Microarray analysis reveals potential target genes of NF-kappaB2/p52 in LNCaP prostate cancer cells.

PURPOSE: Our previous studies showed that NF-kappaB2/p52 is involved in the castration-resistant growth of the androgen-sensitive LNCaP prostate cancer cells. The role of NF-kappaB2/p52 in lymphomagenesis has been studied extensively, but its target genes in other cancers remain unknown. In order to identify genes potentially regulated by p52 in prostate cancer cells, we performed a genome-wide microarray analysis of genes differentially up- or down-regulated by the overexpression of p52 by adenoviral-mediated gene delivery in LNCaP cells. EXPERIMENTAL DESIGN: Total RNAs from vector control-infected and Adeno-p52-infected LNCaP cells were used to prepare cDNAs, which were hybridized to the Whole Genome Human 44k Microarray chips (Agilent Technologies). Data analysis was performed using GeneSpring and Ingenuity Pathway Analysis software. Validation of microarray results was performed by real-time quantitative RT-PCR and Western blot analyses. RESULTS: Expression of approximately 130 genes was differentially upregulated by >5-fold, whereas approximately 60 genes were differentially downregulated by >2-fold in p52-expressing LNCaP cells. Pathway analysis revealed that the upregulated genes belong to functional categories like cell growth and proliferation, cellular movement, cell-to-cell signaling and interaction, cancer, cell cycle, etc., whereas the downregulated genes were represented by functional categories like cell movement, antigen presentation, and cell death. Six of the top upregulated genes including annexin A2, PLAU, RND3, Twist2, VEGFC, and CXCL1 were validated by real-time PCR and Western blot analysis. CONCLUSIONS: This study provides a comprehensive analysis of genes potentially regulated by NF-kappaB2/p52 in the LNCaP prostate cancer cell line and provides a rationale for the induction of castration-resistant growth by p52 in LNCaP cells.

Molecular mechanisms of castration-resistant prostate cancer progression.

Hormone-refractory prostate cancer is the result of regrowth of prostate cancer cells that have adapted to the hormone-deprived environment of the prostate. The process by which castration-resistant prostate cancer (CRPC) cells are generated appears to be varied. The complex mechanism of hormone resistance has been the topic of research in most laboratories that have analyzed the process from different angles. This review compiles research findings that explain the methods of development of hormone resistance in prostate cancer. Research data show many different processes to be involved in the acquisition of hormone resistance. Interestingly, one observes interdependence between these processes, indicating a complex network at play in the development of hormone resistance. Cytokines such as IL-6 have been shown to initiate an alternative signaling pathway, compared with the androgen receptor signaling pathway, in CRPC. IL-6 has been proposed to be the effector of the intracrine signaling pathway by influencing the levels of metabolic enzymes. Neuroendocrine cells are present at low levels in normal prostate, and signify the transitory phase of normal hormone-sensitive cells to hormone-refractory cells. IL-6 induces growth of neuroendocrine cells or neuroendocrine-like features in cells in CRPC. The increased presence of neuroendocrine cells in CRPC signifies a change in the prostate cell microenvironment. The stromal microenvironment also influences the development of CRPC in the hormone-refractory stage. In addition, intracrine androgen metabolic enzymes play a significant role in the development of the hormone refractory process. Despite hormone ablation, there is a residual level of hormones in cells due to active intracrine metabolic pathways. It is acknowledged that the androgen receptor plays the most influential role in development of prostate cancer. In addition to mutation and amplification, the androgen receptor has been characterized and shown to differ in sequence in CRPC compared with the androgen-sensitive prostate cancer cells. These variants of the androgen receptor through sequence changes may preserve the basic function of the molecule, but have far-reaching consequences on the cell as a whole. A multicombinatorial drug treatment approach has been suggested to target these multiple pathways in an effort to reduce the possibility of recurrence of CRPC.

Interleukin-6 regulates androgen synthesis in prostate cancer cells.

PURPOSE: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of androgen receptor activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR and Western blotting were done to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6-mediated AKR1C3 transcriptional activity. IL-6-mediated signaling was knocked down using small interfering RNA specific to IL-6 receptor and gp130, and the effect on AKR1C3 expression was examined. Intraprostatic androgen levels in prostate cancer cells in culture and in tumors were measured by an enzyme immunoassay (Testosterone EIA kit). RESULTS: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes, including HSD3B2 and AKR1C3, involved in androgen biosynthesis. Down-regulation of IL-6 receptor and gp130 expression using specific small interfering RNA abolished IL-6-mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6-mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6-overexpressing LNCaP-IL6(+) cells inoculated orthotopically into the prostates of castrated male nude mice. CONCLUSIONS: These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells.

Uniparentalism in sporadic colorectal cancer is independent of imprint status, and coordinate for chromosomes 14 and 18.

Our previous allelotyping studies of 59 sporadic colorectal cancers revealed that loss of heterozygosity is most frequent for regions of chromosomes 14 and 18. Yet subsequent BAC microarray comparative genomic hybridization studies of the same tumor DNAs showed no corresponding pattern of copy number alteration for chromosome 14. To clarify this apparent discrepancy, we utilized hybridization to SNP microarrays; this revealed frequent uniparentalism for chromosome 14 and for chromosome 18. Based on the BAC array results combined with fluorescent in situ hybridization data, it was evident that uniparental disomy was occurring in many colorectal cancers as well as in additional chromosomes, and often coordinately involved chromosomes 14 and 18. Further studies examined the possibility that uniparentalism was directed towards the selection for imprinted genes, but no association with imprinting was observed.

Colorectal cancers in patients with the (9A/6A) polymorphism of TGFBR1 exhibit lesser inter-(simple sequence repeat) PCR genomic instability and present clinically at greater age.

TGFbeta is involved in the response to DNA damage and signaling the cell cycle checkpoint response, in large part achieved by modulating the activity of the ATM kinase. We have investigated if the presence of a common polymorphism in the TGFbeta receptor TGFBR1 might impact genomic instability in human colorectal cancer. In order to obtain statistically significant numbers of patients with the lesser polymorphism, 177 colorectal cancer patients were genotyped for either the major form of the TGFBR1 receptor gene, homozygous for an internal segment of 9 alanines (9A/9A), or the lesser form, heterozygous for the polymorphism containing 6 alanines (9A/6A). Intrachromosomal genomic instability in the tumors was then quantified by the robust inter-(simple sequence repeat) PCR method. Tumors from all 26 patients heterozygous with the (9A/6A) polymorphism in TGFBR1 exhibited significantly lower genomic instability than from a randomly selected set [the first identified] of 37 patients with the (9A/9A) polymorphism (p=0.0002, Mann-Whitney). The median age of onset for the (9A/6A) patients was 70 years, compared with a median age of onset of 63 years for the patients carrying the (9A/9A) form (p=0.031, Mann-Whitney). These results are consistent with the model wherein genomic instability facilitates tumor progression, with lesser instability associated with later disease presentation. Clinically, our findings may be developed into improved screening guidelines with respect to the age at which colonoscopy is initiated in carriers of the TGFBR1*6A allele.

Development of an androgen-deprivation induced and androgen suppressed human prostate cancer cell line.

BACKGROUND: Emergence of recurrent cells during androgen-deprivation therapy and intermittent androgen suppression therapy suggest that a subset of prostate cancer cells survive and proliferate in the androgen deprivation condition. Some of the recurrent cells that emerge during the androgen-deprivation therapy and intermittent androgen suppression therapy could be suppressed by replacement of androgen. In an attempt to recapitulate the clinical phenomenon, we developed an androgen-deprivation induced and androgen suppressed human prostate cancer cell line. METHODS: LNCS, an androgen-deprivation induced and androgen suppressed human prostate cancer cell line, was generated from an androgen-sensitive LNCaP cells cultured in PRMI-1640 media containing charcoal-stripped FBS for a prolong period for more than a year. The responsiveness to androgen in vitro and in vivo was examined. The characteristics of this subline including activation of signaling pathways were investigated. RESULTS: LNCS, an androgen-deprivation induced and androgen suppressed human prostate cancer cell line, was developed. LNCS cells express considerably lower levels of androgen receptor than LNCaP cells and grow vigorously in androgen deprived condition in vitro, and develop tumors in castrated male mice in vivo. Addition of androgen inhibits cell growth in vitro and tumor growth in vivo. LNCS cells are more resistant to etoposide, a typical apoptotic inducing agent. Although AR signaling is less active in LNCS cells, Stat3 is constitutively activated. Down regulation of Stat3 activation inhibits LNCS cell growth in vitro. CONCLUSIONS: We have developed an androgen-deprivation induced and androgen suppressed human prostate cancer cell line. This cell line is a valuable tool to study molecular mechanisms of the progression of androgen refractory prostate cancer and intermittent androgen suppression therapy.

LIGHT, a member of the TNF superfamily, activates Stat3 mediated by NIK pathway.

Stat3, a member of the signal transducers and activators of transcription (STAT) family, is a key signal transduction protein activated by numerous cytokines, growth factors, and oncoproteins that controls cell proliferation, differentiation, development, survival, and inflammation. Constitutive activation of Stat3 has been found frequently in a wide variety of human tumors and induces cellular transformation and tumor formation. In this study, we demonstrated that LIGHT, a member of tumor necrosis factor superfamily, activates Stat3 in cancer cells. LIGHT induces dose-dependent activation of Stat3 by phosphorylation at both the tyrosine 705 and serine 727 residues. The activation of Stat3 by LIGHT appears to be mediated by NIK phosphorylation. Expression of a kinase-inactive NIK mutant abolished LIGHT induced Stat3 activation. Overexpression of an active NIK induces Stat3 activation by phosphorylation at the both tyrosine 705 and serine 727 residues. Activation of Stat3 by NIK requires NIK kinase activity as showed by kinase assays. In addition, LIGHT increases the expression of Stat3 target genes including cyclin D1, survivin, and Bcl-xL, and stimulates human LNCaP prostate cancer cell growth in vitro which can be blocked by expression of a dominant-negative Stat3 mutant. Taken together, these results indicate that in addition to activating NF-kappaB/p52, LIGHT also activates Stat3. Activation of Stat3 together with activating non-canonical NF-kappaB/p52 signaling by LIGHT may maximize its effects on cellular proliferation, survival, and inflammation.

Comparative genomic instabilities of thyroid and colon cancers.

OBJECTIVES: To assess the forms and extent of genomic instability in thyroid cancers and colorectal neoplasms and to determine if such measurements could explain the generally excellent prognosis of thyroid malignant neoplasms compared with colon carcinoma. DESIGN: Tumor genome analyses. Genomic instability was measured by the following 4 methods, listed in ascending order based on the size of events detected: inter-simple sequence repeat polymerase chain reaction (ISSR-PCR), fractional allelic loss (FAL) analysis, array-based comparative genomic hybridization (aCGH), and spectral karyotyping (SKY). RESULTS: The genomic instability index of 32 thyroid carcinomas, 59 colon carcinomas, and 11 colon polyps was determined by ISSR-PCR; no difference was seen among the 3 groups by this method. Fractional allelic loss rates were comparable in thyroid cancers and colon polyps and lower than FAL rates in colorectal cancers. Indolent papillary thyroid carcinomas were essentially diploid with no large-scale alterations in chromosome number or structure when evaluated by aCGH or SKY. In anaplastic thyroid cancers, aCGH revealed abundant chromosome alterations. Colorectal carcinomas showed extensive copy number changes and chromosomal rearrangements when analyzed by aCGH and SKY. CONCLUSIONS: Genomic alterations in papillary thyroid carcinoma, such as in benign colon polyps, are principally smaller events detected by ISSR-PCR. With the more aggressive tumor types (ie, anaplastic thyroid and colorectal carcinomas), larger events detected by FAL analysis, aCGH, and SKY were revealed. We hypothesize that mutations caused by smaller genomic alterations enable thyroid cells to achieve a minimal malignant phenotype. Mutations for aggressive biological behavior appear with larger genomic events.

Genomic profiles of colorectal cancers differ based on patient smoking status.

Human sporadic colorectal cancer is the result of a lengthy somatic evolutionary process facilitated by various forms of genomic instability. Such instability arises endogenously from mutations in genes whose role is to preserve genomic integrity, and exogenously from environmental agents that generate genomic damage. We have found that cigarette smoking shifts the genomic profiles and genomic instability patterns of colorectal carcinomas. The genomic profiles of 57 consecutive cancers were examined; 31 cases were current or former smokers and 26 were nonsmokers. Genome-wide allelotypes of 348 markers were examined, along with comparative genomic hybridization (CGH) on ordered BAC microarrays, microsatellite instability, and inter-(simple sequence repeat) polymerase chain reaction instability. Tumors from nonsmokers exhibited losses of heterozygosity, particularly on chromosomes 14 and 18, whereas tumors from smokers exhibited a more diffuse pattern of allelic losses. Tumors from smokers exhibited higher overall rates of loss of heterozygosity, but showed lower rates of background microsatellite instability (MSI-L). On BAC array CGH, higher levels of generalized amplifications and deletions were observed in tumors from smokers, differentially affecting male smokers. In the transforming growth factor-beta signaling pathway, MADH4 mutations were more common in tumors from smokers, whereas transforming growth factor-beta RII mutations were more common among nonsmokers.

Analysis of inter-(simple sequence repeat) PCR products from human colorectal cancers.

Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic instability should significantly clarify the process of tumorigenesis. We adapted the sampling technique of inter-(simple sequence repeat) PCR [inter-(SSR) PCR] to measure genetic alterations between simple sequence repeats in colorectal tumors. It becomes important to precisely define both normal and altered inter-(SSR) PCR products. BLAT searches of 131 cloned inter-(SSR) PCR sequences reveal that inter-(SSR) PCR products are located on almost all the chromosomes except chromosome Y, indicating that inter-(SSR) PCR samples a representative diverse range of the genome. We confirm that a change in the pattern of the inter-(SSR) PCR products as seen on gel electrophoresis reflects a true alteration within the genome.