in all three regions and were designated as founder mutations. Variant allele frequencies (VAFs) of founder mutations were significantly higher than those of non-founder mutations. Phylogenetic analysis also demonstrated a good concordance between founder and truncal mutations, defined as mutations shared by all simulated clones at the trunk of the tumor phylogenetic tree. These findings led us to prioritize founder mutations for quantitative ctDNA monitoring by digital PCR with individually-designed primer/probe sets. In preoperative plasma, the average ctDNA VAF of founder mutations was significantly higher than that of non-founder mutations (p = 0.039). Proteomic heterogeneity was present across the tumor regions both within and between patients independent of mutational status. Our results suggest that, in practice, mutations having high VAF identified without multi-regional sequencing may be immediately useful for quantitative ctDNA monitoring but do not provide sufficient information to predict the proteomic composition of tumors.

Analytical Platforms 3: Processing Samples via the RPPA Pipeline to Generate Large-Scale Data for Clinical Studies.

Reverse phase protein array (RPPA) is a functional proteomics technology amenable to moderately high throughputs of samples and antibodies. The University of Texas MD Anderson Cancer Center RPPA Core Facility has implemented various processes and techniques to maximize RPPA throughput; key among them are maximizing array configuration and relying on database management and automation. One major tool used by the RPPA Core is a semi-automated RPPA process management system referred to as the RPPA Pipeline. The RPPA Pipeline, developed with the aid of MD Avnderson's Department of Bioinformatics and Computational Biology and InSilico Solutions, has streamlined sample and antibody tracking as well as advanced quality control measures of various RPPA processes. This chapter covers RPPA Core processes associated with the RPPA Pipeline workflow from sample receipt to sample printing to slide staining and RPPA report generation that enables the RPPA Core to process at least 13,000 samples per year with approximately 450 individual RPPA-quality antibodies. Additionally, this chapter will cover results of large-scale clinical sample processing, including The Cancer Genome Atlas Project and The Cancer Proteome Atlas.

Suppression of Nuclear Factor-κB by Glucocorticoid Receptor Blocks Estrogen-Induced Apoptosis in Estrogen-Deprived Breast Cancer Cells.

Our clinically relevant finding is that glucocorticoids block estrogen (E2)-induced apoptosis in long-term E2-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E2, but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E2, leading to the blockade of E2-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E2-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in patients with aromatase inhibitor-resistant breast cancer.

GPNMB augments Wnt-1 mediated breast tumor initiation and growth by enhancing PI3K/AKT/mTOR pathway signaling and ß-catenin activity.

Glycoprotein Nmb (GPNMB) is overexpressed in triple-negative and basal-like breast cancers and its expression is predictive of poor prognosis within this aggressive breast cancer subtype. GPNMB promotes breast cancer growth, invasion, and metastasis; however, its role in mammary tumor initiation remains unknown. To address this question, we overexpressed GPNMB in the mammary epithelium to generate MMTV/GPNMB transgenic mice and crossed these animals to the MMTV/Wnt-1 mouse model, which is known to recapitulate features of human basal breast cancers. We show that GPNMB alone does not display oncogenic properties; however, its expression dramatically accelerates tumor onset in MMTV/Wnt-1 mice. MMTV/Wnt-1 × MMTV/GPNMB bigenic mice also exhibit a significant increase in the growth rate of established primary tumors, which is attributable to increased proliferation and decreased apoptosis. To elucidate molecular mechanisms underpinning the tumor-promoting effects of GPNMB in this context, we interrogated activated pathways in tumors derived from the MMTV/Wnt-1 and MMTV/Wnt-1 × MMTV/GPNMB mice using RPPA analysis. These data revealed that MMTV/Wnt-1 × MMTV/GPNMB bigenic tumors exhibit a pro-growth signature characterized by elevated PI3K/AKT/mTOR signaling and increased ß-catenin activity. Furthermore, we extended these observations to an independent Wnt-1 expressing model of aggressive breast cancer, and confirmed that GPNMB enhances canonical Wnt pathway activation, as evidenced by increased ß-catenin transcriptional activity, in breast cancer cells and tumors co-expressing Wnt-1 and GPNMB. GPNMB-dependent engagement of ß-catenin occurred, in part, through AKT activation. Taken together, these data ascribe a novel, pro-growth role for GPNMB in Wnt-1 expressing basal breast cancers.

Development of a robust classifier for quality control of reverse-phase protein arrays.

MOTIVATION: High-throughput reverse-phase protein array (RPPA) technology allows for the parallel measurement of protein expression levels in approximately 1000 samples. However, the many steps required in the complex protocol (sample lysate preparation, slide printing, hybridization, washing and amplified detection) may create substantial variability in data quality. We are not aware of any other quality control algorithm that is tuned to the special characteristics of RPPAs. RESULTS: We have developed a novel classifier for quality control of RPPA experiments using a generalized linear model and logistic function. The outcome of the classifier, ranging from 0 to 1, is defined as the probability that a slide is of good quality. After training, we tested the classifier using two independent validation datasets. We conclude that the classifier can distinguish RPPA slides of good quality from those of poor quality sufficiently well such that normalization schemes, protein expression patterns and advanced biological analyses will not be drastically impacted by erroneous measurements or systematic variations. AVAILABILITY AND IMPLEMENTATION: The classifier, implemented in the "SuperCurve" R package, can be freely downloaded at http://bioinformatics.mdanderson.org/main/OOMPA:Overview or http://r-forge.r-project.org/projects/supercurve/. The data used to develop and validate the classifier are available at http://bioinformatics.mdanderson.org/MOAR.

LKB1 is a central regulator of tumor initiation and pro-growth metabolism in ErbB2-mediated breast cancer.

BACKGROUND: Germline and somatic mutations in STK11, the gene encoding the serine/threonine kinase LKB1, are strongly associated with tumorigenesis. While loss of LKB1 expression has been linked to breast cancer, the mechanistic role of LKB1 in regulating breast cancer development, metastasis, and tumor metabolism has remained unclear. METHODS: We have generated and analyzed transgenic mice expressing ErbB2 in the mammary epithelium of LKB1 wild-type or LKB1-deficient mice. We have also utilized ErbB2-expressing breast cancer cells in which LKB1 levels have been reduced using shRNA approaches. These transgenic and xenograft models were characterized for the effects of LKB1 loss on tumor initiation, growth, metastasis and tumor cell metabolism. RESULTS: We demonstrate that loss of LKB1 promotes tumor initiation and induces a characteristic shift to aerobic glycolysis ('Warburg effect') in a model of ErbB2-mediated breast cancer. LKB1-deficient breast cancer cells display enhanced early tumor growth coupled with increased cell migratory and invasive properties in vitro. We show that ErbB2-positive tumors deficient for LKB1 display a pro-growth molecular and phenotypic signature characterized by elevated Akt/mTOR signaling, increased glycolytic metabolism, as well as increased bioenergetic markers both in vitro and in vivo. We also demonstrate that mTOR contributes to the metabolic reprogramming of LKB1-deficient breast cancer, and is required to drive glycolytic metabolism in these tumors; however, LKB1-deficient breast cancer cells display reduced metabolic flexibility and increased apoptosis in response to metabolic perturbations. CONCLUSIONS: Together, our data suggest that LKB1 functions as a tumor suppressor in breast cancer. Loss of LKB1 collaborates with activated ErbB2 signaling to drive breast tumorigenesis and pro-growth metabolism in the resulting tumors.

Small molecule ErbB inhibitors decrease proliferative signaling and promote apoptosis in philadelphia chromosome-positive acute lymphoblastic leukemia.

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph(+)ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph(+)ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ~30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph(+)ALL as compared to just 4.8% of Ph(-)ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph(+)ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.

Tumor epidermal growth factor receptor and EGFR PY1068 are independent prognostic indicators for head and neck squamous cell carcinoma.

PURPOSE: To assess the prognostic value of epidermal growth factor receptor (EGFR) molecular characteristics of head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N = 154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N = 39) were evaluated for EGFR gene amplification by FISH and EGFR protein by immunohistochemical staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (n = 67). Tumor (n = 50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR. RESULTS: EGFR expression by immunohistochemistry (IHC) was significantly higher in the EDRN tumors with EGFR gene amplification (P < 0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (P = 0.019 and P = 0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort [HR = 2.75; 95% confidence interval (CI) = 1.26-6.00 and HR = 3.29; 95% CI = 1.34-8.14, respectively]. CONCLUSIONS: In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.

Proteomic profiling identifies pathways dysregulated in non-small cell lung cancer and an inverse association of AMPK and adhesion pathways with recurrence.

INTRODUCTION: The identification of key pathways dysregulated in non-small cell lung cancer (NSCLC) is an important step toward understanding lung pathogenesis and developing new therapeutic approaches. METHODS: Toward this goal, reverse-phase protein lysate arrays (RPPA) were used to compare signaling pathways between NSCLC tumors and paired normal lung tissue from 46 patients and assess their association with clinical outcome. RESULTS: After RPPA quantification of 63 proteins and phosphoproteins, tissue pairs were randomized to a training set (n = 25 pairs) and test set (n = 21 pairs). In the training set, 15 protein markers were differentially expressed between tumors and normal lung (p ≤ 0.01), including markers in the PI3K/AKT and p38 MAPK signaling pathways (e.g., p70S6K, S6, p38, and phospho-p38), as well as caveolin-1 and ß-catenin. A four-protein signature (p70S6K, cyclin B1, pSrc(Y527), and caveolin-1) independent of histology classified specimens as tumor versus normal with a predicted accuracy of 83%, sensitivity of 67%, and specificity of 100%. The signature was validated in the test set, correctly classifying all normal tissues and 14 of 21 tumor tissues. RPPA results were confirmed by immunohistochemistry for caveolin-1 and p70S6K. In tumors from patients with resected NSCLC, expression of proteins in the energy-sensing AMPK pathway (pLKB1, AMPK, p-Acetyl-CoA, pTSC2), adhesion, EGFR, and Rb signaling pathways was inversely associated with NSCLC recurrence. CONCLUSIONS: These data provide evidence for dysregulation of several pathways including those involving energy sensing and adhesion that are potentially associated with NSCLC pathogenesis and disease recurrence.

Targeting the epidermal growth factor receptor in epithelial ovarian cancer: current knowledge and future challenges.

The epidermal growth factor receptor is overexpressed in up to 60% of ovarian epithelial malignancies. EGFR regulates complex cellular events due to the large number of ligands, dimerization partners, and diverse signaling pathways engaged. In ovarian cancer, EGFR activation is associated with increased malignant tumor phenotype and poorer patient outcome. However, unlike some other EGFR-positive solid tumors, treatment of ovarian tumors with anti-EGFR agents has induced minimal response. While the amount of information regarding EGFR-mediated signaling is considerable, current data provides little insight for the lack of efficacy of anti-EGFR agents in ovarian cancer. More comprehensive, systematic, and well-defined approaches are needed to dissect the roles that EGFR plays in the complex signaling processes in ovarian cancer as well as to identify biomarkers that can accurately predict sensitivity toward EGFR-targeted therapeutic agents. This new knowledge could facilitate the development of rational combinatorial therapies to sensitize tumor cells toward EGFR-targeted therapies.

Cetuximab attenuates metastasis and u-PAR expression in non-small cell lung cancer: u-PAR and E-cadherin are novel biomarkers of cetuximab sensitivity.

Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non-small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif -190/-171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab.

Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition.

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.

Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic Ras is a dominant predictor for resistance.

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Ras-dependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

Validation of reverse phase protein array for practical screening of potential biomarkers in serum and plasma: accurate detection of CA19-9 levels in pancreatic cancer.

The current study analyzed reverse phase protein arrays (RPPA) as a means to experimentally validate biomarkers in blood samples. One microliter samples of sera (n = 71), and plasma (n = 78) were serially diluted and printed on NC-coated slides. CA19-9 levels from RPPA results were compared with identical patient samples as measured by ELISA. There was a strong correlation between RPPA and ELISA (r = 0.87) as determined by scatter plots. Sample reproducibility of CA19-9 levels was excellent (interslide correlation r = 0.88; intraslide correlation r = 0.83). The ability of RPPA to accurately distinguish CA19-9 levels between cancer and noncancer samples were determined using receiver operating characteristic curves and compared with ELISA. The AUC for RPPA and ELISA was comparable (0.87 and 0.86, respectively). When the mean CA19-9 levels of normal samples was used as a cutoff for RPPA and compared with the standard clinical ELISA cutoff, comparable specificities (71% for both) were observed. Notably, RPPA samples normalized to albumin showed increased sensitivity compared to ELISA (90% vs. 75%). As RPPA is a high-throughput method that shows results comparable to that of ELISA, we propose that RPPA is a viable technique for rapid experimental screening and validation of candidate biomarkers in blood samples.

Telomere 3' overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells.

Telomere 3' overhang-specific DNA oligonucleotides (T-oligos) induce cell death in cancer cells, presumably by mimicking telomere loop disruption. Therefore, T-oligos are considered an exciting new therapeutic strategy. The purpose of this study was to elucidate how T-oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T-oligos inhibited the proliferation of malignant glioma cells through induction of nonapoptotic cell death and mitochondria hyperpolarization, whereas normal astrocytes were resistant to T-oligos. Tumor cells treated with T-oligos developed features compatible with autophagy, with development of autophagic vacuoles and conversion of an autophagy-related protein, microtubule-associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reverse-phase protein microarray analysis and Western blotting revealed that treatment with T-oligos inhibited the mammalian target of the rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment with T-oligos significantly prolonged the survival time of mice inoculated intracranially with malignant glioma cells compared with that of untreated mice and those treated with control oligonucleotides (P=0.0065 and P=0.043, respectively). These results indicate that T-oligos stimulate the induction of nonapoptotic autophagic also known as type II programmed cell death and are thus promising in the treatment of malignant glioma.

Antitumor mechanisms of combined gastrin-releasing peptide receptor and epidermal growth factor receptor targeting in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is characterized by epidermal growth factor receptor (EGFR) overexpression, where EGFR levels correlate with survival. To date, EGFR targeting has shown limited antitumor effects in head and neck cancer when administrated as monotherapy. We previously identified a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) aurocrine regulatory pathway in HNSCC, where GRP stimulates Src-dependent cleavage of EGFR proligands with subsequent EGFR phosphorylation and mitogen-activated protein kinase (MAPK) activation. To determine whether GRPR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a GRPR antagonist (PD176252) in conjunction with an EGFR tyrosine kinase inhibitor (erlotinib). Combined blockade of GRPR and EGFR pathways significantly inhibited HNSCC, but not immortalized mucosal epithelial cell, proliferation, invasion, and colony formation. In addition, the percentage of apoptotic cells increased upon combined inhibition. The enhanced antitumor efficacy was accompanied by increased expression of cleaved poly(ADP-ribose) polymerase (PARP) and decreased phospho-EGFR, phospho-MAPK, and proliferating cell nuclear antigen (PCNA). Using reverse-phase protein microarray (RPPA), we further detected decreased expression of phospho-c-Jun, phospho-p70S6K, and phospho-p38 with combined targeting. Cumulatively, these results suggest that GRPR targeting can enhance the antitumor effects of EGFR inhibitors in head and neck cancer.

A module of negative feedback regulators defines growth factor signaling.

Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.