Sigurdur Ingvarsson

Loss of heterozygosity at chromosome 1p in different solid human tumours: Association with survival

SummaryThe distal half of chromosome 1p was analysed with 15 polymorphic microsatellite markers in 683 human solid tumours at different locations. Loss of heterozygosity (LOH) was observed at least at one site in 369 cases or 54% of the tumours. LOHs detected ranged from 30–64%, depending on tumour location. The major results regarding LOH at different tumour locations were as follows: stomach, 20/38 (53%); colon and rectum, 60/109 (55%); lung, 38/63 (60%); breast, 145/238 (61%); endometrium, 18/25 (72%); ovary, 17/31 (55%); testis, 11/30 (37%); kidney, 22/73 (30%); thyroid, 4/14 (29%); and sarcomas, 9/14 (64%). High percentages of LOH were seen in the 1p36.3, 1p36.1, 1p35–p34.3, 1p32 and 1p31 regions, suggesting the presence of tumour-suppressor genes. All these regions on chromosome 1p show high LOH in more than one tumour type. However, distinct patterns of LOH were detected at different tumour locations. There was a significant separation of survival curves, with and without LOH at chromosome 1p, in the breast cancer patients. Multivariate analysis showed that LOH at 1p in breast tumours is a better indicator for prognosis than the other variables tested in our model, including nodal metastasis.

Mapping loss of heterozygosity at chromosome 13q: loss at 13q12-q13 is associated with breast tumour progression and poor prognosis.

Several chromosome regions exhibit loss of heterozygosity (LOH) in human breast carcinoma and are thought to harbour tumour suppressor genes (TSG). At chromosome 13q, two TSGs have been identified, RB1 at 13q14 and BRCA2 at 13q12-q13. In this study, 139 sporadic breast tumours were analysed with 18 polymorphic microsatellite markers for detailed mapping of LOH at chromosome 13q and evaluation of an association with known progression factors. LOH with at least one marker was observed in 71 (51%) of the tumours analysed. The deletion mapping indicated three LOH target regions, 13q12-q13, 13q14 and 13q31-q34. LOH at chromosome 13q12-q13 was associated with low progesterone receptor content, a high S phase fraction and aneuploidy. Multivariate analysis adjusting for lymph node involvement and S phase fraction showed that patients with tumours exhibiting LOH at 13q12-q13 have a 3-4-fold increased risk of recurrence and death compared with other patients. Our results suggest there are at least three separate LOH target regions at chromosome 13q and inactivation of one or more genes at chromosome 13q12-q13 results in poor prognosis for breast cancer patients.

Chromosome alterations and E-cadherin gene mutations in human lobular breast cancer

SummaryWe have studied a set of 40 human lobular breast cancers for loss of heterozygosity (LOH) at various chromosome locations and for mutations in the coding region plus flanking intron sequences of the E-cadherin gene. We found a high frequency of LOH (100%, 31/31) at 16q21–q22.1. A significantly higher level of LOH was detected in ductal breast tumours at chromosome arms 1p, 3p, 9p, 11q, 13q and 18q compared to lobular breast tumours. Furthermore, we found a significant association between LOH at 16 q containing the E-cadherin locus and lobular histological type. Six different somatic mutations were detected in the E-cadherin gene, of which three were insertions, two deletions and one splice site mutation. Mutations were found in combination with LOH of the wild type E-cadherin locus and loss of or reduced E-cadherin expression detected by immunohistochemistry. The mutations described here have not previously been reported. We compared LOH at different chromosome regions with E-cadherin gene mutations and found a significant association between LOH at 13 q and E-cadherin gene mutations. A significant association was also detected between LOH at 13q and LOH at 7q and 11q. Moreover, we found a significant association between LOH at 3 p and high S phase, LOH at 9p and low ER and PgR content, LOH at 17p and aneuploidy. We conclude that LOH at 16q is the most frequent chromosome alteration and E-cadherin is a typical tumour suppressor gene in lobular breast cancer.

Loss of RALT/MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin

An emerging paradigm holds that loss of negative signalling to receptor tyrosine kinases (RTKs) is permissive for their oncogenic activity. Herein, we have addressed tumor suppression by RALT/MIG-6, a transcriptionally controlled feedback inhibitor of ErbB RTKs, in breast cancer cells. Knockdown of RALT expression by RNAi enhanced the EGF-dependent proliferation of normal breast epithelial cells, indicating that loss of RALT signalling in breast epithelium may represent an advantageous condition during ErbB-driven tumorigenesis. Although mutational inactivation of the RALT gene was not detected in human breast carcinomas, RALT mRNA and protein expression was strongly and selectively reduced in ERBB2-amplified breast cancer cell lines. Reconstitution of RALT expression in ERBB2-amplified SKBr-3 and BT474 cells inhibited ErbB-2-dependent mitogenic signalling and counteracted the ability of ErbB ligands to promote resistance to the ErbB-2-targeting drug Herceptin. Thus, loss of RALT expression cooperates with ERBB2 gene amplification to drive full oncogenic signalling by the ErbB-2 receptor. Moreover, loss of RALT signalling may adversely affect tumor responses to ErbB-2-targeting agents.

Loss of heterozygosity at chromosome 11 in breast cancer: association of prognostic factors with genetic alterations

We examined DNA from 116 female and four male breast cancer patients for loss of heterozygosity (LOH). DNA was analysed by polymerase chain reaction using ten microsatellite markers on chromosome 11. Three distinct regions of LOH were identified: 11p15.5, 11q13 and 11q22-qter with a LOH frequency of 19, 23 and 37-43% respectively. The marker D11S969 showing the highest frequency of LOH (43%) is located at the 11q24.1-q25 region. No previous molecular genetic studies have shown frequent LOH at the region telomeric to q23 on chromosome 11. Southern analysis revealed that LOH at 11q13 was due to amplification, whereas LOH at 11q22qter was due to deletion. LOH at 11p15.5 was associated with paucity of hormone receptor proteins, high S-phase and positive node status. An association was found between LOH at 11q13 and positive node status. LOH at the 11q22-qter region correlated with a high S-phase fraction. A significant association was found between LOH at 11p15 and chromosome regions 17q21 (the BRCA1 region) and 3p.

Altered expression of E-cadherin in breast cancer

Reduced cell adhesion brought about by altered surface expression of E-cadherin has been implicated in invasive and metastatic malignant growth. We investigated the patterns of immunohistochemical E-cadherin expression in 120 breast carcinomas. Furthermore, we analysed DNA from the same samples for loss of heterozygosity (LOH) using three separate microsatellite markers on chromosome 16q22.1. Finally, the clinical outcome was ascertained for 108 patients. 19% (18/97) of infiltrating ductal carcinomas showed complete loss of E-cadherin expression compared with 64% (9/14) of infiltrating lobular carcinomas. LOH was detected in 46% (24/52) of infiltrating ductal carcinomas and 89% (8/9) of infiltrating lobular carcinomas. In the infiltrating lobular carcinomas, LOH was associated with complete loss of cell membrane expression of E-cadherin, although a cytoplasmic expression pattern was evident. In contrast, this association was not seen in the infiltrating ductal carcinomas. In a multivariate analysis, loss of E-cadherin expression was shown to be a significant independent risk factor for a poorer disease-free survival (P=0.019), in particular in the node-negative subset of patients (P=0.029). Significance was also approached for breast cancer corrected survival (P=0.056). We conclude that different mechanisms are involved in the altered E-cadherin expression seen in different subtypes of breast carcinomas. Furthermore, we implicate loss of E-cadherin, regardless of the genetic causes, as an independent prognostic marker for disease recurrence, especially in node-negative breast cancer patients, irrespective of the histological type.

Mutation analysis of the CHK2 gene in breast carcinoma and other cancers.

BackgroundMutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways.MethodsWe screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene.ResultsSeventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5 splice site mutation at site +3 in intron 8 (TTgt [a → c]atg) was also detected.ConclusionWe conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth.

Alterations of E-cadherin and β-catenin in gastric cancer

BackgroundThe E-cadherin-catenin complex plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. Perturbation in the expression or function of this complex results in loss of intercellular adhesion, with possible consequent cell transformation and tumour progression.MethodsWe studied the alterations of E-cadherin and β-catenin in a set of 50 primary gastric tumours by using loss of heterozygosity (LOH) analysis, gene mutation screening, detection of aberrant transcripts and immunohistochemistry (IHC).ResultsA high frequency (75%) of LOH was detected at 16q22.1 containing E-cadherin locus. Three cases (6%) showed the identical missense mutation, A592T. This mutation is not likely to contribute strongly to the carcinogenesis of gastric cancer, because a low frequency (1.6%) of this mutation was also found in 187 normal individuals. We also detected a low frequency (0.36%, 0%) of this mutation in 280 breast tumours and 444 other tumours, including colon and rectum, lung, endometrium, ovary, testis, kidney, thyroid carcinomas and sarcomas, respectively. We also analyzed the aberrant E-cadherin mRNAs in the gastric tumours and found that 7 tumours (18%) had aberrant mRNAs in addition to the normal mRNA. These aberrant mRNAs may produce abnormal E-cadherin molecules, resulting in weak cell-cell adhesion and invasive behaviour of carcinoma cells. Reduced expression of E-cadherin and β-catenin was identified at the frequency of 42% and 28%, respectively. Specially, 11 tumours (22%) exhibited positive cytoplasmic staining for β-catenin IHC. An association was found between reduced expression of E-cadherin and β-catenin. Moreover, an association was detected between reduced expression of E-cadherin and diffuse histotype.ConclusionOur results support the hypothesis that alterations of E-cadherin and β-catenin play a role in the initiation and progression of gastric cancer.

Loss of heterozygosity on chromosome arm 3p in nasopharyngeal carcinoma

We have examined 17 primary undifferentiated nasopharyngeal carcinoma biopsies for allelic loss on 3p, comparing the findings in tumors with those in normal lymphocyte DNA from the same patients. Ten polymorphic microsatellite markers were used between 3p13 and 3p26. Allelic loss was observed in 12 samples (70%). Two loci were most frequently affected: D3S1067 (3p21.1‐14.3) in 60% and D3S1217 (3p14.2‐14.1) in 58%. One tumor seemed to have a homozygous deletion at 3p26, detected by the D3S1297 marker. Analysis of the clinical data showed that an increased number of aberrations in 3p was correlated with more advanced tumor stages. Genes Chromosom Cancer 17:118–126 (1996).

The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O2 tension. In high O2 tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel–Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin–ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD–LIMD1–VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxicxa0regulators.