Jamileh Hashemi

Haplotype analysis and age estimation of the 113insR CDKN2A founder mutation in Swedish melanoma families

Germline mutations in the CDKN2A tumor suppressor gene located on 9p21 have been linked to development of melanomas in some families. A germline 3‐bp insertion in exon 2 of CDKN2A, leading to an extra arginine at codon 113 (113insR), has been identified in 17 Swedish melanoma families. Analysis of 10 microsatellite markers, spanning approximately 1 Mbp in the 9p21 region, showed that all families share a common allele for at least one of the markers closest to the CDKN2A gene, suggesting that the 113insR mutation is an ancestral founder mutation. Differences in the segregating haplotypes, due to meiotic recombinations and/or mutations in the short‐tandem‐repeat markers, were analyzed further to estimate the age of the mutation. Statistical analysis using a maximum likelihood approach indicated that the mutation arose 98 generations (90% confidence interval: 52–167 generations), or approximately 2,000 years, ago. Thus, 113insR would be expected to have a more widespread geographic distribution in European and North American regions with ancestral connections to Sweden. Alternatively, CDKN2A may lie in a recombination hot spot region, as suggested by the many meiotic recombinations in this narrow ∼1‐cM region on 9p21.

Melanoma development in relation to non-functional p16/INK4A protein and dysplastic naevus syndrome in Swedish melanoma kindreds.

The CDKN2A gene encodes the cell cycle inhibitor p16/ INK4A, which is involved in familial cutaneous melanoma. We have studied five Swedish familial melanoma kindreds characterized by germline mutations in CDKN2A and dysplastic naevus syndrome (DNS). We found significant correlations between germline CDKN2A mutations and melanoma and between DNS phenotype and melanoma, respectively. There was also a correlation between mutation status and the presence of DNS. In CDKN2A mutation carriers, all cases of early-onset melanoma occurred in DNS individuals, and the mean age at melanoma diagnosis was significantly lower in individuals with DNS than in those without a confirmed DNS phenotype. In one family where the proband had a P48L mutation in CDKN2A exon 1, the DNS phenotype was studied in detail. In vitro binding experiments established that the P48L mutant protein does not bind to cdk4 or cdk6 and thus is functionally abnormal. Furthermore, we demonstrated loss of heterozygosity at markers on chromosome 9p flanking the CDKN2A locus in a primary melanoma and a metastasis from the proband. Our results are consistent with the hypothesis that germline CDKN2A mutations and DNS both contribute to the predisposition to melanoma and may lead to the development of early-onset melanoma when present in the same individual.

Genetic characterization of large parathyroid adenomas

In this study, we genetically characterized parathyroid adenomas with large glandular weights, for which independent observations suggest pronounced clinical manifestations. Large parathyroid adenomas (LPTAs) were defined as the 5% largest sporadic parathyroid adenomas identified among the 590 cases operated in our institution during 2005–2009. The LPTA group showed a higher relative number of male cases and significantly higher levels of total plasma and ionized serum calcium (P<0.001). Further analysis of 21 LPTAs revealed low MIB1 proliferation index (0.1–1.5%), MEN1 mutations in five cases, and one HRPT2 (CDC73) mutation. Total or partial loss of parafibromin expression was observed in ten tumors, two of which also showed loss of APC expression. Using array CGH, we demonstrated recurrent copy number alterations most frequently involving loss in 1p (29%), gain in 5 (38%), and loss in 11q (33%). Totally, 21 minimal overlapping regions were defined for losses in 1p, 7q, 9p, 11, and 15q and gains in 3q, 5, 7p, 8p, 16q, 17p, and 19q. In addition, 12 tumors showed gross alterations of entire or almost entire chromosomes most frequently gain of 5 and loss of chromosome 11. While gain of 5 was the most frequent alteration observed in LPTAs, it was only detected in a small proportion (4/58 cases, 7%) of parathyroid adenomas. A significant positive correlation was observed between parathyroid hormone level and total copy number gain (r=0.48, P=0.031). These results support that LPTAs represent a group of patients with pronounced parathyroid hyperfunction and associated with specific genomic features.

A melanoma-predisposing germline CDKN2A mutation with functional significance for both p16 and p14ARF

The CDKN2A locus on human chromosome 9p21 encodes two proteins, p16 and p14ARF, that mainly regulate cell cycle progression and cell survival via the pRb and p53 pathways, respectively. Germline mutations in CDKN2A have been linked to development of cutaneous melanoma in some families with hereditary melanoma. Due to overlapping open reading frames in exon 2, some mutations in this exon affect both p16 and p14ARF. We previously reported a 24bp deletion in CDKN2A exon 2 in a patient with multiple primary melanomas and melanoma heredity. To further clarify the possible role of the 24bp deletion for melanoma development, especially with respect to p14ARF, we have studied the cellular distribution and function of the resulting p14ARF del (77-84) and p16 del (62-69) mutant proteins. We found that p14ARF del (77-84) had decreased nucleolar localization, and was less efficient than wt p14ARF in stabilizing p53, inducing G1 cell cycle arrest, and inhibiting colony formation. The p16 del (62-69) mutant localized predominantly to the cytoplasm, did not induce G1 cell cycle arrest, and failed to suppress colony formation. We conclude that p14ARF del (77-84) has retained the ability to stabilize MDM2 and p53, but that it is less potent than wt p14ARF. This partial functional defect may complement the clearly defective p16 del (62-69) mutant and thus contribute to melanoma development in patients carrying the 24bp deletion in CDKN2A.

Analysis of Mechanisms and Frequency of CDKN2A/B Gene Loss During Progression of RAS-Transformed Rat Embryo Fibroblast Clones

Rat embryo fibroblasts (REFs) are inefficiently transformed by RAS-oncogenes. Induction of p16INK4Aexpression by RAS has been suggested to contribute to this resistance. Glucocorti-coid hormones, (DEX), enhance REF transformation by RAS and facilitates the isolation of transformed and immortal cell lines. We show that DEX induced cell proliferation is paralleled by a decrease in Cdkn2a gene transcripts, suggesting a mechanism for hormone promotion. The mechanisms of progression into hormone independent cell lines were examined. Twenty-two of 30 clones which reached a population size of approximately 106cells could be established as cell lines. All lines studied showed homozygous deletions of the Cdkn2 loci (Cdkn2a and Cdkn2b) on RNO5. LOH was found for all RNO5 genetic markers examined in 7 of 19 cell lines, suggesting non-disjunction events. In the remaining 12 cell lines, both copies of Cdkn2 appeared to be lost by deletions/rearrangements, some of which could by demonstrated by karyotype analysis. We conclude that (i) clonal expansion of RAS-transfected REF by DEX is paralleled by down-regulation of Cdkn2a expression; (ii) homozygous deletion of Cdkn2 were estimated to occur at a frequency of 2 × 10−8/cell/generation or higher, and (iii) deletion/rearrangements and non-disjunction appear to be the main mechanisms leading to deletion of Cdkn2.

Abstract 3462: Proteomic profiling reveals novel targets for combination treatment with lanreotide in neuroendocrine tumors

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnSomatostatin analogues (SSAs) are well established in the treatment of neuroendocrine tumors (NETs), including small intestinal NET (SI-NET) due to their tumor growth arrest and symptom relieving characteristics. Here we determined the direct effects of lanreotide treatment on NET cell lines and a primary SI-NET culture. The cell lines HC45 and H727 were treated with a pharmacological concentration of 10 nM lanreotide for different time periods and the proteome, analyzed by in-depth HiRIEF LC-MS/MS, was compared with the proteome of non-treated cells. We quantified 6 451 and 7 801 proteins in HC45 and H727. Differential expression of the candidate proteins APC, BIRC5/survivin, BMPER, C14orf142, FYN, INSM1 and SPAG16 was confirmed by Western blotting. The possible direct anti-proliferative effect of lanreotide was evaluated in the primary SI-NET culture and the HC45, H727 and BON1 cell lines, which showed only a slight inhibition of proliferation. However, based on proteomics driven pathway analysis a significant synergy in anti-proliferative effect of lanreotide was observed when the small molecule IGF1R inhibitor, NVP-AEW541, was combined with lanreotide .This effective combination also represents an indirect model of suppressed IGF1 activity in the NET patients treated with SSA. Targeting of survivin with the small molecule YM155 dramatically reduced the proliferation of HC45. We also studied the effect of lanreotide alone or in combination with IGF-1R inhibitor on signaling pathways PI3K/Akt, Erk1/2 and P38. P38, Erk1/2 and GSK3β phosphorylation was equally increased by lanreotide and/or NVP-AEW541, however, lanreotide-induced AktS473 dephosphorylation was reversed after6 hours, unlike consistent AktS473 dephosphorylation after treating with NVP-AEW451 alone or in combination. In conclusion, we report growth inhibitory and potential feedback pro-growth signaling of the SSA lanreotide on NET cells, highlighting the role of SSAs in modulation of IGF1 in NET cell growth arrest and propose combination treatment candidate targets for improved efficacy of this agent.nnCitation Format: Omid Fotouhi, Hanna Kjellin, Jamileh Hashemi, Ming Lu, Christofer Juhlin, Anders Hoog, Jan Zedenius, Catharina Larsson, Janne Lehtio, Magnus Kjellman. Proteomic profiling reveals novel targets for combination treatment with lanreotide in neuroendocrine tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2015-3462