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Baseline characteristics of the patients included (PIK3CA cohort) or not (no-PIK3CA cohort) in the PIK3CA analysis.

Purpose:We explored the prognostic effect of PIK3CA mutation in HER2+ patients enrolled in the ShortHER trial.Patients and Methods:The ShortHER trial randomized 1,253 patients with HER2+ breast cancer to 9 weeks or 1 year of adjuvant trastuzumab combined with chemotherapy. PIK3CA hotspot mutations in exon 9 and 20 were analyzed by pyrosequencing. Expression of 60 genes, including PAM50 genes was measured using the nCounter platform.Results:A mutation of the PIK3CA gene was detected in 21.7% of the 803 genotyped tumors. At a median follow-up of 7.7 years, 5-year disease-free survival (DFS) rates were 90.6% for PIK3CA mutated and 86.2% for PIK3CA wild-type tumors [HR, 0.84; 95% confidence interval (CI), 0.56–1.27; P = 0.417]. PIK3CA mutation showed a favorable prognostic impact in the PAM50 HER2-enriched subtype (n = 232): 5-year DFS 91.8% versus 76.1% (log-rank P = 0.049; HR, 0.46; 95% CI, 0.21–1.02). HER2-enriched/PIK3CA mutated versus wild-type tumors showed numerically higher tumor-infiltrating lymphocytes (TIL) and significant upregulation of immune-related genes (including CD8A, CD274, PDCD1, and MYBL2, a proliferation gene involved in immune processes). High TILs as well as the upregulation of PDCD1 and MYBL2 were associated with a significant DFS improvement within the HER2-enriched subtype (HR, 0.82; 95% CI, 0.68–0.99; P = 0.039 for 10% TILs increment; HR, 0.81; 95% CI, 0.65–0.99; P = 0.049 for PDCD1 expression; HR, 0.72; 95% CI, 0.53–0.99; P = 0.042 for MYBL2 expression).Conclusions:PIK3CA mutation showed no prognostic impact in the ShortHER trial. Within the HER2-enriched molecular subtype, patients with PIK3CA mutated tumors showed better DFS versus PIK3CA wild-type, which may be partly explained by upregulation of immune-related genes.

Purpose:The role of immunotherapy in hormone receptor (HR)-positive, HER2-negative breast cancer is underexplored.Patients and Methods:The neoadjuvant phase II GIADA trial (NCT04659551, EUDRACT 2016-004665-10) enrolled stage II–IIIA premenopausal patients with Luminal B (LumB)-like breast cancer (HR-positive/HER2-negative, Ki67 ≥ 20%, and/or histologic grade 3). Patients received: three 21-day cycles of epirubicin/cyclophosphamide followed by eight 14-day cycles of nivolumab, triptorelin started concomitantly to chemotherapy, and exemestane started concomitantly to nivolumab. Primary endpoint was pathologic complete response (pCR; ypT0/is, ypN0).Results:A pCR was achieved by 7/43 patients [16.3%; 95% confidence interval (CI), 7.4–34.9]; the rate of residual cancer burden class 0–I was 25.6%. pCR rate was significantly higher for patients with PAM50 Basal breast cancer (4/8, 50%) as compared with other subtypes (LumA 9.1%; LumB 8.3%; P = 0.017). Tumor-infiltrating lymphocytes (TIL), immune-related gene-expression signatures, and specific immune cell subpopulations by multiplex immunofluorescence were significantly associated with pCR. A combined score of Basal subtype and TILs had an AUC of 0.95 (95% CI, 0.89–1.00) for pCR prediction. According to multiplex immunofluorescence, a switch to a more immune-activated tumor microenvironment occurred following exposure to anthracyclines. Most common grade ≥3 treatment-related adverse events (AE) during nivolumab were γ-glutamyltransferase (16.7%), alanine aminotransferase (16.7%), and aspartate aminotransferase (9.5%) increase. Most common immune-related AEs were endocrinopathies (all grades 1–2; including adrenal insufficiency, n = 1).Conclusions:Luminal B-like breast cancers with a Basal molecular subtype and/or a state of immune activation may respond to sequential anthracyclines and anti–PD-1. Our data generate hypotheses that, if validated, could guide immunotherapy development in this context.

Consort diagram of ShortHER patients for the different analyses described in the present work.

Supplementary Data from Neoadjuvant Chemotherapy and Immunotherapy in Luminal B-like Breast Cancer: Results of the Phase II GIADA Trial

Supplementary Figure Legend from miR-212 Increases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Sensitivity in Non–Small Cell Lung Cancer by Targeting the Antiapoptotic Protein PED

Supplementary Figure 1 from miR-212 Increases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Sensitivity in Non–Small Cell Lung Cancer by Targeting the Antiapoptotic Protein PED

Purpose: The role of immunotherapy in hormone receptor (HR)-positive, HER2-negative breast cancer is underexplored. Patients and Methods: The neoadjuvant phase II GIADA trial (NCT04659551, EUDRACT 2016-004665-10) enrolled stage II–IIIA premenopausal patients with Luminal B (LumB)-like breast cancer (HR-positive/HER2-negative, Ki67 ≥ 20%, and/or histologic grade 3). Patients received: three 21-day cycles of epirubicin/cyclophosphamide followed by eight 14-day cycles of nivolumab, triptorelin started concomitantly to chemotherapy, and exemestane started concomitantly to nivolumab. Primary endpoint was pathologic complete response (pCR; ypT0/is, ypN0). Results: A pCR was achieved by 7/43 patients [16.3%; 95% confidence interval (CI), 7.4–34.9]; the rate of residual cancer burden class 0–I was 25.6%. pCR rate was significantly higher for patients with PAM50 Basal breast cancer (4/8, 50%) as compared with other subtypes (LumA 9.1%; LumB 8.3%; P = 0.017). Tumor-infiltrating lymphocytes (TIL), immune-related gene-expression signatures, and specific immune cell subpopulations by multiplex immunofluorescence were significantly associated with pCR. A combined score of Basal subtype and TILs had an AUC of 0.95 (95% CI, 0.89–1.00) for pCR prediction. According to multiplex immunofluorescence, a switch to a more immune-activated tumor microenvironment occurred following exposure to anthracyclines. Most common grade ≥3 treatment-related adverse events (AE) during nivolumab were γ-glutamyltransferase (16.7%), alanine aminotransferase (16.7%), and aspartate aminotransferase (9.5%) increase. Most common immune-related AEs were endocrinopathies (all grades 1–2; including adrenal insufficiency, n = 1). Conclusions: Luminal B-like breast cancers with a Basal molecular subtype and/or a state of immune activation may respond to sequential anthracyclines and anti–PD-1. Our data generate hypotheses that, if validated, could guide immunotherapy development in this context.

Background: A combined biomarker based on HER2-enriched subtype (HER2-E) and ERBB2 mRNA predicts response and survival in HER2+ breast cancer following trastuzumab +/- lapatinib in the absence of chemotherapy (Prat et al. ASCO 2018). Here, we tested the ability of the combined biomarker to predict pathological complete response (pCR) following neoadjuvant trastuzumab, pertuzumab and endocrine therapy. Methods: RNA from 40 baseline tumor samples from the phase II PerELISA trial were evaluated. PerELISA evaluated the efficacy of a de-escalated, chemotherapy-free neoadjuvant regimen based on dual HER2 blockade with trastuzumab and pertuzumab in combination with letrozole in HER2+/hormone receptor-positive breast cancer selected on the basis of Ki67 response after short course letrozole-alone (Guarneri ASCO 2018). Ki67 response was defined by protocol as relative Ki67 reduction ≥20% from baseline at day 14. Gene-expression was measured using the nCounter platform. Intrinsic subtypes and ERBB2 levels were determined by the PAM50 gene expression predictor. A pre-specified ERBB2 cutoff was determined to define ERBB2-high. Univariate and multivariable logistic regression analyses were performed. Results: The proportion of HER2-E disease within the ERBB2-high and ERBB2-low groups was 46.2% (6/13) and 18.5% (5/27), respectively. The discordance rate at the individual level was 30% (12/40). A total of 6 (15%) and 34 (85%) samples were HER2-E/ERBB2-high and others, respectively. The magnitude of Ki67 reduction of the HER2-E/ERBB2-high and others groups was 64.8% and 63.2%, respectively (p=0.88). The pCR rate of HER2-E/ERBB2-high was 66.7%. The pCR rate of the others group was 14.7%. The univariate odds ratio between HER2-E/ERBB2-high tumors and the others groups was 11.60 (95% CI 1.66-81.10; p=0.014). No other clinical-pathological variable was significantly associated with pCR. Conclusion: The combined HER2-E/ERBB2-high biomarker can identify patients who might be good candidates to receive dual HER2 blockade alone without chemotherapy. Citation Format: Prat A, Griguolo G, Dieci MV, Bisagni G, Frassoldati A, Bianchi GV, Pascual T, Pare L, Galvan P, Urso L, Conte P, Guarneri V. Independent validation of a combined biomarker based on the PAM50 HER2-enriched subtype and ERBB2 mRNA levels following HER2 blockade without chemotherapy in the PerELISA phase II trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-05.

Background In human epidermal growth factor receptor 2 (HER2+) breast cancers, neoadjuvant trials of chemotherapy plus anti-HER2 treatment consistently showed lower pathologic complete response (pCR) rates in hormone receptor (HR) positive versus negative tumors. The PerELISA study was aimed to evaluate the efficacy of a de-escalated, chemotherapy-free neoadjuvant regimen in HR+/HER2+ breast cancer patients selected on the basis of Ki67 inhibition after 2-week letrozole. Patients and methods PerELISA is a phase II, multicentric study for postmenopausal patients with HR+/HER2+ operable breast cancer. Patients received 2-week letrozole, and then underwent re-biopsy for Ki67 evaluation. Patients classified as molecular responders (Ki67 relative reduction >20% from baseline) continued letrozole and started trastuzumab-pertuzumab for five cycles. Patients classified as molecular non-responders started weekly paclitaxel for 13 weeks combined with trastuzumab-pertuzumab. Primary aim was breast and axillary pCR. According to a two-stage Simon’s design, to reject the null hypothesis, at least 8/43 pCR had to be documented. Results Sixty-four patients were enrolled, 44 were classified as molecular responders. All these patients completed the assigned treatment with letrozole-trastuzumab-pertuzumab and underwent surgery. A pCR was observed in 9/44 cases (20.5%, 95% confidence interval 11.1% to 34.5%). Among molecular non-responders, 16/17 completed treatment and underwent surgery, with pCR observed in 81.3% of the cases. PAM50 intrinsic subtype was significantly associated with Ki67 response and pCR. Among molecular responders, the pCR rate was significantly higher in HER2-enriched than in other subtypes (45.5% versus 13.8%, P = 0.042). Conclusions The primary end point of the study was met, by reaching the pre-specified pCRs. In patients selected using Ki67 reduction after short-term letrozole exposure, a meaningful pCR rate can be achieved without chemotherapy. PAM50 intrinsic subtyping further refines our ability to identify a subset of patients for whom chemotherapy might be spared. EUDRACT number 2013-002662-40 ClinicalTrials.gov Identifier NCT02411344

Simple Summary MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression by blocking translation or inducing degradation of specific gene transcripts. The miR-200 family controls the expression of many genes that play important roles in cancer cells. One of the main pathways controlled by these miRNAs, termed epithelial-mesenchymal transition (EMT), is an essential component of the invasive growth program of solid tumors. The miR-200 family has thus been the focus of many studies aimed at discovering strategies to block cancer cell growth and disease progression. In addition, the miR-200 family miRNAs have been investigated as possible circulating cancer biomarkers. Here we provide an overview of factors that influence miR-200 family expression and target genes relevant to tumor development, followed by a summary of their potential utility as noninvasive biomarkers for selected cancers. Abstract The miR-200 family of microRNAs (miRNAs) includes miR-200a, miR-200b, miR-200c, miR-141 and miR-429, five evolutionarily conserved miRNAs that are encoded in two clusters of hairpin precursors located on human chromosome 1 (miR-200b, miR-200a and miR-429) and chromosome 12 (miR-200c and miR-141). The mature -3p products of the precursors are abundantly expressed in epithelial cells, where they contribute to maintaining the epithelial phenotype by repressing expression of factors that favor the process of epithelial-to-mesenchymal transition (EMT), a key hallmark of oncogenic transformation. Extensive studies of the expression and interactions of these miRNAs with cell signaling pathways indicate that they can exert both tumor suppressor- and pro-metastatic functions, and may serve as biomarkers of epithelial cancers. This review provides a summary of the role of miR-200 family members in EMT, factors that regulate their expression, and important targets for miR-200-mediated repression that are involved in EMT. The second part of the review discusses the potential utility of circulating miR-200 family members as diagnostic/prognostic biomarkers for breast, colorectal, lung, ovarian, prostate and bladder cancers.

Background In early-stage HER2-positive breast cancer, escalation or de-escalation of systemic therapy is a controversial topic. As an aid to treatment decisions, we aimed to develop a prognostic assay that integrates multiple data types for predicting survival outcome in patients with newly diagnosed HER2-positive breast cancer. Methods We derived a combined prognostic model using retrospective clinical-pathological data on stromal tumourinfiltrating lymphocytes, PAM50 subtypes, and expression of 55 genes obtained from patients who participated in the Short-HER phase 3 trial. The trial enrolled patients with newly diagnosed, node-positive, HER2-positive breast cancer or, if node negative, with at least one risk factor (ie, tumour size >2 cm, histological grade 3, lymphovascular invasion, Ki67 >20%, age

This abstract was withdrawn by the authors.

// Loredana Urso 1 , Ilaria Cavallari 2, * , Micol Silic-Benussi 2, * , Lorena Biasini 2 , Giulia Zago 3 , Fiorella Calabrese 4 , Pier Franco Conte 1, 3 , Vincenzo Ciminale 1, 2 and Giulia Pasello 3 1 Department of Surgery, Oncology and Gastroenterology, University of Padova, 35128, Padova, Italy 2 Immunology and Molecular Oncology Unit, Veneto Institute of Oncology, IRCCS, 35128, Padova, Italy 3 Medical Oncology Unit 2, Veneto Institute of Oncology, IRCCS, 35128, Padova, Italy 4 Department of Cardio-Thoracic and Vascular Sciences, University of Padova, 35128, Padova, Italy * These authors contributed equally to this work Correspondence to: Vincenzo Ciminale, email: v.ciminale@unipd.it Keywords: mesothelioma, MDM2, p53, RG7112, TRAIL Received: March 10, 2017 Accepted: April 24, 2017 Published: May 11, 2017 ABSTRACT Malignant Pleural Mesothelioma (MPM) is a chemoresistant tumor characterized by low rate of p53 mutation and upregulation of Murine Double Minute 2 (MDM2), suggesting that it may be effectively targeted using MDM2 inhibitors. In the present study, we investigated the anticancer activity of the MDM2 inhibitors Nutlin 3a ( in vitro ) and RG7112 ( in vivo ), as single agents or in combination with rhTRAIL. In vitro studies were performed using MPM cell lines derived from epithelioid (ZL55, M14K), biphasic (MSTO211H) and sarcomatoid (ZL34) MPMs. In vivo studies were conducted on a sarcomatoid MPM mouse model. In all the cell lines tested (with the exception of ZL55, which carries a biallelic loss-of-function mutation of p53), Nutlin 3a enhanced p21, MDM2 and DR5 expression, and decreased survivin expression. These changes were associated to cell cycle arrest but not to a significant induction of apoptosis. A synergistic pro-apoptotic effect was obtained through the association of rhTRAIL in all the cell lines harboring functional p53. This synergistic interaction of MDM2 inhibitor and TRAIL agonist was confirmed using a mouse preclinical model. Our results suggest that the combined targeting of MDM2 and TRAIL might provide a novel therapeutic option for treatment of MPM patients, particularly in the case of sarcomatoid MPM with MDM2 overexpression and functional inactivation of wild-type p53.

Background Clinical-pathologic predictors of acquired T790M epidermal growth factor receptor (EGFR) mutation in Caucasian patients with non–small-cell lung cancer (NSCLC) progressing after first-/second-generation tyrosine kinase inhibitors (TKIs) is an open field for research. Similarly, the best time point for T790M detection by liquid or tissue biopsy after disease progression is currently matter of debate. Patients and Methods This is an observational study at 7 Italian centers enrolling patients with EGFR-mutant NSCLC progressing after first-/second-generation EGFR TKIs, between 2014 and 2018, aiming at comparing baseline clinical-pathologic features and progression patterns in acquired T790M-positive compared with T790M-negative cases. Results A total of 235 patients received first-line treatment with gefitinib (N = 126; 53%), erlotinib (N = 51; 22%), or afatinib (N = 58; 25%). In 120 (51%) cases, T790M was detected in liquid biopsy, tissue biopsy, or both. Age younger than 65 years (P = .037), the presence of common mutations (P = .004), and better response to first-line TKI (P = .023) were correlated with T790M positivity. T790M detection was associated with higher number of new progressing sites (P = .04), liver progression (P = .002), and a lower frequency of lung metastases (P = .027). When serial liquid biopsies were performed (N = 15), an oligoprogressive disease was correlated with a negative test outcome, whereas systemic progression was observed at the time of T790M positivity. Conclusion This study on a Caucasian population showed that age, type of EGFR mutation at diagnosis, response to first-line treatment, and peculiar progression pattern are associated with T790M status. Serial liquid biopsy might be useful for treatment selection, especially when tissue rebiopsy is not feasible.

Purpose: We explored the prognostic effect of PIK3CA mutation in HER2+ patients enrolled in the ShortHER trial. Patients and Methods: The ShortHER trial randomized 1,253 patients with HER2+ breast cancer to 9 weeks or 1 year of adjuvant trastuzumab combined with chemotherapy. PIK3CA hotspot mutations in exon 9 and 20 were analyzed by pyrosequencing. Expression of 60 genes, including PAM50 genes was measured using the nCounter platform. Results: A mutation of the PIK3CA gene was detected in 21.7% of the 803 genotyped tumors. At a median follow-up of 7.7 years, 5-year disease-free survival (DFS) rates were 90.6% for PIK3CA mutated and 86.2% for PIK3CA wild-type tumors [HR, 0.84; 95% confidence interval (CI), 0.56–1.27; P = 0.417]. PIK3CA mutation showed a favorable prognostic impact in the PAM50 HER2-enriched subtype (n = 232): 5-year DFS 91.8% versus 76.1% (log-rank P = 0.049; HR, 0.46; 95% CI, 0.21–1.02). HER2-enriched/PIK3CA mutated versus wild-type tumors showed numerically higher tumor-infiltrating lymphocytes (TIL) and significant upregulation of immune-related genes (including CD8A, CD274, PDCD1, and MYBL2, a proliferation gene involved in immune processes). High TILs as well as the upregulation of PDCD1 and MYBL2 were associated with a significant DFS improvement within the HER2-enriched subtype (HR, 0.82; 95% CI, 0.68–0.99; P = 0.039 for 10% TILs increment; HR, 0.81; 95% CI, 0.65–0.99; P = 0.049 for PDCD1 expression; HR, 0.72; 95% CI, 0.53–0.99; P = 0.042 for MYBL2 expression). Conclusions: PIK3CA mutation showed no prognostic impact in the ShortHER trial. Within the HER2-enriched molecular subtype, patients with PIK3CA mutated tumors showed better DFS versus PIK3CA wild-type, which may be partly explained by upregulation of immune-related genes.

Mesothelin (MSLN) is a cell surface glycoprotein expressed by mesothelial cells of the pleural, peritoneum, and pericardium. Its function in promoting tumor growth and its overexpression in malignant mesothelioma make this protein as a promising target for treatment of affected patients. Several treatment strategies have been explored in latest years. SS1P immunotoxin has the main limitation of early serum neutralizing antibodies production, thus leading to immunotoxin combinations and new-generation immunotoxins currently under investigation. Among mesothelin-targeted antibodies, both monoclonal antibodies such as amatuximab and antibody–drug conjugates such as anetumab–ravtansine are currently under investigation in combination with the standard of care platinum-based doublet plus pemetrexed chemotherapy. Anti-mesothelin vaccines and intrapleural administration of CAR-T meso therapies represent promising future strategies for malignant pleural mesothelioma patients even though these deserve further investigation.

Malignant pleural mesothelioma (MPM) is a rare malignancy of mesothelial cells with increasing incidence, and in many cases, dismal prognosis due to its aggressiveness and lack of effective therapies. Environmental and occupational exposure to asbestos is considered the main aetiological factor for MPM. Inhaled asbestos fibres accumulate in the lungs and induce the generation of reactive oxygen species (ROS) due to the presence of iron associated with the fibrous silicates and to the activation of macrophages and inflammation. Chronic inflammation and a ROS-enriched microenvironment can foster the malignant transformation of mesothelial cells. In addition, MPM cells have a highly glycolytic metabolic profile and are positive in 18F-FDG PET analysis. Loss-of-function mutations of BRCA-associated protein 1 (BAP1) are a major contributor to the metabolic rewiring of MPM cells. A subset of MPM tumours show loss of the methyladenosine phosphorylase (MTAP) locus, resulting in profound alterations in polyamine metabolism, ATP and methionine salvage pathways, as well as changes in epigenetic control of gene expression. This review provides an overview of the perturbations in metabolism and ROS homoeostasis of MPM cells and the role of these alterations in malignant transformation and tumour progression.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p < 0.001), HIF1alpha (p = 0.013), necrosis (p = 0.013) and proliferation index (p < 0.001) were seen mainly in sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features.

Tumor immune microenvironment (TME) plays a key role in malignant pleural mesothelioma (MPM) pathogenesis and treatment outcome, supporting a role of immune checkpoint inhibitors as anticancer approach. This study retrospectively investigated TME and programmed death ligand 1 (PD-L1) expression in naïve MPM cases and their change under chemotherapy.Diagnostic biopsies of MPM patients were collected from four Italian and one Slovenian cancer centers. Pathological assessment of necrosis, inflammation, grading, and mitosis was carried out. Ki-67, PD-L1 expression, and tumor infiltrating lymphocytes were detected by immunohistochemistry. When available, the same paired sample after chemotherapy was analyzed. Pathological features and clinical characteristics were correlated to overall survival.TME and PD-L1 expression were assessed in 93 and 65 chemonaive MPM samples, respectively. Twenty-eight samples have not sufficient tumor tissue for PD-L1 expression. Sarcomatoid/biphasic samples were characterized by higher CD8+ T lymphocytes and PD-L1 expression on tumor cells, while epithelioid showed higher peritumoral CD4+ T and CD20+ B lymphocytes. Higher CD8+ T lymphocytes, CD68+ macrophages, and PD-L1 expression were associated with pathological features of aggressiveness (necrosis, grading, Ki-67). MPM cases characterized by higher CD8+ T-infiltrate showed lower response to chemotherapy and worse survival at univariate analysis. Patients stratification according to a combined score including CD8+ T lymphocytes, necrosis, mitosis, and proliferation index showed median overall survival of 11.3 months compared with 16.4 months in cases with high versus low combined score (P 0.003). Subgroup exploratory analysis of 15 paired samples before and after chemotherapy showed a significant increase in cytotoxic T lymphocytes in MPM samples and PD-L1 expression in immune cells.TME enriched with cytotoxic T lymphocytes is associated with higher levels of macrophages and PD-L1 expression on tumor cells and with aggressive histopathological features, lower response to chemotherapy and shorter survival. The role of chemotherapy as a tumor immunogenicity inducer should be confirmed in a larger validation set.

Background Tumor Associated Macrophages (TAMs) and ARF-MDM2 pathway seem to be interconnected actors in malignant pleural mesothelioma (MPM) pathogenesis, treatment outcome and prognosis. On the basis of such evidence, this prospective study aims at comparing tumor immune microenvironment (TME) and checkpoint expression (PD-L1) in naive tumor samples and immune cells in blood samples of ARF+ vs. ARF- MPM. Methods Tumor samples were collected at the baseline (T0) in all enrolled patients and at the time of surgery (T1) in resectable patients. Immunohistochemistry was carried out to evaluate p14/ARF, PD-L1, CD3+, CD4+, CD8+ T lymphocytes; CD20+ B-lymphocytes; CD68+ macrophages. Blood samples were collected in a subset of patients at T0 and T1 and analyzed for CD3+CD4+, CD3+CD8+, CD4+CD25+FoxP3+ peripheral T lymphocytes, CD68 + (total) and CD163 + (M2-like) peripheral monocytes by flow cytometry analysis. Results Preliminary data on the first 52 chemonaive MPM samples (32 epithelioid, 16 biphasic and 4 sarcomatoid) were evaluated. P14/ARF was detected in 11 patients (21%). P14/ARF+ cases were characterized by higher CD4+ T-lymphocyte percentage (median value 10% vs 1%, p = 0.05), mainly in peritumoral areas, while p14/ARF – cases were characterized by higher CD163+ M2 macrophages percentage (50% vs 40% in p14/ARF+ cases) and higher CD163+/CD68+ ratio (1.3 vs. 1 in p14/ARF+ cases), even though without statistical significance. Paired (T0 and T1) blood samples of five patients were also assessed for CD8+ and CD4+CD25+FoxP3+ T-reg lymphocytes levels change before and after chemotherapy. We observed a T-reg decrease in 2 patients achieving a disease control to first-line platinum-based chemotherapy, while an increase in 3 progressing patients, without major difference between p14/ARF + and p14/ARF- cases. Conclusions p14ARF+ MPM samples seem to be characterized by lower CD163+ M2-polarized macrophages and higher CD4+ T lymphocytes. The amount of T-reg in blood samples may be useful to anticipate radiological and/or clinical progression to chemotherapy. Larger case series and wider TME characterization in tissue and immune cells quantification in blood samples are needed to validate our preliminary findings and will be presented at the conference. Legal entity responsible for the study Istituto Oncologico Veneto IRCCS. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background Tumor infiltrating lymphocytes (TILs) and PD-L1 expression have been previously associated with early stage NSCLC prognosis. Most data refer to intra-tumoral (IT) lymphocytes, while evidence on the prognostic role of the tumor immune microenvironment (TME) as a whole is lacking. Moreover, a combined immuno-score (IS) including more than two markers, is currently not available. We investigated the prognostic impact of PD-L1 expression and different immune cell infiltrates (CD3+CD4+, CD3+CD8+ T-lymphocytes and CD68+ macrophages) at peritumoral (PT) and IT level. Methods Surgical specimens from chemo-naive stage II-III NSCLC patients radically resected between 2015 and 2017 were analyzed. Immunohistochemistry was carried out to evaluate PD-L1 expression (low Results Preliminary data of 79 patients are reported; eligible cases were 51(65%) adenocarcinoma and 28(35%) squamous cell carcinoma, 31(39%) stage II and 48(61%) stage III. Median follow-up was 2.5 years. Higher PD-L1 expression identified cases with worse prognosis (2.5 years OS 58% in high compared with 74% in low expressing tumors), even though without statistical significance. Shorter OS was observed in cases with higher PT CD3+CD8 + (p = 0.015), CD3+CD4 + (p = 0.047) and CD68 + (p = 0.047). These three parameters were put together into a combined IS (2/3 low: low, 2/3 high: high) which confirmed a prognostic stratification of evaluated patients (2.5 years OS 96% vs 63% respectively in low and high IS, p = 0.004), also at multivariate analysis. The prognostic impact of combined IS within each pathologic stage was confirmed. IT T-lymphocytes and macrophages infiltrate did not show a negative prognostic impact; 2.5 years OS of 94% vs 67% (p = 0,033) was observed in highe and lower PT-CD8+/IT-CD8+ ratio, respectively. Conclusions The amount of PT T-lymphocytes and macrophages might allow early-stage NSCLC patients prognostic stratification for adjuvant strategy plan, especially within a combined immuno-score including multiple TME actors. Legal entity responsible for the study Istituto Oncologico Veneto IRCCS. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

544 Background: We investigated the prognostic role of the PAM50 HER2-enriched (HER2-E) subtype in HER2+ early breast cancer enrolled in the randomized Phase III ShortHER trial. Methods: The ShortHER study randomized 1254 HER2+ early breast cancer patients to receive 9 weeks vs 1 year of adjuvant trastuzumab combined with chemotherapy. Gene expression measured using nCounter platform was available for 438 surgical samples. Intrinsic subtyping was determined using the research-based PAM50 predictor. Metastasis-free survival (MFS) was calculated from randomization to distant disease recurrence or death (median follow up 72 months). Uni- and multi-variable analysis were performed using Cox models. Results: PAM50 subtype distribution was: HER2-E 53% (N = 233), Luminal A 20% (N = 87), Luminal B 10% (N = 43), Normal-like 11% (N = 48) and Basal-like 6% (N = 27). HER2-E subtype was associated with hormone receptor-negative status (p < 0.001) and TILs ≥20% (p < 0.001), but not with stage and age ( < or ≥60 yrs). HER2-E subtype was associated with worse MFS vs other PAM50 subtypes overall (HR 2.78, p = 0.001), in the short (HR 2.24, p = 0.046), and in the long arm (HR 4.04, p = 0.011). Multivariable Cox model confirmed the independent prognostic value of HER2-E subtype (Table). HER2-E subtype added significant prognostic value on top of clinicopathological variables (Likelihood ratio test p < 0.001). Conclusions: HER2-E intrinsic subtype is an independent prognostic factor for HER2+ early breast cancer patients treated with adjuvant chemotherapy and trastuzumab. Integration of PAM50 subtype in prognostic algorithms can help refine risk stratification. These findings warrant independent validation. Clinical trial information: NCT00629278. [Table: see text]

e20612 Background: Acquired T790M EGFR mutation (mut) is not predictable by any clinical-pathological feature. The best time point for T790M mut detection by liquid or tissue biopsy is currently undefined. Methods: This is an observational study at 6 Italian Centers enrolling EGFR mutant NSCLC patients (pts) progressing after first/second generation EGFR TKI, between 2014 and 2018. The primary endpoint of the study was to compare clinical features in acquired T790M+ compared with T790M- cases. The secondary endpoint was to assess different progression (PD) patterns between the two groups. We also explored the PD pattern at the time of cfDNA negativity and subsequent positivity, in a subgroup of pts receiving serial liquid biopsies. Statistical analysis was performed by the Chi-square test to correlate clinical features with T790M status, and by the Kaplan-Meier estimator to evaluate median progression free (mPFS) and overall survival (mOS). Multiple logistic regression and log-rank tests were applied. Results: 219 pts were included. Median follow-up since diagnosis was 25 months. First line treatment was gefitinib (N = 119, 54%), erlotinib (N = 48, 22%) or afatinib (N = 52, 24%). In 108 (49%) cases a T790M acquired mut was detected in liquid (70), tissue (31) biopsy or both (7). Age younger than 65 years ( p= 0.05) and presence of sensitizing exon 19 deletion ( p= 0.04) were correlated with T790M mut; this association was confirmed at multivariate analysis ( p= 0.010 and p= 0.006, respectively). At the time of PD, new PD sites ( p= 0.005) and liver PD ( p< 0.001) were more commonly observed in T790M+ group; at multivariate analysis statistical significance was confirmed ( p= 0.01 and p= 0.008, respectively). Longer mOS was observed in T790M+ cases at univariate (53 versus 22 months, p < 0.0001) and multivariate analysis. In 13 pts undergoing serial liquid biopsies, an oligoprogressive disease was correlated with a negative test outcome, while PS/symptoms worsening, higher number of new lesions and PD sites were observed at the time of T790M positivity, although without statistical significance. Conclusions: This is the first caucasian series showing different clinical features and progression patterns of T790M+ versus T790M- EGFR mutant NSCLC.

Type 2 diabetes mellitus patients are at higher cancer risk, probably because of hyperinsulinemia and insulin growth factor 1 pathway activation. The effects of antidiabetic drugs on cancer risk have been described and discussed in several studies suggesting opposite effects of the biguanide metformin and sulfonylureas on cancer incidence and mortality. The anticancer mechanisms of metformin have been clarified, and some clinical studies, particularly in breast cancer patients, have been published or are currently ongoing; however, data about the effects of sulfonylureas on cancer growth are less consistent. The aims of this work are to review preclinical evidence of second-generation sulfonylureas effects on tumor growth, to clarify the potential mechanisms of action, and to identify possible metabolic targets for patient selection. Most evidence is on the adenosine triphosphate-sensitive potassium channels inhibitor glibenclamide, which interacts with reactive oxygen species production thus inducing cancer cell death. Among diarylsulfonylureas, next-generation DW2282 derivatives are particularly promising because of the proapoptotic activity in multidrug-resistant cells.

Background and purpose: We showed previously that a new Pt(II) complex ([Pt(O,O′-acac)(γ-acac)(DMS)]) exerted high and fast apoptotic processes in MCF-7 cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′-acac)(γ-acac)(DMS)] is also able to exert anoikis and alter the migration ability of MCF-7 cells, and to show some of the signalling events leading to these alterations. Experimental approach: Cells were treated with sublethal doses of [Pt(O,O′-acac)(γ-acac)(DMS)], and the efficiency of colony initiation and anchorage-independent growth was assayed; cell migration was examined by in vitro culture wounding assay. Gelatin zymography for MMP-2 and -9 activities, Western blottings of MMPs, MAPKs, Src, PKC-e and FAK, after [Pt(O,O′-acac)(γ-acac)(DMS)] treatment, were also performed. Key results: Sub-cytotoxic drug concentrations decreased the: (i) anchorage-dependent and -independent growth; (ii) migration ability; and (iii) expression and activity of MMP-2 and MMP-9. [Pt(O,O′-acac)(γ-acac)(DMS)] provoked the generation of reactive oxygen species (ROS), and the activation of p38MAPK, Src and PKC-e. p38MAPK phosphorylation, cell anoikis and migration due to [Pt(O,O′-acac)(γ-acac)(DMS)] were blocked by PKC-e inhibition. Furthermore, Src inhibition blocked the [Pt(O,O′-acac)(γ-acac)(DMS)]-provoked activation of PKC-e, while ROS generation blockage inhibited the activation of Src, and also the decrement of phosphorylated FAK observed in detached [Pt(O,O′-acac)(γ-acac)(DMS)]-treated cells. Conclusions and implications: Sublethal concentrations of [Pt(O,O′-acac)(γ-acac)(DMS)] induced anoikis and prevented events leading to metastasis via alterations in cell migration, anchorage independency, stromal interactions and MMP activity. Hence, [Pt(O,O′-acac)(γ-acac)(DMS)] may be a promising therapeutic agent for preventing growth and metastasis of breast cancer.

Background and purpose: We showed previously that cisplatin inititates a signalling pathway mediated by PKC-δ/extracellular signal-regulated kinase (ERK), important for maintaining viability in PC Cl3 thyroid cells. The studies described herein examined whether c-fos was associated with cisplatin resistance and the signalling link between c-fos and PKC-δ/ERK. Experimental approach: Cells were treated with various pharmacological inhibitors of PKCs and ERK, or were depleted of c-fos, PKC-δ, PKC-e and caspase-3 by small interfering RNA (siRNA), then incubated with cisplatin and cytotoxicity assessed. Key results: Cisplatin provokes the induction of c-fos and the activation of conventional PKC-β, and novel PKC-δ and -e. The cisplatin-provoked c-fos induction was decreased by Go6976, a PKC-β inhibitor; by siRNA for PKC-δ- but not that for PKC-e or by PD98059, a mitogen-activated protein kinase/ERK kinase inhibitor. Expression of c-fos was abolished by GF109203X, an inhibitor of all PKC isoforms, or by PD98059 plus Go6976 or by PKC-δ-siRNA plus Go6976. When c-fos expression was blocked by siRNA, cisplatin cytotoxicity was strongly enhanced with increased caspase-3 activation. In PKC-δ-depleted cells treated with cisplatin, caspase-3 activation was increased and cell viability decreased. In these PKC-δ-depleted cells, PD98059 did not affect caspase-3 activation. Conclusions and implications: In PC Cl3 cells, in the cell signalling pathways that lead to cisplatin resistance, PKC-δ controls ERK activity and, together with PKC-β, also the induction of c-fos. Hence, the protective role of c-fos in thyroid cells has the potential to provide new opportunities for therapeutic intervention.

Aims: In this study we investigated the effect of cisplatin on endothelial cell migration, an essential process for vascular remodeling and regeneration in several physiological and pathological situations. Material and Methods: Human umbilical vein endothelial cells (HUVEC) were treated with cisplatin and endothelial cell migration analyzed by fluorescence and scratch-wound migration assay. MMP2 and MMP9 activity were determined by zymographic assay, and MAPK activation by Western blotting analysis. Results: We demonstrated that cisplatin provoked a time- and dose-dependent decrease of HUVEC migration; this effect was clearly independent from its well known cytotoxic activity. In addition, cisplatin markedly reduced MMP2 activity in both conditioned media and cell lysates, increased p38 MAPK and JNK phosphorylation, but did not affect ERK phosphorylation. Endothelial cell migration was attenuated by treatment of cells with GM6001, a non-specific inhibitor of MMPs, or by a selective anti-MMP2 antibody. However, treatment of cells with SB202190 or SP600125, inhibitors of p38 MAPK and JNK respectively, did not affect HUVEC migration. Conclusion: These results suggested that cisplatin induced a reduction of endothelial cell migration through an inhibition of MMP2 activity by downstream signal transduction pathways independent of JNK and p38 MAPK activation.

The tumor suppressor p53 regulates genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis and senescence. p53 is mutated in about 50% of the human cancers, while in tumors with wild-type p53 gene, the protein function may be lost because of overexpression of Murine Double Minute 2 (MDM2). MDM2 targets p53 for ubiquitylation and proteasomal degradation. p53 reactivation through MDM2 inhibitors seems to be a promising strategy to sensitize p53 wild-type cancer cells to apoptosis. Moreover, additional p53-independent molecular functions of MDM2, such as neoangiogenesis promotion, have been suggested. Thus, MDM2 might be a target for anticancer treatment because of its antiapoptotic and proangiogenetic role. Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related tumor where wild-type p53 might be present. The present review gives a complete landscape about the role of MDM2 in cancer pathogenesis, prognosis and treatment, with particular focus on Malignant Pleural Mesothelioma.

The effects of cisplatin ( cis Pt) on the extra cellular signal-regulated kinase (ERK) and the protein kinase B (PKB/Akt), known to play important roles in promoting cell survival and in down regulating apoptosis, were investigated in thyroid cell lines. The cytotoxic effect of cis Pt was highest in normal PC-Cl3 cells, intermediate in dedifferentiated PC-E1A and PC-raf cells and lowest in fully transformed and tumorigenic PC-E1Araf cells. Cis Pt provoked ERK phosphorylation; such phosphorylation was unaltered by Go6976, a conventional PKC inhibitor, whilst blocked by low doses (0.1 μM) or high doses (10 μM) of GF109203X, an inhibitor of all PKC isozymes, in PC-Cl3 and in PC-E1Araf cells, respectively. In PC-E1Araf, but not in PC-Cl3 cells, the cis Pt-provoked ERK phosphorylation was also blocked by a myristoylated PKC-ζ pseudo substrate peptide (PS-ζ). The cytotoxic effects of cis Pt increased when cells were pre-incubated with the mitogen-activated protein kinase (MEK) inhibitor PD98059. Cis Pt provoked the phosphorylation of PKB/Akt and this effect was blocked by LY294002, a PI3K inhibitor. In PC-Cl3 cells pre-incubated with LY294002 the effects of cis Pt on ERK phosphorylation and cell mortality resulted unaffected; conversely, LY294002 reduced the ERK phosphorylation and increased cis Pt cytotoxity of in PC-E1Araf cells. Furthermore, in PC-E1Araf cells pre-incubated with LY294002 and PS-ζ ERK phosphorylation was abolished and cis Pt cytotoxicity was highest. Altogether results highlight a role for PKCs in the upstream regulation of ERK pathway facing the cell response to cis Pt treatments. Understanding the mechanisms by which cells process cis Pt provides important insights for designing more efficient platinum-based drugs.

PED/PEA-15 (PED) is a death effector domain family member of 15 kDa with a broad antiapoptotic function found overexpressed in a number of different human tumors, including lung cancer. To date, the mechanisms that regulate PED expression are unknown. Therefore, we address this point by the identification of microRNAs that in non–small cell lung cancer (NSCLC) modulate PED levels. In this work, we identify miR-212 as a negative regulator of PED expression. We also show that ectopic expression of this miR increases tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced cell death in NSCLC cells. In contrast, inhibition of endogenous miR-212 by use of antago-miR results in increase of PED protein expression and resistance to TRAIL treatment. Besides, in NSCLC, we show both in vitro and in vivo that PED and miR-212 expressions are inversely correlated, that is, PED is upregulated and miR-212 is rarely expressed. In conclusion, these findings suggest that miR-212 should be considered as a tumor suppressor because it negatively regulates the antiapoptotic protein PED and regulates TRAIL sensitivity. Cancer Res; 70(9); 3638–46. ©2010 AACR.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.

Epidermal growth factor receptor (EGFR) signal transduction pathway has been reported to play a vital role in the biologic progression of several tumours and as targets for therapeutic intervention. We have investigated the role of EGFR in the thyroid PC Cl3 cells response to the chemo-therapeutic agent cisplatin. It was found that cisplatin provoked (1) the activation (phosphorylation) and internalization of EGFR, (2) the phosphorylation of mitogen-activated protein kinase (MAPK)/p38, (3) the activation of PKC-epsilon, (4) the enhancement of matrix metalloproteinase-2 (MMP-2) expression and activity, (5) the generation of reactive oxygen species (ROS) and (6) the activation of the apoptotic intrinsic pathway. Inhibition or down regulation of EGFR reduced (1) the phosphorylation of MAPK/p38, (2) the cisplatin-provoked activation of PKC-varepsilon, and (3) the activation of caspase-7 and PARP cleavage and the overall cells sensitivity to cisplatin. PKC-varepsilon inhibition achieved by siRNA blocked MAPK/p38 activation and significantly increased the cell resistance to cisplatin. Finally, when the cisplatin-induced ROS generation was blocked by using NAD(P)H oxidase inhibitors, a decrease in cisplatin-induced MMP-2 enhancement, MAPK/p38 and EGFR activation, and caspase-7 proteolysis occurred. In conclusion, these findings supported a model in which cisplatin provokes an oxidant-induced MMP-2-dependent EGFR transactivation responsible for the induction of cell apoptosis, a process ascribable to the intracellular signalling of PKC-epsilon and MAPK/p38.

Background and purpose: We showed previously that a new Pt complex containing an O,O′-chelated acetylacetonate ligand (acac) and a dimethylsulphide in the Pt coordination sphere, [Pt(O,O′-acac)(γ-acac)(DMS)], induces apoptosis in HeLa cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′-acac)(γ-acac)(DMS)] is also cytotoxic in a MCF-7 breast cancer cell line relatively insensitive to cisplatin, and to gain a more detailed analysis of the cell death pathways. Experimental approach: Cells were treated with Pt compounds and cytotoxicity tests were performed, together with Western blotting of various proteins involved in apoptosis. The mitochondrial membrane potential was assessed by fluorescence microscopy and spectrofluorometry and the Pt bound to cell fractions was measured by atomic absorption spectrometry. Key results: In contrast to cisplatin, the cytotoxicity of [Pt(O,O′-acac)(γ-acac)(DMS)] correlated with cellular accumulation but not with DNA binding. Also, the Pt content in DNA bases was considerably higher for cisplatin than for [Pt(O,O′-acac)(γ-acac)(DMS)], thus excluding DNA as a target of [Pt(O,O′-acac)(γ-acac)(DMS)]. [Pt(O,O′-acac)(γ-acac)(DMS)] exerted high and fast apoptotic processes in MCF-7 cells since it provoked: (a) mitochondria depolarization; (b) cytochrome c accumulation in the cytosol; (c) translocation of Bax and truncated-Bid from cytosol to mitochondria and decreased expression of Bcl-2; (d) cleavage of caspases -7 and -9, and PARP degradation; (e) chromatin condensation and DNA fragmentation. Conclusions and implications: [Pt(O,O′-acac)(γ-acac)(DMS)] is highly cytotoxic for MCF-7 cells, cells relatively resistant to many chemotherapeutic agents, as it activates the mitochondrial apoptotic pathway. Hence, [Pt(O,O′-acac)(γ-acac)(DMS)] has the potential to provide us with new opportunities for therapeutic intervention. British Journal of Pharmacology (2008) 153, 34–49; doi:10.1038/sj.bjp.0707576; published online 19 November 2007

We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC C13 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. (125)1 pre-loaded cells showed an (125)1 efflux accelerated in chloride-containing buffer with respect to chloride-free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists-stimulated cells, whereas it was barely detectable in un-stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8-Br-cAMP and forskolin. Stimulation with insulin ((125)1 mu g/ml: 0-40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I content in 25 1 pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-delta and -epsilon. Inhibition of both PKC-delta and -epsilon. activities by GF 109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-delta by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-epsilon, translocation inhibitor peptide and also by PKC-epsilon clownregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-epsilon, activity. In conclusion, our study demonstrates that, in PC C13 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-epsilon depenclent intracellular pathway.

We report the cytotoxic effects obtained in HeLa cells of three newly synthesized platinum complexes containing both an O,O'-chelated acetylacetonate ligand and a sulfur ligand in the platinum coordination sphere, which show, by (1)H NMR, negligible reactivity with purine bases. These compounds induce cell death with [Pt(O,O'-acac)(gamma-acac)(DMS)] being the most effective (IC(50)=0.98+/-0.056 and 1.82+/-0.023 microM for [Pt(O,O'-acac)(gamma-acac)(DMS)] and cisplatin, respectively). About 50% of cells died after 5h treatment with 100 microM [Pt(O,O'-acac)(gamma-acac)(DMS)] whilst a 16 h incubation was required to get the same results using 100 microM cisplatin. Cellular accumulation measurements, after treatment with equimolar drug concentrations, indicated the major lipophilicity and cellular uptake of the new compounds. While the cytotoxicity of cisplatin was due to both intracellular accumulation and DNA binding, that of [Pt(O,O'-acac)(gamma-acac)(DMS)] was associated with intracellular Pt accumulation only, since it has low reactivity to DNA in intact cells and in vitro. The reaction of the new complexes with guanosine and 5'-GMP was negligible, whereas the L-methionine instantly reacted with the initial Pt complexes. Both cisplatin and [Pt(O,O'-acac)(gamma-acac)(DMS)] induced apoptosis in HeLa cells. [Pt(O,O'-acac)(gamma-acac)(DMS)] provoked the early signs of apoptosis induction (cleavage of PARP and activation of caspases-9, -3 and -7) only 1h after addition of the drug. However, in cisplatin-treated cells, cleavage of PARP was seen after 9h with activation of caspases also proceeding more slowly. In conclusion, these results indicate that the newly synthesized platinum(II) complexes have high and rapid cytotoxic activity in vitro, and suggest that DNA may not be their primary target.

We investigated the effects of cisplatin (cisPt) in normal PC Cl3 and in transformed and tumourigenic PC E1Araf cells. cisPt cytotoxicity was higher in PC Cl3 than in PC E1Araf cells. In both cell lines, cisPt provoked the ERK1/2 phosphorylation; this was unaltered by Go6976, a conventional PKC inhibitor, whilst it was blocked by low doses (0.1 microM) or high doses (10 microM) of GF109203X, an inhibitor of all PKC isozymes, in PC Cl3 and in PC E1Araf cells, respectively. In PC E1Araf, the cisPt-provoked ERK phosphorylation was also blocked by the use of a myristoylated PKC-zeta pseudosubstrate peptide. Conversely, in PC Cl3 the cisPt-provoked ERK phosphorylation was blocked by the use of rottlerin, a PKC-delta inhibitor. Results show that cisPt activates both PKC (the -delta and the -zeta isozymes in PC Cl3 and in PC E1Araf cells, respectively) and ERK in association with prolonged survival of thyroid cell lines.

Introduction: Malignant pleural mesothelioma (MPM) is an aggressive, currently incurable tumor with increasing incidence in industrialized countries. Tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which induces cancer cell death through extrinsic apoptotic pathway, while sparing normal cells. The aim of this study was to investigate the antitumor activity of recombinant human Apo2L/TRAIL (dulanermin) in combination with chemotherapy in MPM in vitro and in vivo. Methods: In the present studies, we employed a panel of MPM cell lines to test the antitumor activity of recombinant human Apo2L/TRAIL (T) in combination with carboplatin and pemetrexed (CP) in vitro and SCID mice. Results: Results demonstrated a significant increase of apoptosis in cell lines treated with CPT compared with those receiving CP or T as single agents. This synergistic effect was dependent on the ability of CP to increase the expression of the TRAIL receptors DR4 and DR5 in a p53 manner. The CPT combination was also effective in blocking the growth of MPM cell lines in a SCID mice preclinical model. Conclusions: CPT increases MPM cell death in vitro and in vivo compared with CP. In vitro results suggest that chemotherapy sensitizes MPM to TRAIL-dependent apoptosis through p53 activation and subsequent upregulation of DRs.