Downregulation of nuclear expression of the p33(ING1b) inhibitor of growth protein in invasive carcinoma of the breast

BACKGROUND/Aims: The inhibitor of growth gene 1 (ING1) is a modulator of cell cycle checkpoints, apoptosis, and cellular senescence. The most widely expressed ING1 isoform is p33(ING1b), which can modulate p53, a molecule that is frequently altered in breast cancer. Reduced ING1 mRNA expression has been observed in primary breast cancer expressing wild-type p53. METHODS: p33(ING1b), p53, oestrogen receptor (ER), and progesterone receptor (PgR) expression was studied in 86 primary invasive breast cancers using immunohistochemistry. RESULTS: Reduced nuclear expression of p33(ING1b) was found in cancer cells, both in intensity and the proportion of cells staining. This was associated with enhanced cytoplasmic p33(ING1b) expression in a proportion of cases. Analysis of several known biological factors indicated that high grade tumours were of larger size and more often negative for ER and PgR expression. However, larger tumours were more frequently p53 negative. These results provide evidence that p33(ING1b) alterations are associated with more poorly differentiated tumours. Positive correlations were found between nuclear p33(ING1b) expression and both ER and PgR expression. CONCLUSIONS: Optimum function of p53 is dependent on p33(ING1b) so that a reduction of nuclear p33(ING1b) expression, as seen in this series, would be predicted to compromise p53 function. This study showed that p33(ING1b) alterations were associated with more poorly differentiated tumours. Therefore, p33(ING1b) expression could be used as a marker of differentiation in invasive breast cancer. These results support the view that loss of p33(ING1b) may be an important molecular event in the differentiation and pathogenesis of invasive breast cancer.     

Profiling bladder cancer using targeted antibody arrays

Bladder cancer is a common malignancy requiring a high degree of surveillance because of the frequent recurrences and the poor clinical outcome of invasive disease. To date, serum biomarkers for bladder cancer lack optimal sensitivity and specificity to assist in diagnosis and disease categorization. Here, we designed antibody arrays for bladder cancer by selecting antibodies against targets differentially expressed in bladder tumors. Serum protein profiles measured by an antibody array containing 254 antibodies discriminated bladder cancer patients from controls (n = 95) with a correct classification rate of 93.7%. A second independent antibody array containing 144 antibodies revealed that protein profiles provide predictive information by stratifying patients with bladder tumors (n = 37) based on their overall survival (P = 0.0479). In addition, serum proteins, such as c-met, that were top ranked at identifying bladder cancer patients were associated with pathological stage, tumor grade, and survival when validated by immunohistochemistry of tissue microarrays containing bladder tumors (n = 173). This study provides experimental evidence for the use of several integrated technologies strengthening the process of biomarker discovery. Serum protein profiles obtained by antibody arrays represent comprehensive means for bladder cancer diagnosis and clinical outcome stratification, which could potentially assist in selection of cancer patients who would benefit from early, individualized therapeutic intervention.     

Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer

Many molecular alterations are known to occur in urothelial carcinoma of the bladder, but their significance for tumor progression is poorly understood. Deletions of chromosome 8p are frequently found in several tumor types and are often associated with progressive disease. In all, 99 bladder tumors were screened for deletions at 8p using loss of heterozygosity (LOH) and multicolor fluorescence in situ hybridization FISH analyses. Allelic loss on chromosome 8p in at least one marker was found in 25/99 (25%) tumors. There was a significant correlation of 8p deletions with invasive tumor growth and a highly significant association with papillary growth pattern in patients with invasive disease. cDNA array analyses revealed that secreted Frizzled-related protein 1 (sFRP1), an antagonist of Frizzled receptors and Wnt pathway activation on chromosome 8p12-11.1, is frequently downregulated in bladder cancer. To investigate sFRP1 as a candidate for a putative progression-related gene on 8p, urothelial cell lines and primary urothelial carcinomas were screened for sFRP1 expression using quantitative real-time PCR, Northern blot, immunofluorescence and immunohistochemistry (IHC). Of the investigated bladder cancers, 38% showed loss of sFRP1 expression by quantitative RT-PCR. Evaluation of the protein expression by IHC using tissue microarrays containing 776 bladder cancers revealed loss or strong reduction of sFRP1 expression in 66% of cases. SFRP1 loss was associated with higher tumor stage and grade and shorter overall survival. In addition, loss of sFRP1 was an independent indicator of poor survival in patients with papillary but not with muscle invasive bladder cancer. There were neither mutations in the coding region of sFRP1 nor homozygous deletions at 8p12-11.21. However, promoter methylation was detected using methylation-specific PCR in 29% of cases. In conclusion, we could show a close correlation of chromosome 8p deletions and progression of papillary bladder tumors. The sFRP1 gene on chromosome 8p12-11.1 could be a candidate gene for the predicted, progression-related tumor suppressor gene in bladder cancer and could contribute to urothelial carcinogenesis.     

Molecular classification of nodal metastasis in primary larynx squamous cell carcinoma

Classification and prognosis of larynx squamous cell carcinoma (LSCC) depends on clinical and histopathological examination. Currently, expression profiling harbors the potential to investigate, classify, and better manage cancer. Gene expression profiles of 22 primary LSCCs were analyzed by microarrays containing 19,200 cDNAs. GOAL functionally classified differentially expressed genes, and a novel "in silico" procedure identified physical gene clusters differentially transcribed. A signature of 158 genes differentiated tumors with nodal metastasis. A novel statistical method allowed categorization of metastatic tumors into 2 distinct subgroups of differential gene expression patterns. Among genes correlated to nodal metastatic progression, we verified in vitro that NM23-H3 reduced cell motility and TRIM8 were a growth suppressor. Six chromosomal regions were specifically downregulated in metastatic tumors. This large-scale gene expression analysis in LSCC provides information on changes in genomic activity associated with lymphonodal metastasis and identifies molecules that might prove useful as novel therapeutic targets.     

Alterations in transcription clusters underlie development of bladder cancer along papillary and nonpapillary pathways

Bladder cancer develops in the urothelial lining from intraurothelial preneoplasia via two pathways, papillary and nonpapillary, which correspond to nonaggressive and aggressive forms of the disease. Because these two forms of cancer may develop via distinct molecular events, we examined the gene expression patterns in the development of bladder cancer from preneoplasia along papillary and nonpapillary pathways. The expression profiles of 19 pairs of RNA samples from adjacent urothelium and tumors were analyzed using cDNA microarrays. For selected genes their expressions were verified on a cohort of 251 bladder cancer patients using tissue microarray and immunohistochemistry and were related to clinicopathological parameters including follow-up data. We identified alterations in seven gene clusters controlling proliferation, differentiation, and programmed cell death that were common for papillary and nonpapillary cancer. In contrast, genes controlling cellular and stromal interactions were altered in the nonpapillary cancer. The expression levels of only two genes from this group could be used to define an aggressive form of the disease. Tumors characterized by the low expression of e-cadherin and the high expression of DNA alpha-topoisomerase II had a high propensity for distant metastasis and were associated with poor survival.     

Identification of androgen-responsive genes that are alternatively regulated in androgen-dependent and androgen-independent rat prostate tumors

The vast majority of androgen-dependent prostate tumors progress toward incurable, androgen-independent tumors. The identification of androgen-responsive genes, which are still actively transcribed in the tumors of patients who have undergone androgen ablation, may shed light on the molecular mechanisms underlying this phenomenon. To address this question, we chose the Dunning R3327 rat model system, in which the progression from androgen-dependent to -independent tumors is represented by several transplantable prostate-derived tumors. Gene expression profiles were analyzed in normal rat prostates and in the prostates of rats 14 days after castration by use of microarrays containing approximately 5,000 oligonucleotides, together representing more than 4,800 known rat genes. These expression profiles were compared with similarly obtained expression profiles of androgen-dependent and androgen-independent rat prostate tumors. By doing so, a series of known and novel prostate cancer-associated androgen-responsive genes was identified. Within this series, we were able to identify several clusters of genes that are differentially regulated in the various prostate tumors. These genes may serve as (i) novel prognostic identifiers and (ii) novel therapeutic targets.     

Nuclear to cytoplasmic compartment shift of the p33ING1b tumour suppressor protein is associated with malignancy in melanocytic lesions

AIMS: Cutaneous malignant melanoma is an unpredictable neoplasm. Studies of cell cycle and proliferation-associated proteins may help in the understanding of the genesis of melanomas. The tumour suppressor gene TP53 has been shown to be involved in melanomas. However, the incidence of TP53 malfunction in cutaneous melanoma is unclear, and other regulators of cell cycle control are likely to be involved in both the development and progression of melanocytic neoplasia. A candidate is the ING1 gene, which co-operates with TP53 in growth suppression and apoptosis. Thus loss of ING1 function may have similar consequences to loss of TP53 function and may contribute to tumorigenesis. Therefore we have studied the expression of p33ING1b protein in cutaneous melanocytic neoplasia. METHODS AND RESULTS: Sixty-seven melanocytic lesions were studied by immunohistochemistry for the expression of p33ING1b. In our series there was loss of nuclear p33ING1b expression in invasive malignant melanoma compared with normal cutaneous melanocytes or the melanocytes of benign melanocytic naevi. This was associated with an enhancement of cytoplasmic p33ING1b expression which was particularly prominent in invasive malignant melanoma. CONCLUSIONS: Cytoplasmic immunostaining for p33ING1b using MAb GN2 is strongly associated with 'activated' melanocytic lesions; therefore it is possible that this MAb could be of value in diagnostic practice. Furthermore, the reduction in p33ING1b expression and perhaps translocation from the nucleus to the cytoplasm may play a central role in the development and progression of melanomas.     

Molecular profiles of invasive mucinous and ductal carcinomas of the breast: a molecular case study

Expression profiling using cDNA microarrays have redefined the molecular classification of some cancers. The comprehensive genetic analysis also permits the identification of novel pathways that might determine subtle differences in tumor phenotype. Herein, we analyzed the tissues from a patient with bilateral cancer of different histologies in each breast (pure invasive mucinous and pure invasive ductal), thus providing a unique opportunity to assess the expression profiles determined by histology in an isogenic human background. Our results show that the mucinous phenotype is associated with the expression of immunostimulatory and inhibitory genes, consistent with the cellular infiltration of lymphocytes and with the expression of enzymes involved in mucin production. Moreover, the panel of matrix metallo-proteinases are distinctly different between the mucinous and the invasive tumors, suggesting that therapeutic targets to this class of compounds may need to be tailored for the varying histologies. Taken together, these data suggest that expression profiling can be used diagnostically to distinguish individual histologic subclassifications and may guide the selection of target therapeutics.     

Expression of classic cadherins type I in urothelial neoplastic progression

Loss or reduced expression of E-cadherin has been shown to be associated with poor survival in patients with bladder cancer. In numerous cases, loss of E-cadherin expression in bladder tumors has been accompanied by continued association of catenins with the membrane, suggestive of the expression of an alternative cadherin member. In this study we examined 75 bladder tumors using immunohistochemistry for the expression of E-, P-cadherin, and alpha-, beta-, and gamma-catenins. As reported previously, loss or reduced E-cadherin expression is a frequent event in late stage bladder cancer, accompanied by less frequent alterations associated with different catenin family members. Analysis of 51 tumors for expression of E-, P-, and N-cadherin showed P-cadherin localized to the basal cell layers of normal urothelium, with retention of expression in the majority of tumors. In low-grade tumors P-cadherin was found localized to an expanded basal cell compartment, contrasting with the more extensive staining observed in late stage tumors. Membranous P-cadherin staining was often found in the absence of E-cadherin staining. N-cadherin is not expressed in normal bladder mucosa, but detection of this cadherin member was recorded in 39% (20/51) of bladder tumors. Unlike P-cadherin, membranous N-cadherin was detected in focal regions within tumors, representing novel expression in urothelial neoplastic progression. Although focal N-cadherin staining was observed in 3 noninvasive lesions, the majority of tumors expressing N-cadherin were invasive (17/20). Coexpression of E-, P-, and N-cadherin was recorded in 5 grade 2 bladder tumors. Expression of P-cadherin is maintained throughout bladder tumorigenesis, accompanied by aberrant expression of N-cadherin. Clearly, neither P- nor N-cadherin act in an invasive-suppressor mode in bladder cancer, but whether they have a primary role to play in urothelial neoplastic progression has yet to be established.     

Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance

The androgen-signaling pathway is critical to the development and progression of prostate cancer and androgen ablation is a mainstay of therapy for this disease. We performed a genome-wide expression analysis of human prostate cancer during androgen ablation therapy to identify genes regulated by androgen and genes differentially expressed after the development of resistance. Six hundred and fifty-four of 63,175 probe sets detected significant expression changes after 3 months of treatment with goserelin and flutamide. This included 149 genes that were also differentially expressed 36 hours after androgen withdrawal in LNCaP cells. These genes reflect the physiological changes that occur in treated tumors and include potential direct targets of the androgen receptor. Expression profiles of androgen ablation-resistant tumors demonstrated that many of the gene expression changes detected during therapy were no longer present suggesting a reactivation of the androgen response pathway in the absence of exogenous hormone. Therapy resistance was associated with differential expression of a unique set of genes that reflect potential mechanisms of reactivation. Specifically an up-regulation of the androgen receptor and key enzymes for steroid biosynthesis suggest that resistant tumors have increased sensitivity to and endogenous synthesis of androgenic hormones. The specific pathways of reactivation provide opportunities for classification of resistant tumors and targeted therapies.     

Loss of nuclear expression of the p33(ING1b) inhibitor of growth protein in childhood acute lymphoblastic leukaemia

BACKGROUND/AIMS: p33(ING1b) is a tumour suppressor protein involved in growth control and apoptosis. Suppression of p33(ING1b) expression is associated with the loss of cellular growth control and immortalisation, whereas its overexpression causes cell cycle arrest. Moreover, normal p33(ING1b) expression is essential for optimal function of p53. Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood, accounting for one third of all childhood malignancies. A variety of cytogenetic abnormalities have been described but there is no single abnormality common to all cases. Deregulation of the TP53 pathway is a common genetic abnormality in human malignancies. However, TP53 mutations are uncommon in ALL. It is possible that alternative mechanisms of regulation of the TP53 apoptosis pathway, such as modulation of p33(ING1b) expression, may be important in ALL. The aim of this study was to assess the expression of p33(ING1b) in childhood ALL. METHODS: One hundred and forty five patients with childhood ALL were investigated in this immunohistochemical study of the expression of p33(ING1b). RESULTS: Loss of nuclear expression of p33(ING1b) was seen in 78% of cases. This was associated with increased cytoplasmic expression of the protein. Kaplan Meier survival analysis demonstrated a trend towards a better prognosis for patients with tumours that had lost nuclear p33(ING1b). CONCLUSION: These results suggest that the loss of nuclear p33(ING1b) expression may be an important molecular event in the pathogenesis of childhood ALL.     

Deletion of the inhibitor of growth 4 (ING4) tumor suppressor gene is prevalent in human epidermal growth factor 2 (HER2)-positive breast cancer

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor gene that was shown to be deleted in 10% to 20% of breast cancers by array comparative genome hybridization analysis. We developed fluorescent in situ hybridization to detect the ING4 gene directly in the tissue samples on tumor tissue microarrays. We evaluated the ING4 gene status in 1033 breast cancer tissue samples and observed that ING4 was deleted in 16.5% (170/1033) of all breast cancers. ING4 deletion was significantly associated with Her2 overexpression: of the tumors with ING4 deletion, 23.8% (39/164) were human epidermal growth factor 2 (HER2) positive, as compared with 14.1% (115/814) of the tumors without ING4 deletion (P = .002). In addition, the tumors with ING4 deletion were more likely to belong to the HER2 molecular subtype (estrogen receptor negative/progesterone receptor negative/human epidermal growth factor positive) of breast cancer, compared with the other subtypes (28.4% HER2 versus 15.7% all, P = .002). ING4 deletion did not affect survival outcome of all patients with breast cancer (P = .797) or of the patients with HER2-positive tumors (P = .792). We conclude that ING4 deletion in breast cancer is relatively common, as 1 in 6 breast cancer harbors ING4 deletion. Furthermore, ING4 deletion is more prevalent in HER2-positive tumors, suggesting a functional antagonistic relationship between the ING4 tumor suppressor and the HER2 oncogene. These results sustain the view that ING4 is a tumor suppressor in breast cancer and suggest that ING4 deletion may contribute to the pathogenesis of HER2-positive breast cancer.     

Analysis of whole genomic expression profiles and screening of the key signaling pathways associated with pancreatic cancer

The tumorigenesis of pancreatic cancer is thought to be a complex process. Investigation of the molecular mechanism of pancreatic cancer and exploring the specific markers for early diagnosis and specific targets of therapy is a key point to prevent and treat pancreatic cancer effectively and to improve their prognosis. In this study, expression profiles experiment was performed using Agilent human whole genomic oligonucleotide microarrays with 41,000 genes. Differentially expressed genes related with pancreatic cancer were screened, and analyzed further by GO term analysis and KEGG Pathway analysis. Our results showed that there were 1276 differentially expressed genes associated with pancreatic cancer. 691 genes were up regulated and 585 were down regulated in pancreatic cancer group. The present study confirmed that the occurrence of pancreatic cancer was involved in multiple-gene interaction. In addition, our study found that pancreatic cancer was related to an activation of the mTOR signaling pathway and renal cell carcinoma pathway.     

Expression characteristics of prostate-derived Ets factor support a role in breast and prostate cancer progression

The purpose of this study was to understand the characteristics of prostate-derived Ets factor (PDEF) protein expression in breast and prostate cancer progression. A polyclonal antibody specific to PDEF was raised and reacted with tissue microarrays consisting of benign breast, in situ ductal, invasive ductal, and invasive lobular breast carcinomas. The antibody was also reacted with tissue microarrays, including benign prostate, prostate intraepithelial neoplasias (PINs), and prostate carcinomas. Increased expression of PDEF was identified in 18%, 50%, 46%, and 51% of benign breast tissues, intraductal, invasive ductal, and invasive lobular carcinomas, respectively. Importantly, in matched samples of benign breast vs tumor, 90% showed higher expression of PDEF in the tumor tissue. Moreover, in invasive breast carcinomas, increased PDEF expression tended to correlate with Her2/neu overexpression. Increased expression of PDEF was also found in 27%, 33%, and 40% of benign prostate tissues, PIN samples, and prostate adenocarcinomas, respectively. Again, in matching samples of cancer vs benign and cancer vs PIN, 68% and 70%, respectively, showed increased expression in the malignant tissue. Moreover, PDEF was found to be more highly expressed in tumors with intermediate or high Gleason score compared with low-grade tumors (P < .01). In addition, R1881 treatment induced PDEF expression in the LNCaP prostate tumor cell line, suggesting regulation of PDEF by androgens in vivo. Together, these results for the first time show frequent increased expression of PDEF protein in breast and prostate tumors and support a role for PDEF in breast and prostate cancer progression.     

Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.