Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer.

Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the Polyoma Middle-T antigen genetically engineered mouse model (MMTV-PyMT) demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.

Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer.

The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.

The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.

Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity.

Development and validation of prognostic gene signature for basal-like breast cancer and high-grade serous ovarian cancer.

PURPOSE: Molecular similarities have been reported between basal-like breast cancer (BLBC) and high-grade serous ovarian cancer (HGSOC). To date, there have been no prognostic biomarkers that can provide risk stratification and inform treatment decisions for both BLBC and HGSOC. In this study, we developed a molecular signature for risk stratification in BLBC and further validated this signature in HGSOC. METHODS: RNA-seq data was downloaded from The Cancer Genome Atlas (TCGA) project for 190 BLBC and 314 HGSOC patients. Analyses of differentially expressed genes between recurrent vs. non-recurrent cases were performed using different bioinformatics methods. Gene Signature was established using weighted linear combination of gene expression levels. Their prognostic performance was evaluated using survival analysis based on progression-free interval (PFI) and disease-free interval (DFI). RESULTS: 63 genes were differentially expressed between 18 recurrent and 40 non-recurrent BLBC patients by two different methods. The recurrence index (RI) calculated from this 63-gene signature significantly stratified BLBC patients into two risk groups with 38 and 152 patients in the low-risk (RI-Low) and high-risk (RI-High) groups, respectively (p = 0.0004 and 0.0023 for PFI and DFI, respectively). Similar performance was obtained in the HGSOC cohort (p = 0.0131 and 0.004 for PFI and DFI, respectively). Multivariate Cox regression adjusting for age, grade, and stage showed that the 63-gene signature remained statistically significant in stratifying HGSOC patients (p = 0.0005). CONCLUSION: A gene signature was identified to predict recurrence in BLBC and HGSOC patients. With further validation, this signature may provide an additional prognostic tool for clinicians to better manage BLBC, many of which are triple-negative and HGSOC patients who are currently difficult to treat.

Genome-wide DNA methylation analysis of colorectal adenomas with and without recurrence reveals an association between cytosine-phosphate-guanine methylation and histological subtypes.

Aberrant methylation of DNA is supposed to be a major and early driver of colonic adenoma development, which may result in colorectal cancer (CRC). Although gene methylation assays are used already for CRC screening, differential epigenetic alterations of recurring and nonrecurring colorectal adenomas have yet not been systematically investigated. Here, we collected a sample set of formalin-fixed paraffin-embedded colorectal low-grade adenomas (n = 72) consisting of primary adenomas without and with recurrence (n = 59), recurrent adenomas (n = 10), and normal mucosa specimens (n = 3). We aimed to unveil differentially methylated CpG positions (DMPs) across the methylome comparing not only primary adenomas without recurrence vs primary adenomas with recurrence but also primary adenomas vs recurrent adenomas using the Illumina Human Methylation 450K BeadChip array. Unsupervised hierarchical clustering exhibited a significant association of methylation patterns with histological adenoma subtypes. No significant DMPs were identified comparing primary adenomas with and without recurrence. Despite that, a total of 5094 DMPs (false discovery rate <0.05; fold change >10%) were identified in the comparisons of recurrent adenomas vs primary adenomas with recurrence (674; 98% hypermethylated), recurrent adenomas vs primary adenomas with and without recurrence (241; 99% hypermethylated) and colorectal adenomas vs normal mucosa (4179; 46% hypermethylated). DMPs in cytosine-phosphate-guanine (CpG) islands were frequently hypermethylated, whereas open sea- and shelf-regions exhibited hypomethylation. Gene ontology analysis revealed enrichment of genes associated with the immune system, inflammatory processes, and cancer pathways. In conclusion, our methylation data could assist in establishing a more robust and reproducible histological adenoma classification, which is a prerequisite for improving surveillance guidelines.

Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7.

Accumulating evidence supports the role of an aberrant transcriptome as a driver of metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. Previously, we demonstrated that the CCR4-NOT scaffold component Cnot2 is an inherited metastasis susceptibility gene. In this study, using orthotopic metastasis assays and genetically engineered mouse models, we show that one of the enzymatic subunits of the CCR4-NOT complex, Cnot7, is also a metastasis modifying gene. We demonstrate that higher expression of Cnot7 drives tumor cell autonomous metastatic potential, which requires its deadenylase activity. Furthermore, metastasis promotion by CNOT7 is dependent on interaction with CNOT1 and TOB1. CNOT7 ribonucleoprotein-immunoprecipitation (RIP) and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3'UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis.

An Integrated Genome-Wide Systems Genetics Screen for Breast Cancer Metastasis Susceptibility Genes.

Metastasis remains the primary cause of patient morbidity and mortality in solid tumors and is due to the action of a large number of tumor-autonomous and non-autonomous factors. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways in breast cancer metastasis. This analysis implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. Moreover, the analysis identified novel or FDA-approved drugs as potentially useful for anti-metastatic therapy. Further explorations implementing this strategy may therefore provide a variety of information for clinical applications in the control and treatment of advanced neoplastic disease.

Association of high-evidence gastric cancer susceptibility loci and somatic gene expression levels with survival.

Eleven high-evidence single-nucleotide polymorphisms (SNPs) at nine loci for gastric cancer (GC) risk were reported, but their associations with survival remain unknown. In this study, we examined associations between SNP and GC survival by anatomic location and histology among 1147 incident cases from the Shanxi Upper Gastrointestinal Genetics Project. We further examined whether SNPs were expression quantitative trait loci in normal and tumor gastric tissues, and whether tumor versus normal somatic mRNA differences in 126 cases were associated with survival. No SNPs were associated with GC survival overall. However, subtype-specific associations were observed for gastric cardia adenocarcinomas at MUC1/TRIM46/1q22 rs2070803 [HRAA versus GA+GG = 2.16; 95% confidence interval (CI) = 1.24-3.78; P = 0.0068] and LTA/TNF/6p21.33 rs1799724 (HRTT+CT versus CC = 1.30; 95% CI = 1.07-1.57; P = 0.0077), and for diffuse-type GC at PSCA/8q24.3 rs2294008 (HRTT versus CT+CC = 1.99; 95% CI = 1.33-2.97; P = 7.8E-04). Rs2294008T was a cis-expression quantitative trait loci for PSCA, upregulating mRNA in normal gastric (ß = 0.60; P = 5.7E-21) and GC (ß = 0.30; P = 0.0089) tissues. Cases in the highest quartile (the smallest downregulation of tumor PSCA) had shortest survival than cases with the most downregulated PSCA (median survival of 0.47 years in the highest quartile versus 3.73 years in the lowest quartile; hazard ratio = 9.70; 95% CI = 2.46-38.4; P = 0.0012). Less striking effects for mRNA levels were observed for MTX1 at 1q22 in gastric cardia adenocarcinoma and for JRK at 8q24.3 in diffuse GC. Our results suggest three high-evidence GC risk loci have prognostic importance in GC subtypes. Future studies in well-characterized independent populations are warranted to validate our findings and further investigate the clinical utility of these variants in predicting GC prognosis.

p53 loss increases the osteogenic differentiation of bone marrow stromal cells.

The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild-type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild-type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild-type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence.

Integrative genomics analysis of genes with biallelic loss and its relation to the expression of mRNA and micro-RNA in esophageal squamous cell carcinoma.

BACKGROUND: Genomic instability plays an important role in human cancers. We previously characterized genomic instability in esophageal squamous cell carcinomas (ESCC) in terms of loss of heterozygosity (LOH) and copy number (CN) changes in tumors. In the current study we focus on biallelic loss and its relation to expression of mRNA and miRNA in ESCC using results from 500 K SNP, mRNA, and miRNA arrays in 30 cases from a high-risk region of China. RESULTS: (i) Biallelic loss was uncommon but when it occurred it exhibited a consistent pattern: only 77 genes (<0.5%) showed biallelic loss in at least 10% of ESCC samples, but nearly all of these genes were concentrated on just four chromosomal arms (i.e., 42 genes on 3p, 14 genes on 9p, 10 genes on 5q, and seven genes on 4p). (ii) Biallelic loss was associated with lower mRNA expression: 52 of the 77 genes also had RNA expression data, and 41 (79%) showed lower expression levels in cases with biallelic loss compared to those without. (iii) The relation of biallelic loss to miRNA expression was less clear but appeared to favor higher miRNA levels: of 60 miRNA-target gene pairs, 34 pairs (57%) had higher miRNA expression with biallelic loss than without, while 26 pairs (43%) had lower miRNA expression. (iv) Finally, the effect of biallelic loss on the relation between miRNA and mRNA expression was complex. Biallelic loss was most commonly associated with a pattern of elevated miRNA and reduced mRNA (43%), but a pattern of both reduced miRNA and mRNA was also common (35%). CONCLUSION: Our results indicate that biallelic loss in ESCC is uncommon, but when it occurs it is localized to a few specific chromosome regions and is associated with reduced mRNA expression of affected genes. The effect of biallelic loss on miRNA expression and on the relation between miRNA and mRNA expressions was complex.

An integrated genomic approach identifies persistent tumor suppressive effects of transforming growth factor-ß in human breast cancer.

INTRODUCTION: Transforming growth factor-ßs (TGF-ßs) play a dual role in breast cancer, with context-dependent tumor-suppressive or pro-oncogenic effects. TGF-ß antagonists are showing promise in early-phase clinical oncology trials to neutralize the pro-oncogenic effects. However, there is currently no way to determine whether the tumor-suppressive effects of TGF-ß are still active in human breast tumors at the time of surgery and treatment, a situation that could lead to adverse therapeutic responses. METHODS: Using a breast cancer progression model that exemplifies the dual role of TGF-ß, promoter-wide chromatin immunoprecipitation and transcriptomic approaches were applied to identify a core set of TGF-ß-regulated genes that specifically reflect only the tumor-suppressor arm of the pathway. The clinical significance of this signature and the underlying biology were investigated using bioinformatic analyses in clinical breast cancer datasets, and knockdown validation approaches in tumor xenografts. RESULTS: TGF-ß-driven tumor suppression was highly dependent on Smad3, and Smad3 target genes that were specifically enriched for involvement in tumor suppression were identified. Patterns of Smad3 binding reflected the preexisting active chromatin landscape, and target genes were frequently regulated in opposite directions in vitro and in vivo, highlighting the strong contextuality of TGF-ß action. An in vivo-weighted TGF-ß/Smad3 tumor-suppressor signature was associated with good outcome in estrogen receptor-positive breast cancer cohorts. TGF-ß/Smad3 effects on cell proliferation, differentiation and ephrin signaling contributed to the observed tumor suppression. CONCLUSIONS: Tumor-suppressive effects of TGF-ß persist in some breast cancer patients at the time of surgery and affect clinical outcome. Carefully tailored in vitro/in vivo genomic approaches can identify such patients for exclusion from treatment with TGF-ß antagonists.

Protein phosphatase 6 activates NF-κB to confer sensitivity to MAPK pathway inhibitors in KRAS- and BRAF-mutant cancer cells.

The Ras-mitogen-activated protein kinase (MAPK) pathway is a major target for cancer treatment. To better understand the genetic pathways that modulate cancer cell sensitivity to MAPK pathway inhibitors, we performed a CRISPR knockout screen with MAPK pathway inhibitors on a colorectal cancer (CRC) cell line carrying mutant KRAS. Genetic deletion of the catalytic subunit of protein phosphatase 6 (PP6), encoded by PPP6C, rendered KRAS- and BRAF-mutant CRC and BRAF-mutant melanoma cells more resistant to these inhibitors. In the absence of MAPK pathway inhibition, PPP6C deletion in CRC cells decreased cell proliferation in two-dimensional (2D) adherent cultures but accelerated the growth of tumor spheroids in 3D culture and tumor xenografts in vivo. PPP6C deletion enhanced the activation of nuclear factor κB (NF-κB) signaling in CRC and melanoma cells and circumvented the cell cycle arrest and decreased cyclin D1 abundance induced by MAPK pathway blockade in CRC cells. Inhibiting NF-κB activity by genetic and pharmacological means restored the sensitivity of PPP6C-deficient cells to MAPK pathway inhibition in CRC and melanoma cells in vitro and in CRC cells in vivo. Furthermore, a R264 point mutation in PPP6C conferred loss of function in CRC cells, phenocopying the enhanced NF-κB activation and resistance to MAPK pathway inhibition observed for PPP6C deletion. These findings demonstrate that PP6 constrains the growth of KRAS- and BRAF-mutant cancer cells, implicates the PP6-NF-κB axis as a modulator of MAPK pathway output, and presents a rationale for cotargeting the NF-κB pathway in PPP6C-mutant cancer cells.

Apoptosis-induced nuclear expulsion in tumor cells drives S100a4-mediated metastatic outgrowth through the RAGE pathway.

Most tumor cells undergo apoptosis in circulation and at the metastatic organ sites due to host immune surveillance and a hostile microenvironment. It remains to be elucidated whether dying tumor cells have a direct effect on live tumor cells during the metastatic process and what the underlying mechanisms are. Here we report that apoptotic cancer cells enhance the metastatic outgrowth of surviving cells through Padi4-mediated nuclear expulsion. Tumor cell nuclear expulsion results in an extracellular DNA-protein complex that is enriched with receptor for advanced glycation endproducts (RAGE) ligands. The chromatin-bound RAGE ligand S100a4 activates RAGE receptors in neighboring surviving tumor cells, leading to Erk activation. In addition, we identified nuclear expulsion products in human patients with breast, bladder and lung cancer and a nuclear expulsion signature correlated with poor prognosis. Collectively, our study demonstrates how apoptotic cell death can enhance the metastatic outgrowth of neighboring live tumor cells.

Melanoma clonal subline analysis uncovers heterogeneity-driven immunotherapy resistance mechanisms.

Intratumoral heterogeneity (ITH) can promote cancer progression and treatment failure, but the complexity of the regulatory programs and contextual factors involved complicates its study. To understand the specific contribution of ITH to immune checkpoint blockade (ICB) response, we generated single cell-derived clonal sublines from an ICB-sensitive and genetically and phenotypically heterogeneous mouse melanoma model, M4. Genomic and single cell transcriptomic analyses uncovered the diversity of the sublines and evidenced their plasticity. Moreover, a wide range of tumor growth kinetics were observed in vivo , in part associated with mutational profiles and dependent on T cell-response. Further inquiry into melanoma differentiation states and tumor microenvironment (TME) subtypes of untreated tumors from the clonal sublines demonstrated correlations between highly inflamed and differentiated phenotypes with the response to anti-CTLA-4 treatment. Our results demonstrate that M4 sublines generate intratumoral heterogeneity at both levels of intrinsic differentiation status and extrinsic TME profiles, thereby impacting tumor evolution during therapeutic treatment. These clonal sublines proved to be a valuable resource to study the complex determinants of response to ICB, and specifically the role of melanoma plasticity in immune evasion mechanisms.

Modified eQTL and Somatic DNA Segment Alterations in Esophageal Squamous Cell Carcinoma for Genes Related to Immunity, DNA Repair, and Inflammation.

We integrated ESCC expression and GWAS genotyping, to investigate eQTL and somatic DNA segment alterations, including somatic copy number alteration, allelic imbalance (AI), and loss of heterozygosity (LOH) in ESCC. First, in eQTL analysis, we used a classical approach based on genotype data from GWAS and expression signals in normal tissue samples, and then used a modified approach based on fold change in the tumor vs. normal samples. We focused on the genes in three pathways: inflammation, DNA repair, and immunity. Among the significant (p < 0.05) SNP-probe pairs from classical and modified eQTL analyses, 24 genes were shared by the two approaches, including 18 genes that showed the same numbers of SNPs and probes and 6 genes that had the different numbers of SNPs and probes. For these 18 genes, we found 28 SNP−probe pairs were correlated in opposite directions in the two approaches, indicating an intriguing difference between the classical and modified eQTL approaches. Second, we analyzed the somatic DNA segment alterations. Across the 24 genes, abnormal gene expression on mRNA arrays was seen in 19−95% of cases and 26−78% showed somatic DNA segment alterations on Affymetrix GeneChip Human Mapping Arrays. The results suggested that this strategy could identify gene expression and somatic DNA segment alterations for biological markers (genes) by combining classical and modified eQTLs and somatic DNA evaluation on SNP arrays. Thus, this study approach may allow us to understand functionality indicative of potentially relevant biomarkers in ESCC.

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

DNA Methyltransferase 3B-Mediated Intratumoral Heterogeneity and Therapeutic Targeting in Breast Cancer Recurrence and Metastasis.

The mechanisms of how cancer cells are selected and evolve to establish distant metastatic colonies remain unclear. Tumor heterogeneity and lack of biomarkers are some of the most difficult challenges in cancer biology and treatment. Here using mouse models for triple-negative breast cancer (TNBC) metastasis, we report heterogeneous expression of DNA methyltransferase 3B (DNMT3B) in both mouse and human primary tumors. High levels of DNMT3B were correlated with poor clinical outcomes in multiple human breast cancer datasets. Mechanistically, clonal cells with high DNMT3B (DNMT3BH) showed higher vimentin (VIM) expression and displayed enhanced epithelial-to-mesenchymal transition capacity. Deletion of VIM diminished the metastatic phenotype of DNMT3BH cells. Importantly, in preclinical mouse models in which the primary tumors were surgically removed, perioperative targeting of DNMT3B in combination with chemotherapy markedly suppressed tumor recurrence and metastasis. Our studies identify DNMT3B-mediated transcription regulation as an important mediator of tumor heterogeneity and show that DNMT3B is critical for tumor invasion and metastasis, reinforcing its potential as a target for treating metastatic disease. IMPLICATIONS: Our findings of transcriptome changes mediated by DNMT3B provide new mechanistic insight for intratumor heterogeneity and chemoresistance, and therapeutic targeting of DNMT3B in combination with chemotherapy offer additional treatment options for metastatic disease especially for patients with TNBC.