Effect of cachexia on bone turnover in cancer patients: a case-control study.

BACKGROUND: Increased bone turnover is frequently observed in advanced cancer and predominantly related to bone metastases or therapy. Cachexia represents an important cause of morbidity and mortality in cancer patients. Key features are weight loss, muscle wasting and chronic inflammation, which induce profound metabolic changes in several organs, including the bone. However, whether cachexia contributes to abnormal bone metabolism in cancer patients is unknown. Aim of the present study was to determine the potential correlation of bone turnover markers with body composition and laboratory parameters in treatment-naïve cancer patients. METHODS: In this cross-sectional study we measured the levels of carboxy terminal telopeptide of collagen (CTX), an indicator of bone resorption, as well as osteocalcin (Ocn) and procollagen type I N-terminal propeptide (PINP), indicators of bone formation, in 52 cancer patients and correlated with body composition and laboratory parameters. Univariate and multivariate logistic analysis were performed to identify determinants of negative bone remodeling balance, estimated by CTX/Ocn and CTX/PINP ratio. RESULTS: Based on weight loss, body mass index and muscle mass, patients were divided into a cachectic (59.6%) and a control (40.4%) group. After correcting for the presence of bone metastases, our results showed a significant upregulation of CTX in cachectic patients compared to non-cachectic cancer patients (median 0.38 vs 0.27 ng/mL, p < 0.05), with no difference in Ocn and PINP levels (mean 14 vs. 16 ng/ml, p = 0.2 and median 32 vs. 26 µg/L, p = 0.5, respectively). In addition, the CTX/Ocn and the CTX/PINP ratio were indicative of bone resorption in 68% and 60% of cachexia patients, respectively (vs. 20% and 31% in the control group, p = 0.002 and p = 0.06). The main determinants of the unbalanced bone turnover were hypoalbuminemia for the CTX/Ocn ratio (OR 19.8, p < 0.01) and high CRP for the CTX/PINP ratio (OR 5.3, p < 0.01) in the multivariate regression analysis. CONCLUSIONS: CTX is substantially higher in cachectic patients compared to non-cachectic oncological patients and hypoalbuminemia as well as elevated CRP concentrations are independent predictors of a negative bone remodeling balance in cancer patients. These results strongly indicate that cachexia correlates with exacerbated bone turnover in cancer.

Concentration-Dependent Pro- and Antitumor Activities of Quercetin in Human Melanoma Spheroids: Comparative Analysis of 2D and 3D Cell Culture Models.

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

Promoter Methylation of Selected Genes in Non-Small-Cell Lung Cancer Patients and Cell Lines.

Specific gene promoter DNA methylation is becoming a powerful epigenetic biomarker in cancer diagnostics. Five genes (CDH1, CDKN2Ap16, RASSF1A, TERT, and WT1) were selected based on their frequently published potential as epigenetic markers. Diagnostic promoter methylation assays were generated based on bisulfite-converted DNA pyrosequencing. The methylation patterns of 144 non-small-cell lung cancer (NSCLC) and 7 healthy control formalin-fixed paraffin-embedded (FFPE) samples were analyzed to evaluate the applicability of the putative diagnostic markers. Statistically significant changes in methylation levels are shown for TERT and WT1. Furthermore, 12 NSCLC and two benign lung cell lines were characterized for promoter methylation. The in vitro tests involved a comparison of promoter methylation in 2D and 3D cultures, as well as therapeutic tests investigating the impact of CDH1/CDKN2Ap16/RASSF1A/TERT/WT1 promoter methylation on sensitivity to tyrosine kinase inhibitor (TKI) and DNA methyl-transferase inhibitor (DNMTI) treatments. We conclude that the selected markers have potential and putative impacts as diagnostic or even predictive marker genes, although a closer examination of the resulting protein expression and pathway regulation is needed.

ErbB Family Signalling: A Paradigm for Oncogene Addiction and Personalized Oncology.

ErbB family members represent important biomarkers and drug targets for modern precision therapy. They have gained considerable importance as paradigms for oncoprotein addiction and personalized medicine. This review summarizes the current understanding of ErbB proteins in cell signalling and cancer and describes the molecular rationale of prominent cases of ErbB oncoprotein addiction in different cancer types. In addition, we have highlighted experimental technologies for the development of innovative cancer cell models that accurately predicted clinical ErbB drug efficacies. In the future, such cancer models might facilitate the identification and validation of physiologically relevant novel forms of oncoprotein and non-oncoprotein addiction or synthetic lethality. The identification of genotype-drug response relationships will further advance personalized oncology and improve drug efficacy in the clinic. Finally, we review the most important drugs targeting ErbB family members that are under investigation in clinical trials or that made their way already into clinical routine. Taken together, the functional characterization of ErbB oncoproteins have significantly increased our knowledge on predictive biomarkers, oncoprotein addiction and patient stratification and treatment.

Organotypic three-dimensional cancer cell cultures mirror drug responses in vivo: lessons learned from the inhibition of EGFR signaling.

Complex three-dimensional (3D) in vitro models that recapitulate human tumor biology are essential to understand the pathophysiology of the disease and to aid in the discovery of novel anti-cancer therapies. 3D organotypic cultures exhibit intercellular communication, nutrient and oxygen gradients, and cell polarity that is lacking in two-dimensional (2D) monolayer cultures. In the present study, we demonstrate that 2D and 3D cancer models exhibit different drug sensitivities towards both targeted inhibitors of EGFR signaling and broad acting cytotoxic agents. Changes in the kinase activities of ErbB family members and differential expression of apoptosis- and survival-associated genes before and after drug treatment may account for the differential drug sensitivities. Importantly, EGFR oncoprotein addiction was evident only in the 3D cultures mirroring the effect of EGFR inhibition in the clinic. Furthermore, targeted drug efficacy was strongly increased when incorporating cancer-associated fibroblasts into the 3D cultures. Taken together, we provide conclusive evidence that complex 3D cultures are more predictive of the clinical outcome than their 2D counterparts. In the future, 3D cultures will be instrumental for understanding the mode of action of drugs, identifying genotype-drug response relationships and developing patient-specific and personalized cancer treatments.

The combinatorial approach of laser-captured microdissection and reverse transcription quantitative polymerase chain reaction accurately determines HER2 status in breast cancer.

BACKGROUND: HER2 expression in breast cancer correlates with increased metastatic potential, higher tumor recurrence rates and improved response to targeted therapies. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are two methods commonly used for the analysis of HER2 in the clinic. However, lack of standardization, technical variability in laboratory protocols and subjective interpretation are major problems associated with these testing procedures. METHODS: Here we evaluated the applicability of reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for HER2 testing in breast cancer. We tested thirty formaldehyde-fixed and paraffin-embedded tumor samples by RT-qPCR, FISH and IHC and analysed and compared the data from the three methods. RESULTS: We found that laser-captured microdissection is essential for the accurate determination of HER2 expression by RT-qPCR. When isolating RNA from total tumor tissue we obtained a significant number of false negative results. However, when using RNA from purified cancer cells the RT-qPCR data were fully consistent with FISH and IHC. In addition we provide evidence that ductal carcinomas might be further classified by the differential expression of HER3 and HER4. CONCLUSIONS: Laser-captured microdissection in combination with RT-qPCR is a precise and cost-effective diagnostic approach for HER2 testing in cancer. The PCR assay is simple, accurate and robust and can easily be implemented and standardized in clinical laboratories.

Alpha-catulin contributes to drug-resistance of melanoma by activating NF-κB and AP-1.

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.

A combined impedance and AlphaLISA-based approach to identify anti-inflammatory and barrier-protective compounds in human endothelium.

Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.