Triple-negative breast cancers (including basal-like and claudin-low molecular subtypes) represent 20% to 25% of all breast cancers, but disproportionately contribute to breast cancer-associated death. We have identified a novel fundamental biological property of triple-negative breast cancers: most triple-negative breast cancers express aberrant DNA hypermethylation due to overexpression of DNA methyltransferase 3b (and hyperactivity of the DNA methyltransferase enzymes). DNA methyltransferase 3b overexpression occurs secondary to loss of miRNA-mediated post-transcriptional regulation. The resulting hyperactivity of DNA methyltransferase 3b produces concurrent DNA methylation-dependent silencing of numerous critical gene targets (including tumor suppressors and pro-apoptotic genes) and resistance to cytotoxic chemotherapy. This observation presents new opportunities for development of innovative treatment strategies on the basis of the epigenome as a novel therapeutic target in triple-negative breast cancers. Epigenetic therapy represents a new principle in cancer treatment in which restoration of critical molecular pathways occurs secondary to reexpression of silenced genes that encode negative mediators of cancer cell growth.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase reported to be overexpressed in human leukemia. Though much regarding the function of LTK remains unknown, it shares a high degree of similarity with anaplastic lymphoma kinase (ALK), which is found mutated in human cancer. In order to determine if LTK has transforming potential, we created two LTK mutants, F568L and R669Q, that correspond to two well-characterized activating mutations of ALK (F1174L and R1275Q). LTK-F568L, but not wildtype LTK or LTK-R669Q, transformed hematopoietic cells to cytokine independence. LTK-F568L exhibited a stronger ability to induce loss of contact inhibition and anchorage-independent growth of epithelial cells compared to LTK-R669Q, while wildtype LTK was non-transforming in the same cells. Likewise, LTK-F568L induced greater neurite outgrowth of PC12 cells than R669Q, while wildtype LTK could not. Correlating with transforming activity, LTK-F568L displayed significantly enhanced tyrosine phosphorylation compared to wildtype LTK and LTK-R668Q and induced activation of various signaling proteins including Shc, ERK and the JAK/STAT pathway. Expression of wildtype LTK or LTK-R669Q generally led to weaker activation of signaling proteins than expression of LTK-F568L, or no activation at all. Thus, mutating LTK at residue F568, and to a lesser extent at R669, activates the receptor tyrosine kinase, inducing cell signaling that results in transforming properties. These studies suggest that aberrant activation of LTK may contribute to neoplastic cell growth.

Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR-ABL (breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.

Expression of cytokine receptor-like factor 2 (CRLF2) has recently been shown to be upregulated as well as mutated in populations of B-progenitor acute lymphoblastic leukemia (B-ALL), including Down syndrome (DS-ALL) patients, lacking recurring chromosomal translocations. Increased CRLF2 expression associates with JAK2 mutation, a combination that transforms hematopoietic cells, suggesting that mutant JAK2 and CRLF2 may cooperate to contribute to B-ALL formation. Importantly, elevated CRLF2 expression correlates with poor outcome in high-risk B-ALL patients. Therefore, CRLF2 may provide a new prognostic marker for high-risk B-ALL, and inhibition of CRLF2/JAK2 signaling may represent a therapeutic approach for this population of ALL patients.

A subset of breast cancer cell lines express a hypermethylation defect characterized by DNMT hyperactivity, overexpression of DNMT3b, and silencing of numerous genes. To investigate the role of DNMT3b in the hypermethylation defect, we examined the effect of RNAi‐mediated DNMT3b knockdown on expression of methylation‐sensitive genes in model hypermethylator cell lines: MDA‐MB‐453 and BT549. Western blot analysis confirmed reduction of DNMT3b in cells transfected with the DNMT3b RNAi targeting construct. RT‐PCR was used to analyze expression of six methylation‐sensitive genes (CEACAM6, CST6, ESR1, SCNN1A, GNA11, CDH1 and/or MUC1) that are methylated and exhibit diminished or no expression in parent cell lines compared to MCF12A. In MDA‐MB‐453 cells, all six genes were reexpressed or increased expression in response to DNMT3b knockdown. CEACAM6, CST6, ESR1, and GNA11 were expressed at normal levels, while MUC1 and SCNN1A were expressed at low but detectable levels. In BT549 cells, CEACAM6, CST6, ESR1, GNA11, and SCNN1A were reexpressed or increased expression in response to DNMT3b knockdown, whereas CDH1 was not expressed. These results strongly suggest that CEACAM6, CST6, ESR1, GNA11, SCNN1A, and MUC1 are direct methylation targets for DNMT3b. Further, these results suggest that DNMT3b overexpression governs the hypermethylator defect associated with some breast cancer cell lines.Support: NIH CA78343

A subset of breast cancers express a hypermethylation defect characterized by DNMT hyperactivity, overexpression of DNMT3b, and concurrent silencing of numerous genes. This hypermethylation defect is a feature of most basal breast cancers. Our goal was to determine the mechanism responsible for DNMT3b overexpression in model breast cancer cell lines. Several microRNAs (miRs) have been implicated in the regulation of DNMT3b. To investigate the role of miRs in DNMT3b overexpression, we analyzed miRs‐29a, 29b, 29c, 148a, and 148b in a panel of 16 breast cancer cell lines classified as hypermethylators (n=10) or low frequency methylators (n=6). 80% of hypermethylator cell lines express low levels of >3 miRs and 83% of low frequency methylator cell lines express >3 miRs at normal levels. Cell lines with high DNMT3b levels (SUM185, SUM102, SUM149) expressed all miRs at low or negligible levels, whereas cell lines with low DNMT3b levels (MDA‐MB‐415, MDA‐MB‐468, BT20, ZR‐75‐1) expressed all miRs normally. Further, miR expression patterns correlated with methylation‐sensitive gene expression among these cell lines (R=0.54, p=0.012). These findings suggest that dysregulation of miR expression drives DNMT3b overexpression in hypermethylator cell lines, and identifies DNMT3b and its regulatory miRs as targets for development of novel therapeutic strategies for treating basal breast cancers.Support: NIH CA78343

Background DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines. Results The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes ( CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A ), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1 , CEACAM6, CST6, ESR1, LCN2 , and SCNN1A . This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers. Conclusion These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.