Prostate Cancer Surface Antigenicity and the Interferon Induction Hypothesis in LNCaP Cells Treated with Antisense Oligonucleotides

Antisense oligonucleotides (oligos) have been employed in both in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. Genes targeted include protein growth factors, androgens, receptors for stimulating factors, inhibitors of apoptosis (bcl-2 and clusterin) and oncogenes. Some (developed by Oncogenex Pharmaceuticals) have entered clinical trials (OGX-011) while others are in preclinical development (OGX225). [1] Oligos act through a variety of mechanisms and provide a specific and relatively non-toxic method for translational arrest by degradation of annealed mRNA: oligo duplexes by RNase H [2], protein binding and DNA triplex formation [3,4].


Introduction
Antisense oligonucleotides (oligos) have been employed in both in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines.Genes targeted include protein growth factors, androgens, receptors for stimulating factors, inhibitors of apoptosis (bcl-2 and clusterin) and oncogenes.Some (developed by Oncogenex Pharmaceuticals) have entered clinical trials (OGX-011) while others are in preclinical development (OGX-225). [1]Oligos act through a variety of mechanisms and provide a specific and relatively non-toxic method for translational arrest by degradation of annealed mRNA: oligo duplexes by RNase H [2] , protein binding and DNA triplex formation [3,4] .
Our laboratory has attempted to increase the efficacy of oligos by evaluating bispecific derivatives [5][6][7][8][9][10][11] which contain more than one binding site on a single DNA strand.Bispecifics, have been evaluated against genes involved in both a single autocrine loop [5] as well as those found in different growth regulatory pathways . [6,9,10,11]Initial studies measured inhibition of in vitro growth, but more direct methods use reverse transcriptase-polymerase chain reaction (RT-PCR) to measure suppression of gene specific mRNA.
In LNCaP cells we find that monospecific oligos targeting bcl-2 and bispecifics targeting both bcl-2 and the epidermal growth factor receptor (EGFR) have comparable activities in suppressing bcl-2 expression as determined by both in vitro growth and bcl-2 expression measured by RT-PCR. [11]Therefore, the presence of a second binding site did not diminish activity of the other.This finding is significant because it's naive to believe that cancer cell growth can be controlled by oligos through knockdown of a single gene product.Instead multiple genes must be down regulated, and it would be useful if more than one therapeutic activity could be combined onto a single entity.
In these experiments we evaluated the effect of bispecific oligos directed against bcl-2 and EGFR employing RT-PCR, by measuring the expression of four prostate specific gene products

Abstract
Antisense oligonucleotides (oligos) have been administered against prostatic LNCaP cells in both in vivo and in vitro models.While most oligos consist of a single mRNA binding site targeting a single gene product, or others having sequence homology, we evaluate bispecifics directed towards two unrelated proteins.In LNCaP cells we identified bispecifics which increase the expression of prostate specific membrane antigen (PSMA) while not affecting secreted prostate specific antigen (PSA) or prostatic acid phosphatase (PAP).We then postulated that surface antigen expression is increased by bispecifics able to induce interferon (IFN-γ) through complementary intrastrand regions.To further test this hypothesis we measured the effect of oligo treatment on another secreted product, prostatic cancer antigen-3 (PCA-3).
We initially evaluated the inhibition of in vitro propagating LNCaP cells employing mono-and bispecific oligos directed against bcl-2 [the second bispecific binding site was against the epidermal growth factor receptor (EGFR)].These oligos were administered with lipofectin in a nanoparticle delivery system.Employing RT-PCR, the expression of non-targeted genes products encoding PSMA, PSA, PAP, PCA-3 and IFN-γ have now been evaluated.When LNCaP prostate tumor cells were incubated with oligos and compared to lipofectin containing controls significant growth inhibition resulted.
Employing RT-PCR, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with a monospecific directed solely against bcl-2.No increased expression in PSA, PAP or PCA-3 following treatment with either oligo type were detected.Like PSMA, IFN-γ was significantly induced only by bispecific oligos.These data support the hypothesis that double strand forming bispecific oligos induce IFN-γ, enhancing cell surface antigen expression (PSMA) but not secretory products.Expression of tumor associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.not directly targeted, as well as the hypothetical role of induced IFN-γ in their expression.These (three) differentiation antigens and (non-protein) PCA-3 are surrogates for tumor growth and regression, and as biochemical markers they are early indicators for tumor recurrence (biochemical relapse).The bispecifics administered have a unique base sequence which computer models suggest could lead to intrastand binding Figure 1.Double stranded nucleic acids (poly I:C) are known to induce IFN-γ which increases the expression of some cell surface antigens (HLA antigens and receptors for tumor necrosis factor).Any increase in antigen encoding mRNA could be a rather unique result of (the hypothesized) IFN-γ induction produced by the unusual secondary structure of these oligos and support an additional theory that the prostate retains an interferon based antiviral defense system. [12]MA is a trans membrane bound receptor with folate hydrolase activity.In contrast, prostate specific antigen (PSA) is a secreted kallikrein related peptidase marker associated with prostate cancer recurrence and progression.It's often employed as a screening tool and has been evaluated as a target for directed therapy.PAP was the original serum marker for prostate cancer, distinguishable from other types of acid phosphatase by formalin resistance.PSMA and PAP are considered targets for an activated immune system and have been included in prostate cancer vaccines.ALPHAVAX ARV developed by the consortium of Cytogen and Progenics targets PSMA, and Provenge developed by Dendrion immunizes against PAP.Administration of therapy which inhibits growth while enhancing surface antigenicity would be of interest, as the field of immunotherapy develops.PCA-3 is being evaluated as a more specific marker for prostate cancer.Also known as DD3, this marker is a non-coding RNA which when measured in urine is considered less sensitive, but more specific than PSA when managing disease.Enhanced expression by any surface protein would presumably aid cytotoxic (CD8+) T cell targeting and increase (anti-PSA) antibodies as reported by Lubaroff. [13]sed on their secondary structure, these oligos have the capacity to form double stranded nucleic acids, and could act as an IFN-γ inducer in a similar manner as poly I:C.The enhanced expression of HLA and tumor necrosis factor receptors following IFN-γ exposure has long been recognized as a promoting factor for immune mediated cytotoxicity. [14,15]In these experiments we evaluated whether bispecific oligos MR 24 and MR 42 alter the expression of a more recently identified and studied prostate specific gene product (PCA-3) and determine whether and its expression pattern further supports the IFN-γ induction hypothesis.

Oligonucleotides
Oligos (mono-or bispecific) were purchased from Eurofins MWG Operon (Huntsville, AL).Each was phosphorothioated on three terminal bases at 5' and 3' positions.Stock solutions were made to a final concentration of 625 μM in sterile Dulbecco PBS.

Base Sequences
Each oligo contained at least one CAT sequence and targeted the area adjacent to the mRNA AUG initiation codon for the respective targeted protein (EGFR or bcl-2).

Cell Culture
LNCaP cells were grown in RPMI 1640 supplemented with 10% bovine serum, 1% L-glutamine and 1% penicillin/ streptomycin in a 5% CO 2 incubator.Log phase cells were harvested using EDTA/Trypsin and equally distributed into 75 cm 2 flasks (Corning, NY).At intervals media was either supplemented or replaced with fresh media.

Determination of Growth
Four days prior to the addition of oligos 1 X 10 4 LNCaP cells were added, in a total 200 μl volume of media, to each depression of a 96 well plate and incubated at 37 °C in a 5% CO 2 incubator.On the day of transfection the following solutions were prepared: A) 1 μl of buffer containing either oligo or a diluent was added to 50 μl of OPTI-MEM and gently mixed.One dilution was made for each well.Cells were incubated for 24-48 hrs before solutions were aspirated and re-incubated for an additional 48 hrs in 200 μl of media.Cell counts were determined following the addition of WST-1 reagent to each well, and after 2 hrs the color intensity was measured by a micro-plate reader at a wavelength of 450 nm, using a reference of 650 nm.Values obtained were determined after the subtraction of paired blank samples from the experimental wells and were multiplied by a constant to give whole integers for analysis.Microsoft Excel software was utilized to calculate means and standard deviations and Students t tests were used to determine significance.

Oligo Treatment Prior to PCR
Fours days prior to oligo addition, when cell density approached 75% confluence, 10 ml of fresh media was added.Cells were incubated for an additional 3 days before 5 ml of media was replaced with fresh the day before oligos were added.100 μl of stock oligos were added to bring the final concentration to 6.25 μM.Incubation proceeded for an additional 24 hours in the presence or absence of monospecific MR 4 , or the MR 24 and MR 42 bispecifics.

RNA Extraction
Following treatment, media was removed, a single ml of cold (4 °C) RNAzol B was added to each 75 cm 2 culture flask and the monolayer lysed by repeated passage through a pipette.All procedures were performed at 4 °C.The lysate was removed, placed in a centrifuge tube to which 0.2 ml of chloroform was added, and shaken.The mixture stayed on ice for 5 min, was spun at 12,000 g for 15 min, and the upper aqueous volume removed and placed in a fresh tube.An equal volume of isopropanol was added, the tube shaken, and allowed to stay at 4 °C for 15 min before similar centrifugation to pellet the RNA.The supernatant was removed, the pellet washed in a single ml of 75% ethanol, then spun for 8 min at 7500 g.The ethanol was pipetted off and the formed pellet air dried at -20 °C.

RNA Quantitation
RNA was resuspended in 250 μl of DEPC treated H 2 O, and quantitated using a Qubit fluorometer and Quant-iT RNA assay kit (invitrogen).DEPC is an inhibitor of RNase activity.

RT-PCR
Extracted RNA was diluted to 40 μg/μl in DEPC treated water.1-4 μl of this RNA was added to1 μl of both sense and antisense primers (forward and reverse sequences) for human actin (used as a control), PSMA, PSA, IFN-γ and PAP.From a kit purchased from invitrogen the following reactants were added for RT-PCR: 25 μl of 2X reaction mixture, 2 μl SuperScript III RT / platinum Taq mix, tracking dye, and MgSO 4 (3 μl of a stock concentration of 5mM, used for bcl-2 vials only).DEPC treated water was added to yield a final volume of 50 μl.As a control for RT-PCR product production, human actin expression was tested in RNA extracted from HeLa cells which was provided in a kit purchased from invitrogen.RT-PCR was performed for 2 X 25 cycles using the F54 program in a Sprint PCR Thermocycler.

Detection and Quantitation of Product
Agarose Gel Electrophoresis: 1.5% agarose gels were prepared in a 50 ml volume of TBE buffer (1X solution: 0.089 M Tris borate and 0.002M EDTA, P H 8.3), containing 3 μl of ethidium bromide in a Fisher Biotest electrophoresis system.Samples were run for 2 hours at a constant voltage of 70 using a BioRad 1000/500 power supply source.To locate the amplified PCR product, 3 μl of a molecular marker (invitrogen) which contained a sequence of bases in 100 base pair increments (invitrogen) as well as 2 μl of a sucrose based bromophenol blue tracking dye were run in each gel.For actin product localization, the tracking dye was included in each sample run; for all others the tracking dye was run separately.
Quantitation: Gels were visualized under UV light and photographed using a Canon 800 digital camera.Photos were converted to black and white format and bands quantitated using Mipav software provided by NIH.

Prostate Cancer Surface Antigenicity and the Interferon Induction Hypothesis in LNCaP Cells Treated with Antisense Oligonucleotides
Copyright: © 2014 Rubenstein et al.

Cell Culture Experiments
LNCaP cells were incubated with MR 4 , MR 24 and MR 42 and compared to lipofectin containing controls Figure 2. In an initial experiment each oligo significantly inhibited the growth of LNCaP cells: MR 4 by 23.8% (p = 0.0004); MR 24 by 31.2% (p < 0.001); and MR 42 by 31.7% (p < 0.001).
In a repeat experiment LNCaP cells were similarly incubated and compared to lipofectin containing controls.Bispecific oligos MR 24, and MR 42 produced significant respective inhibitions of 49.5% (p < 0.001) and 56.8% (p < 0.001), and were at least as effective as the monospecific MR 4 directed only towards bcl-2 in the inhibition of in vitro cell growth.

RT-PCR Experiments
Comparable amounts of extracted RNA from LNCaP cells treated with either mono-or bispecific oligos directed against bcl-2 (and EGFR in the bispecifics).In a series of control experiments (data not shown) to validate RNA extraction and RT-PCR procedures, the expression of human actin in HeLa cells was identified. [11]This RNA was then evaluated by RT-PCR using primers directed against PSMA, PSA, PAP, PCA-3 and IFN-γ.Product was run on agarose gels and digitally photographed.The identified product bands were cropped and scanned with Mipav software.Representative bands for PSMA, PSA, PAP, PCA-3 and IFN-γ are presented in Figures 3-7.

PSMA
When background intensity was subtracted, the intensity of the bands containing PSMA product (Table 1) were 19.3% ± 10.5, 20.9% ± 14.7, 33.7% ± 11.4 and 33.0% ± 10.8.The amount of PSMA product was significantly enhanced by the MR 24 (EGFR/ bcl-2) and MR 42 (bcl-2/EGFR) bispecifics (p = 0.03 and p = 0.02 respectively).These results were pooled from both duplicate PCR runs and gels.In contrast, no difference was seen in cells treated with the MR 4 monospecific oligo.A representative band is seen on agarose gel in Figure 3.

PSA
When background intensity was subtracted, the intensity of bands containing PSA product Table 1 were 60.9% ± 11.2, 60.4% ± 8.1,69.6%± 4.6 and 66.1% ± 7.3 respectively for the untreated cells and those treated with monospecific MR 4 and bispecific MR 24 and MR 42 .Treated cells were not significantly different from the untreated controls.A representative band is seen on agarose gel in Figure 4.

PAP
The intensity of bands containing PAP product, when compared to controls Table 1, were decreased 7.58% ± 10.6 (NS), 3.96% ± 23.1 and 17.27% ± 15.9 (p = 0.042) respectively for those treated with monospecific MR 4 and bispecifics MR 24 and MR 42 .Expression of secretory PAP was therefore similar to PSA (not increased) compared to surface antigen PSMA.In cells treated with MR 42 the decrease may be significant.This supports the  PSA expression in LNCaP cells treated with mono and bispecific oligos directed against BCL-2 and EGFR hypothesis that double stranded oligos can act as an interferon inducer and enhance surface antigen expression which could have immunological significance if it highlights transformed cells for attack by cytotoxic T cells.A representative band is seen on agarose gel in Figure 5.

PCA-3
The intensity of bands containing PCA-3 product, when compared to controls Table 1, were not significantly altered 55.7% ± 61.3 (NS), 51.2% ± 106.5 and 68.7% ± 104.0 (NS) respectively for those treated with monospecific MR 4 and bispecifics MR 24 and MR 42 .Expression of secretory PCA-3 was not increased compared to surface antigen PSMA and therefore its expression pattern resembles PSA and PAP with no increase Table 1, further supporting the IFN-γ induction hypothesis.A representative band is seen on agarose gel in Figure 6.

IFN-γ
When background intensity was subtracted, cells treated with the bispecifics had significantly greater IFN-γ expression (p < 0.05) than the untreated controls and bands were visually more intense than those seen in the monospecific treated group.The intensity of bands containing IFN-γ product, when compared to controls, were increased 3.12% ± 10.6 (NS), 28.5% ± 23.1 (p = 0.025) and 19.2% ± 15.9 p < 0.05) respectively for those treated with monospecific MR 4 and bispecifics MR 24 and MR 42 .This supports the hypothesis that double stranded oligos can act as an interferon inducer.A representative band is seen on agarose gel in Figure 7.

Discussion
The hypothesis that double stranded oligos could act as an IFN-γ inducer was first proposed following the discovery that PSMA and IFN-γ were enhanced following treatment of LNCaP cells with bispecific oligos having the capability of forming double stranded regions.The lack of similar enhancement in subsequent experiments evaluating PSA and PAP supported this hypothesis.In this study we evaluated yet another secretory product (nonprotein PCA-3) and found further support for this theory.
Gene therapy is a complex process which requires both hormone and protein stimulated as well as the apoptotic pathway (with all their regulatory gene products) to be simultaneously regulated.For over expressed gene products there are methods to suppress their translation several of which have been clinically evaluated; for those which are diminished or lacking, methods and (viral) vehicles for their insertion must be more safely developed.Gene therapy can also be improved when combined with chemo-or immunotherapy.Translational arrest can be accomplished by antisense strategies and a cocktail of antisense oligos, could theoretically shut down many of the overexpressed genes.Rather than employ a pool of separate oligos, each targeting a different gene [16], it would be desirable if several activities could be regulated by bispecific [5] or proposed multispecific oligos [17].While some investigators refer to "bispecific" oligos targeting genes which share a region of sequence homology and have similar biologic activity [18,19], what we define as bispecific oligos are those which target more than one protein (unrelated in sequence), capable of binding two protein encoding mRNAs from (even) different regulatory pathways. [20] have now evaluated the effect of oligo mediated growth suppression on four seemingly unrelated markers of prostatic tumor progression (PSMA, PSA, PAP, PCA-3) and one induced cytokine (IFN-γ).Although LNCaP cell growth is inhibited by bispecific oligos, it appears that when activity is normalized for the amount of extracted RNA, PSA, PAP and PCA-3 expression does not increase.In contrast, the expression of PSMA and IFN-γ is enhanced by the bispecifics (targeting EGFR and bcl-2) and unchanged from controls when treated with the monospecific (targeting only bcl-2).Enhanced PSMA expression may be due to IFN-γ induction by the uniquely configured bispecific oligos and its effect on cell surface antigen expression.The recent determination that PCA-3 also remains unchanged further supports this hypothesis.Double stranded nucleic acids are known inducers of IFN-γ, similar to poly I:C and we have shown this cytokine to be induced in LNCaP cells by the bispecific oligos tested here.IFN-γ promotes the expression of HLA antigens [14] (often diminished on tumor cells) as well as increasing the number of tumor necrosis factor receptors [15], making both targets for immune destruction by cytotoxic (CD8+) T cells.When

Prostate Cancer Surface Antigenicity and the Interferon Induction Hypothesis in LNCaP
induced, PSMA expression on prostate tumor cells could similarly enhance tumor recognition.Therefore, in addition to a bispecific oligo's potential for growth inhibition, its effect on enhanced antigenic expression could also be clinically (immunologically) significant, particularly if combined with vaccines like Provenge.
The development of ALPHAVAX ARV and Provenge, directed respectively against PSMA and PAP, would suggest the need to evaluate synergism between immune potentiating vaccines and specifically directed antisense oligos containing double stranded regions.This would appear particularly likely for ALPHAVAX ARV which targets membrane bound PSMA, and which has been deemed suitable for monoclonal targeting by oligos [21].
We predict that for antisense therapy to take the next step forward, more complex forms of antisense must be formulated and delivery mechanisms improved.Multichain (and fat soluble) oligos have already been proposed [17] which could be constructed specifically to target specific gene combinations uniquely expressed or activated in individual patients or tumor types, based on microarray analysis and expression patterns [22].Furthermore, mechanisms for enhanced delivery and stability, some of which employ polymalic acid [23] or nanoparticle formation with polypropylin-imine dendrimeres [24], have been developed.The combination of (oligo based) gene therapy, enhanced delivery and immune potentiation could provide an additional tier of therapy to biochemically relapsed prostate cancer patients.

B) 1
μl of Lipofectin was diluted in 50 μl of OPTI-MEM and mixed gently for 5 minutes at room temperature.C) Oligo dilutions were mixed with 50 μl of Lipofectin and gently mixed for 20 minutes at room temperature.

Figure 1 :
Figure 1: Suggested complementary binding conformation of MR 24 and MR 42.
μl of the Lipofectin and oligos were added to 100 μl of RPMI medium and mixed.

Figure 2 :
Figure 2: Inhibition of in vitro growth of LNCaP cells by mono-and bispecific oligos.

Figure 5 :Figure 6 :Figure 7 :
Figure 5:The intensity of bands containing PAP product, when compared to controls were decreased 7.58% ± 10.6, 3.96% ± 23.1 and 17.27% ± 15.9 (p = 0.042) respectively for those treated with monospecific MR4 and bispe cifics MR24 and MR42.Expression of secretory PAP was therefore similar to PSA (not increased) compared to surface antigen PSMA.PAP expression in LNCap cells treated with mono and bispecific oligos treated against BCL-2 and EGFR