DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis.

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.

JUN mediates the senescence associated secretory phenotype and immune cell recruitment to prevent prostate cancer progression.

BACKGROUND: Prostate cancer develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although activator protein-1 transcription factors such as JUN have been implicated as potential oncogenic drivers, the molecular programs contributing to prostate cancer progression are not fully understood. METHODS: We analyzed JUN expression in clinical prostate cancer samples across different stages and investigated its functional role in a Pten-deficient mouse model. We performed histopathological examinations, transcriptomic analyses and explored the senescence-associated secretory phenotype in the tumor microenvironment. RESULTS: Elevated JUN levels characterized early-stage prostate cancer and predicted improved survival in human and murine samples. Immune-phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of STAT3 activation and IL-1ß production. Jun depletion in a Pten-deficient background prevented immune cell attraction which was accompanied by significant reduction of active STAT3 and IL-1ß and accelerated prostate tumor growth. Comparative transcriptome profiling of prostate epithelial cells revealed a senescence-associated gene signature, upregulation of pro-inflammatory processes involved in immune cell attraction and of chemokines such as IL-1ß, TNF-α, CCL3 and CCL8 in Pten-deficient prostates. Strikingly, JUN depletion reversed both the senescence-associated secretory phenotype and senescence-associated immune cell infiltration but had no impact on cell cycle arrest. As a result, JUN depletion in Pten-deficient prostates interfered with the senescence-associated immune clearance and accelerated tumor growth. CONCLUSIONS: Our results suggest that JUN acts as tumor-suppressor and decelerates the progression of prostate cancer by transcriptional regulation of senescence- and inflammation-associated genes. This study opens avenues for novel treatment strategies that could impede disease progression and improve patient outcomes.

Comparison of discovery rates and prognostic utility of [68Ga]Ga-PSMA-11 PET/CT and circulating tumor DNA in prostate cancer-a cross-sectional study.

BACKGROUND: Circulating-tumor DNA (ctDNA) and prostate-specific membrane antigen (PSMA) ligand positron-emission tomography (PET) enable minimal-invasive prostate cancer (PCa) detection and survival prognostication. The present study aims to compare their tumor discovery abilities and prognostic values. METHODS: One hundred thirty men with confirmed PCa (70.5 ± 8.0 years) who underwent [68Ga]Ga-PSMA-11 PET/CT (184.8 ± 19.7 MBq) imaging and plasma sample collection (March 2019-August 2021) were included. Plasma-extracted cell-free DNA was subjected to whole-genome-based ctDNA analysis. PSMA-positive tumor lesions were delineated and their quantitative parameters extracted. ctDNA and PSMA PET/CT discovery rates were compared, and the prognostic value for overall survival (OS) was evaluated. RESULTS: PSMA PET discovery rates according to castration status and PSA ranges did differ significantly (P = 0.013, P < 0.001), while ctDNA discovery rates did not (P = 0.311, P = 0.123). ctDNA discovery rates differed between localized and metastatic disease (P = 0.013). Correlations between ctDNA concentrations and PSMA-positive tumor volume (PSMA-TV) were significant in all (r = 0.42, P < 0.001) and castration-resistant (r = 0.65, P < 0.001), however not in hormone-sensitive patients (r = 0.15, P = 0.249). PSMA-TV and ctDNA levels were associated with survival outcomes in the Logrank (P < 0.0001, P < 0.0001) and multivariate Cox regression analysis (P = 0.0023, P < 0.0001). CONCLUSION: These findings suggest that PSMA PET imaging outperforms ctDNA analysis in detecting prostate cancer across the whole spectrum of disease, while both modalities are independently highly prognostic for survival outcomes.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Performance of clinical risk scores and prediction models to identify pathogenic germline variants in patients with advanced prostate cancer.

PURPOSE: Determining the frequency and distribution of pathogenic germline variants (PGVs) in Austrian prostate cancer (PCa) patients and to assess the accuracy of different clinical risk scores to correctly predict PGVs. METHODS: This cross-sectional study included 313 men with advanced PCa. A comprehensive personal and family history was obtained based on predefined questionnaires. Germline DNA sequencing was performed between 2019 and 2021 irrespective of family history, metastatic or castration status or age at diagnosis. Clinical risk scores for hereditary cancer syndromes were evaluated and a PCa-specific score was developed to assess the presence of PGVs. RESULTS: PGV presence was associated with metastasis (p = 0.047) and castration resistance (p = 0.011), but not with personal cancer history or with relatives with any type of cancer. Clinical risk scores (Manchester score, PREMM5 score, Amsterdam II criteria or Johns Hopkins criteria) showed low sensitivities (3.3-20%) for assessing the probability of PGV presence. A score specifically designed for PCa patients stratifying patients into low- or high-risk regarding PGV probability, correctly classified all PGV carriers as high-risk, whereas a third of PCa patients without PGVs was classified as low risk of the presence of PGVs. CONCLUSION: Application of common clinical risk scores based on family history are not suitable to identify PCa patients with high PGV probabilities. A PCa-specific score stratified PCa patients into low- or high-risk of PGV presence with sufficient accuracy, and germline DNA sequencing may be omitted in patients with a low score. Further studies are needed to evaluate the score.

KMT2C methyltransferase domain regulated INK4A expression suppresses prostate cancer metastasis.

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

Thyroid and androgen receptor signaling are antagonized by µ-Crystallin in prostate cancer.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3'-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein µ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRß) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3- and androgen-mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.

Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value.

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes' single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.

Genome amplification and cellular senescence are hallmarks of human placenta development.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) ß-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.

Oncogenic role of miR-155 in anaplastic large cell lymphoma lacking the t(2;5) translocation.

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non-Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin-anaplastic lymphoma tyrosine kinase (NPM-ALK) fusion protein (ALCL ALK(+)). However, little is known about the molecular features and tumour drivers in ALK-negative ALCL (ALCL ALK(-)), which is characterized by a worse prognosis. We found that ALCL ALK(-), in contrast to ALCL ALK(+), lymphomas display high miR-155 expression. Consistent with this, we observed an inverse correlation between miR-155 promoter methylation and miR-155 expression in ALCL. However, no direct effect of the ALK kinase on miR-155 levels was observed. Ago2 immunoprecipitation revealed miR-155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPß and SOCS1. To investigate its function, we over-expressed miR-155 in ALCL ALK(+) cell lines and demonstrated reduced levels of C/EBPß and SOCS1. In murine engraftment models of ALCL ALK(-), we showed that anti-miR-155 mimics are able to reduce tumour growth. This goes hand-in-hand with increased levels of cleaved caspase-3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR-155 induces IL-22 expression and suppresses the C/EBPß target IL-8. These data suggest that miR-155 can act as a tumour driver in ALCL ALK(-) and blocking miR-155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1).

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers.

BACKGROUND: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. METHODS: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. RESULTS: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. CONCLUSIONS: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer.

Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island.

Epigenetic silencing of tumor suppressor genes is generally thought to involve DNA cytosine methylation, covalent modifications of histones, and chromatin compaction. Here, we show that silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells involves distinct changes in nucleosomal occupancy. Three nucleosomes, almost completely absent from the start sites in normal cells, are present on the methylated and silenced promoter, suggesting that epigenetic silencing may be accomplished by the stable placement of nucleosomes into previously vacant positions. Activation of the promoter by demethylation with 5-aza-2'-deoxycytidine involves nucleosome eviction. Epigenetic silencing of tumor suppressor genes may involve heritable changes in nucleosome occupancy enabled by cytosine methylation.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans.

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells.

Expression profiling of T24 cells revealed that 17 out of 313 human miRNAs were upregulated more than 3-fold by simultaneous treatment with the chromatin-modifying drugs 5-aza-2'-deoxycytidine and 4-phenylbutyric acid. One of these, miR-127, is embedded in a CpG island and is highly induced from its own promoter after treatment. miR-127 is usually expressed as part of a miRNA cluster in normal cells but not in cancer cells, suggesting that it is subject to epigenetic silencing. In addition, the proto-oncogene BCL6, a potential target of miR-127, was translationally downregulated after treatment. These results suggest that DNA demethylation and histone deacetylase inhibition can activate expression of miRNAs that may act as tumor suppressors.

A systematic review on the culture methods and applications of 3D tumoroids for cancer research and personalized medicine.

Cancer is a highly heterogeneous disease, and thus treatment responses vary greatly between patients. To improve therapy efficacy and outcome for cancer patients, more representative and patient-specific preclinical models are needed. Organoids and tumoroids are 3D cell culture models that typically retain the genetic and epigenetic characteristics, as well as the morphology, of their tissue of origin. Thus, they can be used to understand the underlying mechanisms of cancer initiation, progression, and metastasis in a more physiological setting. Additionally, co-culture methods of tumoroids and cancer-associated cells can help to understand the interplay between a tumor and its tumor microenvironment. In recent years, tumoroids have already helped to refine treatments and to identify new targets for cancer therapy. Advanced culturing systems such as chip-based fluidic devices and bioprinting methods in combination with tumoroids have been used for high-throughput applications for personalized medicine. Even though organoid and tumoroid models are complex in vitro systems, validation of results in vivo is still the common practice. Here, we describe how both animal- and human-derived tumoroids have helped to identify novel vulnerabilities for cancer treatment in recent years, and how they are currently used for precision medicine.

Examining the Relationship and Prognostic Significance of Cell-Free DNA Levels and the PSMA-Positive Tumor Volume in Men with Prostate Cancer: A Retrospective-Prospective [68Ga]Ga-PSMA-11 PET/CT Study.

Functional imaging with prostate-specific membrane antigen (PSMA) ligands has emerged as the standard imaging method for prostate cancer (PCA). In parallel, the analysis of blood-derived, cell-free DNA (cfDNA) has been shown to be a promising quantitative biomarker of PCA aggressiveness and patient outcome. This study aimed to evaluate the relationship and prognostic value of cfDNA concentrations and the PSMA-positive tumor volume (PSMA-TV) in men with PCA undergoing [68Ga]Ga-PSMA-11 PET/CT imaging. Methods: We recruited 148 men with histologically proven PCA (mean age, 70.7 ± 7.7 y) who underwent [68Ga]Ga-PSMA-11 PET/CT (184.9 ± 18.9 MBq) and blood sampling between March 2019 and August 2021. Among these, 74 (50.0%) had hormone-sensitive PCA and 74 (50.0%) had castration-resistant PCA (CRPC). All patients provided written informed consent before blood sample collection and imaging. The cfDNA was extracted and quantified, and PSMA-expressing tumor lesions were delineated to extract the PSMA-TVs. The Spearman coefficient assessed correlations between PSMA-TV and cfDNA concentrations and cfDNA's relation with clinical parameters. The Kruskal-Wallis test examined the mean cfDNA concentration differences based on PSMA-TV quartiles for significantly correlated patient groups. Log-rank and multivariate Cox regression analyses evaluated the prognostic significance of high and low cfDNA and PSMA-TV levels for overall survival. Results: Weak positive correlations were found between cfDNA concentration and PSMA-TV in the overall group (r = 0.16, P = 0.049) and the CRPC group (r = 0.31, P = 0.007) but not in hormone-sensitive PCA patients (r = -0.024, P = 0.837). In the CRPC cohort, cfDNA concentrations significantly differed between PSMA-TV quartiles 4 and 1 (P = 0.002) and between quartiles 4 and 2 (P = 0.016). Survival outcomes were associated with PSMA-TV (P < 0.0001, P = 0.004) but not cfDNA (P = 0.174, P = 0.12), as per the log-rank and Cox regression analysis. Conclusion: These findings suggest that cfDNA might serve as a biomarker of advanced, aggressive CRPC but does not reliably reflect total tumor burden or prognosis. In comparison, [68Ga]Ga-PSMA-11 PET/CT provides a highly granular and prognostic assessment of tumor burden across the spectrum of PCA disease progression.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer.

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

High STAT5 levels mediate imatinib resistance and indicate disease progression in chronic myeloid leukemia.

In BCR-ABL1(+) leukemia, drug resistance is often associated with up-regulation of BCR-ABL1 or multidrug transporters as well as BCR-ABL1 mutations. Here we show that the expression level of the transcription factor STAT5 is another parameter that determines the sensitivity of BCR-ABL1(+) cells against tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, or dasatinib. Abelson-transformed cells, expressing high levels of STAT5, were found to be significantly less sensitive to TKI-induced apoptosis in vitro and in vivo but not to other cytotoxic drugs, such as hydroxyurea, interferon-ß, or Aca-dC. The STAT5-mediated protection requires tyrosine phosphorylation of STAT5 independent of JAK2 and transcriptional activity. In support of this concept, under imatinib treatment and with disease progression, STAT5 mRNA and protein levels increased in patients with Ph(+) chronic myeloid leukemia. Based on our data, we propose a model in which disease progression in BCR-ABL1(+) leukemia leads to up-regulated STAT5 expression. This may be in part the result of clonal selection of cells with high STAT5 levels. STAT5 then accounts for the resistance against TKIs, thereby explaining the dose escalation frequently required in patients reaching accelerated phase. It also suggests that STAT5 may serve as an attractive target to overcome imatinib resistance in BCR-ABL1(+) leukemia.