Melanoma 2.0. Skin cancer as a paradigm for emerging diagnostic technologies, computational modelling and artificial intelligence.

We live in an unprecedented time in oncology. We have accumulated samples and cases in cohorts larger and more complex than ever before. New technologies are available for quantifying solid or liquid samples at the molecular level. At the same time, we are now equipped with the computational power necessary to handle this enormous amount of quantitative data. Computational models are widely used helping us to substantiate and interpret data. Under the label of systems and precision medicine, we are putting all these developments together to improve and personalize the therapy of cancer. In this review, we use melanoma as a paradigm to present the successful application of these technologies but also to discuss possible future developments in patient care linked to them. Melanoma is a paradigmatic case for disruptive improvements in therapies, with a considerable number of metastatic melanoma patients benefiting from novel therapies. Nevertheless, a large proportion of patients does not respond to therapy or suffers from adverse events. Melanoma is an ideal case study to deploy advanced technologies not only due to the medical need but also to some intrinsic features of melanoma as a disease and the skin as an organ. From the perspective of data acquisition, the skin is the ideal organ due to its accessibility and suitability for many kinds of advanced imaging techniques. We put special emphasis on the necessity of computational strategies to integrate multiple sources of quantitative data describing the tumour at different scales and levels.

Novel integrated workflow allows production and in-depth quality assessment of multifactorial reprogrammed skeletal muscle cells from human stem cells.

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Emerging functional markers for cancer stem cell-based therapies: Understanding signaling networks for targeting metastasis.

Metastasis is one of the most challenging issues in cancer patient management, and effective therapies to specifically target disease progression are missing, emphasizing the urgent need for developing novel anti-metastatic therapeutics. Cancer stem cells (CSCs) gained fast attention as a minor population of highly malignant cells within liquid and solid tumors that are responsible for tumor onset, self-renewal, resistance to radio- and chemotherapies, and evasion of immune surveillance accelerating recurrence and metastasis. Recent progress in the identification of their phenotypic and molecular characteristics and interactions with the tumor microenvironment provides great potential for the development of CSC-based targeted therapies and radical improvement in metastasis prevention and cancer patient prognosis. Here, we report on newly uncovered signaling mechanisms controlling CSC's aggressiveness and treatment resistance, and CSC-specific agents and molecular therapeutics, some of which are currently under investigation in clinical trials, gearing towards decisive functional CSC intrinsic or surface markers. One special research focus rests upon subverted regulatory pathways such as insulin-like growth factor 1 receptor signaling and its interactors in metastasis-initiating cell populations directly related to the gain of stem cell- and EMT-associated properties, as well as key components of the E2F transcription factor network regulating metastatic progression, microenvironmental changes, and chemoresistance. In addition, the study provides insight into systems biology tools to establish complex molecular relationships behind the emergence of aggressive phenotypes from high-throughput data that rely on network-based analysis and their use to investigate immune escape mechanisms or predict clinical outcome-relevant CSC receptor signaling signatures. We further propose that customized vector technologies could drastically enhance systemic drug delivery to target sites, and summarize recent progress and remaining challenges. This review integrates available knowledge on CSC biology, computational modeling approaches, molecular targeting strategies, and delivery techniques to envision future clinical therapies designed to conquer metastasis-initiating cells.

A web platform for the network analysis of high-throughput data in melanoma and its use to investigate mechanisms of resistance to anti-PD1 immunotherapy.

Cellular phenotypes are established and controlled by complex and precisely orchestrated molecular networks. In cancer, mutations and dysregulations of multiple molecular factors perturb the regulation of these networks and lead to malignant transformation. High-throughput technologies are a valuable source of information to establish the complex molecular relationships behind the emergence of malignancy, but full exploitation of this massive amount of data requires bioinformatics tools that rely on network-based analyses. In this report we present the Virtual Melanoma Cell, an online tool developed to facilitate the mining and interpretation of high-throughput data on melanoma by biomedical researches. The platform is based on a comprehensive, manually generated and expert-validated regulatory map composed of signaling pathways important in malignant melanoma. The Virtual Melanoma Cell is a tool designed to accept, visualize and analyze user-generated datasets. It is available at: https://www.vcells.net/melanoma. To illustrate the utilization of the web platform and the regulatory map, we have analyzed a large publicly available dataset accounting for anti-PD1 immunotherapy treatment of malignant melanoma patients.

Epigenetic factor EPC1 is a master regulator of DNA damage response by interacting with E2F1 to silence death and activate metastasis-related gene signatures.

Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Recently, it has been shown that aberrant E2F1 expression often detectable in advanced cancers contributes essentially to cancer cell propagation and characterizes the aggressive potential of a tumor. Conceptually, this requires a subset of malignant cells capable of evading apoptotic death through anticancer drugs. The molecular mechanism by which the pro-apoptotic activity of E2F1 is antagonized is widely unclear. Here we report a novel function for EPC1 (enhancer of polycomb homolog 1) in DNA damage protection. Depletion of EPC1 potentiates E2F1-mediated apoptosis in response to genotoxic treatment and abolishes tumor cell motility. We found that E2F1 directly binds to the EPC1 promoter and EPC1 vice versa physically interacts with bifunctional E2F1 to modulate its transcriptional activity in a target gene-specific manner. Remarkably, nuclear-colocalized EPC1 activates E2F1 to upregulate the expression of anti-apoptotic survival genes such as BCL-2 or Survivin/BIRC5 and inhibits death-inducing targets. The uncovered cooperativity between EPC1 and E2F1 triggers a metastasis-related gene signature in advanced cancers that predicts poor patient survival. These findings unveil a novel oncogenic function of EPC1 for inducing the switch into tumor progression-relevant gene expression that may help to set novel therapies.

MiR-182 promotes cancer invasion by linking RET oncogene activated NF-κB to loss of the HES1/Notch1 regulatory circuit.

BACKGROUND: Dominant-activating mutations in the RET proto-oncogene, a receptor tyrosine kinase, are responsible for the development of medullary thyroid carcinoma (MTC) and causative for multiple endocrine neoplasia (MEN) type 2A and 2B. These tumors are highly aggressive with a high propensity for early metastasis and chemoresistance. This attribute makes this neoplasia an excellent model for probing mechanisms underlying cancer progression. METHODS: The expression level of miR-182 was measured in MTC tumor specimens and in TT cells by real-time RT-PCR. TT cells and modified NThy-ori 3.1 that stably express RETM918T were used to investigate RET-dependent regulation of miR-182. Identification and validation of miR-182 targets and pathways was accomplished with luciferase assays, qRT-PCR, Western blotting and immunofluorescence. In vitro, overexpression and knockdown experiments were carried out to examine the impact of miR-182 and HES1 on invasion and migration. RESULTS: We found that miR-182 expression is significantly upregulated in MTC patient samples and tumor-derived cell lines harboring mutated RET. Inhibition of RET oncogenic signaling through a dominant-negative RET∆TK mutant in TT cells reduces miR-182, whereas overexpression of RETM918T in NThy-ori 3.1 cells increases miR-182 levels. We further show that overexpression of this miRNA in NThy.miR-182 cells promotes the invasive and migratory properties without affecting cell proliferation. MiR-182 is upregulated after RET induced NF-κB translocation into the nucleus via binding of NF-κB to the miR-182 promoter. Database analysis revealed that HES1, a repressor of the Notch pathway, is a target of miR-182, whose upregulation correlates with loss of HES1 transcription in MTC tissue samples and mutant RET cell lines. Moreover, we demonstrated that the 3'UTR of the HES1 mRNA bearing the targeting sequence for miR-182 clearly reduced luciferase reporter activity in cells expressing miR-182. Decreased expression of HES1 promotes migration by upregulating Notch1 inhibitor Deltex1 and consequent repression of Notch1. CONCLUSION: We demonstrate a novel mechanism for MTC aggressiveness in which mutated RET/NF-κB-driven expression of miR-182 impedes HES1 activation in a negative feedback loop. This observation might open new possibilities to treat RET oncogene associated metastatic cancer.

Non-canonical pathway induced by Wnt3a regulates ß-catenin via Pyk2 in differentiating human neural progenitor cells.

Wnt/ß-catenin and Wnt/Ca2+ pathways are involved in cellular processes during embryonic development and the interaction between them in the same cell decides the outcome of cellular functions. In this study, we showed that Wnt3a triggers the Wnt/Ca2+ signaling pathway, indicated by an increase of cytosolic free calcium ([Ca2+]i) and activation of calmodulin dependent kinase II (CaMKII) during the differentiation of human neuronal progenitor cells (hNPCs). Wnt3a via the increase of [Ca2+]i activates proline-rich tyrosine kinase 2 (Pyk2), which subsequently regulates phosphorylation of glycogen synthase kinase 3ß (GSK3ß) and ß-catenin stabilization. Our findings suggest that Pyk2 plays an important role in the coordination of stabilization of ß-catenin in the crosstalk between Wnt/ß-catenin and Wnt/Ca2+ signaling pathways upon Wnt3a stimulation in differentiating hNPCs.

Integrated Loss of miR-1/miR-101/miR-204 Discriminates Metastatic from Nonmetastatic Penile Carcinomas and Can Predict Patient Outcome.

PURPOSE: Penile squamous cell carcinoma is a rare but aggressive cancer. Little is known about pivotal events in tumor pathogenesis and metastasis. Lymph node metastasis is the prevailing prognostic factor while clinical detection in patients remains difficult. Our aim was to identify distinct miRNAs that are differentially expressed in metastatic vs nonmetastatic penile carcinoma, which may serve as diagnostic biomarkers for disease progression. MATERIALS AND METHODS: TaqMan® arrays and quantitative polymerase chain reaction were applied to analyze miRNA profiles in penile squamous cell carcinoma specimens and glans tissue from 24 patients. The prognostic value of deregulated miRNAs was analyzed using the Kaplan-Meier method. The Spearman test was applied to determine a potential linkage between distinctive miRNAs in individual patients. RESULTS: Loss of miR-1 (p = 0.0048), miR-101 (p = 0.0001) and miR-204 (p = 0.0004) in metastasizing tumors and associated metastases (p = 0.0151, 0.0019 and 0.0003, respectively) distinguished patients with metastatic and nonmetastatic penile squamous cell carcinoma. These 3 miRNAs showed a coherent expression pattern. Consistently, patients with low levels of all 3 miRNAs had worse survival (p = 0.03). We identified a coordinately regulated miRNA target hub that is over expressed in penile squamous cell carcinoma and associated with lymphovascular invasion. CONCLUSIONS: Our results provide evidence of a novel multiple miRNA based signature associated with lymph node metastasis and unfavorable prognosis of penile squamous cell carcinoma. The integrated loss of miR-1, miR-101 and miR-204 may predict the formation of metastases in penile cancer at an early stage.

Effect of 10 different polymorphisms on preoperative volumetric characteristics of glioblastoma multiforme.

There is a distinct diversity between the appearance of every glioblastoma multiforme (GBM) on pretreatment magnetic resonance imaging (MRI) with a potential impact on clinical outcome and survival of the patients. The object of this study was to determine the impact of 10 different single nucleotide polymorphisms (SNPs) on various volumetric parameters in patients harboring a GBM. We prospectively analyzed 20 steroid-naïve adult patients who had been treated for newly diagnosed GBM. The volumetry was performed using MRI with the help of a semiautomated quantitative software measuring contrast enhancing tumor volume including necrosis, central necrosis alone and peritumoral edema (PTE). We calculated ratios between the tumor volume and edema (ETR), respectively necrosis (NTR). SNP analysis was done using genomic DNA extracted from peripheral blood genotyped via PCR and sequencing. There was a strong correlation between tumor volume and PTE (p < 0.001), necrosis (p < 0.001) and NTR (p = 0.003). Age and sex had no influence on volumetric data. The Aquaporin 4-31G > A SNP had a significant influence on the ETR (p = 0.042) by decreasing the measured edema compared with the tumor volume. The Interleukin 8-251A > T SNP was significantly correlated with an increased tumor (p = 0.048), PTE (p = 0.033) and necrosis volume (p = 0.028). We found two SNPs with a distinct impact on pretreatment tumor characteristics, presenting a potential explanation for the individual diversity of GBM appearance on MRI and influence on survival.

E2F1 induces miR-224/452 expression to drive EMT through TXNIP downregulation.

Malignant melanoma is highly lethal due to its aggressive invasive properties and metastatic dissemination. The transcription factor E2F1 is crucial for melanoma progression through poorly understood mechanisms. Here, we show that the miR-224/miR-452 cluster is significantly increased in advanced melanoma and invasive/metastatic cell lines that express high levels of E2F1. miR-224/miR-452 expression is directly activated by E2F1 through transactivation of the GABRE gene. Ectopic expression of miR-224/miR-452 in less aggressive cells induces EMT and cytoskeletal rearrangements and enhances migration/invasion. Conversely, miR-224/miR-452 depletion in metastatic cells induces the reversal of EMT, inhibition of motility, loss of the invasive phenotype and an absence of lung metastases in mice. We identify the metastasis suppressor TXNIP as new target of miR-224/miR-452 that induces feedback inhibition of E2F1 and show that miR-224/452-mediated downregulation of TXNIP is essential for E2F1-induced EMT and invasion. The E2F1-miR-224/452-TXNIP axis constitutes a molecular signature that predicts patient survival and may help to set novel therapies.

Hybrid modeling of the crosstalk between signaling and transcriptional networks using ordinary differential equations and multi-valued logic.

A decade of successful results indicates that systems biology is the appropriate approach to investigate the regulation of complex biochemical networks involving transcriptional and post-transcriptional regulations. It becomes mandatory when dealing with highly interconnected biochemical networks, composed of hundreds of compounds, or when networks are enriched in non-linear motifs like feedback and feedforward loops. An emerging dilemma is to conciliate models of massive networks and the adequate description of non-linear dynamics in a suitable modeling framework. Boolean networks are an ideal representation of massive networks that are humble in terms of computational complexity and data demand. However, they are inappropriate when dealing with nested feedback/feedforward loops, structural motifs common in biochemical networks. On the other hand, models of ordinary differential equations (ODEs) cope well with these loops, but they require enormous amounts of quantitative data for a full characterization of the model. Here we propose hybrid models, composed of ODE and logical sub-modules, as a strategy to handle large scale, non-linear biochemical networks that include transcriptional and post-transcriptional regulations. We illustrate the construction of this kind of models using as example a regulatory network centered on E2F1, a transcription factor involved in cancer. The hybrid modeling approach proposed is a good compromise between quantitative/qualitative accuracy and scalability when considering large biochemical networks with a small highly interconnected core, and module of transcriptionally regulated genes that are not part of critical regulatory loops. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology & Clinical Implications. Guest Editor: Yudong Cai.

Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.

Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1-dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluronan-mediated motility receptor RHAMM. E2F1 directly up-regulates RHAMM, which in turn acts as a co-activator of E2F1 to stimulate expression of the extracellular matrix protein fibronectin. Enhanced fibronectin secretion links E2F1/RHAMM transcriptional activity to integrin-ß1-FAK signalling associated with cytoskeletal remodelling and enhanced tumour cell motility. RHAMM depletion abolishes fibronectin expression and cell transmigration across the endothelial layer in E2F1-activated cells. In a xenograft model, knock-down of E2F1 or RHAMM in metastatic cells protects the liver parenchyma of mice against extravasation of CTCs, whereas the number of transmigrated cells increases in response to E2F1 induction. Expression data from clinical tissue samples reveals high E2F1 and RHAMM levels that closely correlate with malignant progression. These findings suggest a requirement for RHAMM in late-stage metastasis by a mechanism involving cooperative stimulation of fibronectin, with a resultant tumourigenic microenvironment important for enhanced extravasation and distant organ colonization. Therefore, stimulation of the E2F1-RHAMM axis in aggressive cancer cells is of high clinical significance. Targeting RHAMM may represent a promising approach to avoid E2F1-mediated metastatic dissemination.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC.

Novel Viral Vectors for Gene Therapy.

Viral vectors are gene transfer tools assembled from the backbones of naturally occurring viruses [...].

Chikungunya virus capsid protein contains nuclear import and export signals.

BACKGROUND: Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. METHODS: EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. RESULTS: Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. CONCLUSIONS: We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.

The E2F1-miRNA cancer progression network.

The transcription factor E2F1 exhibits dual properties, acting as a tumor suppressor and oncogene. Cellular stress such as DNA damage or mitogenic signaling leads to the activation of E2F1 as a mediator of apoptosis in the context of a conserved cellular anti-tumorigenic safeguard mechanism. However in highly aggressive chemoresistant tumors like malignant melanoma and prostate/bladder cancer it switches off this role and acts as promoter of cancer progression. Possible reasons for E2F1 mediated aggressiveness are defects in cell death pathways caused by epigenetic inactivation of important tumor suppressor genes, which often occur in late stage cancer and contribute to chemoresistance. Nevertheless exact mechanisms underlying E2Fs role in invasiveness and metastasis are largely unknown. Different reports hint towards the existence of feedback loops between E2F1 and microRNAs (miRNAs or miRs). MiRs are activated by E2F1 and either the transcription factor itself or cellular genes necessary for the growth regulating function of E2F1 are inhibited by different miRNAs. This mutual regulation possibly influences the balance between E2F1s proapoptotic versus prosurvival function. In the following we will summarize some miRNA-E2F1-interactions contributing to a complex regulatory network.

Senescence determines the fate of activated rat pancreatic stellate cells.

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long-term cultures and by applying chemical triggers, using senescence-associated ß-Galactosidase (SA ß-Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell-mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride-induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long-term culture and exposure of PSC to stressors (doxorubicin, H(2) O(2) and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)-6, but displayed low levels of α-smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA ß-Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual-specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.

Drug Repurposing at the Interface of Melanoma Immunotherapy and Autoimmune Disease.

Cancer cells have a remarkable ability to evade recognition and destruction by the immune system. At the same time, cancer has been associated with chronic inflammation, while certain autoimmune diseases predispose to the development of neoplasia. Although cancer immunotherapy has revolutionized antitumor treatment, immune-related toxicities and adverse events detract from the clinical utility of even the most advanced drugs, especially in patients with both, metastatic cancer and pre-existing autoimmune diseases. Here, the combination of multi-omics, data-driven computational approaches with the application of network concepts enables in-depth analyses of the dynamic links between cancer, autoimmune diseases, and drugs. In this review, we focus on molecular and epigenetic metastasis-related processes within cancer cells and the immune microenvironment. With melanoma as a model, we uncover vulnerabilities for drug development to control cancer progression and immune responses. Thereby, drug repurposing allows taking advantage of existing safety profiles and established pharmacokinetic properties of approved agents. These procedures promise faster access and optimal management for cancer treatment. Together, these approaches provide new disease-based and data-driven opportunities for the prediction and application of targeted and clinically used drugs at the interface of immune-mediated diseases and cancer towards next-generation immunotherapies.

Germline RET sequence variation I852M and occult medullary thyroid cancer: harmless polymorphism or causative mutation?

OBJECTIVE: Rearranged during transfection (RET) gene analysis, widely used to identify carriers at risk of medullary thyroid cancer (MTC), occasionally uncovers novel sequence 'variants of unknown clinical significance' including RET I852M. This study aimed to clarify whether RET I852M represents a harmless polymorphism or a pathogenic mutation. DESIGN: Clinical investigation supported by functional characterization of I852M mutant cells in vitro. PATIENTS AND MEASUREMENTS: Genotype-phenotype correlation including five kindreds from a three-generational Caucasian I852M RET family. RESULTS: A node-negative occult MTC was found in the 64-year-old index patient who had increased basal and stimulated peak calcitonin levels of 190 and 13 307 ng/l, respectively. Her 4-year-old grandson had no histopathological evidence of C-cell disease although his serum calcitonin levels had increased within 5 months from 3·2 to 6·3 ng/l basally and from 17·2 to 24·5 ng/l after pentagastrin stimulation. His mother and two 11- and 1·5-year-old siblings, also carrying the gene, had normal basal and stimulated calcitonin levels and hence did not undergo surgery. Functional characterization of transfected NIH3T3 cells in vitro (cell proliferation rate; cell viability; anchorage-independent cell growth; cell migration; and invasion) indicated that I852M mutant cells have transforming and migratory activities similar to American Thyroid Association (ATA) class A V804M mutants. I852M mutants demonstrated a weaker proliferative potential than fast-proliferating ATA class C C634R mutants and revealed a weaker migratory activity compared with aggressively growing ATA class D A883F mutants. CONCLUSIONS: I852M sequence variations represent genuine RET mutations, falling into ATA class A of weakly activating RET germline mutations.

Functional interplay between E2F1 and chemotherapeutic drugs defines immediate E2F1 target genes crucial for cancer cell death.

The E2F1 transcription factor enhances apoptosis by DNA damage in tumors lacking p53. To elucidate the mechanism of a potential cooperation between E2F1 and chemotherapy, whole-genome microarrays of chemoresistant tumor cell lines were performed focusing on the identification of cooperation response genes (CRG). This gene class is defined by a synergistic expression response upon endogenous E2F1 activation and drug treatment. Cluster analysis revealed an expression pattern of CRGs similar to E2F1 mono-therapy, suggesting that chemotherapeutics enhance E2F1-dependent gene expression at the transcriptional level. Using this approach as a tool to explore E2F1-driven gene expression in response to anticancer drugs, we identified novel apoptosis genes such as the tumor suppressor TIEG1/KLF10 as direct E2F1 targets. We show that TIEG1/KLF10 is transcriptionally activated by E2F1 and crucial for E2F1-mediated chemosensitization of cancer cells. Our results provide a broader picture of E2F1-regulated genes in conjunction with cytotoxic treatment that allows the design of more rational therapeutics.