Hormone Therapy Failure in Human Prostate Cancer: Analysis by Complementary DNA andTissue Microarrays

BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P = .0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P = .0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cance

Genome-wide characterization of genetic aberrations in pancreatic cancer

Haimasyöpä on haiman ulkoeritteisten eli eksokriinisten rauhasten pahanlaatuinen kasvain, joka saa alkunsa rauhastiehyeiden pintakerroksesta. Haimasyöpään sairastuu Suomessa vuosittain noin 700 ihmistä. Taudin ennuste on erittäin huono, sillä lähes kaikki sairastuneet kuolevat viiden vuoden kuluessa diagnoosista. Huono ennuste johtuu pääasiallisesti siitä, että haimasyöpä aiheuttaa yleensä oireita vasta taudin varsin myöhäisessä vaiheessa. Tällöin syöpä on tavallisesti jo ehtinyt levitä paikallisesti ja lähettää etäpesäkkeitä muualle elimistöön. Tässä väitöskirjatyössä selvitettiin erilaisin solu- ja molekyyligeneettisin menetelmin haimasyövän syntyyn ja kehittymiseen liittyviä geneettisiä muutoksia. Käytetyt menetelmät kattavat koko genomin erilaisella herkkyydellä ja selventävät haimasyövän syntyyn liittyviä tapahtumia lähtien kromosomitason muutoksista aina yksittäisten geenien osallisuuden tutkimiseen. Tarkimmalla menetelmällä, jossa 12232 kloonilla testattiin geenien monistuma-astetta ja ilmentymistä, löydettiin statistisella testillä 105 monistunutta ja yliekspressoitunutta kloonia. Suurin osa näistä edustaa geenejä, jotka ovat osallisena DNA:n jakautumisessa, transkriptiossa ja solun sisäisessä tiedonvälityksessä, jolla ajatellaan olevan merkitystä syövän synnyssä ja leviämisessä. Tämän väitöskirja aloitettiin käyttämällä kromosomaalista vertailevaa genomista hybridisaatiota, jolla menetelmällä nähdään 10-20 megaemäsparin tasolla monistuneet ja hävinneet kromosomialueet. Menetelmällä analysoitiin 31 haimasyöpäsolulinjaa ja 13 haimasyöpänäytettä. Tutkimuksessa löydettiin useita kromosomialueita, kuten kromosomikäsivarret 8q, 11q, 12p, 17q ja 20q, joissa esiintyi yleisesti perimäaineksen lisääntymistä eli monistumaa. Samalla tavoin tunnistettiin useita kromosomialueita, joissa esiintyy yleisesti perimäaineksen häviämistä. Näihin kuuluivat mm. kromosomialueet 18q, 9p, 4q, 3p ja 8p. Tutkimuksessa tunnistetuilla monistuvilla kromosomialueilla sijaitsee useita geenejä, joiden on jo aiemmin osoitettu olevan osallisena muiden kiinteän kudoksen kasvainten, kuten rintasyövän, synnyssä. Tutkimuksessa selvitettiinkin 14 tällaisen geenin mahdollista osuutta haimasyövän synnyssä käyttäen fluoresenssi in situ hybridisaatio -menetelmää. Tunnetuista syöpägeeneistä MYC todettiin monistuneeksi 54 %:ssa solulinjoista ja CCND1 28%:ssa. Sen lisäksi 17q-kromosomikäsivarressa sijaitsevat ERBB2-, TBX2- ja BIRC5-geenit olivat monistuneita 20 %:ssa, 50 %:ssa ja 58 %:ssa tapauksista. Kromosomi 20q - alueelta löytyi useita geenejä, jotka olivat erittäin yleisesti monistuneita haimasyöpäsolulinjoissa. Näistä CTSZ-geeni oli kaikkein useimmin monistunut 83 %:ssa solulinjoista. Tutkimuksessa etsittiin lisäksi tarkemmin 12p-kromosomialueen monistuman mahdollisia kohdegeenejä. Saadut tulokset osoittivat, että monistuma-alue on kooltaan 3,5 megaemäsparia. Tällä alueella sijaitsevien geenien ilmenemistasoja tutkittiin käyttäen mikrosirumenetelmää ja nämä tutkimukset osoittivat, että KRAS2-, DEC2- ja PPFIBP1-geenit olivat yliekspressoituneita monistuneissa tapauksissa ja siten edustavat mahdollisia monistuman kohdegeenejä. Viimeisessä osatyössä tutkittiin genomin laajuisella mikrosirutekniikalla 12232 geenin monistuma- ja ekspressiotasot 13 haimasyöpäsolulinjassa. Tutkimuksessa paikannettiin 24 erillistä monistuma-aluetta, joiden sijainti voitiin määrittää erittäin tarkasti. Statistisen testin avulla tunnistettiin 105 geeniä, jotka olivat sekä monistuneita että yliekspressoituneita haimasyövässä. Näistä osa oli aiemmin syövässä monistuneiksi todettuja geenejä, kuten AURKA(STK15) ja MLN51, tai tunnettuja onkogeenejä, kuten RAB4A ja RELA. Lisäksi oli joukko geenejä, joiden ei ole aiemmin tiedetty monistuvan syövässä. Näihin kuuluu mm. PAK4-geeni, joka toimii mm. solujen migraatiossa ja adheesiossa. Suuri osa tutkimuksessa tunnistetuista 105 kloonista (67 geeniä, 64 %) on osallisena sellaisissa solun sisäisissä prosesseissa, kuten DNA:n jakautumisessa, transkriptiossa ja solun signaloinnissa, joilla voidaan olettaa olevan merkitystä syövän synnyssä.Pancreatic cancer was the fourth most common cause for cancer deaths in males, and the third most common in females in 2001 (Finnish Cancer Registry, 2003). The prognosis of this cancer is poor and almost all patients die within five years of diagnosis. Although several genes have been implicated in the pathogenesis of pancreatic cancer, the entire spectrum of genetic aberrations leading to the development of this disease is poorly characterized. The main aim of this study was to identify genetic aberrations and specific genes having a crucial role in the pathogenesis of pancreatic cancer. Copy number aberrations were analyzed in 31 pancreatic cancer cell lines and 13 tumor biopsies by chromosomal CGH. Several common regions of gain and amplification were detected, including those at 3q, 7p, 8q, 11q, 17q, 19q, and 20q. Similarly, frequent losses were observed at 4q, 8p, 9p, 18q, and 21q. The chromosomal regions involved in frequent copy number gains contain several genes, such as the MYC, CCND1, and ERBB2 oncogenes, with an established role in cancer pathogenesis. The potential involvement of these oncogenes as well as that of several genes from 17q and 20q -regions, was explored in 30 pancreatic cancer cell lines. Amplification of the MYC oncogene was observed in 54% of the cell lines and CCND1 in 28%. At the 17q region, ERBB2, TBX2 (17q23), and BIRC5 (17q25) were amplified in 20%, 50%, and 58% of the cell lines respectively. At 20q, the CTSZ gene (20q13) was most commonly amplified in 83%, NCOA6 (20q11) in 71%, and PTPN1 in 70% of cases. Detailed characterization of the commonly observed 12p amplicon was performed in 15 pancreatic cancer cell lines to identify possible amplification target genes. FISH analysis using YAC clones allowed delineation of the region of interest to an approximately 5 Mb segment at 12p11-p12. Semiquantitative PCR was then used to further narrow down the amplification to a 3.5 Mb segment, between markers D12S1617 and sts-N38796. A chromosome segment-specific cDNA microarray containing 29 expressed sequences from the D12S1617 and sts-N38796 interval was constructed to explore expression levels of genes from this region in eight pancreatic cancer cell lines. This expression survey revealed overexpression of four ESTs, including the DEC2 and PPFIBP1 genes. In addition, increased expression of the KRAS2 gene, located in the distal part of the amplicon, was observed in all cell lines with amplification. A genome-wide 12 232 clone cDNA microarray was used for high-resolution mapping of copy number increases and for the identification of putative amplification target genes in 13 pancreatic cancer cell lines. The CGH microarray analysis implicated 24 independent amplified regions. These included several chromosomal segments, such as 3q, 5p, 7q, 8q24, 11q13, 15q, 17q, 19q, and 20q, previously shown to be gained or amplified by chromosomal CGH or by FISH, whose exact boundaries were now delineated on a base-pair scale. A statistical analysis revealed 105 genes that were systematically overexpressed when amplified. These included previously described amplified genes, such as STK15 and MLN51, as well as novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4) involved in cell migration, cell adhesion, and anchorage-independent growth. Functional characterization indicated that 67 genes (64 %) of the 105 genes are associated with cellular processes, such as signal transduction, transcription, and DNA replication, that could be directly associated with cancer pathogenesis. The 105 genes identified in this study to be activated by increased copy number are therefore likely to be part of the tumorigenesis of pancreatic cancer

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High-Resolution Genomic and Expression Profiling Reveals 105 Putative Amplification Target Genes . . .

Comparative genomic hybridization (CGH) studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4), which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer

Detailed genomic mapping and expression analyses of 12p amplifications in pancreatic carcinomas reveal a 3.5-Mb target region for amplification.

Previous cytogenetic and comparative genomic hybridization (CGH) analyses have shown that the gain of chromosome arm 12p is frequent in pancreatic carcinomas. We investigated 15 pancreatic carcinoma cell lines using CGH, fluorescence in situ hybridization (FISH), and semiquantitative polymerase chain reaction (PCR) to characterize 12p amplifications in detail. The CGH analysis revealed gains of 12p in four of the cell lines and local amplification within 12p11-12 in six cell lines. By FISH analysis, using precisely mapped YAC clones, the commonly amplified region was found to be approximately 5 Mb. The amplified segment extended from YAC 753f12, covering the KRAS2 locus, to YAC 891f1, close to the centromere. A semiquantitative PCR methodology was used to estimate genomic copy numbers of 14 precisely mapped expressed sequence tags (ESTs) and sequence-tagged sites, located within this interval. The level of amplification ranged from two- to 12-fold. The produced gene copy profiles revealed a 3.5-Mb segment with various local amplifications. This region includes KRAS2 and ranges from D12S1617 to sts-N38796. Two of the cell lines (primary and metastatic tumor from the same patient) showed amplification peaks within the distal region of this segment, two had peaks within the proximal region, one showed subpeaks in both regions, and one displayed amplification of the entire region. Chromosome segment-specific cDNA array analysis of 29 expressed sequences within the whole interval between D12S1617 and sts-N38796 indicated overexpression of four ESTs, two corresponding to DEC2 and PPFIBP1, and two to ESTs with unknown function. Expression analysis of these and of KRAS2 showed specific overexpression in the six cell lines with local 12p amplifications. These findings indicate two target regions within the 3.5-Mb segment in 12p11-12, one proximal including PPFIBP1, and one distal including KRAS2