Epigenetic deregulation of gene expression plays a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma Set Domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared to immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, while depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar, and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial to mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus, leading to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET plays a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3′-untranslated region (3′-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

Purpose: To understand the changes in gene expression in polycythemia vera (PV) progenitor cells and their relationship to JAK2V617F.Experimental Design: Messenger RNA isolated from CD34 + cells from nine PV patients and normal controls was profiled using Affymetrix arrays. Gene expression change mediated by JAK2V617F was determined by profiling CD34 + cells transduced with the kinase and by analysis of leukemia cell lines harboring JAK2V617F, treated with an inhibitor.Results: A PV expression signature was enriched for genes involved in hematopoietic development, inflammatory responses, and cell proliferation. By quantitative reverse transcription-PCR, 23 genes were consistently deregulated in all patient samples. Several of these genes such as WT1 and KLF4 were regulated by JAK2, whereas others such as NFIB and EVI1 seemed to be deregulated in PV by a JAK2-independent mechanism. Using cell line models and comparing gene expression profiles of cell lines and PV CD34 + PV specimens, we have identified panels of 14 JAK2-dependent genes and 12 JAK2-independent genes. These two 14-and 12-gene sets could separate not only PV from normal CD34 + specimens, but also other MPN such as essential thrombocytosis and primary myelofibrosis from their normal counterparts.Conclusions: A subset of the aberrant gene expression in PV progenitor cells can be attributed to the action of the mutant kinase, but there remain a significant number of genes characteristic of the disease but deregulated by as yet unknown mechanisms. Genes deregulated in PV as a result of the action of JAK2V617F or independent of the kinase may represent other targets for therapy.

The WTX, Wilms tumor-associated tumor-suppressor gene, is present on the X chromosome and a single WTX mutation may be sufficient to promote carcinogenesis. Unlike the WT1 tumor suppressor, a transcription factor, WTX lacks conserved functional protein domains. To study the function of WTX, we constructed inducible cell lines expressing WTX and tumor-associated WTX mutants. Induction of WTX inhibited cell growth and caused G 1 /G 0 arrest. In contrast, a short, tumorassociated truncation mutant of WTX358 only slightly inhibited cell growth without a significant cell-cycle arrest, although expression of a longer truncation mutant WTX565 led to the growth inhibition and cell-cycle arrest to a similar extent as wild-type WTX. Like WT1, WTX slowed growth and caused cell-cycle arrest through p21 induction. Gene expression profiling showed that these two tumorsuppressors regulated genes in similar pathways, including those implicated in control of the cellular growth, cell cycle, cell death, cancer and cardiovascular system development. When gene expression changes mediated by wild-type WTX were compared with those affected by mutant forms, WTX565 showed a 55% overlap (228 genes) in differentially regulated genes, whereas WTX358 regulated only two genes affected by wild-type WTX, implying that amino-acid residues 358-561 are critical for WTX function.

Cancer cell resistance to chemotherapy may be mediated by defects in apoptotic pathways. A prior study showed that in vivo apoptosis of Acute Lymphoblastic Leukemia (ALL) blasts in response to chemotherapy could occur through diverse pathways including both p53-dependent and -independent mechanisms. In this study we investigated the apoptotic response in more detail by using a panel of ALL cell lines that differed in respect to p53 status. Upon exposure to a uniform stimulus, expression of apoptotic proteins, including the effector caspase-3, varied among ALL cell lines partly depending on p53 transcriptional activity and caspase-8 activation. Although the expression and contribution to apoptosis differed among known members of the apoptotic pathway, apoptosis was universally mediated by mitochondrial depolarization. The NFkappaB pathway was activated in response to chemotherapy but NFkappaB inhibition appeared to not influence chemosensitivity. This study further documents the highly variable nature of cell death programs in ALL and provides the foundation for cell death pathway modulation to improve ALL cure rates without increasing chemotherapy-related toxicity.

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In this issue of Cancer Cell, Rui et al. identify JAK2 and JMJDC2 as two contiguous, co-amplified oncogenes in primary mediastinal B-cell and Hodgkin lymphoma. Together JAK2 and JMJD2C induce major changes in chromatin structure and gene expression. Targeting theses protein with small molecules represents a new avenue for therapy.

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