Anil Wali

Gene expression profiles predict survival and progression of pleural mesothelioma

Purpose: Clinical outcomes for malignant pleural mesothelioma (MPM) patients having surgery are imprecisely predicted by histopathology and intraoperative staging. We hypothesized that gene expression profiles could predict time to progression and survival in surgically cytoreduced pleural mesothelioma of all stages. Experimental Design: Gene expression analyses from 21 MPM patients having cytoreductions and identical postoperative adjuvant therapy were performed using the U95 Affymetrix gene chip. Using both dChip and SAM, neural networks constructed a common 27 gene classifier, which was associated with either the high-risk and low-risk group of patients. Data were validated using real-time PCR and immunohistochemical staining. The 27 gene classifier was also used for validation in a separate set of 17 MPM patients from another institution. Results: The groups predicted by the gene classifier recapitulated the actual time to progression and survival of the test set with 95.2% accuracy using 10-fold cross-validation. Clinical outcomes were independent of histology, and heterogeneity of progression and survival in early stage patients was defined by the classifier. The gene classifier had a 76% accuracy in the separate validation set of MPMs. Conclusions: These data suggest that pretherapy gene expression analysis of mesothelioma biopsies may predict which patients may benefit from a surgical approach.

Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437.

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes ∼130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.

Cell Cycle- and Apoptosis-regulatory Protein-1 Is Involved in Apoptosis Signaling by Epidermal Growth Factor Receptor

CARP-1, a novel apoptosis inducer, regulates apoptosis signaling by diverse agents, including adriamycin and growth factors. Epidermal growth factor receptor (EGFR)-related protein (ERRP), a pan-ErbB inhibitor, inhibits EGFR and stimulates apoptosis. Treatments of cells with ERRP or Iressa (an EGFR tyrosine kinase inhibitor) results in elevated CARP-1 levels, whereas antisense-dependent depletion of CARP-1 causes inhibition of apoptosis by ERRP. CARP-1 is a tyrosine-phosphorylated protein, and ERRP treatments cause elevated tyrosine phosphorylation of CARP-1. CARP-1 contains multiple, nonoverlapping apoptosis-inducing subdomains; one such subdomain is present within amino acids 1–198. Wild-type or CARP-1-(1–198) proteins that have substitution of tyrosine 192 to phenylalanine abrogate apoptosis by ERRP. In addition, apoptosis mediated by wild type or CARP-1-(1–198), and not CARP-1-(1–198Y192F), results in activation of caspase-9 and increased phosphorylation of p38 MAPK. However, the expression of dominant-negative forms of p38 MAPK activators MKK3 or MKK6 proteins inhibits apoptosis induced by both the full-length and truncated (amino acids 1–198) proteins. Together, data demonstrate that tyrosine 192 of CARP-1 is a target of apoptosis signaling, and CARP-1, in turn, promotes apoptosis by activating p38 MAPK and caspase-9.

Common EGFR mutations conferring sensitivity to gefitinib in lung adenocarcinoma are not prevalent in human malignant mesothelioma

Dear Sir, Gefitinib (Iressa) is a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) that produces markedly favorable responses in a subset of patients with nonsmall cell lung cancer (NSCLC). Activating somatic mutations in the EGFR tyrosine-kinase domain (TKD) are strongly predictive of both clinical and in vitro sensitivity to gefitinib. To date, gefitinib-sensitizing EGFR mutations have been detected almost exclusively in lung adenocarcinomas, with only 1 missense TKD mutation identified in a colorectal carcinoma. The paucity of gefitinib-sensitizing mutations in cancers exclusive of the lung is consistent with the modest therapeutic responses gefitinib has generally shown in early clinical studies of nonlung cancers. However, not all cancers have been clinically evaluated for gefitinib responsiveness, and screening for gefitinib-sensitizing mutations may be an efficient means of detecting additional responsive cancers. Patients with mesothelioma have a notoriously poor prognosis, with few therapeutic options beyond palliative surgery. Although mesotheliomas have been shown to overexpress EGFR and have been considered as targets for EGFR inhibitors, a screen for EGFR TKD mutations in this cancer has not been reported. In this letter, we describe our screening of human malignant mesothelioma for the most common EGFR TKD mutations identified in gefitinib-responsive NSCLC. A point mutation at EGFR nucleotide 2573 (TfiG) in exon 21, conferring an arginine for leucine substitution at position 858 (L858R), and a series of in-frame deletions in exon 19 have together comprised 70–100% of the TKD mutations detected in gefitinib-responsive NSCLC in the 4 relevant studies published to date. Three additional point mutations, at nucleotide 2582 (TfiA) in exon 21 for L861Q and 2 exon 19 substitutions at nucleotide 2155 (GfiT or A) for G719S and G719C, respectively, have been identified with a 0–20% combined frequency. We developed PCR-based assays to rapidly screen tumor DNA for these 5 somatic mutations One primer in each PCR was labeled with a cyanine-based fluorescent dye (D3-PA or D4-PA) for subsequent fragment analysis using the CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA). For detection of deletions in the EGFR exon 19, primers 50-D3-PA-GCTGGTAACATCCACCCAGA-30 and 50-GAGAAAAGGTGGGCCTGAG-30 were designed to amplify a 247 bp region encompassing the entire EGFR exon 19, which was directly sized by fragment analysis. The EGFR point mutations were identified by endonuclease digestion and restriction fragment analysis of their respective amplicons. Primers utilized for genotyping the 858 and 861 codons were 50-D3-PA-GCAGAGCTTCTTCCCATGAT-30 and 50-CTGACCTAAAGCCACCTCCTT-30 and for the 760 codon, 50-D4-PA-GCTGAGGTGACCCTTGTCTC-30 and 50-CCTGTGCCAGGGACCTTA-30. For codon 858, Fau I specifically cleaves the TfiG mutation, such that the 235 bp 858/ 861 amplicon yields a 154 bp labeled restriction fragment. For codon 861, the mutant TfiA is cut by Pvu II, reducing the 858/861 amplicon to a 170 bp labeled fragment. The 182 bp codon 760 amplicon was digested with BsiHKA1, which cleaves at 135 bp both GfiT and GfiA mutants, can be subsequently discriminated by Sac I and cuts exclusively the GfiA. PCRs were performed in 5 ll or 10 ll reactions using PCR Master Mix (Promega, Madison, WI) and 5 ng of tumor DNA purified with the DNeasy 96 Tissue Kit (Qiagen, Valencia, CA), both according to manufacturer’s recommendations. Cycling parameters for all PCRs were as follows: primary denaturation at 95 C for 5 min, then 25 to 30 cycles of 95 C denaturation for 30 sec, 62 C annealing for 30 sec and 72 C extension for 30 sec, followed by a final extension for 5 min. All endonucleases were purchased from New England Biolabs (Beverly, MA) and used with appropriate controls according to manufacturer’s recommendations. One microliter of unpurified PCR was digested for 4–6 hours in a 10 ll reaction volume, 5 ll of which was subsequently analyzed in the CEQ8000 using the standard fragment analysis parameters. Sixty-six patients underwent resection for mesothelioma at the Karmanos Cancer Institute at Wayne State University in Detroit, Michigan, from 2001–2004. The case series is an expansion of that described elsewhere and includes 56 males and 10 females, all Caucasian and with a mean age of 66 years (range 41–87). Mesothelioma DNA from each subject was successfully extracted and screened for EGFR mutations. No size polymorphisms in EGFR exon 19 were detected, nor were point mutations found in any of the evaluated codons: 858, 861 and 719. In a concurrent screen of 99 lung adenocarcinoma specimens, 19 (19%) were found to harbor either a heterozygous inframe deletion in exon 19 (9 samples) or a heterozygous L858R (10 samples). Direct DNA sequencing of the amplicons derived from the lung cancer mutants and an equal number of wild-type specimens confirmed the results of our screening assays.

Consensus Report of the 2015 Weinman International Conference on Mesothelioma

ABSTRACT On November 9 and 10, 2015, the International Conference on Mesothelioma in Populations Exposed to Naturally Occurring Asbestiform Fibers was held at the University of Hawaii Cancer Center in Honolulu, Hawaii. The meeting was cosponsored by the International Association for the Study of Lung Cancer, and the agenda was designed with significant input from staff at the U.S. National Cancer Institute and National Institute of Environmental Health Sciences. A multidisciplinary group of participants presented updates reflecting a range of disciplinary perspectives, including mineralogy, geology, epidemiology, toxicology, biochemistry, molecular biology, genetics, public health, and clinical oncology. The group identified knowledge gaps that are barriers to preventing and treating malignant mesothelioma (MM) and the required next steps to address barriers. This manuscript reports the groups efforts and focus on strategies to limit risk to the population and reduce the incidence of MM. Four main topics were explored: genetic risk, environmental exposure, biomarkers, and clinical interventions. Genetics plays a critical role in MM when the disease occurs in carriers of germline BRCA1 associated protein 1 mutations. Moreover, it appears likely that, in addition to BRCA1 associated protein 1, other yet unknown genetic variants may also influence the individual risk for development of MM, especially after exposure to asbestos and related mineral fibers. MM is an almost entirely preventable malignancy as it is most often caused by exposure to commercial asbestos or mineral fibers with asbestos‐like health effects, such as erionite. In the past in North America and in Europe, the most prominent source of exposure was related to occupation. Present regulations have reduced occupational exposure in these countries; however, some people continue to be exposed to previously installed asbestos in older construction and other settings. Moreover, an increasing number of people are being exposed in rural areas that contain noncommercial asbestos, erionite, and other mineral fibers in soil or rock (termed naturally occurring asbestos [NOA]) and are being developed. Public health authorities, scientists, residents, and other affected groups must work together in the areas where exposure to asbestos, including NOA, has been documented in the environment to mitigate or reduce this exposure. Although a blood biomarker validated to be effective for use in screening and identifying MM at an early stage in asbestos/NOA‐exposed populations is not currently available, novel biomarkers presented at the meeting, such as high mobility group box 1 and fibulin‐3, are promising. There was general agreement that current treatment for MM, which is based on surgery and standard chemotherapy, has a modest effect on the overall survival (OS), which remains dismal. Additionally, although much needed novel therapeutic approaches for MM are being developed and explored in clinical trials, there is a critical need to invest in prevention research, in which there is a great opportunity to reduce the incidence and mortality from MM.

Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR

AbstractBackground: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. Methods and Results: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. Conclusion: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Withaferin A Inhibits the Proteasome Activity in Mesothelioma In Vitro and In Vivo

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.

Schlafen 3, a novel gene, regulates colonic mucosal growth during aging

Although aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa of Fischer 344 rats, the regulatory mechanisms are poorly understood. Gene expression profiling (Illumina platform) was carried out in freshly isolated colonic mucosal cells from young (4-6 mo old) and aged (22-24 mo old) Fischer 344 rats. Sixty-six genes were differentially expressed in the colonic mucosa between young and old animals (P<0.05). In particular, the expression of schlafen 3, a negative regulator of proliferation, was decreased by 8- to 10-fold in the colonic mucosa of aged rats. Administration of wortmannin, which inhibited colonic mucosal proliferation in the colonic mucosa of aged rats, stimulated the expression of schlafen 3, indicating a growth regulatory role of this gene. To further determine the growth regulatory properties of schlafen 3 gene, schlafen 3 cDNA was transfected in colon cancer HCT-116 cells. This resulted in a 30-40% inhibition of cellular growth, accompanied by decreased expression of PCNA and cyclin D1 and reduced phosphorylation of retinoblastoma protein. In conclusion, our present study demonstrates that several genes involved in proliferation and apoptosis are differentially expressed in the colonic mucosa of young and aged rats. Schlafen 3, a novel negative regulator of growth, which is markedly downregulated in the colonic mucosa of the aged, may play a role in regulating colonic mucosal growth during aging.

Targeted proteasome inhibition by Velcade induces apoptosis in human mesothelioma and breast cancer cell lines

IntroductionThoracic malignancies and human breast cancer (HBC) continue to be aggressive solid tumors that are poor responders to the existing conventional standard chemotherapeutic approaches. Malignant pleural mesothelioma (MPM) is an asbestos-related tumor of the thoracic pleura that lacks effective treatment options. Altered ubiquitin proteasome pathway is frequently encountered in many malignancies including HBC and MPM and thus serves as an important target for therapeutic intervention strategies. Although proteasome inhibitor Velcade (Bortezomib) has been under clinical investigation for a number of cancers, limited preclinical studies with this agent have thus far been conducted in HBC and MPM malignancies.PurposeTo study the biological and molecular responses of MPM and HBC cells to Velcade treatments, and to identify mechanisms involved in transducing growth inhibitory effects of this agent.MethodsFlow-cytometric analyses coupled with western immunoblotting and gene-array methodologies were utilized to determine mechanisms of Velcade-dependent growth suppression of five MPM (H2595, H2373, H2452, H2461, and H2714) and two breast cancer (MDA MB-468, SKBR-3) cell lines.ResultsOur data revealed significant reduction in cell growth properties that were dose and time dependent. Velcade treatment resulted in G2M phase arrest, increased expression of cyclin-dependent kinase inhibitor p21 and pro-apoptotic protein Bax. Pretreatment of mesothelioma cells with Velcade showed synergistic effect with cisplatin combination regimens. High-throughput gene expression profiling among Velcade treated and untreated mesothelioma cell lines resulted in identification of novel transducers of apoptosis such as CARP-1, XAF1, and Troy proteins.ConclusionsVelcade targets cell cycle and apoptosis signaling to suppress MPM and HBC growth in part by activating novel transducers of apoptosis. This pilot study has paved way for further in-depth analysis of the downstream target molecules associated with presensitization of mesothelioma cells in finding effective therapeutic treatment options for both mesothelioma and recalcitrant breast cancers.

Transactivator of transcription–tagged cell cycle and apoptosis regulatory protein-1 peptides suppress the growth of human breast cancer cells in vitro and in vivo

Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)–tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1–198, 197–454, and 896–1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1–198Y192/F) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1–198Y192/F), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1–198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1–198Y192/F), inhibits growth of human breast cancer cell–derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing. [Mol Cancer Ther 2007;6(5):1661–72]