Δημοφιλείς αναρτήσεις

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Τετάρτη, 10 Φεβρουαρίου 2016

Hypoxic signaling and the cellular redox tumor environment determine sensitivity to MTH1 inhibition

Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific non-oncogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in non-malignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathological upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pre-treatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance

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Genomic landscape of somatic alterations in esophageal squamous cell carcinoma and gastric cancer

Gastric cancer and esophageal cancer are the 2nd and 6th leading causes of cancer death worldwide. Multiple genomic alterations underlying gastric cancer and esophageal squamous cell carcinoma (ESCC) have been identified, but the full spectrum of genomic structural variations and mutations have yet to be uncovered. Here we report the results of whole genome sequencing of 30 samples comprising tumor and blood from 15 patients, four of whom presented with ESCC, seven with gastric cardia adenocarcinoma (GCA), and four with gastric noncardia adenocarcinoma (GNCA). Analyses revealed that an A>C mutation was common in GCA, and in addition to the preferential nucleotide sequence of A located 5 prime to the mutation as noted in previous studies, we found enrichment of T in the 5 prime base. The A>C mutations in GCA suggested that oxidation of guanine may be a potential mechanism underlying cancer mutagenesis. Furthermore, we identified genes with mutations in gastric cancer and ESCC, including well-known cancer genes, TP53, JAK3, BRCA2, FGF2, FBXW7, MSH3, PTCH, NF1, ERBB2, and CHEK2, and potentially novel cancer-associated genes, KISS1R, AMH, MNX1, WNK2, and PRKRIR. Finally, we identified recurrent chromosome alterations in at least 30% of tumors in genes including MACROD2, FHIT, and PARK2 that were often intragenic deletions. These structural alterations were validated using the TCGA dataset. Our studies provide new insights into understanding the genomic landscape, genome instability, and mutation profile underlying gastric cancer and ESCC development.

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A Preclinical Model of Chronic Alcohol Consumption Reveals Increased Metastatic Seeding of Colon Cancer Cells in the Liver

Liver metastasis is the main cause of death from colorectal cancer. Alcohol consumption impacts liver function and is suggested to be an independent risk factor for liver metastasis of colorectal cancer, but no experimental evidence supporting this hypothesis has been demonstrated to date. In this study, we investigated the effect of alcohol intake on liver metastasis. We examined colon cancer cell spread from the spleen in mice provided with water (control group), alcohol for 4 weeks before tumor injection (pre-alcohol), alcohol for 3 weeks after tumor injection (post-alcohol), or alcohol throughout the 7-week study (alcohol). Alcohol intake significantly increased hepatic metastatic burden in the pre-alcohol (2.4-fold, p < 0.001), post-alcohol (2.0-fold, p < 0.01), and alcohol groups (2.2-fold, p < 0.001). A fluorescence-based metastasis tracking assay also confirmed an alcohol-induced increase in the abundance of tumor cells in the liver (2.5-fold, p < 0.001). Investigation of the host microenvironment revealed an alcohol-induced inflammatory response marked by elevated TNF-α, IL-1β, IL-6, and IFN-γ protein levels, as well as increased expression of intercellular molecule-1 (ICAM1) in hepatic tissues after 4 weeks of alcohol consumption. Moreover, the peripheral blood of mice provided with alcohol for 4 weeks exhibited reduced natural killer and CD8+ T cell counts. Collectively, our findings suggest that chronic alcohol consumption accelerates liver metastasis of colorectal cancer cells through alterations to the liver microenvironment and inactivation of immune surveillance.

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Distinct subtypes of gastric cancer defined by molecular characterization include novel mutational signatures with prognostic capability

Gastric cancer is not a single disease and its subtype classification is still evolving. Next-generation sequencing studies have identified novel genetic drivers of gastric cancer, but their use as molecular classifiers or prognostic markers of disease outcome have yet to be established. In this study, we integrated somatic mutational profiles and clinicopathological information from 544 gastric cancer patients by previous genomics studies to identify significantly mutated genes with prognostic relevance. Gastric cancer patients were classified into regular (86.8%) and hyper-mutated (13.2%) subtypes based on mutation burden. Notably, TpCpW mutations occurred significantly more frequently in regular, but not hyper-mutated, gastric cancers where they were associated with APOBEC expression. In the former group, six previously unreported (XIRP2, NBEA, COL14A1, CNBD1, ITGAV, AKAP6) and 12 recurrent mutated genes exhibited high mutation prevalence (≥ 3.0%) and an unexpectedly higher incidence of nonsynonymous mutations. We also identified two molecular subtypes of regular-mutated gastric cancer that were associated with distinct prognostic outcomes, independently of disease staging, as confirmed in a distinct patient cohort by targeted capture sequencing. Lastly, in diffuse-type gastric cancer, CDH1 mutation was found to be associated with shortened patient survival, independently of disease staging. Overall, our work identified previously unreported significantly mutated genes and a mutation signature predictive of patient survival in newly classified subtypes of gastric cancer, offering opportunities to stratify patients into optimal treatment plans based on molecular subtyping.

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Activated KRAS cooperates with MLLAF4 to promote extramedullary engraftment and migration of cord blood CD34+ HSPC but is insufficient to initiate leukemia

The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematological malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood (CB)-derived CD34+ hematopoietic stem/progenitor cells (HSPCs). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34+ HSPCs. Intra-bone marrow transplantation into immunodeficient mice further showed that MA4 and KRASG12V alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34+ HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)+B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS impacts the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4+ B-ALL is important for tumor maintenance rather than initiation.

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Cystine deprivation triggers programmed necrosis in VHL-deficient renal cell carcinomas

Oncogenic transformation may reprogram tumor metabolism and render cancer cells addicted to extracellular nutrients. Deprivation of these nutrients may therefore represent a therapeutic opportunity, but predicting which nutrients cancer cells become addicted to remains difficult. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear-cell renal cancer cells (ccRCC), with or without VHL, upon the deprivation of individual amino acids. We found that cystine deprivation triggered rapid programmed necrosis in VHL-deficient cell lines and primary ccRCC tumor cells, but not in VHL-restored counterparts. Blocking cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status, suggesting that metabolic responses alone are not sufficient to explain the observed distinct fates of VHL-deficient and restored cells. Instead, we found that increased levels of TNFα (TNF) associated with VHL loss forced VHL-deficient cells to rely on intact RIPK1 to inhibit apoptosis. However, the pre-existing elevation in TNFα expression rendered VHL-deficient cells susceptible to necrosis triggered by cystine deprivation. We further determined that reciprocal amplification of the Src-p38 (MAPK14)-Noxa (PMAIP1) signaling and TNFα-RIP1/3 (RIPK1/RIPK3)-MLKL necrosis pathways potentiated cystine deprived-necrosis. Together, our findings reveal that cystine deprivation in VHL-deficient RCCs presents an attractive therapeutic opportunity that may bypass the apoptosis-evading mechanisms characteristic of drug-resistant tumor cells.

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Calreticulin expression in human non-small cell lung cancers correlates with increased accumulation of antitumor immune cells and favorable prognosis

A high density of tumor-infiltrating mature dendritic cells (DC) and CD8+ T cells correlates with a positive prognosis in a majority of human cancers. The recruitment of activated lymphocytes to the tumor microenvironment, primed to recognize tumor-associated antigens, can occur in response to immunogenic cell death (ICD) of tumor cells. ICD is characterized by the pre-apoptotic translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the cell surface as a result of an ER stress response accompanied by the phosphorylation of eukaryotic initiation factor 2α (eIF2α). We conducted a retrospective study on two independent cohorts of patients with non-small cell lung cancer (NSCLC) to investigate the prognostic potential of CRT. We report that the level of CRT expression on tumor cells, which correlated with eIF2α phosphorylation, positively influenced the clinical outcome of NSCLC. High CRT expression on tumor cells was associated with a higher density of infiltrating mature DC and effector memory T cell subsets, suggesting that CRT triggers the activation of adaptive immune responses in the tumor microenvironment. Accordingly, patients with elevated CRT expression and dense intratumoral infiltration by DC or CD8+ T lymphocytes had the best prognosis. We conclude that CRT expression constitutes a new powerful prognostic biomarker that reflects enhanced local antitumor immune responses in the lung.

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Aspirin suppresses the acquisition of chemo-resistance in breast cancer by disrupting an NF{kappa}B-IL6 signaling axis responsible for the generation of cancer stem cells

Acquired chemo-resistance has curtailed cancer survival since the dawn of the chemotherapy. Accumulating evidence suggests a major role for cancer stem cells (CSCs) in chemo-resistance, though their involvement in acquired resistance is still unknown. The use of aspirin has been associated with reduced cancer risk and recurrence, suggesting that the anti-inflammatory drug may exert effects on CSCs. In this study, we investigated the contribution of CSCs to acquired chemo-resistance of breast cancer and the avenues for reversing such effects with aspirin. We observed that the residual risk of recurrence was higher in breast cancer patients who had acquired chemo-resistance. Treatment of pre-existing CSCs with a genotoxic drug combination (5-fluorouracil, adriamycin, and cyclophosphamide) generated an NFκB-IL6-dependent inflammatory environment that imparted stemness to non-stem cancer cells, induced multi-drug resistance, and enhanced the migration potential of CSCs. Treatment with aspirin prior to chemotherapy suppressed the acquisition of chemo-resistance by perturbing the nuclear translocation of NFκB in pre-existing CSCs. Therefore, disruptions to the NFκB-IL6 feedback loop prevented CSC induction and sensitized pre-existing CSCs to chemotherapy. Collectively, our findings suggest that combining aspirin and conventional chemotherapy may offer a new treatment strategy to improve recurrence-free survival of breast cancer patients.

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Phytoestrogen suppresses efflux of the diagnostic marker protoporphyrin IX (PpIX) in lung carcinoma

One promising method to visualize cancer cells is based on dection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method can not be used in cancers which exhibiti poor PpIX accumulation. PpIX appears to pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by ~2-fold. ABCG2 depletion of overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pre-treatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells, and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies.

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Stomach-specific activation of oncogenic KRAS and STAT3-dependent inflammation cooperatively promote gastric tumorigenesis in a preclinical model

About 5-10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre-recombinase-mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg) comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the Tamoxifen-inducible Cre recombinase (CreERT2) coding sequence. The resulting Tg(Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf. Administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);KrasLSL-G12D/+ and Tg(Tff1-CreERT2);BrafV600E/+ mice resulted in gastric metaplasia, inflammation, and adenoma development characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130F/F mice, which carry a knockin mutation in the Il6 signal transducer (Il6st), we generated Tg(Tff1-CreERT2);Stat3fl/fl;gp130F/F mice that also harbor a conditional Stat3 knock-out allele and found that tamoxifen administration conferred a significant reduction in their tumor burden. Conversely, excessive Kras activity in Tg(Tff1-CreERT2);KrasLSL-G12D/+;gp130F/F mice promoted more extensive gastric inflammation, metaplastic transformation, and tumorigenesis than observed in Tg(Tff1-CreERT2);KrasLSL-G12D/+ mice. Collectively, our findings demonstrate that advanced gastric tumorigenesis requires oncogenic KRAS or BRAF in concert with aberrant STAT3 activation in epithelial precursor cells of the glandular stomach, providing a new conditional model of gastric cancer in which to investigate candidate therapeutic targets and treatment strategies.

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Galectin-3 cleavage alters bone remodeling: Different outcomes in breast and prostate cancer skeletal metastasis

Management of bone metastasis remains clinically challenging and requires the identification of new molecular target(s) that can be therapeutically exploited to improve patient outcome. Galectin-3 (Gal-3) has been implicated as a secreted factor that alters the bone tumor microenvironment. Proteolytic cleavage of Gal-3 may also contribute to malignant cellular behaviors, but has not been addressed in cancer metastasis. Here, we report that Gal-3 modulates the osteolytic bone tumor microenvironment in the presence of RANKL. Gal-3 was localized on the osteoclast cell surface, and its suppression by RNAi or a specific antagonist markedly inhibited osteoclast differentiation markers, including TRAP, and reduced the number of mature osteoclasts. Structurally, the 158-175 amino acid sequence in the carbohydrate recognition domain (CRD) of Gal-3 was responsible for augmented osteoclastogenesis. During osteoclast maturation, Gal-3 interacted and co-localized with myosin-2A along the surface of cell-cell fusion. Pathologically, bone metastatic cancers expressed and released an intact form of Gal-3, mainly detected in breast cancer bone metastases, as well as a cleaved form, more abundant in prostate cancer bone metastases. Secreted intact Gal-3 interacted with myosin-2A, leading to osteoclastogenesis, whereas a shift to cleaved Gal-3 attenuated the enhancement in osteoclast differentiation. Thus, our studies demonstrate that Gal-3 shapes the bone tumor microenvironment through distinct roles contingent on its cleavage status, and highlight Gal-3 targeting through the CRD as a potential therapeutic strategy for mitigating osteolytic bone remodeling in the metastatic niche.

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ILK induction in lymphoid organs by a TNF{alpha}-NF-{kappa}B-regulated pathway promotes the development of chronic lymphocytic leukemia

The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell cycle progression, but is poorly characterized in hematological malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high risk markers such as unmutated IGHV genes, high Zap70 or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes (LNs) from CLL patients. Using co-culture conditions mimicking the proliferative LN microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein co-localized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B cell-specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα-NF-κB-mediated mechanism by which ILK expression is induced in the LN microenvironment, and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation.-

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Pharmacological inhibition of the histone lysine demethylase KDM1A suppresses the growth of multiple acute myeloid leukemia subtypes

Lysine specific demethylase 1 (KDM1A) is a transcriptional co-regulator that can function in both the activation and repression of gene expression, depending upon context. KDM1A plays an important role in hematopoiesis and was identified as a dependency factor in leukemia stem cell populations. Therefore, we investigated the consequences of inhibiting KDM1A in a panel of cell lines representing all acute myeloid leukemia (AML) subtypes using selective, reversible and irreversible KDM1A small molecule inhibitors. Cell models of AML, CML and T-ALL were potently affected by KDM1A inhibition, and cells bearing RUNX1-RUNX1T1 (AML1-ETO) translocations were especially among the most sensitive. RNAi-mediated silencing of KDM1A also effectively suppressed growth of RUNX1-RUNX1T1-containing cell lines. Furthermore, pharmacological inhibition of KDM1A resulted in complete abrogation of tumor growth in an AML xenograft model harboring RUNX1-RUNX1T1 translocations. We unexpectedly found that KDM1A-targeting compounds not only inhibited the catalytic activity of the enzyme, but evicted KDM1A from target genes. Accordingly, compound-mediated KDM1A eviction was associated with elevated levels of local histone H3 lysine 4 dimethylation, and increased target gene expression, which was further accompanied by cellular differentiation and induction of cell death. Finally, our finding that KDM1A inhibitors effectively synergize with multiple conventional as well as candidate anti-AML agents affords a framework for potential future clinical application.

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G0S2 suppresses oncogenic transformation by repressing a Myc-regulated transcriptional program

Methylation-mediated silencing of G0S2 has been detected in a variety of solid tumors, whereas G0S2 induction is associated with remissions in patients with acute promyelocytic leukemia, implying that G0S2 may possess tumor suppressor activity. In this study, we clearly demonstrate that G0S2 opposes oncogene-induced transformation using G0S2-null immortalized mouse embryonic fibroblasts (MEFs). G0S2-null MEFs were readily transformed with HRAS or EGFR treatment compared to wildtype MEFs. Importantly, restoration of G0S2 reversed HRAS-driven transformation. G0S2 is known to regulate fat metabolism by attenuating adipose triglyceride lipase (ATGL), but repression of oncogene-induced transformation by G0S2 was independent of ATGL inhibition. Gene expression analysis revealed that an upregulation of gene signatures associated with transformation, proliferation, and MYC targets in G0S2-null MEFs. RNAi-mediated ablation and pharmacologic inhibition of MYC abrogated oncogene-induced transformation of G0S2-null MEFs. Furthermore, we found that G0S2 was highly expressed in normal breast tissues compared to malignant tissue. Intriguingly, high levels of G0S2 were also associated with a decrease in breast cancer recurrence rates, especially in estrogen receptor-positive subtypes, and overexpression of G0S2 repressed the proliferation of breast cancer cells in vitro. Taken together, these findings indicate that G0S2 functions as a tumor suppressor in part by opposing MYC activity, prompting further investigation of the mechanisms by which G0S2 silencing mediates MYC-induced oncogenesis in other malignancies.

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Ranking Anticancer Drugs

Whole chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a distinguishing feature of most cancer types. CIN is generally considered to drive tumorigenesis, but a threshold level exists whereby further increases in CIN frequency in fact hinder tumor growth. While this attribute is appealing for therapeutic exploitation, drugs that increase CIN beyond this therapeutic threshold are currently limited. In our previous work, we developed a quantitative assay for measuring CIN based on the use of a nonessential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Here, we used this assay to rank 62 different anticancer drugs with respect to their effects on chromosome transmission fidelity. Drugs with various mechanisms of action, such as antimicrotubule activity, histone deacetylase inhibition, mitotic checkpoint inhibition, and targeting of DNA replication and damage responses, were included in the analysis. Ranking of the drugs based on their ability to induce HAC loss revealed that paclitaxel, gemcitabine, dactylolide, LMP400, talazoparib, olaparib, peloruside A, GW843682, VX-680, and cisplatin were the top 10 drugs demonstrating HAC loss at a high frequency. Therefore, identification of currently used compounds that greatly increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target and leverage the CIN phenotype in cancer cells. Cancer Res; 76(4); 1–10. ©2016 AACR.

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CD74-NRG1 Fusion Gene Product Confers Cancer Stem Cell Properties

The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucinous adenocarcinoma, a malignant form of lung cancer. However, the function of the CD74-NRG1 fusion gene in adenocarcinoma pathogenesis and the mechanisms by which it may impart protumorigenic characteristics to cancer stem cells (CSC) is still unclear. In this study, we found that the expression of the CD74-NRG1 fusion gene increased the population of lung cancer cells with CSC-like properties. CD74-NRG1 expression facilitated sphere formation not only of cancer cells, but also of nonmalignant lung epithelial cells. Using a limiting dilution assay in a xenograft model, we further show that the CD74-NRG1 fusion gene enhanced tumor initiation. Mechanistically, we found that CD74-NRG1 expression promoted the phosphorylation of ErbB2/3 and activated the PI3K/Akt/NF-κB signaling pathway. Furthermore, the expression of the secreted insulin-like growth factor 2 (IGF2) and phosphorylation of its receptor, IGF1R, were enhanced in an NF-κB–dependent manner in cells expressing CD74-NRG1. These findings suggest that CD74-NRG1–induced NF-κB activity promotes the IGF2 autocrine/paracrine circuit. Moreover, inhibition of ErbB2, PI3K, NF-κB, or IGF2 suppressed CD74-NRG1–induced tumor sphere formation. Therefore, our study provides a preclinical rationale for developing treatment approaches based on these identified pathways to suppress CSC properties that promote tumor progression and recurrence. Cancer Res; 76(4); 1–10. ©2016 AACR.

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Major Advance in Hypoxia Imaging



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Hypoxia-Induced EMT Creates Immunosuppression

Portal vein tumor thrombosis (PVTT) is a significant risk factor for metastasis in hepatocellular carcinoma (HCC) patients and is therefore associated with poor prognosis. The presence of PVTT frequently accompanies substantial hypoxia within the tumor microenvironment, which is suggested to accelerate tumor metastasis, but it is unclear how this occurs. Recent evidence has shown that the hypoxia-inducible factor HIF-1α induces epithelial-to-mesenchymal transition (EMT) in tumor cells to facilitate metastasis. In this study, we investigated whether hypoxia-induced EMT in cancer cells also affects immune cells in the tumor microenvironment to promote immunosuppression. We found that hypoxia-induced EMT increased the expression of the CCL20 cytokine in hepatoma cells. Furthermore, coculture of monocyte-derived macrophages with hypoxic hepatoma cells revealed that the expression of indoleamine 2, 3-dioxygenase (IDO) was induced in monocyte-derived macrophages in a CCL20-dependent manner. In turn, these IDO-expressing monocyte-derived macrophages suppressed T-cell proliferation and promoted the expansion of immunosuppressive regulatory T cells. Moreover, high CCL20 expression in HCC specimens was associated with PVTT and poor patient survival. Collectively, our findings suggest that the HIF-1α/CCL20/IDO axis in hepatocellular carcinoma is important for accelerating tumor metastasis through both the induction of EMT and the establishment of an immunosuppressive tumor microenvironment, warranting further investigation into the therapeutic effects of blocking specific nodes of this signaling network. Cancer Res; 76(4); 1–13. ©2016 AACR.

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Requirement of HMGA2 in TSC

Tuberous sclerosis (TSC) is a tumor suppressor gene syndrome that is associated with the widespread development of mesenchymal tumor types. Genetically, TSC is said to occur through a classical biallelic inactivation of either TSC genes (TSC1, hamartin or TSC2, tuberin), an event that is implicated in the induction of the mTOR pathway and subsequent tumorigenesis. High Mobility Group A2 (HMGA2), an architectural transcription factor, is known to regulate mesenchymal differentiation and drive mesenchymal tumorigenesis in vivo. Here, we investigated the role of HMGA2 in the pathogenesis of TSC using the TSC2+/− mouse model that similarly mirrors human disease and human tumor samples. We show that HMGA2 expression was detected in 100% of human and mouse TSC tumors and that HMGA2 activation was required for TSC mesenchymal tumorigenesis in genetically engineered mouse models. In contrast to the current dogma, the mTOR pathway was not activated in all TSC2+/− tumors and was elevated in only 50% of human mesenchymal tumors. Moreover, except for a subset of kidney tumors, tuberin was expressed in both human and mouse tumors. Therefore, haploinsufficiency of one TSC tumor suppressor gene was required for tumor initiation, but further tumorigenesis did not require the second hit, as previously postulated. Collectively, these findings demonstrate that tissue-specific genetic mechanisms are employed to promote tumor pathogenesis in TSC and identify a novel, critical pathway for potential therapeutic targeting. Cancer Res; 76(4); 1–11. ©2016 AACR.

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