Suppression of BCL2 by Antisense Oligonucleotides and Compensation by Non-Targeted Genes May Enhance Tumor Proliferation.

Antisense oligonucleotides have been used to target regulatory proteins in both in vivo and in vitro models of prostate cancer. Our previous studies showed that oligonucleotide-treated LNCaP prostate cancer cells compensate for diminished expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), an apoptosis inhibitor, by suppressing the expression of caspase-3 (an apoptosis promoter) while enhancing that of serine/threonine protein kinase (AKT1) (another apoptosis inhibitor). In addition, we found an enhanced expression of the androgen receptor (AR), its p300 and interleukin-6 (IL6) co-activators, polymerase transcription mediator (MED12), and growth-regulating signal transducer (STAT3). The net result was an altered pattern of gene expression often associated with more aggressive and proliferative tumors. To further evaluate adaptive compensatory mechanisms related to tumor resistance, aggression and proliferation, herein we evaluated the level of expression of a proliferation antigen (KI-67) and mitosis-regulating cyclins (B1 and D1). Compared to the relative levels of compensation detailed above, we found the expression of KI-67 to be statistically the most enhanced non-targeted protein yet identified in compensation for suppression of BCL2. Expression of cyclin D1 was also significantly enhanced, although to a much lesser extent. As a result, we propose that oligonucleotide-mediated treatment could be more effective when directed towards KI-67 and BCL2. This could be accomplished by dual monospecific targeting KI-67 and BCL2, or with a bispecific (or proposed multispecific) oligonucleotide simultaneously targeting both.

Additional compensatory mechanisms altering antisense oligonucleotide suppression of BCL2: effects upon AKT1 and STAT3.

Antisense oligonucleotides have targeted regulatory proteins in both in vivo and in vitro prostate cancer models. We evaluated mono- and bispecific oligonucleotides which targeted and comparably suppressed B-cell lymphoma-2 BCL-2 (an apoptosis-inhibitory protein) expression in LNCaP cells. These oligonucleotides were administered with lipofectin as part of a nanoparticle delivery system. Treated cells compensated by suppressing caspase-3 (an apoptosis promoter) and enhancing expression of the androgen receptor and its co-activating p300 and IL-6 proteins. This suggests a progression to increased androgen sensitivity (in LNCaP) accompanies BCL-2 suppression and a gene activation pattern associated with more advanced prostate tumors. To further evaluate compensatory mechanisms related to tumor resistance in the present study we evaluate the expressed levels of the AKT1 oncogene and STAT3 transcription factor, finding both to be enhanced.

Oligonucleotide suppression of bcl-2 in LNCaP cells is compensated by increased androgen sensitivity, p53 and oncogene activity, and suppressed caspase-3.

Antisense oligonucleotides (oligos) have been employed against prostate cancer models in both in vivo and in vitro systems. While most target growth factors or their receptors, other oligos are directed against inhibitors of apoptosis or mediators of androgen action. Those which suppress bcl-2 activity (in prostate cancer patients) have even reached clinical trials. We evaluated a set of oligos which targeted and comparably suppressed the expression of bcl-2, an apoptosis inhibitory protein. Our first study reported that LNCaP cells were adapted by suppression of caspase-3 (a promoter of apoptosis). In this study we evaluated additional proteins associated with tumor progression and found the expression of the androgen receptor, its p300 and IL-6 co-activators, as well as v-myc (oncogenic) and (unexpectedly) tumor suppressor p53 genes to be enhanced. We conclude that oligo treatment directed against bcl-2, intended to stimulate apoptosis, can be evaded through compensatory changes in gene activity associated with additional regulators of apoptosis, androgen sensitivity and oncogenesis. This suggests that therapeutic suppression of bcl-2 can promote tumor resistance and transformation to a more aggressive (androgen and oncogene driven) phenotype.

Increased expression of the androgen receptor with p300 and interleukin-6 coactivators compensate for oligonucleotide suppression of bcl-2: no increased CREB binding protein or interleukin-4 expression.

BACKGROUND: Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth regulatory proteins. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis and mediators of androgen action. We previously evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein bcl-2. LNCaP cells adapted to this restoration of apoptosis with suppression of caspase 3 (an apoptosis promoter) and an enhanced expression of the androgen receptor (AR), suggesting an increased sensitivity to androgens. METHODS AND RESULTS: In a continuation of this study, we evaluated the expression of AR coactivators p300, its homolog CREB binding protein (CREBBP) and cytokines interleukin (IL)-4 and IL-6, finding p300 and IL-6 similarly enhanced. CONCLUSIONS: LNCaP cells are hormone sensitive and untreated cells express minimal p300 activity. Therefore, the enhanced expression which followed oligo treatment makes its induction more impressive and implies a pattern of gene expression more associated with later stage (androgen insensitive) disease. This suggests that oligo treatment directed against bcl-2 can be evaded through compensatory changes in AR expression and some coactivators, promoting tumor growth, and may promote transformation of the tumor to a more aggressive phenotype.

Review: description of the developmental patterns of epithelial cells and cancers originating from them will provide many sites for therapy.

Many types of cancers represent different forms of blocked differentiation, but the relationship with major, currently identified cancer driver mutational and epigenetic events is not well defined. Normal cellular differentiation depends upon hierarchically-organized sequences of control and synthetic events coupled with cellular proliferation. An ability to identify and possibly correct or otherwise modify defective cancer stem or daughter cell differentiation, or even non-malignant precursor cell differentiation occurring prior to the effect of oncogene and suppressor genomic activity should provide many new approaches to circumventing the oncogenic process. The use of whole-genome sequencing and related procedures coupled with computer-based analyses of developmental event trees, and with the numerous reports from the ENCODE (Encyclopedia of DNA elements) project that has uncovered the large number of potential underlying regulatory and other functions in the junk DNA of cells should eventually define the temporal progression of normal and aberrant differentiating events as they modulate cellular proliferation. This information should identify many new differentiation sites and their coupling with cellular proliferation as new potential targets susceptible to therapeutic manipulation.

No compensation in CD44 stem cell marker following BCL-2 suppression by antisense oligonucleotides.

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.

No compensation in VEGF expression follows antisense suppression of BCL-2 activity.

Antisense oligonucleotides (oligos) have been employed against prostate cancer models targeting growth-regulatory proteins, and at least one oligo (against bcl-2) has reached clinical trial. We previously found that, in LNCaP cells, mono- and bispecific oligos, which comparably suppressed the expression of bcl-2, compensated with suppression of caspase-3 (apoptosis promoter) activity, and enhanced the expression of the androgen receptor (AR) and its p300 and IL-6 co-activators. In addition, prostate-specific membrane antigen (PSMA) and (possibly its regulator) interferon (IFN) were elevated. A total of 14 proteins distributed between regulators of apoptosis, androgen regulation, differentiation antigens and autocrine-mediated growth have previously been examined. We extend these findings to include vascular endothelial growth factor (VEGF), a promoter of angiogenesis, which is not significantly altered through compensation, and therefore would not need additional regulation for suppressive bcl-2 therapy to be effective (like caspase-3).

Order-of-magnitude estimates of latency (time to appearance) and refill time of a cancer from a single cancer 'stem' cell compared by an exponential and a logistic equation.

BACKGROUND/AIM: The time required before a mass of cancer cells considered to have originated from a single malignantly transformed cancer 'stem' cell reaches a certain number has not been studied. Applications might include determination of the time the cell mass reaches a size that can be detected by X-rays or physical examination or modeling growth rates in vitro in order to compare with other models or established data. MATERIALS AND METHODS: We employed a simple logarithmic equation and a common logistic equation incorporating 'feedback' for unknown variables of cell birth, growth, division, and death that can be used to model cell proliferation. It can be used in association with free or commercial statistical software. RESULTS: Results with these two equations, varying the proliferation rate, nominally reduced by generational cell loss, are presented in two tables. The resulting equation, instructions, examples, and necessary mathematical software are available in the online appendix, where several parameters of interest can be modified by the reader www.uic.edu/nursing/publicationsupplements/tobillion_Anderson_Rubenstein_Guinan_Patel1.pdf. CONCLUSION: Reducing the proliferation rate by whatever alterations employed, markedly increases the time to reach 10(9) cells originating from an initial progenitor. In thinking about multistep oncogenesis, it is useful to consider the profound effect that variations in the effective proliferation rate may have during cancer development. This can be approached with the proposed equation, which is easy to use and available to further peer fine-tuning to be used in future modeling of cell growth.

Differentiated prostatic antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against BCL-2 and EGFR.

Antisense oligonucleotides (oligos) have been administered against in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. While most oligos consist of a single mRNA binding site targeting a single gene product or those with sequence homology, our lab has developed bispecific oligos directed toward two unrelated proteins. In LNCaP cells, we initially identified bispecifics that increased the expression of prostate-specific membrane antigen (PSMA) while not affecting secreted prostate-specific antigen (PSA). We postulated that surface antigen expression is increased by bispecifics able to form double-stranded regions, acting as interferon (IFN-γ) inducers. In other systems, when induced, IFN-γ promotes cell surface antigen expression, including HLA and receptors for tumor necrosis factor. To test this hypothesis, we measured the effect of oligo treatment on both IFN-γ induction and the expression of another secreted product of differentiated prostate cells, prostatic acid phosphatase (PAP). This study 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). Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for PSMA, PSA, PAP, and IFN-γ was subsequently valuated. 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 differences were detected in mRNA levels encoding PSA following treatment with either oligo type. IFN-γ was significantly induced only by bispecific oligos, and PAP expression was similar to PSA. These data support the hypothesis that double strand-forming bispecific oligos induce IFN-γ that enhances cell surface PSMA expression. Expression of tumor-associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.

Compensatory and non-compensatory effects on protein expression following BCL-2 suppression by antisense oligonucleotides.

Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth regulatory proteins. In LNCaP cells, we evaluated both monospecific and bispecific oligos that targeted and comparably suppressed the expression of bcl-2, an apoptosis inhibitory protein. Cells compensated with both suppressed caspase-3 (an apoptosis promoter) activity, and an enhancement of both androgen receptor (AR) and p300 expression. This suggests that a progression to increased androgen sensitivity accompanies bcl-2 suppression, in this tumor line. To further evaluate mechanisms of adaptation, we now evaluate the effects upon the expression of insulin-like growth factor (IGF1) and another AR coactivator, IL-4, thought to increase prostate cancer growth. IGF1 expression was not significantly altered suggesting this pathway need not be regulated when bcl-2 directed gene therapy is employed. In contrast to increased AR and p300 expression that compensated for bcl-2 suppression, the AR coactivator IL-4 expression was not increased, suggesting no role in any increased androgen sensitivity.

In LNCaP cells enhanced expression of both androgen receptor and costimulatory protein p300 compensate for antisense oligonucleotide suppression of bcl-2.

BACKGROUND: Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth stimulatory gene products. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis and mediators of androgen action. In LNCaP cells we evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein bcl-2. LNCaP cells adapted to this restoration of apoptosis with an enhanced expression of the androgen receptor (AR) suggesting an increased sensitivity to androgens. In a continuation of this study, we now evaluate the expression of p300, an AR coactivating protein expressed in the later stages of prostate cancer. METHOD AND RESULTS: In previous experiments, monospecific and bispecific oligos directed against bcl-2 suppressed both the targeted bcl-2 protein (an inhibitor of apoptosis) and the nontargeted caspase-3 (a promoter of apoptosis), potentially negating the effect on apoptosis produced by specific inhibition of bcl-2. In a further study we reported that expression of the AR was significantly enhanced by these oligos. We now report that expression of p300 is similarly enhanced. LNCaP cells are hormone sensitive and the untreated cells expressed minimal p300 activity. CONCLUSIONS: The enhanced expression which followed oligo treatment makes its induction more impressive, and implies a pattern of gene expression more associated with later stage (androgen-insensitive) disease. This suggests that oligo treatment directed against bcl-2 not only can be evaded through compensatory changes in AR expression which promotes tumor growth, but the induced expression of p300 may transition the tumor to a more dedifferentiated and aggressive phenotype.

In LNCaP cells enhanced expression of the androgen receptor compensates for Bcl-2 suppression by antisense oligonucleotides.

BACKGROUND AND METHODS: Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth stimulatory gene products. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis. In LNCaP cells we evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein Bcl-2. LNCaP cells adapted to this restoration of apoptosis with a compensatory suppression of caspase-3 expression, a nontargeted promoter of this process. In a continuation of this study we now evaluate the expression of the androgen receptor (AR) following oligo mediated regulation of apoptosis with suppression of Bcl-2. RESULTS: Monospecific and bispecific oligos directed against Bcl-2 suppressed both the targeted Bcl-2 protein (an inhibitor of apoptosis) and the nontargeted caspase-3 (a promoter of apoptosis), potentially negating the effect on apoptosis produced by specific inhibition of Bcl-2. In contrast, the expression of the AR was significantly enhanced by each type of oligo. CONCLUSIONS: This suggests that when Bcl-2 expression is inhibited there are compensatory changes in the expression of additional proteins which regulate tumor growth, apoptosis and cell survival, and in this scenario might increase or re-establish hormonal sensitivity. If tumors variants are selected which evade gene therapy additional mechanisms of compensation must be identified and subsequently suppressed. These experiments identify pathways by which tumors can develop resistance to gene therapy and suggests additional targets for intervention.

Effects of BCL-2 suppression by antisense oligonucleotides on additional regulators of apoptosis compensatory change in non-targeted protein expression.

Antisense oligonucleotides (oligos) have been employed against prostate cancer in both in vivo and in vivo models. While most oligos contain a single mRNA binding site, our laboratory has developed bispecifics directed towards two. Previous work has determined that when oligos are used to suppress the expression of individual proteins in highly regulated physiologic processes, additional proteins can be affected. These non-targeted (non-specific effects) regulators can compensate for proteins specifically targeted, reverse the intended effect and promote tumor growth. To evaluate specific and compensatory non-specific effects on growth inhibition of LNCaP cells employing mono- and bispecific oligos directed against BCL-2, LNCaP cells were incubated in the presence of oligos specifically directed against BCL-2 [the second binding site was directed against epidermal growth factor receptor (EGFR)] and compared to lipofectin containing controls. Significant, but comparable, growth inhibition was produced by mono- and bispecific forms. Employing RT-PCR to determine BCL-2 expression, mRNA suppression approached 100% for each oligo type: monospecific MR4 (directed only against BCL-2), 100%; and bispecifics MR24 and MR42, 86% and 100% respectively. Based upon inhibition of in vivo growth and BCL-2 expression, bispecific antisense oligos directed against EGFR and BCL-2 mRNAs are at least as effective as a monospecific directed towards BCL-2. To identify a compensatory response to evade apoptosis in the presence of BCL-2 suppression, levels of mRNA encoding non-targeted BAX, caspase-3 and clusterin were evaluated. We initially found that specific suppression of the apoptosis inhibitor BCL-2 in LNCaP cells does not affect (non-targeted) BAX expression and (non-targeted) caspase-3 expression was suppressed. This suggested that tumor cell variants develop which resist apoptosis through diminished expression of this promoter. This study suggests that compensatory changes in the regulation of apoptosis may not be widespread or be limited to apoptosis promoters (caspase-3), since the expression of the non-targeted apoptosis inhibitor clusterin is not affected. Should BCL-2 suppression be clinically employed with antisense oligos, it may only require maintainance (or replacement) of caspase-3 activity.

Bispecific oligonucleotides may induce interferon expression in LNCaP cells enhancing surface antigen expression: effect of intrastrand base pair complementarity.

Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models. Most oligos consist of a single mRNA binding site, targeting a single gene product or others sharing sequence homology. However, our lab has developed bispecifics directed towards two (including unrelated) proteins. Previously we have shown that mono- and bispecific oligos targeting BCL-2 significantly inhibit LNCaP cell growth. Employing reverse transcriptase-polymerase chain reaction we found comparable suppression of expressed BCL-2. Computer models suggested that this activity could, in part, be enhanced by the formation of siRNA-like double-stranded regions, generated by intrastrand base pair complementarity. We hypothesize that these regions could be interferon inducers (like poly I:C) and enhance the expression of prostate specific cell surface antigens. The expression of cell surface prostate-specific membrane antigen (PSMA) and the secreted prostate-specific antigen (PSA) were candidates for evaluation. To test this theory, we evaluated the effects of mono- and bispecific oligos (with intrastrand complementarity), targeting BCL-2, upon the expression of non-targeted proteins PSMA, PSA and interferon-gamma (IFN-γ) in LNCaP cells. Levels of mRNA encoding PSMA were significantly elevated following treatment with the bispecific oligos (directed against both BCL-2 and the epidermal growth factor receptor) but not by the monospecific directed solely against BCL-2. Furthermore, no differences were detected in mRNA levels encoding PSA following treatment with either mono- or bispecific forms. IFN-γ expression was also significantly increased by the bispecific and not by the monospecific oligos, supporting the hypothesis of interferon induction. This suggests that prostate cells (including LNCaP) retain an endogenous interferon-based antiviral defense mechanism (similar to that found in the testes) which is induced by double stranded oligos. Enhanced expression of cell surface differentiation antigens (such as PSMA) could increase targeting by cytotoxic T-cells and potentiate prostate cancer vaccines directed against tumor-associated cell surface antigens.

Does the prostate retain an endogenous antiviral defense system suggesting a past viral etiology for cancer?

Evidence of viral infection of the prostate is presented, and consists of viral isolation as well as antigen and genomic detection of integrated or episomal forms. Prior infection from "ancient" exogenous retroviruses is suggested by residual endogenous forms. The presence of an antiviral defense system based upon interferon induction has been suggested for the testes. I suggest that a similar system is also present in the prostate. Prostate cell lines (or tissue) can be tested directly for expression of interferon-γ (IFN-γ). However, enhanced expression should be induced by IFN inducers such as bispecific oligonucleotides having double stranded regions or poly I:C. RNA can be extracted and RT-PCR performed using commercially available primers for gamma interferon. Evidence for the presence of an interferon based anti-viral system would be indicated not only by interferon expression, but also its enhanced expression following exposure to the inducers. Gamma interferon is primarily associated with lymphocytic expression and immune regulation. Demonstration of its controlled expression in non-immune cells would support the hypotheses suggested here.

Bax expression remains unchanged following antisense treatment directed against BCL-2.

Antisense oligonucleotides (oligos) have been evaluated in both in vivo and in vitro prostate cancer models. Although most contain a single mRNA binding site, our laboratory has also evaluated bispecific types directed toward two proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of two apoptosis regulating proteins, bcl-2 and non-targeted bax, was then evaluated. LNCaP prostate tumor cells were initially incubated for 24 h in the presence of oligos (6.25 µM) directed against bcl-2 and compared to lipofectin containing controls. Comparable and significant growth inhibition was produced by both mono- and bispecific forms. Employing RT-PCR to determine the expression of bcl-2, we found that the greatest amount of mRNA suppression approached 100% for each oligo type: monospecific MR4 (directed only against bcl-2), 100%; and bispecifics MR24 and MR42, 86 and 100%, respectively. We conclude, based upon both inhibition of in vitro growth and bcl-2 expression, that bispecific antisense oligos directed against EGFR and bcl-2 mRNAs are at least as effective as a monospecific directed solely toward bcl-2. In an effort to determine a compensatory response by cells evading apoptosis in the presence of bcl-2 suppression, the levels of mRNA encoding the non-targeted apoptosis activating protein bax were evaluated. Non-targeted protein suppression by these bispecifics has previously been demonstrated against prostate-specific membrane antigen (PSMA). However, in contrast to effects against bcl-2 and PSMA, no significant alteration in bax expression was produced by either oligo type. In LNCaP cells, bcl-2 suppression does not influence bax expression and, at least for this protein, there is no compensatory change in bax expression regulating apoptosis at this level. Identifying changes in the expression of proteins which regulate apoptosis is important if gene therapy targets bcl-2.

Bispecific antisense oligonucleotides have activity comparable to monospecifics in inhibiting expression of BCL-2 in LNCaP cells.

Antisense oligonucleotides (oligos) have been employed against prostate cancer models in both in vivo and in vitro systems. Most oligos employed by investigators include only a single mRNA-binding site, and target only a single gene. However, some target multiple genes which share sequence homology. Recently, our lab has developed bispecific oligos, which target two different genes not related by sequence homology, and which are able to regulate activity in either the same or in different biological pathways. To date, the effectiveness of bispecific oligos has been evaluated solely by in vitro cell growth inhibition studies. This study evaluates the suppression of targeted mRNA by both mono- and bispecific oligos directed towards the apoptosis regulatory protein BCL-2. The bispecifics used contain binding sites for both the epidermal growth factor receptor (EGFR) and BCL-2 mRNA and differ only in the 5' to 3' tandem orientation. LNCaP cells incubated for 24 hours in the presence of 6.25 muM of oligos suppress the expression of BCL-2 mRNA and support the finding that there is comparable biologic activity produced by both mono- and bispecific oligos in in vitro cell inhibition experiments. For each type of oligo (mono- or bispecific) evaluated, the greatest amount of BCL-2 mRNA suppression approached 100% as produced by the monospecific MR(4) (directed only against BCL-2) and for the bispecifics MR2(4) and MR(42), 86% and 100%, respectively. Suppression was found in duplicate PCR runs employing BCL-2 primers, as well as in multiple agarose gel quantifications. Based upon both inhibition of in vitro growth and bCL-2 expression measured by PCR, we conclude that bispecific antisense oligos directed against both EGFR and BCL-2 mRNAs are at least as effective as a monospecific oligo directed solely towards BCL-2. Therefore the addition of a second mRNA-binding site to these oligos does not prevent activity at the initial site specific for BCL-2.

Treatment of prostate and breast tumors employing mono- and bi-specific antisense oligonucleotides targeting apoptosis inhibitory proteins clusterin and bcl-2.

Antisense oligonucleotides (oligos) have demonstrated their efficacy in inhibiting the growth of prostate and breast tumor cells. Previous studies employed first generation, phosphorothioated, cDNA oligos synthesized complimentary to mRNA encoding transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), the anti-apoptosis protein bcl-2, and the androgen receptor (AR). In an effort to construct oligos with greater than one mRNA binding site, bi-specifics have been developed which target combinations of the above proteins, and these have been shown at least as effective as the mono-specific oligos from which their sequences were derived. While all bi-specifics have inhibitory effects, which can be enhanced by the combined administration of an additional chemotherapeutic agent, those bi-specifics which target bcl-2 and EGFR were reported to be the most effective. The experiments presented here are an effort to evaluate a new group of bi-specifics whose targets include the chaperone protein clusterin, whose expression is up regulated in many tumors and activity is known to inhibit apoptosis. Of particular interest were those bi-specifics constructed to target both clusterin and bcl-2 (also an apoptosis inhibitory protein). Cell lines targeted included both prostate LNCaP and PC-3, as well as the breast derived MCF-7. In order to identify agents which enhance oligo activity, but contribute less toxicity, oligos were tested both alone and in combination with either the immune inhibitor Rapamycin, or the chemotherapeutic (and more toxic) Taxol. Results indicate that bi-specifics targeting clusterin are statistically effective, and are similarly enhanced by Rapamycin, or Taxol. When bi-specifics including clusterin as a target, were tested against LNCaP and MCF-7 cells, the level of activity was intermediate between that of the mono-specific compounds tested separately. In experiments which compared both, bi-specifics which included a target for clusterin had inhibitory activity similar to the previously described bi-specifics directed towards bcl-2 and EGFR.

Increased prostate-specific membrane antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against bcl-2 and EGFR.

Antisense oligonucleotides have been employed against in vivo and in vitro prostate cancer models. While most oligos consist of a single mRNA binding site, targeting a single gene product or others sharing sequence homology, our laboratory has developed bispecific oligos directed toward even unrelated proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second bispecific binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) were subsequently evaluated. When LNCaP prostate tumor cells were incubated with bispecific oligos (directed against bcl-2 and EGFR) and compared to lipofectin-containing controls significant growth inhibition resulted. In subsequent experiments, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with the monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either mono- or bispecific oligos. Previously, we suggested that cell growth inhibition produced by some bispecifics could be attributed to complementary double-stranded regions formed by intra-strand base pairs. Double-stranded nucleic acids are known inducers of interferon, which promote expression of cell surface HLA type antigens. If induced, perhaps this cytokine also enhances PSMA expression, making prostate tumor cells a more recognizable target for cytotoxic T cells.

Multigene targeting of signal transduction pathways for the treatment of breast and prostate tumors: comparison between combination therapies employing bispecific oligonucleotides with either Rapamycin or Paclitaxel.

Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-alpha (MR(1))], its binding site the epidermal growth factor receptor [EGFR (MR(2))] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR(4))], and the androgen receptor [AR (MR(5))]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-alpha and EGFR mRNA [TGF-alpha/EGFR (MR(12)) and EGFR/TGF-alpha (MR(21))]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR(24)) and bcl-2/EGFR (MR(42))]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4(45)) and AR/bcl-2 (MR(54))]. Oligo pairs differ in their 5'-3' linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD(50) dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.