Patricia Keith

Expression analysis of delta-catenin and prostate-specific membrane antigen: Their potential as diagnostic markers for prostate cancer

The current approach to prostate cancer diagnosis has major limitations including the inability of prostate‐specific antigen (PSA) assays to accurately differentiate between prostate cancer and benign prostate hyperplasia (BPH) and the imprecision of transrectal ultrasound (TRUS) biopsy sampling. We have employed cDNA microarray screening to compare gene expression patterns in BPH and tumour samples to identify expression markers that may be useful in discriminating between these conditions. Screening of 3 individual cDNA arrays identified 8 genes with expression 3‐fold greater in 6 tumour tissues than in 1 nontumour sample and 1 BPH sample. Real‐time PCR was used to confirm the overexpression of these 8 genes and 12 genes selected from the literature against a panel of 17 tumours and 11 BPH samples. Two genes, δ‐catenin (delta‐catenin; CTNND2) and prostate‐specific membrane antigen (PSMA; FOLH1), were significantly overexpressed in prostate cancer compared to BPH. Prostate epithelial cells stained positively for δ‐catenin and PSMA in our prostate cancer tissues, whereas the majority of our BPH tissues were negative for both markers. Thus we have identified δ‐catenin (not previously associated with prostatic adenocarcinoma) and confirmed the potential of PSMA as potential candidates for the diagnosis and management of prostate cancer.

Aberrant expression of E-cadherin in lobular carcinomas of the breast.

Invasive lobular carcinoma (ILC) and lobular carcinoma in situ characteristically show loss of E-cadherin expression and so immunohistochemistry for E-cadherin is being increasingly used as a tool to differentiate between lobular and ductal lesions in challenging situations. However, misinterpretation of “aberrant” positive staining may lead some to exclude a diagnosis of lobular carcinoma. E-cadherin and β-catenin immunohistochemistry was analyzed in 25 ILCs. E-cadherin “positive” ILCs were subjected to molecular analysis including comparative genomic hybridization. Different morphologic components of case 25, showing heterogenous E-cadherin expression, were analyzed by E-cadherin gene sequencing, methylation, and DASL gene expression profiling. Four ILCs were positive for E-cadherin, but each also had neoplastic cells with aberrant staining. Two of these ILCs were positive for β-catenin, again with some aberrantly stained neoplastic cells, and 2 were negative. The solid component of case 25 was positive for E-cadherin whereas the classic and alveolar areas were negative. All components harbored an in-frame deletion in exon 7 (867del24) of the E-cadherin gene and loss of the wild type allele. Comparative genomic hybridization demonstrated evidence of clonal evolution from E-cadherin–positive to E-cadherin–negative components. E-cadherin down-regulation seems to be through transcriptional repression via activation of transforming growth factor-β/SMAD2 rather than methylation. Positive staining for E-cadherin should not preclude a diagnosis of lobular in favor of ductal carcinoma. Molecular evidence suggests that even when E-cadherin is expressed, the cadherin-catenin complex maybe nonfunctional. Misclassification of tumors may lead to mismanagement of patients in clinical practice, particularly in the context of in situ disease at margins.

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer

IntroductionMetastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.MethodsWe used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.ResultsMost brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.ConclusionsDeregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.

Subtypes of familial breast tumours revealed by expression and copy number profiling

Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism–comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

Disease-associated polymorphisms in ERAP1 do not alter endoplasmic reticulum stress in patients with ankylosing spondylitis.

The mechanism by which human leukocyte antigen B27 (HLA-B27) contributes to ankylosing spondylitis (AS) remains unclear. Genetic studies demonstrate that association with and interaction between polymorphisms of endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 influence the risk of AS. It has been hypothesised that ERAP1-mediated HLA-B27 misfolding increases endoplasmic reticulum (ER) stress, driving an interleukin (IL) 23-dependent, pro-inflammatory immune response. We tested the hypothesis that AS-risk ERAP1 variants increase ER-stress and concomitant pro-inflammatory cytokine production in HLA-B27+ but not HLA-B27− AS patients or controls. Forty-nine AS cases and 22 healthy controls were grouped according to HLA-B27 status and AS-associated ERAP1 rs30187 genotypes: HLA-B27+ERAP1risk, HLA-B27+ERAP1protective, HLA-B27−ERAP1risk and HLA-B27−ERAP1protective. Expression levels of ER-stress markers GRP78 (8 kDa glucose-regulated protein), CHOP (C/EBP-homologous protein) and inflammatory cytokines were determined in peripheral blood mononuclear cell and ileal biopsies. We found no differences in ER-stress gene expression between HLA-B27+ and HLA-B27− cases or healthy controls, or between cases or controls stratified by carriage of ERAP1 risk or protective alleles in the presence or absence of HLA-B27. No differences were observed between expression of IL17A or TNF (tumour necrosis factor) in HLA-B27+ERAP1risk, HLA-B27+ERAP1protective and HLA-B27−ERAP1protective cases. These data demonstrate that aberrant ERAP1 activity and HLA-B27 carriage does not alter ER-stress levels in AS, suggesting that ERAP1 and HLA-B27 may influence disease susceptibility through other mechanisms.

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers

Introduction Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Methods Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. Results There was no significant difference in HLA-class I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Discussion Large differences were not seen in HLA-B27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.

Genetic association of ankylosing spondylitis with TBX21 influences T-bet and pro-inflammatory cytokine expression in humans and SKG mice as a model of spondyloarthritis.

Objectives Ankylosing spondylitis (AS) is a highly heritable immune-mediated arthropathy. Inflammation in AS is poorly understood. TBX21 encodes T-bet, a transcription factor, lying within a locus with genome-wide significant association with AS. T-bet is implicated in innate and adaptive immunity. However, the role of T-bet in AS pathogenesis is unclear. Methods We assessed the importance of T-bet in disease development and progression in peripheral blood mononuclear cells from 172 AS cases and 83 healthy controls carrying either risk or protective alleles of the peak AS-associated TBX21 single nucleotide polymorphism. Kinetics and localisation of T-bet expression in the SKG mouse model of spondyloarthropathy was examined, along with the impact of Tbx21 knockout on arthritis development in SKG mice. Results Patients with AS had higher T-bet expression than healthy individuals, driven predominantly by natural killer and CD8+ T cells, with expression levels in CD8+ T cells completely distinguishing AS cases from healthy controls. T-bet expression was increased in AS cases carrying risk compared with protective alleles of rs11657479. In curdlan-treated SKG mice, T-bet expression increased early after disease initiation and persisted throughout the course of disease. There was marked reduction in gut and peripheral joint inflammation, and less IFNγ-producing and IL-17-producing CD8+ T cells, in Tbx21−/− compared with wild-type SKG mice. Conclusions AS-associated variants in TBX21 influence T-bet expression. T-bet+ innate and adaptive immune cells have altered IL-17 and IFNγ, and early activation marker CD69 expression than T-bet cells. This indicates that T-bet is a major component of inflammatory pathways of spondyloarthropathy in humans and mice.

Dual loss of Rb1 and Trp53 in melanocytes perturbs melanocyte homeostasis and genetic stability in vitro but does not cause melanoma or pigmentation defects in vivo.

Dear Sir, We have previously shown that the loss of Rb1 is insufficient to deregulate melanocyte homeostasis in vivo (Tonks et al., 2005), suggesting that further genetic lesions apart from the loss of this important tumour suppressor gene may be required to facilitate spontaneous melanoma. Deregulation of Trp53 appears to be an obvious candidate as the principal melanoma susceptibility gene, CDKN2A, encodes via alternative reading frames p16 and Arf (Quelle et al., 1995), which can regulate the pocket protein (PP) and Trp53 pathways respectively (Sherr and Roberts, 1999; Zhang et al., 1998). Consequently, to test whether the co-ablation of the main tumour suppressor genes of each regulatory pathway was able to yield spontaneous melanoma in vivo, we generated mice with compound ablation of Rb1 and Trp53 in Tyrosinase (Tyr) transcriptional domains using the Cre ⁄ loxP system. In this case, Trp53 ⁄ F2)10 mice (Jonkers et al., 2001) were bred with Rb1 ⁄ :TEC1 mice (Tonks et al., 2005) to yield ultimately Rb1 ⁄ :Trp53 ⁄ :TEC1 pups, where the TEC1 transgene drives Cre recombinase expression and ablation of floxed alleles in a variety of neuroepithelial and neural crest derived tissues, including the melanocytes (Tonks et al., 2003). The Rb1 ⁄ :Trp53 ⁄ :TEC1 pups were born at the expected Mendelian ratios but, in contrast to their control littermates, the Rb1 ⁄ :Trp53 ⁄ :TEC1 mice survived no longer than 6 months. In all cases, mortality was attributable to phaeochromocytomas, but not melanomas (Tonks, I.D., Mould, A., Nurcombe, V., Cool, S.M., Cotterill, A., Keith, P., Walker, G.J., Hayward, N.K. and Kay, G.F., unpublished data). Indeed, at no point did the Rb1 ⁄ :Trp53 ⁄ :TEC1 cohorts manifest any hyperor hypopigmentation defects, even though the floxed alleles were appropriately deleted (Figure S1). Despite the lack of melanoma, the loss of Trp53 alone or in combination with Rb1 does have ramifications on melanocyte homeostasis in vitro. Wild type and Rb1 ⁄ DX2 melanocytes have doubling times of 30 h and 20 h respectively (Tonks et al., 2005). In contrast, the Rb1 ⁄ :Trp53 ⁄ :TEC1 (Rb1 ⁄ :Trp53 ⁄ ) and Rb1 ⁄ :Trp53 ⁄ :TEC1 (Rb1 ⁄ :Trp53 ⁄ ) cultures were found to have an average doubling time of 23.7 h and 17.5 h respectively (Figure S2A). Furthermore, Rb1 ⁄ :Trp53 ⁄ D and Rb1 ⁄ :Trp53 ⁄ D cultures both readily proliferated in substantially reduced levels of mitogens (Figure S2B). The faster growth rates of Rb1 ⁄ :Trp53 ⁄ D and Rb1 ⁄ DX2 cultures compared with Rb1 ⁄ :Trp53 ⁄ D equivalents was unexpected, but was not attributable to increased levels of apoptosis in Rb1 ⁄ :Trp53 ⁄ D cultures. Instead Fluorescent Activated Cell Sorting (FACS) analysis indicated that the differences were underpinned by the marked tendency of Rb1 ⁄ :Trp53 ⁄ D melanocytes to become aneuploid. Indeed, while Rb1 ⁄ :Trp53 ⁄ D melanocytes possessed a relatively constant proportion of hypoploid cells over the passages examined, the Rb1 ⁄ :Trp53 ⁄ D melanocytes showed not only a comparatively and progressively larger population of hypoploid cells for a given passage number but, at higher passage, a significant hyperploid population of cells (Table 1). To assess the potential defects that lead to aneuploidy, melanocytes were probed with anti-a-microtubule and anti-c-tubulin antibodies and counterstained with 4¢,6-diamidino-2-phenylindole (DAPI) to examine their microtubule ⁄ spindle structure, centrosome

Melanocyte transformation requires complete loss of all pocket protein function via a mechanism that mitigates the need for MAPK pathway activation

Deregulation of p16INK4A is a critical event in melanoma susceptibility and progression. It is generally assumed that the major effect of loss of p16 function is mediated through the CDK−cyclin pathway via its influence on the pocket protein (PP) pRb. However, there are also two other PPs, p107 and p130, which, when phosphorylated by CDK–cyclin complexes, play a role in permitting cell progression. Cohorts of mice carrying melanocyte-specific knockouts (KOs) of various combinations of the three PPs were generated. Mice null for pRb, p107, p130 or any combination of double mutants did not develop melanoma. Surprisingly, melanocyte-specific loss of all three PPs facilitated melanoma development (median age of onset 308 days, penetrance 40% at 1 year). Tumorigenesis was exacerbated by Trp53 co-deletion (median age of onset 275 days, penetrance 82% at 1 year), with cell culture studies indicating that this difference may result from the apoptotic role of Trp53. Melanomas in PP;Trp53-deficient mice lacked either Ras or Braf mutations, and hence developed in the absence of constitutive MAPK pathway activation. The lag period between induction of total PP or PP/Trp53 KO and melanoma development indicates that additional genetic or epigenetic alterations may account for neoplastic progression. However, exome sequencing of PP;Trp53 KO melanomas failed to reveal any additional recurrent driver mutations. Analysis of the putative mutation signature of the PP;Trp53 KO melanomas suggests that melanocytes are primed for transformation via a mutagenic mechanism involving an excess of T>G substitutions, but not involving a preponderance of C>T substitutions at CpG sites, which is the case for most spontaneous cancers not driven by a specific carcinogen. In sum, deregulation of all three PPs appears central to neoplastic progression for melanoma, and the customary reference to the p16INKA/CDK4/pRB pathway may no longer be accurate; all PPs are potentially critical targets of CDK-cyclins in melanoma.