Prostate cancer is the most commonly diagnosed internal malignancy in men and the second leading cause of cancer-related deaths. The etiology of PCa is uncertain with environmental, hormonal and hereditary factors implicated. The initiation of PCa (ie. the formation of a histologically identifiable lesion) is a common event, being detected at autopsy series in nearly one-third of men over age 45.
A diagnosis of prostate cancer (from prostatic biopsies) is initiated typically following an elevation in serum measurements of prostate specific antigen, also known as kallikrein III, seminin, semenogelase, γ-seminoprotein and P-30 antigen); it is a 34 kD glycoprotein manufactured almost exclusively by the prostate gland; PSA is produced for the ejaculate where it liquifies the semen and allows sperm to swim freely. It is also believed to be instrumental in dissolving the cervical mucous cap, allowing the entry of sperm. Biochemically it is a serine protease enzyme, the gene of which is located on the nineteenth chromosome (19q13).
PSA is not a test for cancer and there is no threshold level of this enzyme providing a high sensitivity and specificity with a continuum of risk for all PSA values. A raised serum PSA so often commits men to the invasive and imprecise procedure of transrectal ultrasound guided biopsies. A further indictment of the limitations of PSA in PCa detection is the disparity between TRUS biopsy findings and those from radical prostatectomy.
Diagram showing effect of PSA testing, which began in the mid-1980s, on volume of index (largest) cancer in the prostate over ensuing decades.
In the early 1990s, at about the same time that PSA testing was starting to gain widespread adoption, a young molecular biologist from Holland, Marion Bussemakers studies on human prostate tissue using the technique of differential display, a then newly described method to identify gene expression in different tissues. During this series of experiments, an mRNA was discovered that appeared to be highly specific for prostate cancer.
Northern blot analysis using probes for DD3,now called PCA3 (upper lane) and PSA (middle lane),with rRNA (28S) as a control (lower lane).
This gene could not be found in any of the existing gene databases. Bussemakers called his new gene DD3, and he concluded that it might be useful in prostate cancer detection. Further study revealed the new gene to be a noncoding RNA, which could be mapped to chromosome 9q21-22.
The gene has 4 distinct exons, which can give rise to a number of differently sized transcripts (open reading frame analysis has confirmed that the PCA3 exons are populated by an unusual number of stop codons). It was upregulated in 53 of 56 prostate cancers when compared with non-malignant prostate tissue.
Upregulation of the major PCA3 transcript, was shown to be a sensitive and specific marker for the diagnosis of Pca. It is established that a class of very small ncRNAs known as microRNAs are altered in different tumour types and can act as oncogenes or tumour suppressor gene.
Comparative genomic analyses demonstrated that PCA3 has only recently evolved in an anti-sense orientation within a second gene, BMCC1/PRUNE2. BMCC1 has been shown to interact with RhoA and RhoC (RhoA participates in oncogenic transformation whereas RhoC promotes tumor metastasis and cell migration), determinants of cellular transformation and metastasis, respectively. Using RT-PCR it was demonstrated that the longer BMCC1-1 isoform - like PCA3 – is upregulated in PCa tissues and metastases and in PCa cell lines. Expression of BMCC1-1 was evident in normal prostate and BPH specimens and was upregulated in PCa and metastases. Both of these gene are androgen responsive.
New genomic structure for prostate cancer specific gene PCA3 within BMCC1: implications for prostate cancer detection and progression
WHY IS PCA3 OVEREXPRESSED?
Its overexpression in PCa, suggested that PCA3 mRNA production is regulated by a unique prostate cancer-specific transcriptional mechanism. That could be a promising tool for the treatment of prostate cancer.
Nucleotide sequence analysis did not reveal any obvious promoter elements, but it was demonstrated that there are many TATA -less promoters which may initiate transcription at multiple start sites.
It was shown that the PCA3 promoter activity was restricted to the PCA3-positive cell line LNCaP; PCA3-negative prostatic and non-prostatic cell lines only marginally initiated transcription from the PCA3 promoter. So the PCA3 gene promoter is tissue and cell-type specific and, therefore, is a genuine prostate cancer-specific promoter.
The most interesting result in studying PCA3 promoter seems to be the discover of footprinted regions, such as FP2, that might represent some of the components of the transcription initiation complex (infact mutation of this region, resulted in a decreased transcription rate, suggesting a positive role for this element in PCA3 transcription). Proteins I and Y (HMG-I(Y)) bind FP2 region in the PCA3 promoter; the expression of HMG-I(Y) was shown to be up-regulated in prostate cancer cells and a significant correlation with tumor grade and stage was found. Binding of HMG-I(Y) could activate transcription initiation by altering the chromatin and/or by creating recognition sites for other acting factors.
These data suggest that the up-regulation of transcription in vivo (prostate cancer) requires specific mechanisms to overcome repression, such as inactivation of the repressor by mutation, transcriptional inactivation, binding of an inhibitor, or otherwise. Further studies are required.
Isolation and Characterization of the Promoter of the Human Prostate Cancer-specific DD3 Gene
At present the use of RT-PCR to detect expression of PCA3 in post-prostatic, massage urine is available commercially as a test for prostate cancer:
1. Perform a DRE (3 strokes per lobe) to release a sufficient number of prostate cells into the urine
2. Post-DRE, collect 20-30 mL first-catch urine from the patient
3. Transfer 2.5 mL of urine to the transport tube and send it as soon as possible to the PCA3 testing laboratory.
4. Using transcription-mediated amplification (TMA) technology, PCA3 mRNA molecules are amplified and the PCA3 Score (ratio of PCA3 and PSA mRNA) is calculated.
The ratio is used because the denominator, PSA mRNA, establishes the amount of prostate- specific nuclear material in the specimen. A low level of PCA3 is expressed by normal prostate cells, and if absolute concentration of PCA3 were used, a high Score might be obtained from a specimen rich only in normal prostate cells. Thus, the PCA3 Score tells the expression of PCA3 corrected for the background of normal or BPH epithelial cells present in the specimen.
PCA3: A Genetic Marker of Prostate Cancer
In comparison with serum levels of PSA, the urinary PCA3 score appears to be highly specific for prostate cancer. While serum PSA levels are known to be influenced by volume of BPH tissue, age, inflammation, trauma, and use of 5-alpha reductase (finasteride, dutasteride), preliminary data indicate that these factors do not appear to influence PCA3 scores.
PCA3 Score may provide prognostic information regarding the pathological stage and significance of PCa. In this respect, the PCA3 Score, and specifically its combination with the biopsy Gleason Score and pre-operative PSA level, may aid in treatment decisions.
PCA3 Score before radical prostatectomy predicts extracapsular extension and tumor volume
The PCA3 test performs well in clinical practice, showing a high informative rate and a strong agreement between laboratories as well as a high sensitivity and specificity for detecting prostate cancer. In patients with a high PSA level and prior negative biopsies, the PCA3 test may be useful in choosing between a repeat biopsy and a more conservative follow-up.
PCA3 urine mRNA testing for prostate carcinoma: patterns of use by community urologists and assay performance in reference laboratory setting