Invasion leading to the formation of metastasis is one of the hallmarks of cancer. Analysis of different human cancers has led to the identification of the PPFIA1 gene encoding the protein liprin-α1, a possible player in cancer. The PPFIA1 gene is amplified in malignant tumors, including about 20% of breast cancers. Also the liprin-α1 protein is found overexpressed in tumors. Liprin-α1 belongs to the liprin family of cytosolic scaffold proteins that includes four liprin-α, two liprin-β members, and liprin-γ/kazrinE. In this review we will discuss the available evidence on the role of different members of the liprin family in distinct aspects of tumor cell migration and invasion. Evidence from in vitro studies indicates that the widely expressed liprin-α1 protein regulates the migration and invasion of human breast cancer cells. Liprin-α1 affects cell migration and invasion by regulating the organization of lamellipodia and invadopodia, two structures relevant to cell invasion. In the cell liprin-α1 forms a complex with liprin-β1, ERC1/ELKS and LL5 proteins, which localizes at the front of migrating cells and positively regulates lamellipodia stability, and integrin-mediated focal adhesions. On the other hand, liprin-β2 appears to play a role as tumor suppressor by inhibiting breast cancer cell motility and invasion. The available data indicate that liprins are central players in the regulation of tumor cell invasion, therefore representing interesting targets for anti-metastatic therapy.

Menopause timing has a substantial impact on infertility and risk of disease, including breast cancer, but the underlying mechanisms are poorly understood. We report a dual strategy in ∼70,000 women to identify common and low-frequency protein-coding variation associated with age at natural menopause (ANM). We identified 44 regions with common variants, including two regions harboring additional rare missense alleles of large effect. We found enrichment of signals in or near genes involved in delayed puberty, highlighting the first molecular links between the onset and end of reproductive lifespan. Pathway analyses identified major association with DNA damage response (DDR) genes, including the first common coding variant in BRCA1 associated with any complex trait. Mendelian randomization analyses supported a causal effect of later ANM on breast cancer risk (∼6% increase in risk per year; P = 3 × 10(-14)), likely mediated by prolonged sex hormone exposure rather than DDR mechanisms.

Reactive oxygen species (ROS) are thought to be among the initiating insults that drive carcinogenesis; however, beyond the mutagenic properties of ROS, it is unclear how reactive oxygen species and response to redox imbalance may shape cancer phenotype. We have previously observed that basal activity of the powerfully oncogenic transcription factor NF-κB in cultured breast cancer and other tumor cell lines is dependent upon the DNA damage-responsive kinase ATM. Here we show that, in MDA-MB-231 and HeLa cells, basal ATM-dependent NF-κB activation occurs through a canonical DNA damage-responsive signaling pathway as knockdown of two proteins involved in this signaling pathway, ERC1 and TAB1, results in loss of NF-κB basal activity. We further show that knockdown of ATM in MDA-MB-231, a breast cancer line with a pronounced mesenchymal phenotype, results in the reversion of these cells to an epithelial morphology and gene expression pattern. Culture of MDA-MB-231 and HeLa cells on the antioxidant N-acetyl cysteine (NAC) blunted NF-κB transcriptional activity, and long-term culture on low doses of NAC resulted in coordinate reductions in steady-state ROS levels, acquisition of an epithelial morphology, as well as upregulation of epithelial and downregulation of mesenchymal marker gene expression. Moreover, these reversible effects are attributable, at least in part, to downregulation of ATM-dependent NF-κB signaling in MDA-MB-231 cells as RNAi-mediated knockdown of the NF-κB subunit RelA or its upstream activator TG2 produced similar alterations in phenotype. We conclude that chronic activation of ATM in response to persistent ROS insult triggers continual activation of the oncogenic NF-κB transcriptional complex that, in turn, promotes aggressive breast cancer phenotype.

Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition.

The pubertal height growth spurt is a distinctive feature of childhood growth reflecting both the central onset of puberty and local growth factors. Although little is known about the underlying genetics, growth variability during puberty correlates with adult risks for hormone-dependent cancer and adverse cardiometabolic health. The only gene so far associated with pubertal height growth, LIN28B, pleiotropically influences childhood growth, puberty and cancer progression, pointing to shared underlying mechanisms. To discover genetic loci influencing pubertal height and growth and to place them in context of overall growth and maturation, we performed genome-wide association meta-analyses in 18 737 European samples utilizing longitudinally collected height measurements. We found significant associations (P < 1.67 × 10(-8)) at 10 loci, including LIN28B. Five loci associated with pubertal timing, all impacting multiple aspects of growth. In particular, a novel variant correlated with expression of MAPK3, and associated both with increased prepubertal growth and earlier menarche. Another variant near ADCY3-POMC associated with increased body mass index, reduced pubertal growth and earlier puberty. Whereas epidemiological correlations suggest that early puberty marks a pathway from rapid prepubertal growth to reduced final height and adult obesity, our study shows that individual loci associating with pubertal growth have variable longitudinal growth patterns that may differ from epidemiological observations. Overall, this study uncovers part of the complex genetic architecture linking pubertal height growth, the timing of puberty and childhood obesity and provides new information to pinpoint processes linking these traits.

Rare breast neoplasms resembling the tall-cell variant of papillary thyroid carcinoma have been reported. In addition, papillary carcinoma of the breast occasionally displays nuclear features reminiscent of papillary thyroid carcinoma. In this study, we evaluated 33 intraductal/intracystic papillary carcinomas of the breast for the presence and extent of nuclear overlap, grooves, clearing, and inclusions, as well as features of the tall-cell or columnar-cell variants of papillary thyroid carcinoma. RET rearrangements were assessed in a subset of these cases. Paired probes localizing to the centromeric and telomeric ends of the RET gene on chromosome 10 were used for FISH using a break-apart approach. Single round and nested PCR was performed to detect RET/PTC1, RET/PTC2, RET/PTC3 and ELKS-RET fusion transcripts. Nuclear overlap, grooves, stratification, and clearing were identified in 24 (73%), 14 (42%), 11 (33%), and 9 (27%) cases respectively, whereas nuclear inclusions and 'tall-cell' features were each seen in only one (3%) and two (6%) cases, respectively. Four of 19 tested cases displayed split FISH signals in a low percentage of cells and were considered borderline for RET rearrangement. All 19 tested cases with amplifiable RNA were negative for the four RET fusion transcripts evaluated by RT-PCR. Although papillary carcinomas of breast often display one or more cytoarchitectural features of papillary thyroid carcinoma, there is no evidence that RET rearrangements are involved.

BACKGROUND: The MDM2 gene encodes a negative regulator of the p53 tumour suppressor protein. A single nucleotide polymorphism (SNP) in the MDM2 promoter (a T to G exchange at nucleotide 309) has been reported to produce accelerated tumour formation in individuals with inherited p53 mutations. We have investigated the effect of the MDM2 SNP309 on clinical outcome in a cohort of patients with germline mutations of BRCA1.
METHODS: Genomic DNA was obtained for 102 healthy controls and 116 patients with established pathogenic mutations of BRCA1 and Pyrosequencing technologytrade mark was used to determine the genotype at the MDM2 SNP309 locus.
RESULTS: The polymorphism was present in 52.9% of the controls (G/T in 37.3% and G/G in 15.6%) and 58.6% of the BRCA1 mutation carriers (47.4% G/T and 11.2% G/G). Incidence of malignancy in female BRCA1 carriers was not significantly higher in SNP309 carriers than in wildtype (T/T) individuals (72.7% vs. 75.6%, p = 1.00). Mean age of diagnosis of first breast cancer was 41.2 years in the SNP309 G/G genotype carriers, 38.6 years in those with the SNP309 G/T genotype and 39.0 years in wildtype subjects (p = 0.80).
CONCLUSION: We found no evidence that the MDM2 SNP309 accelerates tumour development in carriers of known pathogenic germline mutations of BRCA1.

Somatic rearrangement of the tyrosine kinase receptor RET is restricted to papillary thyroid carcinoma (PTC). The prevalence of RET/PTC1, RET/PTC2, and RET/PTC3 has been found to vary between 0% and 20% in most series of sporadic (nonradiation-induced) PTCs analyzed by type-specific reverse transcription-polymerase chain reaction (RT-PCR) alone. However, high prevalence reported from Taiwan (6 out of 11, 55%) indicates RET rearrangement is an important genetic lesion underlying the development of PTC in Taiwan. Because the high prevalence of RET rearrangements in Chinese patients was particularly striking, we were prompted to reexamine chimeric transcripts of RET/PTC1, RET/PTC2, and RET/PTC3 using the same experimental designs in a larger number of cases in the same population. RT-PCR was performed to amplify fusion products of RET/PTC1, RET/PTC2, RET/PTC3, and ELKS-RET from frozen tissue of 105 sporadic PTCs. RT-PCR was also performed with two different primer sets for RET/PTC1, RET/PTC2, and RET/PTC3 followed by Southern hybridization in the first 62 tumors. In our study, RET/PTC1, RET/PTC2, and RET/PTC3 oncogenes were found in only 7 of 105 (7%) sporadic PTCs. Of these tumors, 3 involved RET/PTC1 and 4 involved RET/PTC3. No RET/PTC2 rearrangements were observed. In the first 62 tumor samples, another two different primer sets for each rearrangement also gave concordant results. Furthermore, application of Southern hybridization in these 62 PTCs did not identify additional tumor harboring RET chimeric transcripts. We identified one tumor as having an ELKS-RET rearrangement (1 of 105, 1%). In conclusion, we detected RET rearrangements in 8 of 105 (8%) sporadic PTCs in Taiwan, a much lower prevalence than previously reported for this population but comparable to those reported in other nations using similar methodology. RET chimeric oncogenes only account for a small fraction of PTCs in Taiwan.

The RET proto-oncogene is responsible for the development of several human inherited and non-inherited diseases. Germline point mutations were identified in multiple endocrine neoplasia types 2A and 2B, and familial medullary thyroid carcinoma. More than 10 rearranged forms of RET, referred to as RET/PTC 1-9, ELKS/RET and RFP/RET, have been cloned from sporadic and radiation-associated papillary thyroid carcinomas. These mutations induced oncogenic activation of RET tyrosine kinase by different mechanisms. To date, various kinds of therapeutic approaches have been developed for the treatment of RET-associated cancers, including tyrosine kinase inhibitors, gene therapy with dominant negative RET mutants, and RNA interference to abrogate oncogenic mutant RET expression. RET and some signaling molecules that function downstream of RET could be potential targets for the development of selective cancer therapeutics.

A novel gene, ELKS, whose 5' portion was fused to the RET gene, was found in a papillary thyroid carcinoma. A cDNA of this gene obtained from a human-brain cDNA library revealed that it encoded a peptide of 948 amino acids, termed ELKSalpha. We identified four other isoforms, which encoded ELKSbeta, ELKSgamma, ELKSdelta, and ELKSepsilon proteins consisting, respectively, of 992, 720, 1088, and 1116 amino acid residues. Analysis of the gene structure revealed that the isoforms were generated by alternative splicing. Isoforms beta, gamma, delta, and epsilon all contain an optional exon (exon14a), but ELKSgamma, -delta, and -epsilon lack exon 1b. ELKSgamma lacks exons 3 to 6. ELKSdelta and -epsilon lack exons 12 and 17; ELKSepsilon contains an optional exon (exon 6a). Analysis by RT-PCR suggested that ELKSalpha and ELKSbeta mRNAs are abundant in the brain, ELKSdelta and ELKSepsilon mRNAs predominate in testis and thyroid, and ELKSepsilon mRNA predominates in other tissues. To prove whether the fusion of different ELKS isoforms to RET (between ELKS coiled-coil domains and the RET kinase domain) could produce chimeric proteins that could be autophosphorylated, we synthesized ELKSgamma-RET, ELKSdelta-RET, and ELKSepsilon-RET fusion proteins in vitro. Immunoblotting with anti-ELKS, anti-RET, and anti-phosphotyrosine antibodies demonstrated that the chimeric proteins were constitutively phosphorylated at tyrosine residues, whereas native RET protein was not. These results indicate that the ELKS gene is alternatively spliced, and that every type of ELKS-RET chimeric protein having oligomerization domains can activate RET's cytoplasmic tyrosine kinase.

We recently isolated a novel cDNA, designated ELKS, that was fused to RET cDNA in a papillary thyroid carcinoma. Its encoded polypeptide sequence was rich in glutamic acid (E), leucine (L), lysine (K), and serine (S), and was characterized by the presence of nine alpha-helical coiled-coil domains consisting of periodic heptad repeats. We have now cloned the entire structure of the human ELKS gene from within a 700-kb genomic region represented by overlapping bacteriophage P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones, and localized it to chromosomal band 12p13.3 by fluorescence in situ hybridization. The gene is approximately 500 kb long, with 19 exons and 18 introns; the transcription initiation site within exon 1 is separate from the initiation codon (in exon 2). Analysis of the exon/intron structure revealed that introns interrupt the coding sequence in such a way that many functional segments of the protein are encoded by distinct exons. Exon 1 encodes the 5' non-coding region; exons 2, 3, 6, 7, 8, 9, 11, 14, and 15 encode the nine coiled-coil domains. Exons 17-19 constitute the 3' non-coding region. Analysis of the region immediately upstream of exon 1 showed that it was extremely rich in G/C nucleotides and contained multiple Sp-1 and AP2 binding sequences. The ELKS-RET gene fusion rearrangement we had observed in a papillary thyroid carcinoma occurred between intron 10 of the ELKS gene and intron 11 of RET.

In papillary thyroid carcinomas, the genes for receptor-type tyrosine kinase, RET or TRKA, are sometimes rearranged, resulting in fusion of its tyrosine kinase domain to 5' portions of several activating genes. In a papillary thyroid carcinoma, we identified a novel gene (ELKS), the 5' portion of which is fused to the RET gene by gene rearrangement due to the translocation t(10;12)(q11;p13). Subsequent cloning of the ELKS cDNA revealed that ELKS encodes a novel 948 amino acid peptide and is expressed ubiquitously in human tissues. The presence of multiple coiled-coil domains in the ELKS product suggests that the ELKS protein forms dimers. Since the tyrosine kinase of RET is activated by dimerization that occurs when its ligands bind to the receptor, fusion of RET with the 5' dimerization domains of ELKS would activate its cytoplasmic tyrosine kinase constitutively in papillary thyroid carcinomas.

We have previously shown by reverse transcriptase-PCR (rtPCR) that CML CD34+ HLA-DR- cells are enriched for BCR/ABL(-) hematopoietic progenitor cells (HPC) while leukemic HPC reside predominately within CML CD34+ HLA-DR+ cells. We investigated whether the 30/35 kDa fragment of fibronectin (FN) could be used to enhance retroviral-mediated gene transfer (RMGT) in chronic phase CML marrow HPC. CML CD34+ HLA-DR- and CD34+ HLA-DR+ cells were transduced with vector supernate containing the neomycin resistance gene on plates coated with either FN or bovine serum albumin (BSA) as control, then assayed for transduced HPC in progenitor cell assays in the presence or absence of G418. Transduction efficiency of CML CD34+ HLA-DR- cells over BSA ranged from 0.09 to 7.2% (mean 3.3 +/- 1.5%), while that over FN plates ranged from 3.8 to 23% (mean 11.0 +/- 4.5%) (n = 4). Transduction efficiencies of CML CD34+ HLA-DR+ cells ranged from 0.4 to 9.8% (mean 3.7 +/- 1.7%) and 6.0 to 26% (mean 17.3 +/- 4.5%) (n = 5) over BSA and FN, respectively. rtPCR analysis for BCR/ABL mRNA of individual G418-resistant HPC generated from CD34+ HLA-DR- cells revealed that normal BCR/ABL(-) HPC were successfully transduced under these experimental conditions. These results demonstrate the feasibility of transducing normal CML primitive HPC, and illustrate the potential clinical use of FN in the setting of gene therapy for CML, as well as other diseases.