Abstract
Antisense oligonucleotides have previously been used to target regulatory proteins in prostate cancer models. We evaluated mono- and bispecific oligonucleotides which comparably suppressed expression of B-cell lymphoma-2 (BCL-2) in LNCaP cells. Cells compensated by suppressing caspase-3 (an apoptosis promoter), and enhancing the expression of androgen receptor and co-activating p300 and interleukin-6 (IL-6) proteins. This suggests that increased androgen sensitivity accompanies BCL-2 suppression with a pattern associated with more advanced tumors. To further evaluate tumor resistance and compensatory mechanisms we evaluated the stem cell-associated CD44 expression and found it to be unaffected by treatment, suggesting that this tumor population is not activated or expanded by suppressive BCL-2 therapy.
Gene therapy is in theory specific, but tumors can alter their dependence on targeted genes by relying upon others through compensation (1). Although targets are found in many pathways, and tumors exhibit altered gene expression patterns, the activity of most growth-regulatory genes are similar to the normal condition. Resistance develops because biochemical pathways are complex, regulated by stimulatory and inhibitory factors, each affected by therapy. Bacteria and viruses mutate to evade antimicrobial agents; tumor cells are similarly selected to evade chemotherapy, and the unintended consequences of intervention are poorly understood.
Gene therapy has been clinically applied to treat human prostatic tumors and together with radio- (2, 3) or chemotherapy (4) antisense oligonucleotides (oligos) have been administered to act against inhibitors of apoptosis [e.g. B-cell lymphoma-2 (BCL-2) and clusterin] in attempts to restore that process. If such therapy is to be successful, it is important to examine mechanisms by which tumors compensate and become resistant. We previously reported, in LNCaP cells, that inhibition of BCL-2 with antisense oligos suppressed the apoptotic promoter caspase-3 (1) and enhanced expression of the androgen receptor (AR) (5), p300 (6), interleukin-6 (IL-6) (7) and viral myelocytomatosis (v-MYC) (8) proteins. This suggests that following BCL-2-suppressive therapy, there could be selective pressure for a more aggressive (androgen-sensitive) phenotype. Unexpectedly altered gene expression also affects proteins not associated with growth. In an earlier study, we reported on enhanced expression of prostate-specific membrane antigen (PSMA) (9) by bispecific oligos directed against BCL-2. The unique capacity of these bispecifics to produce such changes is, unlike compensation, attributed to an unusual double strand conformation and induction of interferon (an enhancer of surface antigen expression) (10). Such expression could enable better recognition and targeting by cytotoxic-T cells (10).
Previous studies evaluated apoptosis, androgen regulation, angiogenesis, autocrine and oncogene activities. We now evaluate cluster of differentiation-44 (CD44), a stem cell marker associated with tumor growth, to evaluate whether BCL-2 compensation activates or expands this cell population.
Materials and Methods
Oligonucleotides. Oligos (mono- and bispecific) were purchased from Eurofins MWG Operon (Huntsville, AL, USA). Each was phosphorothioated on three terminal bases at the 5’ and 3’ positions. Stock solutions were prepared to a final concentration of 625 μM in sterile Dulbecco phosphate buffered saline (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 [epidermal growth factor receptor (EGFR) or BCL-2].
MR4: (monospecific, targeting BCL-2): T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T; MR24: (bispecific, targeting EGFR/BCL-2): G-A-G-G-G-T-C-G-C-A-T-C-G-C-T-G-C-T-C-T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T; MR42: (bispecific, targeting BCL-2/EGFR) T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T-G-A-G-G-G-T-C-G-C-A-T-C-G-C-T-G-C-T-C.
Cell culture. LNCaP cells were grown in RPMI-1640 supplemented with 10% bovine serum, 1% L-glutamine and 1% penicillin/streptomycin in an incubator with 5% CO2. Log-phase cells were harvested using EDTA/trypsin and equally-distributed into 75 cm2 flasks (Corning, NY, USA). At intervals, media were either supplemented or replaced with fresh.
Oligo treatment prior to polymerase chain reaction (PCR). Four days prior to oligo addition, when cell density approached 75% confluence, 10 ml of fresh medium were added. Cells were incubated for an additional three days before 5 ml of media was replaced with fresh the day before oligos were added. Stock oligos (100 μl) were added to bring the final concentration to 6.25 μM. Incubation proceeded for an additional 24 h in the presence or absence of monospecific MR4, or the bispecifics MR24 and MR42.
RNA extraction. Following treatment, the medium was removed, a single milliliter of cold (4°C) RNAzol B (Sigma-Aldrich, St. Louis, MO) was added to each 75 cm2 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 was placed on ice for 5 min, was spun at 12,000 ×g for 15 min, and the upper aqueous volume was removed and placed in a fresh tube. An equal volume of isopropanol was added, the tube shaken, and kept at 4°C for 15 min before similar centrifugation to pellet the RNA. The supernatant was then removed, the pellet washed in a single milliliter of 75% ethanol, then spun for 8 min at 7,500 ×g. The ethanol was pipetted-off and the formed pellet air dried at −20°C.
RNA quantitation. RNA was resuspended in 250 μl of diethylpyrocarbonate (DEPC)-treated H2O, and quantified using a Qubit florometer and Quant-iT RNA assay kit (Invitrogen, Carlsbad, CA, USA). DEPC is an inhibitor of RNase activity.
RT-PCR. Extracted RNA was diluted in DEPC-treated water to 40 μg/μl, then 1-4 μl of this RNA was added to1 μl of both sense and antisense primers (forward and reverse sequences) for actin, BCL-2 and CD44. From a kit purchased from Invitrogen, the following reactants were added for RT-PCR: 25 μl of 2× reaction mixture, 2 μl SuperScript III RT/platinum Taq mix, tracking dye, and 3 μl MgSO4 (of a 5 mM stock concentration). DEPC-treated water was added to yield a final volume of 50 μl. RT-PCR was performed for two rounds of 25 cycles using the F54 program in a Sprint PCR Thermocycler (BioRad, Hercules, CA, USA). As a control for RT-PCR product formation, human actin expression was tested in RNA extracted from HeLa cells which was provided in a kit purchased from Invitrogen (in the reaction mixture, no MgSO4 was included, the difference compensated for by 3 μl of DEPC treated water).
Primers: Actin: Forward primer sequence: 5’ CAA ACA TGA TCT GGG TCA TCT TCT C 3’, reverse primer sequence: 5’ GCT CGT CGT CGA CAA CGG CTC 3’. The PCR product was 353 base pairs in length.
BCL-2: Forward primer sequence: 5’ GAG ACA GCC AGG AGA AAT CA 3’, reverse primer sequence: 5’ CCT GTG GAT GAC TGA GTA CC 3’. The PCR product was 127 base pairs in length.
CD44: Forward primer sequence: 5’ ACT TCA CCC CAC AAT CTT GA 3’, reverse primer sequence: 5’ GTG GCT TGT TGC TTT TCA GT 3’. The PCR product was 245 base pairs in length.
Detection and quantification of product. Agarose gel electrophoresis: Agarose gels (1.5%) were prepared in a 50 ml volume of TBE buffer (1× solution: 0.089 M Tris borate and 0.002M EDTA, pH 8.3), containing 3 μl of ethidium bromide in a Fisher Biotest electrophoresis system (Fisher, Houston, TX, USA). Samples were run for 2 h at a constant voltage of 70 V, using a BioRad 1000/500 power supply. 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 bromphenol blue tracking dye, were run in each gel.
Quantification: Gels were visualized under UV light and photographed using a Canon 800 digital camera. Photos were converted to black and white format and bands quantified using Medical Image Processing and Visualization (Mipav) software provided by the National Institute of Health (Bethesda, MD, USA). Means and standard deviations were compared using Student t-tests to determine significance.
Results
BCL-2 expression. As a control (see reference 11) for RT-PCR product formation, human actin expression was assessed in RNA extracted from HeLa cells (11).
LNCaP cells incubated for 24 h in the presence of 6.25 μM of oligos suppressed BCL-2 expression, and supporting the finding of comparable biological activity of both mono- and bispecific oligos measured in the in vitro cell growth inhibition experiments (11). When identified product bands on agarose gels were photographed and Mipav quantified, the greatest BCL-2 expression was always found in untreated LNCaP cells. Those treated with mono- or bispecific oligos produced bands with obvious (to the naked eye) suppression. For each evaluated oligo, the greatest amount of suppression measured approached 100% for monospecific MR4; and 86% and 100% for bispecifics MR24 and MR42, respectively. Suppression was found in both repeat PCR runs with BCL-2 primers, as well as in repetitive agarose gel quantifications.
CD44 expression. Comparable amounts of extracted RNA from LNCaP cells treated with either mono- or bispecific oligos directed against BCL-2 (and EGFR in the bispecifics) were then evaluated by RT-PCR using primers directed against CD44. When the background intensity was subtracted, the relative intensity of all bands corresponding to CD44 representing cells treated with MR4, MR24 and MR42, compared to controls, were non-significantly increased by 3.0%±33.6, suppressed 16.4%±49.1 and suppressed 9.2%±26.5 respectively. These results were pooled from both duplicate PCR runs and gels, and indicate that no significant changes in CD44 expression were produced by any oligo type. Figure 1 shows a typical agarose gel.
Agarose gel showing the expression of CD44 in LNCaP cells following treatment with oligonucleotides directed against BCL-2.
Discussion
Gene therapy is often promoted as a highly-specific treatment to control aberrant gene expression by tumor cells, particularly when growth factors, receptors or apoptosis inhibitors are excessively produced. However, it is apparent that it is not as specific as thought at first. Antisense oligos consist of nucleotide bases synthesized complimentary in sequence to mRNA. When hybridized to mRNA, they produce a translational arrest of the targeted gene's mRNA expression. Now in clinical trials against a variety of solid tumors, this method is effective, relatively non-toxic and inexpensive. While it's understandable that genes which share sequence homology would also be susceptible to antisense oligos directed at common sequences, what is not expected is compensatory effects on non-targeted genes.
Tumors consist of genetically-unstable heterogenous cells capable of rapid mutation and selection. Just as bacteria and viral agents develop resistance to chemotherapeutics, tumor cells have similar capability. In normal cells, growth and death pathways are highly regulated, but tumor cells can develop resistance through both an increased production or sensitivity to positive growth factors, or an evasion of apoptosis. These growth and death pathways provide the targets for much of the gene therapy being commercially developed and mediated via antisense oligos.
Oligos produced by Oncogenex Pharmaceuticals (Bothell, WA, USA) have reached clinical trials for the treatment of prostate cancer (OGX-011), while others remain in pre-clinical development (OGX-225). Often administered in combination with traditional chemotherapy, these oligos target BCL-2, clusterin (OGX-011 in phase II testing), heat-shock protein 27 (OGX-427), and insulin growth factor-binding proteins (OGX-225) (12). Genta has also conducted a phase III test using oligos (Genasense; oblimersen) directed against BCL-2 for treating melanoma, chronic lymphocytic leukemia and various types of solid tumors (13), but compensatory effects produced by this agent have not yet been reported.
Our laboratory at the Hektoen Institute has developed and tested bispecific oligos containing dual mRNA-binding sites. We first reported that mono- and bispecific oligos directed against bcl-2 had comparable activity when evaluated using RT-PCR (11). Subsequent experiments (Table I) tested their activity against other proteins associated with growth and development, in an effort to identify compensatory changes in other pathways which could influence BCL-2 suppression
The first series of experiments evaluated compensatory changes in apoptosis and involved measuring the activity of BCL-2 (11), BCL-2-associated X protein (bax) (14), clusterin (15) and caspase-3 (1) in LNCaP cells. Only caspase-3 exhibited compensation (1). Since the level of expression for this promoter was suppressed, it suggests that one way for tumors to overcome BCL-2 suppression is to reduce the promotion of apoptosis. It would also suggest that when employing oligos to suppress BCL-2, caspase-3 activity should be maintained or restored.
The second group of experiments evaluated androgen regulation, specifically the AR (5), and its co-activators p300 (6), IL-4 (7) and IL-6 (7). While AR and p300 were increased by mono- and bispecific oligos, no such alterations were seen for IL-4. The AR plays a principle role in prostate function, cancer progression and targeted treatment strategies. It acts as a transcription factor to enhance the synthesis of growth-stimulating proteins, including insulin-like growth factor (IGF) (16). Although disruption of this process by androgen deprivation provides the rationale for most types of prostate cancer treatment, most tumors recur in an androgen insensitive form within a few years. At this stage, genes are driven towards transcription by both AR and co-activating transcription factors such as p300 and cAMP response element-binding (CREB) binding protein (CREBBP). p300 is essential for cell growth and governs the expression of the cyclins regulating the G1, S, G2 and M phases of mitosis (17). Acting with IL-6, p300/CBP plays a role in the androgen-independent expression of prostate-specific antigen (PSA) (18). In the LNCaP model, administration of R1881 reduces both CREBBP mRNA and the encoded CREBBP protein suggesting that following androgen ablation, the expression of some co-activators increases and contributes to a state of AR hypersensitivity (19). Treatment of prostate cancer cells with siRNA directed against p300 reduces cancer cell growth (19), and eliminates the ability of IL-6 to induce PSA (17). Since both transcriptional co-activator proteins p300 and CREBBP are expressed to a greater extent in advanced prostate cancer (19), well-differentiated, androgen-sensitive, LNCaP cells would be expected to have relatively low expression of p300, and we have shown this to be true (6). In untreated LNCaP cells, p300 expression is barely detectable. The enhanced expression seen following oligo treatment makes its induction appear more impressive, and implies a transition to a pattern of gene expression more associated with later stage disease. This suggests that oligo treatment directed against BCL-2 can not only be evaded through compensatory changes in expression which encourage tumor growth, but may also contribute to hormone hypersensitivity mediated through enhanced AR and p300 expression.
Summary of gene expression findings from use of oligonucleotides directed against BCL-2
The third group of experiments evaluated the expression of cell surface PSMA and secreted PSA and prostatic acid phosphatase (PAP) differentiation (proteins) antigens. Only PSMA expression was altered, and only by these bispecific oligos (9, 20). This peculiarity reminded us that we previously postulated that these bispecifics were capable of forming double-stranded binding between complementary intra-strand bases. Such double-stranded nucleic acids are thought to be interferon (IFN) inducers, capable of enhancing the expression of cell surface antigens such as tumor necrosis factor-alpha (TNF-α) and human leukocyte antigen (HLA) groupings. To test this hypothesis, we evaluated IFN expression (10), and found it to be significantly elevated by the bispecifics. Surface expression of PSMA could enhance targeting by cytotoxic T-cell against prostate cancer cells.
The last group of experiments described here evaluated effects upon the insulin-like growth factor-1 (IGF1) autocrine loop, angiogenesis (21) and the v-MYC oncogene (8). Neither IGF1 nor vascular endothelial growth factor (VEGF) were affected. However, the monospecific and MR24 bispecific oligos significantly enhanced v-MYC activity. MR42 produced no such increase, possibly due to the 5’ position of its BCL-2 binding site and steric hindrance. Gene therapy is a complex process requiring multiple pathways (and the regulatory proteins) to be simultaneously regulated. Clinically these types of experiments are important because they suggest that for oligo-mediated BCL-2 suppression to be effective, caspase-3 activity should be either maintained or restored (1). As a result of the experiments described here no compensatory effects involving IGF1 or IL-4 are noted. Therefore, it would not appear that further manipulation of these genes is required.
Tumors are resilient in their efforts to overcome even newly-developed therapeutics and become resistant to therapy. If gene therapy is to be effective, we must understand how primary effects evoke compensatory changes.
In 2012, the American Cancer Society estimated that in spite of early detection, screening for PSA and effective treatments for localized disease, in the United States there were 28,170 deaths from prostate cancer with 241,740 newly-diagnosed cases (22). New types of treatment, including gene therapy and translational inhibition must be developed and employed (probably in combination with traditional androgen ablation).
Acknowledgements
The Cellular Biology Laboratory at the Hektoen Institute is supported, in part, by the Blum Kovler Foundation, the Cancer Federation, Safeway/Dominicks Campaign for Breast Cancer Awareness, Lawn Manor Beth Jacob Hebrew Congregation, the Max Goldenberg Foundation, the Sternfeld Family Foundation, and the Herbert C. Wenske Foundation.
- Received November 26, 2012.
- Revision received January 23, 2013.
- Accepted January 24, 2013.
- Copyright © 2013 The Author(s). Published by the International Institute of Anticancer Research.
