Splice variant PRKC-ζ(-PrC) is a novel biomarker of human prostate cancer

Abstract

Background: Previously, using gene-knockdown techniques together with genome expression array analysis, we showed the gene protein Kinase C (PKC)-zeta (PRKCZ) to mediate the malignant phenotype of human prostate cancer. However, according to NCBI, the gene has undergone several major iterations. Therefore, to understand the relationship between its structure and biological activities, we have analysed its expressed sequence in prostate cancer cell lines and tissues.

Methods: Transcriptome-walking and targeted PCR were used to sequence the mRNA transcribed from PRKCZ. Hydropathy analysis was employed to analyse the hypothetical protein sequence subsequently translated and to identify an appropriate epitope to generate a specific monoclonal antibody.

Results: A novel sequence was identified within the 3'-terminal domain of human PRKCZ that, in prostate cancer cell lines and tissues, is expressed during transcription and thereafter translated into protein (designated PKC-ζ(-PrC)) independent of conventional PKC-ζ(-a). The monoclonal antibody detected expression of this 96 kD protein only within malignant prostatic epithelium.

Interpretation: Transcription and translation of this gene sequence, including previous intronic sequences, generates a novel specific biomarker of human prostate cancer. The presence of catalytic domains characteristic of classic PKC-β and atypical PKC-ι within PKC-ζ(-PrC) provides a potential mechanism for this PRKCZ variant to modulate the malignant prostatic phenotype out-with normal cell-regulatory control.

Figure 1

(A) Full exonic sequence of NM variant ‘a’ (NCBI database Build 36, April 2011). Genome exon numbers are in square brackets. Sizes of individual exons (blue boxes) and intervening introns are as shown. (B) Comparative structure of the 3′-terminal region of PRKCZ NCBI database Build 36 (November 2011) between bases #124751 and #136201. Horizontal bars indicate the relative size and location of each exon. Preceding numbers (square brackets and italics) denote the genome number assigned to each exon. Letters above each bar identify the splice variants that include the particular exon. Numbers following each bar specify the length (bp) of each exon. Pink rectangles identify the size and location of individual coding regions. Yellow rectangles denote the location of the gene sequence corresponding to the antigenic site identified by polyclonal antiserum sc-216. The upstream (5′) extension (253 bp) of exon 89 into the adjacent intronic region is identified in red.

Figure 2

(A) Sequence of the antigenic peptide identified by polyclonal antiserum sc-216, provided by Santa Cruz. (B) Back translation of the antigenic peptide to identify its location (bold italics) in the 3′-terminal expressed sequence of exon 98 PRKCZ variant ‘a’ according to the NCBI database (build 36, 2011). Upper-case letters signify expressed sequence, whereas lower case letters are exonic but untranslated. (C) Immunohistochemical detection of PKC-ζ_-a protein in (i) non-neoplastic, (ii) hyperplastic, (iii) prostatic carcinoma (Gleason 3+3) and (iv) prostatic carcinoma (Gleason 5+5). (D) Western blot of PKC-ζ_-a in human prostate cell lines. (E) Western blot data standardised to β-actin expression. (F) PCR analysis of human prostate cell lines confirming expression of PKC-ζ_-a mRNA. (G) Relative levels of PKC-ζ_-a mRNA standardised to β-actin expression.

Figure 3

Consensus sequences derived from three replicates for each specimen obtained by genome walking in a 5′←3′ direction from exon 98 comparing expression sequence data from cell line PC-3M with that from primary carcinomas #4667T and #4327T and their alignment with the published sequence for exon 89 (variant ‘vq’). The sequence originating at nucleotide #223 corresponds to the start of the 5′ UTR of exon 89 with the obtained sequence 1–222 located within the upstream intronic sequence. Data obtained from the other cell lines and prostate cancer specimens revealed similar matching sequences and alignments (data not shown).

Figure 4

PCR amplification of mRNA from PC-3M cells. (A) Schematic diagram of the three contiguous regions, including intronic sequences, identified by genome walking to be expressed in prostate cancer cells. Red and green rectangles identify the positions of forward and reverse PCR primers, respectively (Supplementary Table 4). (B) Control gel confirming absence of contaminating genomic DNA. (C) Region 1: Three products at ∼700, ∼1400 and ∼1600 bp obtained using primer pairs 1A/B spanning exon 87 to the 5′ extension of exon 89 within the intervening intronic sequence. (D) Region 2: Single product at ∼600 bp spanning exons 89–98 including intervening intronic 223 bp. (E) Region 3: Single product of ∼594 bp from primer pair 3A spanning exons 98–104, including intronic 288 bp. A smaller product at ∼554 bp (data not shown) was derived from primer pair 3B.

Figure 5

Expression of genes PRKC-ζ and PRKC-ι in prostate epithelial cells. (A) Real-time PCR analysis of PRKC-ζ variants ‘a’ (white bars) and ‘PrC’ (dark bars) in prostate cell lines using the ΔΔCT method. (B) Expression of variants ‘a’ (white bars) and ‘PrC’ (dark bars) in the parental PC-3M cells and their knockdown derivative si-PRKC-ζ-PC-3 M_T1-6 cells relative to that in PNT-2 cells (C) 1% (w/v) agarose gel electrophoresis of PRKC-ι (top) and β-actin (bottom) in malignant and non-malignant prostate cancer cell lines and tissues Amplification cycle number was × 40 for Real-time PCR or × 35 for RT-PCR. –RT (Reverse transcriptase not included) and –cDNA (template not included) represent negative controls. (D) Expression of PRKC-ι quantified in prostate epithelial cells.

Figure 6

Confirmation of novel sequence expression in prostatic carcinomas. (A) Tissue morphology of primary prostatic tissues (i) Primary prostatic carcinoma (#29110T), (ii) predominantly benign prostatic tissue (#31105T) but containing small clusters of carcinoma cells infiltrating between the benign glands. (B) Amplification of novel sequence using Check 2 primer set (Supplementary Table 4) in PC-3M cells (top) and in three primary prostatic tissues (middle). Abbreviations: B=predominantly benign tissue; T=tumour tissue. Contamination of benign tissue by prostate cancer cells revealed as a weak band in the benign lane (#31105B). (C) Nucleotide sequence of the products from the gels illustrated in panel B aligned with the consensus sequence. Yellow arrows indicate location of the Check 2 primer pair.

Figure 7

Analysis of novel peptide epitope expression. (A) Transcript sequence obtained from the 3′-UTR of exon 98 amplified as part of Region 2 (Figure 4). The sequence encoding the epitope peptide (below) is shown in red. (B) Translation of DNA consensus sequence to a hypothetical peptide. The subsequent immunising epitope sequence used as immunogen selected by hydropathy analysis is indicated in red. (C) Kyte and Doolittle hydropathy analysis of hypothecated peptide indicating (arrows) the hydrophilic immunogenic region. (D) Western blot of human epithelial cell lines using monoclonal antibody 5A6. Cell lines: PNT-2: non-malignant prostate; PC-3M, LNCaP, Du145: malignant prostate; . Beas 2b, COR-L88: malignant lung; HT1197: malignant bladder; PANC: malignant pancreas; MCF-7: malignant breast). (E) Western blot comparing the relative motilities of the bands identified in benign (PNT-2) and malignant (PC-3 M) prostatic epithelial cell lines using monoclonal antibody 5A6 (variant ‘PrC’) and polyclonal antiserum sc-216 (variant ‘a’). (F) Immunohistochemistry of human prostate tissues using monoclonal antibody 5A6: (i) benign epithelium (ii) malignant epithelium.