Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature.

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.

Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma.

Background High resistance to therapy and poor prognosis characterizes malignant pleural mesothelioma (MPM). In fact, the current lines of treatment, based on platinum and pemetrexed, have limited impact on the survival of MPM patients. Adaptive response to therapy-induced stress involves complex rearrangements of the MPM secretome, mediated by the acquisition of a senescence-associated-secretory-phenotype (SASP). This fuels the emergence of chemoresistant cell subpopulations, with specific gene expression traits and protumorigenic features. The SASP-driven rearrangement of MPM secretome takes days to weeks to occur. Thus, we have searched for early mediators of such adaptive process and focused on metabolites differentially released in mesothelioma vs mesothelial cell culture media, after treatment with pemetrexed. METHODS: Mass spectrometry-based (LC/MS and GC/MS) identification of extracellular metabolites and unbiased statistical analysis were performed on the spent media of mesothelial and mesothelioma cell lines, at steady state and after a pulse with pharmacologically relevant doses of the drug. ELISA based evaluation of arachidonic acid (AA) levels and enzyme inhibition assays were used to explore the role of cPLA2 in AA release and that of LOX/COX-mediated processing of AA. QRT-PCR, flow cytometry analysis of ALDH expressing cells and 3D spheroid growth assays were employed to assess the role of AA at mediating chemoresistance features of MPM. ELISA based detection of p65 and IkBalpha were used to interrogate the NFkB pathway activation in AA-treated cells. RESULTS: We first validated what is known or expected from the mechanism of action of the antifolate. Further, we found increased levels of PUFAs and, more specifically, arachidonic acid (AA), in the transformed cell lines treated with pemetrexed. We showed that pharmacologically relevant doses of AA tightly recapitulated the rearrangement of cell subpopulations and the gene expression changes happening in pemetrexed -treated cultures and related to chemoresistance. Further, we showed that release of AA following pemetrexed treatment was due to cPLA2 and that AA signaling impinged on NFkB activation and largely affected anchorage-independent, 3D growth and the resistance of the MPM 3D cultures to the drug. CONCLUSIONS: AA is an early mediator of the adaptive response to pem in chemoresistant MPM and, possibly, other malignancies.

Synergistic effect of gefitinib and rofecoxib in mesothelioma cells.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive tumor that is resistant to conventional modes of treatment with chemotherapy, surgery or radiation. Research into the molecular pathways involved in the development of MM should yield information that will guide therapeutic decisions. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are involved in the carcinogenesis of MM. Combination of COX-2 and EGFR inhibitors, therefore, could be an effective strategy for reducing cell growth in those lines expressing the two molecular markers. RESULTS: In order to verify the effect of COX-2 and EGFR inhibitors, five MM cell lines NCI-2452, MPP89, Ist-Mes-1, Ist-Mes-2 and MSTO-211 were characterized for COX-2 and EGFR and then treated with respective inhibitors (rofecoxib and gefitinib) alone and in combination. Only MPP89, Ist-Mes-1 and Ist-Mes-2 were sensitive to rofecoxib and showed growth-inhibition upon gefitinib treatment. The combination of two drugs demonstrated synergistic effects on cell killing only in Ist-Mes-2, the cell line that was more sensitive to gefitinib and rofecoxib alone. Down-regulation of COX-2, EGFR, p-EGFR and up-regulation of p21 and p27 were found in Ist-Mes-2, after treatment with single agents and in combination. In contrast, association of two drugs resulted in antagonistic effect in Ist-Mes-1 and MPP89. In these cell lines after rofecoxib exposition, only an evident reduction of p-AKT was observed. No change in p-AKT in Ist-Mes-1 and MPP89 was observed after treatment with gefitinib alone and in combination with rofecoxib. CONCLUSIONS: Gefitinib and rofecoxib exert cell type-specific effects that vary between different MM cells. Total EGFR expression and downstream signalling does not correlate with gefitinib sensitivity. These data suggest that the effect of gefitinib can be potentiated by rofecoxib in MM cell lines where AKT is not activated.

Effect of a 2-month treatment with Klamin, a Klamath algae extract, on the general well-being, antioxidant profile and oxidative status of postmenopausal women.

BACKGROUND AND AIM: Because of a growing demand for alternative treatments of the psychological and somatic/vasomotor symptoms related to menopausal transition, in this study we aimed to investigate the effect of a 2-month supplementation period with the Klamath algae extract (Klamin, Nutratec Srl, Urbino, Italy) on the general and psychological well-being of a group of 21 menopausal women not treated with hormonal therapy, as well as on their oxidative stress status and level of antioxidants. Klamin is an extract naturally rich in powerful algal antioxidant molecules (AFA-phycocyanins) and concentrated with Klamath algae's natural neuromodulators (phenylethylamine as well as natural selective MAO-B inhibitors). CONCLUSIONS: At the end of the Klamin supplementation period, plasma lipid peroxidation significantly decreased (as proven by a significant lowering of plasma MDA levels), while the overall antioxidant system improved thanks to the significant increase in the plasma levels of carotenoids, tocopherols and retinol. Furthermore, the average Green Scale score, which evaluates menopausal symptoms and thus by contrast the overall and psychological well-being of menopausal women, was significantly reduced. As it did not show the steroid-like effects on the hormonal parameters, Klamin could be proposed both as a valid natural remedy for women seeking an alternative to hormonal therapy, as well as as a complementary treatment for many climacteric symptoms.

Isolation of Chemoresistant Cell Subpopulations.

Chemoresistance is a major challenge for cancer therapy and drives tumor relapse. The emergence, within the treated tumor mass, of specific cancer cell subpopulations endowed with high tolerance to the microenvironment stress induced by therapy is being growingly recognized as a mechanism of tumor progression. To obtain detailed information with regard to the pathways underlying survival, expansion, and microenvironmental cross talk of such chemoresistant cell subpopulations may be instrumental for cancer chemoprevention. Additionally, the obtained cell subpopulations may be used for direct screening of cancer chemopreventive compounds, in appropriate experimental settings. Here we report detailed experimental procedures that we and others have setup in order to obtain cell cultures enriched for chemoresistant cells from both malignant pleural mesothelioma specimens and primary cell cultures. We provide indications for the purification and characterization of those chemoresistant cell populations and to generally validate the obtained enriched cell populations for their chemoresistance.

Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin.

In the present work we show that multiple lung cancer cell lines contain cisplatin resistant cell subpopulations expressing the Colony-Stimulating-Factor-Receptor-1 (CSF-1R) and surviving chemotherapy-induced stress. By exploiting siRNA-mediated knock down in vitro and the use of an investigational CSF-1R TKI (JNJ-40346527) in vitro and in vivo, we show that expression and function of the receptor are required for the clonogenicity and chemoresistance of the cell lines. Thus, inhibition of the kinase activity of the receptor reduced the levels of EMT-associated genes, stem cell markers and chemoresistance genes. Additionally, the number of high aldehyde dehydrogenase (ALDH) expressing cells was reduced, consequent to the lack of cisplatin-induced increase of ALDH isoforms. This affected the collective chemoresistance of the treated cultures. Treatment of tumor bearing mice with JNJ-40346527, at pharmacologically relevant doses, produced strong chemo-sensitizing effects in vivo. These anticancer effects correlated with a reduced number of CSF-1Rpos cells, in tumors excised from the treated mice. Depletion of the CD45pos cells within the treated tumors did not, apparently, play a major role in mediating the therapeutic response to the TKI. Thus, lung cancer cells express a functional CSF-1 and CSF-1R duo which mediates pro-tumorigenic effects in vivo and in vitro and can be targeted in a therapeutically relevant way. These observations complement the already known role for the CSF-1R at mediating the pro-tumorigenic properties of tumor-infiltrating immune components.

HMGB1 and Its Hyperacetylated Isoform are Sensitive and Specific Serum Biomarkers to Detect Asbestos Exposure and to Identify Mesothelioma Patients.

PURPOSE: To determine whether serum levels of high mobility group box protein 1 (HMGB1) could differentiate malignant mesothelioma patients, asbestos-exposed individuals, and unexposed controls. EXPERIMENTAL DESIGN: Hyperacetylated and nonacetylated HMGB1 (together referred to as total HMGB1) were blindly measured in blood collected from malignant mesothelioma patients (n = 22), individuals with verified chronic asbestos exposure (n = 20), patients with benign pleural effusions (n = 13) or malignant pleural effusions not due to malignant mesothelioma (n = 25), and healthy controls (n = 20). Blood levels of previously proposed malignant mesothelioma biomarkers fibulin-3, mesothelin, and osteopontin were also measured in nonhealthy individuals. RESULTS: HMGB1 serum levels reliably distinguished malignant mesothelioma patients, asbestos-exposed individuals, and unexposed controls. Total HMGB1 was significantly higher in malignant mesothelioma patients and asbestos-exposed individuals compared with healthy controls. Hyperacetylated HMGB1 was significantly higher in malignant mesothelioma patients compared with asbestos-exposed individuals and healthy controls, and did not vary with tumor stage. At the cut-off value of 2.00 ng/mL, the sensitivity and specificity of serum hyperacetylated HMGB1 in differentiating malignant mesothelioma patients from asbestos-exposed individuals and healthy controls was 100%, outperforming other previously proposed biomarkers. Combining HMGB1 and fibulin-3 provided increased sensitivity and specificity in differentiating malignant mesothelioma patients from patients with cytologically benign or malignant non-mesothelioma pleural effusion. CONCLUSIONS: Our results are significant and clinically relevant as they provide the first biomarker of asbestos exposure and indicate that hyperacetylated HMGB1 is an accurate biomarker to differentiate malignant mesothelioma patients from individuals occupationally exposed to asbestos and unexposed controls. A trial to independently validate these findings will start soon. Clin Cancer Res; 22(12); 3087-96. ©2016 AACR.

A STAT3-NFkB/DDIT3/CEBPß axis modulates ALDH1A3 expression in chemoresistant cell subpopulations.

Here we studied the relevance and modulation of aldehyde dehydrogenase (ALDH) expression in malignant pleural mesothelioma (MPM) chemoresistant cell subpopulations (ALDH(bright) cells), which survive pemetrexed + cisplatin treatment in vitro and in vivo. Expression of the ALDH1A3 isoform was invariably enriched in purified ALDH(bright) cells from multiple MPM cell lines and accounted for the enzymatic activity of those cells. RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH(bright) cells at steady state and, much more, after pemetrexed + cisplatin treatment. We demonstrated, for the first time, that a pSTAT3(tyr705)-NFkB(p65) complex is required for the repression of DDIT3 mRNA and this ensures high levels of CEBPß-dependent ALDH1A3 promoter activity. Inhibition of STAT3-NFkB activity allowed high levels of DDIT3 expression with increased formation of a DDIT3-CEBPß complex. This reduced the occupancy of the ALDH1A3 promoter by CEBPß, thus largely reducing the ALDH1A3 expression. Consequently, survival of ALDH(bright) cells in pemetrexed + cisplatin-treated cultures was impaired, following increased apoptosis. We show that such a mechanism is relevant in vivo and underlies the action of butein, a dual STAT3-NFkB inhibitor capable of abating the chemoresistance of mesothelioma cells in vivo. The possible broad translational relevance of the described mechanism is discussed.

Butein impairs the protumorigenic activity of malignant pleural mesothelioma cells.

Chronic inflammation appears to be a driving force behind cancer progression. NFκB and STAT3 activation plays a pertinent role in this process by mediating chemoresistance and the acquisition of mesenchymal features of protumorigenic cells. Epidemiological data and experimental observations suggest that Malignant Pleural Mesothelioma (MPM) is a prototype of chronic inflammation-driven cancer. Chemoresistance is a major feature of MPM. Thus, this paper explores the effect of butein (3,4,2',4'-tetrahydroxychalcone), a naturally occurring NFκB and STAT3 inhibitor, on the tumorigenic properties of MPM cells. MPM cells harbor high nuclear levels of NFκB and pSTAT3(Y(705)). Butein inhibits pSTAT3(Y(705)) phosphorylation, nuclear localization of NFκB and the physical interaction of NFκB and pSTAT3. This correlates with a downregulation of several genes involved in cancer progression (such as ICAM1, Vimentin, MMP9, Twist) of proangiogenic cytokines (VEGF) and of IL-6 and IL-8, key growth factors for MPM. Hence, butein inhibits the migration of MPM cells and strongly affects the clonogenicity of MPM cells in vitro. Finally, we show that the in vitro actions of butein translate into anticancer effects in vivo. In fact, butein treatment severely affects tumor engraftment and potentiates the anticancer effects of pemetrexed in mouse xenograft models. Butein does not significantly affect the viability of human, untransformed mesothelial cells in vitro, nor does it affect survival of tumor-free mice in vivo. The possibility of using butein as an additional treatment to current MPM therapies is discussed here.

Biomarkers of oxidation, inflammation and cartilage degradation in osteoarthritis patients undergoing sulfur-based spa therapies.

OBJECTIVES: To investigate the effects of sulfur-based spa therapies on oxidation, inflammation and cartilage degradation biomarkers in osteoarthritis (OA) patients. DESIGN AND METHODS: Analyses were performed before therapy (T0), after therapy (T1) and 1 month after its suspension (T2), in OA subjects undergoing mud bath treatments in combination (group A) or not (group B) with hydropinotherapy, and compared with those of patients not subjected to spa therapies (group C). RESULTS: No modifications in plasma/serum biomarker concentrations were observed throughout the study in non-treated patients, while a significant reduction in oxidation, inflammation and cartilage degradation parameters was evidenced in patients of group A. Group B presented a favorable biochemical profile at T1 but not at T2. CONCLUSIONS: To ensure the long term preservation of the chondroprotective effects of sulfur-based therapies, standard mud bath treatments should be associated with hydropinotherapy in order to maintain reduced oxidative, inflammatory and degradative stimuli longer.