Cross-resistance between taxanes and new hormonal agents abiraterone and enzalutamide may affect drug sequence choices in metastatic castration-resistant prostate cancer.

INTRODUCTION: Treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) have expanded in recent years with the introduction of cabazitaxel, abiraterone and enzalutamide. With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. As shown recently, patients who had previously been treated with abiraterone showed impaired responses to docetaxel, suggesting clinical cross-resistance [1]. In the present study, we aimed to identify cross-resistance between taxanes (docetaxel and cabazitaxel) and the new hormonal agents abiraterone and enzalutamide. As a potential mechanism for cross-resistance, we investigated the effects on androgen receptor (AR) nuclear translocation of these compounds. METHODS: To identify cross-resistance, we determined the effects of docetaxel, cabazitaxel, abiraterone and enzalutamide on cell viability in prostate cancer cell lines with acquired resistance to abiraterone and enzalutamide. Time-lapse confocal microscopy was used to study the dynamics of AR nuclear translocation. RESULTS: We observed impaired efficacy of docetaxel, cabazitaxel and enzalutamide in the abiraterone-resistant cell line, compared to the non-resistant cell line, providing evidence for in vitro cross-resistance. Impaired efficacy of docetaxel, cabazitaxel and abiraterone was observed in the enzalutamide-resistant cell line. Furthermore, docetaxel and cabazitaxel inhibited AR nuclear translocation, which was also observed for abiraterone and enzalutamide. CONCLUSIONS: In conclusion we found substantial preclinical evidence for cross-resistance between the taxanes docetaxel and cabazitaxel, and AR targeting agents abiraterone and enzalutamide. Since these compounds all interfere with AR-signalling, this strongly suggests a common mechanism of action, and thus a potential mechanism for cross-resistance in mCRPC.

Characterization of chromosome 8 aberrations in the prostate cancer cell line LNCaP-FGC and sublines.

In two androgen-dependent (FGC and P70) and two androgen-independent (LNO and R) sublines of the prostate cancer model LNCaP numerical and structural aberrations of chromosome 8 were investigated in detail. The techniques used were whole chromosome paint (WCP) and fluorescence in situ hybridization (FISH) with three cosmid probes mapping to different parts of the p-arm (D8S7 (8p23.3), LPL (8p22) and PLAT (8p11.1)). By WCP all four cell lines showed four copies of chromosome 8 in most cells. However, FISH demonstrated that in all sublines deletions in the 8p region were present. The majority of both FGC and P70 had two copies of cosmids D8S7 and LPL. The cosmid PLAT showed a broader distribution (1-4 copies), especially in P70. Compared with FGC and P70, both LNO and R showed a larger number of copies (3 or 4) of all three cosmid loci. It is discussed that this difference is probably the result of nondisjunction as a reaction to loss of other sequences on 8p, possibly the tumor suppressor gene (TSG) mapping to 8p21. The fact that both sublines LNO and R are androgen-independent raises the possibility of a link between TSG loss on 8p and androgen independence.

Cytogenetic analysis of 39 prostate carcinomas and evaluation of short-term tissue culture techniques.

Karyotypic analysis was performed on 102 prostate cancer specimens which were obtained through radical prostatectomy, transurethral resection, or regional lymph node dissection. Short term tissue culture was applied in all cases. Of the media and growth factors evaluated, F12/DMEM, supplemented with 2% fetal calf serum, insulin, epidermal growth factor, hydrocortisone, and cholera toxin produced the largest increase of in vitro proliferation. Such in vitro cultured cells were all phenotypically acinar epithelial cells, the supposed targets for neoplastic transformation. Stromal cell growth appeared to be completely suppressed. Of the three culture techniques investigated, the method developed in Lund, Sweden, was the most successful: 11/15 cultures yielded metaphases and, in three of these, clonal aberrations were identified. All 39 karyotypes obtained essentially had a 46,XY karyotype with clonal aberrations (eight cases) and/or nonclonal aberrations (30 cases). Clonal structural aberrations involved 2p, 3q, 11p, 17p, and 21q. The clonal numerical aberrations found were: + 8, + dmin, and -Y. The most frequently observed nonclonal aberrations were 8p deletions (five cases) and loss of 6, 7, 8, 15, 17, 18, 21, and/or Y (> or = five cases). In summary, clonal aberrations were observed in 20% of the evaluable PC cell cultures, and nonclonal aberrations in 77%. So, although diploid cells without clonal abnormalities still had a growth advantage, under optimal conditions PC cells were able to proliferate in primary in vitro culture.

Specific cytogenetic aberrations in two novel human prostatic cell lines immortalized by human papillomavirus type 18 DNA.

Using chromosome banding and fluorescence in situ hybridization (FISH) with painting probes, sequential cytogenetic analysis was performed of two novel prostate cell lines, PZ-HPV-7 and CA-HPV-10, established by human papillomavirus (HPV) 18 DNA transformation. PZ-HPV-7 originates from a normal diploid prostate epithelial cell strain. PZ-HPV-7 progressed from an initial diploid to a hypertetraploid chromosome number with a relative gain of chromosomes 5 and 20 (7 to 8 copies each). Structural changes were limited; 3p- (2 copies), 3q- (1 copy), and possibly a der(16p;12q). CA-HPV-10 originates from an epithelial cell strain derived from a high-grade human prostate cancer specimen, which showed several karyotypic abnormalities including an extra Y chromosome and double minutes (dmin). In early passage the karyotype of CA-HPV-10 appeared unstable with a decreasing number of cells exhibiting dmin. In late passage the dmin were replaced by a large homogeneously staining region (hsr) on 9p+ marker. The hsr was shown by FISH to be of chromosome 1 origin. The modal number was mainly hypertriploid (72, range 69 to 75). Loss of Y was remarkable (0 to 1 copy). Consistent markers included two copies each of del(1)(q12q31) and der(9)t(1;9)(?;p22), and one der(11)t(4;11) (?;q21). HPV type 18 genomic integration sites were identified on 1p for PZ-HPV-7 and on the 9p+ marker for CA-HPV-10. In conclusion, both PZ-HPV-7 and CA-HPV-10 showed clonal cytogenetic changes. These two cell lines constitute a novel in vitro model to study the mechanisms involved in human prostate carcino-genesis.

Gain and loss of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate cancers.

Fluorescence in situ hybridization (FISH) with centromere probes was used to investigate numerical aberrations of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate carcinoma (PC) and 11 benign prostatic hyperplasia (BPH) samples. None of the benign specimens showed any chromosomal aberration. Forty-one of 46 PC specimens showed numerical aberrations of one or more chromosomes. All investigated chromosomes showed numerical aberrations in at least 30% of the specimens, gain being more frequent than loss. Comparison of DNA flow cytometry (FCM) and FISH results showed that not only aneuploid tumors but also most diploid tumors harbored numerical chromosome aberrations. Chromosome 10 was the most frequently gained (65%), and Y the most frequently lost chromosome (14%). Nonmetastatic and metastatic tumors differed significantly (P < .05) in the number of copies for chromosomes 7, 8, and 10, but not for 1, 18, and Y. These results suggest strongly that gains of chromosomes 7, 8, and 10 are involved in PC progression.

Loss and gain of chromosomes 1, 18, and Y in prostate cancer.

Nuclear suspensions of 42 prostate carcinoma specimens obtained at surgery were used to investigate loss and gain chromosomes 1, 18, and Y by fluorescence in situ hybridization (FISH) with centromere-specific probes. The outcome of FISH analysis was correlated with clinical parameters and the relationship between DNA-FCM (ploidy at cellular level) and FISH (ploidy of individual chromosomes) was assessed. Significant loss of chromosomes 1 and 18 was infrequent (respectively, three and five cases), but 53% of the tested specimens showed loss of Y. Loss was not correlated with DNA ploidy. Significant gain occurred in 36% (chromosome 1), 63% (chromosome 18), and 28% (Y) of the specimens. Gain of chromosome 18 was shown in DNA diploid (7/14) and aneuploid tumors (18/26), while gain of chromosomes 1 and Y was nearly restricted to DNA aneuploid specimens. Significant unbalance between these chromosomes occurred in 11 cases. Most cases which had significant gain of chromosome 1 or 18 showed trisomic as well as tetrasomic cells. Simultaneous loss of some and gain of other investigated chromosomes is suggestive of clonal heterogeneity and/or multiclonality. This was observed in eight tumors. Correlation between DNA-FCM and FISH was best for the Y chromosome. DNA-FCM showed more aberrant histograms with increasing stage and grade of tumors. The presence of numerical aberrations of the investigated chromosomes however, seemed independent of clinical grade or stage.

Tissue culture loss of aneuploid cells from carcinomas of the prostate.

The frequency of aneuploid cells in cultured prostate carcinoma specimens was investigated. Ploidy distribution of the original tissue was established by flow cytometry (FCM). Fluorescence in situ hybridization (FISH) of chromosome I was applied to directly isolated and cultured cells to investigate whether any modifications in the ploidy distribution of chromosome I took place during tissue culture. In six tumor specimens that were diploid by FCM and FISH, no differences were found in the ploidy distribution of chromosome I before and after tissue culture. In eight tumors that were aneuploid by FISH, the percentage of aneuploid nuclei was significantly reduced from 28.0 +/- 15.0 (range 13-59%) in uncultured cells to 9.1 +/- 4.4 (range 4-18%) after tissue culture. The reduction of aneuploid nuclei ranged from 44 to 85%, which means that the majority of the aneuploid cell populations that were observed in the original specimens were undetectable in cultured samples. This suggests a preferential growth of normal epithelial cells. The data presented can explain the high percentage of diploid karyotypes usually found in short-term cultured prostate carcinoma specimens.

Characterization of the human spr2 promoter: induction after UV irradiation or TPA treatment and regulation during differentiation of cultured primary keratinocytes.

We have isolated genomic clones from several members of the UV and TPA inducible human spr2 gene-family in order to analyse the regulation of these genes at a molecular level. From one of these members, the spr2-1 gene, we have identified and sequenced the regulatory region. By using CAT fusion plasmids and a liposome mediated transfection procedure we show that the isolated promoter region contains all the cis-elements necessary for induced expression after UV irradiation or phorbolester treatment of cultured human keratinocytes. Additionally the spr2-1 promoter is shown to be regulated aswell during the normal process of keratinocyte differentiation. This makes the spr2-1 promoter sequence an ideal tool to study the molecular mechanisms by which environmental agents such as UV radiation and chemical tumor promoters interfere with normal gene expression during cell proliferation and differentiation.

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells.

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.