Functional expression of voltage-gated sodium channel α-subunits (VGSCαs), specifically Nav1.7, is associated with strong metastatic potential in prostate cancer (CaP) in vitro. Furthermore, VGSC activity in vitro directly potentiates processes integral to metastasis. To investigate VGSCα expression in CaP in vivo, immunohistochemistry and real-time PCR were performed on human prostate biopsies (n>20). VGSCα immunostaining was evident in prostatic tissues and markedly stronger in CaP vs non-CaP patients. Importantly, RT-PCRs identified Nav1.7 as the VGSCα most strikingly upregulated (∼20-fold) in CaP, and the resultant receiver-operating characteristics curve demonstrated high diagnostic efficacy for the disease. It is concluded that VGSCα expression increases significantly in CaP in vivo and that Nav1.7 is a potential functional diagnostic marker.
Prostate-specific antigen (PSA), in combination with morphology-based factors such as clinical stage and biopsy Gleason sum, is used most commonly to diagnose prostate cancer (CaP) and monitor disease progression but is of limited efficacy (specificity and sensitivity).1, 2, 3, 4, 5, 6 Several other CaP diagnostic and prognostic markers have recently been discovered and are currently being evaluated as potential adjuncts to existing techniques, including MIB-1/Ki-67 LI, Her-2, EGR-1, bcl-2, DD3.7, 8 Advances in genomic and proteomic high-throughput array strategies promise to identify yet more markers.9 Currently, however, there is no independent technique that can reliably diagnose early-stage CaP and determine tumour aggressiveness. Consequently, there is an urgent need for the identification and evaluation of new genotypic and phenotypic markers to assist early diagnosis and disease prognosis, and thus guide the clinician in choosing the treatment that is most appropriate for each individual case.
In previous studies, we demonstrated a robust positive relationship between voltage-gated sodium channel (VGSC) expression and metastatic potential in both rat and human CaP in vitro.10, 11, 12 Furthermore, VGSC activity could directly enhance metastatic ability by potentiating essential cellular behaviours integral to the metastatic cascade, including process extension,13 adhesion,14 lateral motility,15 directional movement in a small direct-current electric field (galvanotaxis),16, 17 secretory membrane activity18 and transverse invasion.10, 11, 12 A more recent study has shown that overexpression of VGSC alone is sufficient to increase the invasive potential of nonmetastatic and moderately metastatic human CaP cells in vitro.19 These results raised the possibility that VGSC expression could be a novel marker of cancer and metastatic potential in human CaP.
VGSCs consist of two generic subunit types: a single, large (190–260 kDa), heavily glycosylated α-subunit (VGSCα) and a variable number of auxillary β-subunits (VGSCβs). Expression of the VGSCα alone has been demonstrated to be sufficient for functional channel formation,20 while the β-subunits have been attributed with several modulatory roles.21, 22 VGSCαs constitute a multigene family with more than 10 members identified to date.23 Utilizing RT-PCR approaches, we have previously determined the basal expression of multiple VGSCα genes in both strongly and weakly metastatic rat and human CaP cell lines.24 Most importantly, these results showed that expression of the Nav1.7 VGSCα gene product was predominant in strongly metastatic cell lines, at levels approximately three orders of magnitude greater than in the corresponding weakly metastatic cells.24
The objectives of the present in vivo study were two-fold: (i) to verify that VGSCα (mRNA and protein) upregulation occurs in CaP, and (ii) to evaluate the diagnostic potential of Nav1.7 specifically using a quantitative real-time PCR approach.
Materials and methods
For immunohistochemistry, sections of human prostate from 20 different patients (from radical prostatectomies), fixed in 10% formalin and embedded in paraffin, provided by the Royal Liverpool University Hospital, were used. The patients varied in age from 40 to 71 y. Those with diagnosed CaP had Gleason gradings ranging from 4 to 9. For RT-PCR studies, 22 prostate tissue samples, consisting of five benign prostatic hyperplasia (BPH) and 17 CaP (from patients undergoing needle-biopsy or radical prostatectomy for biopsy-proven adenocarcinoma at St Mary's Hospital by a single surgeon) were used. These specimens were snap-frozen in liquid nitrogen in the operating room and stored at −80°C until used for RNA extraction. Patients with BPH or low-grade prostatic intraepithelial neoplasia (PIN) with no evident CaP (following thorough histopathological examination25) were defined as ‘non-CaP’. ‘PIN’ was defined morphologically, and distinguished from carcinoma, according to established criteria.26 All tissues were obtained with local ethics approval and after signature of informed consent forms.
Antigen retrieval was achieved by boiling the sections in freshly made 0.01 M citrate buffer (pH 6) in a microwave oven for 10 min. To enhance the staining, sections were also treated with 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 10 min prior to the inhibition of endogenous peroxidase activity by 3% H2O2/methanol application. Processed sections were incubated with 2 μg/ml rabbit anti-pan VGSCα antibody (Upstate Biotechnology Inc., Dundee, UK) at 4°C overnight. This VGSC antibody recognized the highly conserved D3–D4 intracellular inactivation loop and thus could bind to all VGSCα proteins.27 Sections were then washed and unspecific secondary antibody sites blocked by applying swine serum (DAKO, Ely, UK) for 15 min at room temperature. Signal detection was achieved using biotinylated anti-rabbit swine secondary antibody (DAKO), avidin–biotin complex, avidin–biotin block (Vector, Peterborough, UK) and diaminobenzidine (Vector) as the chromogen. Negative controls in which (i) the primary antibody was omitted or (ii) the primary antibody was preabsorbed with the immunizing peptide were also carried out. In this study, prostatic ducts and glands were not resolved and both were commonly referred to as ‘epithelial structures’ (ESs). The pathological character of each ES was determined by a single pathologist (CSF) following counterstaining of adjacent sections with hemotoxylin.
RNA extraction and conventional (‘fixed end point’) RT-PCR
RNA was extracted using StrataPrep Total RNA Miniprep kits (Stratagene, Cambridge, UK). Thus, up to 0.5 g of frozen tissue was homogenized (using an electric homogenizer) in a solution containing 1200 μl Lysis Buffer and 8.4 μl β-mercaptoethanol. As a final step in the StrataPrep kit protocol, extracted RNA was subjected to a DNase incubation in order to remove contaminating genomic DNA. Approximate RNA quality was rapidly verified visually by gel electrophoresis and spectrophotometrically from 260/280 nm absorbance ratios. In all, 1 μg of total RNA (as determined by spectrophotometric measurement at 260 nm) was then used as the substrate for single-stranded cDNA synthesis (primed with 250 ng random hexamer mix) using Superscript II reverse-transcriptase (Invitrogen, Paisley, UK) for each of the 22 patients. Reactions were performed in a final volume of 20 μl, which was made up to 100 μl with H2O once the reverse-transcription process was completed. PCRs specific for Nav1.2, Nav1.3, Nav1.5, Nav1.6, Nav1.7 and Nav1.9, which we have recently shown to be the major VGSCα types expressed in CaP cells in vitro,24 were undertaken on 12 biopsy samples (six non-CaP and six CaP).
β-Actin was also amplified in order to control for variables including sample-to-sample differences in RNA input, RNA quality and reverse-transcription efficiency that might contribute to apparent intersample VGSCα expression differences. β-Actin has been used widely as a control gene in real-time PCR studies on CaP samples, with its mRNA expression level consistently found to be highly stable during CaP tumorigenesis and progression.28, 29, 30 Additionally, preliminary experiments we performed on four different human transformed prostate epithelial cell lines (PNT2-C2, LNCaP, PC-3 and PC-3 M) of varying metastatic potential indicated that β-actin expression in in vitro models of CaP progression was indeed much less variable than that of commonly used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hydroxymethylbilane synthase (HMBS) controls (data not shown). GAPDH expression has previously been shown to alter markedly with disease state.31, 32
The sequences of the β-actin primer pair used were: IndexTerm5′-AGCCTCGCCTTTGCCGA-3′; IndexTerm5′-CTGGTGCCTGGGGCG-3′; the annealing temperature was 67°C. This primer pair has previously been shown to yield products of the correct size only from mRNA since the primers span intron positions and do not amplify associated β-actin pseudogenes.33. VGSCα primer pairs and conditions were as previously described;24 reactions were performed on 5 μl of cDNA template using Taq DNA polymerase (Amersham Pharmacia, Little Chalfont, UK) with a manual hot-start technique. Duplicate reactions for each VGSCα were carried out simultaneously for different fixed numbers of cycles (eg 30 and 35 cycles for Nav1.7) in order to permit more effective comparison of relative expression levels in all samples (by gaining some measure of the kinetics of reaction product accumulation). In all, 5 μl of each PCR reaction was run on agarose gels and band intensities of products derived from CaP and non-CaP samples were compared as an approximate measure of relative mRNA expression levels.
Real-time PCR and data analysis
Real-time PCR utilizing SYBR I Green technology was performed on cDNA derived from all 22 patient tissue samples using the DNA Engine Opticon system (MJ Research) to more accurately determine Nav1.7 expression levels. β-Actin was also measured in each sample as the control/reference gene and used to normalize the respective measured Nav1.7 expression. PCR reactions were performed in a final volume of 20 μl, containing 5 μl cDNA, 500 nM of each specific primer and 1 × QuantiTect SYBR Green PCR mix (Qiagen, Crawley, UK), according to the manufacturer's instructions. Amplification was via an initial denaturation at 95°C for 15 min to activate the HotStar Taq, with subsequent three-step cycling of 95°C for 30 s, 59 or 67°C (depending on the primer pair) for 30 s and 72°C for 30 s. A final melt curve was also carried out from 65 to 95°C with 0.3°C steps in order to verify product composition. Triplicate reactions on each sample cDNA were carried out simultaneously for Nav1.7 and β-actin. Each set of reactions also included a standard calibration curve for each target, using six serial dilutions of a PC-3 CaP cell line cDNA template (covering a four orders of magnitude range). In each PCR run, blank reactions without added cDNA were also performed in order to control for cross-contamination from other sources.
The number of threshold amplification cycles (CNt) representing the first discernable amplification cycle above which product fluorescence was greater than background noise was determined for each reaction using the Opticon software. The values of CNt were then analysed by two different methods: the standard curve fit and the 2−ΔΔCt method34 to determine target expression levels, which were expressed as mean percentages (relative to the level in the PC-3 cell line) ± standard errors. Subsequently, the Nav1.7 expression in each sample was normalized relative to the corresponding sample-matched expression level of β-actin. Differences in Nav1.7 expression levels between (i) non-CaP and CaP, and (ii) low-grade and higher-grade CaP (corresponding to Gleason scores of ⩾6 and <6, respectively) were determined by testing normalized medians for statistical significance with the nonparametric Mann–Whitney U-test and the Kruskal–Wallis test (for differences between non-CaP, low- and high-grade tumours).7 To visualize the efficacy of Nav1.7 to discriminate non-CaP from CaP tissue, the data were also summarized as a receiver-operating characteristics (ROC) curve, generated using the Statistical Package for Social Sciences (SPSS). When a test has a strong discriminative value, the ROC curve is in the upper left-hand corner of the plot; if the marker is not discriminative the curve lies close to the diagonal.7
Localization of VGSCα protein in human prostatic tissue sections with a range of pathological character was studied in order to verify VGSCα upregulation with CaP onset in vivo, as previously determined in vitro.24 In non-CaP ESs (BPH and low-grade PIN), the overall level of VGSCα staining was low (Figure 1a and b). VGSCα staining was significantly increased in ESs of high-grade PIN (the earliest discernable carcinoma precursor) and adenocarcinoma (Figure 1c and d). However, the most marked increase in VGSCα protein expression accompanied the transition from low- to high-grade PIN (Figure 1a vs c). There was some heterogeneity in the staining within the ESs and some staining was noted outside the ES regions; however, these aspects were not studied further. There was no staining at all in the control sections, stained with preabsorbed primary antibody or with the secondary antibody alone (data not shown). Thus, immunohistochemistry indicated that VGSCα expression was increased in CaP in vivo, as previously determined in vitro.24
Conventional (fixed end point) RT-PCR was used for an initial screen of VGSCα subtype(s) upregulated in CaP. Results for Nav1.2, Nav1.3, Nav1.5, Nav1.6, Nav1.7 and Nav1.9 (previously found in vitro24) from six non-CaP and six CaP patient samples are shown in Figure 2. All these VGSCαs were detected in all biopsies following an extensive number of amplification cycles. Two splice forms of Nav1.2, Nav1.3, Nav1.5 and Nav1.6 were detected in most samples and in each case could be identified by size as the full-length and exon-skipped isoforms (Figure 2b–e), previously also described in rat and human-derived CaP cell lines.24 Partial kinetic analysis of product amplification (by performing each reaction for two different amplification cycle numbers) permitted an approximation of the relative level of expression of each VGSCα in each patient sample to be made, since β-actin expression levels were very similar (Figure 2g). Increased VGSCα product intensity in CaP compared to non-CaP patient samples was most evident for Nav1.7 RT-PCRs (Figure 2a), as in strongly metastatic human CaP cells in vitro.24 Thus, Nav1.7 was a candidate VGSCα upregulated in CaP and was therefore subjected to more accurate measurement of sample-based mRNA expression levels in each sample using the real-time PCR technique.
Nav1.7 expression levels (normalized with respective sample-matched β-actin, relative to PC-3 levels) ranged from 2.2 to 1093.3, and were higher in CaP tissues (median=132.5; range=2.6–1093.2; mean=234.2±68.2; n=17) compared to non-CaP samples (median=6.7; range=2.2–27.2; mean=11.6±4.5; n=5). Median differences were statistically significant (Mann–Whitney U-test, P<0.01) and corresponded to a 19.8-fold higher expression in CaP compared with non-CaP (Figure 3a). Normalized Nav1.7 expression was also analysed within the CaP samples in relation to Gleason score divided into high (sum ⩾6) and low (sum <6) grades (Figure 3b). This analysis revealed that Nav1.7 expression was greater in high-grade CaP with the median 3.6-fold (63.6 vs 228.3) and mean 2.3-fold (138±62.4 vs 319.7±112.3) higher than low grade. These differences were also significant (Kruskal–Wallis test, P<0.05). The 2−ΔΔCt method of analysis, also performed on the real-time PCR data to generate normalized Nav1.7 expression levels, led essentially to the same statistical conclusions as regards upregulation of Nav1.7 by (i) 19.1-fold (Mann–Whitney U-test, P<0.01) in CaP (median, 120.4) vs non-CaP (median, 6.3) and (ii) 3.8-fold (Kruskal–Wallis test, P<0.05) in higher Gleason grade (median, 193.8) vs low-grade CaP (median, 50.6).
All CaP patients in the present study were surgically treated and did not subsequently develop evident metastases in the 1-y follow-up period. Therefore, Nav1.7 expression could not be correlated with patient relapse and metastatic disease. Serum PSA data at diagnosis (just before surgical intervention) were available for 14 CaP patients (six low-grade CaP, eight higher-grade CaP), with levels ranging from 2.1 to 5000 ng/ml, but (i) were not significantly different in low- vs high-grade CaP (median, 3.1 vs 6.9, P>0.33), and (ii) were not correlated with Nav1.7 expression (Spearman's rank correlation coefficient, R=−0.199, P>0.50).
An ROC curve was constructed, representing the diagnostic efficacy of Nav1.7 to distinguish CaP from non-CaP tissue (Figure 3c). The area under the curve was 0.94 (95% confidence interval, 0.84–1.04), indicating good discriminating power. This analysis also suggested a threshold Nav1.7 mRNA expression value (normalized to β-actin) of 30.1% of the level in PC-3 cells (≡1.48 log unit difference; Figure 3a), clearly distinguishing CaP from non-CaP in the patient samples studied (n=22).
The present study extends the concept of VGSCα upregulation with increased CaP metastatic potential, which we have previously developed in vitro, to disease onset and progression in ex vivo patient tissues.
We found a marked upregulation of VGSCα protein levels in prostatic epithelial cells at the transition from low-grade to high-grade PIN, presently the earliest morphologically discernable disease stage used as a marker for prostatic adenocarcinoma.35 This is consistent with our RT-PCR studies, which independently showed that VGSCα (Nav1.7) mRNA expression was strongly (∼20-fold) upregulated in CaP compared with non-CaP prostate tissues. Since VGSCα protein levels increased in epithelial structures with CaP development and progression, upregulated Nav1.7 expression is likely to be localized to these epithelial cancer cells. VGSCα protein expression in normal and tumour prostatic tissues was also studied using multitumour tissue arrays.36 In that study, the majority of tissue samples taken from CaP patients also displayed greater levels of VGSCα proteins than ‘normal’ tissues, in agreement with our present in vivo and earlier in vitro findings. In a separate study, it was shown that voltage-gated K+ (Kv1.3) channel (VGPC) expression changed in the opposite direction, being reduced in CaP compared with normal tissue.37 Taking the available evidence together, it would seem that progression to CaP is accompanied by upregulation of VGSC and concomitant downregulation of VGPC expression, as shown earlier to occur in vitro.10, 11, 38 This is consistent with the membranes of metastatic CaP cells being ‘excitable’ in line with the cells’ hyperactive behaviour.39
For the first time, we have investigated prostatic VGSCα subtype-specific expression in vivo, using conventional (fixed end point) and real-time PCR techniques and identified Nav1.7 as the VGSCα most likely to account for this increase in VGSC protein, consistent with our earlier in vitro model findings.24 It is not yet known why this particular VGSCα specifically is upregulated in CaP, although there is precedent for its expression to be specifically elevated to high levels in another carcinoma type.40 Functional characteristics of Nav1.7 such as novel electrophysiology, for example, ramp current production;41 subcellular localization, for example, at the tips of cellular processes42 and regulation by growth factors, including those that may be involved in CaP (eg epidermal growth factor, nerve growth factor) may relate to its potentiating role.43, 44
The functional role of the ‘minor’ VGSCαs present in the samples is not clear at present. Interestingly, Bennett et al19 have stated that transfection of a normally nondominant VGSCα (Nav1.4) into the weakly metastatic LNCaP cells would greatly increase their invasive capacity, implying that different VGSCαs could subserve the enhanced metastatic potential, perhaps under different conditions.
VGSC activity could contribute to CaP metastasis in several different ways, as follows: (i) Secretion: VGSC activity has been shown to modulate (enhance) both the quantity18 and quality/pattern45 of secretory membrane activity. Interestingly, release of both PSA and interleukin-6 was suppressed by VGSC-blocker anticonvulsants in vitro.46 Other possible secretagogues include proteolytic enzymes and growth factors.1, 47 (ii) Process extension. VGSC activity was shown earlier to enhance the morphological characteristics, including process extension, specifically of strongly metastatic Mat-LyLu rat CaP cells.13 (iii) Motility and adhesion. Directional motility,15 including galvanotaxis,16 was found to be enhanced by VGSC activity, which, also, modulated adhesion.14 (iv) Invasion. CaP cell transverse (‘Matrigel’) invasiveness was strongly potentiated by VGSC activity.10, 11, 12 Indeed, Bennett et al19 have stated that VGSC expression is ‘necessary and sufficient’ for the invasiveness of CaP cells.19 The underlying molecular mechanisms through which VGSC expression could potentiate these cellular behaviours may involve the cytoskeleton (directly via VGSCβ interaction and/or indirectly via local Ca2+ fluxes), protein kinase activity and gene expression.
VGSCα/Nav1.7 expression would fulfil many of the properties that would be required of a readily utilizable, reliable marker for CaP, and could offer several advantages, as follows, over other potential markers: (i) The ROC analysis suggested that Nav1.7 is a highly sensitive and specific diagnostic marker. The threshold mRNA expression determined would clearly distinguish CaP from non-CaP and could ultimately form the basis of a clinical test. (ii) Nav1.7 is functionally involved in metastatic cell behaviour, and hence, it is also of therapeutic value. Indeed, VGSC blockers have recently been used to inhibit hormone-insensitive CaP.46, 48, 49, 50 (iii) This functional role is consistent with Nav1.7 upregulation being an early event in disease progression. (iv) The contributions of the VGSC activity to metastatic cell behaviour is such as to account for a significant part of the difference between slow and fast progressing tumours—a major problem in CaP management. These characteristics, taken together, suggest strongly that Nav1.7 could be an efficient predictor (as well as a blocker) of CaP onset and progression.
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This study was supported by PCaSO (Prostate Cancer Network) and the Pro-Cancer Research Fund (PCRF). We thank Dr Graham Wilkin for use of OPTIMAS 5.2 image analysis system and Professor Norman Maitland (supported by Yorkshire Cancer Research) for the PNT2-C2 cell line.
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Diss, J., Stewart, D., Pani, F. et al. A potential novel marker for human prostate cancer: voltage-gated sodium channel expression in vivo. Prostate Cancer Prostatic Dis 8, 266–273 (2005). https://doi.org/10.1038/sj.pcan.4500796
- real-time PCR
- receiver-operating characteristics curve
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