Inhibition of progression to androgen-independence by combined adjuvant treatment with antisense BCL-XL and antisense Bcl-2 oligonucleotides plus taxol after castration in the Shionogi tumor model

We have reported that antisense Bcl-2 oligodeoxynucleotide (ODN) delays progression to androgen independence in the androgen-dependent (AD) mouse Shionogi tumor model. Here, we characterize changes in bcl-xL, another important anti-apoptotic gene, and test the efficacy of adjuvant antisense Bcl-xL ODN therapy either alone or in combination with antisense Bcl-2 ODN and chemotherapy after castration in the Shionogi tumor model. Bcl-xL mRNA levels increased up to 3-fold postcastration and remained 1. 5-fold higher in androgen-independent (AI) recurrent tumors compared with AD tumors before castration. Treatment of Shionogi cells with antisense Bcl-xL ODN inhibited Bcl-xL expression in a dose-dependent and sequence-specific manner. Systemic administration of antisense Bcl-xL ODN in mice bearing Shionogi tumors after castration delayed emergence of AI recurrent tumors. We then examined whether combined adjuvant antisense Bcl-xL and/or Bcl-2 ODNs plus taxol (paclitaxel) therapy further delays time to AI progression. Combined treatment of Shionogi cells with antisense Bcl-xL and Bcl-2 ODNs significantly enhanced taxol chemosensitivity compared with either agent alone, reducing the IC(50) of taxol by more than 1 log. Apoptotic DNA laddering and cleavage of poly(ADP-ribose) polymerase were more substantial after treatment with combined antisense Bcl-2 and Bcl-xL ODNs plus taxol than that with either 2 agents. Adjuvant administration of antisense Bcl-xL and Bcl-2 ODNs plus micellar taxol resulted in a significantly delayed time to AI recurrence compared with administration of either 2 agents. Our findings suggest that Bcl-xL represents a suitable molecular target for antisense ODN strategy and illustrate the potential additive effects of multi-target pharmacology for cancer therapy.     

Antisense Bcl-2 oligodeoxynucleotides inhibit progression to androgen-independence after castration in the Shionogi tumor model.

Progression to androgen-independence remains the main obstacle to improving survival for patients with advanced prostate cancer. Although Bcl-2 expression in normal prostatic epithelial cells is low or absent, Bcl-2 is highly up-regulated in prostate cancer cells after androgen withdrawal and during progression to androgen-independence. Here, we test the efficacy of antisense Bcl-2 oligodeoxynucleotide (ODN) therapy administered adjuvantly after castration to delay time to androgen-independent recurrence in the androgen-dependent mouse Shionogi tumor model. Treatment of Shionogi tumor cells in vitro with antisense Bcl-2 ODN inhibited Bcl-2 expression in a dose-dependent and sequence-specific manner. Systemic administration of antisense Bcl-2 ODN in mice bearing Shionogi tumors beginning 1 day postcastration resulted in a more rapid regression of tumors and a significant delay of emergence of androgen-independent recurrent tumors. Furthermore, despite significant reduction of Bcl-2 expression in tumor tissues, antisense Bcl-2 ODN had no effect on Bcl-2 expression in normal mouse organs. These findings illustrate the potential utility of antisense Bcl-2 therapy for prostate cancer in an adjuvant setting with androgen ablation.     

Chemosensitization and delayed androgen-independent recurrence of prostate cancer with the use of antisense Bcl-2 oligodeoxynucleotides

BACKGROUND: Increased expression of the bcl-2 gene has been observed in prostate cancer cells after androgen withdrawal and has been associated with the development of androgen independence and chemoresistance. The objective of this study was to determine whether antisense Bcl-2 oligodeoxynucleotides could enhance paclitaxel cytotoxicity and delay androgen-independent progression. METHODS: Northern and western blot analyses were used to measure changes in Bcl-2 expression in mouse Shionogi tumor cells after treatment with antisense Bcl-2 oligodeoxynucleotides and/or paclitaxel. Growth inhibition and induction of apoptotic cell death were assessed with the use of standard methods. All P values are two-sided. RESULTS: Treatment of Shionogi tumor cells with 500 nM antisense Bcl-2 oligodeoxynucleotides decreased expression of Bcl-2 messenger RNA (mRNA) by approximately 85%. Paclitaxel treatment induced Bcl-2 protein phosphorylation but did not alter Bcl-2 mRNA expression. Antisense Bcl-2 oligodeoxynucleotide treatment substantially enhanced paclitaxel chemosensitivity in a dose-dependent manner. Characteristic apoptotic DNA laddering and cleavage of poly(adenosine diphosphate-ribose) polymerase were demonstrated only after combined treatment. Adjuvant in vivo administration of antisense Bcl-2 oligodeoxynucleotides and micellar paclitaxel following castration resulted in a statistically significant delay of androgen-independent, recurrent tumors compared with administration of either agent alone (P<.001, Mantel-Cox log-rank test). Combination therapy also statistically significantly inhibited the growth of established hormone-refractory tumors compared with treatment with either agent alone (P<.001, Student's t test). CONCLUSIONS. Combined treatment with antisense Bcl-2 oligodeoxynucleotides and paclitaxel could be a novel and attractive strategy to inhibit progression to androgen-independent disease as well as growth of hormone-refractory prostate cancer through deprivation of Bcl-2 function.     

Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor

We have shown recently that a 15-mer phosphorothioate oligodeoxynucleotide (ODNas750/15) that hybridizes to the (CAG)n polyglutamine region of mRNA encoding human androgen receptor (AR) inhibits the expression of AR in LNCaP prostate cancer cells in vitro. This AR downregulation was accompanied by significant cell growth inhibition and reduced PSA secretion. In the present study we investigated the effects of this antisense AR ODN on prostate tumor growth in vivo using a mouse xenograft model. Via subcutaneously implanted diffusion pumps, either ODNas750/15 or a scrambled control sequence ODNsr750/15 was continuously administered into LNCaP tumor-bearing male nude mice for 7 weeks. Compared with untreated control animals, treatment with ODNas750/15 resulted in significant tumor growth inhibition. Retardation of tumor growth was also significant in castrated mice, whereas the scrambled control ODN did not exert any effects. No side effects such as loss of body weight were observed at any time of treatment. ODN treatment was well tolerated and, in contrast to castration, did not induce shrinkage of mouse prostates. Both AR expression in the tumor and PSA levels in mouse serum correlated with tumor size. However, we failed to demonstrate a correlation between tumor retardation and Ki-67 antigen expression and the number of apoptotic cells, respectively. Testing of antisense-treated LNCaP cells revealed that expression levels of other proteins that contain shorter polyglutamine sequence stretches such as HDAC2, TFIID, and c-jun were not affected. The present study demonstrates that downregulation of AR with antisense ODNas750/15 causes prostate tumor growth inhibition. These results further point out the important role of the AR in prostate tumors and support further testing of AR downregulation for treatment of prostate cancer.     

Testosterone-repressed prostate message-2 is an antiapoptotic gene involved in progression to androgen independence in prostate cancer

Although initially reported as an androgen-repressed gene in the rat prostate, the functional role of testosterone-repressed prostate message-2 (TRPM-2) in apoptosis remains undefined. Inhibition of castration-induced apoptosis by calcium channel blocker treatment in androgen-dependent Shionogi tumors resulted in the prevention of TRPM-2 gene up-regulation, suggesting that TRPM-2 is not directly androgen-repressed, but is regulated by apoptotic stimuli. The overexpression of the TRPM-2 gene in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to androgen ablation in vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucleotide (ODN) therapy in the Shionogi tumor model and demonstrated that the systemic administration of antisense TRPM-2 ODNs in mice bearing Shionogi tumors after castration resulted in a more rapid onset of apoptosis and time to complete regression, as well as a significant delay of emergence of androgen-independent recurrent tumors compared to control ODN treatment. Collectively, these findings illustrate that TRPM-2 is an antiapoptotic rather than an androgen-repressed gene that confers resistance to androgen ablation and thereby helps accelerate the progression to androgen independence.     

Comparative RNA-seq analysis reveals potential mechanisms mediating the conversion to androgen independence in an LNCaP progression cell model

The androgen-independent phenotype is an important symptom of refractory prostate cancer. However, the molecular mechanisms underlying this phenotypic conversion remain unclear. Using RNA-seq analysis of androgen-dependent prostate cancer cells (LNCaP) vs. androgen-independent cancer cells (LNCaP-AI-F), we identified 788 differentially expressed genes, 315 alternative splicing events, and eight novel LNCaP-AI-F-specific fusion genes. The fusion genes EIF2AK1-ATR and GLYR1-SLC9A8 were predicted to be damaging and oncogenic. We also observed dramatic changes in androgen receptor (AR)-mediated pathway molecules, including prostate-specific antigen (PSA, a major biomarker of prostate cancer) and AR variants, as well as neuroendocrine-like (NE-like) and tumor stem cell-like characteristics, during androgen-independent phenotype progression. Our findings provide new insights into the regulatory complexities of refractory prostate cancers.     

TENB2, a proteoglycan identified in prostate cancer that is associated with disease progression and androgen independence.

TENB2 encodes a putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, which has been identified through the application of a differential display technique to a xenograft model of prostate cancer. Northern analysis and competitive PCR were used to demonstrate significantly increased TENB2 expression (p = 0.0003) on the acquisition of androgen independence in the model system. TENB2 is also overexpressed in clinical prostate carcinoma vs. its benign counterpart (p < 0.0001), with particular prominence in high-grade tumours, and shows a high degree of tissue specificity, being detected on a multitissue Northern array exclusively in brain and prostate material. Studies of recombinant protein expression demonstrate that TENB2 is a chondroitin sulphate proteoglycan. The presence of an EGF and 2 follistatin domains suggests a role in the regulation of growth factor signalling either as a ligand precursor, a membrane-bound receptor or as a binding protein for growth factors. These data are indicative of a significant role for TENB2 in the progression of poorly differentiated tumour types, with implications for prostate cancer detection, prognosis and therapy.     

Optimal timing and dosage of chemotherapy as a combined treatment with androgen withdrawal in the human prostate LNCaP tumour model

Although numerous chemotherapeutic agents have been evaluated for patients with advanced prostate cancer, none have demonstrated improved survival benefits. Here, in order to determine whether the efficacy of chemotherapy can be enhanced by changing the regimen, we evaluated the effect of the varied timing and dosage of chemotherapy in combination with androgen withdrawal on time to androgen-independent (AI) progression in the human androgen-dependent LNCaP tumour model. We first demonstrated the synergistic effect of mitoxantrone on induction of apoptosis in LNCaP cells maintained in the steroid hormone-depleted charcoal-stripped media (CSM) compared to those in the standard media. In addition, this synergy was most remarkable during the simultaneous treatment of LNCaP cells with mitoxantrone and CSM compared to the pre- or post-use of mitoxantrone with CSM. LNCaP tumour growth in athymic nude mice and their increase in serum PSA levels were significantly inhibited by the simultaneous injection of mitoxantrone with castration, compared to the administration of mitoxantrone 2 weeks before or after castration. The TUNEL staining revealed that apoptotic cell death was extensively induced in LNCaP tumours treated with simultaneous castration and mitoxantrone compared to tumours treated with the other schedules. Furthermore, nude mice bearing LNCaP tumours were injected with a total of 0.5 mg mitoxantrone divided into 2, 5 and 10 days, with the most significant therapeutic effect and delayed AI progression observed in mice given injection of mitoxantrone for 2 days. These findings suggest that this might be the optimal way to perform cytotoxic chemotherapy and androgen withdrawal simultaneously in patients with advanced prostate cancer and to administer chemotherapeutic agents at high dosage within short intervals.     

Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells

Calreticulin has been identified previously as one of the androgen-response genes in the prostate. The role of calreticulin in androgen action was studied using androgen-sensitive LNCaP and androgen-insensitive PC-3 human prostate cancer cell lines. Calreticulin appears to be a primary androgen-response gene in cultured LNCaP cells because androgen induction of calreticulin mRNA resists protein synthesis inhibition. Calreticulin is a high capacity intracellular Ca2+ binding protein, suggesting that calreticulin expression is likely to be associated with the intracellular Ca2+ buffering capacity that could regulate the sensitivity to cytotoxic intracellular Ca2+ overload. As expected, androgen protects androgen-sensitive LNCaP but not androgen-insensitive PC-3 cells from cytotoxic intracellular Ca2+ overload induced by Ca2+ ionophore A23187. To provide evidence for the role of calreticulin in reducing cytotoxic effect of Ca2+ influx in prostatic cells, we have shown that calreticulin antisense oligonucleotide down-regulates calreticulin protein level and significantly increases the sensitivity to A23187-induced apoptosis in both LNCaP and PC-3 cells. Furthermore, calreticulin antisense oligonucleotide reverses the androgen-induced resistance to A23187 in LNCaP cells. The above observations collectively suggest that calreticulin mediates androgen regulation of the sensitivity to Ca2+ ionophore-induced apoptosis in LNCaP cells.     

Docetaxel followed by castration improves outcomes in LNCaP prostate cancer-bearing severe combined immunodeficient mice.

PURPOSE: Androgen ablation is the standard initial treatment for advanced prostate cancer; however, tumors eventually develop androgen independence and become incurable. Chemotherapy is commonly used after hormone treatment fails but has not shown significant survival benefit. Studies suggest that androgen ablation can select for a population of hormone-independent cells that are also relatively chemotherapy resistant. Thus, it may be therapeutically advantageous to target prostate cancer with chemotherapy before hormone ablation. This study was undertaken to determine the relative efficacy of such an approach in a preclinical model of prostate cancer. EXPERIMENTAL DESIGN: Severe combined immunodeficient mice bearing human LNCaP prostate tumors were treated with docetaxel and/or surgical castration applied singly, concurrently, or in different sequences. Treatment efficacy was determined by tumor volume and growth delay measurements. The extent of apoptosis in tumors in response to treatments was assessed via terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. In addition, Western blots were done to study the relative expression of Bcl-2 and Bax in the tumors. RESULTS: Docetaxel followed by castration showed the most potent antitumor effects. In contrast, with the exception of castration alone, castration followed by docetaxel produced the least antitumor activity. TUNEL assays confirmed that the density of apoptotic tumor cells was significantly greater for docetaxel followed by castration than for any other treatment. In tumors of mice treated with single modality therapies, Bax to Bcl-2 ratios decreased significantly after castration, whereas this ratio remained high after docetaxel treatment. CONCLUSION: A treatment sequence of docetaxel followed by hormone ablation may be more effective in treating prostate cancer than concurrent docetaxel/hormone therapy or hormone ablation followed by docetaxel.     

HER-2/neu receptor in prostate cancer development and progression to androgen independence

Development of prostate cancer and progression to androgen-independent disease is correlated with increased expression of growth factors and receptors capable of establishing autocrine and/or paracrine growth-stimulatory loops. A thorough review was made of the current literature and recent abstract presentations at scientific meetings focusing on the role of the HER-2/neu (c-erbB2) receptor in prostate cancer and the potential clinical usefulness of its specific inhibitors. In the past 10 years, conflicting results on HER-2/neu expression in prostate cancer have been reported. More recently, four studies have shown experimental evidence of HER-2/neu in the development of prostate cancer and, more specifically, in the progression to a hormone-refractory clinical behavior. Furthermore, it has been proposed that HER-2 family and androgen receptors function synergistically in the absence of androgen, which suggests a cross-talk between the HER-2/neu and androgen receptor pathways. Finally, clinical trials are in progress in prostate cancer patients to test novel agents that selectively interfere with HER-2/neu activity.     

Antisense-MDM2 sensitizes LNCaP prostate cancer cells to androgen deprivation, radiation, and the combination in vivo

PURPOSE: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. METHODS: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging-based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. RESULTS: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. CONCLUSIONS: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30-40% of men with high-risk disease.     

Combining doxorubicin and liposomal anti-HER-2/NEU antisense oligodeoxynucleotides to treat HER-2/NEU-expressing MDA-MB-435 breast tumor model

This study assessed the in vivo therapeutic activity of an antisense molecule targeted against HER-2/neu expressing mRNA. Antisense activity was evaluated in female SCID/Rag2m mice bearing subcutaneous tumors derived from HER-2/neu-transfected MDA-MB-435 (MDA-MB-435(HER2)) cells, a transfected line derived from the human breast cancer MDA-MB-435 cell line. Animals were treated with free or liposome-encapsulated antisense. The area under the curve (AUC(0-24h)) of the liposomal formulated antisense was demonstrated to be more than 30-fold greater than that of free antisense following intravenous administration. Efficacy was determined by assessing changes in tumor growth rate as well as by an immunohistological end-point evaluating HER-2/neu expression. HER-2/neu protein expression was reduced in mice bearing HER-2/neu-transfected MDA-MB-435 tumors when treated with liposomal antisense. However, tumors in these mice grew at a faster rate than the control, a result that was interpreted to be a consequence of selection of a more rapidly proliferating HER-2/neu-negative subpopulation of cells. Effective control of the MDA-MB-435(HER2) tumors was achieved when antisense treatment was combined with doxorubicin. Tumors derived from animals treated with the combination of doxorubicin and the liposomal antisense against HER-2/neu exhibited no detectable levels of HER-2/neu expression. Antisense targeted against HER-2/neu mRNA was effective in reducing or eliminating HER-2/neu protein expression, and when combined wtih doxorubicin treatment was efficacious in the treatment of mice bearing HER-2/neu-overexpressing human xenograft tumors.     

Growth inhibition of DU-145 prostate cancer cells by a Bcl-2 antisense oligonucleotide is enhanced by N-(2-hydroxyphenyl)all-trans retinamide.

Hormonally insensitive prostate cancer is a relatively slow-growing, but usually fatal, disease with no long-term treatment options. Transformation of normal prostate cells to a malignant phenotype often involves corruption of the apoptotic machineries. Bcl-2 protein is one of the key inhibitors of apoptosis and is often unregulated in advanced prostate cancer. The prostate cancer cell line DU-145 was used as a model of a hormonally insensitive, advanced prostate cancer. Cell growth in liquid culture was significantly inhibited by antisense Bcl-2 oligonucleotides compared with control sense oligonucleotides; inhibition by these oligonucleotides was significantly enhanced on combination with the synthetic retinoid N-(2-hydroxyphenyl)all-trans-retinamide (2-HPR). Interestingly, growth inhibition occurred in the absence of apoptosis as measured using two assay techniques. We hypothesize that in these recalcitrant cells the apoptotic pathway is compromised at several levels, and Bcl-2 may play another role in promoting cell growth. The use of Bcl-2 antisense oligonucleotides plus 2-HPR may provide a novel approach to therapy of hormone-resistant prostate cancer.     

Adrenomedullin, an autocrine/paracrine factor induced by androgen withdrawal, stimulates 'neuroendocrine phenotype' in LNCaP prostate tumor cells

Neuroendocrine (NE) differentiation in prostate cancer (CaP) has been reported to be an early marker associated with the development of androgen independence. The mechanisms by which CaP acquires NE properties are poorly understood. In this study, a putative role of adrenomedullin (AM) in the NE differentiation was investigated. The expression of AM and AM receptors (calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein-2 and -3 (RAMP2 and RAMP3) was evaluated after experimental manipulation of androgen status. Levels of AM mRNA and immunoreactive AM (ir-AM) increased four- to sevenfold in androgen-sensitive LNCaP cells after androgen withdrawal in vitro and in LNCaP xenografts in animals after castration. Treatment of LNCaP cells with androgen analogue (dihydrotestosterone; 10(-9) M) prevented the increase in AM mRNA and ir-AM levels. Interestingly, the expression of CRLR, RAMP2 and RAMP3 is not regulated by androgen status. We demonstrate that in the presence of serum, AM is able to induce an NE phenotype in LNCaP cells via CRLR/RAMP2 and RAMP3, which includes extension of neuritic processes and expression of the neuron-specific enolase (NSE), producing cGMP in a dose-dependent manner, which is mediated by a pertussis toxin-sensitive GTP-binding protein. 8-Bromo-cGMP mimicked the effects of AM on cell differentiation. We demonstrate that AM induces a G-kinase Ialpha translocation to the nucleus. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both AM and 8-bromo-cGMP. In noncastrated animals, administration of AM enhanced expression of NSE and chromogranin A in LNCaP xenografts with a significant increase of NSE levels in serum and no changes in tumor growth. In castrated animals, intraperitoneal injection of AM resulted in a 240+/-18% (P<0.001) increase in tumor volume 36 days after treatment, indicating that the nature of effect of AM in CaP depends on the presence or absence of endogenous androgen. Together, these results demonstrate that AM may function as a mediator of NE-like differentiation in culture as well as in vivo and indicate that its production may be important for tumor resurgence following androgen ablation.     

Lack of toxicity associated with the systemic administrat of antisense oligonucleotides for treatment of rats bearing LNCaP prostate tumors

Antisense oligonucleotides (oligos) are now in clinical trials for the treatment of a variety of diseases. However, concern is sometimes expressed as to the toxicity of such compounds, particularly those with phosphorothioated backbones. We have previously reported (J. Surg. Oncol. 62, 194, 1996) our experience in treating nude mice bearing human PC-3 prostate tumors with phosphorothioated antisense oligos directed against mRNA encoding transforming growth factor-alpha (TGF-α) and the epidermal growth factor receptor (EGFR). This therapy resulted in a 75% (9/12) response rate for the intralesional treatment and a 100% (3/3) response rate for the systemic administration utilizing Alzet diffusion pumps. In the current study, athymic nude rats bearing orthotopically implanted LNCaP tumors, whose establishment was confirmed by the expression of human PSA, were implanted subcutaneously with Alzet diffusion pumps and treated systemically for 14 days with a total of 1 mg of each cligo (2 mg total). Controls consisted of five untreated rats similarly inoculated with LNCaP cells, but which did not receive antisense oligos. After 2 weeks the rats were sacrificed and serum samples were evaluated for BUN, creatinine, LDH and SGOT. Lungs, kidneys, livers, spleens and prostates were also removed for pathologic evaluation. There were no serum marker differences between groups nor was there histologie evidence of oligo toxicity seen in any evaluated tissue. Of interest was the observation that the livers and spleens, as well as prostates, of treated animals revealed mild lymphocytic infiltration compared to controls. We conclude that at this level of administration, there is no toxicity associated with 14-day oligo treatment.