Identification of nucleolar protein No55 as a tumour-associated autoantigen in patients with prostate cancer

Four different genes were identified by immunoscreening of a cDNA expression library from the human prostate cancer cell line DU145 with allogeneic sera from four prostate cancer patients. A cDNA encoding the nucleolar protein No55 was further analysed and shown to be expressed at the mRNA level in several normal tissues, including ovaries, pancreas and prostate and in human prostate cancer cell lines PC-3, PC-3m and LNCaP. By reverse transcriptase/polymerase chain reaction, expression of No55 was several-fold higher in two out of nine prostate cancer primary tumours and two out of two metastatic lesions, compared to normal prostate tissue. Antibodies to No55 were detected in sera from seven out of 47 prostate cancer patients but not in sera from 20 healthy male controls. Sequence analysis of the No55 open reading frame from normal and tumour tissues revealed no tumour-specific mutations. The No55 gene was located to chromosome 17q21, a region reported to be partially deleted in prostate cancer. Considering the immunogenicity of the No55 protein in the tumour host, the expression profile and chromosomal localization of the corresponding gene, studies evaluating No55 as a potential antigen for immunological studies in prostate cancer may be warranted. © 2000 Cancer Research Campaig

Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer

INTRODUCTION: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response. METHODS: Tumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Akt(ser473 )phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy. RESULTS: Using a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAkt(ser473 )than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAkt(ser473 )and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAkt(ser473 )level. CONCLUSION: The activity of the PI3K pathway can be determined in tissue sections by quantitative imaging using an antibody towards pAkt(ser473). Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts. This indicates that PI3K inhibitors may have selective efficacy in basal-like breast cancer with increased PI3K signaling, and identifies lactate, phosphocholine and glycerophosphocholine as potential metabolic biomarkers for early therapy monitoring. In human biopsies, variable pAkt(ser473 )levels were observed, suggesting heterogeneous PI3K signaling activity in BLBC

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control

Distinct choline metabolic profiles are associated with differences in gene expression for basal-like and luminal-like breast cancer xenograft models

<p>Abstract</p> <p>Background</p> <p>Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood.</p> <p>Methods</p> <p>The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS).</p> <p>The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses.</p> <p>The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer.</p> <p>Results</p> <p>In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (<it>CHKA</it>, <it>CHKB</it>) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (<it>PLA2G4A</it>) and phospholipase B1 (<it>PLB1</it>) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer.</p> <p>Conclusions</p> <p>The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer.</p

Cystatin E/M suppresses legumain activity and invasion of human melanoma

<p>Abstract</p> <p>Background</p> <p>High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.</p> <p>Methods</p> <p>A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.</p> <p>Results</p> <p>Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.</p> <p>Conclusions</p> <p>These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.</p

Germline HOXB13 mutations p.G84E and p.R217C do not confer an increased breast cancer risk

In breast cancer, high levels of homeobox protein Hox-B13 (HOXB13) have been associated with disease progression of ER-positive breast cancer patients and resistance to tamoxifen treatment. Since HOXB13 p.G84E is a prostate cancer risk allele, we evaluated the association between HOXB13 germline mutations and breast cancer risk in a previous study consisting of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls from the Netherlands. Although both recurrent HOXB13 mutations p.G84E and p.R217C were not associated with breast cancer risk, the risk estimation for p.R217C was not very precise. To provide more conclusive evidence regarding the role of HOXB13 in breast cancer susceptibility, we here evaluated the association between HOXB13 mutations and increased breast cancer risk within 81 studies of the international Breast Cancer Association Consortium containing 68,521 invasive breast cancer patients and 54,865 controls. Both HOXB13 p.G84E and p.R217C did not associate with the development of breast cancer in European women, neither in the overall analysis (OR = 1.035, 95% CI = 0.859-1.246, P = 0.718 and OR = 0.798, 95% CI = 0.482-1.322, P = 0.381 respectively), nor in specific high-risk subgroups or breast cancer subtypes. Thus, although involved in breast cancer progression, HOXB13 is not a material breast cancer susceptibility gene.Peer reviewe