Smyd2 is a Myc-regulated gene critical for MLL-AF9 induced leukemogenesis.

The Smyd2 protein (Set- and Mynd domain containing protein 2) is a methyl-transferase that can modify both histones and cytoplasmic proteins. Smyd2 is over-expressed in several cancer types and was shown to be limiting for tumor development in the pancreas. However, genetic evidence for a role of Smyd2 in other cancers or in mouse development was missing to date. Using germ line-deleted mouse strains, we now show that Smyd2 and the related protein Smyd3 are dispensable for normal development. Ablation of Smyd2 did not affect hematopoiesis, but retarded the development of leukemia promoted by MLL-AF9, a fusion oncogene associated with acute myeloid leukemia (AML) in humans. Smyd2-deleted leukemic cells showed a competitive disadvantage relative to wild-type cells, either in vitro or in vivo. The Smyd2 gene was directly activated by the oncogenic transcription factor Myc in either MLL9-AF9-induced leukemias, Myc-induced lymphomas, or fibroblasts. However, unlike leukemias, the development of lymphomas was not dependent upon Smyd2. Our data indicate that Smyd2 has a critical role downstream of Myc in AML.

Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism.

The ability to generate senescent progeny as a mechanism underlying breast cancer cell heterogeneity.

BACKGROUND: Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. METHODOLOGY/PRINCIPAL FINDINGS: A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21(Cip1) correlated with senescence in these cell lines. p21(Cip1) knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. CONCLUSIONS/SIGNIFICANCE: Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.

Senescence and immortality in hepatocellular carcinoma.

Cellular senescence is a process leading to terminal growth arrest with characteristic morphological features. This process is mediated by telomere-dependent, oncogene-induced and ROS-induced pathways, but persistent DNA damage is the most common cause. Senescence arrest is mediated by p16(INK4a)- and p21(Cip1)-dependent pathways both leading to retinoblastoma protein (pRb) activation. p53 plays a relay role between DNA damage sensing and p21(Cip1) activation. pRb arrests the cell cycle by recruiting proliferation genes to facultative heterochromatin for permanent silencing. Replicative senescence that occurs in hepatocytes in culture and in liver cirrhosis is associated with lack of telomerase activity and results in telomere shortening. Hepatocellular carcinoma (HCC) cells display inactivating mutations of p53 and epigenetic silencing of p16(INK4a). Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment.

Extremely skewed X-chromosome inactivation patterns in women with recurrent spontaneous abortion.

BACKGROUND: The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of recurrent spontaneous abortion (RSA) but the results obtained were conflicting. AIMS: We therefore investigated the XCI patterns in peripheral blood DNA obtained from 80 patients who had RSA and 160 age-matched controls. METHODS: Pregnancy history, age, karyotype, and disease information was collected from all subjects. The methylation status of a highly polymorphic cytosine-adenine-guanine repeat in the androgen-receptor (AR) gene was determined by use of methylation-sensitive restriction enzyme HpaII and polymerase chain reaction. RESULTS: Skewed XCI (> 85% skewing) was observed in 13 of the 62 patients informative for the AR polymorphism (20.9%), and eight of the 124 informative controls (6.4%) (P = 0.0069; chi2 test). More importantly, extremely skewed XCI, defined as > 90% inactivation of one allele, was present in 11 (17.7%) patients, and in only two controls (P = 0.0002; chi2 test). CONCLUSIONS: These results support the interpretation that disturbances in XCI mosaicism may be involved in the pathogenesis of RSA.

Endothelial progenitor cells display clonal restriction in multiple myeloma.

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (> or = 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (> or = 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.

Evidence from autoimmune thyroiditis of skewed X-chromosome inactivation in female predisposition to autoimmunity.

The etiologic factors in the development of autoimmune thyroid diseases (AITDs) are not fully understood. We investigated the role of skewed X-chromosome inactivation (XCI) mosaicism in female predisposition to AITDs. One hundred and ten female AITDs patients (81 Hashimoto's thyroiditis (HT), 29 Graves' disease (GD)), and 160 female controls were analyzed for the androgen receptor locus by the HpaII/polymerase chain reaction assay to assess XCI patterns in DNA extracted from peripheral blood cells. In addition, thyroid biopsy, buccal mucosa, and hair follicle specimens were obtained from five patients whose blood revealed an extremely skewed pattern of XCI, and the analysis was repeated. Skewed XCI was observed in DNA from peripheral blood cells in 28 of 83 informative patients (34%) as compared with 10 of 124 informative controls (8%, P<0.0001). Extreme skewing was present in 16 patients (19%), but only in three controls (2.4%, P<0.0001). The buccal mucosa, and although less marked, the thyroid specimens also showed skewing. Analysis of two familial cases showed that only the affected individuals demonstrate skewed XCI patterns. Based on these results, skewed XCI mosaicism may play a significant role in the pathogenesis of AITDs.

Skewed X chromosome inactivation in blood cells of women with scleroderma.

OBJECTIVE: Scleroderma (SSc) is an autoimmune disease of unknown etiology. The disease is 3-8 times more frequent in women than in men. The role of X chromosome inactivation (XCI) in the predisposition of women to autoimmunity has been questioned. Until now this has not been illustrated experimentally. This study was undertaken to test the hypothesis that disturbances in XCI mosaicism may be involved in the pathogenesis of the disease in female patients with SSc. METHODS: Seventy female SSc patients and 160 female controls were analyzed for the androgen receptor locus by the Hpa II/polymerase chain reaction assay to assess XCI patterns in DNA extracted from peripheral blood cells. Furthermore, skin biopsy samples were obtained from 5 patients whose blood revealed an extremely skewed pattern of XCI, and the analysis repeated. Since microchimerism in SSc was reported, Y chromosome sequences were investigated in all samples. RESULTS: Skewed XCI was observed in DNA from peripheral blood cells in 35 of 55 informative patients (64%), as compared with 10 of 124 informative controls (8%) (P < 0.0001). Extreme skewing was present in 27 of the patient group (49%), as compared with only 3 of the controls (2.4%) (P < 0.0001). However, XCI was random in all skin biopsy samples. The potential contribution of microchimerism to the random XCI pattern is highly unlikely based on the medical histories of the patients. CONCLUSION: Skewed XCI mosaicism may play a significant role in the pathogenesis of SSc.