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Large-cell neuroendocrine lung carcinoma (LCNEC) is a high-grade neoplasm with median survival of 1 year and limited therapeutic options. Here, we report the unusual case of a 47-year-old female smoker with stage IV LCNEC featuring EML4-ALK variant 2 (E20:A20), wild-type TP53/RB1 and low tumor mutational burden of 3.91 mut/Mb. Despite early progression within 3 months under crizotinib, a durable response was achieved with alectinib. Oligoprogression in the left breast 10 months later was treated by surgery, followed by switch to ceritinib upon multifocal progression and detection of ALK:p.V1180L in the mastectomy specimen, but without success. Another rebiopsy revealed ALK:p.L1196M, but the tumor did not respond to brigatinib or carboplatin/pemetrexed, before stabilization under lorlatinib. Diffuse progression 8 months later with detection of ALK:p.L1196M/p.G1202R and p.L1196M/p.D1203N evolving from the previous p.L1196M did not respond to chemoimmunotherapy, and the patient succumbed with an overall survival (OS) of 37 months. This case illustrates the importance of molecular profiling for LCNEC regardless of smoking status, and the superiority of next-generation ALK inhibitors compared to crizotinib for ALK+ cases. Lorlatinib retained efficacy in the heavily pretreated setting, while its upfront use could possibly have prevented the stepwise emergence of compound ALK mutations. Furthermore, the disease course was more aggressive and OS shorter compared to the V2/TP53wt ALK+ lung adenocarcinoma, while crizotinib, ceritinib and brigatinib did not confer the benefit expected according to NGS results, which also underline the need for more potent drugs against ALK in the high-risk setting of neuroendocrine histology.

The advancement of efficient technologies to comply with the needs of systems biology and drug discovery has so far not received adequate attention. A substantial bottleneck for the time-resolved quantitative description of signaling networks is the limited throughput and the inadequate sensitivity of currently established methods. Here, we present an improved protein microarray-based approach towards the sensitive detection of proteins in the fg-range which is based on signal detection in the near-infrared range. The high sensitivity of the assay permits the specific quantification of proteins derived from as little as only 20,000 cells with an error rate of only 5%. The capacity is limited to the analysis of up to 500 different samples per microarray. Protein abundance is determined qualitatively, and quantitatively, if recombinant protein is available. This novel approach was called IPAQ (infrared-based protein arrays with quantitative readout). IPAQ offers a highly sensitive experimental approach superior to the established standard protein quantification technologies, and is suitable for quantitative proteomics. Employing the IPAQ approach, a detailed analysis of activated signaling networks in biopsy samples and of crosstalk between signaling modules as required in drug discovery strategies can easily be performed.

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

Terpyridineplatinum(II) complexes (TPCs) efficiently inhibit the proliferation of glioblastoma cells in vitro and have been tested successfully in a rodent glioblastoma model. Apart from intercalation with DNA, the major mechanism of action of TPCs is a very potent and specific interaction with the human selenoprotein thioredoxin reductase (TrxR). TrxR plays a crucial role in cellular redox homeostasis and protection against oxidative damage. In many malignant cells the thioredoxin system is upregulated, promoting tumor growth and progression. Thus, the thioredoxin system has been proposed to be an attractive target for cancer therapy. This study gives the first comprehensive overview of the effects of TPCs on the transcriptome and proteome of glioblastoma cells. We reveal that under TPC treatment, mechanisms countersteering TrxR inhibition are activated in parallel to DNA-damage-responsive pathways. TPC pressure results in long-term compensatory upregulation of TrxR expression. In parallel, p53 is activated, leading to a range of regulations typical for cell-cycle-arrested cells such as upregulation of CDKN1A, induction of GADD45, inhibition of eIF5A maturation, and reduced phosphorylation of stathmin. We also show that TPCs induce endoplasmic reticulum stress, as they activate the unfolded protein response. This profiling study provides a thorough insight into the spectrum of cellular events resulting from specific TrxR inhibition and characterizes the TPC mode of action.

Protein expression of osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC), RANKL and PTHrP was determined by use of immunohistochemical analysis on tissue arrays (48 cases of PVNS, 20 cases of active (a-RA), non-active rheumatoid arthritis (na-RA), and osteoarthritis (OA)). Additionally, gene expression was analysed using complimentary DNA (cDNA) microarrays. All PVNS cases showed a higher level of both protein and gene expression of RANKL, OPN and BSP in comparison with OA cases. Expression of OPG was not significantly different in PVNS compared to OA. The RANKL/OPG expression ratio was significantly higher in PVNS than in OA. High expressions level of proteins involved in bone degradation in PVNS may promote an intra-osseous propagation of the lesion. This evidence suggests that PVNS might respond to treatment using specific inhibitors of RANKL, OPN and BSP.

At present, most non-small cell lung cancer (NSCLC) treatments are not adapted to the individual response of a patient. The stratification of patients for the most efficient response to conventional chemotherapeutics and targeted therapies will improve established therapy schemes and patient's perspectives. Our project aims at unraveling the influence of specific signaling molecules on the response to common NSCLC drugs. We searched for predictive markers for the response of NSCLC tumors to certain drugs and novel combinations of treatments, which may be useful for therapy decisions towards a more efficient personalized treatment. Expression of 77 proteins described to be involved in EGFR signaling (e.g. MAPK, JAK/STAT, PI3K/AKT pathways) and NSCLC disease in general were quantified in 53 patient derived NSCLC xenograft models using the reverse-phase protein array technology (RPPA). The tumor models are characterized by different response rates upon treatment with different chemotherapeutics and EGFR-targeted therapies. The response rates were obtained directly in the xenograft models. After RPPA analysis we associated the protein expression with the response to established chemotherapeutics (e.g. Vinorelbine, Paclitaxel, Carboplatin) and EGFR-targeted therapies (Cetuximab, Erlotinib). Another goal is to analyze the protein expression changes after treatment in dependence on the response rate. Therefore, protein profiles were raised in NSCLC xenograft models before and after treatment with Cetuximab, a chimeric (mouse/human) monoclonal antibody that inhibits EGFR. The major goal in this experimental subset is to find links between the activities of specific proteins and the response to Cetuximab treatment. A similar experiment with Erlotinib (an EGFR inhibitor) treated samples is ongoing. Statistical analysis indicated significant associations between the expression of distinct proteins and the response rate to certain drugs. Proteins of the ErbB signaling pathway were differentially expressed in Carboplatin responders and non-responders. MEK1 upregulation was observed upon Cetuximab treatment in responders. We revealed an association between higher P-SRC expression and increased Gemcitabine response rates. This result fits to observations that describe coherence between SRC inhibition and reduced suppression of cell growth and survival in the presence of Gemcitabine in cancer cell lines. We are now strengthening our results in various validation experiments. First, we selected seven target proteins based on RPPA analysis. Predictive targets for drug sensitivity are being analyzed to investigate the dependency between target protein activity, downstream signaling and response to distinct drugs in tumor cell lines in a pharmacogenomic approach. Therefore, we knocked down target genes in several NSCLC cell lines and unraveled changes in the drug sensitivity. Microarray and protein analyzes were carried out to study the mechanisms of target-drug association by investigating the downstream signaling upon target knockdown. In summary, our study suggests predictive markers for the response of NSCLC tumors to certain drugs, which may be useful for therapy decisions towards a more efficient personalized treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A185.

Udgivelsesdato: 2009-Sep In vitro systems for monitoring safety of nutritional additives are desirable for high-throughput screenings and as a substitute for animal models. Carrageenan (CGN) is a sulfated polysaccharide widely used as a thickener and texturizer in human nutrition and is intensely discussed regarding its pathophysiological potential. Low molecular weight (lm) variants of CGN are considered to exert more profound pathophysiological effects in vivo than high molecular weight (hm) variants. We used a systematic approach to construct reporter systems allowing distinction between CGN-variants with different pathophysiological potential. Reporter systems utilizing segments of the CGN-responsive DMBT1 promoter did not display substantial activity in SW620 cells of intestinal epithelial origin. Genome-wide profiling revealed stronger qualitative and quantitative changes in global gene activities for hm-CGN than for lm-CGN (824 versus 91 genes; -6.64 to 22.33-fold for hm-CGN versus the range of -2.65 to 2.96-fold for lm-CGN). Reporter systems with segments of the IL-8 promoter showed a specific activation in response to hm-sulfated polysaccharides with lower pathophysiological potential in vivo and provided a better classification of CGN-variants than cytotoxicity assays in vitro. IL-8 reporter systems can be used for discerning between the effects of sulfated polysaccharides in vivo. Our data further provide initial insights into the molecular mechanisms that may play a role in the different effects of CGN-variants.