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WO2003006690A1 - Use of urokinase gene amplification as a marker - Google Patents

Use of urokinase gene amplification as a marker Download PDF

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Publication number
WO2003006690A1
WO2003006690A1 PCT/FI2002/000636 FI0200636W WO03006690A1 WO 2003006690 A1 WO2003006690 A1 WO 2003006690A1 FI 0200636 W FI0200636 W FI 0200636W WO 03006690 A1 WO03006690 A1 WO 03006690A1
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upa
amplification
gene
cancer
invasive
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PCT/FI2002/000636
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French (fr)
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Merja Helenius
Outi SARAMÄKI
Marika Linja
Teuvo Tammela
Tapio Visakorpi
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Fit Biotech Oyj Plc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the use of urokinase gene amplification as a marker for evaluating the treatment sensitivity and specificity of carcinomas, especially invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer and breast cancer.
  • the present invention also relates to methods of identification of carcinomas profiled by urokinase gene amplification. Background of the invention The incidence of carcinomas is steadily increasing in Western countries, although the mortality rates have inevitably not risen as much as the incidence rates mainly due to early diagnosis and effective treatment of these diseases.
  • prognostic clinicopathological factors which can be associated with invasive forms of carcinomas.
  • the prognosis of the disease and the selection of the post-operational treatment of cancer are currently mainly based on the evaluation of the clinical stage of the disease and the histological, especially nuclear, gradus of the tumors. These methods do not, however, predict well the progression rate of the disease.
  • the determination of steroid hormone receptor content is additionally used in the prognosis of the disease and the selection of adjuvant chemotherapy in hormone-dependent carsinomas, such as in breast cancer or prostate cancer.
  • Other means are also used experimentally to supplement the evaluation of treatment recommendations. They include the determination of the growth rate of the cancer, DNA flow cytometric analysis, and immunohistochemical analysis of prognostic markers, such as various oncogenes, for example erbb-2 oncogene, oncogenic products and tumor suppressor genes.
  • the long arm of chromosome 8 (8q) is one of the most common regions of amplification in cancers of several organs, such as bladder and ovarian cancer, but especially carcinomas in the breast and the prostate [Vi- sakorpi, T., et al., Cancer Res. 55 (1995) 342-7; Cher et al., Cancer Res. 56 (1996) 3091-102; Nupponen, N. N., et al., Am. J. Pathol. 153 (1998) 141-8; Tir- kkonen et al., Genes Chromosomes Cancer 21 (1998) 177-84].
  • chromosome 10 Another region of amplification associated to carcinomas is chromosome 10 (10q). It has been shown by CGH that approximately 15% of the hormone-refractory recurrent prostate carcinomas contain a gain or amplification at 10q (Nupponen, N. N., et al., supra) and that prostate cancer cell line PC-3 shows high level-amplification at 10p12-q23 [Nupponen, N. N., et al., Cancer Genet. Cytogenet. 101 (1998) 53-57]. However, a specific relationship between an identified potential target gene in 10q and certain specific forms of cancer has not been shown. The identification of such a relationship would lead in much desired improvements in the postoperative treatment of invasive recurrent forms of cancer and would be greatly benefit cancer patients.
  • uPA urokinase-type
  • tPA tissue-type plasminogen activator
  • uPA has restricted substrate specificity, its main function being the cleavage of plasminogen to plasmin (Liotta, L, et al., supra), which degrades several ECM components and also activates many pro-MMPs (matrix-metalloproteases) [Liotta, L, et al., supra; Murphy, G., et al., Ann. NY Acad. Sci. 667 (1992) 1- 12].
  • the urokinase gene is one of the genes mapped to chromosome 10q.
  • the uPA is believed to play a key role in the cancer invasion and metastasis [Dano, K. et al., 1999, supra; Festuccia, C, et al., Clin. Exp. Metastasis 16 (1998) 513-528].
  • Both the uPA secretion and the presence of the receptor bound uPA at the cell surface characterize prostate cancer cells that have invasive phenotype [Gaylis, F., et al., J. Urol. 742 (1989) 193-198].
  • An increased expression of urokinase has been reported in various malignancies including prostate cancer [Gaylis, F.
  • uPA is one of the target genes for the 10q gain.
  • the amplification profile of uPA provides novel approaches, which can be utilized in the development and improvement of the therapy of these carcinomas.
  • An object of the invention is thus to provide novel means that are useful in predicting and identifying optimal treatment of recurrent invasive forms of carcinoma, especially of hormone-refractory forms of carcinoma, such as invasive prostate cancer.
  • Another object of the invention is to provide novel means that are useful in the identification of particular types of carcinomas, which could respond to specifically targeted chemotherapy.
  • Another object of the invention is to provide novel means that are useful in the identification of cancer patients that would benefit of specifically targeted chemotherapy.
  • Yet another object of the invention is to provide means that are useful in the identification of novel drugs for the treatment of recurrent invasive forms of carcinoma, especially of hormone-refractory forms of carcinoma, such as invasive hormone-refractory breast cancer or prostate cancer.
  • the present invention relates to the use of urokinase gene amplification as a marker for evaluating the treatment sensitivity and specificity of carcinomas, especially invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer.
  • the present invention also relates to methods of identification of carcinomas profiled by urokinase gene amplification by detecting the presence or absence of amplification of uPA in a tumor sample.
  • the present invention further relates to a method of identifying of cancer patients, especially those suffering from invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer, that would benefit of targeted chemotherapy by detecting the presence or absence of amplification of uPA in a biological sample obtained from said patients.
  • the present invention further related to a method of the identification of novel drugs for the treatment of recurrent invasive forms of carcinoma, especially of hormone-refractory forms of carcinoma, such as invasive hormone-refractory breast cancer or prostate cancer by determining the inhibiting ability of the uPA activity of said drug.
  • Figure 1 shows (A) the mean ( ⁇ SD) fluorescence ratio profile from p- to q-telomere for chromosome 10 in prostate cancer cell line PC-3 obtained by CGH; a two color-FISH analyses of normal (B) and PC-3 cell lines metaphase chromosome preparations (C), and interphase nuclei of two hormone-refractory prostate carsinomas (D, E) with BAC probe for the uPA gene (red signals) and the chromosome 10 centromere specific probe (green signals).
  • Figure 2 shows (A) the results of a Northern hybridization of the uPA demonstrating that invasive prostate cancer cell lines PC-3 and DU-145 express high levels of uPA whereas the non-invasive LNCaP cell line does not; and (B) the relative expression level of uPA (uPA/ ⁇ -actin) after quantification with phosphoimager.
  • Figure 3 shows the expression level of uPA measured by real-time
  • RT-PCR (A) the PCR curves of the serially diluted standard containing cDNA transcribed from 500, 100, 20, 4, 0.8 and 0.16 ng of totRNA, (B) the standard curve blotting fractional cycle number at the fluorescent threshold for each standard curve presented in panel A, and (C) the relative expression of uPA genes in prostate tumor samples: • represents a tumor containing high-level
  • AR gene amplification • represents tumors with gain of the uPA gene and o represents tumors without uPA gene copy number alterations.
  • Horizontal scattered line shows the median value of expression.
  • Figure 4 shows the effects of amiloride on the invasive ability of
  • PC-3, DU145 and LNCaP cells as examined by the Matrigel invasion assay.
  • the present invention is based on the studies in which the uPA gene was re-mapped to 10q22 and shown to amplify in the PC-3 cell line and, for the first time, also in vivo.
  • the frequency of the uPA gene amplification in hormone-refractory prostate carcinomas was studied by fluorescence in situ hybridization (FISH) and the expression of the gene by Northern analysis and real-time RT-PCR (LightCycler methodology).
  • FISH fluorescence in situ hybridization
  • the association between the uPA gene amplification and the sensitivity of the cells to the uPA inhibition were studied by an invasion assay.
  • a genomic clone for uPA was obtained by screening a human BAC library with specific primers specific for the gene.
  • FISH fluorescence in situ hybridization
  • the DU145 and LNCaP cell lines showed only 3 and 4 copies of both uPA and chromosome 10 centromere by FISH, respectively.
  • the findings are consistent with the CGH data indicating no high-level amplification at 10q in these cell lines [Nupponen, N.N., et al., Cancer Genet. Cytogenet. 101 (1998) 53-57].
  • uPA urokinase
  • Fig.3 normal gene copy number
  • Fig.3 normal gene copy number
  • the association between the amplification and over- expression of the gene both in the cell lines and the tumors suggests that uPA is a strong candidate target gene for the 10q amplification in prostate cancer.
  • the presence or absence of the uPA gene can be detected from a biological sample by any known detection method suitable for detecting a gene copy number or expression, i.e. methods based on detecting the copy number of the gene (or DNA) and/or those based on detecting the gene expression products (mRNA or protein).
  • detection methods are easily recognized by those skilled in the art and include in situ hybridizations, such as fluorescence in situ hybridization (FISH) and mRNA in situ hybridization, Southern analysis, RT-PCR, Northern and Western analyses, immunohistochemistry, and other immunoassays.
  • FISH fluorescence in situ hybridization
  • mRNA in situ hybridization Southern analysis, RT-PCR, Northern and Western analyses, immunohistochemistry, and other immunoassays.
  • Preferable methods are those suitable for use in routine clinical laboratories, such as FISH and immunohistochemistry.
  • the biological sample can be any sample containing tumor cells, such as a biopsy sample from the breast, prostate, a lymph node or other tissues containing metastatic lesions, including circulating cancer cells.
  • the biological sample can also be a body fluid, such as whole blood, serum, plasma, urine, lymph, and a cerebrospinal fluid sample.
  • the biological sample can be pretreated, if necessary, in a suitable manner known to those skilled in the art.
  • the invention will be elucidated below by the following non-limiting examples.
  • the cell lines and tumors used in the Examples were as follows: Prostate cancer cell lines PC-3, DU145, and LNCaP were obtained from ATCC (Manassas, VA, USA) and cultured according to manufacturer's protocols. Freshly frozen transurethral resection (TURP) specimens from 13 patients diagnosed with locally recurrent hormone-refractory prostate cancer were obtained from Tampere University Hospital. The TURPs were performed on the patients due to urethral obstruction during hormonal therapy.
  • the endocrine therapy modalities were orchiectomy (4 cases), luteinizing hormone- releasing hormone analogue (4 case), estrogen (1 case), maximal androgen blockade (3 cases), or unknown (1 case).
  • a genomic clone for uPA was obtained by screening human BAC library with primers specific for the gene (5'-atc age tgt aag aag age tgg g-3', sequence id. number 3, and 5'- atg ccc tgc cct ttt taa ct -3', sequence id. number 4). The authenticity of the clone (BAC-46i3) was verified by partial sequencing.
  • the BAC-uPA probe was labelled with digoxigenin-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation. SpectrumGreen labelled chromosome 10 centromere specific probe (CEP10, Vysis Inc, Downers Grove, IL) was used as a reference probe.
  • Metaphase chromosome preparations from the prostate cancer cell lines and normal blood lymphocytes were prepared using routine techniques. Five micrometers' tissue sections from the freshly frozen tumor blocks were fixed on objective slides in a series of 50%, 75%, and 100% Carnoy's solution (3:1 methanol-acetic acid) for 10 min each at room temperature.
  • the dual-color FISH was performed essentially as described by Hyytinen et al., Cytometry 16 (1994) 93-99]. Briefly, the slides were denatured in 70% formamide/ 2X SSC at 70.5 °C for 2.5 min and dehydrated through an ethanol series. Hybridizations were carried out at 37 °C for 48 hours. After stringent washes, the slides were stained with anti-digoxigenin-rhodamine (Roche Diagnostics) and counter-stained with an antifade solution (Vectashied, Vector Laboratories, Burlingame, CA) containing 4,6-diamidino 2- phenylindole (DAPI).
  • DAPI 4,6-diamidino 2- phenylindole
  • the FISH signals were scored from non-overlapped epithelial cells using Olympus BX50 epifluorescence microscope (Tokyo, Japan).
  • the criteria for high-level amplification was either the presence of the tight cluster of uPA signals or more than 5 copies of the gene.
  • the results are set forth in Figure 1.
  • the profile in Figure 1A indicates amplification at 10p12-q23 [cf.
  • FISH analysis maps the uPA to chromosome 10q22 ( Figure 2B). Multiple copies (20 to 30) of uPA in the rearranged chromosomes are seen as a sign of high-level amplification in the PC-3 cell line ( Figure 1C). Three copies of uPA and 1 copy of the chromosome 10 centromere were seen in a hormone-refractory tumor ( Figure 1 D). Approximately 10 copies of uPA and 2 copies of the chromosome 10 centromere indicate 5-fold amplification of uPA gene ( Figure 1 E).
  • uPA expression level of uPA in prostate tumors
  • the amplification of the putative target genes is thought to lead to their over-expression.
  • the expression of uPA in 12 locally recurrent hormone-refractory prostate tumors was evaluated by quantitative real-time RT-PCR analysis.
  • One to three 20 ⁇ m frozen sections were cut from the tumor blocks using a cryotome.
  • Total RNAs were isolated from the sections with Qiagen RNeasy MiniKit (Qiagen Inc, Valencia, CA) and used for the first-strand cDNA synthesis with SuperscriptTM II reverse transcriptase and oligo d(T)i 2 - ⁇ primer according to manufacturer's protocol (Life Technologies).
  • totRNA For the standard curve, 5 ⁇ g of totRNA from normal mammary tissue (Clontech) was reverse transcribed as described above. After the first strand cDNA synthesis, serial dilutions were made corresponding to cDNA transcribed from 500, 100, 20, 4, 0.8, and 0.16 ng of totRNA.
  • uPA a primer sequence (5'-3'):
  • the primers were designed to avoid amplification of any genomic DNA by choosing the forward and reverse primers for each of the genes from different exons.
  • the PCR reactions were performed in the LightCyclerTM apparatus [Wittwer, C. T., et al., Biotechniques 22 (1997) 176-181] using LC DNA Hybridization Probes Kit (Roche Diagnostics).
  • Thermocycling for each reaction was done in a final volume of 20 ⁇ l containing 2 ⁇ l of cDNA sample (or standard), 4mM MgCI , 0.5 ⁇ M of each primer, 0.2 ⁇ M fluorescein and 0.4 ⁇ M LC Red640 labelled probes as well as 1x ready-to-use reaction mix including Taq DNA polymerase, the reaction buffer, and the dNTP mix.
  • the cycling conditions of 55 cycles consisted of denaturation at 95° for 0 s, annealing at 57° (for uPA) or 58° (for TBP) for 13 s, and elongation at 72° for 9 (uPA) or 10 (TBP) seconds.
  • the LightCyclerTM apparatus measured the fluorescence of each samples in every cycle at the end of the annealing step. After proportional background adjustment, the fit point method was used to determine the cycle, in which the log-linear signal is distinguished from the background, and that cycle number was used as a crossing-point value.
  • the software produced the standard curve by measuring the crossing point of each standard and plotting them against the logarithmic value of concentrations. The concentrations of unknown samples were then calculated by setting their crossing points to standard curve.
  • TBP TBP was selected for the reference gene because there are no known retropseudogenes for it and the expression of TBP is lower than in many commonly used abundantly expressed reference genes [Bieche, I., et al., Cancer Res. 59 (1999) 2759-2765]. After the PCR, all samples were also run in 1.2% agarose gel electrophoresis to ensure that right size product was amplified in the reaction.
  • 1x10 5 cells (PC-3, DU145, or LNCaP) in the culture medium containing 1 % FBS were plated onto an insert. Half of the inserts were treated with 151 ⁇ M amiloride (Sigma Chemicals CO., St.Louis, MO) or mock-treated for 22 hours. Subsequently, the cells were removed and the invaded cells were fixed with methanol and stained with crystal violet. The number of invaded cells was counted under a microscope. The experiments were repeated four times per each cell line and treatment. The results are shown in Fig. 4. The data represent mean ⁇ SEM of four experiments performed.
  • the uPA gene amplification but not the uPa expression, was associated with cells sensitive to the uPA inhibitors.
  • the finding further indicates that the uPA is one of the target genes for the 10q gain and that it may well be a potential treatment target.
  • a high-level amplification of uPA gene in hormone-refractory prostate carcinoma in vivo The increased copy number was associated with increased expression of the gene and with the sensitivity of the cells to uPA inhibition.
  • the findings support the suggestion that uPA is involved in the acquisition of invasive phenotype in prostate cancer cells.

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Abstract

The present invention relates to the use of urokinase gene amplification as a marker for evaluating the treatment sensitivity and specificity of carcinomas, especially invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer and breast cancer. The present invention also relates to methods of identification of carcinomas profiled by urokinase gene amplification.

Description

Use of urokinase gene amplification as a marker
Field of the invention
The present invention relates to the use of urokinase gene amplification as a marker for evaluating the treatment sensitivity and specificity of carcinomas, especially invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer and breast cancer. The present invention also relates to methods of identification of carcinomas profiled by urokinase gene amplification. Background of the invention The incidence of carcinomas is steadily increasing in Western countries, although the mortality rates have fortunately not risen as much as the incidence rates mainly due to early diagnosis and effective treatment of these diseases. However, because of the wide variability in the course of the carcinomas, it is difficult to assess the prognosis and to select an optimal treat- ment for individual patients, especially for those suffering from invasive recurrent forms of cancer, such as invasive recurrent forms of hormone-refractory cancers like prostate cancer and breast cancer.
Various means have been developed for identifying the prognostic clinicopathological factors, which can be associated with invasive forms of carcinomas. Generally in clinical practice, the prognosis of the disease and the selection of the post-operational treatment of cancer are currently mainly based on the evaluation of the clinical stage of the disease and the histological, especially nuclear, gradus of the tumors. These methods do not, however, predict well the progression rate of the disease. The determination of steroid hormone receptor content is additionally used in the prognosis of the disease and the selection of adjuvant chemotherapy in hormone-dependent carsinomas, such as in breast cancer or prostate cancer. Other means are also used experimentally to supplement the evaluation of treatment recommendations. They include the determination of the growth rate of the cancer, DNA flow cytometric analysis, and immunohistochemical analysis of prognostic markers, such as various oncogenes, for example erbb-2 oncogene, oncogenic products and tumor suppressor genes.
None of the present methods, however, fulfills the requirements for accurate prognostic evaluation for the sensitivity and specificity of specific cancer types to chemotherapy. They are also expensive and require special equipment, reagents and skills to perform. Thus, novel means for identifying particular types of carcinomas, which could respond to certain chemotherapy, are still needed. Such novel means would benefit especially patients with recurrent invasive forms of carcinomas in terms of targeted treatment. They should reduce time spent on attempts to choose a suitable chemotherapy and thus to increase the patients' chances of recovery or at least to extend the remission period of the disease and thus improve the status of the disease and the expected survival time of the patients.
It has been suggested that the development and progression of cancer are induced by multiple genetic alterations, such as gene amplification. Several amplified oncogenes have been identified in cancer [Alitalo, K. and Schwab, M., Adv. Cancer Res. 47 (1986) 235-81 ; Brison, O., Biochim Biophys Acta 1155 (1993) 25-41], but studies by comparative genomic hybridization (CGH) [Kallioniemi, O. P., et al., Science 258 (1992) 818-21] have recently indi- cated that known oncogenes account for only a part of the detected amplifications in human neoplasias [Forozan et al., Trends Genet. 13 (1997) 405-9].
The long arm of chromosome 8 (8q) is one of the most common regions of amplification in cancers of several organs, such as bladder and ovarian cancer, but especially carcinomas in the breast and the prostate [Vi- sakorpi, T., et al., Cancer Res. 55 (1995) 342-7; Cher et al., Cancer Res. 56 (1996) 3091-102; Nupponen, N. N., et al., Am. J. Pathol. 153 (1998) 141-8; Tir- kkonen et al., Genes Chromosomes Cancer 21 (1998) 177-84].
Another region of amplification associated to carcinomas is chromosome 10 (10q). It has been shown by CGH that approximately 15% of the hormone-refractory recurrent prostate carcinomas contain a gain or amplification at 10q (Nupponen, N. N., et al., supra) and that prostate cancer cell line PC-3 shows high level-amplification at 10p12-q23 [Nupponen, N. N., et al., Cancer Genet. Cytogenet. 101 (1998) 53-57]. However, a specific relationship between an identified potential target gene in 10q and certain specific forms of cancer has not been shown. The identification of such a relationship would lead in much desired improvements in the postoperative treatment of invasive recurrent forms of cancer and would be greatly benefit cancer patients. In order to invade and metastasize, the cancer cells must effectively degrade extra cellular matrix (ECM) components. Plasminogen activation has been implicated as one of the mechanisms of the ECM degradation. Mammalian cells contain two types of plasminogen activators, the urokinase-type (uPA) and the tissue-type (tPA) plasminogen activator, of which uPA is primarily involved in ECM degradation [Dano, K., et al., APMIS 107 (1999) 120-127; Andreasen, P. A., et al., Int. J. Cancer 72 (1997) 1-22]. uPA has restricted substrate specificity, its main function being the cleavage of plasminogen to plasmin (Liotta, L, et al., supra), which degrades several ECM components and also activates many pro-MMPs (matrix-metalloproteases) [Liotta, L, et al., supra; Murphy, G., et al., Ann. NY Acad. Sci. 667 (1992) 1- 12].
The urokinase gene (uPA) is one of the genes mapped to chromosome 10q. The uPA is believed to play a key role in the cancer invasion and metastasis [Dano, K. et al., 1999, supra; Festuccia, C, et al., Clin. Exp. Metastasis 16 (1998) 513-528]. Both the uPA secretion and the presence of the receptor bound uPA at the cell surface characterize prostate cancer cells that have invasive phenotype [Gaylis, F., et al., J. Urol. 742 (1989) 193-198]. An increased expression of urokinase has been reported in various malignancies including prostate cancer [Gaylis, F. et al., supra; Van Veldhuisen, et al., Am. J. Med Sci. 312 (1996) 8-11], breast cancer [Look, M. P., and Foeckens, J. A., APMIS 107 (1999) 150-159], colon cancer [Pyke, C, et al., Am. J. Pathol. 738 (1991 ) 1059-1067], and lung cancer [Skriver, L., et al., J. Cell. Biol. 99 (1984) 752-757]. In many cases, its increased expression seems to be associated with an increased metastatic potential and poor survival [Hsu, D. W., et al., Am. J. Path. 747 (1995) 114-123; Miyake, H., et al., Int. J. Cancer 74 (1999) 535-541 ; Yang, J. L., et al., Int. J. Cancer 20 (2000) 431-439]. The mechanisms of the increased expression of uPA have, however, remained unknown.
Summary of the invention
We have now verified the location of the uPA gene to 10q22-qter and found that it is amplified in a significant portion of invasive recurrent carcinomas, such as hormone-refractory cancers, like prostate cancer. This implies that uPA is one of the target genes for the 10q gain. The amplification profile of uPA provides novel approaches, which can be utilized in the development and improvement of the therapy of these carcinomas. An object of the invention is thus to provide novel means that are useful in predicting and identifying optimal treatment of recurrent invasive forms of carcinoma, especially of hormone-refractory forms of carcinoma, such as invasive prostate cancer.
Another object of the invention is to provide novel means that are useful in the identification of particular types of carcinomas, which could respond to specifically targeted chemotherapy.
Another object of the invention is to provide novel means that are useful in the identification of cancer patients that would benefit of specifically targeted chemotherapy.
Yet another object of the invention is to provide means that are useful in the identification of novel drugs for the treatment of recurrent invasive forms of carcinoma, especially of hormone-refractory forms of carcinoma, such as invasive hormone-refractory breast cancer or prostate cancer.
The present invention relates to the use of urokinase gene amplification as a marker for evaluating the treatment sensitivity and specificity of carcinomas, especially invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer.
The present invention also relates to methods of identification of carcinomas profiled by urokinase gene amplification by detecting the presence or absence of amplification of uPA in a tumor sample. The present invention further relates to a method of identifying of cancer patients, especially those suffering from invasive forms of carcinomas, and in particular invasive forms of hormone-dependent cancers, such as prostate cancer, that would benefit of targeted chemotherapy by detecting the presence or absence of amplification of uPA in a biological sample obtained from said patients.
The present invention further related to a method of the identification of novel drugs for the treatment of recurrent invasive forms of carcinoma, especially of hormone-refractory forms of carcinoma, such as invasive hormone-refractory breast cancer or prostate cancer by determining the inhibiting ability of the uPA activity of said drug.
Brief description of the drawings
Figure 1 shows (A) the mean (±SD) fluorescence ratio profile from p- to q-telomere for chromosome 10 in prostate cancer cell line PC-3 obtained by CGH; a two color-FISH analyses of normal (B) and PC-3 cell lines metaphase chromosome preparations (C), and interphase nuclei of two hormone-refractory prostate carsinomas (D, E) with BAC probe for the uPA gene (red signals) and the chromosome 10 centromere specific probe (green signals).
Figure 2 shows (A) the results of a Northern hybridization of the uPA demonstrating that invasive prostate cancer cell lines PC-3 and DU-145 express high levels of uPA whereas the non-invasive LNCaP cell line does not; and (B) the relative expression level of uPA (uPA/β-actin) after quantification with phosphoimager.
Figure 3 shows the expression level of uPA measured by real-time
RT-PCR: (A) the PCR curves of the serially diluted standard containing cDNA transcribed from 500, 100, 20, 4, 0.8 and 0.16 ng of totRNA, (B) the standard curve blotting fractional cycle number at the fluorescent threshold for each standard curve presented in panel A, and (C) the relative expression of uPA genes in prostate tumor samples: • represents a tumor containing high-level
AR gene amplification, • represents tumors with gain of the uPA gene and o represents tumors without uPA gene copy number alterations. Horizontal scattered line shows the median value of expression.
Figure 4 shows the effects of amiloride on the invasive ability of
PC-3, DU145 and LNCaP cells as examined by the Matrigel invasion assay.
Data represent mean ±SEM of four experiments. * p<0.001 , n.s. =statistically non-significant difference (Student's t test).
Detailed description of the invention
The present invention is based on the studies in which the uPA gene was re-mapped to 10q22 and shown to amplify in the PC-3 cell line and, for the first time, also in vivo. The frequency of the uPA gene amplification in hormone-refractory prostate carcinomas was studied by fluorescence in situ hybridization (FISH) and the expression of the gene by Northern analysis and real-time RT-PCR (LightCycler methodology). The association between the uPA gene amplification and the sensitivity of the cells to the uPA inhibition were studied by an invasion assay. To map the gene precisely, a genomic clone for uPA was obtained by screening a human BAC library with specific primers specific for the gene.
Using fluorescence in situ hybridization (FISH), the uPA gene was localized to
10q22. Previously the uPA gene has been located to 10q24-qter [Tripputi, P., et al., Proc. Nat. Acad. Sci. 82 (1985) 4448-4452]. To show that the gain or the amplification in 10q22 in prostate cancer cell line PC-3 is actually due to the amplification of the uPA gene, the copy number of uPA in this cell line and in two other prostate cancer cell lines, DU145 and LNCaP, was studied by FISH. High-level amplification, i.e. about 20-30 copies of the uPA gene, was found in PC-3 (Figure 1). However, the DU145 and LNCaP cell lines showed only 3 and 4 copies of both uPA and chromosome 10 centromere by FISH, respectively. The findings are consistent with the CGH data indicating no high-level amplification at 10q in these cell lines [Nupponen, N.N., et al., Cancer Genet. Cytogenet. 101 (1998) 53-57].
To evaluate the possible alterations of the uPA gene copy number in prostate tumors in vivo, the gene copy number in 13 locally recurrent hormone-refractory prostate carcinomas were studied. Androgen-dependent prostate tumors rarely show 10q gains [Nupponen, N. N. and Visakorpi, T., Microsc. Res. Tech. 51 (2000) 456-463]. FISH analyses of the tumors showed an increased copy number of the uPA gene in 3 out of 13 cases. These included one high-level amplification (8%) and two moderately increased copy numbers (15%) (Fig. 1 D and 1 E). The FISH analysis of the tumor with high- level amplification showed tight clusters of signals making scoring the exact copy number difficult. However, at least 10 copies of the gene and 2 copies of the centromere were estimated to be present indicating a 5-fold amplification. In one of the cases with a moderate gain of uPA, the copy numbers of the uPA and centromere were 3 and 1 , respectively, whereas in the other case both uPA and centromere probes showed 4 copies suggesting tetraploidization.
To study whether the increased copy number of the uPA is associated with increased expression of the gene, Northern hybridization of the three prostate cancer cell lines, PC-3, DU-145, and LNCaP was performed. The expression of urokinase was increased in the invasive PC-3, containing the high level amplification of the gene. Also, the invasive DU145 cell line expressed high level of the uPA. On the contrary, no expression was found in the non-invasive LNCaP cell line.
The relative expression level of uPA (uPA/β-actin) in PC-3 and in DU145 was analysed by quantification with phosphoimager. uPA was expressed 2-times more in PC-3 than in DU145. The findings are in consistent with a previous report of the levels of uPA mRNA in these cell lines (Hollas, W., et al., supra).
The expression of uPA in the locally recurrent hormone-refractory prostate tumors was also evaluated by quantitative real-time RT-PCR analysis. The results showed that the expression of urokinase was increased in 2 out of 3 samples with the increased copy number of uPA as compared to uPA expression levels in tumors with normal gene copy number (Fig.3). The case with the high-level amplification expressed two times more uPA than the other tumors on average. The association between the amplification and over- expression of the gene both in the cell lines and the tumors suggests that uPA is a strong candidate target gene for the 10q amplification in prostate cancer.
The high-level amplification of the uPA gene was found both in an androgen-independent prostate cancer cell lines and native tumor. The emergence of androgen-independent disease is considered to be a late event in the progression of prostate cancer. Additionally, it almost always requires the presence of hormonal therapy (Kallioniemi, O. P. and Visakorpi, T., supra). Based on the function of the uPA, it is likely that the processes related to invasiveness rather than hormonal therapy are associated with the selection of the uPA amplification. Due to the indications that uPA plays a central role in the invasion of cancer, it has been suggested that uPA could be therapeutically targeted. Several compounds that inhibit the activity of uPA in prostate cancer in vitro have been demonstrated. These include maspin and retinoic acid [McGowen, R., et al., Cancer Res. 60 (2000) 4771-4778; Webber, M. M. and Waghray, A., Clin. Cancer Res. 7 (1995) 755-761 (32,33)]. In addition, using prostate cancer xenografts established by implanting DU145 and LNCaP cells into nude mice, Jankun and co-authors [Jankun, G., et al., Cancer Res. 57 (1997) 559-563] showed that both PAI-1 and several small molecule uPA inhibitors, not just inhibit the invasion, but also reduce tumor growth. In order to evaluate, whether the amplification of uPA gene could be associated with treatment sensitivity, the inhibition of uPA in three prostate cancer cell lines (PC-3, DU145, LNCaP) was analyzed with Matrigel invasion assay (Figure 4). Both PC-3 and DU145 cells, which express uPA, invaded through Matrigel coated filters, whereas LNCaP, which does not express uPA, had only very weak invasive capability. When known uPA inhibitor, amiloride, was added to the growth medium, the invasiveness of PC-3, containing the gene amplification, reduced about 10-fold, whereas the number of invasive cells of DU145 was only marginally reduced. The reduction of invading cells in PC-3 was statistically significant. Thus, the uPA gene amplification was associated with cells sensitive to the uPA inhibitors. According to the detection method of the present invention, the presence or absence of the uPA gene can be detected from a biological sample by any known detection method suitable for detecting a gene copy number or expression, i.e. methods based on detecting the copy number of the gene (or DNA) and/or those based on detecting the gene expression products (mRNA or protein). Such methods are easily recognized by those skilled in the art and include in situ hybridizations, such as fluorescence in situ hybridization (FISH) and mRNA in situ hybridization, Southern analysis, RT-PCR, Northern and Western analyses, immunohistochemistry, and other immunoassays. Preferable methods are those suitable for use in routine clinical laboratories, such as FISH and immunohistochemistry.
In the detection method of the invention, the biological sample can be any sample containing tumor cells, such as a biopsy sample from the breast, prostate, a lymph node or other tissues containing metastatic lesions, including circulating cancer cells. The biological sample can also be a body fluid, such as whole blood, serum, plasma, urine, lymph, and a cerebrospinal fluid sample. The biological sample can be pretreated, if necessary, in a suitable manner known to those skilled in the art.
The invention will be elucidated below by the following non-limiting examples. The cell lines and tumors used in the Examples were as follows: Prostate cancer cell lines PC-3, DU145, and LNCaP were obtained from ATCC (Manassas, VA, USA) and cultured according to manufacturer's protocols. Freshly frozen transurethral resection (TURP) specimens from 13 patients diagnosed with locally recurrent hormone-refractory prostate cancer were obtained from Tampere University Hospital. The TURPs were performed on the patients due to urethral obstruction during hormonal therapy. The endocrine therapy modalities were orchiectomy (4 cases), luteinizing hormone- releasing hormone analogue (4 case), estrogen (1 case), maximal androgen blockade (3 cases), or unknown (1 case). The time from the beginning of the therapy to TURP varied from 16 to 60 months. The presence of more than 60% of tumor cells in the tissue samples was histologically confirmed by hematoxylin-eosin stained frozen section slides. Example 1 Re-mapping of uPA The uPA gene has previously been mapped to 10q24-qter
[Tripputi, P., et al., Proc. Nat. Acad. Sci. 82 (1985) 4448-4452], a region, which often is deleted in prostate cancer. Since it has been found that old mapping data is sometimes inaccurate, the uPA gene was re-mapped by a FISH analysis.
A genomic clone for uPA was obtained by screening human BAC library with primers specific for the gene (5'-atc age tgt aag aag age tgg g-3', sequence id. number 3, and 5'- atg ccc tgc cct ttt taa ct -3', sequence id. number 4). The authenticity of the clone (BAC-46i3) was verified by partial sequencing. The BAC-uPA probe was labelled with digoxigenin-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation. SpectrumGreen labelled chromosome 10 centromere specific probe (CEP10, Vysis Inc, Downers Grove, IL) was used as a reference probe.
Metaphase chromosome preparations from the prostate cancer cell lines and normal blood lymphocytes were prepared using routine techniques. Five micrometers' tissue sections from the freshly frozen tumor blocks were fixed on objective slides in a series of 50%, 75%, and 100% Carnoy's solution (3:1 methanol-acetic acid) for 10 min each at room temperature.
The dual-color FISH was performed essentially as described by Hyytinen et al., Cytometry 16 (1994) 93-99]. Briefly, the slides were denatured in 70% formamide/ 2X SSC at 70.5 °C for 2.5 min and dehydrated through an ethanol series. Hybridizations were carried out at 37 °C for 48 hours. After stringent washes, the slides were stained with anti-digoxigenin-rhodamine (Roche Diagnostics) and counter-stained with an antifade solution (Vectashied, Vector Laboratories, Burlingame, CA) containing 4,6-diamidino 2- phenylindole (DAPI). The FISH signals were scored from non-overlapped epithelial cells using Olympus BX50 epifluorescence microscope (Tokyo, Japan). The criteria for high-level amplification was either the presence of the tight cluster of uPA signals or more than 5 copies of the gene. The results are set forth in Figure 1. The profile in Figure 1A indicates amplification at 10p12-q23 [cf.
Nupponen, N.N., et al., Cancer Genet. Cytogenet. 707 (1998) 53-57]. FISH analysis maps the uPA to chromosome 10q22 (Figure 2B). Multiple copies (20 to 30) of uPA in the rearranged chromosomes are seen as a sign of high-level amplification in the PC-3 cell line (Figure 1C). Three copies of uPA and 1 copy of the chromosome 10 centromere were seen in a hormone-refractory tumor (Figure 1 D). Approximately 10 copies of uPA and 2 copies of the chromosome 10 centromere indicate 5-fold amplification of uPA gene (Figure 1 E).
In the analysis of the metaphase chromosomes, uPA signals were seen in several different chromosomes consistent with the spectral karyotyping
(SKY) data, which have earlier shown that chromosome 10 is involved in at least 6 different chromosomal rearrangements in the PC-3 cell line [Pan, Y., et al., Cytogen. Cell Genet. 87 (1999) 225-232].
Example 2
Amplification of uPA in prostate cancer cell lines
To study whether the increased copy number of the uPA is associated with increased expression of the gene, Northern hybridization of three prostate cancer cell lines was performed. Total RNA was isolated from each of the cell lines using Trizol reagent (Life Technologies, Rockville, MD,
USA). Ten micrograms of total RNA was separated in 1.2% denaturing formaldehyde-agarose gel electrophoresis and blotted as described in Ausubel, F. M., et al., (Eds), Current Protocols in Molecular Biology, John
Wiley & Sons, 1987. The 32P-dCTP labelled PCR amplified insert of IMAGE-
EST (GenBank ace. AI818478) was used as a probe and hybridized over night. After washing, the blot was visualized and quantified with Storm
Phosphoimager (Molecular Dynamics Inc.). To verify equal loading of the gel, the blot was stripped and rehybridized using 32P-labelled probe for β-actin
(Clontech, Palo Alto, CA) as described above.
The results are set forth in Figure 2. The expression of urokinase is increased in the invasive PC-3 cell line containing the high level amplification of the gene. Also the invasive DU145 cell line expressed high level of the uPA. On the contrary, no expression was found in the non-invasive LNCaP cell line. According to quantification with phosphoimager uPA was expressed 2-times more in PC-3 than in DU145 (cf. Example 3). Example 3
Expression level of uPA in prostate tumors The amplification of the putative target genes is thought to lead to their over-expression. To show that uPA is amplified in native tumors obtained from patients suffering from prostate cancer, the expression of uPA in 12 locally recurrent hormone-refractory prostate tumors was evaluated by quantitative real-time RT-PCR analysis. One to three 20μm frozen sections were cut from the tumor blocks using a cryotome. Total RNAs were isolated from the sections with Qiagen RNeasy MiniKit (Qiagen Inc, Valencia, CA) and used for the first-strand cDNA synthesis with Superscript™ II reverse transcriptase and oligo d(T)i2-ιβ primer according to manufacturer's protocol (Life Technologies). For the standard curve, 5 μg of totRNA from normal mammary tissue (Clontech) was reverse transcribed as described above. After the first strand cDNA synthesis, serial dilutions were made corresponding to cDNA transcribed from 500, 100, 20, 4, 0.8, and 0.16 ng of totRNA.
The primer and probe sequences used in the analyses of the expression of uPA and TATA-box binding protein (TBP) genes were as follows. For uPA, a primer sequence (5'-3'):
TCACCACCAAAATGCTGTGTAGGCCATTCTCTTCCTTGGT
(SEQUENCE ID. NO. 1 ) and a hybridization probe sequence (5'-3'):
TCCCCCTGAGTCTCCCTGGCA-FluoresceinRed640- AATCTGTTTTCCACTGTGGGTCAGCAG;
For TBP, a primer sequence (5'-3'):
GAATATAATCCCAAGCGGTTTGACTTCACATCACAGCTCCCC
(SEQUENCE ID. NO. 1 ) and a hybridization probe sequence (5'-3'): TTTCCCAGAACTGAAAATCAGTGCC-FluoresceinRed640-
TGGTTCGTGGCTCTCTTATCCTCATG
The primers were designed to avoid amplification of any genomic DNA by choosing the forward and reverse primers for each of the genes from different exons. The PCR reactions were performed in the LightCycler™ apparatus [Wittwer, C. T., et al., Biotechniques 22 (1997) 176-181] using LC DNA Hybridization Probes Kit (Roche Diagnostics). Thermocycling for each reaction was done in a final volume of 20 μl containing 2 μl of cDNA sample (or standard), 4mM MgCI , 0.5 μM of each primer, 0.2 μM fluorescein and 0.4 μM LC Red640 labelled probes as well as 1x ready-to-use reaction mix including Taq DNA polymerase, the reaction buffer, and the dNTP mix. After an initial denaturation for 30 s at 95°, the cycling conditions of 55 cycles consisted of denaturation at 95° for 0 s, annealing at 57° (for uPA) or 58° (for TBP) for 13 s, and elongation at 72° for 9 (uPA) or 10 (TBP) seconds.
The LightCycler™ apparatus measured the fluorescence of each samples in every cycle at the end of the annealing step. After proportional background adjustment, the fit point method was used to determine the cycle, in which the log-linear signal is distinguished from the background, and that cycle number was used as a crossing-point value. The software produced the standard curve by measuring the crossing point of each standard and plotting them against the logarithmic value of concentrations. The concentrations of unknown samples were then calculated by setting their crossing points to standard curve.
The expression level of uPA was normalized by TBP. TBP was selected for the reference gene because there are no known retropseudogenes for it and the expression of TBP is lower than in many commonly used abundantly expressed reference genes [Bieche, I., et al., Cancer Res. 59 (1999) 2759-2765]. After the PCR, all samples were also run in 1.2% agarose gel electrophoresis to ensure that right size product was amplified in the reaction.
The results are set forth in Figure 3. The results show that the expression of urokinase was increased in 2 out of 3 tumor samples with the increased copy number of uPA as compared to uPA expression levels in tumors with normal gene copy number (Fig.3). The case with the high-level amplification expressed two times more uPA than the other tumors on average. The association between the amplification and over-expression of the gene both in the cell lines and the tumours suggests that uPA is, indeed, a strong candidate target gene for the 10q amplification in prostate cancer. However, based just on the gene copy number and expression data is not possible to rule out completely the presence of other target genes for the 10q gain. Example 4
Evaluation of the association between the amplification of the uPA gene and treatment sensitivity
In order to evaluate, whether the amplification of uPA gene could be associated with treatment sensitivity, the inhibition of uPA was studied in three prostate cancer cell lines (PC-3, DU145, and LNCaP) by Matrigel invasion assay with Matrigel (BD Biosciences, Bedford, MA, USA) coated PET membrane of 8μm pore size (BD Biosciences). One hundred microliters of Matrigel (1 mg/ml in phosphate buffered saline) was applied to a 24-well cell culture plate insert and allowed to solidify. The culture medium in the wells contained 5% FBS and fibronectin (5 μg/ml) as chemoattractant. 1x105 cells (PC-3, DU145, or LNCaP) in the culture medium containing 1 % FBS were plated onto an insert. Half of the inserts were treated with 151 μM amiloride (Sigma Chemicals CO., St.Louis, MO) or mock-treated for 22 hours. Subsequently, the cells were removed and the invaded cells were fixed with methanol and stained with crystal violet. The number of invaded cells was counted under a microscope. The experiments were repeated four times per each cell line and treatment. The results are shown in Fig. 4. The data represent mean ±SEM of four experiments performed.
Both the PC-3 and DU145 cells, which express uPA, invaded through Matrigel coated filters, whereas the LNCaP cells, which do not express uPA, had only a very weak invasive capability. In the presence of amiloride, a known uPA inhibitor [Jankun, G., et al., Cancer Res. 57 (1997) 559-563], the invasiveness of the PC-3 cells, which contain the uPA gene amplification, was reduced about 10-fold. However, the number of DU145 cells was only marginally reduced. The reduction of invading cells in PC-3 was statistically significant (p<0.001 in Student's t test).
Thus, the uPA gene amplification, but not the uPa expression, was associated with cells sensitive to the uPA inhibitors. The finding further indicates that the uPA is one of the target genes for the 10q gain and that it may well be a potential treatment target. In conclusion, we report here, for the first time, a high-level amplification of uPA gene in hormone-refractory prostate carcinoma in vivo. The increased copy number was associated with increased expression of the gene and with the sensitivity of the cells to uPA inhibition. The findings support the suggestion that uPA is involved in the acquisition of invasive phenotype in prostate cancer cells.

Claims

CLAIMS 1. The use of urokinase gene (uPA) amplification as a marker for evaluating the treatment sensitivity and specificity of an invasive form of carcinoma.
2. The use of claim ^ characterized in that the invasive form of carcinoma is an invasive form of a hormone-dependent cancer, such as prostate cancer.
3. A method for the identification of a carcinoma profiled by urokinase gene amplification, characterized by detecting the presence or absence of amplification of uPA in a tumor sample.
4. A method for the identification of a cancer patient suffering from an invasive form of a carcinoma that would benefit of targeted chemotherapy, characterized by detecting the presence or absence of amplification of urokinase gene (uPA) in a biological sample obtained from said patient.
5. A method for the identification of a novel drug for the treatment of recurrent invasive forms of carcinoma, characterized by determining the inhibiting ability on the uPA activity of said drug.
6. A method of any one of claims 3 to 5, characterized in that the carcinoma is an invasive form of carcinoma, specifically an invasive form of a hormone-dependent cancer, such as prostate cancer.
7. A method of claim 3 or 4, characterized in that the detection is performed using a gene-technological method.
8. A method of claim 7, characterized in that the detection is performed using an in situ hybridization method, such as fluorescence in situ hybridization (FISH) or mRNA in situ hybridization, a Southern analysis, an RT- PCR, or a Northern analysis.
9. A method of claim 3 or 4, characterized in that the detection is performed using an immunological method, such as a Western analysis, immunohistology and an immunoassay.
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