Subhadra V. Nandula

Mutations of multiple genes cause deregulation of NF-kB in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-κB transcription complex. However, except for a small fraction of cases, it remains unclear whether NF-κB activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A (RANK)) regulators of NF-κB. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-κB responses, is most commonly affected, with ∼30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-κB. Thus, our results demonstrate that NF-κB activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-κB responses.

Inactivation of the PRDM1/BLIMP1 gene in diffuse large B cell lymphoma

PR domain containing 1 with zinc finger domain (PRDM1)/B lymphocyte–induced maturation protein 1 (BLIMP1) is a transcriptional repressor expressed in a subset of germinal center (GC) B cells and in all plasma cells, and required for terminal B cell differentiation. The BLIMP1 locus lies on chromosome 6q21-q22.1, a region frequently deleted in B cell lymphomas, suggesting that it may harbor a tumor suppressor gene. We report here that the BLIMP1 gene is inactivated by structural alterations in 24% (8 out of 34) activated B cell–like diffuse large cell lymphoma (ABC-DLBCL), but not in GC B cell–like (n = 0/37) or unclassified (n = 0/21) DLBCL. BLIMP1 alterations included gene truncations, nonsense mutations, frameshift deletions, and splice site mutations that generate aberrant transcripts encoding truncated BLIMP1 proteins. In all cases studied, both BLIMP1 alleles were inactivated by deletions or mutations. Furthermore, most non–GC type DLBCL cases (n = 20/26, 77%) lack BLIMP1 protein expression, despite the presence of BLIMP1 mRNA. These results indicate that a sizable fraction of ABC-DLBCL carry an inactive BLIMP1 gene, and suggest that the same gene is inactivated by epigenetic mechanisms in an additional large number of cases. These findings point to a role for BLIMP1 as a tumor suppressor gene, whose inactivation may contribute to lymphomagenesis by blocking post–GC differentiation of B cells toward plasma cells.

The NF-κB negative regulator TNFAIP3 (A20) is inactivated by somatic mutations and genomic deletions in marginal zone lymphomas

Unique and shared cytogenetic abnormalities have been documented for marginal zone lymphomas (MZLs) arising at different sites. Recently, homozygous deletions of the chromosomal band 6q23, involving the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) gene, a negative regulator of NF-kappaB, were described in ocular adnexal MZL, suggesting a role for A20 as a tumor suppressor in this disease. Here, we investigated inactivation of A20 by DNA mutations or deletions in a panel of extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Inactivating mutations encoding truncated A20 proteins were identified in 6 (19%) of 32 MZLs, including 2 (18%) of 11 EMZLs, 3 (33%) of 9 NMZLs, and 1 (8%) of 12 SMZLs. Two additional unmutated nonsplenic MZLs also showed monoallelic or biallelic A20 deletions by fluorescent in situ hybridization (FISH) and/or SNP-arrays. Thus, A20 inactivation by either somatic mutation and/or deletion represents a common genetic aberration across all MZL subtypes, which may contribute to lymphomagenesis by inducing constitutive NF-kappaB activation.

Identification of copy number gain and overexpressed genes on chromosome arm 20q by an integrative genomic approach in cervical cancer: potential role in progression.

Recurrent karyotypic abnormalities are a characteristic feature of cervical cancer (CC) cells, which may result in deregulated expression of important genes that contribute to tumor initiation and progression. To examine the role of gain of the long arm of chromosome 20 (20q), one of the common chromosomal gains in CC, we evaluated CC at various stages of progression using single nucleotide polymorphism (SNP) array, gene expression profiling, and fluorescence in situ hybridization (FISH) analyses. This analysis revealed copy number increase (CNI) of 20q in >50% of invasive CC and identified two focal amplicons at 20q11.2 and 20q13.13 in a subset of tumors. We further demonstrate that the acquisition of 20q gain occurs at an early stage in CC development and the high‐grade squamous intraepithelial lesions (HSIL) that exhibit 20q CNI are associated (P = 0.05) with persistence or progression to invasive cancer. We identified a total of 26 overexpressed genes as consequence of 20q gain (N = 14), as targets of amplicon 1 (N = 9; two genes also commonly expressed with 20q gain) and amplicon 2 (N = 6; one gene also commonly expressed with 20q gain). These include a number of functionally important genes in cell cycle regulation (E2F1, TPX2, KIF3B, PIGT, and B4GALT5), nuclear function (CSEL1), viral replication (PSMA7 and LAMA5), methylation and chromatin remodeling (ASXL1, AHCY, and C20orf20), and transcription regulation (TCEA2). Our findings implicate a role for these genes in CC tumorigenesis, represent an important step toward the development of clinically significant biomarkers, and form a framework for testing as molecular therapeutic targets.

Promoter Hypermethylation of FANCF Disruption of Fanconi Anemia-BRCA Pathway in Cervical Cancer

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methylation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hypersensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimental therapeutic protocols.

Gene dosage alterations revealed by cDNA microarray analysis in cervical cancer: Identification of candidate amplified and overexpressed genes

Cervical cancer (CC) cells exhibit complex karyotypic alterations, which is consistent with deregulation of numerous critical genes in its formation and progression. To characterize this karyotypic complexity at the molecular level, we used cDNA array comparative genomic hybridization (aCGH) to analyze 29 CC cases and identified a number of over represented and deleted genes. The aCGH analysis revealed at least 17 recurrent amplicons and six common regions of deletions. These regions contain several known tumor‐associated genes, such as those involved in transcription, apoptosis, cytoskeletal remodeling, ion‐transport, drug metabolism, and immune response. Using the fluorescence in situ hybridization (FISH) approach we demonstrated the presence of high‐level amplifications at the 8q24.3, 11q22.2, and 20q13 regions in CC cell lines. To identify amplification‐associated genes that correspond to focal amplicons, we examined one or more genes in each of the 17 amplicons by Affymetrix U133A expression arrays and semiquantitative reverse‐transcription PCR (RT‐PCR) in 31 CC tumors. This analysis exhibited frequent and robust upregulated expression in CC relative to normal cervix for genes EPHB2 (1p36), CDCA8 (1p34.3), AIM2 (1q22‐23), RFC4, MUC4, and HRASLS (3q27‐29), SKP2 (5p12‐13), CENTD3 (5q31.3), PTK2, RECQL4 (8q24), MMP1 and MMP13 (11q22.2), AKT1 (14q32.3), ABCC3 (17q21‐22), SMARCA4 (19p13.3) LIG1 (19q13.3), UBE2C (20q13.1), SMC1L1 (Xp11), KIF4A (Xq12), TMSNB (Xq22), and CSAG2 (Xq28). Thus, the gene dosage and expression profiles generated here have enabled the identification of focal amplicons characteristic for the CC genome and facilitated the validation of relevant genes in these amplicons. These data, thus, form an important step toward the identification of biologically relevant genes in CC pathogenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

3-Phosphoinositide-dependent kinase 1 potentiates upstream lesions on the phosphatidylinositol 3-kinase pathway in breast carcinoma.

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP(3)). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP(3) recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer.

The basal-like mammary carcinomas induced by Brca1 or Bard1 inactivation implicate the BRCA1/BARD1 heterodimer in tumor suppression

Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either Bard1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional Bard1- and Brca1-mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional Bard1/Brca1-mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are “triple negative” for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of Bard1-, Brca1-, and Bard1/Brca1-mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.

Integrative genomics analysis of chromosome 5p gain in cervical cancer reveals target over-expressed genes, including Drosha.

BackgroundCopy number gains and amplifications are characteristic feature of cervical cancer (CC) genomes for which the underlying mechanisms are unclear. These changes may possess oncogenic properties by deregulating tumor-related genes. Gain of short arm of chromosome 5 (5p) is the most frequent karyotypic change in CC.MethodsTo examine the role of 5p gain, we performed a combination of single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and gene expression analyses on invasive cancer and in various stages of CC progression.ResultsThe SNP and FISH analyses revealed copy number increase (CNI) of 5p in 63% of invasive CC, which arises at later stages of precancerous lesions in CC development. We integrated chromosome 5 genomic copy number and gene expression data to identify key target over expressed genes as a consequence of 5p gain. One of the candidates identified was Drosha (RNASEN), a gene that is required in the first step of microRNA (miRNA) processing in the nucleus. Other 5p genes identified as targets of CNI play a role in DNA repair and cell cycle regulation (BASP1, TARS, PAIP1, BRD9, RAD1, SKP2, and POLS), signal transduction (OSMR), and mitochondrial oxidative phosphorylation (NNT, SDHA, and NDUFS6), suggesting that disruption of pathways involving these genes may contribute to CC progression.ConclusionTaken together, we demonstrate the power of integrating genomics data with expression data in deciphering tumor-related targets of CNI. Identification of 5p gene targets in CC denotes an important step towards biomarker development and forms a framework for testing as molecular therapeutic targets.

Promoter methylation-mediated inactivation of PCDH10 in acute lymphoblastic leukemia contributes to chemotherapy resistance.

PCDH10 has been implicated as a tumor suppressor, since epigenetic alterations of this gene have been noted in multiple tumor types. However, to date, studies regarding its role in acute and chronic leukemias are lacking. Here, we have investigated the presence of promoter hypermethylation of two CpG islands of the PCDH10 gene by methylation‐specific PCR in 215 cases of various subsets of myeloid‐ and lymphoid‐lineage leukemias. We found that PCDH10 promoter hypermethylation was frequent in both B‐cell (81.9%) and T‐cell (80%) acute lymphoblastic leukemia (ALL), while it was present in low frequency in most subtypes of myeloid leukemias (25.9%) and rare in chronic myeloid leukemia (2.2%). PCDH10 expression was downregulated via promoter hypermethylation in primary ALL samples (N = 4) and leukemia cell lines (N = 11). The transcriptional repression caused by PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases. ALL cell lines harboring methylation‐mediated inactivation of PCDH10 were less sensitive to commonly used leukemia‐specific drugs suggesting that PCDH10 methylation might serve as a biomarker of chemotherapy response. Our results demonstrate that PCDH10 is a target of epigenetic silencing in ALL, a phenomenon that may impact lymphoid‐lineage leukemogenesis, serve as an indicator of drug resistance and may also have potential implications for targeted epigenetic therapy.