Luca Cavallone

Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155

BRCA1, a well-known tumor suppressor with multiple interacting partners, is predicted to have diverse biological functions. However, so far its only well-established role is in the repair of damaged DNA and cell cycle regulation. In this regard, the etiopathological study of low-penetrant variants of BRCA1 provides an opportunity to uncover its other physiologically important functions. Using this rationale, we studied the R1699Q variant of BRCA1, a potentially moderate-risk variant, and found that it does not impair DNA damage repair but abrogates the repression of microRNA-155 (miR-155), a bona fide oncomir. Mechanistically, we found that BRCA1 epigenetically represses miR-155 expression via its association with HDAC2, which deacetylates histones H2A and H3 on the miR-155 promoter. We show that overexpression of miR-155 accelerates but the knockdown of miR-155 attenuates the growth of tumor cell lines in vivo. Our findings demonstrate a new mode of tumor suppression by BRCA1 and suggest that miR-155 is a potential therapeutic target for BRCA1-deficient tumors.

Do men and women follow different trajectories to reach extreme longevity

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.

Association between the interleukin-1β polymorphisms and Alzheimer's disease: A systematic review and meta-analysis

The pro-inflammatory cytokine interleukin(IL)-1beta is a main component in inflammatory pathways and is overexpressed in the brain of Alzheimers disease (AD) patients. Several studies report associations between IL-1beta polymorphisms and AD, but findings from different studies are controversial. Our aim was to verify the correlation between the single nucleotide polymorphisms (SNPs) of the IL-1beta, at sites -511 and +3953, and AD by meta-analysis. Computerized bibliographic searches of PUBMED and AlzGene database (http://www.alzgene.org) were supplemented with manual searches of reference lists. There is evidence for association between IL-1beta +3953 SNP and AD, with an OR=1.60 (95% C.I.: 1.16-2.22; Z=2.83 p=0.005) for TT genotype. No significant difference in genotype distribution of the IL-1beta -511 SNP in AD was obtained, but high between-study heterogeneity was found. To reduce heterogeneity, subgroup analyses were performed using, as stratifying variables, characteristics of the population under study (age, gender, type of AD diagnosis, Mini Mental State Examination of the controls) and characteristics related to the study design (statistical power of individual studies). The frequency of the IL-1beta -511 TT genotype resulted significantly higher than other genotypes only when the Caucasian studies with the highest statistical power were included in the subgroup analysis (OR=1.32; 95% C.I.: 1.03-1.69; p=0.03), with no evidence of between-study heterogeneity. Our data support an association between the TT genotype of IL-1beta +3953 SNP and AD, and suggest a possible association of the -511 TT genotype. Unreplicability of the results seems to be due mainly to the lack of statistical power of the individual studies.

Haplotype analysis of TP53 polymorphisms, Arg72Pro and Ins16, in BRCA1 and BRCA2 mutation carriers of French Canadian descent

BackgroundThe TP53 polymorphisms Arg72Pro (Ex4+199 G>C) and Ins16 (IVS3+24 ins16) have been proposed to modify risk of breast cancer associated with germline BRCA1 and BRCA2 mutations. Allele frequencies of these polymorphisms were investigated to determine if they modify risk in BRCA mutation carriers in breast cancer cases drawn from French Canadian cancer families, a population shown to exhibit strong founder effects.MethodsThe frequencies of the TP53 alleles, genotypes and haplotypes of 157 index breast cancer cases comprised of 42 BRCA1 mutation carriers, 57 BRCA2 mutation carriers, and 58 BRCA mutation-negative cases, where each case was drawn from independently ascertained families were compared. The effect of TP53 variants on the age of diagnosis was also investigated for these groups. The TP53 polymorphisms were also investigated in 112 women of French Canadian descent with no personal history of cancer.ResultsThe BRCA mutation-positive groups had the highest frequency of homozygous carriers of the 72Pro allele compared with mutation-negative group. The TP53 polymorphisms exhibited linkage disequilibrium (p < 0.001), where the 72Arg and Ins16minus alleles occurred in strong disequilibrium. The highest frequency of carriers of Ins16minus-72Arg haplotype occurred in the BRCA mutation-negative groups. The BRCA1 mutation carriers homozygous for the 72Pro allele had the youngest ages of diagnosis of breast cancer. However none of these observations were statistically significant. In contrast, the BRCA2 mutation carriers homozygous for the 72Pro allele had a significantly older age of diagnosis of breast cancer (p = 0.018). Moreover, in this group, the mean age of diagnosis of breast cancer in carriers of the Ins16minus-72Arg haplotype was significantly younger than that of the individuals who did not this carry this haplotype (p = 0.009).ConclusionWe observed no significant association of breast cancer risk with TP53 genetic variants based on BRCA1/2 mutation carrier status. Although the small sample size did not permit analysis of all possible haplotypes, we observed that BRCA2 mutation carriers harboring the Ins16minus-72Arg haplotype had a significantly younger mean age of diagnosis of breast cancer. These observations suggest that investigations in a larger French Canadian sample are warranted to further elucidate the effects of TP53 variants on age of diagnosis of breast cancer among BRCA1 and BRCA2 mutation carriers.

The Estrogen Receptor Cofactor SPEN Functions as a Tumor Suppressor and Candidate Biomarker of Drug Responsiveness in Hormone-Dependent Breast Cancers

The treatment of breast cancer has benefitted tremendously from the generation of estrogen receptor-α (ERα)-targeted therapies, but disease relapse continues to pose a challenge due to intrinsic or acquired drug resistance. In an effort to delineate potential predictive biomarkers of therapy responsiveness, multiple groups have identified several uncharacterized cofactors and interacting partners of ERα, including Split Ends (SPEN), a transcriptional corepressor. Here, we demonstrate a role for SPEN in ERα-expressing breast cancers. SPEN nonsense mutations were detectable in the ERα-expressing breast cancer cell line T47D and corresponded to undetectable protein levels. Further analysis of 101 primary breast tumors revealed that 23% displayed loss of heterozygosity at the SPEN locus and that 3% to 4% harbored somatically acquired mutations. A combination of in vitro and in vivo functional assays with microarray-based pathway analyses showed that SPEN functions as a tumor suppressor to regulate cell proliferation, tumor growth, and survival. We also found that SPEN binds ERα in a ligand-independent manner and negatively regulates the transcription of ERα targets. Moreover, we demonstrate that SPEN overexpression sensitizes hormone receptor-positive breast cancer cells to the apoptotic effects of tamoxifen, but has no effect on responsiveness to fulvestrant. Consistent with these findings, two independent datasets revealed that high SPEN protein and RNA expression in ERα-positive breast tumors predicted favorable outcome in patients treated with tamoxifen alone. Together, our data suggest that SPEN is a novel tumor-suppressor gene that may be clinically useful as a predictive biomarker of tamoxifen response in ERα-positive breast cancers.

Exome profiling of primary, metastatic and recurrent ovarian carcinomas in a BRCA1 -positive patient

BackgroundOvarian carcinoma is a common, and often deadly, gynecological cancer. Mutations in BRCA1 and BRCA2 genes are present in at least a fifth of patients. Uncovering other genes that become mutated subsequent to BRCA1/BRCA2 inactivation during cancer development will be helpful for more effective treatments.MethodsWe performed exome sequencing on the blood, primary tumor, omental metastasis and recurrence following therapy with carboplatin and paclitaxel, from a patient carrying a BRCA1 S1841R mutation.ResultsWe observed loss of heterozygosity in the BRCA1 mutation in the primary and subsequent tumors, and somatic mutations in the TP53 and NF1 genes were identified, suggesting their role along with BRCA1 driving the tumor development. Notably, we show that exome sequencing is effective in detecting large chromosomal rearrangements such as deletions and amplifications in cancer. We found that a large deletion was present in the three tumors in the regions containing BRCA1, TP53, and NF1 mutations, and an amplification in the regions containing MYC. We did not observe the emergence of any new mutations among tumors from diagnosis to relapse after chemotherapy, suggesting that mutations already present in the primary tumor contributed to metastases and chemotherapy resistance.ConclusionsOur findings suggest that exome sequencing of matched samples from one patient is a powerful method of detecting somatic mutations and prioritizing their potential role in the development of the disease.

Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits

Ultrasensitive methods for rare allele detection are critical to leverage the full potential offered by liquid biopsies. Here, we describe a novel molecular barcoding method for the precise detection and quantification of circulating tumor DNA (ctDNA). The major benefits of our design include straightforward and cost-effective production of barcoded adapters to tag individual DNA molecules before PCR and sequencing, and better control over cross-contamination between experiments. We validated our approach in a cohort of 24 patients with a broad spectrum of cancer diagnoses by targeting and quantifying single-nucleotide variants (SNVs), indels and genomic rearrangements in plasma samples. By using personalized panels targeting a priori known mutations, we demonstrate comprehensive error-suppression capabilities for SNVs and detection thresholds for ctDNA below 0.1%. We also show that our semi-degenerate barcoded adapters hold promise for noninvasive genotyping in the absence of tumor biopsies and monitoring of minimal residual disease in longitudinal plasma samples. The benefits demonstrated here include broad applicability, flexibility, affordability and reproducibility in the research and clinical settings.

Genome sequencing of multiple primary tumors reveals a novel PALB2 variant

Introduction PALB2 (partner and localizer of BRCA2) has been implicated in hereditary breast cancer susceptibility, with estimates of breast cancer risk up to 91% (95% CI, 44% to 100%) to age 70 years for particular mutations. Germline mutations in PALB2 have also been identified in individuals with pancreatic cancer and ovarian carcinoma, both with and without familial breast cancer, suggesting a role in susceptibility to breast and ovarian cancer. PALB2 acts in the double-strand DNA break repair pathway recruiting RAD51 and BRCA2 to DNA breaks via its WD40 domain. Biallelic germline mutations cause Fanconi anemia, complementation group N(FANCN). Pathogenic germline variants of PALB2 causing loss of normal function may be substitutions or insertions/ deletions. Tumors in germline PALB2 mutation carriers show loss of the wild-type allele consistent with a tumor suppressor function. Structural variants deleting or duplicating multiple exons of PALB2 have been reported in association with familial breast cancer and in FANCN, and founder and recurrent mutations in PALB2 have been identified in several populations.

Abstract LB-47: SPEN is a novel candidate tumor suppressor gene that regulates response to tamoxifen in estrogen receptor positive breast cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The majority of breast cancers are hormone-responsive and are treated with anti-estrogens, such as tamoxifen. However, most of the 30 and 50% of estrogen receptor positive (ER+) patients that initially respond to tamoxifen eventually become resistant to the drug. Although there are several mechanisms responsible for resistance of breast cancers to tamoxifen, no predictive biomarkers for tamoxifen resistance are in clinical use besides the estrogen receptor (ER) and the progesterone receptors (PR). Using a novel integrative genomic method based on the discovery of nonsense mutations in deleted chromosomal fragments, we identified a nonsense mutation in the SPEN gene in the T47D breast cancer cell line. SPEN is a transcriptional repressor of the estrogen-signaling pathway, which is recruited to estrogen-responsive elements upon activation of the ER. We found 4 somatic mutations (2 nonsense and 2 missense) in 23 breast tumors showing loss of heterozygosity at the SPEN locus. Moreover, tissue microarrays showed that SPEN was frequently over-expressed in the nucleus of normal breast epithelial cells, but in only 10% of breast tumor cells, suggesting that inactivation of SPEN in breast cancer contributes to disease progression. In vitro, overexpression of SPEN in the T47D breast cancer cell line, in which SPEN is mutated and endogenous levels of the protein are very low, resulted in significantly decreased cell proliferation and anchorage-independent growth, decreased PR expression as well as increased sensitivity to tamoxifen. Remarkably, tamoxifen treatment induced 5-fold higher levels of apoptosis in SPEN-overexpressing compared to control T47D cells. In addition, using a tissue microarray of 100 tumor samples from ER+ breast cancer patients treated with tamoxifen only, we found that patients whose tumors express high levels of SPEN had a much better prognosis than patients whose tumors express low or no levels of the protein. To identify transcriptional targets of SPEN besides the PR, we performed gene expression profiling on a panel of breast cancer cell lines in which SPEN was either overexpressed or knocked-down. We found an inverse relationship between SPEN and Apolipoprotein D (APOD) expression, suggesting that SPEN potently repressed transcription of the APOD gene. APOD encodes a glycoprotein from the lipocalin family, which can chelate multiple molecules including progesterone, arachidonic acid as well as tamoxifen itself. Hence, our analysis shows that the loss or mutation of SPEN in ER+ breast cancers has the potential to affect tumor growth as well as sensitivity to tamoxifen, in part through upregulation of APOD expression. Together, our results highlight the role of SPEN as a novel putative tumor suppressor gene in breast cancer and suggest that SPEN is a candidate predictive biomarker of tamoxifen resistance in ER+ breast cancer patients. Citation Format: Stephanie Legare, Luca Cavallone, Aline Mamo, Catherine Chabot, Dana Keilty, Anthony Magliocco, Alexander Klimowicz, Patricia Tonin, Mark Basik. SPEN is a novel candidate tumor suppressor gene that regulates response to tamoxifen in estrogen receptor positive breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-47. doi:10.1158/1538-7445.AM2013-LB-47