Drug resistance is a major problem in cancer therapy. A growing body of evidence demonstrates that the tumor microenvironment, including cancer-associated fibroblasts (CAFs), can modulate drug sensitivity in tumor cells. We examined the effect of primary human CAFs on p53 induction and cell viability in prostate cancer cells on treatment with chemotherapeutic drugs. Co-culture with prostate CAFs or CAF-conditioned medium attenuated DNA damage and the p53 response to chemotherapeutic drugs and enhanced prostate cancer cell survival. CAF-conditioned medium inhibited the accumulation of doxorubicin, but not taxol, in prostate cancer cells in a manner that was associated with increased cancer cell glutathione levels. A low molecular weight fraction (<3 kDa) of CAF-conditioned medium had the same effect. CAF-conditioned medium also inhibited induction of reactive oxygen species (ROS) in both doxorubicin- and taxol-treated cancer cells. Our findings suggest that CAFs can enhance drug resistance in cancer cells by inhibiting drug accumulation and counteracting drug-induced oxidative stress. This protective mechanism may represent a novel therapeutic target in cancer.

Cancer-associated fibroblasts (CAFs) promote tumor growth and progression, and increase drug resistance through several mechanisms. We have investigated the effect of CAFs on the p53 response to doxorubicin in prostate cancer cells. We show that CAFs produce interleukin-6 (IL-6), and that IL-6 attenuates p53 induction and upregulation of the pro-apoptotic p53 target Bax upon treatment with doxorubicin. This is associated with increased levels of MDM2 mRNA, Mdm2 protein bound to p53, and ubiquitinated p53. IL-6 also inhibited doxorubicin-induced cell death. Inhibition of JAK or STAT3 alleviated this effect, indicating that IL-6 attenuates p53 via the JAK/STAT signaling pathway. These results suggest that CAF-derived IL-6 plays an important role in protecting cancer cells from chemotherapy and that inhibition of IL-6 could have significant therapeutic value.

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.

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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.

The mTOR pathway is in the process of establishing itself as a key access-point of novel oncological drugs and targeted therapies. This is also reflected by the growing number of mTOR pathway genes included in commercially available next-generation sequencing (NGS) oncology panels. This review summarizes the portfolio of medium sized diagnostic, as well as research destined NGS panels and their coverage of the mTOR pathway, including 16 DNA-based panels and the current gene list of Foundation One as a major reference entity. In addition, we give an overview of interesting, mTOR-associated somatic mutations that are not yet incorporated. Especially eukaryotic translation initiation factors (eIFs), a group of mTOR downstream proteins, are on the rise as far as diagnostics and drug targeting in precision medicine are concerned. This review aims to raise awareness for the true coverage of NGS panels, which should be valuable in selecting the ideal platform for diagnostics and research.

The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.

Tumorigenesis as well as the molecular orchestration of cancer progression are very complex mechanisms that comprise numerous elements of influence and regulation. Today, many of the major concepts are well described and a basic understanding of a tumor's fine-tuning is given. Throughout the last decade epigenetics has been featured in cancer research and it is now clear that the underlying mechanisms, especially DNA and histone modifications, are important regulators of carcinogenesis and tumor progression. Another key regulator, which is well known but has been neglected in scientific approaches as well as molecular diagnostics and, consequently, treatment conceptualization for a long time, is the subtle influence patient gender has on molecular processes. Naturally, this is greatly based on hormonal differences, but from an epigenetic point of view, the diverse susceptibility to stress and environmental influences is of prime interest. In this review we present the current view on which and how epigenetic modifications, emphasizing DNA methylation, regulate various tumor diseases. It is our aim to elucidate gender and epigenetics and their interconnectedness, which will contribute to understanding of the prospect molecular orchestration of cancer in individual tumors.