Upregulation of BMI1-suppressor miRNAs (miR-200c, miR-203) during terminal differentiation of colon epithelial cells.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties. METHODS: "Bottom-of-the-crypt" (EPCAM+/CD44+/CD66alow) and "top-of-the-crypt" (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in "top-of-the-crypt" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest "top-of-the-crypt" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX). RESULTS: Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in "top-of-the-crypt" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells. CONCLUSION: In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.

High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry.

We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/µL and 0.31 ng/µL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/µL and 2.5 ng/µL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%-7% (R2 > 0.98) and 0.13%-1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.

Mouse Models of Germinal Center Derived B-Cell Lymphomas.

Over the last decades, the revolution in DNA sequencing has changed the way we understand the genetics and biology of B-cell lymphomas by uncovering a large number of recurrently mutated genes, whose aberrant function is likely to play an important role in the initiation and/or maintenance of these cancers. Dissecting how the involved genes contribute to the physiology and pathology of germinal center (GC) B cells -the origin of most B-cell lymphomas- will be key to advance our ability to diagnose and treat these patients. Genetically engineered mouse models (GEMM) that faithfully recapitulate lymphoma-associated genetic alterations offer a valuable platform to investigate the pathogenic roles of candidate oncogenes and tumor suppressors in vivo, and to pre-clinically develop new therapeutic principles in the context of an intact tumor immune microenvironment. In this review, we provide a summary of state-of-the art GEMMs obtained by accurately modelling the most common genetic alterations found in human GC B cell malignancies, with a focus on Burkitt lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma, and we discuss how lessons learned from these models can help guide the design of novel therapeutic approaches for this disease.

PHLDA2 is a key oncogene-induced negative feedback inhibitor of EGFR/ErbB2 signaling via interference with AKT signaling.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is located within the tumor suppressor region of 11p15, and its expression is suppressed in several malignant tumor types. We recently identified PHLDA2 as a robustly induced, novel downstream target of oncogenic EGFR/ErbB2 signaling. In an immunohistochemical study, we find that PHLDA2 protein expression correlates positively with AKT activation in human lung cancers corroborating our data that PHLDA2 is induced upon oncogenic activation and might serve as a biomarker for AKT pathway activation. We show that PHLDA2 overexpression inhibits AKT phosphorylation while decreased PHLDA2 expression increases AKT activity. We further find that PHLDA2 competes with the PH domain of AKT for binding of membrane lipids, thereby directly inhibiting AKT translocation to the cellular membrane and subsequent activation. Indeed, PHLDA2 overexpression suppresses anchorage-independent cell growth and decreased PHLDA2 expression results in increased cell proliferation and reduced sensitivity to targeted agents of EGFR/ErbB2-driven cancers demonstrating functional relevance for this interaction. In summary, our studies demonstrate that PHLDA2 is strongly regulated by EGFR/ErbB2 signaling and inhibits cell proliferation via repressing AKT activation in lung cancers in a negative feedback loop. We highlight a novel action for PHLDA2 as a potential biomarker for AKT pathway activation.

Next-Generation Sequencing of Pulmonary Sarcomatoid Carcinoma Reveals High Frequency of Actionable MET Gene Mutations.

PURPOSE: To further understand the molecular pathogenesis of pulmonary sarcomatoid carcinoma (PSC) and develop new therapeutic strategies in this treatment-refractory disease. MATERIALS AND METHODS: Whole-exome sequencing in a discovery set (n = 10) as well as targeted MET mutation screening in an independent validation set (n = 26) of PSC were performed. Reverse transcriptase polymerase chain reaction and Western blotting were performed to validate MET exon 14 skipping. Functional studies for validation of the oncogenic roles of MET exon 14 skipping were conducted in lung adenosquamous cell line H596 (MET exon 14 skipped and PIK3CA mutated) and gastric adenocarcinoma cell line Hs746T (MET exon 14 skipped). Response to MET inhibitor therapy with crizotinib in a patient with advanced PSC and MET exon 14 skipping was evaluated to assess clinical translatability. RESULTS: In addition to confirming mutations in known cancer-associated genes (TP53, KRAS, PIK3CA, MET, NOTCH, STK11, and RB1), several novel mutations in additional genes, including RASA1, CDH4, CDH7, LAMB4, SCAF1, and LMTK2, were identified and validated. MET mutations leading to exon 14 skipping were identified in eight (22%) of 36 patient cases; one of these tumors also harbored a concurrent PIK3CA mutation. Short interfering RNA silencing of MET and MET inhibition with crizotinib showed marked effects on cell viability and decrease in downstream AKT and mitogen-activated protein kinase activation in Hs746T and H596 cells. Concurrent PIK3CA mutation required addition of a second agent for successful pathway suppression and cell viability effect. Dramatic response to crizotinib was noted in a patient with advanced chemotherapy-refractory PSC carrying a MET exon 14 skipping mutation. CONCLUSION: Mutational events of MET leading to exon 14 skipping are frequent and potentially targetable events in PSC.

Therapeutic effects of an anti-Myc drug on mouse pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is frequently driven by oncogenic KRAS(KRAS*) mutations. We developed a mouse model of KRAS*-induced PDA and, based on genetic results demonstrating that KRAS* tumorigenicity depends on Myc activity, we evaluated the therapeutic potential of an orally administered anti-Myc drug. METHODS: We tested the efficacy of Mycro3, a small-molecule inhibitor of Myc-Max dimerization, in the treatment of mouse PDA (n = 9) and also of xenografts of human pancreatic cancer cell lines (NOD/SCID mice, n = 3-12). Tumor responses to the drug were evaluated by PET/CT imaging, and histological, immunohistochemical, molecular and microarray analyses. The Student's t test was used for differences between groups. All statistical tests were two-sided. RESULTS: Transgenic overexpression of KRAS* in the pancreas resulted in pancreatic intraepithelial neoplasia in two-week old mice, which developed invasive PDA a week later and became moribund at one month. However, this aggressive form of pancreatic tumorigenesis was effectively prevented by genetic ablation of Myc specifically in the pancreas. We then treated moribund, PDA-bearing mice daily with the Mycro3 Myc-inhibitor. The mice survived until killed at two months. PET/CT image analysis (n = 5) demonstrated marked shrinkage of PDA, while immunohistochemical analyses showed an increase in cancer cell apoptosis and reduction in cell proliferation (treated/untreated proliferation index ratio: 0.29, P < .001, n = 3, each group). Tumor growth was also drastically attenuated in Mycro3-treated NOD/SCID mice (n = 12) carrying orthotopic or heterotopic xenografts of human pancreatic cancer cells (eg, mean tumor weight ± SD of treated heterotopic xenografts vs vehicle-treated controls: 15.2±5.8 mg vs 230.2±43.9 mg, P < .001). CONCLUSION: These results provide strong justification for eventual clinical evaluation of anti-Myc drugs as potential chemotherapeutic agents for the treatment of PDA.

AXL kinase as a novel target for cancer therapy.

The AXL receptor tyrosine kinase and its major ligand, GAS6 have been demonstrated to be overexpressed and activated in many human cancers (such as lung, breast, and pancreatic cancer) and have been correlated with poor prognosis, promotion of increased invasiveness/metastasis, the EMT phenotype and drug resistance. Targeting AXL in different model systems with specific small molecule kinase inhibitors or antibodies alone or in combination with other drugs can lead to inactivation of AXL-mediated signaling pathways and can lead to regained drug sensitivity and improved therapeutic efficacy, defining AXL as a promising novel target for cancer therapeutics. This review highlights the data supporting AXL as a novel treatment candidate in a variety of cancers as well as the current status of drug development targeting the AXL/GAS6 axis and future perspectives in this emerging field.

New-onset headache in an elderly man with uremia that improved only after correction of hyperphosphatemia ("uremic headache"): a case report.

INTRODUCTION: New-onset headaches in the elderly are usually secondary and rarely primary. We present the case of an elderly man with recent-onset headache due to uremic hyperphosphatemia and hypocalcemia. To the best of our knowledge, this is the first case report of its kind in the literature. CASE PRESENTATION: We present the case of a 70-year-old Indian man with chronic kidney disease whose new-onset headache improved only when his hyperphosphatemia and hypocalcemia were corrected. He had diffuse, dense calcification of tentorium cerebelli and falx due to hyperphosphatemia. CONCLUSIONS: This case report reinforces the importance of identifying the cause of a new-onset headache, particularly in the elderly, and treating it before blaming a tension headache or primary headache as the cause.

Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells.

The mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34(+) MPN cells than normal CD34(+) HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells.

The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34(+) bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.

Co-treatment with heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG) and vorinostat: a highly active combination against human mantle cell lymphoma (MCL) cells.

Heat shock protein (hsp) 90 inhibitors promote proteasomal degradation of pro-growth and pro-survival hsp90 client proteins, including CDK4, c-RAF and AKT, and induce apoptosis of human lymphoma cells. The pan-histone deacetylase inhibitor vorinostat has also been shown to induce growth arrest and apoptosis of lymphoma cells. Here, we determined the effects of the more soluble, orally bio-available, geldanamycin analogue 17-NN-dimethyl ethylenediamine geldanamycin (DMAG, Kosan Biosciences Inc.) and/or vorinostat in cultured and primary human MCL cells. While vorinostat induced accumulation in the G(1) phase, treatment with DMAG arrested MCL cells in the G(2)/M phase of the cell cycle. Both agents dose-dependently induced apoptosis of MCL cells. Vorinostat also induced hyperacetylation of hsp90 and disrupted the association of hsp90 with its co-chaperones p23 and cdc37, as well as with its client proteins CDK4 and c-RAF. Treatment of MCL cells with vorinostat or 17-DMAG was associated with the inductionof p21 and p27, as well as with depletion of c-Myc, c-RAF, AKT and CDK4. Compared to treatment with either agent alone, co-treatment with DMAG and vorinostat markedly attenuated the levels of cyclin D1 and CDK4, as well as of c-Myc, c-RAF and AKT. Combined treatment with DMAG and vorinostat synergistically induced apoptosis of the cultured MCL cells, as well as induced more apoptosis of primary MCL cells than either agent alone. Therefore, these findings support the rationale to determine the in vivo efficacy of co-treatment with vorinostat and DMAG against human MCL cells.

Panobinostat treatment depletes EZH2 and DNMT1 levels and enhances decitabine mediated de-repression of JunB and loss of survival of human acute leukemia cells.

The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34(+) bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.

Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors.

BACKGROUND: Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown. RESULTS: We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents. CONCLUSIONS: Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT.

Genetic analysis identifies putative tumor suppressor sites at 2q35-q36.1 and 2q36.3-q37.1 involved in cervical cancer progression.

We performed comparative genomic hybridization (CGH) and high-resolution deletion mapping of the long arm of chromosome 2 (2q) in invasive cervical carcinoma (CC). The CGH analyses on 52 CCs identified genetic losses at 2q33-q36, gain of 3q26-q29, and frequent chromosomal amplifications. Characterization of 2q deletions by loss of heterozygosity (LOH) in 60 primary tumors identified two sites of minimal deleted regions at 2q35-q36.1 and 2q36.3-q37.1. To delineate the stage at which these genetic alterations occur in CC progression, we analysed 33 cervical intraepithelial neoplasia (CIN) for LOH. We found that 89% of high-grade (CINII and CINIII) and 40% of low-grade (CINI) CINs exhibited LOH at 2q. To identify the target tumor suppressor gene (TSG), we performed an extensive genetic and epigenetic analyses of a number of candidate genes mapped to the deleted regions. We did not find inactivating mutations in CASP10, BARD1, XRCC5, or PPP1R7 genes mapped to the deleted regions. However, we did find evidence of downregulated gene expression in CFLAR, CASP10 and PPP1R7 in CC cell lines. We also found reactivated gene expression in CC cell lines in vitro after exposure to demethylating and histone deacetylase (HDAC) inhibiting agents. Thus, these data identify frequent chromosomal amplifications in CC, and sites of TSGs at 2q35-q36.1 and 2q36.3-q37.1 that are critical in CC development.

Frequent promoter methylation of CDH1, DAPK, RARB, and HIC1 genes in carcinoma of cervix uteri: its relationship to clinical outcome.

BACKGROUND: Cervical cancer (CC), a leading cause of cancer-related deaths in women worldwide, has been causally linked to genital human papillomavirus (HPV) infection. Although a host of genetic alterations have been identified, molecular basis of CC development is still poorly understood. RESULTS: We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 16 gene promoters in 90 CC cases. We found a high frequency of promoter methylation in CDH1, DAPK, RARB, and HIC1 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed the following: a) overall promoter methylation was higher in more advanced stage of the disease, b) promoter methylation of RARB and BRCA1 predicted worse prognosis, and c) the HIC1 promoter methylation was frequently seen in association with microsatellite instability. Promoter methylation was associated with gene silencing in CC cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression. CONCLUSIONS: These results may have implications in understanding the underlying epigenetic mechanisms in CC development, provide prognostic indicators, and identify important gene targets for treatment.

Characteristic promoter hypermethylation signatures in male germ cell tumors.

BACKGROUND: Human male germ cell tumors (GCTs) arise from undifferentiated primordial germ cells (PGCs), a stage in which extensive methylation reprogramming occurs. GCTs exhibit pluripotentiality and are highly sensitive to cisplatin therapy. The molecular basis of germ cell (GC) transformation, differentiation, and exquisite treatment response is poorly understood. RESULTS: To assess the role and mechanism of promoter hypermethylation, we analyzed CpG islands of 21 gene promoters by methylation-specific PCR in seminomatous (SGCT) and nonseminomatous (NSGCT) GCTs. We found 60% of the NSGCTs demonstrating methylation in one or more gene promoters whereas SGCTs showed a near-absence of methylation, therefore identifying distinct methylation patterns in the two major histologies of GCT. DNA repair genes MGMT, RASSF1A, and BRCA1, and a transcriptional repressor gene HIC1, were frequently methylated in the NSGCTs. The promoter hypermethylation was associated with gene silencing in most methylated genes, and reactivation of gene expression occurred upon treatment with 5-Aza-2' deoxycytidine in GCT cell lines. CONCLUSIONS: Our results, therefore, suggest a potential role for epigenetic modification of critical tumor suppressor genes in pathways relevant to GC transformation, differentiation, and treatment response.

Genomic and expression analysis of the 12p11-p12 amplicon using EST arrays identifies two novel amplified and overexpressed genes.

We performed parallel array comparative genomic hybridization and array expression analysis of the 12p11-p12 amplicon in human testicular seminomas and an ovarian carcinoma cell line using an expressed sequence tags (ESTs) array spotted with 8254 ESTs. The data were normalized using a robust statistical modeling and the significance inferred from the local SD. We identified two ESTs within the chromosomal amplicon that were amplified and overexpressed in > or =75-100 percent of analyzed tumors with the 12p11-p12 amplicon. These sequences, belonging to coding regions of two novel genes designated here as GCT1 and GCT2, were broadly expressed in a panel of human tissues, including testis and ovary. GCT1 and GCT2 were overexpressed in 92 and 71 percent, respectively, of a panel of seminomas tested. Combined array comparative genomic hybridization and array expression analysis is a valid approach for gene discovery in large chromosomal amplicons.