Metabolic dysregulation and cancer risk program (MeDOC): a transdisciplinary approach to Obesity-Associated cancers.

With the escalating prevalence of obesity, the association between obesity and cancer is a growing public health concern. Obesity will soon surpass tobacco smoking as the most important preventable cause of cancer. Obesity-driven mechanisms can alter cell functions to induce metabolic changes, chronic inflammation, and insulin resistance that are believed to contribute to cancer risk and development; yet the specific underlying biological mechanisms of obesity-related cancer development are largely unknown. The Metabolic Dysregulation and Obesity Cancer Risk (MeDOC) Program is a trans-NCI research program supported by the Division of Cancer Control and Population Sciences, the Division of Cancer Biology, the Division of Cancer Prevention, and the Center to Reduce Cancer Health Disparities. The overall purpose of the MeDOC Program is to advance our understanding of the underlying mechanisms that connect obesity, metabolic dysregulation, and increased obesity cancer risk, as well as identify markers that will enhance cancer risk prediction, improve screening for high-risk individuals, and identify targets for preventive and therapeutic interventions for cancer interception or treatment. This report describes the funded research projects, the Coordinating Center, and the goals of the MeDOC Program.

Integrating sleep health into resilience research.

Sleep is a biological necessity that is a critical determinant of mental and physical well-being. Sleep may promote resilience by enhancing an individual's biological preparedness to resist, adapt and recover from a challenge or stressor. This report analyzes currently active National Institutes of Health (NIH) grants focussed on sleep and resilience, specifically examining the design of studies that explore sleep as a factor that promotes health maintenance, survivorship, or protective/preventive pathways. A search of NIH R01 and R21 research project grants that received funding in Fiscal Years (FY) 2016-2021 and focussed on sleep and resilience was conducted. A total of 16 active grants from six NIH institutes met the inclusion criteria. Most grants were funded in FY 2021 (68.8%), used the R01 mechanism (81.3%), were observational studies (75.0%), and measured resilience in the context of resisting a stressor/challenge (56.3%). Early adulthood and midlife were most commonly studied and over half of the grants focussed on underserved/underrepresented populations. NIH-funded studies focussed on sleep and resilience, or the ways in which sleep can influence an individual's ability to resist, adapt, or recover from a challenging event. This analysis highlights an important gap and the need to expand research focussed on sleep as a promotor of molecular, physiological, and psychological resilience.

Antagonists of anaphase-promoting complex (APC)-2-cell cycle and apoptosis regulatory protein (CARP)-1 interaction are novel regulators of cell growth and apoptosis.

CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Although CARP-1 is a regulator of chemotherapy-dependent apoptosis, it is also a part of the NF-κB proteome and a co-activator of steroid/thyroid nuclear receptors as well as ß-catenin signaling. Our yeast two-hybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. CARP-1 also binds with anaphase-promoting complex/cyclosome co-activators Cdc20 and Cdh1. Following mapping of the minimal epitopes involved in CARP-1 binding with APC-2, a fluorescence polarization assay was established that indicated a dissociation constant (K(d)) of 480 nm for CARP-1/APC-2 binding. Fluorescence polarization assay-based high throughput screening of a chemical library yielded several small molecule antagonists of CARP-1/APC-2 binding, termed CARP-1 functional mimetics. CFM-4 (1(2-chlorobenzyl)-5'-phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one), a lead compound, binds with and stimulates CARP-1 expression. CFM-4 prevents CARP-1 binding with APC-2, causes G(2)M cell cycle arrest, and induces apoptosis with an IC(50) range of 10-15 µm. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc20 proteins. Depletion of CARP-1, however, interferes with CFM-4-dependent cell growth inhibition, activation of caspases, and apoptosis. Because CFM-4 also suppresses growth of drug-resistant human breast cancer cells without affecting the growth of human breast epithelial MCF-10A cells, elevating CARP-1 by CFM-4 and consequent apoptosis could in principle be exploited to further elucidate, and perhaps effectively target, often deregulated cell cycle pathways in pathological conditions, including cancer.

Curcumin suppresses growth of mesothelioma cells in vitro and in vivo, in part, by stimulating apoptosis.

Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-related malignancy of the thoracic pleura. Although, platinum-based agents are the first line of therapy, there is an urgent need for second-line therapies to treat the drug-resistant MPM. Cell cycle as well as apoptosis pathways are frequently altered in MPM and thus remain attractive targets for intervention strategies. Curcumin, the major component in the spice turmeric, alone or in combination with other chemotherapeutics has been under investigation for a number of cancers. In this study, we investigated the biological and molecular responses of MPM cells to curcumin treatments and the mechanisms involved. Flow-cytometric analyses coupled with western immunoblotting and gene-array analyses were conducted to determine mechanisms of curcumin-dependent growth suppression of human (H2373, H2452, H2461, and H226) and murine (AB12) MPM cells. Curcumin inhibited MPM cell growth in a dose- and time-dependent manner while pretreatment of MPM cells with curcumin enhanced cisplatin efficacy. Curcumin activated the stress-activated p38 kinase, caspases 9 and 3, caused elevated levels of proapoptotic proteins Bax, stimulated PARP cleavage, and apoptosis. In addition, curcumin treatments stimulated expression of novel transducers of cell growth suppression such as CARP-1, XAF1, and SULF1 proteins. Oral administration of curcumin inhibited growth of murine MPM cell-derived tumors in vivo in part by stimulating apoptosis. Thus, curcumin targets cell cycle and promotes apoptosis to suppress MPM growth in vitro and in vivo. Our studies provide a proof-of-principle rationale for further in-depth analysis of MPM growth suppression mechanisms and their future exploitation in effective management of resistant MPM.

Protumorigenic role of HAPLN1 and its IgV domain in malignant pleural mesothelioma.

PURPOSE: Tumor extracellular matrix (ECM) plays a crucial role in cancer progression mediating and transforming host-tumor interactions. Targeting the ECM is becoming an increasingly promising therapeutic approach in cancer treatment. We find that one of the ECM proteins, HAPLN1, is overexpressed in the majority of mesotheliomas. This study was designed to characterize the protumorigenic role of HAPLN1 in mesothelioma. EXPERIMENTAL DESIGN: Overexpression of HAPLN1 was assessed and validated on a large set of normal/mesothelioma specimens on the RNA and protein levels. We also analyzed DNA copy number alterations in the HAPLN1 genomic locus using the array-based comparative genomic hybridization representational oligonucleotide microarray analysis tool. Tumorigenic activities of the HAPLN1 domains were evaluated in vitro on mesothelioma cells transfected with HAPLN1-expressing constructs. RESULTS: We found that HAPLN1 is 23-fold overexpressed in stage I mesothelioma and confirmed it for 76% samples (n = 53) on RNA and 97% (n = 40) on protein levels. The majority of lung cancers showed no differential expression of HAPLN1. Analysis of DNA copy number alterations identified recurrent gain in the 5q14.3 HAPLN1 locus in approximately 27% of tumors. Noteworthy, high expression of HAPLN1 negatively correlated with time to progression (P = 0.05, log-rank test) and overall survival (P = 0.006). Proliferation, motility, invasion, and soft-agar colony formation assays on mesothelioma cells overexpressing full-length HAPLN1 or its functional domains strongly supported the protumorigenic role of HAPLN1 and its SP-IgV domain. CONCLUSION: Overexpression of HAPLN1 and its SP-IgV domain increases tumorigenic properties of mesothelioma. Thus, targeting the SP-IgV domain may be one of the therapeutic approaches in cancer treatment.

Asbestos exposure, pleural mesothelioma, and serum osteopontin levels.

BACKGROUND: We investigated the presence of osteopontin in pleural mesothelioma and determined serum osteopontin levels in three populations: subjects without cancer who were exposed to asbestos, subjects without cancer who were not exposed to asbestos, and patients with pleural mesothelioma who were exposed to asbestos. METHODS: A group of 69 subjects with asbestos-related nonmalignant pulmonary disease were compared with 45 subjects without exposure to asbestos and 76 patients with surgically staged pleural mesothelioma. Tumor tissue was examined for osteopontin by immunohistochemical analysis, and serum osteopontin levels were measured by an enzyme-linked immunosorbent assay. RESULTS: There were no significant differences in mean (+/-SE) serum osteopontin levels between age-matched subjects with exposure to asbestos and subjects without exposure to asbestos (30+/-3 ng per milliliter and 20+/-4 ng per milliliter, respectively; P=0.06). In the group with exposure to asbestos, elevated serum osteopontin levels were associated with pulmonary plaques and fibrosis (56+/-13 ng per milliliter) but not with normal radiographic findings (21+/-5 ng per milliliter), plaques alone (23+/-3 ng per milliliter), or fibrosis alone (32+/-7 ng per milliliter) (P=0.004). Serum osteopontin levels were significantly higher in the group with pleural mesothelioma than in the group with exposure to asbestos (133+/-10 ng per milliliter vs. 30+/-3 ng per milliliter, P<0.001). Immunohistochemical analysis revealed osteopontin staining of the tumor cells in 36 of 38 samples of pleural mesothelioma. An analysis of serum osteopontin levels comparing the receiver-operating-characteristic curve in the group exposed to asbestos with that of the group with mesothelioma had a sensitivity of 77.6 percent and a specificity of 85.5 percent at a cutoff value of 48.3 ng of osteopontin per milliliter. Subgroup analysis comparing patients with stage I mesothelioma with subjects with exposure to asbestos revealed a sensitivity of 84.6 percent and a specificity of 88.4 percent at a cutoff value of 62.4 ng of osteopontin per milliliter. CONCLUSIONS: Serum osteopontin levels can be used to distinguish persons with exposure to asbestos who do not have cancer from those with exposure to asbestos who have pleural mesothelioma.

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 (1-198(Y192/F)) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1-198(Y192/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 (1-198(Y192/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.

DNA methylation profile of 28 potential marker loci in malignant mesothelioma.

Patients with malignant mesothelioma (MM), an aggressive cancer associated with asbestos exposure, usually present clinically with advanced disease and this greatly reduces the likelihood of curative treatment. MM is difficult to diagnose without invasive techniques; the development of non-invasively detectable molecular markers would therefore be highly beneficial. DNA methylation changes in cancer cells provide powerful markers that are potentially detectable non-invasively in DNA shed into bodily fluids. Here we examined the methylation status of 28 loci in 52 MM tumors to investigate their potential as molecular markers for MM. To exclude candidate MM markers that might be positive in biopsies/pleural fluid due to contaminating surrounding non-tumor lung tissue/DNA, we also examined the methylation of these markers in lung samples (age- or environmentally induced hypermethylation is frequently observed in non-cancerous lung). Statistically significantly increased methylation in MM versus non-tumor lung samples was found for estrogen receptor 1 (ESR1; p = 0.0002), solute carrier family 6 member 20 (SLC6A20; p = 0.0022) and spleen tyrosine kinase (SYK; p=0.0003). Examination of associations between methylation levels of the 28 loci and clinical parameters suggest associations of the methylation status of metallothionein genes with gender, histology, asbestos exposure, and lymph node involvement, and the methylation status of leucine zipper tumor suppressor 1 (LZTS1) and SLC6A20 with survival.

EGF receptor-related protein (ERRP) inhibits invasion of colon cancer cells and tubule formation by endothelial cells in vitro.

Activation of the epidermal growth factor receptor (EGFR) and/or its family member(s) stimulates many processes of carcinogenesis, including cell invasion and the formation of new blood vessels, events that are critically involved in angiogenesis. Interference with the activation of EGFRs, therefore, represents a promising strategy for the development of novel and selective anticancer therapies. Previously, we reported that EGFR-related protein (ERRP), which we have isolated and characterized as a pan-erbB inhibitor, is a potential therapeutic agent for colorectal and other epithelial cancers. The present investigation was undertaken to determine whether ERRP would affect the invasion of colon cancer cells and formation of tubules, and the regulation of these processes. ERRP inhibited tubule formation by aortic endothelial cells and invasion of HCT-116 colon cancer cells through matrigel. These changes were associated with marked reductions in the synthesis and secretion of bFGF, VEGF and TGF-alpha by HCT-116 cells. Secretion of bFGF and VEGF by aortic endothelial cells was also inhibited by ERRP. Microarray analysis of ERRP-treated HCT-116 cells showed reduced levels of several growth regulatory proteins such as p21Rac1, Stratifin (14-3-3 Sigma), focal adhesion kinase (FAK) and mediators of the Ras-Raf-ERK pathway. ERRP treatments resulted in reduced expression of p21Rac1 and inhibited the constitutive activation of FAK and MEK2 in HCT-116 cells. Transfection of constitutively activate p21Rac1 or MEK2 into HCT-116 cells abrogated ERRP-induced inhibition of growth. In summary, it was demonstrated that ERRP not only inhibits cell growth, but also the processes of cell invasion and blood vessel formation that are critical for the development and progression of carcinogenesis.

Consensus Report of the 2015 Weinman International Conference on Mesothelioma.

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 group's 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.

Identification of intelectin overexpression in malignant pleural mesothelioma by serial analysis of gene expression (SAGE).

Malignant pleural mesothelioma (MPM) is a fatal neoplasm with no acceptable curative approaches. We used serial analysis of gene expression (SAGE) to compare the gene expression pattern of a surgically resected MPM to the autologous normal mesothelium. Intelectin gene overexpression (>139-fold) was found in the tumor. Online SAGE datasets revealed intelectin to be consistently present in mesothelioma(s), ovarian cancer, and colon cancer. Intelectin mRNA expression was found by RT-PCR in 4 of 5 resected MPM tumors, and Intelectin protein expression was confirmed by immunohistochemistry in 28 of 53 MPM tumors, and in 4 of 4 mesothelioma cell lines studied by Western blot. A marked induction in intelectin gene expression was observed among human primary mesothelial cells as a consequence of crocidolite asbestos exposure and simian virus 40 infection. Intelectin overexpression in mesothelioma could have potential screening, and therapeutic implications.

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.

Methylation profile landscape in mesothelioma: possible implications in early detection, disease progression, and therapeutic options.

Malignant Pleural Mesothelioma (MPM) is an aggressive malignancy of the pleura associated with asbestos exposure. Incidence of MPM is expected to increase over the course of next decade in both Europe and the developing countries. Although significant progress has been made in terms of etiology and pathogenesis of this disease, currently available therapeutic options have not significantly improved the survival outcome of patients on standard chemotherapeutic regimens. Integrity of the cellular DNA is often altered in many cancers. Understanding of the molecular mechanisms that regulate cellular DNA alterations to facilitate cancer initiation and development has potential to allow better design of cancer cell inhibitory strategies. In this context, there is a need to explore the gamut of "omics" strategies to provide a comprehensive epigenetics profile for MPM. This chapter discusses the functional genomics and epigenetic patterns observed by various investigators studying MPM patient populations on global fronts, and attempts to present a holistic approach in combating this insidious disease. Here we provide investigators in this field with novel insights and methodologies used in other types of cancers that might have profound impact in the early detection, prognosis and potential therapeutic strategies for MPM.

Increased osteonectin expression is associated with malignant transformation and tumor associated fibrosis in the lung.

Chemical transformation of the SV-40 immortalized bronchial epithelial cell line BEAS2-B induces alterations in molecules involved in cell cycle control, including up-regulation of EGFR and cyclin E [Oncogene 13 (1996) 1983; Clin Cancer Res 8 (2002) 54]. The finding that these changes also occur in vivo, in both pre-invasive and invasive lung cancer [Cancer Res 55 (1995) 1365; Cancer Res 59 (1999) 2470], proves this to be a suitable model to study lung carcinogenesis. The current study tested the hypothesis that chemical treatment of BEAS2-B with Cigarette Smoke Condensate (CSC) may affect levels of gene products involved in cell adhesion and tissue remodeling. To this end, we studied the extent of changes in osteonectin (ON) protein levels induced in BEAS 2 B-cells by CSC treatment and its timing to changes occurring in the anchorage independent cloning efficiency. ON, a multimodular protein component of the extra-cellular matrix, has been implicated in tissue remodeling occurring in neoplastic and non-neoplastic conditions, but its role in lung carcinogenesis is incompletely characterized. To validate the in vitro findings, as in our previous reports, we studied resected lung tissue, to assess whether ON expression in neoplastic lung tissue differs from normal, and to determine its cellular localization. We found that CSC treatment of BEAS2-B cells results in a 7-16-fold increase in ON protein levels, that is associated with increased colony forming efficiency. ON is absent in normal lung; in contrast it is present in the majority (39/52) of non-small cell lung cancer (NSCLC). Here, its expression is restricted to peritumoral fibroblasts in squamous cell carcinoma and adenocarcinoma. In contrast, it is localized to tumor cells in pulmonary sarcomatoid carcinoma (8/10). Thus, up-regulated ON is linked in vitro to cell transformation and in vivo, it is frequently expressed in tumor-associated fibrosis, compatible with its proposed role in tissue remodelling. Increased ON expression by tumor cells appears to represent a marker of sarcomatoid NSCLC.

Human lung cancer cells and tissues partially recapitulate the homeobox gene expression profile of embryonic lung.

The fetal cell features of tumor cells suggest that neoplasia arises through a process of defective ontogeny. Homeobox (HOX) genes code for transcription factors that orchestrate organogenesis patterning and maintain tissue homeostasis. Thus, if detective ontogeny is a mechanism in cancer development, it can be hypothesized that tumor cells should express the HOX genes normally expressed by the embryonic cells of that tissue. Our data herein indicate that some HOX genes, whose expression is normally restricted to pulmonary embryogenesis, are re-expressed in lung cancer cells. However, lung cancer cells also frequently and inappropriately express HOX genes that are not normally expressed in lung tissue, regardless of developmental stage. Thus, whereas re-expression of some of the embryo-specific HOX genes is a common feature of lung cancer, tumors do not faithfully recapitulate the expression pattern of cells that participate in the early stages of lung development.

CARP-1 functional mimetics are a novel class of small molecule inhibitors of malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is an asbestos-related thoracic malignancy that is characterized by late metastases, and resistance to therapeutic modalities. The toxic side-effects of MPM therapies often limit their clinical effectiveness, thus necessitating development of new agents to effectively treat and manage this disease in clinic. CARP-1 functional mimetics (CFMs) are a novel class of compounds that inhibit growth of diverse cancer cell types. Here we investigated MPM cell growth suppression by the CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited MPM cell growth, in vitro, in part by stimulating apoptosis. Apoptosis by CFM-4 involved activation of pro-apoptotic stress-activated protein kinases (SAPKs) p38 and JNK, elevated CARP-1 expression, cleavage of PARP1, and loss of the oncogene c-myc as well as mitotic cyclin B1. Treatments of MPM cells with CFM-4 resulted in depletion of NF-κB signaling inhibitor ABIN1 and Inhibitory κB (IκB)α and ß, while increasing expression of pro-apoptotic death receptor (DR) 4 protein. CFM-4 enhanced expression of serine-phosphorylated podoplanin and cleavage of vimetin. CFMs also attenuated biological properties of the MPM cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Both podoplanin and vimentin regulate processes of cell motility and invasion, and their expression often correlates with metastatic disease, and poor prognosis. The fact that phosphorylation of serines in the cytoplasmic domain of podoplanin interferes with processes of cellular motility, CFM-4-dependent elevated phosphorylated podoplanin and cleavage of vimentin underscore a metastasis inhibitory property of these compounds, and suggest that CFMs and/or their future analogs have potential as anti-MPM agents.

Disulfiram suppresses growth of the malignant pleural mesothelioma cells in part by inducing apoptosis.

Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and ß proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.

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.