Lack of PTEN sequesters CHK1 and initiates genetic instability.

Pten-/- cells display a partially defective checkpoint in response to ionizing radiation (IR). The checkpoint defect was traced to the ability of AKT to phosphorylate CHK1 at serine 280, since a nonphosphorylated mutant of CHK1 (S280A) complemented the checkpoint defect and restored CDC25A degradation. CHK1 phosphorylation at serine 280 led to covalent binding of 1 to 2 molecules of ubiquitin and cytoplasmic CHK1 localization. Primary breast carcinomas lacking PTEN expression and having elevated AKT phosphorylation had increased cytoplasmic CHK1 and displayed aneuploidy (p <0.005). We conclude that loss of PTEN and subsequent activation of AKT impair CHK1 through phosphorylation, ubiquitination, and reduced nuclear localization to promote genomic instability in tumor cells.

Irs2 inactivation suppresses tumor progression in Pten+/- mice.

Mutations in the phosphatase and tensin homologue (PTEN)/phosphatidylinositol-3 kinase-alpha (PI3K) signaling pathway are frequently found in human cancer. In addition, Pten(+/-) mice develop tumors in multiple organs because of the activation of the PI3K signaling cascade. Because activation of PI3K signaling leads to feedback inhibition of insulin receptor substrate-2 (IRS2) expression, an upstream activator of PI3K, we therefore anticipated that IRS2 expression would be low in tumors that lack PTEN. Surprisingly, however, an elevation of IRS2 was often detected in tumor samples in which PTEN levels were compromised. To determine the potential contribution of Irs2 to tumor progression, Pten(+/-) mice were crossed with Irs2(+/-) mice. Deletion of Irs2 did not affect the initiation of neoplasia found in Pten(+/-) mice but suppressed cancer cell growth, proliferation, and invasion through the basement membrane. Deletion of Irs2 also attenuated the expression of Myc in prostatic intraepithelial neoplasia in Pten(+/-) mice. In addition, the expression levels of IRS2 and MYC were highly correlated in human prostate cancer, and IRS2 could stimulate MYC expression in cultured cells. Our findings provide evidence that the PI3K-activating adaptor Irs2 contributes to tumor progression in Pten(+/-) mice by stimulating both Myc and DNA synthesis.

Immunohistochemical analysis for cytokeratin 7, KIT, and PAX2: value in the differential diagnosis of chromophobe cell carcinoma.

Immunohistochemical staining for cytokeratin 7 (CK7), KIT, and PAX2 expression was performed on 91 renal neoplasms, 37 conventional (clear cell) renal cell carcinomas (CRCCs), 20 papillary RCCs (PRCCs), 11 chromophobe RCCs (ChCs), and 23 oncocytomas, with available karyotypes. All ChCs, 19 PRCCs, 2 CRCCs, and 1 oncocytoma were CK7+; all ChCs, 22 oncocytomas, 2 CRCCs, and no PRCCs expressed KIT; PAX2 was positive in 31 CRCCs, 17 PRCCs, 20 oncocytomas, and 1 ChC. The predominant expression profiles were as follows: CRCC, CK7-/KIT-/PAX2+ (26/37); PRCC, CK7+/KIT-/PAX2+ (17/20); ChC, CK7+/KIT+/PAX2- (10/11); and oncocytoma, CK7-/KIT+/PAX2+ (19/23). Cytogenetic analysis showed that the sole PAX2+ ChC had a retained chromosome 10, and all ChCs with chromosome 10 loss were PAX2-. These results identify specific staining patterns of the 4 major histologic subtypes of renal neoplasms and raise the question of a relationship between chromosome 10 loss and loss of PAX2 expression in ChC.

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.

Genetic analysis identifies putative tumor suppressor sites at 2q35-q36.1 and 2q36.3-q37.1 involved in cervical cancer progression.

We performed comparative genomic hybridization (CGH) and high-resolution deletion mapping of the long arm of chromosome 2 (2q) in invasive cervical carcinoma (CC). The CGH analyses on 52 CCs identified genetic losses at 2q33-q36, gain of 3q26-q29, and frequent chromosomal amplifications. Characterization of 2q deletions by loss of heterozygosity (LOH) in 60 primary tumors identified two sites of minimal deleted regions at 2q35-q36.1 and 2q36.3-q37.1. To delineate the stage at which these genetic alterations occur in CC progression, we analysed 33 cervical intraepithelial neoplasia (CIN) for LOH. We found that 89% of high-grade (CINII and CINIII) and 40% of low-grade (CINI) CINs exhibited LOH at 2q. To identify the target tumor suppressor gene (TSG), we performed an extensive genetic and epigenetic analyses of a number of candidate genes mapped to the deleted regions. We did not find inactivating mutations in CASP10, BARD1, XRCC5, or PPP1R7 genes mapped to the deleted regions. However, we did find evidence of downregulated gene expression in CFLAR, CASP10 and PPP1R7 in CC cell lines. We also found reactivated gene expression in CC cell lines in vitro after exposure to demethylating and histone deacetylase (HDAC) inhibiting agents. Thus, these data identify frequent chromosomal amplifications in CC, and sites of TSGs at 2q35-q36.1 and 2q36.3-q37.1 that are critical in CC development.

Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions.

BACKGROUND: Previous studies have shown that the short arm of chromosome 5 (5p) exhibit frequent genetic changes in invasive cervical carcinoma (CC), and that these changes arise early during the carcinogenesis, in precancerous lesions. These data therefore suggest that loss of candidate tumor suppressor genes located on 5p is associated with the development of CC. However, the precise location of 5p deletions is not known. RESULTS: We performed a detailed deletion mapping of 5p in 60 cases of invasive CC. We found that 60% of the tumors exhibit a 5p loss of heterozygosity (LOH). The patterns of LOH allowed us to identify two minimal regions of deletions, one at 5p15.3 spanning a 5.5 cM genetic distance and a second site of 7 cM at 5p15.2-15.3. In addition, we also identified 5p deletions in 16% lesions of high-grade cervical intraepithelial neoplasia (CIN). 5p LOH was found in 63% of HPV 16 positive tumors, while only 33% tumors with other HPV-types had 5p LOH. The differences in frequency of 5p LOH between tumors harboring HPV16 in combination with other HPV types and tumors harboring HPV16 DNA alone were significantly higher, suggesting a synergistic effect of high-risk types in causing genomic instability. CONCLUSION: These findings implicate the presence of tumor suppressor gene(s) on 5p relevant to CC tumorigenesis.

Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors.

BACKGROUND: Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown. RESULTS: We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents. CONCLUSIONS: Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT.

Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma.

BACKGROUND: Carcinoma of uterine cervix is the second most common cancers among women worldwide. Combined radiation and chemotherapy is the choice of treatment for advanced stages of the disease. The prognosis is poor, with a five-year survival rate ranging from about 20-65%, depending on stage of the disease. Therefore, genetic characterization is essential for understanding the biology and clinical heterogeneity in cervical cancer (CC). METHODS: We used a genome-wide screening method--comparative genomic hybridization (CGH) to identify DNA copy number changes in 77 patients with cervical cancer. We applied categorical and survival analyses to analyze whether chromosomal changes were related to clinico-pathologic characteristics and patients survival. RESULTS: The CGH analysis revealed a loss of 2q33-q37 (57.1%), gain of 3q (54.5%) and chromosomal amplifications (20.77%) as frequent genetic changes. A total of 15 amplified chromosomal sites were detected in 16 cases that include 1p31, 2q32, 7q22, 8q21.2-q24, 9p22, 10q21, 10q24, 11q13, 11q21, 12q15, 14q12, 17p11.2, 17q22, 18p11.2, and 19q13.1. Recurrent amplified sites were noted at 11q13, 11q21, and 19q13.1. The genomic alterations were further evaluated for prognostic significance in CC patients, and we did not find any correlation with a number of clinical or histological parameters. The tumors harboring HPV18 exhibited higher genomic instability compared to tumors with HPV 16. CONCLUSIONS: This study demonstrated that 2q33-q37 deletions, 3q gains and chromosomal amplifications as characteristic changes in invasive CC. These genetic alterations will aid in the identification of novel tumor suppressor gene(s) at 2q33-q37 and oncogenes at amplified chromosomal sites. Molecular characterization of these chromosomal changes utilizing the current genomic technologies will provide new insights into the biology and clinical behavior of CC.

Novel translocation (9;12)(q22;q24) in secondary chondrosarcoma arising from hereditary multiple exostosis.

We report a new translocation in a patient with a history of hereditary multiple exostosis (HME) who developed a recurrent grade I chondrosarcoma involving the sacrum and retroperitoneum. Karyotypic analysis of the tumor revealed a sole chromosome abnormality t(9;12)(q22;q24.3). To our knowledge, this translocation has not been previously identified in either chondrosarcoma, HME, or related tumor types. Our novel translocation may be related to the sarcomatous degeneration of the pre-existing exostosis.

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.

Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair.

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.

Structural requirements for the BARD1 tumor suppressor in chromosomal stability and homology-directed DNA repair.

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.

Two somatic biallelic lesions within and near SMAD4 in a human breast cancer cell line.

Loss of chromosome arm 18q is a common event in human pancreatic, colon, and breast cancers and is often interpreted as representing loss of one or more tumor-suppressor genes. In this article, we describe two novel biallelic deletions at chromosome band 18q21.1 in a recently characterized human breast cancer cell line, HCC-1428. One lesion deletes a fragment of approximately 300 kb between SMAD4 and DCC that encodes no known genes. The second lesion is an in-frame SMAD4 deletion (amino acids 49-51) that affects the level of SMAD4 protein but not the SMAD4 message. This change accelerates 26S proteasome-mediated degradation of both endogenous and exogenous mutant SMAD4. Examination of normal DNA from the same patient demonstrated that both lesions are somatic and associated with loss of both normal alleles. These data support the concept that two independent tumor-suppressor loci exist at chromosome segment 18q21.1, one at SMAD4 and the other potentially at an enhancer of DCC or an unrelated novel gene.

Comprehensive molecular cytogenetic characterization of cervical cancer cell lines.

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH), to characterize the genetic aberrations in eight widely used cervical cancer (CC) cell lines. CGH identified the most frequent chromosomal losses including 2q, 3p, 4q, 6q, 8p, 9p, 10p, 13q, and 18q; gains including 3q, 5p, 5q, 8q, 9q, 11q, 14q, 16q, 17q, and 20q; and high-level chromosomal amplification at 3q21, 7p11, 8q23-q24, 10q21, 11q13, 16q23-q24, 20q11.2, and 20q13. Several recurrent structural chromosomal rearrangements, including der(5)t(5;8)(p13;q23) and i(5)(p10); deletions affecting chromosome bands 5p11, 5q11, and 11q23; and breakpoint clusters at 2q31, 3p10, 3q25, 5p13, 5q11, 7q11.2, 7q22, 8p11.2, 8q11.2, 10p11.2, 11p11.2, 14q10, 15q10, 18q21, and 22q11.2 were identified by SKY. We detected integration of HPV16 sequences by FISH on the derivative chromosomes involving bands 18p10 and 18p11 in cell line C-4I, 2p16, 5q21, 5q23, 6q, 8q24, 10, 11p11, 15q, and 18p11 in Ca Ski, and normal chromosome 17 at 17p13 in ME-180. FISH analysis was also used further to determine the copy number changes of PIKA3CA and MYC. This comprehensive cytogenetic characterization of eight CC cell lines enhances their utility in experimental studies aimed at gene discovery and functional analysis.

Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells.

MAD2 inhibits the anaphase-promoting complex when chromosomes are unattached to the mitotic spindle. It acts as a tumor suppressor gene because MAD2+/-cells enter anaphase early and display chromosome instability, leading to the formation of lung tumors in mice. Complete MAD2 inactivation has not been identified in human tumors, although partial defects are prevalent. By employing RNA interference in human somatic cells, we found that severe reduction of MAD2 protein levels results in mitotic failure and extensive cell death arising from defective spindle formation, incomplete chromosome condensation, and premature mitotic exit leading to multinucleation. Cyclin B is degraded prematurely in the MAD2 short interfering RNA-treated cells but not in MAD2+/- cells, suggesting an explanation for the spindle failure and mitotic catastrophe in the MAD2 knockdown cells. Thus, anaphase-promoting complex substrates exhibit distinct sensitivities in the presence of different MAD2 doses, which in turn determine MAD2's role as either a tumor suppressor or an essential gene.

BCL8 is a novel, evolutionarily conserved human gene family encoding proteins with presumptive protein kinase A anchoring function.

BCL8 is a novel human gene family initially identified through cloning of BCL8A, located at the t(14;15)(q32;q11-q13) translocation breakpoint, in a case of diffuse large B-cell lymphoma. Multiple copies of BCL8A map to the 1-Mb proximal duplicated region at 15q. We identified additional copies on human chromosomes 13 (BCL8B), 22 (BCL8C), 2 (BCL8D), and 10 (BCL8E) by cDNA cloning and sequence analysis. BCL8A, BCL8C, BCL8D, and BCL8E are truncated at the genomic level and are presumably pseudogenes or sterile transcripts. BCL8B is expressed predominantly in human brain and encodes a 327-kDa protein with extensive homology to the Drosophila melanogaster protein kinase A anchoring protein RG. LRBA, a human gene with a ubiquitous expression pattern mapping to 4q32, encodes a protein closely related to BCL8. The phylogenetically conserved BCL8 gene family evolved by transchromosomal and intrachromosomal duplications within the human genome.

CTNNB1 mutations and overexpression of Wnt/beta-catenin target genes in WT1-mutant Wilms' tumors.

Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1.