Cancer by Sfakianakis Alexandros: 05/01/17

Δευτέρα 1 Μαΐου 2017

Comprehensive Transcriptome and Mutational Profiling of Endemic Burkitt Lymphoma Reveals EBV Type-Specific Differences

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein–Barr virus (EBV), unlike sporadic Burkitt lymphoma (sBL) that occurs with a lower incidence in developed countries. Given these differences and the variable clinical presentation and outcomes, we sought to further understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from multiple primary eBL tumors compared with sBL tumors. Within eBL tumors, minimal expression differences were found based on: anatomical presentation site, in-hospital survival rates, and EBV genome type, suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex (PSMB9/β1i, PSMB10/β2i, PSMB8/β5i, and PSME2/PA28β) in eBL tumors carrying type 2 EBV compared with type 1 EBV. Second, in comparison with previously published pediatric sBL specimens, the majority of the expression and pathway differences was related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than geographic designation. Third, common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, a set of new genes mutated in BL, including TFAP4, MSH6, PRRC2C, BCL7A, FOXO1, PLCG2, PRKDC, RAD50, and RPRD2, were identified. Overall, these data establish that EBV, particularly EBV type 1, supports BL oncogenesis, alleviating the need for certain driver mutations in the human genome.

Implications: Genomic and mutational analyses of Burkitt lymphoma tumors identify key differences based on viral content and clinical outcomes suggesting new avenues for the development of prognostic molecular biomarkers and therapeutic interventions. Mol Cancer Res; 15(5); 563–76. ©2017 AACR.

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Dysregulated GPCR Signaling and Therapeutic Options in Uveal Melanoma

Uveal melanoma is the most common primary intraocular malignant tumor in adults and arises from the transformation of melanocytes in the uveal tract. Even after treatment of the primary tumor, up to 50% of patients succumb to metastatic disease. The liver is the predominant organ of metastasis. There is an important need to provide effective treatment options for advanced stage uveal melanoma. To provide the preclinical basis for new treatments, it is important to understand the molecular underpinnings of the disease. Recent genomic studies have shown that mutations within components of G protein–coupled receptor (GPCR) signaling are early events associated with approximately 98% of uveal melanomas.

Implications: This review discusses the alterations in GPCR signaling components (GNAQ and GNA11), dysregulated GPCR signaling cascades, and viable targeted therapies with the intent to provide insight into new therapeutic strategies in uveal melanoma. Mol Cancer Res; 15(5); 501–6. ©2017 AACR.

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Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex

In chronic lymphocytic leukemia (CLL), STAT3 is constitutively phosphorylated on serine 727 and plays a role in the pathobiology of CLL. However, what induces constitutive phosphorylation of STAT3 is currently unknown. Mass spectrometry was used to identify casein kinase 2 (CK2), a serine/threonine kinase that coimmunoprecipitated with serine phosphorylated STAT3 (pSTAT3). Furthermore, activated CK2 incubated with recombinant STAT3 induced phosphorylation of STAT3 on serine 727. Although STAT3 and CK2 are present in normal B- and T cells, STAT3 is not constitutively phosphorylated in these cells. Further study found that CD5 and BLNK coexpressed in CLL, but not in normal B- or T cells, are required for STAT3 phosphorylation. To elucidate the relationship of CD5 and BLNK to CK2 and STAT3, STAT3 was immunoprecipitated from CLL cells, and CK2, CD5, and BLNK were detected in the immunoprecipitate. Conversely, STAT3, CD5, and BLNK were in the immunoprecipitate of CLL cells immunoprecipitated with CK2 antibodies. Furthermore, siRNA knockdown of CD5 or BLNK, or treatment with CD5-neutralizing antibodies significantly reduced the levels of serine pSTAT3 in CLL cells. Finally, confocal microscopy determined that CD5 is cell membrane bound, and fractionation studies revealed that the CK2/CD5/BLNK/STAT3 complex remains in the cytoplasm, whereas serine pSTAT3 is shuttled to the nucleus.

Implications: These data show that the cellular proteins CK2, CD5, and BLNK are required for constitutive phosphorylation of STAT3 in CLL. Whether this protein complex phosphorylates other proteins or inhibiting its activity would have clinical benefit in patients has yet to be determined. Mol Cancer Res; 15(5); 610–8. ©2017 AACR.

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Mutant IDH1 Disrupts the Mouse Subventricular Zone and Alters Brain Tumor Progression

IDH1 mutations occur in the majority of low-grade gliomas and lead to the production of the oncometabolite, D-2-hydroxyglutarate (D-2HG). To understand the effects of tumor-associated mutant IDH1 (IDH1-R132H) on both the neural stem cell (NSC) population and brain tumorigenesis, genetically faithful cell lines and mouse model systems were generated. Here, it is reported that mouse NSCs expressing Idh1-R132H displayed reduced proliferation due to p53-mediated cell-cycle arrest as well as a decreased ability to undergo neuronal differentiation. In vivo, Idh1-R132H expression reduced proliferation of cells within the germinal zone of the subventricular zone (SVZ). The NSCs within this area were dispersed and disorganized in mutant animals, suggesting that Idh1-R132H perturbed the NSCs and the microenvironment from which gliomas arise. In addition, tumor-bearing animals expressing mutant Idh1 displayed a prolonged survival and also overexpressed Olig2, features consistent with IDH1-mutated human gliomas. These data indicate that mutant Idh1 disrupts the NSC microenvironment and the candidate cell-of-origin for glioma; thus, altering the progression of tumorigenesis. In addition, this study provides a mutant Idh1 brain tumor model that genetically recapitulates human disease, laying the foundation for future investigations on mutant IDH1-mediated brain tumorigenesis and targeted therapy.

Implications: Through the use of a conditional mutant mouse model that confers a less aggressive tumor phenotype, this study reveals that mutant Idh1 impacts the candidate cell-of-origin for gliomas. Mol Cancer Res; 15(5); 507–20. ©2017 AACR.

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Tumor-induced Stromal STAT1 Accelerates Breast Cancer via Deregulating Tissue Homeostasis

The tumor microenvironment (TME), the dynamic tissue space in which the tumor exists, plays a significant role in tumor initiation, and is a key contributor in cancer progression; however, little is known about tumor-induced changes in the adjacent tissue stroma. Herein, tumor-induced changes in the TME were explored at the morphologic and molecular level to further understand cancer progression. Tumor-adjacent mammary glands (TAG) displayed altered branching morphology, expansion of myofibroblasts, and increased mammosphere formation, broadly suggesting a tumor-induced field effect. FACS analysis of TAGs demonstrated an increased number of Lin^–CD24⁺/CD49⁺ enriched mammary gland stem cells (MaSC), suggesting deregulated tissue homeostasis in TAGs. Comparative transcriptome analysis of TAGs and contralateral control glands coupled with meta-analysis on differentially expressed genes with two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1. Knockdown of STAT1 in cancer-associated fibroblast (CAF) cocultured with human breast cancer cells altered cancer cell proliferation, indicating a role for STAT1 as a stromal contributor of tumorigenesis. Furthermore, depletion of STAT1 in CAFs significantly reduced periductal reactive fibrosis and delayed early breast cancer progression in vivo. Finally, cotreatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Taken together, these results demonstrate that stromal STAT1 expression promotes tumor progression and is a potential therapeutic target for breast cancer.

Implications: Tumors induce stromal STAT1-dependent cytokine secretion that promotes tumor cell proliferation and can be targeted using clinically-approved inhibitors of STAT1. Mol Cancer Res; 15(5); 585–97. ©2017 AACR.

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Exploiting AR-Regulated Drug Transport to Induce Sensitivity to the Survivin Inhibitor YM155

Androgen receptor (AR) signaling is fundamental to prostate cancer and is the dominant therapeutic target in metastatic disease. However, stringent androgen deprivation therapy regimens decrease quality of life and have been largely unsuccessful in curtailing mortality. Recent clinical and preclinical studies have taken advantage of the dichotomous ability of AR signaling to elicit growth-suppressive and differentiating effects by administering hyperphysiologic levels of testosterone. In this study, high-throughput drug screening identified a potent synergy between high-androgen therapy and YM155, a transcriptional inhibitor of survivin (BIRC5). This interaction was mediated by the direct transcriptional upregulation of the YM155 transporter SLC35F2 by the AR. Androgen-mediated YM155-induced cell death was completely blocked by the overexpression of multidrug resistance transporter ABCB1. SLC35F2 expression was significantly correlated with intratumor androgen levels in four distinct patient-derived xenograft models, and with AR activity score in a large gene expression dataset of castration-resistant metastases. A subset of tumors had significantly elevated SLC35F2 expression and, therefore, may identify patients who are highly responsive to YM155 treatment.

Implications: The combination of androgen therapy with YM155 represents a novel drug synergy, and SLC35F2 may serve as a clinical biomarker of response to YM155. Mol Cancer Res; 15(5); 521–31. ©2017 AACR.

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Highlights of This Issue

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Prognostic Relevance of Tumor Purity and Interaction with MGMT Methylation in Glioblastoma

Promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is a critical biomarker in glioblastoma (GBM), as treatment decisions and clinical trial inclusion rely on its accurate assessment. However, interpretation of results is complicated by poor interassay reproducibility as well as a weak correlation between methylation status and expression levels of MGMT. This study systematically investigates the influence of tumor purity on tissue subjected to MGMT analysis. A quantitative, allele-specific real-time PCR (qAS-PCR) assay was developed to determine genotype and mutant allele frequency of telomerase promoter (pTERT) mutations as a direct measure of tumor purity. We studied tumor purity, pTERT mutation by Sanger sequencing, MGMT methylation by pyrosequencing, IDH1 mutation status, and clinical parameters in a cohort of high-grade gliomas (n = 97). The qAS-PCR reliably predicted pTERT genotype and tumor purity compared with independent methods. Tumor purity positively and significantly correlated with the extent of methylation in MGMT methylated GBMs. Extent of MGMT methylation differed significantly with respect to pTERT mutation hotspot (C228T vs. C250T). Interestingly, frontal lobe tumors showed greater tumor purity than those in other locations. Above all, tumor purity was identified as an independent prognostic factor in GBM. In conclusion, we determined mutual associations of tumor purity with MGMT methylation and pTERT mutations and found that the extent of MGMT methylation reflects tumor purity. In turn, tumor purity is prognostic in IDH1 wild-type GBM.

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Novel Assay to Detect RNA Polymerase I Activity In Vivo

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.

Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577–84. ©2017 AACR.

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Distinctive Histogenesis and Immunological Microenvironment Based on Transcriptional Profiles of Follicular Dendritic Cell Sarcomas

Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumors with variable clinical, morphologic, and phenotypic characteristics. Transcriptome analysis was performed on multiple FDC sarcomas and compared with other mesenchymal tumors, microdissected Castleman FDCs, and normal fibroblasts. Using unsupervised analysis, FDC sarcomas clustered with microdissected FDCs, distinct from other mesenchymal tumors and fibroblasts. The specific endowment of FDC-related gene expression programs in FDC sarcomas emerged by applying a gene signature of differentially expressed genes (n = 1,289) between microdissected FDCs and fibroblasts. Supervised analysis comparing FDC sarcomas with microdissected FDCs and other mesenchymal tumors identified 370 and 2,927 differentially expressed transcripts, respectively, and on the basis of pathway enrichment analysis ascribed to signal transduction, chromatin organization, and extracellular matrix organization programs. As the transcriptome of FDC sarcomas retained similarity with FDCs, the immune landscape of FDC sarcoma was investigated by applying the CIBERSORT algorithm to FDC sarcomas and non-FDC mesenchymal tumors and demonstrated that FDC sarcomas were enriched in T follicular helper (T_FH) and T regulatory (T_REG) cell populations, as confirmed in situ by immunohistochemistry. The enrichment in specific T-cell subsets prompted investigating the mRNA expression of the inhibitory immune receptor PD-1 and its ligands PD-L1 and PD-L2, which were found to be significantly upregulated in FDC sarcomas as compared with other mesenchymal tumors, a finding also confirmed in situ. Here, it is demonstrated for the first time the transcriptional relationship of FDC sarcomas with nonmalignant FDCs and their distinction from other mesenchymal tumors.

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Combined TRAF6 Targeting and Proteasome Blockade Has Anti-myeloma and Anti-Bone Resorptive Effects

TNF receptor–associated factor 6 (TRAF6) has been implicated in polyubiquitin-mediated IL1R/TLR signaling through activation of IB kinase (IKK) to regulate the NF-B and JNK signaling pathways. Here, TRAF6 protein was determined to be overexpressed in bone marrow mononuclear cells (BMMC) from patients with multiple myeloma. TRAF6 expression in BMMCs from patients with progressive disease is significantly elevated as compared with individuals in complete remission, with monoclonal gammopathy of undetermined significance, or healthy subjects. Furthermore, TRAF6 dominant–negative (TRAF6dn) peptides were constructed which specifically reduced TRAF6 signaling and activation of IKK. TRAF6 not only reduced cellular growth but also increased the apoptosis of multiple myeloma tumor cells in a concentration-dependent fashion. Because TRAF6 activates IKK through polyubiquitination, independent of its proteasome activity, a TRAF6dn peptide was combined with the proteasome inhibitors bortezomib or carfilzomib to treat multiple myeloma. Importantly, targeting of TRAF6 in the presence of proteasome inhibition enhanced anti–multiple myeloma effects and also decreased TLR/TRAF6/NF-B–related signaling. Finally, TRAF6dn dose dependently inhibited osteoclast cell formation from CD14⁺ monocytes, induced with RANKL and mCSF, and markedly reduced bone resorption in dentin pits. In all, these data demonstrate that blocking TRAF6 signaling has anti–multiple myeloma effects and reduces bone loss.

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Genome-Wide Analysis Identifies MEN1 and MAX Mutations and a Neuroendocrine-Like Molecular Heterogeneity in Quadruple WT GIST

Quadruple wild-type (WT) gastrointestinal stromal tumor (GIST) is a genomic subgroup lacking KIT/PDGFRA/RAS pathway mutations, with an intact succinate dehydrogenase (SDH) complex. The aim of this work is to perform a wide comprehensive genomic study on quadruple WT GIST to improve the characterization of these patients. We selected 14 clinical cases of quadruple WT GIST, of which nine cases showed sufficient DNA quality for whole exome sequencing (WES). NF1 alterations were identified directly by WES. Gene expression from whole transcriptome sequencing (WTS) and miRNA profiling were performed using fresh-frozen, quadruple WT GIST tissue specimens and compared with SDH and KIT/PDGFRA-mutant GIST. WES identified an average of 18 somatic mutations per sample. The most relevant somatic oncogenic mutations identified were in TP53, MEN1, MAX, FGF1R, CHD4, and CTDNN2. No somatic alterations in NF1 were identified in the analyzed cohort. A total of 247 mRNA transcripts and 66 miRNAs were differentially expressed specifically in quadruple WT GIST. Overexpression of specific molecular markers (COL22A1 and CALCRL) and genes involved in neural and neuroendocrine lineage (ASCL1, Family B GPCRs) were detected and further supported by predicted miRNA target analysis. Quadruple WT GIST show a specific genetic signature that deviates significantly from that of KIT/PDGFRA-mutant and SDH-mutant GIST. Mutations in MEN1 and MAX genes, a neural-committed phenotype and upregulation of the master neuroendocrine regulator ASCL1, support a genetic similarity with neuroendocrine tumors, with whom they also share the great variability in oncogenic driver genes.

Implications: This study provides novel insights into the biology of quadruple WT GIST that potentially resembles neuroendocrine tumors and should promote the development of specific therapeutic approaches. Mol Cancer Res; 15(5); 553–62. ©2017 AACR.

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A Transcriptional Program for Detecting TGF{beta}-Induced EMT in Cancer

Most cancer deaths are due to metastasis, and epithelial-to-mesenchymal transition (EMT) plays a central role in driving cancer cell metastasis. EMT is induced by different stimuli, leading to different signaling patterns and therapeutic responses. TGFβ is one of the best-studied drivers of EMT, and many drugs are available to target this signaling pathway. A comprehensive bioinformatics approach was employed to derive a signature for TGFβ-induced EMT which can be used to score TGFβ-driven EMT in cells and clinical specimens. Considering this signature in pan-cancer cell and tumor datasets, a number of cell lines (including basal B breast cancer and cancers of the central nervous system) show evidence for TGFβ-driven EMT and carry a low mutational burden across the TGFβ signaling pathway. Furthermore, significant variation is observed in the response of high scoring cell lines to some common cancer drugs. Finally, this signature was applied to pan-cancer data from The Cancer Genome Atlas to identify tumor types with evidence of TGFβ-induced EMT. Tumor types with high scores showed significantly lower survival rates than those with low scores and also carry a lower mutational burden in the TGFβ pathway. The current transcriptomic signature demonstrates reproducible results across independent cell line and cancer datasets and identifies samples with strong mesenchymal phenotypes likely to be driven by TGFβ.

Implications: The TGFβ-induced EMT signature may be useful to identify patients with mesenchymal-like tumors who could benefit from targeted therapeutics to inhibit promesenchymal TGFβ signaling and disrupt the metastatic cascade. Mol Cancer Res; 15(5); 619–31. ©2017 AACR.

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"Obesity-Associated" Breast Cancer in Lean Women: Metabolism and Inflammation as Critical Modifiers of Risk

Why is obesity only weakly associated with certain "obesity-driven" cancers? Recent population studies identify cohorts of high body mass index (BMI) subjects with unexpectedly reduced risk for breast and colon cancer, and normal BMI subjects with unexpectedly elevated risk for breast cancer, provoking hard thinking about cellular and molecular mechanisms that most strongly couple obesity to cancer occurrence or progression. Emerging work suggests that abnormal metabolism and its associated chronic inflammation make the difference. Type II diabetes, for example, is a chronic inflammatory disease with specific imbalances in T-cell and myeloid-origin cytokines. Inflammation is elevated systemically, measured through blood biomarkers, and locally in adipose tissue. Here, cytokines and chemokines likely modify tumor microenvironments in dangerous ways. High BMI subjects with low inflammation and less disturbed metabolism appear to have reduced risk for certain obesity-associated cancers, whereas lean or slightly overweight subjects with high inflammation and metabolic abnormalities have elevated risk. This latter phenotype is prevalent among South Asian adults and suggests we are not monitoring certain normal weight adults sufficiently for risks of "obesity-associated" cancers. Profiling of patient metabolism and inflammation should accompany measures of body composition when considering cancer risk; the evidence base for these refinements must be extended through new, prospective observational studies. Cancer Prev Res; 10(5); 267–9. ©2017 AACR.

See related article by Iyengar et al., Cancer Prev Res 2017;10(4):235–43.

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A Phase II Randomized, Double-blind, Presurgical Trial of Polyphenon E in Bladder Cancer Patients to Evaluate Pharmacodynamics and Bladder Tissue Biomarkers

We performed a phase II pharmacodynamic prevention trial of Polyphenon E [a green tea polyphenol formulation primarily consisting of epigallocatechin gallate (EGCG)] in patients prior to bladder cancer surgery. Patients with a bladder tumor were randomized to receive Polyphenon E containing either 800 or 1,200 mg of EGCG or placebo for 14 to 28 days prior to transurethral resection of bladder tumor or cystectomy. The primary objective was to compare the postintervention EGCG tissue levels in patients receiving Polyphenon E as compared with placebo. Secondary objectives included assessments of tissue expression of PCNA, MMP2, clusterin, VEGF, p27, IGF-1, IGFBP-3; correlation of tissue, plasma, and urine levels of EGCG; and EGCG metabolism by catechol-O-methyltransferase and UDP-glucuronosyltransferase pharmacogenomic mutations. Thirty-one patients (male:female, 26:5; mean age, 67.2 years) were randomized and 29 (94%) completed the study. There was not an observed significant difference (P = 0.12) in EGCG tissue levels between two Polyphenon E dosage groups combined versus placebo. However, a dose–response relationship for EGCG levels was observed in both normal (P = 0.046) and malignant bladder tissue (P = 0.005) across the three study arms. In addition, EGCG levels in plasma (P < 0.001) and urine (P < 0.001) increased and PCNA (P = 0.016) and clusterin (P = 0.008) were downregulated in a dose-dependent fashion. No pharmacogenomic relationship was observed. EGCG levels in plasma, urine, and bladder tissue followed a dose–response relationship, as did modulation of tissue biomarkers of proliferation and apoptosis. Despite the limitations of this pilot study, the observed pharmacodynamics and desirable biologic activity warrant further clinical studies of this agent in bladder cancer prevention. Cancer Prev Res; 10(5); 298–307. ©2017 AACR.

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Sessile Serrated Polyps and Colon Cancer Prevention

Evidence suggests that up to one fifth of colorectal carcinomas develop from serrated polyps, named for their pattern of colonic crypts, and include the sessile serrated adenoma/polyp (SSA/P) that has malignant potential. SSA/Ps are typically located in the proximal colon and have molecular features of hypermethylation of CpG islands in gene promoters and activating point mutations (V600E) in the BRAF oncogene. Both of these features are seen in sporadic colorectal carcinomas with microsatellite instability (MSI) which is potentially consistent with an origin of these cancers from precursor SSA/Ps. Dysplasia is detected in a subset of SSA/Ps with a high risk of progression to carcinoma. An uncommon serrated polyp is the traditional serrated adenoma that is typically found in the left colon, has a tubulovillous architecture, and frequently harbors mutant KRAS. To date, the epidemiology of these serrated lesions is poorly understood, and limited observational data suggest a potential chemopreventive benefit of nonsteroidal anti-inflammatory drugs. The current primary strategy to reduce the risk of colorectal carcinoma from serrated polyps is to enhance their detection at colonoscopy and to ensure their complete removal. This review provides insight into the epidemiologic, clinical, histopathologic, and molecular features of serrated polyps and includes data on their endoscopic detection and chemoprevention. Cancer Prev Res; 10(5); 270–8. ©2017 AACR.

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No Decreased Risk of Gastrointestinal Cancers in Users of Metformin in The Netherlands; A Time-Varying Analysis of Metformin Exposure

Previous studies on metformin use and gastrointestinal (GI) cancer risk have yielded inconclusive results on metformin's chemoprotective effects. We aimed to evaluate GI cancer risk in users of metformin in The Netherlands using a time-varying approach in a large population-based database. A cohort study was performed using the NCR-PHARMO database. Patients using ≥1 non-insulin antidiabetic drug (NIAD) during 1998 to 2011 were included (N = 57,621). Exposure to NIADs was modeled time-varyingly. Cox regression analysis estimated HRs of GI cancers in current metformin users versus current users of other NIADs. Covariables included age, sex, drugs known to impact cancer risk, history of hospitalization, and starting year of follow-up. A sensitivity analysis was performed, applying a new-user design. Current use of metformin was not associated with a decreased risk of GI cancer [HR, 0.97; 95% confidence interval (CI), 0.82–1.15] or specific GI cancer sites. The sensitivity analysis yielded comparable results. No decreasing trends were observed with increasing cumulative dose of metformin [HR 1.05, 95% CI, 0.85–1.28; HR 0.89, 95% CI, 0.73–1.10; HR 0.96, 95% CI, 0.77–1.19 for dose tertiles low (<405 g), medium (405–999 g), and high (≥999 g)]. In contrast, an increased risk of pancreatic cancer was found in current users of metformin plus insulin (HR, 4.90; 95% CI, 2.64–9.10). In conclusion, no decreased risk of GI cancer was found in current metformin users compared with current users of other NIADs. Variations in the exposure definition of metformin use may be one of the explanations of previously found reduced cancer risks in metformin users. Cancer Prev Res; 10(5); 290–7. ©2017 AACR.

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Fatty Acid Synthesis Intermediates Represent Novel Noninvasive Biomarkers of Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate

Increased de novo synthesis of fatty acids is a distinctive feature of prostate cancer, which continues to be a leading cause of cancer-related deaths among American men. Therefore, inhibition of de novo fatty acid synthesis represents an attractive strategy for chemoprevention of prostate cancer. We have shown previously that dietary feeding of phenethyl isothiocyanate (PEITC), a phytochemical derived from edible cruciferous vegetables such as watercress, inhibits incidence and burden of poorly differentiated prostate cancer in transgenic adenocarcinoma of mouse prostate (TRAMP) model. The current study was designed to test the hypothesis of whether fatty acid intermediate(s) can serve as noninvasive biomarker(s) of prostate cancer chemoprevention by PEITC using archived plasma and tumor specimens from the TRAMP study as well as cellular models of prostate cancer. Exposure of prostate cancer cells (LNCaP and 22Rv1) to pharmacologic concentrations of PEITC resulted in downregulation of key fatty acid metabolism proteins, including acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyltransferase 1A (CPT1A). The mRNA expression of FASN and CPT1A as well as acetyl-CoA levels were decreased by PEITC treatment in both cell lines. PEITC administration to TRAMP mice also resulted in a significant decrease in tumor expression of FASN protein. Consistent with these findings, the levels of total free fatty acids, total phospholipids, triglyceride, and ATP were significantly lower in the plasma and/or prostate tumors of PEITC-treated TRAMP mice compared with controls. The current study is the first to implicate inhibition of fatty acid synthesis in prostate cancer chemoprevention by PEITC. Cancer Prev Res; 10(5); 279–89. ©2017 AACR.

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The Prolyl Isomerase Pin1 Is a Novel Target of 6,7,4'-Trihydroxyisoflavone for Suppressing Esophageal Cancer Growth

Intake of soy isoflavones is inversely associated with the risk of esophageal cancer. Numerous experimental results have supported the anticancer activity of soy isoflavones. This study aimed to determine the anti-esophageal cancer activity of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF), a major metabolite of daidzein, which is readily metabolized in the human body. Notably, 6,7,4'-THIF inhibited proliferation and increased apoptosis of esophageal cancer cells. On the basis of a virtual screening analysis, Pin1 was identified as a target protein of 6,7,4'-THIF. Pull-down assay results using 6,7,4'-THIF Sepharose 4B beads showed a direct interaction between 6,7,4'-THIF and the Pin1 protein. Pin1 is a critical therapeutic and preventive target in esophageal cancer because of its positive regulation of β-catenin and cyclin D1. The 6,7,4'-THIF compound simultaneously reduced Pin1 isomerase activity and the downstream activation targets of Pin1. The specific inhibitory activity of 6,7,4'-THIF was analyzed using Neu/Pin1 wild-type (WT) and Neu/Pin1 knockout (KO) MEFs. 6,7,4'-THIF effected Neu/Pin1 WT MEFs, but not Neu/Pin1 KO MEFs. Furthermore, the results of a xenograft assay using Neu/Pin1 WT and KO MEFs were similar to those obtained from the in vitro assay. Overall, we found that 6,7,4'-THIF specifically reduced Pin1 activity in esophageal cancer models. Importantly, 6,7,4'-THIF directly bound to Pin1 but not FKBP or cyclophilin A, the same family of proteins. Because Pin1 acts like an oncogene by modulating various carcinogenesis-related proteins, this study might at least partially explain the underlying mechanism(s) of the anti-esophageal cancer effects of soy isoflavones. Cancer Prev Res; 10(5); 308–18. ©2017 AACR.

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"Obesity-Associated" Breast Cancer in Lean Women: Metabolism and Inflammation as Critical Modifiers of Risk

See related article by Iyengar et al., Cancer Prev Res 2017;10(4):235–43.

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A Phase II Randomized, Double-blind, Presurgical Trial of Polyphenon E in Bladder Cancer Patients to Evaluate Pharmacodynamics and Bladder Tissue Biomarkers

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Sessile Serrated Polyps and Colon Cancer Prevention

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No Decreased Risk of Gastrointestinal Cancers in Users of Metformin in The Netherlands; A Time-Varying Analysis of Metformin Exposure

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Fatty Acid Synthesis Intermediates Represent Novel Noninvasive Biomarkers of Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate

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The Prolyl Isomerase Pin1 Is a Novel Target of 6,7,4'-Trihydroxyisoflavone for Suppressing Esophageal Cancer Growth

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A systematic review and network meta-analysis of immunotherapy and targeted therapy for advanced melanoma

Abstract

Immune and BRAF-targeted therapies have changed the therapeutic scenario of advanced melanoma, turning the clinical decision-making a challenging task. This Bayesian network meta-analysis assesses the role of immunotherapies and targeted therapies for advanced melanoma. We retrieved randomized controlled trials testing immune, BRAF- or MEK-targeted therapies for advanced melanoma from electronic databases. A Bayesian network model compared therapies using hazard ratio (HR) for overall survival (OS), progression-free survival (PFS), and odds ratio (OR) for response rate (RR), along with 95% credible intervals (95% CrI), and probabilities of drugs outperforming others. We assessed the impact of PD-L1 expression on immunotherapy efficacy. Sixteen studies evaluating eight therapies in 6849 patients were analyzed. For OS, BRAF-MEK combination and PD-1 single agent ranked similarly and outperformed all other treatments. For PFS, BRAF-MEK combination surpassed all other options, including CTLA-4-PD-1 dual blockade hazard ratio (HR: 0.56; 95% CrI: 0.33–0.97; probability better 96.2%), whereas BRAF single agent ranked close to CTLA-4-PD-1 blockade. For RR, BRAF-MEK combination was superior to all treatments including CTLA-4-PD-1 (OR: 2.78; 1.18–6.30; probability better 97.1%). No OS data were available for CTLA-4-PD-1 blockade at the time of systematic review, although PFS and RR results suggested that this combination could also bring meaningful benefit. PD-L1 expression, as presently defined, failed to inform patient selection to PD-1-based immunotherapy. BRAF-MEK combination seemed an optimal therapy for BRAF-mutated patients, whereas PD-1 inhibitors seemed optimal for BRAF wild-type patients. Longer follow-up is needed to ascertain the role of CTLA-4-PD-1 blockade. Immunotherapy biomarkers remain as an unmet need.

This Bayesian meta-analysis compared immunotherapy and BRAF-MEK-targeted therapy in more than 6000 melanoma patients. Double BRAF-MEK therapy ranked highest in terms of survival and response followed by single agent PD-1 and BRAF inhibitors. Those results should better inform patient selection.

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A systematic review and network meta-analysis of immunotherapy and targeted therapy for advanced melanoma

Abstract

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Ameliorative Effect of VE, IGF-I, and hCG on the Fluoride-Induced Testosterone Release Suppression in Mice Leydig Cells

Abstract

Excessive consumption of fluoride (F) through drinking, eating, and/or environmental contaminants induces chronic toxicity known as fluorosis. Our previous research has shown that fluorosis was associated with male reproductive disorders. The current study is designed to explain the protective effect of vitamin E (VE), insulin-like growth factor-I (IGF-I), and human chorionic gonadotropin (hCG) against natrium fluoride (NaF)-induced alterations in isolated Leydig cells (LCs). These NaF-induced alterations include decreased cell proliferation, steroidogenesis, and relative gene expression. Isolated LCs were incubated with NaF (0, 5, 20 mg/L) and/or 10 μg/ml VE, 100 ng/ml IGF-I, and 100 IU/ml hCG. NaF-treated cells' ability to secrete testosterone (T) was significantly less than other treated groups (P < 0.05). Additionally, in NaF-treated cells, there was a significant upregulation of certain relative mRNA expressions such as Star and Cyp11a, as well as significantly less cell proliferation in a dose-dependent manner (P < 0.05). These data clearly show that VE, IGF-1, and hCG have a protective effect in the LCs functions. Taken together, the final results of this study shown herein are consistent with the assumption that VE, IGF-I, and hCG volunteered ameliorative effects against the deleterious effects of NaF through their protective activity. Although it is hypothesized that ameliorative effects might have been involved, the fundamental pathway(s) remain(s) to be illuminated.

Graphical Abstract

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Ameliorative Effect of VE, IGF-I, and hCG on the Fluoride-Induced Testosterone Release Suppression in Mice Leydig Cells

Abstract

Graphical Abstract

ᅟ

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Role of MAML1 and MEIS1 in Esophageal Squamous Cell Carcinoma Depth of Invasion

Abstract

Homeobox (HOX) transcription factors and NOTCH signaling pathway are critical regulators of stem cell functions, cell fate in development and homeostasis of gastrointestinal tissues. In the present study, we analyzed cross talk between NOTCH pathway and HOX genes through assessment of probable correlation betweenMAML1 and MEIS1 as the main transcription factor of NOTCH pathway and enhancer of HOX transcriptional machinery, respectively in esophageal squamous cell carcinoma (ESCC) patients. Fifty one ESCC cases were enrolled to assess the levels of Meis1 and Maml1 mRNA expression using real-time polymerase chain reaction (PCR). Only 3 out of 51 (5.9%) cases had MEIS1/MAML1 under expression and 2/51 (3.9%) cases had MEIS1/MAML1over expression. Nine out of 51 samples (17.6%) have shown MEIS1 under expression and MAML1 over expression. There was a significant correlation between MAML1and MEIS1mRNA expressions (p ≤ 0.05). There were significant correlations between MEIS1 under/MAML1 over expressed cases and tumor location (p = 0.05), tumor depth of invasion (p = 0.011), and sex (p = 0.04). Our results showed that MEIS1 may have a negative role in regulation of MAML1expression during the ESCC progression.

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Practices and Attitudes Regarding Women Undergoing Fertility Preservation: A Survey of the National Physicians Cooperative

Journal of Adolescent and Young Adult Oncology , Vol. 0, No. 0.

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Practices and Attitudes Regarding Women Undergoing Fertility Preservation: A Survey of the National Physicians Cooperative

Journal of Adolescent and Young Adult Oncology , Vol. 0, No. 0.

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Synthetic lethal targeting of RNF20 through PARP1 silencing and inhibition

Abstract

Purpose

The identification of novel therapeutic targets that exploit the aberrant genetics driving oncogenesis is critical to better combat cancer. RNF20 is somatically altered in numerous cancers, and its diminished expression drives genome instability, a driving factor of oncogenesis. Accordingly, we sought to determine whether PARP1 silencing and inhibition could preferentially kill RNF20-deficient cells using a synthetic lethal strategy.

Methods

RNF20 and PARP1 were silenced using RNAi-based approaches. Direct synthetic lethal tests were performed by silencing RNF20 with and without PARP1 and the impact on cell numbers was evaluated using semi-quantitative imaging microscopy. Next, Olaparib and BMN673 (PARP1 inhibitors) were evaluated for their ability to induce preferential killing in RNF20 silenced cells, while real-time cell analyses were used to distinguish cell cytotoxicity from cell cycle arrest. Finally, quantitative imaging microscopy was employed to evaluate marks associated with DNA double-strand breaks (γ-H2AX) and apoptosis (cleaved Caspase-3).

Results

We found that PARP1 silencing resulted in a decrease in number of RNF20 silenced cells relative to controls. We further found that Olaparib and BMN673 treatments also resulted in fewer RNF20 silenced cells relative to controls. Finally, we found by quantitative imaging microscopy that RNF20 silenced cells treated with BMN673 exhibited significant increases in γ-H2AX and cleaved Caspase-3, suggesting that these treatments induce DNA double-strand breaks that are not adequately repaired within RNF20-silenced cells.

Conclusions

Collectively, our data indicate that RNF20 and PARP1 are synthetic lethal interactors, suggesting that cancers with diminished RNF20 expression and/or function may be susceptible to PARP1 inhibitors.

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Synthetic lethal targeting of RNF20 through PARP1 silencing and inhibition

Abstract

Purpose

Methods

Results

Conclusions

http://ift.tt/2qqMGHm

Synthetic lethal targeting of RNF20 through PARP1 silencing and inhibition

Abstract

Purpose

Methods

Results

Conclusions

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Role of MAML1 and MEIS1 in Esophageal Squamous Cell Carcinoma Depth of Invasion

Abstract

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Role of MAML1 and MEIS1 in Esophageal Squamous Cell Carcinoma Depth of Invasion

Abstract

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Clinicopathological impact of CD20 expression in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL)

Abstract

Recognition of BCR/ABL translocation is obligatory in precursor B cell precursor acute lymphoblastic leukemia (BCP-ALL) patients at diagnosis for categorization of patients. The risk stratification of BCP-ALL especially those negative for Philadelphia chromosome is an area of research. The prognostic impact of CD20 expression in childhood BCP-ALL remains controversial. In pediatric patients with BCP-ALL, some studies have demonstrated that CD20 overexpression correlates with bad overall survival, but some others suggest a better outcome. This study aimed to evaluate the efficacy of CD20 expression as a biological marker in BCP-ALL. For 104 children with newly diagnosed BCP-ALL included in this study, CD20 expression was evaluated by flow cytometry. CD20 was positively expressed in 40 (38.5%) of cases. The CD20^positive subgroup had significantly shorter overall survival and disease free survival as compared to the CD20^negative one (P = 0.018; <0.001, respectively). Combined positivity for CD20 and Philadelphia chromosome was detected in 16/40 of studied cases. This patient group had the poorest overall survival and the shortest disease-free survival. In conclusion, CD20 expression at diagnosis in BCP-ALL is a useful biomarker for risk stratification and subsequently in treatment decision especially in Philadelphia negative BCP-ALL.

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miRNA-34c-5p inhibits amphiregulin-induced ovarian cancer stemness and drug resistance via downregulation of the AREG-EGFR-ERK pathway

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Metformin inhibits SUV39H1-mediated migration of prostate cancer cells

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miRNA-34c-5p inhibits amphiregulin-induced ovarian cancer stemness and drug resistance via downregulation of the AREG-EGFR-ERK pathway

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Metformin inhibits SUV39H1-mediated migration of prostate cancer cells

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Hypercholesterolemia Increases Colorectal Cancer Incidence by Reducing Production of NKT and {gamma}{delta} T Cells from Hematopoietic Stem Cells

Obesity will soon surpass smoking as the most preventable cause of cancer. Hypercholesterolemia, a common comorbidity of obesity, has been shown to increase cancer risk, especially colorectal cancer. However, the mechanism by which hypercholesterolemia or any metabolic disorder increases cancer risk remains unknown. In this study, we show that hypercholesterolemia increases the incidence and pathologic severity of colorectal neoplasia in two independent mouse models. Hypocholesterolemia induced an oxidant stress–dependent increase in miR101c, which downregulated Tet1 in hematopoietic stem cells (HSC), resulting in reduced expression of genes critical to natural killer T cell (NKT) and γδ T-cell differentiation. These effects reduced the number and function of terminally differentiated NKT and γδ T cells in the thymus, the colon submucosa, and during early tumorigenesis. These results suggest a novel mechanism by which a metabolic disorder induces epigenetic changes to reduce lineage priming of HSC toward immune cells, thereby compromising immunosurveillance against cancer. Cancer Res; 77(9); 2351–62. ©2017 AACR.

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A Pyrrole-Imidazole Polyamide Is Active against Enzalutamide-Resistant Prostate Cancer

The LREX' prostate cancer model is resistant to the antiandrogen enzalutamide via activation of an alternative nuclear hormone receptor, glucocorticoid receptor (GR), which has similar DNA-binding specificity to the androgen receptor (AR). Small molecules that target DNA to interfere with protein–DNA interactions may retain activity against enzalutamide-resistant prostate cancers where ligand-binding domain antagonists are ineffective. We reported previously that a pyrrole-imidazole (Py-Im) polyamide designed to bind the consensus androgen response element half-site has antitumor activity against hormone-sensitive prostate cancer. In enzalutamide-resistant LREX' cells, Py-Im polyamide interfered with both AR- and GR-driven gene expression, whereas enzalutamide interfered with only that of AR. Genomic analyses indicated immediate interference with the AR transcriptional pathway. Long-term treatment with Py-Im polyamide demonstrated a global decrease in RNA levels consistent with inhibition of transcription. The polyamide was active against two enzalutamide-resistant xenografts with minimal toxicity. Overall, our results identify Py-Im polyamide as a promising therapeutic strategy in enzalutamide-resistant prostate cancer. Cancer Res; 77(9); 2207–12. ©2017 AACR.

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HuR Small-Molecule Inhibitor Elicits Differential Effects in Adenomatosis Polyposis and Colorectal Carcinogenesis

HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3′ untranslated region of mRNAs encoding oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APCMin mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of Prevotella, Akkermansia, and Lachnospiraceae. Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD. Cancer Res; 77(9); 2424–38. ©2017 AACR.

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Intrinsic Subtypes and Gene Expression Profiles in Primary and Metastatic Breast Cancer

Biological changes that occur during metastatic progression of breast cancer are still incompletely characterized. In this study, we compared intrinsic molecular subtypes and gene expression in 123 paired primary and metastatic tissues from breast cancer patients. Intrinsic subtype was identified using a PAM50 classifier and χ2 tests determined the differences in variable distribution. The rate of subtype conversion was 0% in basal-like tumors, 23.1% in HER2-enriched (HER2-E) tumors, 30.0% in luminal B tumors, and 55.3% in luminal A tumors. In 40.2% of cases, luminal A tumors converted to luminal B tumors, whereas in 14.3% of cases luminal A and B tumors converted to HER2-E tumors. We identified 47 genes that were expressed differentially in metastatic versus primary disease. Metastatic tumors were enriched for proliferation-related and migration-related genes and diminished for luminal-related genes. Expression of proliferation-related genes were better at predicting overall survival in metastatic disease (OSmet) when analyzed in metastatic tissue rather than primary tissue. In contrast, a basal-like gene expression signature was better at predicting OSmet in primary disease compared with metastatic tissue. We observed correlations between time to tumor relapse and the magnitude of changes of proliferation, luminal B, or HER2-E signatures in metastatic versus primary disease. Although the intrinsic subtype was largely maintained during metastatic progression, luminal/HER2-negative tumors acquired a luminal B or HER2-E profile during metastatic progression, likely reflecting tumor evolution or acquisition of estrogen independence. Overall, our analysis revealed the value of stratifying gene expression by both cancer subtype and tissue type, providing clinicians more refined tools to evaluate prognosis and treatment. Cancer Res; 77(9); 2213–21. ©2017 AACR.

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Targeted Degradation of BET Proteins in Triple-Negative Breast Cancer

Triple-negative breast cancers (TNBC) remain clinically challenging with a lack of options for targeted therapy. In this study, we report the development of a second-generation BET protein degrader, BETd-246, which exhibits superior selectivity, potency, and antitumor activity. In human TNBC cells, BETd-246 induced degradation of BET proteins at low nanomolar concentrations within 1 hour of exposure, resulting in robust growth inhibition and apoptosis. BETd-246 was more potent and effective in TNBC cells than its parental BET inhibitor compound BETi-211. RNA-seq analysis revealed predominant downregulation of a large number of genes involved in proliferation and apoptosis in cells treated with BETd-246, as compared with BETi-211 treatment that upregulated and downregulated a similar number of genes. Functional investigations identified the MCL1 gene as a critical downstream effector for BET degraders, which synergized with small-molecule inhibitors of BCL-xL in triggering apoptosis. In multiple murine xenograft models of human breast cancer, BETd-246 and a further optimized analogue BETd-260 effectively depleted BET proteins in tumors and exhibited strong antitumor activities at well-tolerated dosing schedules. Overall, our findings show that targeting BET proteins for degradation represents an effective therapeutic strategy for TNBC treatment. Cancer Res; 77(9); 2476–87. ©2017 AACR.

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Circulating Tumor Cells with Aberrant ALK Copy Number Predict Progression-Free Survival during Crizotinib Treatment in ALK-Rearranged Non-Small Cell Lung Cancer Patients

The duration and magnitude of clinical response are unpredictable in ALK-rearranged non–small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK-FISH patterns [ALK-rearrangement, ALK-copy number gain (ALK-CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK-rearranged patients. Thirty-nine ALK-rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK-FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK-rearrangement and/or ALK-CNG signals. No significant association between baseline numbers of ALK-rearranged or ALK-CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK-CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK-CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543–13.030, P = 0.006). Although not dominant, ALK-CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK-CNG may be a predictive biomarker for crizotinib efficacy in ALK-rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222–30. ©2017 AACR.

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The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription

Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene–amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc–mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522–33. ©2017 AACR.

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Cellular Hierarchy as a Determinant of Tumor Sensitivity to Chemotherapy

Chemotherapy has been shown to enrich cancer stem cells in tumors. Recently, we demonstrated that administration of chemotherapy to human bladder cancer xenografts could trigger a wound-healing response that mobilizes quiescent tumor stem cells into active proliferation. This phenomenon leads to a loss of sensitivity to chemotherapy partly due to an increase in the number of tumor stem cells, which typically respond to chemotherapy-induced cell death less than more differentiated cells. Different bladder cancer xenografts, however, demonstrate differential sensitivities to chemotherapy, the basis of which is not understood. Using mathematical models, we show that characteristics of the tumor cell hierarchy can be crucial for determining the sensitivity of tumors to drug therapy, under the assumption that stem cell enrichment is the primary basis for drug resistance. Intriguingly, our model predicts a weaker response to therapy if there is negative feedback from differentiated tumor cells that inhibits the rate of tumor stem cell division. If this negative feedback is less pronounced, the treatment response is predicted to be enhanced. The reason is that negative feedback on the rate of tumor cell division promotes a permanent rise of the tumor stem cell population over time, both in the absence of treatment and even more so during drug therapy. Model application to data from chemotherapy-treated patient-derived xenografts indicates support for model predictions. These findings call for further research into feedback mechanisms that might remain active in cancers and potentially highlight the presence of feedback as an indication to combine chemotherapy with approaches that limit the process of tumor stem cell enrichment. Cancer Res; 77(9); 2231–41. ©2017 AACR.

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Noncoding Effects of Circular RNA CCDC66 Promote Colon Cancer Growth and Metastasis

Circular RNA (circRNA) is a class of noncoding RNA whose functions remain mostly unknown. Recent studies indicate circRNA may be involved in disease pathogenesis, but direct evidence is scarce. Here, we characterize the functional role of a novel circRNA, circCCDC66, in colorectal cancer. RNA-Seq data from matched normal and tumor colon tissue samples identified numerous circRNAs specifically elevated in cancer cells, several of which were verified by quantitative RT-PCR. CircCCDC66 expression was elevated in polyps and colon cancer and was associated with poor prognosis. Gain-of-function and loss-of-function studies in colorectal cancer cell lines demonstrated that circCCDC66 controlled multiple pathological processes, including cell proliferation, migration, invasion, and anchorage-independent growth. In-depth characterization revealed that circCCDC66 exerts its function via regulation of a subset of oncogenes, and knockdown of circCCDC66 inhibited tumor growth and cancer invasion in xenograft and orthotopic mouse models, respectively. Taken together, these findings highlight a novel oncogenic function of circRNA in cancer progression and metastasis. Cancer Res; 77(9); 2339–50. ©2017 AACR.

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Defining Cancer Subpopulations by Adaptive Strategies Rather Than Molecular Properties Provides Novel Insights into Intratumoral Evolution

Ongoing intratumoral evolution is apparent in molecular variations among cancer cells from different regions of the same tumor, but genetic data alone provide little insight into environmental selection forces and cellular phenotypic adaptations that govern the underlying Darwinian dynamics. In three spontaneous murine cancers (prostate cancers in TRAMP and PTEN mice, pancreatic cancer in KPC mice), we identified two subpopulations with distinct niche construction adaptive strategies that remained stable in culture: (i) invasive cells that produce an acidic environment via upregulated aerobic glycolysis; and (ii) noninvasive cells that were angiogenic and metabolically near-normal. Darwinian interactions of these subpopulations were investigated in TRAMP prostate cancers. Computer simulations demonstrated invasive, acid-producing (C2) cells maintain a fitness advantage over noninvasive, angiogenic (C3) cells by promoting invasion and reducing efficacy of immune response. Immunohistochemical analysis of untreated tumors confirmed that C2 cells were invariably more abundant than C3 cells. However, the C2 adaptive strategy phenotype incurred a significant cost due to inefficient energy production (i.e., aerobic glycolysis) and depletion of resources for adaptations to an acidic environment. Mathematical model simulations predicted that small perturbations of the microenvironmental extracellular pH (pHe) could invert the cost/benefit ratio of the C2 strategy and select for C3 cells. In vivo, 200 mmol/L NaHCO3 added to the drinking water of 4-week-old TRAMP mice increased the intraprostatic pHe by 0.2 units and promoted proliferation of noninvasive C3 cells, which remained confined within the ducts so that primary cancer did not develop. A 0.2 pHe increase in established tumors increased the fraction of C3 cells and signficantly diminished growth of primary and metastatic tumors. In an experimental tumor construct, MCF7 and MDA-MB-231 breast cancer cells were coinjected into the mammary fat pad of SCID mice. C2-like MDA-MB-231 cells dominated in untreated animals, but C3-like MCF7 cells were selected and tumor growth slowed when intratumoral pHe was increased. Overall, our data support the use of mathematical modeling of intratumoral Darwinian interactions of environmental selection forces and cancer cell adaptive strategies. These models allow the tumor to be steered into a less invasive pathway through the application of small but selective biological force. Cancer Res; 77(9); 2242–54. ©2017 AACR.

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Loss of NDRG2 Expression Confers Oral Squamous Cell Carcinoma with Enhanced Metastatic Potential

Loss of the tumor suppressor NDRG2 has been implicated in the development of oral squamous cell carcinoma (OSCC), acting by modulating PI3K/AKT-mediated dephosphorylation of PTEN at S380/S382/T383 (STT). Here, we show that the majority of OSCC tumors with lymph node metastasis, a major prognostic factor, exhibit high levels of phosphorylated AKT-S473 and PTEN-STT and low levels of NDRG2 expression. In Ndrg2-deficient mice, which develop a wide range of tumors, we developed a model of OSCC by treatment with the tobacco surrogate 4-nitroquinoline-1-oxide (4-NQO). In this model, both the number and size of OSCC tumors were increased significantly by Ndrg2 deficiency, which also increased invasion of cervical lymph nodes. 4-NQO treatment of human OSCC cell lines exhibiting low NDRG2 expression induced epithelial–mesenchymal transition via activation of NF-κB signaling. Conversely, ectopic expression of NDRG2 reversed the EMT phenotype and inhibited NF-κB signaling via suppression of PTEN-STT and AKT-S473 phosphorylation. Our results show how NDRG2 expression serves as a critical determinant of the invasive and metastatic capacity of OSCC. Cancer Res; 77(9); 2363–74. ©2017 AACR.

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