A Lentiviral CXCR4 overexpression and knockdown model in colorectal cancer cell lines reveals plerixafor-dependent suppression of SDF-1α-induced migration and invasion.

BACKGROUND: The development of distant metastasis is associated with poor outcome in patients with colorectal cancer (CRC). The stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) have pivotal roles in the chemotaxis of migrating tumor cells during metastasis. Thus, hampering the SDF-1/CXCR4 cross-talk is a promising strategy to suppress metastasis. METHODS: We investigated the invasive behavior of the lentivirally CXCR4 overexpressing CRC cell lines SW480, SW620 and RKO in chemotaxis and invasion assays toward an SDF-1α gradient. Low endogenous CXCR4 expression levels were determined by quantitative realtime polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS) analyses. RESULTS: A lentiviral CXCR4 overexpression and knockdown model was established in these CRC cells. In transwell migration assays, CXCR4 overexpression favored chemotaxis and invasion of cells in all 3 lines depending on an SDF-1α gradient (p < 0.001 vs. untransduced cells). Functional CXCR4 knockdown using lentiviral short hairpin RNA (shRNA) vectors significantly decreased the migration behavior in CRC cell lines (p < 0.001), confirming a CXCR4-specific effect. Pharmacologic inhibition of the SDF-1α/CXCR4 interaction by the bicyclam Plerixafor(TM) at 100 µM significantly abrogated CXCR4-dependent migration and invasion through Matrigel(TM) (SW480, SW620, RKO; p < 0.05). CONCLUSION: Our results indicate that a CXCR4-antagonistic therapy might prevent tumor cell dissemination and metastasis in CRC patients, consequently improving survival.

The ERG-Regulated LINC00920 Promotes Prostate Cancer Cell Survival via the 14-3-3ε-FOXO Pathway.

Numerous noncoding transcripts have been reported to correlate with cancer development and progression. Nevertheless, there remains a paucity of long noncoding RNAs (lncRNA) with well-elucidated functional roles. Here, we leverage the International Cancer Genome Consortium-Early Onset Prostate Cancer transcriptome and identify the previously uncharacterized lncRNA LINC00920 to be upregulated in prostate tumors. Phenotypic characterization of LINC00920 revealed its positive impact on cellular proliferation, colony formation, and migration. We demonstrate that LINC00920 transcription is directly activated by ERG, an oncogenic transcription factor overexpressed in 50% of prostate cancers. Chromatin isolation by RNA purification-mass spectrometry revealed the interaction of LINC00920 with the 14-3-3ε protein, leading to enhanced sequestration of tumor suppressive FOXO1. Altogether, our results provide a rationale on how ERG overexpression, partly by driving LINC00920 transcription, could confer survival advantage to prostate cancer cells and potentially prime PTEN-intact prostate cells for cellular transformation through FOXO inactivation. IMPLICATIONS: The study describes a novel lncRNA-mediated mechanism of regulating the FOXO signaling pathway and provides additional insight into the role of ERG in prostate cancer cells.

Very High Dehydroepiandrosterone Sulfate (DHEAS) in Serum of an Overweight Female Adolescent Without a Tumor.

Introduction: An increase of serum dehydroepiandrosterone (DHEA) sulfate (DHEAS) is observed in premature adrenarche and congenital adrenal hyperplasia. Very high DHEAS levels are typical for adrenal tumors. Approximately 74% of DHEAS is hydrolyzed to DHEA by the steroid sulfatase (STS). The reverse reaction is DHEA sulfation. Besides these two enzyme reactions, the DHEAS transported through the cell membrane is important for its distribution and excretion. Case Presentation: We present a female adolescent with overweight and a very high DHEAS. The presence of a DHEAS-producing tumor was rejected using ultrasonography, Magnetic Resonance Tomography (MRT), and dexamethasone suppression. STS deficiency was suspected. Sequence analysis revealed a heterozygous nonsense mutation which predicts a truncation of the carboxyl region of the STS that is implicated in substrate binding. No partial gene deletion outside exon 5 was detected by multiplex ligation-dependent probe amplification. The bioassay revealed normal enzyme activity in the patient's leukocytes. A defect of transporter proteins was suggested. Both efflux [multidrug-resistance protein (MRP)2 and breast cancer-resistance protein (BCRP)] and uptake [organic anion-transporting polypeptide (OATP) and organic anion transporter (OAT) carriers] transporters were studied. Sequence analysis of exons revealed a heterozygous Q141K variant for BCRP. Conclusions: A novel heterozygous nonsense mutation in the STS gene and a known heterozygous missense variant in the BCRP gene were found. The heterozygous nonsense mutation in the STS gene is not supposed to be responsible for STS deficiency. The BCRP variant is associated with reduced efflux transport activity only in its homozygous state. The combination of the two heterozygous mutations could possibly explain the observed high levels of DHEAS and other sulfated steroids.

Molecular Evolution of Early-Onset Prostate Cancer Identifies Molecular Risk Markers and Clinical Trajectories.

Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.

Ago-RIP-Seq identifies Polycomb repressive complex I member CBX7 as a major target of miR-375 in prostate cancer progression.

Prostate cancer is a heterogeneous disease. MiR-375 is a marker for prostate cancer progression, but its cellular function is not characterized. Here, we provide the first comprehensive investigation of miR-375 in prostate cancer. We show that miR-375 is enriched in prostate cancer compared to normal cells. Furthermore, miR-375 enhanced proliferation, migration and invasion in vitro and induced tumor growth and reduced survival in vivo showing that miR-375 has oncogenic properties in prostate cancer. On the molecular level, we provide the targetome and genome-wide transcriptional changes of miR-375 expression by applying a generalized linear model for Ago-RIP-Seq and RNA-Seq, and show that miR-375 is involved in tumorigenic networks and Polycomb regulation. Integration of tissue and gene ontology data prioritized miR-375 targets and identified the tumor suppressor gene CBX7, a member of Polycomb repressive complex 1, as a major miR-375 target. MiR-375-mediated repression of CBX7 was accompanied by increased expression of its homolog CBX8 and activated transcriptional programs linked to malignant progression in prostate cancer cells. Tissue analysis showed association of CBX7 loss with advanced prostate cancer. Our study indicates that miR-375 exerts its tumor-promoting role in prostate cancer by influencing the epigenetic regulation of transcriptional programs through its ability to directly target the Polycomb complex member CBX7.

Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation.

BACKGROUND: The in vitro analysis of the hypomethylation of imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). To exclude mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and IGF2 and H19 expression were measured, and a microarray expression analysis was performed. RESULTS: Single-cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from the patients and controls (Cnormo). IGF2 expression was below the detection limit of the quantitative real-time PCR (qRT-PCR) assay, whereas H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in SRShypo and Cnormo supernatants. Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035). The microarray analysis revealed gene expression changes in SRS clones, predicting a decrease in cell proliferation and a delay in mitosis. CONCLUSIONS: The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression. A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.

Novel RNA markers in prostate cancer: functional considerations and clinical translation.

The availability of ultra-high throughput DNA and RNA sequencing technologies in recent years has led to the identification of numerous novel transcripts, whose functions are unknown as yet. Evidence is accumulating that many of these molecules are deregulated in diseases, including prostate cancer, and potentially represent novel targets for diagnosis and therapy. In particular, functional genomic analysis of microRNA (miRNA) and long noncoding RNA (lncRNA) in cancer is likely to contribute insights into tumor development. Here, we compile recent efforts to catalog differential expression of miRNA and lncRNA in prostate cancer and to understand RNA function in tumor progression. We further highlight technologies for molecular characterization of noncoding RNAs and provide an overview of current activities to exploit them for the diagnosis and therapy of this complex tumor.

The disparate twins: a comparative study of CXCR4 and CXCR7 in SDF-1α-induced gene expression, invasion and chemosensitivity of colon cancer.

PURPOSE: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. EXPERIMENTAL DESIGN: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. RESULTS: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. CONCLUSIONS: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor.

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells.

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell "dormancy." Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from "sanctuary" niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda"protective niche" for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.