Mikael Lundin

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer‐specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR‐binding sites (ARBs) on LNCaP‐1F5 cell chromatin that was commensurate with changes in androgen‐dependent gene expression signature. We identified three distinct classes of ARBs and androgen‐responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid‐dependent signalling in LNCaP‐1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.

Cleavable ErbB4 Isoform in Estrogen Receptor–Regulated Growth of Breast Cancer Cells

ErbB1 and ErbB2 receptors are well-characterized targets for anticancer drugs, but the clinical relevance of the related ErbB4 receptor is unknown. Here, we have assessed the clinical significance of the proteolytically cleavable ErbB4 isoforms in breast cancer patients and investigated their functions in vitro. The expression of transcripts encoding the cleavable ErbB4 isoforms associated with estrogen receptor-alpha (ER) expression (P < 0.001) and a high histologic grade of differentiation (P </= 0.002) in real-time reverse transcription-PCR analysis of 62 breast cancer samples. Despite high ErbB4 mRNA expression levels in a subset of samples, ErbB4 gene amplification was not observed. High ErbB4 protein expression levels, as assessed by immunohistochemistry, associated with a favorable outcome in ER-positive cases from a series of 458 breast cancer patients (P = 0.01), whereas no association between ErbB4 expression and survival was found among women with ER-negative cancer (P = 0.86). However, nuclear ErbB4 immunoreactivity was associated with poor survival as compared with women whose cancer had membranous ErbB4 staining (P = 0.04). In vitro, overexpression of a cleavable ErbB4 isoform in ER-positive breast cancer cells resulted in translocation of a proteolytically released intracellular ErbB4 receptor fragment into the nucleus, as well as, enhanced proliferation, anchorage-independent growth, and estrogen response element-mediated transcriptional activity. These results suggest that the association of ErbB4 expression with clinical outcome is dependent on the subcellular localization of ErbB4 and that a proteinase-cleavable ErbB4 isoform promotes growth of ER-positive breast cancer and enhances ER-mediated gene transcription.

MYC-Dependent Regulation and Prognostic Role of CIP2A in Gastric Cancer

BACKGROUND Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified human oncoprotein that stabilizes the c-Myc (MYC) protein. However, the clinical relevance of CIP2A to human cancers had not been demonstrated, but the mechanism of its regulation and its clinical role in cancer were completely unknown. METHODS Tissue microarrays consisting of 223 gastric adenocarcinoma specimens were evaluated for the presence of CIP2A using immunohistochemistry, and the association of CIP2A expression with survival was assessed using Kaplan-Meier analysis. The effects of MYC and CIP2A on each others expression and on cell proliferation were investigated in several gastric cancer cell lines using small interfering RNAs to CIP2A and MYC and immunoblotting. To further evaluate the role of MYC in CIP2A regulation, an inhibitor of MYC dimerization, 10058-F4, and an inducible MycER model were used. RESULTS Expression of CIP2A protein was associated with reduced overall survival for gastric cancer patients with tumors 5 cm or smaller, with a 10-year overall survival in the CIP2A-immunopositive group of 8.1% as compared with 37.6% in the CIP2A-negative group (difference = 29.5%, 95% confidence interval = 12.5% to 46.5%, P = .001). In gastric cancer cell lines, CIP2A depletion led to decreased proliferation and anchorage-independent growth of the cells, as well as to reduced stability and expression of MYC protein. Interestingly, MYC depletion led to reduced expression of CIP2A mRNA and protein. Moreover, experiments with an MYC inhibitor and activator suggested that MYC directly promotes CIP2A gene expression. Finally, CIP2A and MYC immunopositivities were associated in gastric cancer specimens (P = .021). CONCLUSIONS CIP2A immunopositivity is a predictor of survival for some subgroups of gastric cancer patients. CIP2A and MYC appear to be regulated in a positive feedback loop, wherein they promote each others expression and gastric cancer cell proliferation.

Basal-like phenotype is not associated with patient survival in estrogen-receptor-negative breast cancers

IntroductionBasal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup.MethodsIHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed.ResultsFrom the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlies intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification.ConclusionBasal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors.

Epithelial and stromal syndecan-1 expression as predictor of outcome in patients with gastric cancer.

The prognostic value of the immunohistochemical expression of epithelial and stromal syndecan‐1 was evaluated in 296 patients with gastric carcinoma. Formalin‐fixed, paraffin‐embedded specimens of gastric adenocarcinomas were stained with mouse monoclonal antibody B‐B4 against human syndecan‐1. Loss of immunoreactivity (≤60% of cancer cells stained) was observed in 197 (67%) patients. Stromal immunoreactivity was observed in 28 (9%) patients. Loss of epithelial syndecan‐1 immunoreactivity correlated with a higher stage of disease (stages II–IV), tumour location in the upper third of the stomach, nodal metastases (N1 or N2), positive stromal syndecan‐1 staining, deep tumour penetration (to subserosa or deeper = T2–T4), larger tumour size (≥5 cm) and intestinal type of cancer. No correlation between epithelial syndecan‐1 immunoreactivity and age, gender, distant metastases, grade of differentiation or Borrmann classification was observed. Positive stromal syndecan‐1 immunoreactivity correlated with decreased epithelial syndecan‐1 expression, intestinal type of cancer and Borrmann type I. Patients with low epithelial syndecan‐1 expression in cancer cells had worse overall survival than patients with strong epithelial syndecan‐1 staining (p = 0.0012). Stromal syndecan‐1‐positive patients had a worse outcome than patients with syndecan‐1‐negative stroma (p = 0.0193). In Cox multivariate analysis, stromal syndecan‐1 immunoreactivity was a prognostic factor independent of TNM stage, surgery for cure and tumour size. Thus, the immunohistochemical expression of syndecan‐1 might be a predictor of outcome in patients with gastric adenocarcinoma.

Breast cancer biological subtypes and protein expression predict for the preferential distant metastasis sites: a nationwide cohort study

IntroductionSome molecular subtypes of breast cancer have preferential sites of distant relapse. The protein expression pattern of the primary tumor may influence the first distant metastasis site.MethodsWe identified from the files of the Finnish Cancer Registry patients diagnosed with breast cancer in five geographical regions Finland in 1991-1992, reviewed the hospital case records, and collected primary tumor tissue. Out of the 2,032 cases identified, 234 developed distant metastases after a median follow-up time of 2.7 years and had the first metastatic site documented (a total of 321 sites). Primary tumor microarray (TMA) cores were analyzed for 17 proteins using immunohistochemistry and for erbB2 using chromogenic in situ hybridization, and their associations with the first metastasis site were examined. The cancers were classified into luminal A, luminal B, HER2+ enriched, basal-like or non-expressor subtypes.ResultsA total of 3,886 TMA cores were analyzed. Luminal A cancers had a propensity to give rise first to bone metastases, HER2-enriched cancers to liver and lung metastases, and basal type cancers to liver and brain metastases. Primary tumors that gave first rise to bone metastases expressed frequently estrogen receptor (ER) and SNAI1 (SNAIL) and rarely COX2 and HER2, tumors with first metastases in the liver expressed infrequently SNAI1, those with lung metastases expressed frequently the epidermal growth factor receptor (EGFR), cytokeratin-5 (CK5) and HER2, and infrequently progesterone receptor (PgR), tumors with early skin metastases expressed infrequently E-cadherin, and breast tumors with first metastases in the brain expressed nestin, prominin-1 and CK5 and infrequently ER and PgR.ConclusionsBreast tumor biological subtypes have a tendency to give rise to first distant metastases at certain body sites. Several primary tumor proteins were associated with homing of breast cancer cells.

Artificial Neural Networks Applied to Survival Prediction in Breast Cancer

In this study, we evaluated the accuracy of a neural network in predicting 5-, 10- and 15-year breast-cancer-specific survival. A series of 951 breast cancer patients was divided into a training set of 651 and a validation set of 300 patients. Eight variables were entered as input to the network: tumor size, axillary nodal status, histological type, mitotic count, nuclear pleomorphism, tubule formation, tumor necrosis and age. The area under the ROC curve (AUC) was used as a measure of accuracy of the prediction models in generating survival estimates for the patients in the independent validation set. The AUC values of the neural network models for 5-, 10- and 15-year breast-cancer-specific survival were 0.909, 0.886 and 0.883, respectively.The corresponding AUC values for logistic regression were 0.897, 0.862 and 0.858. Axillary lymph node status (N0 vs. N+) predicted 5-year survival with a specificity of 71% and a sensitivity of 77%. The sensitivity of the neural network model was 91% at this specificity level. The rate of false predictions at 5 years was 82/300 for nodal status and 40/300 for the neural network. When nodal status was excluded from the neural network model, the rate of false predictions increased only to 49/300 (AUC 0.877). An artificial neural network is very accurate in the 5-, 10- and 15-year breast-cancer-specific survival prediction. The consistently high accuracy over time and the good predictive performance of a network trained without information on nodal status demonstrate that neural networks can be important tools for cancer survival prediction.

Molecular Subtypes of Breast Cancers Detected in Mammography Screening and Outside of Screening

Purpose: The frequency and significance of gene expression profile-derived molecular subtypes of breast cancers found in mammography screening are unknown. Experimental Design: We identified breast cancers diagnosed in women of any age living in defined geographic regions in Finland in 1991 to 1992 and collected clinical and pathologic data. Surrogates for the molecular subtypes were determined for 247 cancers found in organized mammography screening and 989 cancers detected outside of screening using immunohistochemistry or in situ hybridization. Molecular subtypes were defined as luminal A [estrogen receptor (ER) positive and/or progesterone receptor (PR) positive, HER2-], luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-, cytokeratin 5+, and/or HER1+), HER2+/ER- (ER-, PR-, and HER2+), and unclassified. The median follow-up time was 9.4 years. Results: The luminal type A was common (73.7%) and the HER2+/ER- type is rare (5.7%) in screen-detected cancer, and only 16% were HER2 positive. Women with cancer diagnosed in screening at ages 50 to 69 years had similar molecular subtype distribution as women whose cancer was found outside of screening at age >69 years. In a multivariate model, cancer detection at screening independently predicted favorable distant disease-free survival when the molecular subtype was included as a covariate in addition to age, histologic grade, and cancer size. Women with small (pT1N0M0) HER2-positive cancer had similar outcome regardless of the method of detection. Conclusions: Molecular subtype distribution of screen-detected breast cancer differs from that of cancers found outside of screening and accounts in part for the better outcome of screen-detected cancer.

Systematic knockdown of epigenetic enzymes identifies a novel histone demethylase PHF8 overexpressed in prostate cancer with an impact on cell proliferation, migration and invasion

Our understanding of key epigenetic regulators involved in specific biological processes and cancers is still incomplete, despite great progress in genome-wide studies of the epigenome. Here, we carried out a systematic, genome-wide analysis of the functional significance of 615 epigenetic proteins in prostate cancer (PrCa) cells. We used the high-content cell-spot microarray technology and siRNA silencing of PrCa cell lines for functional screening of cell proliferation, survival, androgen receptor (AR) expression, histone methylation and acetylation. Our study highlights subsets of epigenetic enzymes influencing different cancer cell phenotypes. Plant homeo domain (PHD) finger proteins have a key role in cell survival and histone methylation, whereas histone deacetylases were primarily involved in regulating AR expression. In contrast, JumonjiC-domain (JmjC) containing histone lysine demethylases (KDMs) mainly had an impact on cell proliferation. Our results show that the KDMs JARID1B, PHF8, KDM3A, KDM3B and KDM4A were highly expressed in clinical PrCa samples. The PHD-finger protein 8 (PHF8), a transcriptional coactivator with both PHD- and JmjC-domains, was moderately to strongly expressed in 80% of clinical PrCa samples, whereas 76% of normal and benign samples were negative or only showed weak PHF8 expression. Strong PHF8 expression correlated significantly with high Gleason grade and was borderline significant for poor prognosis. The results of functional PHF8 knockdown implicate a role in cell migration and invasion, as shown by cell motility and 3-D invasion assays. Our study suggests that various cellular phenotypes are regulated by distinct subsets of epigenetic enzymes. Proteins interpreting and modifying histone methylation, such as JmjC-domain and particularly PHD-finger proteins like PHF8, are activated in subsets of PrCas and promote cancer relevant phenotypes.

Development and evaluation of a virtual microscopy application for automated assessment of Ki-67 expression in breast cancer

BackgroundThe aim of the study was to develop a virtual microscopy enabled method for assessment of Ki-67 expression and to study the prognostic value of the automated analysis in a comprehensive series of patients with breast cancer.MethodsUsing a previously reported virtual microscopy platform and an open source image processing tool, ImageJ, a method for assessment of immunohistochemically (IHC) stained area and intensity was created. A tissue microarray (TMA) series of breast cancer specimens from 1931 patients was immunostained for Ki-67, digitized with a whole slide scanner and uploaded to an image web server. The extent of Ki-67 staining in the tumour specimens was assessed both visually and with the image analysis algorithm. The prognostic value of the computer vision assessment of Ki-67 was evaluated by comparison of distant disease-free survival in patients with low, moderate or high expression of the protein.Results1648 evaluable image files from 1334 patients were analysed in less than two hours. Visual and automated Ki-67 extent of staining assessments showed a percentage agreement of 87% and weighted kappa value of 0.57. The hazard ratio for distant recurrence for patients with a computer determined moderate Ki-67 extent of staining was 1.77 (95% CI 1.31-2.37) and for high extent 2.34 (95% CI 1.76-3.10), compared to patients with a low extent. In multivariate survival analyses, automated assessment of Ki-67 extent of staining was retained as a significant prognostic factor.ConclusionsRunning high-throughput automated IHC algorithms on a virtual microscopy platform is feasible. Comparison of visual and automated assessments of Ki-67 expression shows moderate agreement. In multivariate survival analysis, the automated assessment of Ki-67 extent of staining is a significant and independent predictor of outcome in breast cancer.